First noncontact millimeter-wave radar measurement of heart rate in great apes: Validation in chimpanzees (Pan troglodytes).

Heart rate is a crucial vital sign and a valuable indicator for assessing the physical and psychological condition of a target animal. Heart rate contributes to (1) fundamental information for cognitive research, (2) an indicator of psychological and physical stress, and (3) improving the animal welfare of captive animals, especially in nonhuman primate studies. Heart rate has been measured using a contact-type device; however, the device burdens the target animals and that there are risks associated with anesthesia during installation. This study explores the application of heartbeat measurement techniques using millimeter-wave radar, primarily developed for humans, as a remote and noninvasive method for measuring the heart rate of nonhuman primates. Through a measurement test conducted on two chimpanzees, we observed a remarkable correspondence between the peak frequency spectrum of heart rate estimated using millimeter-wave radar and the mean value obtained from electrocardiograph data, thereby validating the accuracy of the method. To the best of our knowledge, this is the first demonstration of the precise measurement of great apes' heart rate using millimeter-wave radar technology. Compared to heart rate measurement using video analysis, the method using millimeter-wave radar has the advantage that it is less susceptible to weather and lighting conditions and that measurement techniques for multiple individuals have been developed for human subjects, while its disadvantage is that validation of measurement from long distances has not been completed. Another disadvantage common to both methods is that measurement becomes difficult when the movement of the target individual is large. The possibility of noncontact measurement of heart rate in wild and captive primates will undoubtedly open up a new research area while taking animal welfare into consideration.

Outdoor noncontact respiratory measurements of unrestricted rhesus macaques (Macaca mulatta) using millimeter-wave radar.

Respiration is an invaluable signal that facilitates the real-time observation of physiological dynamics. In recent years, the advancement of noncontact measurement technology has gained momentum in capturing physiological activities in natural settings. This technology is anticipated to be found not only in humans but also in nonhuman primates. Currently, the predominant noncontact approach for nonhuman animals involves measuring vital signs through subtle variations in skin color. However, this approach is limited when addressing areas of the body covered with hair or when working in outdoor settings under fluctuating sunlight. To overcome this issue, we focused on noncontact respiratory measurements using millimeter-wave radar. Millimeter-wave radar systems, which employ millimeter waves that can penetrate animal fur and estimate respiration-derived periodic body motion, exhibit minimal susceptibility to sunlight interference. Thus, this method shows potential for conducting noncontact vital measurements in natural and outdoor settings. In this study, we validated a millimeter-wave radar methodology for capturing respiration in outdoor-housed rhesus macaques (Macaca mulatta). The radar was positioned beyond the captive enclosure and maintained at a distance >5 m from the target. Millimeter waves were transmitted to the target, and the reflected waves were used to estimate skin surface displacement associated with respiration. The results revealed periodic skin surface displacement, and the estimated respiratory rates weres within the reported range of respiratory rates for rhesus macaques. These results suggest the potential applicability of millimeter-wave radar for noncontact respiration monitoring in outdoor-living macaques without anesthesia or immobilization. The continued advancement of noncontact vital measurement technology will contribute to understanding primate mental and physical dynamics during their daily life.

Sustained defense response via volatile signaling and its epigenetic transcriptional regulation.

Plants perceive volatiles emitted from herbivore-damaged neighboring plants to urgently adapt or prime their defense responses to prepare for forthcoming herbivores. Mechanistically, these volatiles can induce epigenetic regulation based on histone modifications that alter the transcriptional status of defense genes, but little is known about the underlying mechanisms. To understand the roles of such epigenetic regulation of plant volatile signaling, we explored the response of Arabidopsis (Arabidopsis thaliana) plants to the volatile ß-ocimene. Defense traits of Arabidopsis plants toward larvae of Spodoptera litura were induced in response to ß-ocimene, through enriched histone acetylation and elevated transcriptional levels of defense gene regulators, including ethylene response factor genes (ERF8 and ERF104) in leaves. The enhanced defense ability of the plants was maintained for 5 d but not over 10 d after exposure to ß-ocimene, and this coincided with elevated expression of those ERFs in their leaves. An array of histone acetyltransferases, including HAC1, HAC5, and HAM1, were responsible for the induction and maintenance of the anti-herbivore property. HDA6, a histone deacetylase, played a role in the reverse histone remodeling. Collectively, our findings illuminate the role of epigenetic regulation in plant volatile signaling.

