Additionally, pain scores at rest during POD 0–3 were not significantly associated with preoperative kidney function. In conclusion, our results suggest that patients with mild to moderate CKD (stage 2–3) did not require reduction of opioid analgesics during POD 0–3, compared to patients with normal preoperative kidney function. Only patients with severe CKD (stage ≥ 4) might require a slight reduction of opioid analgesics.The amount of reduction in opioid doses and its effect on postoperative pain outcomes in chronic kidney disease (CKD) patients in the perioperative setting remains unclear. This study aimed to investigate differences in postoperative pain outcomes after major laparoscopic surgery between patients with CKD and those with normal preoperative kidney function. Medical records of patients who underwent laparoscopic major abdominal surgery from January 2010 to December 2016 were retrospectively reviewed, and 6,612 patients were finally included. During postoperative day (POD) 0–3, patients with an estimated glomerular filtration rate (eGFR) < 30 mL min The most commonly used method of postoperative pain control is systemic opioid analgesia3. A recent study reported that opioids are commonly used until postoperative days (PODs) 4 to 15 among patients after common surgical procedures4. However, opioids are associated with a variety of known adverse effects5 and should be used with caution in patients who are more vulnerable to these adverse effects. Patients with chronic kidney disease (CKD) are particularly at risk of developing serious adverse effects of opioids.It is reported that about 80% of all surgical patients experience postoperative pain−1 1.73 m−2 6, is a global health problem with a worldwide prevalence of 8% to 16%7. Because CKD patients have a reduced ability to clear drugs through the kidney, the dose of opioids should be appropriately reduced to avoid adverse effects in these patients8. A systematic review conducted as part of the European Palliative Care Research Collaborative opioid guidelines project9 reported that, among opioids, morphine has the highest toxicity rate in CKD patients, whereas alfentanil, fentanyl, and methadone have the lowest toxicity rates. However, that study only analysed data from chronic cancer patients and did not include patients with perioperative or acute pain9. Although one study recommended that the dose of transdermal fentanyl should be reduced by 50% in CKD patients10, there is limited evidence regarding the use of fentanyl for postoperative and acute pain control in patients with CKD11. Thus, there is a lack of information regarding the impact of impaired renal function in patients with CKD on requirement for opioids and on pain outcomes in the perioperative setting.CKD, which is defined by an estimated glomerular filtration rate (eGFR) below 60 mL minTherefore, this study aimed to investigate differences in postoperative pain outcomes, including opioid consumption and pain scores, after major laparoscopic surgery, between patients with CKD and those with normal kidney function. Our hypothesis for this study was that a correlation exists between the degree of renal impairment and opioid consumption in patients with CKD stages 1–5, after major laparoscopic surgery−1 1.73 m−2 (25.8). There were no life-threatening opioid-related complications  among the study population.A total of 20,800 patients at the Seoul National University Bundang Hospital (SNUBH) underwent a laparoscopic surgical procedure between January 1, 2010 and December 2016. Of these, 856 patients were excluded for age younger than 20 years, 1,053 were excluded for emergency surgery, 1,390 were excluded for single-port laparoscopy, 9,571 were excluded for surgery lasting less than 2 hours, 343 were excluded for receiving a continuous infusion of remifentanil or dexmedetomidine during the first three PODs, 8 were excluded for being discharged before the third POD, 25 were excluded for undergoing additional surgery during the first three PODs, 42 were excluded for intraoperative conversion to open laparotomy, 96 were excluded for undergoing simple appendectomy or cholecystectomy, 58 were excluded due to preoperative chronic opioid usage, and 746 were excluded for missing or incomplete medical records. As a result, 6,612 patients were included in the final analysis. A flowchart of the patient selection process is shown in Fig. −2), American Society of Anesthesiologists (ASA) physical status, some preoperative comorbidities , surgery time, propofol-based total intravenous anaesthesia (TIVA), intraoperative magnesium infusion, years of surgery, preoperative eGFR were selected with a criterion of P < 0.2 in the univariate model. The results of the multivariate generalized linear regression analysis, including the variables selected from the univariate model, are shown in Table −1 1.73 m−2 had a significant 3.5% lower morphine equivalent consumption on PODs 0 to 3 than the group with a preoperative eGFR ≥ 90 mL min−1 1.73 m−2 . However, the patients with preoperative eGFR: 60–90 mL min−1 1.73 m−2 and 30–60 mL min−1 1.73 m−2 were not associated with differences in morphine equivalent consumption on POD 0–3 .Table P < 0.001), body mass index (kg m−2) , cancer , surgery time (min) , propofol-based TIVA , intraoperative magnesium sulphate infusion  were significantly associated with morphine equivalent consumption during POD 0–3.Additionally, male sex . Among the adjuvant analgesics, acetaminophen consumption during the first three PODs was not significantly associated with preoperative eGFR (P > 0.05); however, ketorolac consumption during the first three PODs was 33.8% lower in the group with an eGFR of 30–60 mL min−1 1.73 m−2 group, compared with the group with a preoperative eGFR ≥ 90 .Table −1 1.73 m−2) than in those with normal kidney function (eGFR ≥ 90 mL min−1 1.73 m−2) during the first three days after major laparoscopic surgery, while it was not significantly associated with mild to moderate CKD (eGFR 30–90 mL min−1 1.73 m−2). In addition, there were no significant differences in NRS pain scores at rest between the patients with CKD and those with normal kidney function during the first three days after major laparoscopic surgery. As a result, this study suggests that preoperative kidney function status did not affect pain outcomes after major laparoscopic surgery, except in severe CKD patients with eGFR < 30 mL min−1 1.73 m−2. Furthermore, considering that the difference in morphine equivalent consumption between severe CKD patients with eGFR < 30 mL min−1 1.73 m−2 and patients with normal kidney function (eGFR ≥ 90 mL min−1 1.73 m−2) was just 3.5%, the clinical significance is questionable.This study showed that opioid consumption was significantly lower in patients with stage 4 or 5 CKD , and analysed their opioid consumption during POD 0–3. Our findings are novel, because no study has evaluated the effect of preoperative CKD stage on the postoperative pain outcome in detail.A recent study by Binhas 13, it is possible that even a reduced dose of fentanyl may have resulted in prolonged analgesic effects in CKD patients. Second, all patients received an IV PCA for pain control, which enabled them to titrate their postoperative pain14. Thus, an opioid dose reduction in CKD patients was unlikely to result in insufficient pain control.Even though the morphine equivalent consumption in our study was 3.5% lower in the patients with severe CKD than in those with normal kidney function, there was no significant difference in postoperative NRS pain scores at rest during the first three PODs between the two groups. There are two reasons that may explain this similarity in pain scores between the groups. First, it is possible that the serum opioid concentration was maintained at a similar level between patients with CKD and those with normal kidney function, regardless of dose reduction, due to delayed excretion of opioids in CKD patients. Moreover, the most commonly used opioid in this study was intravenous (IV) patient-controlled analgesia (PCA) fentanyl and the elimination of fentanyl can be delayed in CKD patients. Because the metabolites of fentanyl are known to be inactive and nontoxic15. However, a recently updated 2017 guideline reported that CKD patients do not show a significant serum accumulation of IV fentanyl16, suggesting that it is not necessary to reduce the dose of IV fentanyl for opioid-naïve CKD patients. The protocol at the SNUBH renders a reduction of the IV PCA fentanyl dose for CKD patients by about 25% to 50%, according to the patient’s eGFR. However, because patients were able to self-titrate and receive other opioid analgesics, the morphine equivalent consumption in severe CKD patients was only 3.5% lower than in patients with normal preoperative kidney function in the present study. Thus, our results suggest that it is not necessary to reduce the IV PCA fentanyl dose by more than 25% for CKD patients.One notable finding of this study pertains to the appropriate dose reduction of IV fentanyl for CKD patients. According to a 2009 guideline, the dose of most opioids, including IV fentanyl, should be reduced by 25% for patients with an eGFR between 10 and 5017 or propofol-based TIVA18 for laparoscopic surgeries. This study showed that intraoperative magnesium sulphate infusion resulted in a significantly lower morphine equivalent consumption (about 8.4%) and propofol-based TIVA resulted in a significantly lower morphine equivalent consumption (about 13.5%) during the first three PODs, which is in line with the findings of previous studies21. This suggests that propofol-based TIVA or magnesium sulphate infusion should be routinely considered for CKD patients. Additionally, surgery time or cancer were associated with morphine equivalent consumption during POD 0–3 in this study. This can be explained by an assumption that more complex and longer duration of surgical procedures are associated with more severe pain than simple surgical procedures. Preoperative BMI is another interesting factor associated with postoperative pain in this study. There were some reports that preoperative BMI was not associated with postoperative pain23; however, according to our study findings, there was positive association between preoperative BMI and morphine equivalent consumption during POD 0–3. At our institution, physicians did not tend to prescribe additional opioids in patients with lower BMI, which affected the results of this study. Therefore, further study is needed to confirm the association between BMI and postoperative pain outcomes at our institution.Another notable finding of this study is that we identified methods that can reduce postoperative pain in CKD patients, including the use of propofol-based TIVA. For several years, the SNUBH has used either an intraoperative magnesium sulphate infusionThis study has a few limitations. First, there is a possibility of selection bias due to the nature of a retrospective cohort design. In order to minimize the risk of selection bias, we used a medical record technician who was blinded to the purpose of this study. Second, this study was performed at a single centre, which