Modeling the marmoset brain using embryonic stem cell-derived cerebral assembloids.

Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their spontaneous neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs containing excitatory and inhibitory neurons showed mature neural activity with an unsynchronized pattern. The CAs represent a powerful in vitro model for studying excitatory and inhibitory neuron interactions, cortical dynamics, and their dysfunction. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.

Subclinical and latent cardiac dysfunction in obstructive sleep apnea and effectiveness of continuous positive airway pressure.

PURPOSE: Obstructive sleep apnea (OSA) is associated with various cardiovascular disorders. This study aimed to investigate the effects of OSA on left ventricular (LV) function in patients with OSA who were at risk for heart failure but who had not yet developed structural heart changes. The study also sought to determine the effects of continuous positive airway pressure (CPAP) in these patients. METHODS: In a retrospective study, consecutive patients with polysomnographic OSA (apnea-hypopnea index [AHI] >5) were categorized into mild (AHI < 15), moderate (15 ≤ AHI < 30), and severe OSA (AHI ≥ 30) groups. The subjects were patients with OSA and at risk for heart failure who had not yet developed structural heart changes. All study participants underwent echocardiography and two-dimensional speckle tracking analysis, and their global longitudinal strain (GLS) was calculated. RESULTS: Of 275 patients, there were 31 with mild, 92 with moderate, and 152 with severe OSA. Of patients with moderate to severe OSA (AHI ≥ 20), 206 started CPAP and 92 patients underwent follow-up echocardiogram and speckle tracking echo analysis (median period of CPAP use: 283 days [258 to 391]). GLS was significantly reduced in patients with moderate and severe OSA compared with mild OSA (-17.8±3.1 vs. -18.0±2.6 vs. -19.3±2.8%, p=0.038). The proportion of patients with GLS ≥ -18% was significantly higher among the patients with moderate to severe OSA than among those with mild OSA. GLS improved after CPAP therapy in patients with moderate to severe OSA (GLS: -18.1±2.7% to -19.0±2.8%, p=0.004). Significant improvement in GLS was confirmed, particularly among patients with good CPAP adherence. CONCLUSION: Moderate to severe OSA is associated with LV dysfunction and can be significantly improved by CPAP therapy.

A novel risk score on admission for predicting death or need for surgery in patients with acute type A intramural hematoma receiving medical therapy.

There has been continuing discussion regarding the treatment strategy for acute type A intramural hematoma (IMH). Most patients are treated conservatively in Japan; hence, predicting fatal events and stratifying risks based on results normally obtained on hospital arrival are important. We aimed to examine the incidences and risk factors of death or need for surgery for acute type A IMH in patients receiving medical treatment and to identify high-risk patients using clinical findings on hospital arrival. From 2011 to 2016, 57 consecutive patients (mean age 72.5 years; male 36.8%) diagnosed with acute type A IMH who were receiving treatment at Shizuoka City Shizuoka Hospital were retrospectively included. Primary endpoint was a composite of cardiovascular death and operation within 1 year after onset. To evaluate sensitivity and specificity of the risk factors and risk score, we estimated the area under the receiver operating characteristic (ROC) curve. Mean follow-up duration was 621 days. Mean systolic blood pressure (SBP) was 129 mmHg. Computed tomography (CT) on arrival showed a mean ascending aorta diameter of 46 mm. Ulcer-like projection (ULP) in the ascending aorta and pericardial effusion (PE) were seen in 33% and 42% of cases, respectively. Twenty-eight patients (49.1%) reached the primary endpoint (cardiovascular death, 7 cases [12.3%]; operation, 21 cases [36.8%]). In univariate analysis of admission values, the primary endpoint group had significantly lower SBP (113.0 ± 28.5 vs 144.3 ± 33.5 mmHg), higher ascending aorta diameter (49.5 ± 8.1 vs 43.6 ± 5.9 mm), and higher frequency of ULP (53.8% vs 13.8%) and PE (58.6% vs 25.0%) than the event-free group. Multivariate analysis showed that ULP on admission CT was a significant predictor of the primary endpoint. The risk score was considered using these risk factors. On admission, the primary endpoint could be predicted with 89.7% sensitivity and 75% specificity (area under the ROC curve 0.823) if the patient had ULP and/or > 2 of the following factors: SBP < 120 mmHg, ascending aorta diameter > 45 mm, and PE. SBP and CT findings on arrival were significantly associated with cardiovascular death and the need for surgery in patients with acute type A IMH receiving initial medical therapy. The novel risk score was useful for predicting cardiovascular death and surgery.

