For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10°C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.
Abstract Background Cardiac lymphatics maintains myocardial fluid and immune cell homeostasis. Downregulation of cardiac lymphatics leads to cardiac dysfunction through impairment of inflammation and edema. On the other hand, it is well known that adipose tissue accumulation causes lymphatic dysfunction and impairs lymphedema. Purpose We evaluated the expression of atrial lymphatics in human left atrial appendage (LAA) histologically and biologically. Subsequently, we examined secretomes from epicardial adipose tissue (EAT)-derived adipocytes and validated if they change atrial lymphatics expression using ex vivo Organo-culture system. Methods Human LAA samples were collected from forty-two patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=16, 73.1±10.4 years), the paroxysmal AF (PAF) group (n=13, 69.8±12.6 years) and the persistent AF (PeAF) group (n=13, 71.2±6.5 years). Human EAT were also excised from patients with and without AF (SR; n=5, AF; n=11) during cardiovascular surgery. Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation and maturation to adipocytes, cell culture supernatant (CCS) was collected, and was dropped onto the epicardial side of left atrial tissues excised from 8 weeks old male rats for 7 days using an ex vivo organo-culture system. Results In human LAA samples, mRNA expression of lymphangiogenesis-related genes (LYVE1, PROX1, VEGFC, and VEGFR3) was significantly lower in the PeAF group compared to the SR group (p=0.0370, <0.0001, 0.0157, and 0.0009). The number of LYVE1-positive atrial lymphatic endothelial cells (LECs) was also significantly lower in the PeAF group compared to the SR group and the PAF group (p=0.0186 and 0.038). There was a negative correlation between the number of LYVE1-positive atrial LECs and the area percentage of atrial myocardial fibrosis (r=-0.5350, p<0.001). In the experiment using organo-culture system, lymphangiogenesis-related genes expression (Lyve1, Prox1, Vegfc, and Vegfr3) of rat atria treated with CCS of adipocytes from AF patients was significantly lower than those from SR patients (p=0.0391, 0.0100, 0.0034, and 0.0338). Furthermore, the CCS of adipocytes from AF patients contained higher protein levels of leptin, which is known to suppress lymphangiogenesis (p=0.0357). Leptin treatment (100ng/ml) on rat atria also decreased lymphangiogenesis-related genes expression such as Lyve1, Prox1, Vegfc, and Vegfr3 (p=0.0413, < 0.0001, <0.0001, and 0.0190). Conclusions Our study with human LAA demonstrated that atrial lymphatics interact with atrial myocardial fibrosis/progression of AF. Furthermore, our findings indicate that secretomes from EAT-derived adipocytes regulate atrial lymphatics expression, and leptin plays an important role in modulating atrial lymphangiogenesis.
Abstract Background Glucose fluctuations (GF) is known to be the significant factor related to poor cardiovascular outcome in the diabetic patients. We investigated the potential impact of GF on paracrine effect of human epicardial adipocyte on cardiomyocyte. Methods Human EAT biopsies obtained during surgery and isolated preadipocyte/adipocytes were used to evaluate the direct impact of GF on the adipocytes. Matured adipocytes were exposed to the media containing normal glucose (100 mg/dl, NG), high glucose (300 mg/dl, HG), or high-low glucose (75-300 mg/dl, GF) for 1 week. Transcriptomic profiling of the cultured matured adipocyte was performed using microarray analysis. The impact of GF on the paracrine effect of adipocyte was evaluated using a co-culture with human iPS-derived atrial cardiomyocytes and the adipocytes. Clinical association study verified the validity of the findings. Results GF for 1 week altered the expression of 595 differentially expressed genes (up-regulated or down-regulated) with an effect size of >50% or `−50% and a significant statistical difference (P < 0.05) in human epicardial adipocytes, while the continuous HG for 1 week altered the expression of only 182 differentially expressed genes. Of the genes affected by GF, top 3 most affected genes included IL1β, IL33, IL8. Regarding the other major pro-inflammatory cytokines, IL1α, CCL2 and IL6 were also significantly increased in the adipocyte treated with GF, however these were not increased in the adipocyte treated with HG. Co-culture of human iPS cell-derived atrial cardiomyocytes and adipocytes treated with NG, HG and GF revealed that GF-treated adipocyte strikingly increased oxidative stress in the cardiomyocyte through the paracrine effect. In the real-world clinical association study, IL1β mRNA expression in human epicardial adipose tissue showed significant positive correlation with the severity of GF during hospitalization and oxidative stress in the left atrial myocardium. Conclusions GF adversely affect the paracrine secretome profile of human epicardial adipocyte. IL1β may be a critical epicardial adipocyte-derived adipokine that is enhanced by GF.
Abstract Background Postoperative atrial fibrillation (POAF) occurs in 20-50% after cardiovascular surgery and is closely associated with the short and long-term morbidity/mortality. The epicardial adipose tissue (EAT) is in contact with the atrial muscle, and may have a potential predisposing factor to induce POAF within it. Purpose We tried to identify the specific gene expression profile of the human epicardial adipocyte associated with POAF. Methods We prospectively evaluated 126 patients without a history of AF (male 86 (68.3%); mean age 68.3 ± 11.7) who underwent scheduled open-heart cardiovascular surgery between May 2020 and November 2022. They were assigned to the POAF group and the Non-POAF group. POAF was defined as AF lasting more than 30 seconds documented by monitor or 12-lead electrocardiogram after the operation. Fresh human EAT samples were obtained from these patients during surgery, and preadipocytes were isolated from these samples. Isolated preadipocytes were terminally differentiated to mature adipocytes and RNAs were extracted. Genome-wide expression profiling of 6 vs. 6 matched samples of the POAF and Non-POAF groups was performed using Human Clariom S Assay-HT 16-Array Plate. The secreted protein levels in the adipocytes-cultured medium were also evaluated using enzyme-linked sorbent assay test to confirm the consistency of mRNA expression levels of adipocytes and its secreted protein levels. Results POAF was observed in 48 patients (38%). Patients in the POAF group were significantly older (p = 0.003) compared to the Non-POAF group. POAF occurred mainly within the first 3 postoperative days (median 2.5 days), and the median duration of POAF was 298 ± 581 minutes. The occurrence of POAF was significantly associated with longer postoperative hospitalization (p = 0.001) and intensive care unit stay (p = 0.04). Microarray analysis revealed that there were 132 down- or up-regulated cording genes (fold change >2.0 or `−2.0, p < 0.05) in epicardial adipocytes of the Non-POAF group compared to the POAF group. Of these genes, 11 genes cording secretable molecules were validated by qPCR, showing that only SPARCL1 had significantly higher expression in the Non-POAF group compared to the POAF group (p = 0.008). The mRNA expression levels of SPARCL1 significantly decreased when the preadipocyte were terminally differentiated to the mature adipocyte (p = 0.0009). SPARCL1 mRNA expression in adipocyte was significantly correlated with the concentration of SPARCL1 protein in the adipocyte-cultured medium (p < 0.0001, r = 0.7476). Conclusion The results of this study suggest that the expression of SPARCL1 in human epicardial adipocytes may play an important role in the suppression of POAF. Our findings suggested that SPARCL1 expression in human epicardial adipocytes may play an important role in the suppression of POAF. SPARCL1 secreted by epicardial adipocytes may be a potential therapeutic target for POAF prevention.Flowchart of patient selectionMicroarray analysis and qPCR varidation
There are several techniques for the treatment of mandibular condylar fractures. This is the first report of the high submandibular anteroparotid approach for open reduction and internal fixation of condylar fracture.A 41-year-old woman fell indoors and injured her face. She was referred to our department for detailed examination and treatment of a suspected mandibular fracture. X-ray and computed tomography showed a right mandibular condylar base fracture and lateral dislocation of the fracture fragment. Open reduction and internal fixation procedures were performed for a right mandibular condylar fracture under general anesthesia. The mandibular ramus was reached by approaching from the inferior margin of the mandible, delaminating the masseter fascia posteriorly, and bypassing the anterior margin of the parotid gland. Once the fractured bone was reached, reduction and fixation were performed.We have achieved good results by the high submandibular anteroparotid approach, which is minimally invasive and simple, to reduce and fix condylar fractures. With this approach, no facial artery or retromandibular vein was encountered, and the mental stress for the surgeon was minimal. Postoperative wound infection, parotid gland complications such as parotitis and salivary fistula, facial nerve dysfunction such as facial paralysis, and esthetic disorders such as scarring were not observed.Although it is necessary to examine more cases in the future, the high submandibular anteroparotid approach may be useful as a new approach for open reduction and internal fixation of condylar fractures.