may limit the generalizability of these findings. Third, most of the opioid analgesia used in this study was IV PCA fentanyl and the use of additional opioids  necessitated the application of a standard conversion ratio, which may have reduced the accuracy of these results. Fourth, the patients’ pain scores at rest were assessed by many different nurses over a period of seven years, which makes it difficult to ensure the accuracy of these scores. In addition, this study did not evaluate NRS pain scores at movement during POD 0–3, which limits the results of this study. Finally, we only focused on laparoscopic abdominal surgery rather than other surgeries, such as laparotomy, thoracotomy, or amputation, which could induce higher postoperative pain than laparoscopic surgery. Therefore, the results of this study might be restricted in its application to other surgeries.In conclusion, our results suggest that patients with mild to moderate CKD (stage 2–3) did not require reduction of opioid analgesics during POD 0–3, compared to patients with normal preoperative kidney function. Only patients with severe CKD (stage ≥ 4) might require a slight reduction of opioid analgesics.This retrospective cohort study was approved by the institutional review board of the SNUBH . The requirement for obtaining informed consents was waived due to the retrospective study design, and this manuscript adheres to the applicable STROBE guidelines. The medical record technician who collected patients’ demographic, clinical, and surgery-related data was blinded to the purpose of this study.24, discharge before POD 3, additional surgery during the first three PODs, intraoperative conversion to open laparotomy, continuous infusion of remifentanil or dexmedetomidine during the first three PODs, preoperative chronic opioid usage, and incomplete or missing medical records.The medical records of patients aged 20 years or older who underwent elective, major laparoscopic surgery at the SNUBH between January 1, 2010 and December 31, 2016 were analysed. Major laparoscopic surgery was defined as any laparoscopic surgical procedure that lasted for more than 2 hours and involved resection of an intraperitoneal organ. Simple appendectomy and cholecystectomy were excluded, even if the duration of surgery was longer than 2 hours. The exclusion criteria also included emergency surgery, single-port laparoscopy (which is known to cause less pain)During the study period, experienced surgical teams at the SNUBH proficiently performed laparoscopic resections of major intraperitoneal organs . Most patients received either balanced anaesthesia with desflurane and remifentanil or total intravenous anaesthesia (TIVA) with propofol and remifentanil.Most patients received IV PCA during the first 2 PODs. Most commonly, the IV PCA contained 8 to 12 µg/mL of fentanyl in normal saline. The background infusion rate (0.8–1.2 mL/h) and bolus dose (0.5–1.5 mL) were adjusted based on the patients’ underlying disease and kidney function. In general, the background infusion rate and bolus dose were reduced by 25% to 50% in patients with low baseline kidney function. After the IV PCA was discontinued, most patients were prescribed an additional opioid analgesic  based on the physician’s judgment or patient request. Adjuvant analgesics, such as acetaminophen and ketorolac, were also used for postoperative pain control. In general, surgeons at the SNUBH did not perform local anaesthetic infiltration of the wound site during the study period.−1) test is performed within one month of surgery as part of the routine preoperative laboratory screening for all elective surgical procedures at the SNUBH. We computed each patient’s preoperative eGFR using this baseline serum creatinine value and the Modification of Diet in Renal Disease Formula25: eGFR (mL min−1 1.73 m−2) = 186 × (preoperative serum creatinine)−1.154 × (age)−0.203 × .A serum creatinine  at rest by registered nurses at 4- to 6-hour intervals and at least 5 times a day. The mean NRS pain scores at rest were computed for PODs 0, 1, 2, and 3. The total opioid consumption, including IV PCA and additional opioid analgesics used on PODs 0 to 3, were computed using the oral morphine equivalent consumption and summed. A standard conversion ratio was used for the conversion and calculation−1 1.73 m−2).The primary endpoint measured in this study was the morphine equivalent consumption during POD 0–3 according to the preoperative kidney function status. The secondary endpoints measured in this study were NRS pain scores and adjuvant analgesic consumption during the first three days after major laparoscopic surgery according to the preoperative kidney function status. We classified the preoperative kidney function into four categories . For secondary endpoints, we also performed the same multivariate generalized linear regression analysis with six postoperative outcomes (NRS pain score at rest on PODs 0 to 3 and use of acetaminophen and/or ketorolac on PODs 0 to 3) as the dependent variables. Results are presented as exp. regression coefficient and 95% CI, and there was no significant multi-collinearity between variables in any of the multivariate generalized linear regression models . Additionally, Pearson chi-square statistics were used to test goodness of fit of multivariable models. All statistical analyses were performed using SPSS software , and statistical significance was set at P < 0.05. Considering that the mean (SD) morphine equivalent consumption during POD 0–3 was 516.6 (303.3) mg in this study, 143 patients were required in each group to detect 100 mg of difference in the morphine equivalent consumption during POD 0–3 between patients with normal kidney function (eGFR ≥ 90 mL min−1 1.73 m−2) and those with CKD (eGFR < 90 mL min−1 1.73 m−2) with a 0.05 chance of type 1 error and 80% power.Patients’ baseline characteristics are presented as number and percentage or mean value and SD. We examined the normality of distribution in dependent variables, such as morphine equivalent consumption, NRS at rest on POD 0–3, acetaminophen consumption, and ketorolac consumption, using the Kolmogorov-Smirnov test. After confirming that these variables did not follow a normal distribution, we decided to perform generalized linear regression analysis. First, we investigated variables associated with morphine equivalent consumption on PODs 0 to 3 using univariate generalized linear regression analysis. In this generalized linear regression model, a gamma distribution was assumed for the dependent variable , and the log link function was used. The variables with Supplmentary table 1. Equianalgesic opioid conversion table1


LAMP2), leading to failure of lysosome binding to autophagosomes, accumulation of glycogen in the heart, and abnormal cardiac function.Danon disease is an X‐linked dominant hereditary condition caused by mutations in the gene encoding lysosomal‐associated membrane protein 2 . Numerous autophagic vacuoles accumulated in muscle cells were detected by electron microscopy, indicating abnormal autophagy function.Pathology examination of patient skeletal muscle biopsy showed myogenic damage and autophagic vacuoles with sarcolemmal features  insertion, where the latter was a stop codon, leading to early termination of LAMP2 protein translation. The resulting truncated protein lacks an important transmembrane domain, which will impair lysosome/autophagosome fusion, damage autophagy function, and result in the clinical manifestations of Danon disease.The mutation did not result in loss of  LAMP2) gene leading to a lysosomal disorder  is an X‐linked dominant genetic condition caused by mutations in the lysosomal‐associated membrane protein 2  had a slightly enlarged left atrium and left ventricle, with no obvious abnormalities in left ventricular systolic function. The electrocardiogram waveform indicated atrial fibrillation. The patient's younger brother  showed an ST‐T change in electrocardiogram and ventricular pre‐excitation. Echocardiography revealed a non‐uniform myocardial echo and normal contractile activity. Blood analysis showed liver and muscle enzyme abnormalities, CK, 2,217 U/L; lactate dehydrogenase, 1,155 U/L (109–245 U/L); ALT, 307 U/L; and AST, 377 U/L. There was no obvious abnormality in the limb muscles, nor was there atrophy of the skeletal muscle. The patient's father (II‐1) had normal electrocardiogram and echocardiography  of the patient revealed left and right ventricular wall hypertrophy, accompanied by a decrease in overall contractile activity, left atrioventricular enlargement, and decreased myocardial T1 value Figure . DiseaseGiven the findings from the examination of the index patient and his family, we considered that the disease was likely to be caused by a familial genetic mutation; therefore, we conducted genetic testing of the family and collect detailed clinical data from all family members. All subjects provided signed informed consent. Peripheral blood samples were collected from the subjects. The study was approved by the Ethics Committee of Zhongshan Hospital, affiliated to Fudan University (NO.2016‐16B).http://www.hgvs.org/mutnomen/).Peripheral blood genomic DNA was extracted from blood samples using a TruSeq DNA Sample Preparation kit. DNA was sheared by sonication and ligated to sequencing adaptors to construct a library. The entire exome was captured using the Roche Nimblegen SeqCap EZ v5.1 kit for high‐throughput sequencing on the Illumina XTEN platform. The resulting data quality Q30 ratio was >90%, the median sequencing depth across the exon target region was >50×, the sequencing depth of 95% of the target region was >30×, and the exon loss rate was <0.2%. Raw genome sequence data were analyzed by comparison using the bwamem/disco vardenovo/freebayes process to extract single nucleotide variant (SNV) and structural variant (SV) data. Copy number variant (CNV) data were evaluated using EulerCNV and CNVnator. SNV data were annotated using data from our in‐house cohort, dbSNP138, and 1000G_OMNI2.5, and the annotated data analyzed using snpEff and deep neural networks developed in‐house. Mutation locus prediction was conducted using an in‐house C++/R deep neural network model workflow. CNV/SV data were filtered by comparison with in‐house cohort data, along with 1000G and public CNV database data, and are reported using Langya. Signal transduction pathway analysis was performed using an in‐house R/Python/Perl package. OMIM/Monarch/ExAC database analysis was conducted using an in‐house Perl/R application program interface. Neural network analysis of phenotype–genotype relationships was performed using Langya/Marshes. Data interpretation was conducted in accordance with the relevant guidelines of the American College of Medical Genetics and Genomics  cardiac syndrome. These conditions all cause cardiac hypertrophy resulting from mutations in non‐sarcomeric genes (Yardeni et al., To date, there is no specific treatment for Danon disease. Malignant ventricular arrhythmias and heart failure are the leading causes of death in patients with this condition (Cetin et al., The authors have no conflict of interest to declare.