Immunohistochemistry and gene expression of GLUT1, RUNX2 and MTOR in reparative dentinogenesis.

OBJECTIVES: To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. SUBJECTS AND METHODS: Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. RESULTS: Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. CONCLUSIONS: After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.

Aberrant serum polyunsaturated fatty acids profile is relevant with acute coronary syndrome.

Although a robust relationship between aberrant serum polyunsaturated fatty acids (PUFAs) profile and coronary artery disease (CAD) has been reported, the details concerning the association between aberrant PUFAs profile and clinical feature of CAD are not fully discovered. Therefore, we investigated the relationship between serum PUFAs and clinical profiles in CAD patients. We classified 595 consecutive CAD patients, who underwent coronary angiography into 3 groups according to the clinical profiles of CAD (group A: early phase ACS, n = 96; group B: stable CAD with previous history of ACS, n = 259; group C: stable CAD without previous history of ACS, n = 240) and measured serum n-3 [eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)] and n-6 [arachidonic acid (AA)] PUFAs. Serum EPA, DHA, and EPA/AA ratio were significantly low in the order of group A < B < C [EPA; 48.1 (34.1-60.3) µg/ml, 61.7 (41.2-94.5) µg/ml, and 74.4 (52.7-104.9) µg/ml, DHA; 113.1 (92.8-135.1) µg/ml, 125.8 (100.4-167.2) µg/ml, and 140.1 (114.7-177.0) µg/ml, EPA/AA ratio; 0.31 (0.22-0.45), 0.39 (0.26-0.62), and 0.44 (0.31-0.69), medians with interquartile range, p < 0.01]. Multiple regression analysis revealed that EPA (p = 0.009) and EPA/AA ratio (p = 0.023), but not DHA and DHA/AA ratio, were negatively associated with clinical profiles of ACS in CAD patients. Significant correlation was not observed between PUFAs profile and severity of coronary stenosis. Low serum EPA and EPA/AA ratio correlates with clinical profiles of ACS in patients with CAD, regardless of the extent and severity of coronary artery stenosis.

Sex differences in factors of pelvis and lower-limb joint kinematics influencing dynamic knee valgus during single-leg squat.

BACKGROUND: Dynamic knee valgus (DKV) during dynamic activity is associated with patellofemoral pain, one of the most common sports disorders, especially in females. No studies have examined the factors that can lead to DKV in females as compared to males while addressing the influence of factors on each other. OBJECTIVE: To identify factors of pelvis and lower joints kinematics affecting DKV during single-leg squat (SLS) in females compared to males with multiple regression analysis. METHODS: Forty-four healthy young people (24 females, 20 males) performed SLS, and pelvis, hip, knee, and ankle 3D kinematics were recorded. The 2D knee frontal plane projection angle (FPPA) was adopted as the DKV measurement. Multiple regression analysis was performed to determine the relationship between FPPA and the kinematics of each joint and segment. RESULTS: Hip adduction and pelvic contralateral rotation were explanatory factors for FPPA in females. Hip adduction, hip internal rotation, and knee external rotation were explanatory factors in males. CONCLUSIONS: The lower limb or pelvic motion factors affecting DKV during SLS differ between the sexes, with pelvic contralateral rotation being extracted only in women, possibly due to differences in pelvic width.

