[Development of a Deep Learning Model for Judging Late Gadolinium-enhancement in Cardiac MRI].

PURPOSE: To verify the usefulness of a deep learning model for determining the presence or absence of contrast-enhanced myocardium in late gadolinium-enhancement images in cardiac MRI. METHODS: We used 174 late gadolinium-enhancement myocardial short-axis images obtained from contrast-enhanced cardiac MRI performed using a 3.0T MRI system at the University of Tokyo Hospital. Of these, 144 images were used for training, extracting a region of interest targeting the heart, scaling signal intensity, and data augmentation were performed to obtain 3312 training images. The interpretation report of two cardiology specialists of our hospital was used as the correct label. A learning model was constructed using a convolutional neural network and applied to 30 test data. In all cases, the acquired mean age was 56.4±12.1 years, and the male-to-female ratio was 1 : 0.82. RESULTS: Before and after data augmentation, sensitivity remained consistent at 93.3%, specificity improved from 0.0% to 100.0%, and accuracy improved from 46.7% to 96.7%. CONCLUSION: The prediction accuracy of the deep learning model developed in this research is high, suggesting its high usefulness.

Simulation of time-intensity curve based on k-space filling in breast dynamic contrast-enhanced three-dimensional magnetic resonance imaging.

This study elucidated the effects of a three-dimensional k-space trajectory incorporating the partial Fourier (PF) technique on a time-intensity curve (TIC) in a dynamic contrast-enhanced magnetic resonance imaging of a typical malignant breast tumor using a digital phantom. Images were obtained from the Cancer Imaging Archive Open Data for Breast Cancer, and 1-min scans with high temporal resolution were analyzed. The order of the k-space trajectory was set as Linear (sequential), Low-High (centric), PF (62.5%; Z-, Y-, and both directions), and Low-High Radial. k0 (center of the k-space) timing and TIC shape were affected by the chosen k-space trajectory and implementation of the PF technique. A small TIC gradient was obtained using a Low-High Radial order. If the k-space filling method (particularly the radial method) produces a gentle TIC gradient, misinterpretation could arise during the assessment of tumor malignancy status.

[Forefront of AI Applications for COVID-19 Imaging Diagnosis].

The intra- and inter-observer variability in diagnosis of thoracic CT images may affect the diagnosis of COVID-19. Therefore, several studies have been reported to develop artificial intelligence (AI) approaches using deep learning (DL) and radiomics technologies. The difference between them is automatic feature extraction (DL) and hand-crafted one (radiomics). The advantages of the AI-based imaging approaches for the COVID-19 are fast throughput, non-invasion, quantification, and integration of PCR results, CT findings, and clinical information. To the best of my knowledge, three types of the AI approaches have been studied: detection, severity differentiation, and prognosis prediction of COVID-19. AI technologies on assessment of severity/prediction of prognosis for COVID-19 may be more crucial than detection of COVID-19 pneumonia after COVID-19 becomes one of common diseases.

Over-expression of PDE5 in Oral Squamous Cell Carcinoma - Effect of Sildenafil Citrate.

BACKGROUND/AIM: Phosphodiesterase 5 (PDE5) holds clinical relevance in several pathological states, including lung, breast, and prostate cancer. In this study, we examined PDE5 expression in oral squamous cell carcinoma (OSCC)-derived cell lines and tissues, and the anti-tumour effect of PDE5 inhibitor, sildenafil citrate (SC). MATERIALS AND METHODS: Cell proliferation, cell invasion, and gap closure assays were performed in six OSCC-derived cell lines upon treatment with varying concentrations of SC. PDE5 expression was determined in primary OSCC tissues by western blotting and immunohistochemistry. RESULTS: Elevated PDE5 expression was observed in all cell lines. A concentration-dependent decrease in cell viability, invasion rate, and migration was observed after SC treatment. A significant correlation (p=0.05) was observed between elevated PDE5 expression and lymphatic infiltration in OSCC tissues. CONCLUSION: PDE5 plays an important role in carcinogenesis of OSCC, and the specific inhibition of PDE5 may be an effective chemotherapeutic strategy.

Cesium-Catalyzed Hydrogen Production by the Gasification of Woody Biomass for Forest Decontamination.

