Quantitative Imaging of Retinoic Acid Activities in Living Mammalian Cells.

Retinoic acid (RA) is an intriguing metabolite that is necessary for embryonic development and differentiation in vertebrates. The present protocol demonstrates how to image RA activities indirectly in mammalian cells with ligand-activatable single-chain bioluminescence (BL) probes. We introduce 13 different molecular designs for characterizing an efficient single-chain probe that quantitatively visualizes RA activities with significant sensitivity.

Molecular Tension Probe for In Vitro Bioassays.

Cell-free bioassays (CFBs) provide their own distinctive merits over cell-based bioassays (CBBs) including (i) rapid and on-site applicability, (ii) long-term utility, and (iii) bioanalytical versatility. The authors previously introduced a unique bioluminescent imaging probe for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. In this chapter, we exemplify that a full-length artificial luciferase is sandwiched between FRB (FKBP-rapamycin-binding domain of FKBP12-rapamycin-associated protein) and FKBP (FK506-binding protein) via minimal flexible linkers, named FRB-A23-FKBP.

Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments.

The balance of programmed death-1 (PD-1)-expressing CD8 T cells and regulatory T (Treg) cells in the tumor microenvironment (TME) determines the clinical efficacy of PD-1 blockade therapy through the competition of their reactivation. However, factors that determine this balance remain unknown. Here, we show that Treg cells gain higher PD-1 expression than effector T cells in highly glycolytic tumors, including MYC-amplified tumors and liver tumors.

Ligand-Activatable BRET9 Probes for Imaging Molecular Events in Living Mammalian Cells.

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions. The present protocol introduces a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length artificial luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched in between a protein pair of interest, FRB and FKBP, and further linked to a fluorescent protein as the energy acceptor for studying protein-protein interaction. A specific ligand, rapamycin, which initiates intramolecular interactions of FRB and FKBP inside the probe, which develops molecular strain in the sandwiched ALuc23 to complete its folding, thus, the probe system greatly enhances both the overall bioluminescence (BL) spectrum and the BRET signal in the far-red (FR) region.

A New Lineage of Artificial Luciferases for Mammalian Cell Imaging.

The present protocol introduces a new lineage of artificial luciferases (ALucs) with unique optical properties for mammalian cell imaging. The primary candidate sequence was first created with a sequence logo generator, resulting in a total of 11 sibling sequences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools in public databases. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases and from a prior series of ALucs produced by our laboratory using a smaller basis set.

Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve.

Background: Different types of tumors have varying susceptibility to immunotherapy and hence require different treatment strategies; these cover a spectrum ranging from 'hot' tumors or those with high mutational burden and immune infiltrates that are more amenable to targeting to 'cold' tumors that are more difficult to treat due to the fewer targetable mutations and checkpoint markers. We hypothesized that an effective anti-tumor response requires multiple agents that would (1) the immune response and generate tumor-specific effector cells; (2) the number and breadth of the immune effector cells; (3) the anti-tumor activity of these immune cells in the tumor microenvironment; and (4) the tumor response to widen immune effector repertoire.

Methods: A hexatherapy combination was designed and administered to MC38-CEA (warm) and 4T1 (cool) murine tumor models.

Color-tunable bioluminescence imaging portfolio for cell imaging.

The present study describes a color-tunable imaging portfolio together with twelve novel coelenterazine (CTZ) analogues. The three groups of CTZ analogues create diverse hues of bioluminescence (BL) ranging from blue to far red with marine luciferases. We found that the hue completes the whole color palette in the visible region and shows red-shifted BL with a marine luciferase: for example, Renilla luciferase 8 (RLuc8) and Artificial Luciferase 16 (ALuc16) show 187 nm- and 105 nm-redshifted spectra, respectively, by simply replacing the substrate CTZ with 1d.

Therapy of Established Tumors with Rationally Designed Multiple Agents Targeting Diverse Immune-Tumor Interactions: Engage, Expand, Enable.

Immunotherapy of immunologically cold solid tumors may require multiple agents to engage immune effector cells, expand effector populations and activities, and enable immune responses in the tumor microenvironment (TME). To target these distinct phenomena, we strategically chose five clinical-stage immuno-oncology agents, namely, (i) a tumor antigen-targeting adenovirus-based vaccine (Ad-CEA) and an IL15 superagonist (N-803) to activate tumor-specific T cells, (ii) OX40 and GITR agonists to expand and enhance the activated effector populations, and (iii) an IDO inhibitor (IDOi) to enable effector-cell activity in the TME. Flow cytometry, T-cell receptor (TCR) sequencing, and RNA-sequencing (RNA-seq) analyses showed that in the CEA-transgenic murine colon carcinoma (MC38-CEA) tumor model, Ad-CEA + N-803 combination therapy resulted in immune-mediated antitumor effects and promoted the expression of costimulatory molecules on immune subsets, OX40 and GITR, and the inhibitory molecule IDO.

Highly sensitive eight-channel light sensing system for biomedical applications.

We demonstrate the potential of an eight-channel light sensing platform system, named Black Box I (BBI), for rapid and highly sensitive measurement of low-level light using a nonradioactive optical readout. We developed, normalized, and characterized the photon sensitivities of the eight channels of the BBI using placental alkaline phosphatase (PLAP) as a model imaging reporter. We found that the BBI system had a statistically strong linear correlation with the reference IVIS Lumina II system.

Ligand-activated BRET9 imaging for measuring protein-protein interactions in living mice.

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor.

Efficient ADCC killing of meningioma by avelumab and a high-affinity natural killer cell line, haNK.

Meningiomas are the most common adult primary tumor of the central nervous system, but there are no known effective medical therapies for recurrent meningioma, particularly for World Health Organization grade II and III tumors. Meningiomas arise from the meninges, located outside the blood-brain barrier, and therefore may be directly targeted by antibody-mediated immunotherapy. We found that programmed cell death ligand 1 (PD-L1) was highly expressed in multiple human malignant meningioma cell lines and patient tumor samples.

Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes.

Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity.

In vitro Determination of Rapamycin-triggered FKBP-FRB Interactions Using a Molecular Tension Probe.

As protein-protein interactions (PPI) have been mostly investigated in cellulo or in vivo, it is unclear whether the PPI-based imaging schemes are practically valid in a bioanalytical means in vitro. The present study exemplifies the PPI in vitro inside a unique single-chain probe, named TP2.4, which carries a full-length artificial luciferase (ALuc) sandwiched in between two model proteins of interest, e.

An IL-15 superagonist/IL-15Rα fusion complex protects and rescues NK cell-cytotoxic function from TGF-β1-mediated immunosuppression.

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a fundamental role in the immunosurveillance of cancers. NK cells of cancer patients exhibit impaired function mediated by immunosuppressive factors released from the tumor microenvironment (TME), such as transforming growth factor (TGF)-β1. An interleukin (IL)-15 superagonist/IL-15 receptor α fusion complex (IL-15SA/IL-15RA; ALT-803) activates the IL-15 receptor on CD8 T cells and NK cells, and has shown significant anti-tumor activity in several in vivo studies.

A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma.

Different epigenetic mechanisms of ERα implicated in the fate of fulvestrant-resistant breast cancer.

Approximately 70% of breast cancers express estrogen receptor α (ERα), which plays critical roles in breast cancer development. Fulvestrant has been effectively used to treat ERα-positive breast cancer, although resistance remains a critical problem. To elucidate the mechanism of resistance to fulvestrant, we established fulvestrant-resistant cell-lines named MFR (MCF-7 derived fulvestrant resistance) and TFR (T-47D derived fulvestrant resistance) from the ERα-positive luminal breast cancer cell lines MCF-7 and T-47D, respectively.

An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele.

Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells.

Nutritional status of patients with phenylketonuria in Japan.

Accumulating evidence suggests that hyperphenylalaninemia in phenylketonuria (PKU) can cause neuropsychological and psychosocial problems in diet-off adult patients, and that such symptoms improve after resumption of phenylalanine-restricted diet, indicating the need for lifetime low-phenylalanine diet. While limiting protein intake, dietary therapy should provide adequate daily intake of energy, carbohydrates, fat, vitamins, and microelements. We evaluated nutrient balance in 14 patients with classical PKU aged 4-38 years.

Fabrication of Molecular Strain Probes for Illuminating Protein-Protein Interactions.

A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner.

How to Fabricate Functional Artificial Luciferases for Bioassays.

The present protocol introduces fabrication of artificial luciferases (ALuc(®)) by extracting the consensus amino acids from the alignment of copepod luciferase sequences. The made ALucs have unique sequential identities that are phylogenetically distinctive from those of any existing copepod luciferase. Some ALucs exhibited heat stability, and strong and greatly prolonged optical intensities.

Fabrication of molecular tension probes.

A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner.

Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab.

Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells.

Increased androgen receptor activity and cell proliferation in aromatase inhibitor-resistant breast carcinoma.

Aromatase inhibitors (AI) are commonly used to treat postmenopausal estrogen-receptor (ER)-positive breast carcinoma. However, resistance to AI is sometimes acquired, and the molecular mechanisms underlying such resistance are largely unclear. Recent studies suggest that AI treatment increases androgen activity during estrogen deprivation in breast carcinoma, but the role of the androgen receptor (AR) in breast carcinoma is still a matter of controversy.

Dipyrrin Zn(II) complexes with functional aryl groups: formation, characterization, and structures in the solid state.

Formation and characterization by single-crystal X-ray analyses of Zn(II) complexes of various dipyrrins (dipyrromethenes) have been reported. Apart from the ordinary [2+1]-type complexes of aryl-substituted dipyrrins, imidazole-substituted derivative yields the "slipped double-decker" [2+2]-type complexes by using Zn(II) halides.

Micro- and nanometer-scale porous, fibrous, and sheet architectures constructed by supramolecular assemblies of dipyrrolyldiketones.

X-ray analysis of some 1,3-dipyrrolyl-1,3-propanediones synthesized from pyrroles and malonyl chloride derivatives revealed 1D supramolecular networks formed by N-H...