Novel whole-mount FISH analysis for intact root of Arabidopsis thaliana with spatial reference to 3D visualization.

Whole-mount fluorescent in situ hybridization (WM-FISH) is an effective tool to observe chromosome behavior in tissues or organs. However, it is difficult to obtain a precise spatial profile of fluorescent signals in roots using conventional WM-FISH mainly because of the severe damage caused during the processing. To address this problem, we established a novel WM-FISH analysis for intact roots of Arabidopsis thaliana and successfully obtained a precise spatial profile of nuclear size and centromere signals. The two main improvements in the novel WM-FISH analysis are: (i) hybridization was performed directly on MAS-coated glass slides covered with silicon wells and (ii) conditions for enzyme treatment were optimized (37 °C, 45 s). After the WM-FISH using a centromere probe, we analyzed the results by 3D data processing to quantify the nuclear volume and number of centromere signals of the obtained cortical cell files and determined the position of each nucleus in intact roots. Then we plotted the nuclear volume and number of centromere signals versus distance from the quiescent center to evaluate the precise spatial profile of each parameter.

Readability and quality of online patient resources regarding knee osteoarthritis and lumbar spinal stenosis in Japan.

BACKGROUND: This study aimed to quantify the readability and quality of online patient resources on knee osteoarthritis and lumbar spinal stenosis in Japan. METHODS: Three search engines (Google, Yahoo, and Bing) were searched for the terms knee osteoarthritis and lumbar spinal stenosis. The first 30 websites of each search were screened. Duplicate websites and those unrelated to the searched diseases were excluded. The remaining 125 websites (62 on knee osteoarthritis, 63 on lumbar spinal stenosis) were analyzed. The text readability was assessed using two web-based programs (Obi-3 and Readability Research Lab) and lexical density. Website quality was evaluated using the DISCERN score, Clear Communication Index, and Journal of American Medical Association benchmark criteria. RESULTS: Readability scores were high, indicating that the texts were difficult to understand. Only 24 (19%) and six (5%) websites were classified as average difficulty readability according to Obi-3 and Readability Research Lab, respectively. The overall quality of information was low, with only four (3%) being rated as having sufficient quality based on the Clear Communication Index and Journal of American Medical Association benchmark criteria. None of the websites satisfied the DISCERN quality criteria. CONCLUSIONS: Patient information on Japanese websites regarding knee osteoarthritis and lumbar spinal stenosis were difficult to understand. Moreover, the quality of the websites was insufficient. Orthopaedic surgeons should contribute to the creation of high-quality easy-to-read websites to facilitate patient-physician communication.

[The Six Cases of Recurrence of Lung Metastasis after Radical Resection of Pancreatic Cancer Performed at Our Hospital].

Prognosis of pancreatic cancer is extremely poor due to recurrence in the early postoperative period. However, there are some reports that the prognosis for lung recurrence alone is relatively better than that of others. In this study, we report the cases of lung recurrence alone after radical resection of pancreatic cancer performed at our hospital. Among 255 cases of radical resection of pancreatic cancer performed at our hospital between July 2010 and August 2021, 6 cases in which the initial recurrence site of recurrence was lung alone were included in the study. The median age of the patients was 72 years (62-82), and there were 5 males and 1 female. Four patients had undergone pancreaticoduodenectomy and 2 patients underwent distal pancreatectomy. Four patients received adjuvant chemotherapy, 3 with S-1 and 1 with GEM. No patients underwent surgical resection for recurrence of lung metastasis, and all patients were treated with chemotherapy. The median time to recurrence was 351 days. As initial therapy after recurrence, 3 patients received GEM plus nab-PTX combination therapy and 3 patients received S-1. The median overall survival after recurrence was 1,979 days, and the 1-year and 3-year overall survival rates after recurrence were 100% and 100%, respectively. The prognosis of patients with recurrence of lung metastasis after surgery for pancreatic cancer at our hospital was similarly good as the previous reports.

Identification of CD56dim subpopulation marked with high expression of GZMB/PRF1/PI-9 in CD56+ interferon-α-induced dendritic cells.

As the sentinels of innate and adaptive immune system, dendritic cells (DCs) have been considered to hold a great promise for medical application. Among the diverse types of DCs, monocyte-derived DCs (mo-DCs) generated in vitro have been most commonly employed. We have been improving the culture protocol and devised a protocol to produce mature interferon-α-induced DCs (IFN-DCs), hereinafter called (mat)IFN-DCs. While exploring the relationship between the expression of CD56 and the cytotoxic activity of (mat)IFN-DCs, we unexpectedly found that sorting of (mat)IFN-DCs with CD56 antibody-coated microbeads (MB) resulted in fractionating cells with tumoricidal activity into the flow-through (FT) but not MB-bound fraction. We uncovered that the FT fraction contains cells expressing low but substantial level of CD56. Moreover, those cells express granzyme B (GrB), perforin (PFN), and serpin B9 at high levels. By employing a specific inhibitor of PFN, we confirmed that direct tumoricidal activity relies on the GrB/PFN pathway. We designated subpopulation in FT fraction as CD56dim and that in CD56 positively sorted fraction as CD56bright , respectively. This is the first time, to our knowledge, to identify subpopulations of CD56-positive IFN-DCs with distinct tumoricidal activity which is ascribed to high expression of the components of GrB/PFN pathway.

The SMC5/6 Complex Subunit NSE4A Is Involved in DNA Damage Repair and Seed Development.

The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.

Different In Vitro-Generated MUTZ-3-Derived Dendritic Cell Types Secrete Dexosomes with Distinct Phenotypes and Antigen Presentation Potencies.

Human dendritic cell (DC) dexosomes were evaluated for their function and preclinical validation for vaccines. Dexosomes are small DC-secreted vesicles that contain absorbing immune signals. Vaccine manufacturing requires a significant number of monocyte-derived DCs (Mo-DCs) from donor blood; thus, Mo-DC dexosomes are expected to serve as novel materials for cancer vaccination. In this study, we characterized a potential dexosome model using immature and mature MUTZ3-derived DCs (M-imIL-4-DC, M-imIFN-DC, M-mIL-4-DC, and M-mIFN-DC) and their dexosomes (M-imIL-4-Dex, M-imIFN-Dex, M-mIL4-Dex, and M-mIFN-Dex). Despite the lack of significant differences in viability, M-mIFN-DC showed a significantly higher level of yield and higher levels of maturation surface markers, such as CD86 and HLA-ABC, than M-mIL-4-DC. In addition, M-mIFN-Dex expressed a higher level of markers, such as HLA-ABC, than M-mIL-4-Dex. Furthermore, M-mIFN-Dex exhibited a higher level of antigen presentation potency, as evaluated using a MART-1 system, than either M-imIFN-Dex or M-mIL-4-Dex. We found that M-mIFN-Dex is one of the four types of MUTZ3-derived DCs that harbor potential immunogenicity, suggesting that DC dexosomes could be useful resources in cancer immunotherapy.

The Detection of Immunity against WT1 and SMAD4P130L of EpCAM+ Cancer Cells in Malignant Pleural Effusion.

Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8+ T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms' Tumor 1 (WT1) antigen over the disease course (two points at MPE1st and 2nd, two months after MPE1st). Epithelial cell adhesion molecule (EpCAM)+ cancer cells (PD-L1- or T cell immunoglobulin mucin-3, TIM-3-), both PD-1 or TIM-3 positive CD8+ T cells, and CD14+CD68+CD163+TIM-3+ macrophages increased from the MPE1st to MPE2nd. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8+ T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (TCM) of WT1-CTLs was decreased. On the other hand, CD8+ T cells in response to SMAD4P130L, which is homogeneously expressed in EpCAM+ cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8+ T cell response to SMAD4P130L was diminished following remarkably decreased numbers of CD8+ TCM in MPE samples. In conclusion, CD8+ T cells responding to WT1 or SMAD4P130L neoantigen expressed in EpCAM+ pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.

[A Case of Locally Advanced Rectal Cancer Treated by Robot Assisted Intersphincteric Resection after Neoadjuvant Chemotherapy].

We present a case of locally advanced rectal cancer(LARC)treated by robot assisted intersphincteric resection(ISR)and lateral lymph node dissection(LLND)after neoadjuvant chemotherapy(NAC). The patient was a 69-year-old female with the diagnosis of adenocarcinoma of the rectum Rb. The clinical stage diagnosis was cT3N0M0, cStage Ⅱ. NAC with FOLFOXIRI(5-fluorouracil/oxaliplatin/leucovorin/irinotecan)plus bevacizumab(BEV)was inisiated as NAC. Tumor volume reduction of primary lesion was evaluated by CT scan and colonoscopy after 6 courses of FOLFOXIRI plus BEV including omit of BEV on last course and were judged as partial response(PR)and no distant metastasis. With maintenance of tolerability for surgery even after NAC, robot assisted ISR and LLND were safely performed with curative resection. The histopathological treatment effect of post NAC was diagnosed as Grade 1b and the final pathological stage was ypT3pN0cM0, ypStage ⅡA. We experienced a case of LARC was performed FOLFOXIRI plus BEV as NAC followed by robotic ISR and LLND with anal preservation.

[Results of Surgical Treatment for Gastric Tube Cancer after Esophagectomy].

Recent improvements in the survival of patients after esophagectomy have led to an increase in the occurrence of gastric tube cancers(GTC). We retrospectively examined 7 patients who were surgically treated for GTC among 13 patients who were diagnosed between April 2004 and December 2018. Partial gastrectomy with regional lymph node dissection was performed in 6 patients while total resection of the stomach was performed only in 1 patient. Postoperative complications included 1 anastomotic leakage and 1 subcutaneous abscess. We performed subtotal gastrectomy with preservation of the upper region of the gastric tube in 3 patients. In these patients, blood flow was confirmed from the remnant esophagus to the upper region of the gastric tube using indocyanine green fluorescence imaging. The pathological stage of the treated GTCs were 4 cases of Stage ⅠA, 2 of Stage ⅠB, and 1 of Stage ⅡA. Median follow-up time and postoperative survival time were 32 months and 46.5 months, respectively. Most of our surgically treated cases were early gastric carcinomas that could be radically resected.

[A Curatively Resected Case of Quadruple Cancer including Synchronous Gastric and Superficial Esophageal Cancers with Metachronous Sigmoid Colon Cancer and Hepatocellular Carcinoma].

A 80s year old man was referred to our hospital with melena. Colonoscopy revealed an elevated lesion in the sigmoid colon. Laparoscopic sigmoidectomy(D2)was performed in August 2011. Postoperative diagnosis was advanced sigmoid colon cancer(pT2N1M0 and pStage Ⅲa, UICC). In January 2015, He suffered from epigastric discomfort after meals. Gastrointestinal endoscopy revealed advanced gastric cancer and superficial esophageal cancer. For esophageal cancer, endoscopic submucosal dissection was performed with a diagnosis of cStage 0-Ⅱa(UICC). Laparoscopic distal gastrectomy with Billroth Ⅰ reconstruction was performed for gastric cancer with a diagnosis of pT1bN0M0 and pStage ⅠA(UICC). Follow up CT and MRI images in October 2016 showed a liver tumor in S4/S5. Laparoscopic partial liver resection was performed. Postoperative pathological diagnosis was hepatocellular carcinoma pT1N0M0, pStage Ⅰ(UICC). We finished following up period of the sigmoid colon cancer. Gastric cancer and esophageal cancer are followed up by gastrointestinal endoscopy once a year. Hepatocellular carcinoma is followed up every 3 months. He has no recurrence until now.

[A Case of Pancreatic Neuroendocrine Tumor(p-NET)with Long-Term Survival with Multidisciplinary Treatment].

Pancreatic neuroendocrine tumor(p-NET)is a relatively rare disease, and treatment is multidisciplinary with resection, local therapy, radiotherapy, and chemotherapy. We report on a case in which long-term survival was achieved by multidisciplinary treatment. The case is a 47-year-old male. He was referred to our hospital because of the diagnosis of pancreatic tail tumor and underwent distal pancreatectomy in May 2008. And he was diagnosed as p-NET G1 by the pathological results. After 3 TACE treatments and 1 partial liver resection for recurrent liver metastasis, multiple liver metastases and lymph node metastases were found in August 2014. As a result of the everolimus treatment, the determination of efficacy to lymph nodes was CR and liver lesions were CR with the addition of TACE treatment. In July 2017, he had multiple liver metastases and right humeral metastases, and has been treated with radiotherapy for bone metastases and has maintained CR. After TACE in November 2017, he received 9 rounds of lanreotide treatment. In December 2018, he again had liver metastases and lymph node recurrence. He has been treated with everolimus treatment again and is maintaining SD in outpatient treatment.

[A Case of Successful Treatment of Recurrent Peritoneal Dissemination of Hepatocellular Carcinoma with Atezolizumab plus Bevacizumab Combination Therapy].

Atezolizumab plus bevacizumab combination therapy is the first cancer immunotherapy that has shown efficacy in the treatment of hepatocellular carcinoma(HCC). We report a case of HCC with recurrent peritoneal dissemination for which atezolizumab plus bevacizumab combination therapy was effective. The patient, a 63-year-old man, underwent transarterial embolization(TAE)for ruptured HCC, and a mass with dissemination on the caudal side of liver S3 was observed. Laparoscopic lateral hepatic resection plus resection of the dissemination plus cholecystectomy was thus performed in September 2019. However, in November 2019, multiple peritoneal dissemination recurrence was observed, and lenvatinib therapy was initiated. In May 2020, PD was observed, and we had switched to sorafenib therapy. However, in October 2020, further tumor growth and rapid increase in tumor markers(AFP 25,668 ng/mL, PIVKA-Ⅱ 64,960 mAU/mL)were observed, and the patient was judged to have PD. Atezolizumab plus bevacizumab combination therapy was initiated in the same month. Subsequently, a CT scan in January 2021 showed a marked decrease in tumor size, indicating PR. The tumor markers have since normalized(AFP 5 ng/mL, PIVKA-Ⅱ 28 mAU/mL). The patient has been treated with atezolizumab plus bevacizumab combination therapy again and is maintaining PR as an outpatient.

[A Case of Metachronous Quintuple Cancer Involving the Stomach, Rectum, Colon, Liver and Prostate].

Advances and improvements in cancer diagnosis and treatment have made it possible to find multiple primary cancers. We report here a rare case of metachronous quintuple cancer involving the stomach, rectum, colon, liver and prostate. An 80s‒ year‒old male was referred to our hospital with abnormality on upper GI series. He had undergone a distal gastrectomy in May 2005. Postoperative diagnosis was advanced gastric cancer (pT2N1M0, pStage ⅡA). In August 2006, anterior resection was performed with a diagnosis of advanced rectal cancer(pT3N0M0, pStage Ⅱa). For ascending colon polyps, endoscopic submucosal dissection was performed with a diagnosis of adenocarcinoma in adenoma(pTisN0M0, pStage 0)in September 2007. In June 2016, laparoscopic ileocecal resection was performed with a diagnosis of advanced cecum cancer(pT3N0M0, pStage Ⅱa). Follow up CT images showed a liver tumor in S4. Partial liver resection was performed in October 2010. Postoperative pathological diagnosis was hepatocellular carcinoma(pT2N0M0, pStage Ⅱ). Prostate cancer(cT2aN0M0)was treated by androgen deprivation therapy from February 2018. Although he had high‒frequency microsatellite instability, germline mutations in hMLH1 and hMSH2 genes were not detected. Histopathological examination showed that each tumor was an independent tumor and had not metastasized from any others. The patient had a good clinical course after these treatment until now.

[Deviation of Peripherally Inserted Central Catheter(PICC)during Total Parenteral Nutrition(TPN)for a Gastric Cancer Patient-A Case of Report].

A 90s woman was diagnosed as having cT4aN2M0, cStage ⅢA, advanced gastric cancer. As she was severely malnourished owing to pyloric stenosis, a peripherally inserted central catheter (PICC)was placed in her left arm, and total parenteral nutrition(TPN)was initiated. She complained of dyspnea, and radiography revealed right pleural effusion on day 4 of TPN. Contrast computed tomography revealed that the tip of the catheter had perforated the vessel wall of the superior vena cava and had migrated into the mediastinal space. After thoracocentesis, the catheter was removed under fluoroscopic guidance after hemostasis was achieved. Thus, the possibility of catheter deviation should be considered in case of dyspnea and pleural effusion during TPN.

Visualization of extracellular vesicles in the regenerating caudal fin blastema of zebrafish using in vivo electroporation.

Zebrafish have high regenerative ability in several organs including the fin. Although various mechanisms underlying fin regeneration have been revealed, some mechanisms remain to be elucidated. Recently, extracellular vesicles (EVs) have been the focus of research with regard to their role in cell-to-cell communication. It has been suggested that cells in regenerating tissues communicate using EVs. In this study, we examined the involvement of EVs in the caudal fin regeneration of zebrafish using an in vivo electroporation method. The process of regeneration appeared normal after in vivo electroporation, and the transferred plasmid showed mosaic expression in the blastema. We took advantage of this mosaic expression to observe the distribution of exosomal markers in the blastema. We transferred exosomal markers by in vivo electroporation and identified EVs in the regenerating caudal fin. The results suggest that blastemal cells communicate with other cells via EVs during caudal fin regeneration.

The mechanisms and significance of the positional control of centromeres and telomeres in plants.

The centromere and telomere are universal heterochromatic domains; however, the proper positioning of those domains in nuclear space during the mitotic interphase differs among eukaryotes. Consequently, the question arises how and why this difference occurs. Studies over the past 2 decades have identified several nuclear membrane proteins, nucleolar proteins, and the structural maintenance of a chromosome complex as factors involved in the positional control of centromeres and/or telomeres during the mitotic interphase in yeasts, animals, and plants. In this review, with a primary focus on plants, the roles of those factors are summarized, and the biological significance of proper centromere and telomere positionings during the mitotic interphase is discussed in an effort to provide guidance for this question.

[A Case of Enterocutaneous Fistula Caused by Cytomegalovirus Enteritis after Gastrectomy Successfully Treated with Antiviral Therapy-A Case Report].

Cytomegalovirus(CMV)infection is a well-recognized complication of immunodeficiency. We present the case of a 90- year-old female admitted due to gastric cancer. Fifty-seven days after gastrectomy, intestinal juice was observed from the umbilical wound, which was suspected of anastomotic failure or gastrointestinal perforation. Abdominal computed tomography didn't reveal gastrointestinal perforation. CMV enteritis was diagnosed by transanal double-balloon endoscopy from the cecum to the oral side 15 cm of the ileum. Enterocutaneous fistula was considered to be caused by CMV enteritis. The intestinal fluid outflow from the wound disappeared treated with ganciclovir, and the ulcer in the intestinal tract disappeared, too. We report this case to reinforce the importance of considering CMV infection as a differential diagnosis in gastrointestinal perforation of compromised patients.