Cyclina sinensis from their feces. DNA electrophoresis and PCR amplification were used to determine the quality of fecal DNA. And we analyzed the effects of different conditions on the degradation of feces and fecal DNA.To avoid destructive sampling for conservation and genetic assessment, we isolated the DNA of clam The clear fecal DNA bands were detected by electrophoresis, and PCR amplification using clam fecal DNA as template was effective and reliable, suggesting that clam feces can be used as an ideal material for noninvasive DNA isolation. In addition, by analyzing the effects of different environmental temperatures and soaking times on the degradation of feces and fecal DNA, we found that the optimum temperature was 4 °C. In 15 days, the feces maintained good texture, and the quality of fecal DNA was good. At 28 °C, the feces degraded in 5 days, and the quality of fecal DNA was poor.The clam feces can be used as an ideal material for noninvasive DNA isolation. Moreover, the quality of fecal DNA is negatively correlated with environmental temperature and soaking time. Cyclina sinensis is an economically important marine bivalve that is abundant and widely distributed around the maritime coasts of Asia. C. sinensis is a kind of eurythermal and euryhaline filter-feeding clam, and its food source mainly includes planktonic microalgae  [C. sinensis has two hard and symmetrical shells on both sides, and it will quickly close the shells to protect itself from damage when it is stimulated by outside environment. Destructive and nondestructive sampling methods are often applied in scientific researches of clam [The clam na,etc.) , 2 and t of clam , 4. The Noninvasive sampling is a sampling method for genetic analysis by collecting exfoliated hair, feces, and urine without having to catch, handle, or even observe the animals . It has In this study, clam feces was used as an experimental material to isolate DNA noninvasively. Moreover, the effects of environmental temperature and soaking time on the degradation of feces and fecal DNA were analyzed. The results can be used as a basis for developing noninvasive DNA isolation technology of shellfish and provide a reference for optimal conditions of fecal sampling, providing technical support for further research on molecular biology and conservation genetics of shellfish.To determine the quality of fecal DNA, electrophoresis was conducted, and the foot muscle DNA was chosen as the positive control. The results showed that all bands of the fecal DNA were clear but showed a slight tailing phenomenon Fig. . MoreoveC. sinensis. The results revealed that the band size of fecal DNA was the same as that of foot DNA and consistent with the expected length of the target band  were yellowish green in color and cylindrical. They had a length of 700 μm and diameter of 450 μm Fig. . In fecaUnder different soaking times and environmental temperatures, the degradation degree of fecal DNA was determined by agarose gel electrophoresis. At 28 °C, fecal DNA degradation occurred at 5 days after soaking the feces in seawater, but high-quality DNA could still be isolated from few fecal samples , which may cause their feces to contain few inhibitors to Taq DNA polymerase. (ii) The modified phenol/chloroform method was effective in isolating DNA from feces. Taken together, the findings indicate that the modified phenol/chloroform method is effective in isolating DNA from feces, and fecal DNA from herbivorous animals can be used as a template for PCR amplification, which is also supported by the findings of Zhang et al.’s study on fecal DNA of pandas [Feces is a very complex mixture of biotic and abiotic components. In this study, DNA was extracted from clam feces, and the quality of fecal DNA was identical to that of foot DNA. As shown in Fig. activity –36. Furtactivity . In thisf pandas .Canis lupus in winter remained significantly higher than that in summer [Bioflocs are composed of microorganisms, protozoa, algae, filamentous bacteria, and organic matter in water . With thn summer . Moreoven summer . These fn summer –45.Cyclina sinensis is a kind of marine shellfish, and its feces are soaked in seawater. Seawater is a very complicated multicomponent aqueous solution containing various organic, inorganic, dissolved, and suspended substances, which may be the reason for the degradation of feces and fecal DNA from clam. Therefore, the fresher the fecal samples collected, the higher the quality of DNA [The degradation of feces and fecal DNA was also affected by soaking time in seawater. The longer the soaking time, the more serious the degradation Fig. . Severaly of DNA .In this study, clam feces were used as experimental material to isolate DNA noninvasively. The isolation of fecal DNA was found to be successful and reliable by PCR amplification. The effects of different environmental temperatures and soaking times on the degradation of feces and fecal DNA were investigated. The results suggest that fresh fecal samples stored at low environmental temperature (~ 4 °C) were beneficial to the isolation of fecal DNA with good quality. This study provides technical support for further molecular biology research and conservation genetics research of shellfish.C. sinensis were collected from a clam farm in Jiangsu, China. They were cultured in seawater for two weeks at room temperature and fed with 0.005 g/mL of Chlorella once a day. Natural seawater was filtered with a double-layer 500-mesh sieve after precipitation, disinfection, and aeration for culturing clams and replaced once a day.Healthy clams 5 cells/L of Chlorella until waste matter (feces) was completely expelled. During this period, clam defecation was observed every 2 h. The feces were collected from the bottom of the beaker using siphon method.Forty-eight healthy clams  were randomly selected and divided into 12 parallel groups (labeled 1 to 12), each groups containing four clams. Feeding was withheld for 2 days in continuously oxygenated seawater. Thereafter, the clams were fed with 6 × 102O) to remove impurities on the fecal surface.Place the feces on a 200-mesh silk screen, and wash it slowly with double-distilled water .Add 10 μL of 20 mg/mL RNase, and incubate the tubes in a 37 °C water bath for 10 min. Centrifuge for 3 min at 12,000×g, and then transfer the supernatant into a new 1.5 mL tube.Add an equal volume of ice-cold Tris-saturated phenol (pH 7.9), and mix upside down and store at room temperature for 5 min.Centrifuge at 12,000×g for 12 min, and then transfer the supernatant into a new 1.5 mL tube.Add an equal volume of chloroform, and mix upside down. Centrifuge at 12,000×g for 10 min, and then transfer the supernatant into a new 1.5 mL tube.Add an equal volume of isopropanol, mix upside down, and store at room temperature for 3 min. Centrifuge at 12,000×g for 12 min, and then remove the supernatant completely.Wash the DNA pellet twice with 1 mL ice-cold 70% ethanol.Air dry.Resuspend the DNA pellet in 30 μL of TE buffer, and then store at − 40 °C before use.Total DNA was isolated from clam feces and foot tissue (used as positive control). Fecal DNA isolation was performed using the phenol/chloroform method in accordance with a previous study of Sambrook  with somhttps://www.ncbi.nlm.nih.gov/). PCR primers were designed by Primer Premier 5.0 software and are shown in Table 2O. The PCR amplification procedure was conducted as follows: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 10 min. The PCR amplification products were detected by 1.5% agarose gel electrophoresis and captured with a gel imaging system . The purified PCR products were sequenced by Shanghai Map Biotech Co., Ltd. The sequencing results were checked by Chromas software and blasted by BLAST online software (https://blast.ncbi.nlm.nih.gov/Blast.cgi).To determine DNA quality, PCR amplification was conducted with primers designed on the basis of mitochondrial and nuclear genomic DNA sequences. The sequences of mitochondrial (COXI and 16S rRNA) and nuclear genomic DNA  were retrieved and downloaded from NCBI  at 0, 5, 10, 15, 20, and 25 days after soaking in seawater. Fecal DNA isolation was conducted using the modified phenol/chloroform method mentioned above, and the fecal DNA quality was determined by Ultramicro Nucleic Acid Analyser , electrophoresis, and PCR amplification.The DNA purity was confirmed by Ultramicro Nucleic Acid Analyser . The DNA and PCR amplification products were detected by 1 and 1.5% agarose gel electrophoresis respectively, and the gel images were observed and captured with a gel imaging system .Additional file 1: Figure S1. Agarose gel electrophoresis of fecal DNA. Lane M, DNA marker; lane N, negative control; lane F, foot DNA; lanes 1–20, DNA of fresh feces (N = 20).Additional file 2: Figure S2. Agarose gel electrophoresis of PCR amplification products Lane M, DNA marker; lane N, negative control; lane F, PCR amplification products of foot DNA; lanes 1–20, PCR amplification products of fecal DNA (N = 20).Additional file 3: Figure S3. Agarose gel electrophoresis of PCR amplification products from clam feces under different soaking times and environmental temperatures. Samples stored at 28 °C (a), 15 °C (b), and 4 °C (c). Lane M, DNA marker; lane N, negative control; lane F, foot DNA of clam; lanes 1–12, fecal DNA.


Pseudomonas syringae pv. actinidiae in kiwifruit, Xanthomonas arboricola pv. pruni in Prunus and Xanthomonas fragariae in strawberry. A screening procedure based on in vitro and in planta assays of the three bacterial pathogens was successful in selecting potential LAB strains as biological control agents. The antagonistic activity of 55 strains was first tested in vitro and the strains Lactobacillus plantarum CC100, PM411 and TC92, and Leuconostoc mesenteroides CM160 and CM209 were selected because of their broad‐spectrum activity. The biocontrol efficacy of the selected strains was assessed using a multiple‐pathosystem approach in greenhouse conditions. L. plantarum PM411 and TC92 prevented all three pathogens from infecting their corresponding plant hosts. In addition, the biocontrol performance of PM411 and TC92 was comparable to the reference products  in semi‐field and field experiments. The in vitro inhibitory mechanism of PM411 and TC92 is based, at least in part, on a pH lowering effect and the production of lactic acid. Moreover, both strains showed similar survival rates on leaf surfaces. PM411 and TC92 can easily be distinguished because of their different multilocus sequence typing and random amplified polymorphic DNA profiles.The use of lactic acid bacteria (LAB) to control multiple pathogens that affect different crops was studied, namely,  SpontanP. syringae pv. actinidiae (Psa) and two strains of Xap and Xf were used as target bacteria in the in vitro antagonism tests and biocontrol efficacy assays (Table Three strains of ys Table . Psa CFBys Table .8 colony forming units (CFU)/mL.Cultures were prepared for routine use from the isolates preserved at −80°C. LAB were grown on de Man, Rogosa and Sharpe (MRS) agar  at 23°C for 48 h. Psa and Xap were grown on Luria‐Bertani agar at 23°C for 24 hr and Xf was grown on B medium , 15‐ to 30‐day‐old Prunus amygdalus × Prunus persica plantlets (cv. GF‐677), and cold‐stored strawberry plants (Fragaria × ananassa cv. Darselect) were obtained from commercial nurseries . Potted plants with about 8 to 10 leaves per plant were used and were kept in a greenhouse at 26 ± 2°C, 60 ± 10% relative humidity (RH) and a 16:8 hr light:dark photoperiod. Standard nitrogen‐phosphate‐potassium (NPK) fertilisation and irrigation, as well as insecticide and miticide sprays were applied. Plants inoculated with Psa, Xap or Xf were maintained in a class II greenhouse .Two‐year‐old kiwifruit plants and 15‐ to 30‐day‐old kiwifruit plantlets (2.37 CFU/mL was mixed in 4.5 mL of Luria‐Bertani (Psa and Xap) or with B medium (Xf) soft agar (0.7% agar) and overlaid on the plate containing the same media. Plates were incubated at 23°C, and the diameter of the zone of inhibition was measured after 24 and 48 hr.The antagonistic activity of 55 LAB isolates was assayed in vitro on Psa (NCPPB3739 and IVIA 3700‐1), Xap (CFBP3894 and CFBP5563) and Xf (IVIA XF349‐9A and CECT549). The experiment was performed twice using two procedures. One assay was carried out with the agar spot test in lactose‐bromocresol purple agar (LBP) as in Trias, Bañeras, Badosa, and Montesinos , albeit 2.4L. plantarum CC100, PM411, TC92 