The large quantities of contaminated wood produced following the radioactive cesium decontamination of forests after the Fukushima Daiichi Nuclear Power Plant accident in 2011 can be used as a biomass resource for energy production via thermal treatment (e.g., gasification). To store the radioactive Cs ash produced from gasification, the immobilization of Cs in the pollucite structure is possible and requires stable Cs additives. In this study, a Cs additive (Cs2CO3, CsCl, CsNO3, or Cs2SO4) was doped with timber waste sawdust (1-30 wt %). Fixed-bed downdraft-type continuous steam gasification experiments (0.7 g/min) showed that Cs2CO3 enhanced H2 production by 157% at 800 °C. X-ray absorption fine structure analysis and scanning electron microscopy observations revealed that the form of Cs on the surface of the char was Cs2CO3, which provided the active sites for gasification acceleration. Thermogravimetric pyrolysis and CO2 gasification experiments showed that Cs2CO3 lowered the activation energy and frequency factor while also enhancing the reactivity.

Effects of k-space orders on the time-intensity curves in dynamic contrast-enhanced magnetic resonance imaging of the breast based on simulation study.

PURPOSE: This study aims to investigate the influence of time-intensity curves (TICs) on the shapes using a dynamic contrast-enhanced magnetic resonance imaging (MRI) study depending on the Cartesian and radial orders for benign and cancerous breast tumors. METHODS: Based on kinetic curve parameters, the signal intensities of six concentration gradients comprising two benign and four cancer models were used. The study aimed to construct a dynamic simulated image by creating a digital phantom image according to the following steps: (1) creating a simple numerical phantom, (2) setting the signal intensity in the contrast area, (3) creating the k-space in each time phase, (4) extracting data from k-space in each time phase, (5) filling in the k-space and adding data to the k-space assembly, and (6) creating a magnitude image. The TICs of Cartesian (centric and sequential) and radial (full-length [RFL] and half-length [RHL]) orders were created and sigmoid curve fitting was performed to compare these curves. Maximum slope (MS, s-1), width of the response (WOR, s), and primary signal response (PSR) were then calculated. Phase encode steps were set for 512 and 256. RESULTS: MS was significantly decreased by radial order in the cancer model. No change was observed in WOR in Cartesian order, whereas RFL and RHL orders increased in the cancer models. PSR increased remarkably in the radial orders of cancer models. The difference in the fill slope in radial orders was remarkable when the TIC was steeper compared with when it was gentle, especially RHL. In WOR, both radial RFL and RHL were well matched except for the one benign model, and the shape of radial TIC was similar to sequential order as compared to centric order in 256 steps. CONCLUSION: The effects of Cartesian and radial orders on the patterns of TICs in a dynamic contrast-enhanced MRI study of benign and cancerous breast tumors were revealed. Interestingly, the TIC gradient of radial orders became gentler, particularly in the breast cancer MRI.

Ameloblastic Fibroma in Mandibular Anterior Tooth Region: A Case Report.

An ameloblastic fibroma (AF) is a proliferative mixed tumor which includes components of both odontogenic epithelium and mesenchymal tissue. It is a relatively rare neoplasm, accounting for approximately only 1.5－4.5% of odontogenic tumors. This case report describes an AF that occurred in the mandibular anterior tooth region in a 9-year-old girl who presented with the chief complaint of swelling in the left mandibular anterior tooth region. Intraoral examination revealed a swelling along the labial gingiva, extending from the left mandibular lateral incisor region to the left mandibular canine. Upon palpation, the swelling appeared to comprise a hard tissue. Computed tomography revealed a supernumerary impacted tooth; soft tissue density in the bone surrounding the region extending from the left mandibular lateral incisor to the left mandibular canine; labial bone expansion; and thinning of the labial cortical bone. A biopsy was performed under local anesthesia and the lesion subsequently diagnosed as an AF. Tumor resection and extraction of the supernumerary impacted tooth were carried out under general anesthesia. At 2 years postoperatively the prognosis is good. Although relapse with an AF is rarer than that with an ameloblastoma, strict follow-up is required, as malignant transformation to an ameloblastic fibrosarcoma has been reported in relapsed cases.

The Relationship between Dental Findings and Tongue Pressure: A Survey of 745 Community-Dwelling Adults and Elderly Persons in Japan.

