Dotinurad restores exacerbated kidney dysfunction in hyperuricemic patients with chronic kidney disease.

BACKGROUND: In this study, we aimed to clarify the beneficial effects of urate-lowering treatment with the novel agent dotinurad on renal function in patients with chronic kidney disease (CKD) and hyperuricemia (HUA). METHODS: Thirty-five patients with CKD (mean age 65.4 ± 14.8 years, 23 men) diagnosed with HUA were recruited. Changes in eGFR before and after dotinurad administration were assessed. Patients first underwent a 3-month observation period and then 3 months treatment with dotinurad. RESULTS: During the observation period, mean eGFR (mL/min/1.73 m2) declined significantly. The baseline eGFR was 31.8 ± 16.4 and the serum urate level (sUA, mg/dL) was 8.1 ± 1.7. During the treatment period, eGFR recovered to 36.5 ± 17.5 and sUA decreased to 6.7 ± 1.0. The increase in eGFR after dotinurad administration was correlated with a decrease in sUA (R = 0.375, p = 0.0263). CONCLUSION: Dotinurad administration to patients with CKD and HUA appears to be beneficial in restoring kidney function. Dotinurad may represent a potential medication for the prevention of kidney function decline caused by HUA.

[A Case of Long-Term Survival without Recurrence after Completion of Immunotherapy Due to Response of Third- Line Nivolumab for Liver Metastasis Recurrence of Esophagogastric Junction Cancer].

The patient was a 66-year-old man with dysphagia. He underwent total gastrectomy and trans-hiatal abdominal esophagectomy with lymph node dissection, including the inferior mediastinum, for esophagogastric junction cancer. The postoperative pathological examination revealed poorly differentiated adenocarcinoma T4aN2, Stage ⅢA, HER2 negative, and postoperative adjuvant therapy S-1 oral administration was started. Four months after surgery, computed tomography (CT)showed recurrent liver and para-aortic lymph node metastases. First-line XELOX therapy and second-line weekly PTX therapy resulted in PD, and nivolumab administration was started as third-line. The evaluation was PR and CR at 3 and 6 months, respectively. At the same time, he developed acute cholangitis and underwent open lithotripsy drainage. Postoperatively, treatment was terminated according to the patient's wishes. To date, it has been 5 years since the first operation and 3 and a half years since remission with nivolumab, and no recurrence has been observed. There is little evidence regarding the timing of conversion or treatment discontinuation for successful cases of immunotherapy in the salvage line for gastric cancer.

Spot urine protein/creatinine ratio as a reliable estimate of 24-hour proteinuria in patients with immunoglobulin A nephropathy, but not membranous nephropathy.

BACKGROUND: Proteinuria is known to be associated with both kidney function deterioration and cardiovascular diseases. While proteinuria estimation from 24-h urine samples has traditionally been considered as the standard method for assessment of the degree of urinary protein excretion, sample collection is associated with several technical problems such as inaccurate collection and the potential spread of drug-resistant pathogens. Therefore, the spot urine protein/creatinine ratio (PCR) assessment is currently recommended as an alternative. While the utility of PCR has been validated, studies on the association between spot urine PCR and 24-h proteinuria (24HP) in patients with chronic glomerular nephritis (CGN) and nephrotic syndrome (NS) are limited. This study aimed to evaluate whether an estimated result from a spot urine PCR could sufficiently approximate the daily urine protein excretion amount from a 24-h urine sample in patients with immunoglobulin A nephropathy (IgAN), minimal change disease (MCD), and membranous nephropathy- nephrotic syndrome (MN-NS). METHODS: The study participants included 161 patients with IgAN, MCD, or MGN-NS at the Jikei University Kashiwa Hospital and Kanagawa Prefecture Shiomidai Hospital. The correlation between spot urine PCR and a 24-h urine protein was investigated using linear regression analysis with Spearman's correlation (r) coefficient and intraclass correlation coefficient (ICC). RESULTS: While high correlation coefficients (r = 0.86, P < 0.001) and substantial agreement (ICC: 0.806, P < 0.001) were observed in patients with IgAN, similar correlations were not observed in patients with MCD or MN-NS. In the patients with MCD, r was 0.53 (P < 0.001), which signified a slight correlation, and in the patients with MN-NS, r was 0.289 (P = 0.17), which was not statistically significant. CONCLUSIONS: This study revealed that spot urine PCR is a reliable estimate of 24HP value in patients with IgAN. In contrast, there is a considerable difference between the daily urine protein excretion amount based on a 24-h urine sample and that which is calculated from spot urine PCR in patients with NS.