and Leuconostoc mesenteroides CM160 and CM209) were evaluated. Potted two‐year‐old kiwifruit plants, Prunus, and strawberry plants were sprayed to runoff with a 108 CFU/mL suspension of LAB cells using a hand‐sprayer . The plants were then kept in plastic bags in the greenhouse in order to reach high RH conditions. After 24 hr, plants were inoculated with a suspension of the corresponding pathogen at 1–5 × 108 CFU/mL . The pathogen suspensions were mixed with diatomaceous earth (1 mg/mL) and applied to runoff using a hand‐sprayer. Once again, the plants were covered with plastic bags for 24 hr and kept in a class II greenhouse. Streptomycin‐treated  (100 mg/L) and water‐treated plants were included as controls. Disease incidence was calculated as the percentage of infected leaves and was determined in each replicate at 15 to 21 days post inoculation.A total of five LAB isolates (2.58 CFU/mL cell suspension of PM411R (in the case of strawberry and 15‐ to 30‐day‐old kiwifruit plantlets) or TC92R (Prunus), then covered with plastic bags and maintained in the greenhouse as described above. The monitoring of PM411R and TC92R population levels was performed as Roselló, Francés, Daranas, Montesinos, and Bonaterra  to runoff using a hand‐sprayer, covered with plastic bags and maintained in the greenhouse. Streptomycin‐treated (Sigma) (100 mg/L) and water‐treated plants were included as controls. To monitor epiphytic and endophytic Psa populations, three leaves per replicate were sampled at 1 and 4 days post inoculation. Leaves were weighed and vigorously homogenised in 20 mL of 50‐mM sterile phosphate buffer and 0.1% peptone for 5 min to collect the epiphytic population. The same leaves were also used to assess the endophytic population consistent with Cellini et al.  were sprayed with a 10i et al. . Appropr2.7L. plantarum PM411 and TC92 in controlling Psa, Xap and Xf was studied in semi‐field assays and compared to reference products. The semi‐field assays consisted of treatment applications in the field and keeping plants there for 7 days before being transported to a class II greenhouse for pathogen inoculation.The efficacy of 8 CFU/mL prepared as described above, (b) Bacillus amyloliquefaciens D747  at 0.375 g a.i. L−1, (c) copper oxide  at 0.45 g a.i. L−1.Potted two‐year‐old kiwifruit plants were taken to an experimental orchard located in Zevio . The treatments administered were: (a) PM411 cell suspension at 10Prunus and strawberry plants were taken to the experimental orchard located at the Mas Badia Agricultural Experiment Station . The treatments performed were: (a) TC92 or PM411 cell suspension at 108 CFU/mL prepared as described above, (b) Bacillus subtilis QST713  at 0.55 g a.i. L−1, (c) chitosan  at 7.5 g a.i. L−1, (d) ASM  at 0.075 g a.i. L−1, (e) copper hydroxide  at 1.05 g a.i. L−1 and (f) kasugamycin  at 0.16 g/L. In all the experiments, water‐treated plants were included as controls. All the treatments, except for kasugamycin, were applied twice, 7 and 1 days before inoculation, using a hand‐sprayer to runoff.Potted 8 CFU/mL  as described in the greenhouse experiments. After inoculation, plants were covered with plastic bags for 48 hr and maintained in the class II greenhouse. The disease incidence was determined as described earlier (see greenhouse experiments) for each replicate at 15–21 days post inoculation.Plants were spray‐inoculated with the corresponding pathogen suspension at 102.8A. chinensis var. deliciosa, cv. Hayward) located in Sarna, close to Faenza . The disease had been present in this orchard in previous years with a moderate pressure. Standard cultural management  was adopted. For each plant , four shoots without Psa symptoms were selected and tagged at the beginning of the experiment. The treatments performed were: (a) PM411 cell suspension at 108 CFU/mL prepared as described above, (b) B. amyloliquefaciens D747 (Amylo‐X 5 × 1010 CFU/g) at 0.375 g a.i. L−1, and (c) copper oxide (Nordox) at 0.45 g a.i. L−1. Water‐treated plants were included as controls. All the treatments were applied every 14 days or after each rainfall (≥4 mm of rain), in the case the rain event occurred at 7 or more days after the treatment. Copper applications started at bud break  03) and were repeated until fruit grew to 30% of the final size (BBCH 73). BCA applications were performed at 10, 50 and 100% of blooming and were repeated until BBCH 73 was reached. Psa incidence was assessed twice during the season, with the second assessment taking place once disease progression had been halted and was calculated as the percentage of symptomatic leaves on 20 shoots per repetition. Psa symptomatology was confirmed by molecular identification following Gallelli, Talocci, L'aurora, and Loreti   and were filtered . 20 μL of CFS was deposited on the surface of plates containing cultures of the target pathogens  prepared as described above . Plates were incubated at 23°C, and the diameter of zone of inhibition was examined at 24 and 48 hr. Fractions of CFS were exposed to different treatments  as described by Trias, Bañeras, Montesinos, and Badosa (Agar diffusion assays using cell‐free supernatants (CFSs) were performed. PM411 and TC92 strains were grown in MRS broth for 24 hr at 30°C with shaking (100 rpm). CFS were obtained by centrifugation , D‐alanine‐D‐alanine ligase (ddl), B subunit of DNA gyrase (gyrB), ATPase subunit of the phosphoribosylaminoimidazole carboxylase (purK1), glutamate dehydrogenase (gdh) and DNA mismatch repair protein (mutS). The amplification and sequencing primers used have been previously described by de las Rivas et al. . Amplification products were purified with a desalting and concentrating DNA solutions kit QIAEX  for direct sequencing using the Automatic Sequencer 3730XL . Sequence alignments and comparisons were performed using a BioEdit Sequencing Editor (http://www.mbio.ncsu.edu/bioedit/bioedit.html). For each gene, the sequences obtained in this study from the 45 L. plantarum isolates , KT247499 , KT247496 , KT247497 , KT247501  and KT247500 . Each strain was defined by an allele profile or sequence type (ST) as described in de las Rivas et al.  for hierarchical cluster analysis. The simple matching coefficient of similarity using the unweighted pair group method with the arithmetic average (UPGMA) as the cluster algorithm was used . For each target pathogen strain, a value of “0” in both assays was considered as no inhibition activity, a value of “1” in only one assay was considered as moderate inhibition activity, while a value of “1” in both assays was considered as high inhibition activity.p < .05. The analysis was performed with the General Linear Model (GLM) procedure of the Statistical Analysis System for Personal Computers (PC‐SAS) .Biocontrol efficacy assays in greenhouse and semi‐field conditions consisted of three independent biological replicates per treatment with five plants per replicate. Two independent experiments were performed in all three pathosystems in the greenhouse and semi‐field experiments except for the semi‐field and monitoring assays in Psa that were performed once. The biocontrol efficacy assay for Psa in the field consisted of four randomised blocks of five plants per treatment. Each block was repeated in separated rows. Disease incidence and Psa population levels were subjected to analysis of variance, and mean values were compared using the least significant difference test at The monitoring of the LAB populations consisted of three independent biological replicates with five plants per replicate and two independent experiments.L. plantarum strain obtained with MLST analysis was used to investigate clonal complexes (CCs) using the minimum spanning tree (MST) method . Polymorphic DNA bands obtained with RAPD analysis were transformed into a binomial matrix (“1” for presence and “0” for absence of fragments). A dendrogram with the five series of RAPD‐PCR profiles of the 45 L. plantarum isolates was generated using the Dice coefficient of similarity and the UPGMA method (NTSYSpc).The allelic profile of each 33.1Several LAB isolates severely inhibited growth (inhibition zone diameter > 10 mm) of Psa (NCPPB3739 and IVIA 3700‐1), Xap (CFBP3894 and CFBP5563) and Xf (IVIA XF349‐9A and CECT549). However, differences in sensitivity among Psa, Xap and Xf towards LAB were observed  and the leaves of Prunus plants (TC92) under greenhouse conditions was monitored  was not significantly different from B. subtilis QST713, chitosan, ASM and kasugamycin in either experiment or from copper in only one experiment. In both experiments, strawberry plants treated with L. plantarum PM411 showed a lower incidence of infection than the non‐treated controls did. PM411 efficacy (63.6–75%) in controlling Xf on strawberry did not differ significantly from B. subtilis QST713, ASM, copper and kasugamycin in either experiment or from chitosan in only one experiment.The efficacy of TC92 and PM411 in controlling Xap and Xf, respectively, was compared to reference products in semi‐field experiments  in high RH conditions Table . The Psa3.6To understand the antibacterial activity of PM411 and TC92, CFS were analysed. While CFS without pH adjustment (pH 3.8) showed antibacterial activity against the three target pathogens , the inhibitory activity was completely lost after CFS pH neutralisation and enzymatic treatments  had no impact on the inhibitory effect (data not shown). A similar amount of a mixture of D‐ and L‐lactic acid obtained from both strains was quantified in CFS (75.75 ± 0.63 mM for PM411 and 74.86 ± 1.02 mM for TC92), with D‐lactic acid being the predominant optical isomer in the mixture.L. plantarum strains based on MLST data  and ST1‐15 (n = 9). The 21 STs were grouped into three CCs. CC1 consisted of five STs that accounted for 42 strains, including ST5 as the putative primary founder. CC2 consisted of ST1‐15 represented by nine strains and ST17, which included one strain. CC3 was composed of ST20 and ST21 with four strains. The remaining STs were considered singletons. Specifically, PM411 and TC92 were found in two different CCs: TC92 in CC1 and PM411 in CC3. Strain PM411 shared ST21 with two strains from the INTEA‐CIDSAV collection. Strain TC92 shared ST16 with another 35 strains from the INTEA‐CIDSAV collection and with two strains studied by Tanganurat et al.  were selected as they exhibited a broad spectrum of in vitro antagonism against Psa, Xap and Xf. Other reports have also demonstrated the high antagonistic activity of certain LAB strains against phytopathogenic bacteria such as In planta tests based on a multiple‐pathosystem approach were apt for highlighting strains with potential biocontrol ability and broad‐spectrum activity from among those previously selected in the in vitro tests. The biocontrol activity of PM411 and TC92 against Psa, Xap and Xf was confirmed in plants under greenhouse conditions, as pathogen infections were reduced in the same way as streptomycin. In a previous study, both strains were also selected in a screening procedure because they exhibited a suppressive effect against Erwinia amylovora in plant assays  regulatory quarantine restrictions for pathogens, the assessment of disease management strategies in orchards affected with these pathogens is practically unworkable in designated protected zones such as Spain. Therefore, semi‐field experiments have been performed in this study as they have been previously reported as being comparable to field experiments for biocontrol testing  with similar levels of expression  and affecting different hosts . The broad‐spectrum antagonism against bacterial pathogens they exhibited is mainly based on antimicrobial metabolites. Moreover, they inhibited pathogen populations on plant surfaces by suppressing infections. However, population reduction by PM411 and TC92 under nonconductive conditions might compromise plant protection. To achieve the BCA population required for biocontrol, repeated spray applications may be necessary. Therefore, monitoring viable cells could aid the design of a suitable delivery schedule of applications  typing of 71 Lactobacillus plantarum strains, including 45 strains from this study  using the unweighted pair group method with arithmetic averages (UPGMA) and the Kimura two‐parameter model . Bootstrap confidence intervals, origin of the isolates, sequence type (ST) and allelic profiles (in brackets) are indicated.nd Muñoz ; and blund Muñoz . ClusterClick here for additional data file.


