Dual-responsive near-infrared turn-on fluorescent probe for cancer stem cell-specific visualization.

Aldehyde dehydrogenase 1A1 (ALDH1A1) stands out as one of the most reliable intracellular biomarkers for stem cells because it is expressed in both cancer stem cells (CSCs) and normal somatic stem cells (NSCs). Although several turn-on fluorescent probes for ALDH1A1 have been developed to visualize CSCs in cancer cells, the discrimination of CSCs from NSCs is difficult. We here report an AND-type dual-responsive fluorescent probe, CHO_ßgal, the near-infrared fluorescence of which can be turned on after responding to both ALDH1A1 and ß-galactosidase. The AND-type dual responsiveness enables CSCs to be clearly visualized, whereas NSCs are non-emissive in microscopy. CSC-positive metastasis model lungs were successfully discriminated from normal lungs in ex vivo staining experiments using CHO_ßgal, whereas the single-input ALDH1A1-responsive probe failed to achieve this discrimination owing to pronounced false-positive fluorescence output from lung NSCs. In tissue slice staining experiments, even in the presence of adjacent normal tissues, the peripheral region-specific localization of CSCs was clear. The versatility of CHO_ßgal holds promise not only as a fundamental in vitro research tool for visualizing CSCs but also as a valuable asset in practical tissue staining diagnosis, significantly contributing to the assessment of cancer malignancy.

AIE-ESIPT Photoluminescent Probe Based on 3-(3-Hydroxypyridin-2-yl)isoquinolin-4-ol for the Detection of Intracellular Hydrogen Peroxide.

Excited-state intramolecular proton transfer (ESIPT) molecules, which feature large Stokes shifts to avoid self-absorption, play an essential role in photoluminescent bioimaging probes. Herein, we report the development of an ESIPT molecule 3-(3-hydroxypyridin-2-yl)isoquinolin-4-ol (PiQ). PiQ not only undergoes a distinct ESIPT process unlike the symmetrical 2,2'-bipyridyl-3,3'-diol but also exhibits aggregation-induced emission (AIE) characteristics. PiQ self-assembles into aggregates with an average size of 241.0±51.9 nm in aqueous solutions, leading to significantly enhanced photoluminescence. On the basis of the ESIPT and AIE characteristics of PiQ, the latter is functionalized with a hydrogen peroxide-responsive 4-pinacoratoborylbenzyl group (B) and a carboxylesterase-responsive acetyl group (A) to produce a photoluminescent probe B-PiQ-A. The potential of PiQ for applications in bioimaging and chemical sensing is underscored by its efficient detection of both endogenous and exogenous hydrogen peroxide in living cells.

Light-controllable cell-membrane disturbance for intracellular delivery.

Highly polar and charged molecules, such as oligonucleotides, face significant barriers in crossing the cell membrane to access the cytoplasm. To address this problem, we developed a light-triggered twistable tetraphenylethene (TPE) derivative, TPE-C-N, to facilitate the intracellular delivery of charged molecules through an endocytosis-independent pathway. The central double bond of TPE in TPE-C-N is planar in the ground state but becomes twisted in the excited state. Under light irradiation, this planar-to-twisted structural change induces continuous cell membrane disturbances. Such disturbance does not lead to permanent damage to the cell membrane. TPE-C-N significantly enhanced the intracellular delivery of negatively charged molecules under light irradiation when endocytosis was inhibited through low-temperature treatment, confirming the endocytosis-independent nature of this delivery method. We have successfully demonstrated that the TPE-C-N-mediated light-controllable method can efficiently promote the intracellular delivery of charged molecules, such as peptides and oligonucleotides, with molecular weights ranging from 1000 to 5000 Da.

A reductively convertible nickel phthalocyanine precursor as a biological thiol-responsive turn-on photoacoustic contrast agent.