Measurement of daily sodium excretion in patients with chronic kidney disease; special reference to the difference between the amount measured from 24 h collected urine sample and the estimated amount from a spot urine.

It is important to grasp a patient's daily sodium intake in the management of chronic kidney disease, as sodium intake is widely recommended at 6 g/day or less. There are multiple equations widely known for estimating the daily sodium excretion from a spot urine sample, but these are aimed at healthy people. There are few reports that validate equations in patients with chronic kidney disease. The purpose of this study is to evaluate whether the amount of measured daily sodium excretion from a sample collected for 24-h urine (24HU) is equal to that of using an equation from a spot urine sample (SU) in patients with chronic kidney disease. One hundred sixty-two patients with chronic kidney disease from Kanagawa Prefecture Shiomidai Hospital, Japan and the Jikei University Kashiwa Hospital, Japan participated in the study. Daily sodium excretion was measured from 24HU and compared with it from SU by using the formula according to Tanaka et al. Sodium excretion by 24HU was 2744 mg/day and estimating daily sodium excretion from SU was 3315 mg/day. The coefficient of determination was 0.17 (p < .001) in multivariate regression analysis. The coefficient of determination was extremely low. Thus, there is a considerable difference between the amount of sodium excretion calculated from a 24HU and that from a SU in patients with chronic kidney disease.

Downregulation of miR122 by grainyhead-like 2 restricts the hepatocytic differentiation potential of adult liver progenitor cells.

Late fetal and adult livers are reported to contain bipotential liver stem/progenitor cells (LPCs), which share surface markers, including epithelial cell adhesion molecule (EpCAM), with cholangiocytes and differentiate into both hepatocytes and cholangiocytes. However, recent results do not necessarily support the idea that LPCs contribute significantly to cellular turnover and regeneration by supplying new hepatocytes. Here, we examined the colony-forming capability of EpCAM(+) cells isolated from mouse livers between E17 and 11 weeks of age. We found that the number of bipotential colonies was greatly reduced between 1 and 6 weeks, indicating that the number of LPCs decreases during postnatal development. Moreover, bipotential colonies derived from adult LPCs contained substantially fewer albumin(+) cells than those from neonatal LPCs. We further examined the differentiation potential of neonatal and adult LPCs by transplantation and found that neonatal cells differentiated into mature hepatocytes in recipient livers more frequently than adult LPCs. Since we previously reported that the transcription factor grainyhead-like 2 (GRHL2) expressed in EpCAM(+) cells inhibits hepatocytic differentiation, we examined whether targets of GRHL2 might block hepatocytic differentiation. DNA and microRNA microarrays revealed that miR122, the expression of which correlates with hepatocytic differentiation, was greatly reduced in adult as compared with neonatal EpCAM(+) cells. Indeed, GRHL2 negatively regulates the promoter/enhancer activity of the Mir122 gene. Our results indicate that neonatal but not adult EpCAM(+) LPCs have great potential to produce albumin(+) hepatocytes. GRHL2 suppresses transcription of miR122 and thereby restricts the differentiation potential of adult LPCs.

Enhancing the Motivation for Rural Career: The Collaboration between the Local Government and Medical School.

The shortage of medical workforce in rural areas is a global long-standing problem. Due to the severity of shortages in the medical workforce, Mie prefectural government has collaborated with a medical school and the municipal governments to increase the rural medical workforce. Since 2010, this collaboration has led to an annual lecture series on rural practice for medical students. We distributed questionnaires at the beginning and end of the lecture series to examine the effect of this program. The questionnaire consisted of two parts that included an understanding of rural practice and the motivation to work in rural areas. The lecture series significantly improved the responses to the following questions "Rural practice is interesting" (p < 0.001), "Rural practitioners can deliver adequate medical care" (p < 0.01), "Rural practitioners cannot go back to urban areas" (p < 0.001), "I want to be a rural practitioner" (p < 0.001), "Healthcare facilities in rural areas have been developed" (p < 0.001), "Rural practitioners can be a specialist" (p < 0.001), and "Rural residents can be served adequate healthcare service" (p < 0.01). The percentage of students who desired to work in rural areas increased significantly (11.1% vs. 23.9%, p = 0.04). A lecture series on rural practice enhanced the motivation of medical students and their interest in a rural career. While collaboration between the local government and medical school rarely occurs in planning medical education programs, this approach may offer a promising way to foster local health professionals.