Reduced social motivation is hypothesised to underlie social behavioural symptoms of Autism Spectrum Disorder (ASD). The extent to which rewarding social stimuli are granted privileged access to awareness in ASD is currently unknown. We use continuous flash suppression to investigate whether individuals with and without ASD show privileged access to awareness for social over nonsocial rewarding scenes that are closely matched for stimulus features. Strong evidence for a privileged access to awareness for rewarding social over nonsocial scenes was observed in neurotypical adults. No such privileged access was seen in ASD individuals, and moderate support for the null model was noted. These results suggest that the purported deficits in social motivation in ASD may extend to early processing mechanisms. Given the brain’s capacity limitations, incoming sensory information must be selected for further processing. Social stimuli, such as faces and bodies, attract attention  display characteristic abnormalities in their social interactions and communication (American Psychiatric Association break suppression) is measured, and is thought to index stimulus salience  with normal or corrected-to-normal visual acuity participated in Experiment 1. For both experiments, ethical clearance was granted by the University of Reading research ethics committee, and all participants gave informed consent.Thirty-eight naïve adults  and non-social images  were rated similarly positive, and arousal ratings were comparable for social images  and non-social images . All images also had canonical orientation, were presented in grey-scale, and were matched on mean luminance and root-mean-square contrast. The scenes were prepared and presented using MATLAB (Mathworks) with PsychToolbox extensions  were presented under interocular suppression using a mirror stereoscope. On each trial, a scene was presented to one eye, and a high-contrast mask pattern updating at 10 times/s was presented to the other eye  × two stimulus manipulations  × eight images × eight repetitions), balanced across presentation location and eye.Following the CFS experiment, participants rated the normal stimuli for valence and arousal. Each stimulus was presented twice—once with a nine point valence scale presented beneath it, and again with a nine point arousal scale. The scales were represented by manikins, which participants clicked on to convey their response (Bradley and Lang M = 3.46%), and did not differ over presentation conditions . Response times ± 3SDs from the mean for each observer were removed from further analysis  and did not differ over presentation conditions . Bayes factors were computed using JASP  and non-social  scenes similarly for both valence , and arousal . This is unsurprising, given that the images were selected based on their similarity in these dimensions.Our sample rated social  = 12.96, p = .001, F = 4.78, p < .05, F = 16.41, p < .001, M = 2.36, SD = .73; non-social normal, M = 2.64, SD = .81; t(33) = 4.87, p < .001), but only when they were not manipulated  = 1.06, p = .29; see Fig. A 2 × 2 repeated measures ANOVA on the mean response times revealed a main effect of Social Content . Response times ± 3SDs from the mean for each observer were removed from further analysis  and did not differ over presentation conditions .Mean response times were again calculated from correct trials. The number of incorrect trials were small  and non-social  scenes similarly for both valence , and arousal .Our ASD sample rated social  = .06, p = .81, F = .24, p = .63, F = .07, p = .79, A 2 × 2 repeated measures ANOVA on the mean response times revealed no main effect of Social Content [The results of the ANOVA were complemented by a Bayes factor ANOVA , both stimulus types were equally slow to break suppression. Using the same experimental design, procedure, and setup, we then tested individuals with a diagnosis of ASD. In both the normal, and manipulated conditions, we found no effect of social content on time to break suppression. These results provide evidence that social stimuli enjoy privileged access to awareness over non-social images in typical observers, but this is not the case in observers with ASD.The lack of a preference for social information in individuals with ASD was not due to participants having difficulties fusing the stimuli in the stereoscope, as all included participants had good stereoscopic depth perception. As all other methodological details were the same as Experiment 1, these results suggest that individuals with ASD have a deficit in processing social stimuli at an early stage of the visual processing hierarchy. It has been suggested that individuals with ASD process ‘protofaces’ (basic face-like stimuli) in a similar way to typical participants  is not an order of magnitude smaller than the extent motoric slowness in the ASD group (~ 0.6 s). In light of these observations, we suggest that the elevated RTs for the ASD sample relative to the sample in Experiment 1 is likely due to domain general processes associated with age.The sample of ASD participants in the present experiment included, by chance, similar number of males and females. ASD tends to occur more frequently in males (Baron-Cohen et al. Besides the implications of the current results for ASD, these experiments provide new insights into the processing of complex social scenes in neurotypicals. Given that previous studies have found upright faces emerge from suppression more quickly than inverted faces (Gray et al. In conclusion, these two experiments demonstrate an advantage for rewarding social stimuli to receive prioritised visual processing compared to similarly valenced nonsocial stimuli. This difference is not explained by stimulus features, as inverted and negated versions of these stimuli do not show this observed advantage. Crucially, no such advantage for social stimuli is seen in individuals with ASD. These results are informative for the social motivation model of understanding the ASD phenotype, by providing fresh evidence on the processing of social stimuli from early visual mechanisms.


Calanus pacificus and pelagic pteropod mollusk Limacina helicina to ocean temperatures and pH by measuring biomarkers of oxidative stress, antioxidant defences, and the activity of the respiratory electron transport system in organisms collected on the 2016 West Coast Ocean Acidification cruise in the California Current System. Copepods and pteropods exhibited strong but divergent responses in the same habitat; copepods had higher oxygen-reactive absorbance capacity, glutathione-S-transferase, and total glutathione content. The ratio between reduced to oxidised glutathione was higher in copepods than in pteropods, indicating lower oxidative stress in copepods. Pteropods showed higher activities of glutathione reductase, catalase, and lipid peroxidation, indicating increased antioxidant defences and oxidative stress. Thus, the antioxidant defence system of the copepods has a greater capacity to respond to oxidative stress, while pteropods already face severe stress and show limited capacity to deal with further changes. The results suggest that copepods have higher adaptive potential, owing to their stronger vertical migration behaviour and efficient glutathione metabolism, whereas pteropods run the risk of oxidative stress and mortality under high CO2 conditions. Our results provide a unique dataset and evidence of stress-inducing mechanisms behind pteropod ocean acidification responses.We compare physiological responses of the crustacean copepod  The shallow shelf areas off California-Oregon-Washington coasts are expected to become undersaturated with respect to aragonite with increasing frequency over the next few decades5. In addition, the northeast Pacific was subjected to a marine heatwave event that occurred from late 2013 until the end of 20157 overlapping with the strong 2015–16 El Niño7. As a result of these climatic phenomena, surface water temperatures in the northeast Pacific reached anomalies of >3 °C above normal8 with lasting ecological consequences over the following years.The California Current System (CCS) along the west coast of North America from British Columbia to Baja California is a region characterised by seasonal upwelling. Coastal regions of the CCS are currently experiencing pH levels well below those that are expected for the global surface ocean later in this centuryarag), an important parameter affecting the ability for pteropods to form their shells. Ocean warming will add further stress to cool-water pteropods, such as Limacina helicina. Pteropod populations have decreased in abundance9 and are today considered important bio-indicators for ocean acidification3. Pacific pteropods L. helicina are essential players in the marine food web, particularly as prey for many commercial fish species10.With ongoing ocean acidification, marine calcifying organisms such as pteropods will be exposed more frequently to low quality pelagic habitat with respect to aragonite saturation state , such as peroxides and superoxide, formed as a by-product of oxygen  to 90 µmol kg−1 O2 at mid depth (between 45 and 55 m). The highest surface aragonite saturation states were observed in nearshore surface waters where biological drawdown of CO2 was intense. Surface temperatures in May and June varied around 12–13 °C in the northern part of the study area, whereas temperatures were slightly higher (13–15 °C) off the Oregon-Washington coasts  cruise to the northern California Current System , were significantly different between pteropods pCO2 (p < 0.05). The response by species measured as LPX showed a significant effect by pCO2 (p < 0.001). In addition, we found that LPX concentrations in species were different due to temperature and pCO2 . pCO2 concentrations affected also the ORAC activities by the two species (p < 0.01) , whereas differences in SOD activity were significantly affected by 01) Fig. .Figure 4pCO2, aragonite saturation, salinity and total alkalinity (TA) as main environmental drivers. PC2 was positively associated with SOD and negatively associated with GR, LPX, GSH:GSSG ratio and glutathione peroxidase (GP), with temperature and oxygen as the significant environmental variables. Of the total observed variation, 97.9% was explained by the first two principal components, 87.0% by PC1 and 10.9% by PC2 , GST, ORAC, and CAT were positively associated with Principal Component 1 (PC1), with PC2 Fig. .Figure 5arag as second most important factor in the PCA. The two main components explained in total > 90% of the observed variance in the PCA: 75.2% by PC1 and 18.4% by PC2  in pelagic pteropods compared to the copepods, whereas for most antioxidants, especially the glutathione system , copepods have higher activities.We present a unique dataset as first evidence to demonstrate that biomarkers measured in copepods and pteropods in the Northern California Current System show strong, yet differential, relationships with temperature and pH/25, and the ratio was approximately one tenth as high in Limacina pteropods as in Calanus copepods . In the current study Calanus spp. LPX concentrations were low, around 0.5 µM cumene-hydroperoxide equivalents mg−1 , it will lead to shell thinning and dissolution, which increases vulnerability to predation and imposes increased energetic costs34, especially when undersaturation stress is interacting with other in situ stressors, such as thermal stress and hypoxia35. The observed pteropod LPX content also showed a relationship with oxygen concentrations in the PCA  that showed a significant relationship with pH or PCA Fig. , which w30, and thereby elevate the GSH:GSSG ratio, this suggests the enhanced stress in the southern NCCS is exceeding the pteropods’ ability to cope by increasing their GR activity. In mammals, a cell in resting phase has a GSH:GSSG ratio exceeding 100:1, whereas a cell suffering from severe oxidative stress has a ratio ~10:1, or 1:138. A low GSH:GSSG ratio indicates cellular toxicity and is a biomarker that works across different functional groups, from plankton to mammals38. The consequences of a low GSH:GSSG ratio  across fairly similar depth ranges in the California Current (~100 m)42, although relatively limited data on pteropod DVM are available to characterise the depth they regularly reach. A potentially important difference is that Calanus spp. diapause at depths > 1000 m12 whereas Limacina does not diapause. Lewis et al.11 observed that adult Calanus sp. in the Arctic, which migrate daily across a >140 μatm gradient of pCO2 in their natural habitat, exhibit little response to high CO2 in the laboratory, indicating a potential resilience to high CO2 conferred by their daily exposure. Thus, perhaps via carry-over effects , copepods that are exposed to a large range of physico-chemical conditions that elicit production of ROS over their life cycle may have higher base levels of antioxidants for efficient elimination of ROS than do pteropods.Considering differences in habitat use of pteropods vs. copepods as a potential explanation for their differences in biomarkers, 44  than pteropods, indicating a larger capacity for ROS scavenging45. This is also supported by our observations of elevated ORAC in copepods30, detected in warmer conditions and in low pH waters during a laboratory study. Detoxification is a fundamental defence mechanism, which allows the organism to survive or even thrive despite oxidative stress, with the GST superfamily, a set of enzymes involved in the detoxification process, being involved in this44. Previous work suggests that copepods are more sensitive to warming than to acidification47, and shown by using biomarkers in Vehmaa et al.25. Also, a review by Havenhand48 suggests that copepods will likely be resilient to near-future OA.In addition, copepods have an efficient glutathione metabolismsee Fig. , which r2O2), a harmful substance for the cell, to oxygen and water. CAT activities were much higher (33–43 µmol min−1 mg−1) in pteropods than in copepods (4–14 µmol min−1 mg−1), suggesting much more ROS in pteropods to be detoxified by CAT. In contrast, CAT activity increased in copepods when pH decreased, whereas this was not the case in pteropods. This suggests that copepods have the capacity to respond to changes in carbonate chemistry, encountered for example during vertical migration, by increasing their antioxidant capacity49, whereas pteropods show no indication of further increasing their response to additional stress, suggesting combined thermal and acidification stress may push their antioxidative activity to the limits. While SOD was the only biomarker that was not significantly different between copepods and pteropods, SOD activity increased towards the south in both species, again pointing to more severe conditions requiring a response of the antioxidant defence system.Catalase (CAT) is an antioxidant enzyme that directly converts hydrogen peroxide  converts H2O2 into oxygen and water. Vuori et al.30 showed that GP in Baltic Limnocalanus copepods (0.01–0.29 µmol min−1 mg−1) was inversely related to lipid peroxidation, which, in turn, suggests the presence of lingering ROS. GP activities in Calanus in the CCS were 0.003–0.01 µmol min−1 mg−1, not revealing any acute stress in copepods. In the PCA of our data, many of the antioxidant biomarkers , warming can amplify the effects of pCO2 on shell degradation and increase respiration and mortality33.In the present study, the ORAC increased only in pteropods with elevated temperature. Several papers suggest that both copepods and pteropods are sensitive to ocean warmingCalanus copepods at five different stations in the present work. The measured rates of 0.06–1.23 µL O2 h−1 ind.−1 compare well with rates of 0.14–4.46 µL O2 h−1 ind.−1 reported for Calanoides in the northern Benguela upwelling system52. ETS did not show a relationship with in situ temperature, which was opposite to what we had expected53. This result could be due to the fact that the in situ temperature we considered was integrated over the upper 100 m of the water column, but the time the copepods spend over those depths is not distributed evenly as they spend the day at depth and are at the surface feeding during the relatively short nights.ETS reflects maximum potential respiratory rates, and was measured in 52. Calanus is a fast-moving copepod and its fairly high ETS level could partly be explained by general activity.Another result of depth integrating temperature is that the range among stations was only 1 °C, which is much less than they experience during DVM. Many other factors can also influence ETS, such as food availability. Further, differences in body mass, depth of occurrence, and general animal activity could explain up to 90% of the variance in respiratory ratespCO2, pH, and O2, by increasing their antioxidant defence enzymes that scavenge harmful reactive oxygen species. Pteropods also responded to temperature, Ωarag, and pH and were severely affected by oxidative stress that can lead to cell damage and death. Pörtner & Farrell54 demonstrated that synergistic stressors like ocean acidification and hypoxia narrow thermal windows according to species-specific sensitivities, i.e., climate change will differentially favour species with wide thermal windows, short generation times, and a range of genotypes among its populations. Our results suggest that copepods are more tolerant than pteropods to current conditions, which can be related to their vertical migration behaviour11. Copepods also have efficient glutathione metabolism, allowing fast responses to changing ROS conditions31. Our data suggest that pteropods are sensitive to changes to temperature, Ωarag, pH, and oxygen. Further studies are needed to investigate biomarkers of these two groups that both form major prey for commercial fish species.To conclude, copepods and pteropods responded differently to environmental conditions, such as warming, hypoxia, and low pH. Copepods showed responses in measured biomarkers indicative of stress in relation to temperature, https://www.nodc.noaa.gov/oceanacidification/data/0169412.xml), which was conducted throughout the entire CCS in May–June 2016. The samples were collected on-board the NOAA Ship Ronald H. Brown, from which a total of 19 stations were sampled from 39.5 to 53°N, 124 to 131°W  with a non-filtering cod end, towed obliquely over the upper 100 m at a speed of 2 knots, usually for 25–40 min. Additional copepod samples were collected opportunistically from surface tows using a 0.5 × 1.0 m, 333 µm mesh neuston net towed at the surface (0–2 m) for 15 min. The two nets were used to maximise the catch. After each net tow, the animals were transferred to 20L coolers filled with seawater from the subsurface, and stored at ambient temperature. Other organisms  were removed from the cooler to avoid stress to target animals. We sorted both taxa on ice under binocular microscopes, each individual picked with a pipette to a petri dish and transferred with tweezers to a 2 mL plastic Eppendorf tube, then flash-frozen in liquid nitrogen. Limacina helicina and Calanus pacificus (Number of samples = 1–7 per taxonomic group) were picked per station depending on availability. Each copepod sample consisted of ~15 adult females , and each pteropod sample consisted of ~20 individuals. In total, 60 samples were collected , and they were transported by air on dry ice to Finland, and stored in −80 °C until biomarker analysis was conducted.This study took place in the northern California Current System (NCCS) during the fifth NOAA West Coast Ocean Acidification cruise , total alkalinity (TA), pH, chlorophyll a (chla), and oxygen using the methods described in Alin et al.55). The ship-based DIC and TA data are both precise and accurate to within 2 µmol kg−1.As part of the WCOA2016 cruise, which included 17 cross-shelf transects, we obtained measurements of conductivity-temperature-depth (CTD) and oxygen from a Sea-Bird SBE 911 plus CTD system. At each station, water samples were collected in modified Niskin-type bottles, poisoned with HgCl•−2 to oxygen and H2O2, which is further catalysed by CAT to ground state O2 and water by GP. GSH is one of the most important and active non- enzymatic antioxidant defence in biological systems and it acts as cofactor for GP, or react with ROS independently. In the reaction with GP, GSH is oxidised, forming GSSG, which can be reduced by GR. The ratio between the reduced and oxidised forms of glutathione has been considered an important indicator of the redox status of cells. GSH is also part of xenobiotic metabolism, by acting as a conjugant in a reaction catalysed by GST enzymes. Some GSTs can catalyse the reaction of organic peroxides with GSH, thus preventing LPX56.In the present study, we used lipid peroxidation LPX and total GSH (glutathione) concentrations; Catalase CAT, Superoxide dismutase SOD, Glutathione S-transferase GST, Glutathione peroxidase GP, Glutathione reductase GR, ETS activities, Oxygen Radical Absorbance Capacity ORAC; and reduced to oxidised glutathione GSH:GSSG ratio Table , as biomCalanus pacificus) or 100 µL (Limacina helicina) of 0.1 M K2HPO4 + 0.15 M KCl buffer (pH 7.4) using a Tissue Lyser II bead mill (Qiagen). 25 µL of raw homogenate was directly frozen in liquid nitrogen and stored at −80 °C for lipid peroxide determination (LPX). Then, the homogenate was centrifuged at 10,000 g for 15 min at 4 °C and the resulting supernatant was divided into aliquots for GST, GR, GP, CAT and SOD enzyme activity determination, ORAC assay and for glutathione sample preparation . The glutathione sample was deproteinised by adding 5% sulfosalicylic acid (SSA). The sample was incubated on ice for 10 min and centrifuged for 10 min at 10,000 g at 4 °C. The supernatant was divided into two different tubes for reduced (GSH) and oxidised glutathione (GSSG) and 33 mM M2VP  in 0.1 M HCl that is a scavenger of GSH, was added to the GSSG sample. The sample homogenate aliquots and glutathione samples were frozen in liquid nitrogen and stored at −80 °C until further analysis.Zooplankton samples were homogenised on ice in 150 µL  and intracellular soluble antioxidant capacity with OxiSelectTM Oxygen Radical Antioxidant Capacity (ORAC) Activity Assay (Cell Biolabs), following the manufacturers’ instructions, except for adjusting the reaction volumes for 384-well plate when needed. GST, GR, CAT, SOD, and LPX were determined as described in Vuori & KanervaThe enzyme activities, lipid hydroperoxides, and total GSH were normalised to the sample protein content, which was determined with PierceTM BCA Protein Assay (Thermo Scientific) with bovine serum albumin (Sigma) as the standard. The ORAC Assay is a tool for measuring the general antioxidant capacity of samples. Antioxidant systems of the sample work to block the peroxyl radical oxidation  of the fluorescent probe until antioxidant activity in the sample is depleted. All samples, standards and blanks were analysed in triplicate. For all assays in this study, the mean coefficient of variation (CV%) of the technical replicates ranged between 2.77 and 7.30% (copepods) and between 3.43 and 5.33% (pteropods).63, modified by Gómez et al.64, and adapted for a 96-well plate. Briefly, samples were homogenised in a Teflon glass grinder at 2 °C for 1.5 min in 20 mM Tris Buffer (pH 7.8), then centrifuged at 1503 × g for 10 min at 2 °C. ETS activity was measured via INT (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride) reduction to formazan by the change in absorbance, measured kinetically at 490 nm with spectrophotometer . For each assay, 30 µL of the homogenate was added to 90 µL substrate solution (1.7 mM NADH and 0.25 mM NADPH dissolved in phosphate buffer), and the reaction was initiated by adding 30 µL INT . Blank measurements were taken using phosphate buffer  without added substrates. Assays and blanks were measured in triplicate at 24 °C and corrected to in situ temperatures (depth integrated from 0–100 m) using the Arrhenius equation with an activation energy of 15 kcal/mol65; potential respiration was calculated according to Packard & Christensen66.For Respiratory Electron Transport System (ETS), adult female Calanus copepods were identified to genus under the microscope and 7–8 individuals per sample were flash frozen on liquid nitrogen and stored at −70 °C. The ETS activity was measured according to Owens & King67. These candidate models were fitted using maximum likelihood to allow AIC-based comparison of models with different fixed effects. The variable with the lowest AIC was therefore selected for the final model . To facilitate models convergence, only two co-variates were included per model. In cases of unfavourably low ratios of observations to variables, indicating overfitting, we omitted two-way interactions from the model. For LMMs, the package ‘lmerTest’ was used in the free software R68, version 3.4.3 (www.R-project.org). pH was transformed to [H+] prior to analyses, as pH is a logarithmic unit.We used linear mixed models (LMMs), based on restricted maximum likelihood estimation, for comparing biomarker levels between species and for analysing effects of environmental conditions. Biomarker levels were treated as the response variable, species as the fixed-effect  factor and environmental conditions (integrated over the upper 100 m) as fixed-effect co-variates, and CTD  station was a random effect. Multicollinearity was evidenced by correlations between environmental variables exceeding 0.7; such variables were not included in the same model. Selection between collinear variables was based on comparing the Akaike’s information criterion (AIC) of single-variable candidate models explaining biomarker levels, with lower values of AIC being preferred69, was used. This involves the construction of three data tables: a table with variables describing the environmental conditions at the stations (R), a table containing traits  of the species (Q), and a table with presence-absence values for species at a series of stations (L). First, a principal component analysis (PCA) was performed for biomarkers and the environmental data, then a correspondence analysis on the sample groups . These three analyses were passed to the RLQ function of ‘ade4’ package in R, version 3.4.3 (www.R-project.org).In order to visualise the associations among measured antioxidant defence and oxidative stress variables and environmental data, a three-table ordination method, the species traits analysis Supplementary table S1


The aim of this study is to examine whether perilla oil supplementation improves glucolipid metabolism and modulates gut microbiota in diabetic KKAy mice.Blautia is significantly decreased in LPO, MPO, and HPO, compared with DM. Nonmetric multidimensional scaling analysis shows NC and LPO are relatively coherent.The successfully established diabetic KKAy mice are randomized into four groups: diabetic model (DM), low‐dose perilla oil (LPO), middle‐dose perilla oil (MPO), and high‐dose perilla oil (HPO). C57BL/6J mice are fed a chow diet as normal control (NC). At the end of 12 weeks, mice are euthanized and glucolipid indications are analyzed. Gut microbiota analysis is carried out based on the sequencing results on V4 region of 16S rRNA. Although serum glucose, insulin, total cholesterol, low‐density lipoprotein cholesterol, high‐density lipoprotein cholesterol, abundance‐based coverage estimator, and shannon are unchanged, serum triglyceride significantly decreases in LPO compared with DM. The histopathological changes of hepatocellular macrovesicular steatosis and adipocyte hypertrophy are ameliorated by perilla oil supplementation. These findings indicate that dietary supplementation with perilla oil can improve hypertriglyceridemia and gut dysbiosis in diabetic KKAy mice, which can be associated with potential benefits to human health. According to the first WHO “Global report on diabetes,”Diet plays a fundamental role in managing diabetes. In 1976, a survey of the composition of Eskimo food has suggested that the low incidence of diabetes in Greenland Eskimos may partly be explained by the high ω‐3 PUFA content of fish oil, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).Bacteroides coprocola.In recent years, emerging evidence has revolutionized our understanding of the close relationship between gut microbiota and diabetes.Therefore, the aim of this study was to examine whether perilla oil‐derived ω‐3 PUFA would improve glucolipid metabolism and thereby modulate gut microbiota in diabetic KKAy mice.22.1Perilla oil samples were manufactured by Sinolife United Co., Ltd  according to our requirements. The ALA concentration was 60.43% .2.2n = 10) were fed normal chow die as normal control (NC). The composition of the diets is shown in Table –1, the KKAy mice were randomly divided into four groups (n = 10 in each group): 1) diabetic model (DM), 2) low dose perilla oil (LPO), 3) middle dose perilla oil (MPO), and 4) high dose perilla oil (HPO). NC and DM were gavaged with 0.5% sodium carboxy methylcellulose. The animal treatment lasted for 12 weeks, during which both diet and water were consumed ad libitum. At the end of experiment, overnight fasted mice were killed with sodium pentothal. Blood and tissue samples were collected for further analysis. The animal experimental protocols were conducted according to the Institutional Animal Care and Use Committee of Southeast University (no. 2015‐0910‐008).Eight‐week‐old spontaneously diabetic male KKAy mice were obtained from Beijing HFK Bioscience Co., Ltd , and age‐matched male nondiabetic C57BL/6J mice were obtained from Model Animal Research Center of Nanjing University . All mice were housed one per cage at 22 ± 2 °C with a 12‐h light/dark cycle. The KKAy mice were fed a high fat diet, which has applied for national invention patent of China (application number: CN201110127312.5). The C57BL/6J mice (2.3–1 bw d–1 for LPO), 20 (1.33 g kg–1 bw d–1 for MPO), 30 (2.00 g kg–1 bw d–1 for HPO) times of that used in human. Perilla oil supplementation was administered by gavage once a day from 2 to 5 pm. The dose is achievable via available supplements.According to Chinese Dietary Reference Intakes2.4Serum glucose, triglyceride (TG), total cholesterol (TC), LDL cholesterol (LDL‐C), and HDL cholesterol (HDL‐C) were determined by automatic biochemical analyzer . Serum insulin was measured using ELISA kits  according to the manufacturer's protocol.2.5Liver, epididymal white adipose, and interscapular brown adipose tissues from each mouse were immediately fixed with formalin and stained with hematoxylin and eosin staining. The degrees of distribution were used to determine lesion including hepatocellular macrovesicular steatosis (liver tissue), adipocyte hypertrophy (white adipose tissue), and single bubble adipocyte accumulation (brown adipose tissue). The histopathological findings were scored as 0  (absent), 1  (<5%), 2  (5–29%), 3  (30–49%), 4  (50–75%), and 5  (>75%). Three fields were scored for each sample. The viewer was blinded to the sample groups.2.6t‐test.Ten mice were selected from each group for gut microbiota analysis. Bacterial genomic DNA was isolated from mouse colonic feces using the FastDNA SPIN Kit for Feces  according to the manufacturer's instructions. The V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers (515F and 806R). Sequencing was performed on Illumina HiSeq platform by HiSeq2500 PE250 . The sequencing depth in each sample is listed in Table S5, Supporting Information. Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). The alpha diversity was calculated using abundance‐based coverage estimator (ACE),2.7p value of less than 0.05.Data are expressed as mean ± SD. Significant differences among treatment factors were analyzed by one‐way analysis of variance followed by Tukey's post hoc test using PASW statistics 18.0 . The significance threshold was set at a 33.1Table Initial body weight, final body weight, body length, liver weight, perirenal fat, and epididymal fat were significantly increased in DM than that in NC Table . Compare3.2Table Serum glucose, insulin, TG, TC, LDL‐C, and HDL‐C were significantly increased in DM than that in NC Table . Compare3.3Figure Table Histological examination of liver, white adipose, and brown adipose tissue in NC showed normal cell architecture Figure , Table 43.4Table Figure In all detected OTUs, 568 were shared by all groups . The unique OTUs were 25, 49, 18, 7, and 26 in NC, DM, LPO, MPO, and HPO, respectively. There was no significant difference in the value of ACE and shannon among the five groups . Significant difference of beta‐diversity distance was found between NC, LPO, and DM, NC and LPO groups were more similar than NC to the other groups . The heatmap analysis of the top 35 genus is displayed in the Figure Akkermansia was high in NC and low in DM. The percentage of Akkermansia was unaffected by supplementation of perilla oil.At phyla level, Firmicutes, Bacteroidetes and Actinobacteria were the dominant phyla in all groups Figure . At genuC Figure . Compare4Blautia.According to the latest results from the Diabetes UK‐James Lind Alliance Priority Setting Partnership, the issue concerning what role do fats have in the management of type 2 diabetes became one of top ten research priorities for type 2 diabetes.Perilla oil is an edible plant oil extracted from perilla seeds, which is both food and herbal medicine in China. Perilla oil is rich in ALA, a plant‐derived essential ω‐3 PUFA. Although perilla oil‐derived ALA is one of ω‐3 PUFA, its antidiabetic effect is little‐studied relative to fish oil‐derived ω‐3 PUFA. The KKAy mouse, also named yellow KK mouse, carries the yellow obese and diabetes genes. This animal model is characterized by marked obesity, hyperinsulinemia, hyperglycemia, and dyslipidemia.Interestingly, we observe greater perirenal fat pads in animals receiving the perilla oil gavage. However, epididymal fat weights are similar in LPO, MPO, HPO, and DM. This phenomenon was probably caused by the higher dosage in the background of high fat diet, because perilla oil was also the major source of energy production. In addition, there are regional differences in preadipocyte adipogenesis. Tchkonia et al.Blautia is a Gram‐positive, anaerobe bacterium belonging to the family Lachnospiraceae, which was thought to take part in the development of glucose metabolism disturbances.Blautia.Blautia was much higher in DM compared with NC, but in the gut of low, middle, and high dose perilla oil‐fed KKAy mice, Blautia was lower than DM. This finding suggest that Blautia may be involved in the improved gut function. Further animal experiments and human studies will be necessary to determine the specific effects of perilla oil on Blautia in diabetes.Currently, the development of gut microbiota is an emerging area of diabetes research. Impairment of gut microbiota homeostasis plays an essential role in the onset and progression of type 2 diabetes. Lactobacillus was the most abundant genus in HPO. Lactobacillus is generally believed to be a beneficial bacteria.Lactobacillus in the cecal content as compared to control mice.Lactobacillus was increased in fish oil fed mice.Lactobacillus in diet‐induced obese mice.When compared with LPO and MPO, Perilla oil gavage may be affecting the gut microbiota via increased bile secretion. Bile acids are the major effectors of digestion and absorption of fat. After the oil gavage, bile acids are released to solubilize fat and processed by the microbiome, giving rise to secondary metabolites, such as SCFAs, trimethylamine, and secondary bile acids.Our study had several limitations. First, this study lacks positive control and negative control group, so it is difficult to discern the effect of perilla oil on lessening diabetic conditions more precisely. Second, one time point testing failed to monitor the kinetics of gut microbiota. Finally, care must be taken in extrapolating results to human due to the small sample size and the inherent physiological differences between mouse and human. Further studies are warranted to examine whether the metabolic health outcomes are causally related to changes in intestinal flora.In conclusion, perilla oil supplementation are not only central to hypertriglyceridemia, but are linked with improvement in gut microbiota. It is recommended that a follow‐up study with a large sample size should be confirmed in clinical trials.The authors declare no conflict of interest.Table S1. The fatty acid profile of perilla oil.Table S2. The fatty acid profile of the diets.Table S3. The ingredients of high fat diet (per 100g).Table S4. The ingredients of chow diet (per 100g).Table S5. The sequencing depth in each sample.*P < 0.05 vs. NC, #P < 0.05 vs. DM.Figure S1. Effect of perilla oil supplementation on serum triglyceride. NC, normal control; DM, diabetic model; LPO, low dose perilla oil; MPO, middle dose perilla oil; HPO, high dose perilla oil; TG, triglyceride.Figure S2. Venn diagram of operational taxonomic units abundance. NC, normal control; DM, diabetic model; LPO, low dose perilla oil; MPO, middle dose perilla oil; HPO, high dose perilla oil.Figure S3. Comparison of bacterial richness and diversity. ACE, Abundance‐based coverage estimator; NC, normal control; DM, diabetic model; LPO, low dose perilla oil; MPO, middle dose perilla oil; HPO, high dose perilla oil.Figure S4. Ternaryplot displaying the genus with significantly different abundance. NC, normal control; DM, diabetic model; LPO, low dose perilla oil; MPO, middle dose perilla oil; HPO, high dose perilla oil.Click here for additional data file.