A nickel phthalocyanine precursor bearing poly(ethylene glycol) as a turn-on contrast agent for photoacoustic imaging was prepared. The water-soluble polymeric chains were smoothly eliminated through thiol-mediated reductive aromatization in cancer cells, enabling the detection of endogenous biological thiols in vitro and in vivo.

Calcitonin gene-related peptide regulates periodontal tissue regeneration.

Calcitonin gene-related peptide (CGRP), a neuropeptide composed of 37 amino acids secreted from the sensory nerve endings, reportedly possesses various physiological effects, such as vasodilation and neurotransmission. Recently, there have been increasing reports of the involvement of CGRP in bone metabolism; however, its specific role in the pathogenesis of periodontitis, particularly in the repair and healing processes, remains to be elucidated. Therefore, this study aimed to investigate dynamic expression patterns of CGRP during the destruction and regeneration processes of periodontal tissues in a mouse model of experimental periodontitis. We also explored the effects of CGRP on periodontal ligament cells, which can differentiate to hard tissue-forming cells (cementoblasts or osteoblasts). Our findings demonstrated that CGRP stimulation promotes the differentiation of periodontal ligament cells into hard tissue-forming cells. Experimental results using a ligature-induced periodontitis mouse model also suggested fluctuations in CGRP expression during periodontal tissue healing, underscoring the vital role of CGRP signaling in alveolar bone recovery. The study results highlight the important role of nerves in the periodontal ligament not only in sensory reception in the periphery, as previously known, but also in periodontal tissue homeostasis and tissue repair processes.

A case of neuroendocrine tumor at the external urethral meatus after total cystectomy.

Introduction: The histological types of urethral cancer are mainly squamous cell or transitional cell carcinoma. Neuroendocrine tumor is extremely a rare type of urethral cancer. Case presentation: A 72-year-old man visited with an erythema at the external urethral meatus. After 3 months, a 1-cm reddish solid tumor was found on the external urethral meatus. He had a history of bladder cancer (pTa with carcinoma in situ), including the prostatic urethra, and underwent radical cystectomy with urethrectomy and ileal conduit construction 11 years ago. After 3 months, a 1-cm reddish solid tumor was found on the external urethral meatus. The pathological diagnosis was a neuroendocrine tumor. Partial penectomy was performed. Conclusion: Small cell neuroendocrine tumor could occur on urethral remnant after radical cystectomy with urethrectomy for urothelial cancer. Inspection of the penis and urethral meatus is important during regular follow-up of patients after radical cystectomy.

Periodontal Tissue Regeneration by Transplantation of Autologous Adipose Tissue-Derived Multi-Lineage Progenitor Cells With Carbonate Apatite.

We have developed an autologous transplantation method using adipose tissue-derived multi-lineage progenitor cells (ADMPCs) as a method of periodontal tissue regeneration that can be adapted to severe periodontal disease. Our previous clinical study confirmed the safety of autologous transplantation of ADMPCs and demonstrated its usefulness in the treatment of severe periodontal disease. However, in the same clinical study, we found that the fibrin gel used as the scaffold material might have caused gingival recession and impaired tissue regeneration in some patients. Carbonate apatite has a high space-making capacity and has been approved in Japan for periodontal tissue regeneration. In this study, we selected carbonate apatite as a candidate scaffold material for ADMPCs and conducted an in vitro examination of its effect on the cellular function of ADMPCs. We further performed autologous ADMPC transplantation with carbonate apatite as the scaffold material in a model of one-wall bone defects in beagles and then analyzed the effect on periodontal tissue regeneration. The findings showed that carbonate apatite did not affect the cell morphology of ADMPCs and that it promoted proliferation. Moreover, no effect on secretor factor transcription was found. The results of the in vivo analysis confirmed the space-making capacity of carbonate apatite, and the acquisition of significant new attachment was observed in the group involving ADMPC transplantation with carbonate apatite compared with the group involving carbonate apatite application alone. Our results demonstrate the usefulness of carbonate apatite as a scaffold material for ADMPC transplantation.