Image-based evaluation of contraction-relaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: Correlation and complementarity with extracellular electrophysiology.

In this study, we used high-speed video microscopy with motion vector analysis to investigate the contractile characteristics of hiPS-CM monolayer, in addition to further characterizing the motion with extracellular field potential (FP), traction force and the Ca(2+) transient. Results of our traction force microscopy demonstrated that the force development of hiPS-CMs correlated well with the cellular deformation detected by the video microscopy with motion vector analysis. In the presence of verapamil and isoproterenol, contractile motion of hiPS-CMs showed alteration in accordance with the changes in fluorescence peak of the Ca(2+) transient, i.e., upstroke, decay, amplitude and full-width at half-maximum. Simultaneously recorded hiPS-CM motion and FP showed that there was a linear correlation between changes in the motion and field potential duration in response to verapamil (30-150nM), isoproterenol (0.1-10µM) and E-4031 (10-50nM). In addition, tetrodotoxin (3-30µM)-induced delay of sodium current was corresponded with the delay of the contraction onset of hiPS-CMs. These results indicate that the electrophysiological and functional behaviors of hiPS-CMs are quantitatively reflected in the contractile motion detected by this image-based technique. In the presence of 100nM E-4031, the occurrence of early after-depolarization-like negative deflection in FP was also detected in the hiPS-CM motion as a characteristic two-step relaxation pattern. These findings offer insights into the interpretation of the motion kinetics of the hiPS-CMs, and are relevant for understanding electrical and mechanical relationship in hiPS-CMs.

Anti-glomerular Basement Membrane Disease Concomitant with MPO-ANCA Positivity Concurrent with High Serum Levels of Interleukin-26 Following Coronavirus Disease 2019 Vaccination.

As coronavirus disease 2019 (COVID-19) vaccine booster campaigns progress worldwide, new reports of complications following COVID-19 vaccination have emerged. We herein report a case of new-onset anti-glomerular basement membrane (GBM) disease concomitant with myeloperoxidase-antineutrophil cytoplasmic antibody positivity concurrent with high levels of interleukin (IL)-26 following the second dose of the Pfizer-BioNTech COVID-19 vaccine. The temporal association with vaccination in this case suggests that an enhanced neutrophilic immune response through IL-26 may have triggered necrotizing glomerulonephritis and a T-cell-mediated immune response to GBMs, leading to the development of anti-GBM antibodies, with an enhanced B-cell response after the vaccination triggering anti-GBM IgG and the onset of anti-GBM disease.

[Distal gastrectomy for initially unresectable gastric cancer with pyloric stenosis following gastrojejunostomy and S-1 plus CDDP chemotherapy].

We report a case of advanced gastric cancer with pyloric stenosis, in which a curative resection was performed following gastrojejunostomy and S-1 based chemotherapy. A 46-year-old female presenting with vomiting was diagnosed with unresectable gastric cancer with pyloric stenosis after a detailed examination. She underwent laparotomy, which revealed a T4 tumor invading the pancreas head and involving the gallbladder. A gastrojejunostomy was performed. After the operation, chemotherapy of S-1(100mg/body, days 1-21)plus cisplatin(85mg/body, day 8)was administered. After 4 courses, the tumor size was markedly reduced upon imaging examinations. Six months after gastrojejunostomy, distal gastrectomy was curatively performed. The pathological findings were type 3, por1, pT4a(SE), pN1, M0, pStage III A. After 5 courses of S-1(100mg/day, days 1-28)as adjuvant chemotherapy, she had a recurrence at a lymph node behind the pancreas head. Despite irinotecan+cisplatin following docetaxel therapy, she had no effective benefits and died from the cancer 17 months after the first operation. The prognosis of unresectable gastric cancer with pyloric stenosis is not promising; however, gastrojejunostomy following S-1-based chemotherapy could lead such patients to curative resection and a longer survival time.