Effect of lateral wedge length on knee adduction moment reduction mechanics during gait.

Lateral wedge insole (LWI) wear is a well-known conservative treatment for patients with knee osteoarthritis and is expected to decrease knee joint loading. Although the effect of LWI length on knee adduction moment (KAM) has been investigated, the biomechanical mechanism has not been fully investigated. Twelve healthy young subjects walked in the laboratory with and without 2 different lengths of LWIs. Three-dimensional motion analysis was performed to calculate the first and second peaks and impulses of the KAM during the stance phase. In addition, the knee-ground reaction force lever arm (KLA) and center of pressure (COP), ankle eversion moment, and ankle eversion angle were calculated. The first peak of KAM was lower, COP was displaced outward, and KLA was shorter with both LWIs attached. On the other hand, the second peak of KAM was lower with longer LWIs, COP was displaced outward, and KLA was shorter. The KAM impulse was significantly smaller in the condition with longer LWI than in the other conditions with smaller ankle eversion motion; longer LWI induced COP to the lateral side through the stance phase and kept KLA short, thus reducing the KAM impulse.

Wound-healing Processes After Pulpotomy in the Pulp Tissue of Type 1 Diabetes Mellitus Model Rats.

INTRODUCTION: Patients with type 1 diabetes mellitus (DM1) tend to have delayed wound healing, even in the pulp tissue. We hypothesized that hyperglycemia affects odontoblast-like cell (OLC) differentiation and is involved in macrophage polarization. Accordingly, we evaluated dental pulp stem cell differentiation and macrophage phenotypes after pulpotomy. METHODS: After modifying DM1 rat models by streptozotocin, 8-week-old rats' upper left first molars were pulpotomized with mineral trioxide aggregate. Meanwhile, the control group was administered saline. Immunohistochemical localization of nestin, osteopontin, α-smooth muscles (α-SMAs), and CD68 (pan-macrophage marker) was conducted 7 days after pulpotomy. The OLC differentiation stage was determined using double immunofluorescence of nestin and α-SMA. Double immunofluorescence of CD68 and iNOS was counted as M1 macrophages and CD68 and CD206 as M2 macrophages. Proliferating cell nuclear antigen and Thy-1 (CD90) were evaluated by immunofluorescence. RESULTS: In DM1 rats, the reparative dentin bridge was not complete; however, the osteopontin-positive area did not differ significantly from that in controls. Proliferating cell nuclear antigen, indicative of cell proliferation, increased in positive cells in DM1 rats compared with controls. Double-positive cells for α-SMA and nestin indicated many immature OLCs in DM1. CD90 was positive only in controls. CD68-positive cells, especially M1 macrophages, were increased in DM1 rats, allowing the inflammatory stage to continue 7 days after pulpotomy. CONCLUSIONS: The condition of DM1 model rats can interfere at various stages of the wound healing process, altering OLC differentiation and macrophage polarization. These findings highlight the importance of normal blood glucose concentrations during pulp wound healing.

Acute Myocardial Infarction Due to Coronary Artery Embolism during Chemotherapy with mFOLFOX-6 Plus Bevacizumab for Metastatic Colon Cancer.

Bevacizumab, a monoclonal antibody against vascular endothelial growth factor, may be associated with arterial embolisms. We herein report a case of acute myocardial infarction caused by coronary embolism during combination chemotherapy with mFOLFOX-6 and bevacizumab in a patient with metastatic colon cancer. Thromboembolism occurred only in the distal right posterolateral branch without stenotic lesions or plaque rupture in the proximal branch of the right coronary artery. Sole thromboaspiration was successfully performed; the final angiogram demonstrated no stenosis in the right coronary artery. Bevacizumab may be associated with acute coronary syndrome in patients with coronary risk factors, despite no significant coronary narrowing.

A mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain.

Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.