INTRODUCTION: Reduced tongue pressure is one of the causes of dysphagia. The purpose of this study was to investigate the relationship between dental findings and tongue pressure, and whether prosthetic treatment prevents reduced tongue pressure. METHODS: This was a cross-sectional study. Participants were 745 community-dwelling adults and elderly persons in the Goto Islands in Nagasaki, who underwent a health checkup for residents in 2015 and 2016. Data were collected on gender; age; grip strength; hemoglobin; Creatinine (Cr); glycated hemoglobin (HbA1c); history of stroke; smoking, drinking, exercise, and walking habits; number of teeth; wearing of removable dentures; functional units of natural teeth (n-FTUs), fixed prostheses (nif-FTUs), and removable dentures (t-FTUs); and tongue pressure. The associations between each variable and tongue pressure were examined using multiple regression analysis. Next, those with 3 or fewer n-FTUs were selected, and differences in tongue pressure were compared between those with 3 or fewer nif-FTUs and those with 4 or more nif-FTUs, using a propensity score matching method. RESULTS: Male gender, weak grip strength, low HbA1c, no drinking, and a low number of teeth were independent factors significantly associated with lower tongue pressure. Among participants with 3 or fewer n-FTUs, the 43 with 4 or more nif-FTUs showed significantly higher tongue pressure than the 43 with 3 or fewer nif-FTUs after propensity score matching, although the number of t-FTUs was not associated with tongue pressure. DISCUSSION/CONCLUSION: Tooth loss was significantly associated with lower tongue pressure. It was suggested that fixed prosthesis treatment might prevent the reduction of tongue pressure, but removable dentures did not have such an effect.

Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.

A Case of a Stafne Bone Defect Associated with Sublingual Glands in the Lingual Side of the Mandible.

A Stafne bone defect from the mandibular anterior to the premolar region is an extremely rare case. A case of a Stafne bone defect extending from the mandibular anterior to the premolar region was presented. Computed tomography (CT) and magnetic resonance imaging (MRI) suggested that salivary gland tissue connected to the sublingual glands was involved in the formation of the cavity. The patient was a 68-year-old man who was examined at our hospital's emergency outpatient department after a traffic accident. He was referred to our department for the treatment of contusions of the lips and oral cavity. A bone defect in the lingual side of the mandible from the right anterior to the right premolar region was incidentally detected on CT. CT showed a rounded cavity in the lingual side of the mandible that had a lingual opening, was monocystic, and had a cortical margin. The margin of the cavity was relatively dull and regular. MRI showed that the tissue filling the cavity in the lingual side of the mandible had similar signal intensity as the sublingual glands and was contiguous with the normal sublingual glands. Based on these findings, the bone defect was diagnosed as a Stafne bone defect filled with salivary gland tissue connected to the sublingual gland tissue.

Association between high psychological distress and poor oral health-related quality of life (OHQoL) in Japanese community-dwelling people: the Nagasaki Islands Study.

BACKGROUND: We investigated the association between psychological distress and oral health status/oral health-related quality of life (OHQoL) in Japanese community-dwelling people. METHODS: We conducted a cross-sectional study using data from the Nagasaki Islands Study. A total of 1183 (455 men and 728 women) has been analyzed in this study. Psychological distress was measured using the Kessler Psychological Distress Scale (K6). Oral health status was measured by dental examination. The OHQoL was measured using the General Oral Health Assessment Index (GOHAI). We defined the total score of ≥5 points on the K6 as high psychological distress (high-K6 group). RESULTS: The multiple linear regression analysis to identify the GOHAI showed that gender, K6, the total number of teeth, the number of dental caries, and visiting a dental clinic within the past 6 months significantly associated with the GOHAI. Among all of these variables, high-K6 (≥ 5) was a substantial contributing factor of the GOHAI (ß = - 0.23, 95% Cl - 2.31 to -1.41, p < 0.0001). CONCLUSIONS: It is likely that the individual with high psychological distress was strongly related to poor OHQoL even in the general population.

Association between human T cell leukemia virus type-1 (HTLV-1) infection and advanced periodontitis in relation to atherosclerosis among elderly Japanese: a cross-sectional study.

BACKGROUND: Human T cell leukemia virus type-1 (HTLV-1) stimulates inflammation activity. Our previous study revealed a positive association between asymptomatic HTLV-1 infection and advanced periodontitis among elderly Japanese individuals with low levels of hematopoietic activity (reflected by reticulocyte levels). Since low hematopoietic activity has been correlated with low-grade inflammation and low-grade inflammation is associated with atherosclerosis, the status of atherosclerosis could, in turn, determine the nature of this association. METHODS: To this end, a cross-sectional study of 907 elderly Japanese individuals (aged 60-99 years), who had participated in dental health check-up during the period 2016-2018, was conducted. Advanced periodontitis was defined as periodontal pocket ≥ 6.0 mm. RESULTS: Among the study population, 295 (32.5%) were found to have atherosclerosis defined as a carotid intima-media thickness (CIMT) of ≥ 1.1 mm. HTLV-1 infection was positively associated with advanced periodontitis in participants with atherosclerosis, but no significant associations were observed among the participants without atherosclerosis. The known risk factors' (including reticulocyte and CIMT) adjusted odds ratio (OR) and 95% confidence interval (CI) of advanced periodontitis were OR 2.01 and 95% CI 1.06-3.81 for participants with atherosclerosis and OR 0.61 and 95% CI 0.34-1.12 for participants without atherosclerosis. CONCLUSION: This study found a significant association between HTLV-1 infection and advanced periodontitis among elderly Japanese with atherosclerosis. However, this association is absent in individuals without atherosclerosis, suggesting that atherosclerosis might act as a determinant in the association between HTLV-1 infection and advanced periodontitis among elderly Japanese.