Interval laparoscopic appendectomy after laparotomy drainage for acute appendicitis with abscess: A case report.

INTRODUCTION: Immediate appendectomy for acute appendicitis with abscess has a high frequency of ileocecal resection and postoperative complications compared with interval appendectomy after conservative treatment. The optimal approach to acute appendicitis with abscess remains controversial. PRESENTATION OF CASE: A 69-year-old woman was referred to our hospital for abdominal pain. A computed tomography scan revealed an enlarged abscess around the cecum. The diagnosis was perforated appendicitis with abscess, and conservative treatment was performed. Percutaneous drainage was difficult because the abscess was near the intestinal tract. Because of the persistence of symptoms on the fourth day of hospitalization, laparotomy drainage was performed, and the patient's condition improved afterwards. Colonoscopy was performed on an outpatient follow-up to rule out malignant tumors of the colon. Interval laparoscopic appendectomy was performed 3 months after discharge to prevent appendicitis. The postoperative course was uneventful. DISCUSSION: For this case of acute appendicitis with abscess, conservative treatment such as antibiotic therapy and laparotomy drainage was performed. Laparotomy drainage enabled us to approach the abscess directly and minimized the risk of its spread into the abdominal cavity compared to the laparoscopic approach. Interval laparoscopic appendectomy was more effective and easier for this case of appendectomy, wherein adhesions to the abdominal wall were expected compared to laparotomy. CONCLUSION: Conservative treatment approaches, such as drainage and antibiotic therapy, can be first-line for appendicitis with abscesses. Interval laparoscopic appendectomy can be useful to resect the appendix and observe the abdominal cavity.

Laparoscopic appendectomy for acute appendicitis in patients with COVID-19 confirmation: A case report.

Introduction: Strategies to postpone elective surgeries were proposed to maintain the hospital capacity to cater for coronavirus disease 2019 (COVID-19) and emergency non-COVID cases. Non-operative management (NOM) was recommended when possible during the COVID-19 era. However, the optimal approach to acute appendicitis (AA) in patients with COVID-19 remains controversial. Presentation of case: A 25-year-old man who tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) was referred to our institution with a diagnosis of AA with appendicolith. Chest computed tomography did not detect evidence of pneumonia. Laparoscopic appendectomy was performed after strict infection prevention measures were taken. The postoperative course was uneventful. No respiratory symptoms such as cough or sputum production occurred postoperatively. No signs of infection in medical staff or spread in the operating room and infectious disease ward were observed. Discussion: The treatment policy should fully consider the risk of COVID-19 infection to medical staff and the risk of aggravation in patients who tested positive for SARS-Cov-2. Surgery was chosen over NOM for AA with appendicolith because the presence of appendicolith was thought to indicate a high probability of treatment failure in NOM and possible perforation; thus, case more difficult measures were required for SARS-Cov-2-positive cases. Conclusion: Careful assessment of the patient's condition and consideration of the treatment method is important, rather than choosing NOM over operative management based solely on SARS-Cov-2-positive status. Laparoscopic appendectomy with adequate infection control measures can be safely performed in SARS-Cov-2-positive cases.

Minimal change disease soon after Pfizer-BioNTech COVID-19 vaccination.

We report on the onset of minimal change disease (MCD) presenting with anasarca after a second dose of the messenger RNA (mRNA)-based Pfizer-BioNTech vaccine against coronavirus disease 2019 (COVID-19). A 75-year-old previously healthy male was admitted with rapidly progressive anasarca and proteinuria of 7.7 g/day following the second dose. A kidney biopsy revealed MCD with nephrotic syndrome. He was treated with intravenous methylprednisolone followed by prednisolone, leading to complete remission after 35 days in the hospital. Since definite causality between the vaccine and MCD remains unclear, awareness of this potential adverse effect of mRNA vaccines is important to determine its true incidence and frequency.

Laparoscopic repair of inguinal hernia in a patient with a ventriculoperitoneal shunt: A case report.