Emergency Transcatheter Aortic Valve Implantation in a Surgical Aortic Bioprosthetic Valve for Acute Decompensated Heart Failure Caused by Sudden Progression of Structural Valve Deterioration.

Bioprosthetic aortic valves have limited durability. We herein report sudden progression of structural valve deterioration (SVD) and a successful case of emergency transcatheter aortic valve (TAV) implantation for acute decompensated heart failure (ADHF) caused by SVD. A 79-year-old man who had undergone a Bentall operation 11 years prior was diagnosed with ADHF due to suddenly progressive SVD. Emergency TAV implantation in the surgical bioprosthetic valve was selected based on the surgical risk. Ours and previous case reports suggest that SVD can progress suddenly, even after months of stability, and that emergency TAV implantation is effective.

Monosynaptic rabies virus tracing from projection-targeted single neurons.

A single neuron integrates inputs from thousands of presynaptic neurons to generate outputs. Circuit tracing using G-deleted rabies virus (RVΔG) vectors permits the brain-wide labeling of presynaptic inputs to targeted single neurons. However, the experimental procedures are complex, and the success rate of circuit labeling is low because of the lack of validation to increase the accuracy and efficiency of monosynaptic RVΔG tracing from targeted single neurons. We established an efficient RVΔG tracing method from projection target-defined single neurons using TVA950, a transmembrane isoform of TVA receptors, for initial viral infection. Presynaptic neurons were transsynaptically labeled from 80 % of the TVA950-expressing single starter neurons that survived after infection with EnvA-pseudotyped RVΔG in the adult mouse brain. We labeled single neuronal networks in the primary visual cortex (V1) and higher visual areas, namely the posteromedial area (PM) and anteromedial area (AM), as well as the single neuronal networks of PM-projecting V1 single neurons. Monosynaptic RVΔG tracing from projection-targeted single neurons revealed the input-output organization of single neuronal networks. Single-neuron network analysis based on RVΔG tracing will help dissect the heterogeneity of neural circuits and link circuit motifs and large-scale networks across scales, thereby clarifying information processing and circuit computation in the brain.

Young females with hallux valgus show lower foot joint movement stability compared to controls: An investigation of coordination patterns and variability.

BACKGROUND: A kinematic coupling relationship exists between foot joints during gait. In individuals with hallux valgus, forefoot or hallux kinematics may be affected by adjacent or nonadjacent joint motion. Thus, this study aimed to investigate the foot joint coordination pattern and variability during gait in young females with hallux valgus. METHODS: Twenty-five young females with hallux valgus and 25 healthy young females without hallux valgus were enrolled. Reflective markers were attached according to a multisegment foot model. Kinematic data were obtained using a three-dimensional motion analysis system. Joint angles between distal and proximal segments were calculated using analysis software. Foot joint coordination pattern and variability were assessed using a vector-coding technique. FINDINGS: Individuals with hallux valgus had a larger rearfoot relative to shank eversion and forefoot relative to midfoot dorsiflexion during terminal stance and pre-swing compared with those without hallux valgus. There were no significant differences in coordination patterns, but the consistency of coordination between the rearfoot relative to shank motion in the frontal plane and forefoot relative to midfoot motion in the sagittal plane during terminal stance was greater in the hallux valgus group than in the control group. INTERPRETATION: The soft tissue composing the first ray might suffer from more severe stress due to the large motion that occurred with low variability in individuals with hallux valgus. This finding may suggest that the altered kinematics and coordination variability in foot joints are related to hallux valgus biomechanical etiology.

Processing of visual statistics of naturalistic videos in macaque visual areas V1 and V4.