Associations between caries experience, dietary habits, and metabolic syndrome in Japanese adults.

Dental caries could be a risk factor for metabolic syndrome (MetS); however, there is limited evidence of such a relationship in the literature. This cross-sectional study investigated the relationships among dental caries experience, dietary habits, and MetS in Japanese adults. A total of 937 participants aged 40-74 years underwent a health check, including dental examination. Decayed, missing, and filled teeth (DMFT) were used as an index of caries experience. The mean DMFT score was 14, and 12% of the participants had MetS in this study. Multivariate logistic regression analyses showed that the prevalence of MetS was significantly related to DMFT (first vs. fourth quartile, odds ratio [OR] = 1.80; P < 0.05). In addition, the OR of DMFT for MetS was found to be greater in each successively higher DMFT quartile. The prevalence of MetS was significantly related to daily coffee consumption (OR = 0.51, P < 0.01), and the relationship between DMFT and MetS was noted after adjusting for daily coffee consumption. There appears to be a positive association between caries experience and MetS in Japanese adults. This relationship increased with the increase in DMFT regardless of dietary habits.

Association between human T cell leukemia virus 1 (HTLV-1) infection and advanced periodontitis in relation to hematopoietic activity among elderly participants: a cross-sectional study.

BACKGROUND: We reported that human T cell leukemia virus 1 (HTLV-1) infection is positively associated with atherosclerosis. Recent evidence has revealed a close association of periodontitis with atherosclerosis, endothelial dysfunction, and disruption of the microcirculation. However, the association between HTLV-1 and advanced periodontitis has not been investigated to date. Since hematopoietic activity is closely linked to endothelial maintenance activity and is known to decline with age, we hypothesized that the state of hematopoietic activity influenced the association between HTLV-1 and advanced periodontitis in elderly participants. METHODS: A cross-sectional study was performed including 822 elderly participants aged 60-99 years who participated in a dental health check-up. Advanced periodontitis was defined as a periodontal pocket ≥ 6.0 mm. Participants were classified as having low or high hematopoietic activity according to the median values of reticulocytes. RESULTS: HTLV-1 infection was positively related to advanced periodontitis among participants with lower hematopoietic activity (lower reticulocyte count), but not among participants with higher hematopoietic activity (higher reticulocyte count). The adjusted odds ratio (95% confidence interval) considering potential confounding factors was 1.92 (1.05-3.49) for participants with a lower reticulocyte count and 0.69 (0.35-1.36) for participants with a higher reticulocyte count. CONCLUSIONS: Among elderly participants, the association between HTLV-1 infection and advanced periodontitis is influenced by hematopoietic activity. Since hematopoietic activity is associated with endothelial maintenance, these findings provide an efficient tool for clarifying the underlying mechanism of the progression of periodontitis among elderly participants.

Self-reported behavior of eating quickly is correlated with visceral fat area in Japanese non-obese adults.

BACKGROUND AND OBJECTIVES: This cross-sectional study investigated the relationship between eating speed and fat accumulation in Japanese non-obese adults. METHODS AND STUDY DESIGN: In total, 381 non-obese participants aged 35-74 years underwent a health checkup including fat distribution. All participants underwent magnetic resonance imaging to quantify visceral fat area (VFA) and subcutaneous fat area (SFA). Information on eating speed was obtained using a self-administrated questionnaire. RESULTS: The numbers of participants with self-reported behavior of eating slowly, medium, or quickly were 24 (6.3%), 180 (47.2%), and 177 (46.5%), respectively. The prevalence of VFA ≥100 cm2 was higher in the eating quickly group than in the eating slowly (p<0.05) or medium groups (p<0.05). On the other hand, no significant differences in SFA ≥100 cm2 were observed between groups. In addition, multiple stepwise regression analysis showed that eating quickly was positively correlated with VFA (standard ß=0.068, p<0.05), but not with SFA. CONCLUSIONS: Although eating quickly was positively correlated with bigger VFA in Japanese non-obese adults, no associations were found between eating speed and SFA.