Laparoscopic surgery in patients with a ventriculoperitoneal (VP) shunt is reportedly associated with increased intracranial pressure secondary to high intraperitoneal pressure and retrograde infection due to intraperitoneal infection. We herein report the first case of transabdominal preperitoneal (TAPP) inguinal hernia repair without catheter manipulation for a patient with a VP shunt. A 69-year-old man with a VP shunt was suspected to have an inguinal hernia based on symptoms and examination findings. With a pneumoperitoneum pressure of 10 mm Hg, the VP shunt was not clamped and mesh was placed while confirming cerebrospinal fluid outflow from the tip of the catheter. The patient developed no shunt-associated complications and was discharged 3 days postoperatively. TAPP inguinal hernia repair without catheter manipulation is a potential surgical option for patients with a VP shunt.

Prediction of presepsin concentrations through commensurate decline in kidney function in the elderly.

BACKGROUND: Presepsin is a useful biomarker to diagnose sepsis. However, the correlation between plasma presepsin concentrations and kidney function in the elderly with chronic kidney disease (CKD) remains to be elucidated. We determined whether plasma presepsin concentrations were influenced by kidney function decline in the elderly. METHODS: One hundred seventy outpatients with CKD aged ≥65 y were enrolled. Plasma presepsin concentrations were measured using immunoassay analysis. The relationship between plasma presepsin concentration and kidney function was assessed. RESULTS: The median age of patients of this cohort was 778 (72-85) y and the mean estimated glomerular filtration rate was 51.8 ±â€¯28.1 ml/min/1.73 m2. Plasma presepsin concentrations in those with CKD G4-G5 (362 pg/ml [273-553]) were significantly higher than in those with CKD G1-G2 (111 pg/ml [91-113]) and CKD G3 (145 pg/ml [124-205]) (p < 0.001, p < 0.001, respectively). A high correlation between plasma presepsin concentrations and kidney function was observed (R2 = 0.733, p < 0.001). Even after adjusting for confounders, plasma presepsin concentrations were independently associated with kidney function. CONCLUSIONS: Increases in plasma presepsin concentrations were exponentially correlated to kidney function decline in the elderly with CKD.

Intestinal anisakiasis with severe intestinal ischemia caused by extraluminal live larvae: a case report.

BACKGROUND: Anisakiasis is a parasitic infection caused by Anisakis worms found in raw fish. Most cases of anisakiasis occur in the stomach and rarely occur in the intestine. It is extremely rare for live larvae to break through the intestine into the mesentery and cause severe intestinal ischemia. Anisakiasis can be treated conservatively, because the larvae will die in approximately 1 week, but, sometimes, a serious condition can arise, as in this case. We report the first case of extraluminal anisakiasis in which a live Anisakis worm caused severe intestinal ischemia. CASE PRESENTATION: The patient was a 26-year-old woman who ate squid a week prior. She had abdominal pain and was admitted to our emergency department. On physical examination, abdominal guarding and rebound tenderness were present in her lower abdomen. Contrast-enhanced computed tomography showed ascites, the whirl sign, localized submucosal edema of the intestinal wall, and a dilated small bowel segment with edema. We suspected the strangulated small bowel obstruction based on the CT-scan findings. To rule out the strangulated small bowel obstruction, laparoscopic exploration was performed. Bloody ascites in the pouch of Douglas and severe inflammation in 20 cm of the ileum were observed. An Anisakis larva had perforated the intestinal wall and was found alive in the mesentery. The ileum had developed a high degree of ischemia, so the affected section was resected. Histopathological examination revealed that the Anisakis worm body was in the inflamed mesentery and caused a high degree of ischemia in the intestinal tract. The patient was discharged 9 days after surgery. CONCLUSIONS: A living Anisakis larva punctured the mesentery of the small intestine, resulting in severe intestinal ischemia. As seen in this case, intestinal anisakiasis may cause serious symptoms, and a low threshold for performing diagnostic laparoscopy for the early diagnosis of bowel ischemia secondary to anisakiasis can be useful in determining the definite diagnosis and indications for resection.

A novel treatment of hyperinsulinemic hypoglycemia induced by insulin antibodies with alkali administration: a case report.