Lectin histochemistry of posterior lingual glands of developing rats.

The posterior lingual glands are classified as Weber and von Ebner glands. Glycans play an important role in salivary glands. Although the distribution of glycans can explain functional diversity and variation, there are many unknowns in the developing rat posterior lingual glands. The purpose of this study was to elucidate the relationship between the development and function of the posterior lingual gland in rats by histochemical analysis using lectins that bind to sugar residues. In adult rats, Arachis hypogaea (PNA), Glycine maximus (SBA), and Triticum vulgaris (WGA) were associated with serous cells and Dolichos biflorus (DBA) with mucous cells. In both Weber's and von Ebner's glands, all 4 lectins were bound to serous cells in early development, but as development progressed, DBA disappeared in serous cells and only the DBA remained in mucous cells. These results suggest that Galß (1,3) > Galß(1,4) > Gal, αGalNAc > αGal > ßGalNAc, NeuAc > (GalNAc)2-3>>>GlcNAc, and GalNAcα(1,3) are present in the early stage of development, but that GalNAcα(1,3) disappear in serous cells and only GalNAcα(1,3) are localized in mucous cells after maturation. These results indicate that Weber glands function as serous glands in the early postnatal stage when von Ebner glands have not matured.

Steric Control in Activator-Induced Nucleophilic Quencher Detachment-Based Probes: High-Contrast Imaging of Aldehyde Dehydrogenase 1A1 in Cancer Stem Cells.

Turn-on fluorescence probes can visualize the enzyme activity with high contrast. We have established a new turn-on mechanism, activator-induced nucleophilic quencher detachment (AiQd), and developed AiQd-based turn-on fluorescence probes for the detection of enzymes. Herein, we demonstrate that the precise steric control efficiently quenches the fluorescence of AiQd-based turn-on probes before the enzymatic transformation. Theoretical calculation appropriately predicted the ratio of the fluorescence-quenched closed-ring form of probes. ßC5S-A, which has a sterically demanding methyl group at the ß-position of a fluorescence-quenching nucleophilic mercapto group, showed a low background signal. ßC5S-A responded to aldehyde dehydrogenase 1A1 (ALDH1A1) with high selectivity, thereby enabling high-contrast live imaging of cancer stem cells (signal-to-noise ratio >10). The ALDH1A1-responsiveness of ßC5S-A was not significantly affected by amino acids and biological thiols, such as cysteine and glutathione.

An exploratory clinical trial to evaluate the safety and efficacy of combination therapy of REGROTH® and Cytrans® granules for severe periodontitis with intrabony defects.

Introduction: Currently, flap operation (FOP) using REGROTH® (0.3% basic fibroblast growth factor [FGF-2]) is the standard treatment for periodontal regenerative therapy in Japan. However, the periodontal tissue regenerative effect with REGROTH® monotherapy is inadequate for severe alveolar bone defects. Therefore, in this study, we evaluated the safety and effectiveness of periodontal regenerative therapy for patients with severe periodontitis using REGROTH® (test medicine) combined with Cytrans® Granules (test device: carbonated apatite granules), which is a new artificial bone. Methods: The study participants included 10 patients with severe periodontitis (mean age: 47.4 years). All participants provided written informed consents. In each patient, the intrabony defect site (mean bone defect depth: 5.7 mm) was defined as the test site. FOP was performed for the test site after the baseline investigation; moreover, the test medicine and test device were administered simultaneously. Furthermore, the observation of subjects' general condition and test sites was conducted and the blood, urine, and periodontal tissue tests were performed up to 36 weeks after FOP. The rate of bone increase (%), clinical attachment level (CAL), probing pocket depth (PPD), bleeding on probing (BOP), tooth mobility (Mo), width of keratinized gingiva (KG), gingival recession (REC), gingival index (GI), and plaque index (PlI) were evaluated during the periodontal tissue investigation. Results: As the primary endpoint, no adverse events related to the test medicine and test device occurred during the entire observation period of this study. Regarding the secondary endpoints, there was a significant increase in new alveolar bone (p = 0.003) and CAL acquisition (p = 0.001) as well as decrease in PPD (p = 0.002) and BOP (p = 0.016) at 36 weeks after administration of the test medicine and test device compared with the preoperative values. Furthermore, at 36 weeks after surgery, the Mo, GI, and PlI decreased to preoperative levels at 40%, 60%, and 30% of sites, respectively. However, at 36 weeks after surgery, there was no difference in KG and REC compared with their preoperative values. Conclusions: The safety of periodontal regenerative therapy using the test medicine in combination with the abovementioned test device was confirmed. In addition, it was suggested that this periodontal regenerative therapy is effective for tissue regeneration in severe alveolar bone defects.This clinical trial was conducted after registering and publicizing as a specified clinical trial in the Japan registry of clinical trials (jRCTs051190045).