Natural scenes are characterized by diverse image statistics, including various parameters of the luminance histogram, outputs of Gabor-like filters, and pairwise correlations between the filter outputs of different positions, orientations, and scales (Portilla-Simoncelli statistics). Some of these statistics capture the response properties of visual neurons. However, it remains unclear to what extent such statistics can explain neural responses to natural scenes and how neurons that are tuned to these statistics are distributed across the cortex. Using two-photon calcium imaging and an encoding-model approach, we addressed these issues in macaque visual areas V1 and V4. For each imaged neuron, we constructed an encoding model to mimic its responses to naturalistic videos. By extracting Portilla-Simoncelli statistics through outputs of both filters and filter correlations, and by computing an optimally weighted sum of these outputs, the model successfully reproduced responses in a subpopulation of neurons. We evaluated the selectivities of these neurons by quantifying the contributions of each statistic to visual responses. Neurons whose responses were mainly determined by Gabor-like filter outputs (low-level statistics) were abundant at most imaging sites in V1. In V4, the relative contribution of higher order statistics, such as cross-scale correlation, was increased. Preferred image statistics varied markedly across V4 sites, and the response similarity of two neurons at individual imaging sites gradually declined with increasing cortical distance. The results indicate that natural scene analysis progresses from V1 to V4, and neurons sharing preferred image statistics are locally clustered in V4.

Temporally multiplexed dual-plane imaging of neural activity with four-dimensional precision.

Spatiotemporal patterns of neural activity generate brain functions, such as perception, memory, and behavior. Four-dimensional (4-D: x, y, z, t) analyses of such neural activity will facilitate understanding of brain functions. However, conventional two-photon microscope systems observe single-plane brain tissue alone at a time with cellular resolution. It faces a trade-off between the spatial resolution in the x-, y-, and z-axes and the temporal resolution by a limited point-by-point scan speed. To overcome this trade-off in 4-D imaging, we developed a holographic two-photon microscope for dual-plane imaging. A spatial light modulator (SLM) provided an additional focal plane at a different depth. Temporal multiplexing of split lasers with an optical chopper allowed fast imaging of two different focal planes. We simultaneously recorded the activities of neurons on layers 2/3 and 5 of the cerebral cortex in awake mice in vivo. The present study demonstrated the proof-of-concept of dual-plane two-photon imaging of neural circuits by using the temporally multiplexed SLM-based microscope. The temporally multiplexed holographic microscope, combined with in vivo labeling with genetically encoded probes, enabled 4-D imaging and analysis of neural activities at cellular resolution and physiological timescales. Large-scale 4-D imaging and analysis will facilitate studies of not only the nervous system but also of various biological systems.

Cell type- and layer-specific convergence in core and shell neurons of the dorsal lateral geniculate nucleus.

Over 40 distinct types of retinal ganglion cells (RGCs) generate parallel processing pathways in the visual system. In mice, two subdivisions of the dorsal lateral geniculate nucleus (dLGN), the core and the shell, organize distinct parallel channels to transmit visual information from the retina to the primary visual cortex (V1). To investigate how the dLGN core and shell differentially integrate visual information and other modalities, we mapped synaptic input sources to each dLGN subdivision at the cell-type level with G-deleted rabies viral vectors. The monosynaptic circuit tracing revealed that dLGN core neurons received inputs from alpha-RGCs, Layer 6 neurons of the V1, the superficial and intermediate layers of the superior colliculus (SC), the internal ventral LGN, the lower layer of the external ventral LGN (vLGNe), the intergeniculate leaf, the thalamic reticular nucleus (TRN), and the pretectal nucleus (PT). Conversely, shell neurons received inputs from alpha-RGCs and direction-selective ganglion cells of the retina, Layer 6 neurons of the V1, the superficial layer of the SC, the superficial and lower layers of the vLGNe, the TRN, the PT, and the parabigeminal nucleus. The present study provides anatomical evidence of the cell type- and layer-specific convergence in dLGN core and shell neurons. These findings suggest that dLGN core neurons integrate and process more multimodal information along with visual information than shell neurons and that LGN core and shell neurons integrate different types of information, send their own convergent information to discrete populations of the V1, and differentially contribute to visual perception and behavior.