BACKGROUND: Insulin autoimmune syndrome is a rare cause of hyperinsulinemic hypoglycemia characterized by autoantibodies to human insulin without previous insulin use. We report a case of a patient with hyperinsulinemic hypoglycemia possibly caused by insulin antibodies induced by insulin analogs and a novel therapeutic measure for this condition. CASE PRESENTATION: An 84-year-old Japanese man with a 28-year history of type 2 diabetes and chronic kidney disease, treated with biphasic insulin aspart 30, experienced persistent early morning hypoglycemia with daytime hyperglycemia. Despite discontinuation of biphasic insulin aspart 30, the condition persisted even after the patient ate small, frequent meals. Sodium bicarbonate was administered to correct the chronic metabolic acidosis, which then rectified the early morning glucose level. CONCLUSIONS: We believe this to be the first published case of a therapeutic approach to the treatment of hyperinsulinemic hypoglycemia associated with insulin antibodies that factors in blood pH and the correction of acidosis using sodium bicarbonate, which physicians could consider.

Anatomical study of the vertebral artery in Japanese adults.

The aim of the present study was to examine the vertebral arteries. The origins of the right and left vertebral arteries and their entrance points into the cervical transverse foramen were examined in dissections of 515 Japanese cadavers (303 males, 212 females) at Kurume University School of Medicine from 1990 to 2003. There were 515 right vertebral arteries and 514 left vertebral arteries. The right vertebral artery originated from the right subclavian artery in 514 of 515 arteries and one of the arteries arose from the bifurcation of the brachiocephalic trunk. The mean distance between the origin of the right subclavian artery and the right vertebral artery was 20.9 mm. The left vertebral artery originated from the left subclavian artery in 484 of 514 arteries and the mean distance between the origin of the left subclavian artery and the left vertebral artery was 32.1 mm. The remaining 30 arteries (5.8%) originated from the aortic arch between the left common carotid artery and the left subclavian artery and this frequency is similar to previously published data. There was no right-left difference for the entrance point of the vertebral arteries into the cervical transverse foramen and the 6th cervical vertebra (C6) was the most common entrance point. Seventy-eight percent of our cases had right and left vertebral arteries that originated in the subclavian arteries and entered the cervical transverse foramen at C6. Among the 30 left vertebral arteries that originated from the aortic arch, 20 arteries (66.7%) entered a cervical transverse foramen at a level higher than C6. This frequency was higher than that for the left vertebral artery that originated from the subclavian artery.

Geosmin biosynthesis in Streptomyces avermitilis. Molecular cloning, expression, and mechanistic study of the germacradienol/geosmin synthase.

Geosmin (1) is responsible for the characteristic odor of moist soil. The Gram-positive soil bacterium Streptomyces avermitilis produces geosmin (1) as well as its precursor germacradienol (3). The S. avermitilis gene SAV2163 (geoA) is extremely similar to the S. coelicolor A3(2) SCO6073 gene that encodes a germacradienol/geosmin synthase. S. avermitilis mutants with a deleted geoA were unable to produce either germacradienol (3) or geosmin (1). Biosynthesis of both compounds was restored by introducing an intact geoA gene into the mutants. Incubation of recombinant GeoA, encoded by the SAV2163 gene of S. avermitilis, with farnesyl diphosphate (2) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (66%), (7S)-germacrene D (4) (24%), geosmin (1) (8%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (2%). Incubation of this germacradienol/geosmin synthase with [1,1-(2)H2] FPP (2a) gave geosmin-d1 (1a), as predicted. When recombinant GeoA from either S. avermitilis or S. coelicolor A3(2) was incubated with nerolidyl diphosphate (8), only the acyclic elimination products beta3-farnesene (10), (Z)-alpha-farnesene (11), and (E)-alpha-farnesene (12) were formed, thereby ruling out nerolidyl diphosphate as an intermediate in the conversion of farnesyl diphosphate to geosmin, germacradienol, and germacrene D.

A Method for Evaluating the Efficacy of Antifouling Paints Using Mytilus galloprovincialis in the Laboratory in a Flow-Through System.

A laboratory test with a flow-through system was designed and its applicability for testing antifouling paints of varying efficacies was investigated. Six different formulations of antifouling paints were prepared to have increasing contents (0 to 40 wt.%) of Cu2O, which is the most commonly used antifouling substance, and each formulation of paint was coated on just one surface of every test plate. The test plates were aged for 45 days by rotating them at a speed of 10 knots inside a cylinder drum. A behavioral test was then conducted using five mussels (Mytilus galloprovincialis) that were pasted onto the coated surface of each aged test plate. The number of the byssus threads produced by each mussel generally decreased with increasing Cu2O content of the paint. The newly designed method was considered valid owing to the high consistency of its results with observations from the field experiment.