Identification of Breast Cancer Stem Cells Using a Newly Developed Long-acting Fluorescence Probe, C5S-A, Targeting ALDH1A1.

BACKGROUND/AIM: Aldehyde dehydrogenase (ALDH) 1A1 is a well-known marker for cancer stem cells (CSCs), characterized by self-renewal capacity and multidrug resistance in breast cancer. We developed a near-infrared turn-on fluorescence probe for ALDH1A1, C5S-A, which is suitable for observing and analyzing viable cells. Here, we demonstrated the utility of C5S-A in CSC research using breast cancer cell lines. MATERIALS AND METHODS: To evaluate concordance between C5S-A and conventional stem cell markers, breast cancer cells sorted for ALDEFLUOR-positive cells and for CD44+/CD24- cell populations were stained with C5S-A. Tumorigenicity of C5S-A-positive cells was examined by mammosphere formation assay and subcutaneous transplantation to immunodeficient mice. Additionally, to determine how long fluorescence from a single staining remained observable, we cultured breast cancer cells for 5 days after C5S-A staining. We then evaluated whether C5S-A-positive cells possessed resistance to cytotoxic drugs by chronological imaging. RESULTS: C5S-A staining showed good concordance with conventional breast CSC markers, and good utility for research into CSC characteristics in breast cancer cell lines, including tumorigenesis. Additionally, C5S-A was observable for more than 3 days with a single staining. Using this property, we then confirmed that C5S-A-positive cells possessed resistance to cytotoxic drugs, which is one of the characteristics of CSCs. CONCLUSION: We showed that C5S-A is suitable for CSC research using breast cancer cell lines, and confirmed its utility in observing cells over time.

An activator-induced quencher-detachment-based turn-on probe with a cationic substrate moiety for acetylcholinesterase.

We report a choline ester-grafted turn-on fluorescence probe to detect acetylcholinesterase (AChE) in living cells. The AChE-mediated hydrolysis of the choline ester moiety producing carboxylate initiates the activation of the Cy5 fluorophore quenched by an intramolecular nucleophilic mercapto group. The probe has the advantages of high AChE affinity and low cytotoxicity.

Deep-Red/Near-Infrared Turn-On Fluorescence Probes for Aldehyde Dehydrogenase 1A1 in Cancer Stem Cells.

Accumulating evidence supports that cancer stem cells (CSCs) are responsible for cancer proliferation, metastasis, and therapy resistance; therefore, an effective strategy to identify and isolate CSCs is required urgently. Because of their low invasiveness and high signal/noise ratio, "turn-on" fluorescence probes working in the deep-red/near-infrared (DR/NIR) region are one of the most attractive yet undeveloped tools for CSC detection. Herein, we report DR/NIR turn-on fluorescence probes, CS5-A and CS7-A, targeted to aldehyde dehydrogenase 1A1 as an intracellular CSC marker. In contrast to the conventional "always-on" green-fluorescent ALDEFLUOR, we succeeded in generating high-contrast (signal/noise ratio > 8.3) and wash-free in vitro CSC imaging with the DR probe C5S-A. This probe can facilitate CSC isolation with minimal contamination by autofluorescence from other tissues through fluorescence-activated cell sorting. Furthermore, the NIR absorbance/emission and turn-on properties of C7S-A allow simple and rapid CSC detection in vivo within 15 min.

Aluminum naphthalocyanine conjugate as an MMP-2-activatable photoacoustic probe for in vivo tumor imaging.

Matrix metalloproteinase-2 (MMP-2), which is one of MMPs family, is known as an extracellular gelatinase controlling cancer cell adhesion, growth, and metastasis. Because of the great interest in MMP-2 activity, the detailed protocols for evaluating MMP-2-responsive contrast agents, especially photoacoustic probes for in vivo use, are helpful for researchers in the field. We here describe the detailed synthetic procedure of MMP-2-activatable photoacoustic probe AlNc-pep-PEG consisting of aluminum naphthalocyanine, MMP-2-responsive peptide sequence, and poly(ethylene glycol), which has recently been developed in our research group. The detailed measurement protocol of photoacoustic signal intensity in vitro and in vivo by using in-house built photoacoustic signal measurement system and photoacoustic imaging apparatus are also summarized.

Periodontal inflamed surface area is associated with hs-CRP in septuagenarian Japanese adults in cross-sectional findings from the SONIC study.

Periodontal disease is a chronic inflammatory condition that affects various peripheral organs. The periodontal inflamed surface area (PISA) quantifies periodontitis severity and the spread of inflammatory wounds. This study aimed to investigate the association between PISA and high-sensitivity C-reactive protein (hs-CRP), a systemic inflammation marker. This study included 250 community-dwelling septuagenarians (69-71 years). We collected information on their medical (e.g., diabetes and dyslipidemia) and dental examinations (e.g., measurement of the probing pocket depth). Generalized linear model analysis was used to explore the association between PISA and hs-CRP levels. There was a significant difference in hs-CRP levels between groups with PISA ≥ 500 and < 500 (p = 0.017). Moreover, the generalized linear model analysis revealed a significant association between PISA and hs-CRP levels (risk ratio = 1.77; p = 0.033) even after adjusting other factors. Further, we found a correlation between PISA and hs-CRP (Spearman's rank correlation coefficient, rs = 0.181; p = 0.023). Our findings suggest that PISA is an effective index for estimating the effect of periodontitis on the whole body, enabling medical-dental cooperation.

MMP-2-Activatable Photoacoustic Tumor Imaging Probes Based on Al- and Si-Naphthalocyanines.

Enzyme-activatable photoacoustic probes are powerful contrast agents to visualize diseases in which a specific enzyme is overexpressed. In this study, aluminum and silicon naphthalocyanines (AlNc and SiNc, respectively) conjugated with matrix metalloprotease-2 (MMP-2)-responsive PLGLAG peptide sequence and poly(ethylene glycol) (PEG) as an axial ligand were designed and synthesized. AlNc-peptide-PEG conjugates AlNc-pep-PEG formed dimeric species interacting with each other through face-to-face H-aggregation in water, while SiNc-based conjugates SiNc-pep-PEG hardly interacted with each other because of the two bulky hydrophilic axial ligands. Both conjugates formed spherical nanometer-sized self-assemblies in water, generating photoacoustic waves under near-infrared photoirradiation. The treatment of MNc-peptide-PEG conjugates (M = Al, Si) with MMP-2 smoothly induced the cleavage of the PLGLAG sequence to release the hydrophilic PEG moiety, resulting in the aggregation of MNcs. By comparing the PA signal intensity changes at 680 and 760 nm, the photoacoustic signal intensity ratios were shown to be enhanced by 3-5 times after incubation with MMP-2. We demonstrated that MNc-peptide-PEG conjugates (M = Al, Si) could work as activatable photoacoustic probes in the in vitro experiment of MMP-2-overexpressed cell line HT-1080 as well as the in vivo photoacoustic imaging of HT-1080-bearing mice.

Substituted meso-vinyl-BODIPY as thiol-selective fluorogenic probes for sensing unfolded proteins in the endoplasmic reticulum.

A new type of thiol probes based on the meso-vinyl-BODIPY (VB) scaffold were developed. The monochloro-substituted VB1Cl exhibited the largest fluorescence enhancement (>200-fold) as well as high selectivity upon biological thiol sensing. VB1Cl was successfully applied for reporting the protein unfolding process under ER stress in living cells.

Association of periodontal disease with atherosclerosis in 70-year-old Japanese older adults.

Periodontal disease and arteriosclerotic disease are greatly affected by aging. In this study, the association of conventional risk factors and periodontal disease with atherosclerosis was longitudinally examined in Japanese older adults. Subjects in this study were 490 community-dwelling septuagenarians (69-71 years) randomly recruited from the Basic Resident Registry of urban or rural areas in Japan. At the baseline examination, all subjects underwent socioeconomic and medical interviews; medical examinations, including examinations for carotid atherosclerosis, hypertension, diabetes mellitus, and dyslipidemia; and conventional dental examinations, including a tooth count and measurement of probing pocket depth (PPD). After 3 years, 182 septuagenarians who had no atherosclerosis at the baseline examination were registered and received the same examination as at the baseline. In the re-examination conducted 3 years after the baseline survey, 131 (72.0%) of the 182 participants who had no atherosclerosis at the baseline examination were diagnosed with carotid atherosclerosis. Adjusting and analyzing the mutual relationships of the conventional risk factors for atherosclerosis by multiple logistic regression analysis for the 171 septuagenarians with a full set of data, the proportion of teeth with PPD ≥ 4 mm was independently related to the prevalence of atherosclerosis (odds ratio: 1.029, P < 0.022). This longitudinal study of Japanese older adults suggests that periodontal disease is associated with the onset/progression of atherosclerosis. Maintaining a healthy periodontal condition may be an important factor in preventing the development and progression of atherosclerosis.

pH-Activatable Cyanine Dyes for Selective Tumor Imaging Using Near-Infrared Fluorescence and Photoacoustic Modalities.

Photoacoustic (PA) imaging is an emerging molecular imaging modality that complements fluorescence imaging and enables high resolution within deep tissue. Fluorescence/PA multimodality imaging would be a powerful technique to extract more comprehensive information from targets than traditional single-modality imaging. In this paper, we developed a new pH-activatable sensor, CypHRGD, which is applicable to both fluorescence and PA detection. CypHRGD was derived from our previous near-infrared pH-sensing platform, in which substitution with a bulky phenyl group and functionalization with a cRGD peptide remarkably improved the sensor's biocompatibility with attenuated dye aggregation. The multimodality imaging applications of CypHRGD were demonstrated in cultured cells and cancer-xenografted mice with rapid kinetics and high sensitivity and specificity, which relies on cRGD-facilitated tumor targeting, probe accumulation and subsequent activation in the acidic organelles after endocytosis.

An enzyme-triggered turn-on fluorescent probe based on carboxylate-induced detachment of a fluorescence quencher.

We developed a new class of turn-on fluorescent probes for an esterase. After the esterase-mediated hydrolysis produced carboxylate (as a fluorescence activator), the fluorescence intensity was markedly increased through the detachment of a quencher moiety from the quenched Cy5 fluorophore. Because the probes based on this new activator-induced quencher-detachment (AiQd) adopt a non-immolative linker between the cleavable site and the fluorophore, the rate of the enzymatic reaction is greatly improved, without the generation of any by-products.