Association between eating behavior and poor glycemic control in Japanese adults.

This study investigated the relationship between eating behavior and poor glycemic control in 5,479 Japanese adults with hemoglobin A1c (HbA1c) <6.5% who participated in health checks. Respondents to a 2013 baseline survey of eating behavior, including skipping breakfast and how quickly they consumed food were followed up until 2017. We defined poor glycemic control after follow-up as HbA1c ≥6.5%, or increases in HbA1c of ≥0.5% and/or being under medication to control diabetes. We identified 109 (2.0%) respondents who met these criteria for poor glycemic control. After adjusting for sex, age, smoking status, body mass index (BMI), and eating behavior, the risk of poor glycemic control was increased in males (odds ratio [OR], 2.38; 95% confidence interval [CI] 1.37-4.12; p < 0.01), and associated with being older (OR, 1.07; 95% CI, 1.04-1.11; p < 0.001), having a higher BMI (OR, 1.29; 95% CI 1.23-1.35; p < 0.001), skipping breakfast ≥3 times/week (OR, 2.44; 95% CI, 1.35-4.41; p < 0.01), and changing from eating slowly or at medium speed to eating quickly (OR, 2.11; 95% CI, 1.04-4.26; p < 0.05). In conclusion, Japanese adults who were male, older, had a high BMI, skipped breakfast ≥3 times/week and ate quickly were at increased risk for poor glycemic control.

Association between number of pairs of opposing posterior teeth, metabolic syndrome, and obesity.

We investigated the relationship between the number of pairs of posterior teeth and metabolic syndrome (MetS), abdominal obesity (AO), and obesity, among Japanese adults. In 2005, 2,807 Japanese adults aged 25-74 years participated in the Survey of Dental Diseases and the National Health and Nutrition Survey. Based on the survey data, BMI, AO (JAS) determined by the Japan Atherosclerosis Society, AO (IDF) by the International Diabetes Federation, and MetS were assessed. Total functional tooth units (t-FTUs) were scored with pairs of opposing posterior teeth, including artificial teeth. Subjects were divided into three categories of chewing ability based on the score of t-FTUs: Poor (if score ≤ 9), Good (if score = 10-11), and Complete (if score = 12). The relationships between chewing ability and BMI ≥ 25, BMI ≥ 30, AO (JAS), AO (IDF), and MetS were tested using univariate and multivariate logistic regression analysis. The chewing ability was significantly associated with MetS, AO, and obesity in the univariate and multivariate logistic regression analysis. Adjusted OR of "Poor" compared to "Complete" were 1.51 (95% confidence interval (CI), 1.24-1.84) for BMI ≥ 25, 2.10 (95% CI 1.40-3.14) for BMI ≥ 30, 1.31 (95% CI 1.07-1.61) for AO (JAS), 1.40 (95% CI 1.15-1.70) for AO (IDF), and 1.34 (95% CI 1.04-1.72) for MetS. All were statistically significant. Preventing tooth loss and maintaining pairs of good chewing ability may be important factors in preventing MetS, AO, and obesity.

Correlation between ultrasound-diagnosed non-alcoholic fatty liver and periodontal condition in a cross-sectional study in Japan.

This cross-sectional study investigated the relationship between periodontal condition and ultrasound-diagnosed non-alcoholic fatty liver disease (NAFLD) in a Japanese oral health check population. A total of 1226 consecutive participant were enrolled in the study. Abdominal ultrasonography was applied to diagnose NAFLD. Of the study participants, 339 (27.7%) had ultrasonography-diagnosed NAFLD. The participants with NAFLD had a significantly higher prevalence of probing pocket depth (PPD) ≥ 4 mm (86.7%) than those without NAFLD (72.9%) (p < 0.05). After adjusting for gender, age, Brinkman index, regular exercise habits, body mass index, number of teeth present, presence of periodontitis, blood pressure, and serum parameters, there was a statistically significant difference in the adjusted odds ratios of having PPD ≥ 4 mm for NAFLD (Odds ratio = 1.881, 95% confidence interval 1.184-2.987, p < 0.01). Having PPD ≥ 4 mm may be a risk factor for ultrasound-diagnosed NAFLD in this cross-sectional study of a Japanese oral health check population.

CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag.

There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis.

AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis.

Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the "AGIA" tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10-9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis.