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l-Amino acid oxidase (LAAO), an FAD-dependent enzyme, catalyzes the oxidation of l-amino acids (l-AAs) to their corresponding imino acids. While LAAOs, which can oxidize charged or aromatic l-AAs specifically, have been extensively characterized across various species, LAAOs that have high specificity toward alkyl-chain l-AAs, such as l-Met, are hardly characterized for now. In this study, we screened a highly specific l-Met oxidizing LAAOs from Burkholderiales bacterium (BbMetOx) and Undibacterium sp. KW1 (UndMetOx) using sequence similarity network (SSN) analysis. These enzymes displayed an order of magnitude higher specific activity towards l-Met compared to other l-AAs. Enzyme activity assays showed that these LAAOs operate optimally at moderate condition because the optimal pH and Tm values were pH 7.0 and 58-60°C. We determined the crystal structures of wild-type BbMetOx (BbMetOx(WT)) and an inactivated mutant, BbMetOx (K304A), at 2.7 Å and 2.2 Å resolution, respectively. The overall structure of BbMetOx is closely similar to other known LAAOs of which structures were determined. Comparative analysis of the BbMetOx structures revealed significant conformational changes in the catalytic domain, particularly a movement of approximately 8 Å in the Cα atom of residue Y180. Further analysis highlighted four residues, i.e., Y180, M182, F300, and M302, as critical for l-Met recognition, with alanine substitution at these positions resulting in loss of activity. This study not only underscores the utility of SSN for discovering novel LAAOs but also advances our understanding of substrate specificity in this enzyme family.
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The objective of this study was to evaluate the intramammary distribution of MRI-detected mass and focus lesions that were difficult to identify with conventional B-mode ultrasound (US) alone. Consecutive patients with lesions detected with MRI but not second-look conventional B-mode US were enrolled between May 2015 and June 2023. Following an additional supine MRI examination, we performed third-look US using real-time virtual sonography (RVS), an MRI/US image fusion technique. We divided the distribution of MRI-detected mammary gland lesions as follows: center of the mammary gland versus other (superficial fascia, deep fascia, and atrophic mammary gland). We were able to detect 27 (84%) of 32 MRI-detected lesions using third-look US with RVS. Of these 27 lesions, 5 (19%) were in the center of the mammary gland and 22 (81%) were located in other areas. We were able to biopsy all 27 lesions; 8 (30%) were malignant and 19 (70%) were benign. Histopathologically, three malignant lesions were invasive ductal carcinoma (IDC; luminal A), one was IDC (luminal B), and four were ductal carcinoma in situ (low-grade). Malignant lesions were found in all areas. During this study period, 132 MRI-detected lesions were identified and 43 (33%) were located in the center of the mammary gland and 87 (64%) were in other areas. Also, we were able to detect 105 of 137 MRI-detected lesions by second-look conventional-B mode US and 38 (36%) were located in the center of the mammary gland and 67 (64%) were in other areas. In this study, 81% of the lesions identified using third-look US with RVS and 64% lesions detected by second-look conventional-B mode US were located outside the center of the mammary gland. We consider that adequate attention should be paid to the whole mammary gland when we perform third-look US using MRI/US fusion technique.
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Neoplasias da Mama , Imageamento por Ressonância Magnética , Ultrassonografia Mamária , Humanos , Feminino , Imageamento por Ressonância Magnética/métodos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Pessoa de Meia-Idade , Adulto , Ultrassonografia Mamária/métodos , Idoso , Imagem Multimodal/métodos , Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/patologiaRESUMO
Methotrexate may cause methotrexate-associated lymphoproliferative disorder (MTX-LPD); however, this disease is uncommon in the gastrointestinal tract. We examined six patients with MTX-LPD in the gastrointestinal tract at our hospital. All of the lesions were ulcerative. Four cases regressed spontaneously, all of which were positive for Epstein-Barr virus-encoded small RNA by in situ hybridization. Two patients who did not regress were negative for Epstein-Barr virus RNA, one of whom received chemotherapy, and one underwent surgery. The prognosis of this disease is considered to be good. MTX-LPD should considered when ulcerative lesions are observed during endoscopy in patients receiving MTX.
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There is no straightforward method to visualize the intracellular distribution of nuclear receptors, such as retinoid X receptors (RXRs), which are trafficked between the cytosol and nucleus. Here, in order to develop a simple fluorescence labeling method for RXRs, we designed and synthesized compound 4, consisting of an RXR-selective antagonist, CBTF-EE (2), linked via an ether bond to the fluorophore nitrobenzoxadiazole (NBD). Compound 4 is nonfluorescent, but the ether bond (-O-NBD) reacts with biothiols such as cysteine and homocysteine to generate a thioether (-S-NBD), followed by intramolecular Smiles rearrangement with an amino group such as that of lysine to form a fluorescent secondary amine (-NH-NBD) adjacent to the binding site. Fluorescence microscopy of intact or RXR-overexpressing MCF-7 cells after incubation with 4 enabled us to visualize RXR expression as well as nuclear transfer of RXR induced by the agonist bexarotene (1).
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BACKGROUND AND AIMS: Clinicians sometimes encounter papillary thyroid microcarcinoma (PMC) that is less than 10 mm, associated with lymph node metastasis. In this study, we assessed PMC clinicopathologically to clarify risk factors for poor prognosis. PATIENTS AND METHODS: Fifty-one patients who underwent thyroid surgery at Aichi Medical University from September 2009 to October 2016 were included. Patients were divided into two groups, pEX-positive (23 patients) and pEX-negative (28 patients), based on the pathological finding of thyroid capsule invasion. The former indicates that the tumor infiltrated the thyroid capsule and spread to the neighboring tissue, and the latter indicates no capsule invasion. We analyzed factors such as patient characteristics, pathological findings, and serum levels of thyroid hormones in the two groups. RESULTS: No statistical differences were observed between the two groups in gender distribution or age at surgery. Preoperative cancer diagnoses were established for more patients in the pEX-positive group than in the pEX-negative group (n = 21 and 14, respectively; P = 0.004). The mean (±SD) pathological tumor diameter was 5.42 ± 2.77 in the pEX-negative group and 8.32 ± 1.61 in the pEX-positive group (P < 0.001). No significant differences in preoperative serum levels of free T3, free T4, thyroid-stimulating hormone, or thyroglobulin were observed between the two groups. The odds ratio for node positivity in tumors invading thyroid capsules (pEX-positive) compared to those with no capsule invasion (pEX-negative) was 13.20 (95% confidence interval, 3.45-50.42). Immunohistological staining for phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and Akt (protein kinase B) revealed the facilitation of PTEN and suppression of Akt, which might indicate downregulation of the phosphoinositide 3-kinase-Akt (PI3K-Akt) cascade. DISCUSSION: In general, the prognosis of PMC is favorable. However, the prognosis is less favorable in patients with nodal metastasis or extrathyroidal invasion. It is controversial whether resection is required for proven PMCs. For PMCs associated with extrathyroidal invasion, regional lymph node resection with lobectomy should be performed due to the high risk for lymphatic spread. There might be a possibility that the natural progression of PMC seems to be controlled by the facilitation of PTEN. However, a tumor in the lateral peripheral region of the thyroid parenchyma might be associated with capsule invasion followed by lymphatic spread.
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AIM: This study aimed to analyze two cases of marked hypo-high-density lipoprotein (HDL) cholesterolemia to identify mutations in ATP-binding cassette transporter A1 (ABCA1) and elucidate the molecular mechanism by which these novel pathological mutations contribute to hypo-HDL cholesterolemia in Tangier disease. METHODS: Wild type and mutant expression plasmids containing a FLAG tag inserted at the C-terminus of the human ABCA1 gene were generated and transfected into HEK293T cells. ABCA1 protein expression and cholesterol efflux were evaluated via Western blotting and efflux assay. The difference in the rate of change in protein expression was evaluated when proteolytic and protein-producing systems were inhibited. RESULTS: In case 1, a 20-year-old woman presented with a chief complaint of gait disturbance. Her HDL-C level was only 6.2 mg/dL. Tangier disease was suspected because of muscle weakness, decreased nerve conduction velocity, and splenomegaly. Whole-exome analysis showed compound heterozygosity for a W484* nonsense mutation and S1343I missense mutation, which confirmed Tangier disease. Cholesterol efflux decreased by a mixture of W484* and S1343I mutations. The S1343I mutation decreased the protein production rate but increased the degradation rate, decreasing the protein levels. This patient also had Krabbe disease. The endogenous ABCA1 protein level of macrophage cell decreased by knocking down its internal galactocerebrosidase.Case 2, a 51-year-old woman who underwent tonsillectomy presented with peripheral neuropathy, corneal opacity, and HDL-C of 3.4 mg/dL. Whole-exome analysis revealed compound heterozygosity for R579* and R1572* nonsense mutations, which confirmed Tangier disease. CONCLUSION: Case 1 is a new ABCA1 mutation with complex pathogenicity, namely, a W484*/S1343I compound heterozygote with marked hypo-HDL cholesterolemia. Analyses of the compound heterozygous mutations indicated that decreases in ABCA1 protein levels and cholesterol efflux activity caused by the novel S1343I mutation combined with loss of W484* protein activity could lead to marked hypo-HDL cholesterolemia. Galactocerebrosidase dysfunction could also be a potential confounding factor for ABCA1 protein function.
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Food allergies are a significant health issue worldwide. In many countries, labeling of primary allergens in food products has been made mandatory to ensure consumer safety. In food manufacturing settings, the lateral flow immunoassay (LFI)-based on antigen-antibody reactions-is a rapid and accurate method for allergen testing and is widely used. Peptide arrays are tools that enable the synthesis of peptides of any sequence on a substrate and high-throughput analysis of their interactions with chemicals. This study aimed to investigate a new application of peptide arrays in the field of food technology, particularly in the development of antibodies for food allergen testing. First, monoclonal antibodies against hen egg ovalbumin, a major food allergen, were produced. Then, using a peptide array, the epitope and specificity of the antibodies were comprehensively and precisely analyzed. Finally, an LFI kit incorporating the antibodies demonstrated both high specificity and detection sensitivity for food allergen testing. These findings indicate that peptide arrays are valuable tools in the development of antibodies for food allergen testing, ensuring reliability and accuracy at the molecular level.
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Enantiomerically pure D-amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide-based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L-amino acids into their D-isomers. The system integrates four enzymes: ancestral L-amino acid oxidase (AncLAAO-N4), D-amino acid dehydrogenase (DAADH), D-glucose dehydrogenase (GDH), and catalase. AncLAAO-N4 initiates the process by converting L-amino acids to corresponding keto acids, which are then stereo-selectively aminated to D-amino acids by DAADH using NADPH and NH4Cl. Concurrently, any generated H2O2 is decomposed into O2 and H2O by catalase, while GDH regenerates NADPH from D-glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D-amino acids, including a D-Phe derivative, three D-Trp derivatives, and D-phenylglycine, all with high enantiopurity (>99 % ee) at a preparative scale (>100â mg). This system demonstrates a versatile approach for producing a diverse array of D-amino acids.
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Aminoácidos , L-Aminoácido Oxidase , Aminoácidos/química , Catalase , NADP , Peróxido de Hidrogênio , Glucose 1-DesidrogenaseRESUMO
AIM: The aim of this study was to investigate genetic alterations within breast cancer in the setting of recurrent or de novo stage IV disease. PATIENTS AND METHODS: This study included 22 patients with recurrent breast cancer (n = 19) and inoperable de novo stage IV breast cancer (n = 3). For next generation sequencing, FoundationOneCDx (F1CDx) (Foundation Medicine Inc., Cambridge, MA, USA) was performed in 21 patients and FoundationOneLiquid CDx was performed in 1 patient. RESULTS: Median age was 62.9 years (range, 33.4-82.1). Pathological diagnoses of specimens included invasive ductal carcinoma (n = 19), invasive lobular carcinoma (n = 2), and invasive micropapillary carcinoma (n = 1). F1CDx detected a median of 4.5 variants (range, 1-11). The most commonly altered gene were PIK3CA (n = 9), followed by TP53 (n = 7), MYC (n = 4), PTEN (n = 3), and CDH1 (n = 3). For hormone receptor-positive patients with PIK3CA mutations, hormonal treatment plus a phosphoinositide 3-kinase inhibitor was recommended as the treatment of choice. Patients in the hormone receptor-negative and no human epidermal growth factor receptor 2 expression group had significantly higher tumor mutational burden than patients in the hormone receptor-positive group. A BRCA2 reversion mutation was revealed by F1CDx in a patient with a deleterious germline BRCA2 mutation during poly ADP ribose polymerase inhibitor treatment. CONCLUSION: Guidance on tailored precision therapy with consideration of genomic mutations was possible for some patients with information provided by F1CDx. Clinicians should consider using F1CDx at turning points in the course of the disease.
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Neoplasias da Mama , Carcinoma , Humanos , Pessoa de Meia-Idade , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fosfatidilinositol 3-Quinases/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Genômica , Mutação , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
High selectivity of small-molecule drug candidates for their target molecule is important to minimize potential side effects. One factor that contributes to the selectivity is the internal polarity of the ligand-binding pocket (LBP) in the target molecule, but this is difficult to measure. Here, we first confirmed that the retinoid X receptor (RXR) agonist 6-(ethyl(1-isobutyl-2-oxo-4-(trifluoromethyl)-1,2-dihydroquinolin-7-yl)amino)nicotinic acid (NEt-iFQ, 1) exhibits fluorescence solvatochromism, i.e., its Stokes shift depends on the polarity of the solvent, and then we utilized this property to directly measure the internal polarity of the RXRα-LBP. The Stokes shift of 1 when bound to the RXRα-LBP corresponded to that of 1 in chloroform solution. This finding is expected to be helpful for designing RXR-selective ligands. A similar approach should be appliable to evaluate the internal polarity of the LBPs of other receptors.
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Receptores X de Retinoides , Receptores X de Retinoides/metabolismo , LigantesRESUMO
Purpose: Chronic pain and migraines often go untreated despite patient- and economic-related burdens (e.g., impaired quality of life and productivity). Understanding the reasons for non-treatment is important to enable interventions aimed at improving care-seeking behaviors. However, reports on disease-specific justifications for nontreatment in Japan are limited. We aimed to determine the barriers to healthcare access in untreated patients with chronic pain or migraines. Patients and methods: This was a non-interventional, cross-sectional, internet questionnaire survey of patients with chronic pain or migraines. The primary endpoint was to identify the reasons for untreated chronic pain or migraines. Secondary endpoints included factors associated with healthcare access, including patient background, patient-reported outcomes, and awareness of generic or authorized generic drugs (AG). Results: We surveyed 1,089 patients with chronic pain [605 (55.6%) untreated] and 932 patients with migraines [695 (74.6%) untreated] in 2021. The main reasons for not seeking treatment for chronic pain was "my pain is tolerable" and for migraine, "I can manage my pain with over-the-counter drugs." Background factors significantly associated with untreated chronic pain were younger age, less time required to access the nearest medical institution, less pain, higher activities of daily living (ADL) scores, and lower awareness of generic drugs and AG. Among patients with migraine, notable characteristics included being female, having shorter travel times to the nearest medical facility, residing in municipalities with populations under 50,000, experiencing moderate to severe pain, having higher ADL scores, and displaying lower awareness of AG. The AG awareness rate was 2-fold higher in treated patients than in untreated patients. Conclusion: Educating patients regarding the risks associated with pain and its underlying causes, availability of inexpensive treatment options, and location of appropriate treatment facilities may increase treatment rates.
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Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a "ninja-star" feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger kcat/Km values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in kcat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac-forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs.
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BRCA1 and 2 mutations are known to be associated with breast cancer, and olaparib, a poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor, has been shown to be effective in cells carrying these mutations in some studies. Erythema nodosum (EN), which is one adverse event of olaparib and is discussed in this paper, is considered to be a very rare condition. A 69-year-old female patient underwent left breast conservative surgery with axillary lymph node dissection for left invasive ductal breast cancer (stage IIB). Her family history included a sister who developed ovarian cancer at age 63. Five years postoperatively, systemic metastases were discovered in the lung, bone, hilar, and poststernal lymph nodes. The surgically removed metastatic lung nodule was diagnosed as an estrogen receptor (ER)-positive, progesterone receptor (PgR)-negative, and human epidermal growth factor receptor 2 (HER2)-negative metastatic adenocarcinoma of breast cancer origin. And germline mutations of BRCA1/2 were assessed using BRACAnalysis CDx® (Myriad Genetics, Salt Lake City, UT, USA), and BRCA2 1241 delC was identified as a deleterious mutation. Oral administration of olaparib was started. On day 4 of this treatment, numerous erythematous plaques characterized by intense tenderness and infiltration appeared on the extensor surfaces of the bilateral lower legs. On the basis of the clinical findings, the lesions were diagnosed as EN. Oral prednisolone was started at the same time as olaparib discontinuation, and the EN lesions disappeared in one week. EN is an inflammatory lesion characterized by tender subcutaneous induration with a flushed surface, predominantly on the bilateral lower legs. EN occurring after olaparib administration is considered to be very rare. This article describes such a case and reviews the relevant literature.
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The aim of this study is to provide a new perspective on the development of masking agents by examining the application of their time-series sensory profiles. The analysis of the relationship between 14 time-intensity (TI) parameters and the beany flavor masking ability of 100 flavoring materials indicate that the values of AreaInc, DurDec, and AreaDec, TI parameters related to the flavor release in the increasing and decreasing phases, were significantly higher in the top 10 masking score materials than in the bottom 10 materials. In addition to individual analysis, machine learning analysis, which can derive complex rules from large amounts of data, was performed. Machine learning-based principal component analysis and cluster analysis of the flavoring materials presented AreaInc and AreaDec as TI parameters contributing to the classification of flavor materials and their masking ability. AreaDec was suggested to be particularly important for the beany flavor masking in the two different analyses: an effective masking can be achieved by focusing on the TI profiles of flavor materials. This study proposed that time-series profiles, which are mainly used for the understanding of the sensory characteristics of foods, can be applied to the development of masking agents.
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BACKGROUND: Invasive lobular carcinoma (ILC) sometimes presents with unique clinical, pathologic, and radiographic features. In this case report, we describe a patient with ILC, whose initial presentation consisted with symptoms secondary to bone-marrow dissemination. In addition, the breast primary was revealed only by magnetic resonance imaging (MRI) followed by real-time virtual sonography (RVS). CASE PRESENTATION: A 51-year-old woman presented to our outpatient clinic with dyspnea on exertion. She had severe anemia (hemoglobin, 5.3 g/dL) and thrombocytopenia (platelet count, 31 × 103/mL). Bone-marrow biopsy was performed to evaluate hematopoietic function. The pathologic diagnosis was bone-marrow carcinomatosis due to metastatic breast cancer. Initial mammography followed by ultrasonography (US) failed to detect the primary tumor. On MRI, a non-mass-enhancement lesion was observed. While second-look US also did not detect the lesion, it was clearly visualized with RVS. We were finally able to biopsy the breast lesion. The pathologic diagnosis was ILC positive for both estrogen receptor and progesterone receptor, with 1 + immunohistochemical staining for human epidermal growth factor receptor 2. This case of ILC was characterized by bone-marrow metastasis. Due to decreased cell adhesion, the risk of bone-marrow metastasis is higher in ILC than in invasive ductal carcinoma, the most prevalent type of breast cancer. Biopsy of the primary lesion, which was initially only detected with MRI, was successfully performed with clear visualization during RVS, which is based on the fusion of MRI and US images. CONCLUSION: In this case report and literature review, we describe the unique clinical characteristics of ILC and a strategy for identifying primary lesions that are initially only visualized with MRI.
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Retinoid X receptor (RXR), a nuclear receptor (NR) that regulates transcription of target genes in a ligand binding-dependent manner, is of interest as a drug target. RXR agonists have been developed as therapeutic agents for cutaneous invasive T-cell lymphoma (e.g., bexarotene (1)) and investigated as potential anti-inflammatory agents. Screening systems for the binding of RXR alone have been reported. However, although RXRs function as RXR heterodimers, information on systems to evaluate the differential binding of RXR agonists as RXR heterodimers has not been available until recently. Here we show that the fluorescent RXR agonist CU-6PMN (3), designed by our group, can be useful for assessing RXR binding to PPARγ/RXRα, and that the binding data differ from those of RXRα alone. This screening method opens a new avenue for binding assays for RXR heterodimers.
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Sequence-based protein design approaches are being adopted to generate highly functional enzymes; however, screening the enzymes remains a time-consuming task. In this study, by analyzing the enzymatic properties of four ancestral meso-2,6-diaminopimelate dehydrogenases (AncDAPDHs), AncDAPDH-N1, -N2, -N3, and -N4, we attempted to define a new index parameter that is helpful for efficiently screening the enzymes. Biochemical and thermodynamic analyses indicated that only AncDAPDH-N4 exhibited greater thermal stability than and activity similar to those of native DAPDHs. Structural and sequence comparisons between DAPDH from Corynebacterium glutamicum (CgDAPDH) and the AncDAPDHs suggested that "quality of mutations" is a potential index parameter. In fact, the mutations introduced from CgDAPDH to AncDAPDH-N4 correlated highly with the mutations accumulated during the evolution process from mesophiles to thermophiles. These results suggest that, although there are several exceptions, the correlation coefficient can be used as an index parameter for screening high-functioning enzymes from sequence data.