Muscle co-contraction of ankle joint in young adults in functional reach test at different distances.

PURPOSE: It has been reported that young people may be able to modulate simultaneous contraction depending on the task. The functional reach test (FRT) is widely used as a method to assess dynamic balance. Although there are several reports on the center of pressure (COP), there are few reports on muscle activity and no studies focus on muscle co-contraction during FRT at different distances. We aimed to clarify how the differences in reach distance affect the activity of the lower limb muscles by measuring COP and muscle activity during FRT at different distances. METHODS: Eighteen healthy young adults performed FRT at different distances (maximum, 75%, and 50%) and measured COP and muscle activity of tibialis anterior (TA) and soleus (SOL). Postural control variables were calculated from the COP, and mean muscle activity and muscle co-contraction index (CI) were calculated from muscle activity. Each variable was compared between the reach distance conditions and the correlation between the variable was examined. RESULTS: Most COP variables were significantly higher as the reach distance increased. A significant increase in muscle activity and CI was similarly observed with increasing distance. There was no consistent correlation between COP variables and CI, but there was a positive correlation between TA muscle activity and CI. CONCLUSIONS: The results of the present study showed that the young people used task-specific strategies by modulating lower limb muscle contraction and varying the degree of simultaneous muscle contraction during reaching movements at different distances.

Difficulty Diagnosing Retrograde Type A Aortic Dissection with Intramural Hematoma and Risk of Re-dissection and Rupture: A Report of Two Cases.

Acute type A aortic dissection is a potentially fatal disease, and emergency surgery should be considered when it is diagnosed. We herein report two cases of retrograde type A aortic dissection with intramural hematoma, followed by re-dissection, rupture, and cardiac tamponade. The diagnoses in these cases had to be made carefully, as the false lumen of the ascending aorta was sometimes unclear on contrast-enhanced computed tomography.

[Circuit mechanisms of spatial perception and visuomotor integration].

Animals can make appropriate decisions based on sensory information about the environment. Vision is one of the most critical ability for survival in dynamic situations in nature, particularly for mammalian species, such as primates, carnivores, and rodents. Although there is a huge computational cost involved in processing visual information, the brain can perform this task very rapidly using well-organized parallel and hierarchical neural circuits, enabling animals to rapidly sense the environment and, in turn, perform adaptive actions. Physiological, psychophysical, and clinical studies over hundreds of years have delineated the neural circuit mechanisms of the visual system. Artificial intelligence and robotics have also started making progress in this area. However, due to technical limitations, there are still many open questions that elude explanation in understanding the neural mechanism of visuomotor integration. Herein, we initially describe the anatomical structures of occipital cortices related to vision and then provide an overview of the physiological and clinical studies of the dorsal visual pathway related to spatial perception and prediction in non-human primate species. Finally, we introduce recent approaches in which rodents have been used as model species to elucidate the neural circuit mechanism of visually-guided behavior. Uncovering neural implementation of the association between visual-spatial perception and visuomotor function could provide key insights into the engineering of highly active robots and could also contribute to the development of novel therapeutic strategies addressing visual impairment and psychiatric/neurological disorders.

Cyclization Reaction-Based Turn-on Probe for Covalent Labeling of Target Proteins.

Fluorescent molecules have contributed to basic biological research but there are currently only a limited number of probes available for the detection of non-enzymatic proteins. Here, we report turn-on fluorescent probes mediated by conjugate addition and cyclization (TCC probes). These probes react with multiple amino acids and exhibit a 36-fold greater emission intensity after reaction. We analyzed the reactions between TCC probes and nuclear receptors by electrospray ionization mass spectrometry, X-ray crystallography, spectrofluorometry, and fluorescence microscopy. In vitro analysis showed that probes consisting of a protein ligand and TCC could label vitamin D receptor and peroxisome proliferator-activated receptor γ. Moreover, we demonstrated that not only a ligand unit but also a peptide unit can label the target protein in a complex mixture.