Adolescence is the transitional period between childhood and adulthood, characterized by substantial changes in reward‐driven behavior. Although reward‐driven behavior is supported by subcortical‐medial prefrontal cortex (PFC) connectivity, the development of these circuits is not well understood. Particularly, while puberty has been hypothesized to accelerate organization and activation of functional neural circuits, the relationship between age, sex, pubertal change, and functional connectivity has hardly been studied. Here, we present an analysis of resting‐state functional connectivity between subcortical structures and the medial PFC, in 661 scans of 273 participants between 8 and 29 years, using a three‐wave longitudinal design. Generalized additive mixed model procedures were used to assess the effects of age, sex, and self‐reported pubertal status on connectivity between subcortical structures  and cortical medial structures . We observed an age‐related strengthening of subcortico‐subcortical and cortico‐cortical connectivity. Subcortical–cortical connectivity, such as, between the nucleus accumbens—frontal medial cortex, and the caudate—dorsal anterior cingulate cortex, however, weakened across age. Model‐based comparisons revealed that for specific connections pubertal development described developmental change better than chronological age. This was particularly the case for changes in subcortical–cortical connectivity and distinctively for boys and girls. Together, these findings indicate changes in functional network strengthening with pubertal development. These changes in functional connectivity may maximize the neural efficiency of interregional communication and set the stage for further inquiry of biological factors driving adolescent functional connectivity changes. It is particularly relevant to consider these transformations not only in relation to neural structural or localized activation changes, but also in relation to functional connectivity changes in the adolescent brain Casey, . RelativCentral to adolescent brain development is a change in the neural motivational circuitry  functional magnetic resonance imaging (fMRI), which provides an important framework for investigating functional systems in the organization of the adolescent developing brain . Longitudinal accelerated designs consider individual trajectories, thereby allowing for a more accurate estimate of developmental change. RS functional connectivity was examined between a set of subcortical and cortical structures of interest, which included the ventral and dorsal striatum, putamen, hippocampus, amygdala, and all atlas‐based anatomical regions of the medial PFC . We expected a decoupling between subcortical and medial PFC regions with age, which may be particularly driven by pubertal‐related changes. Given the large sample size and intensive longitudinal measurements, this study also allowed us to examine sex differences and age by sex interactions in RS connectivity change.22.1The current study was part of BrainTime, a longitudinal study from Leiden University, Leiden, the Netherlands. Participants were recruited through local schools and advertisements and provided written informed consent for the study at every time point . Participants were screened for MRI contraindications and had no neurological or psychiatric disorders at time point 1 (T1). All anatomical MRI scans were reviewed by a radiologist and no anomalous findings at any of the time points were reported. At each time point, participants received an endowment for participation in a larger scale study. The RS data presented here were collected as the first scan of the BrainTime experimental protocol examining affective and cognitive development via the use of task‐based functional neuroimaging. The study and its procedures were approved by the institutional review board of the Leiden University Medical Center. Cross‐sectional RS analyses have previously been reported in van Duijvenvoorde, Achterberg, et al.  for nuclMage = 13.98 years; SDage = 3.68; range = 8.01–25.95 years; 146 males), who were invited for time point 2 (T2) approximately 2 years after T1 . T2 MRI data were collected from 255 participants . All participants were invited for time point 3 (T3), approximately 2 years after T2 . At T3, 243 participants participated in the MRI session .At T1, MRI data were collected from 299 participants . Second, participants were excluded from all time points if they were diagnosed with a neurological or psychiatric disorder  at T2 and/or T3 (n = 21). Third, participants were excluded when excessive head motion was detected . Motion exclusion was based on having ≥2 mm translation or more than 2° rotation in any direction, having ≥10 volumes . FD and reference RMS outliers were established using the motion outlier tool implemented in FMRIB Software Library (FSL) version 5.0.4 , 211 participants at T2 , and 214 participants at T3 . Table The final sample consisted of 661 observations from 273 participants. Specifically, 236 participants at T1  and T2  and were not significantly related to age at either time point  for included subjects.Intelligence quotient (IQ) was estimated at T1 using the subsets “similarities” and “block design” and at T2 using the subsets “Vocabulary” and “Picture Completion” of the Wechsler Intelligence Scale for Adults (WAIS‐III) or the Wechsler Intelligence Scale for Children, third edition . This questionnaire was only administered to participants up to 18 years of age , because it was assumed that all participants completed pubertal development by 18 years. Other missing data occurred because of administration or technical errors . Longitudinal data of the PDS have been reported on this sample for two waves in Braams et al. (r (185) = .785, p < .001; T2: r (129) = .739, p < .001; T3: r (101) = .638, p < .001.Stage of physical pubertal maturation was assessed at each time point with the Pubertal Development Scale (PDS) for participants under 18 years of age . Two additional dummy scans preceded the scan to allow for equilibration of T1 saturation effects. Participants were instructed to lie still with their eyes closed, without falling asleep.Neuroimaging was conducted using a 3.0 T Philips Achieva MRI scanner with a standard whole‐head coil. The same scanner and settings were used for all participants and at all three time points. The RS scans were acquired as the first scan of a fixed‐order imaging protocol, with T2*‐weighted echo‐planar imaging (EPI)  = 2,200 ms, time echo (TE) = 30 ms; flip angle = 80°; field of view (FOV) = 220 × 220 × 114.67 mm3; in‐plane resolution = 1.96 × 1.96; slice thickness = 2 mm), and a T1‐weighted anatomical scan , at the end of a fixed imaging protocol which included functional tasks.For registration purposes, we additionally obtained a high‐resolution T2*‐weighted gradient EPI scan , part of FSL  = −.142, p = .029 and T3 (r(213) = −.15, p = .025). To minimize these potential influences of head motion, we denoised the preprocessed RS data of the included participants with FIX  using the included standard training data set (threshold 15)  and cerebrospinal fluid (CSF) can be confounding effects that result in overestimated RS connectivity strength. These signals primarily reflect noise from non‐neural origin  and are largely independent from Blood Oxygenation Level‐Dependent (BOLD) signal fluctuations in gray matter , which segments the T1‐weighted anatomical scan into different tissue types . These maps were then FLIRT‐based transformed into functional subject space and eroded by one voxel (3 × 3 × 3 mm) to minimize potential partial volume effects. Global signal time series were calculated in native space as the average signal across all nonzero voxels in the brain. WM, CSF, and global signal time series were used as temporal covariates and removed from the RS time series of each ROI at the individual participant level through linear regression in MATLAB. We then calculated Pearson correlations between all ROI time series and transformed them into 2.7y = 30 based on Bush, Luu, and Posner (Regions of interest (ROIs) were selected from the Harvard–Oxford probabilistic anatomical brain atlas  in FSL, with a thresholded probability of ≥0.5. Although based on anatomical parcellation, the Harvard–Oxford atlas is an often used and well‐known atlas in functional brain analyses. Given our focus on age‐related change in connectivity between frontal midline and subcortical structures, we included four cortical midline structures in this atlas  that spanned a ventral to dorsal cortical midline. Considering the extent and functional specificity of the ACC, this anatomical structure was divided in a more posterior‐dorsal and a more anterior‐ventral part with a cutoff of d Posner  (see ford Posner . We incl2.8https://www.r-project.org/). GAMM is similar to a generalized linear mixed model where predictors can be replaced by smooth functions of themselves, offering efficient and flexible estimation of nonlinear effects. Smooth splines can capture important nonlinear changes that are easily missed with polynomials, prevent biased fits at the extreme ranges of the data, while controlling Type 1 error rate in AIC/ Bayesian Information Criterion (BIC) values  the developmental age‐related pattern over the whole group; (b) a main effect of sex; and (c) differences in developmental trajectories between sexes. These models were compared to test which model provided the best fit for each connection. In short, first a simple age model of , and a Sex by Age interaction  was selected as the best fitting model.The dimension used to represent the smooth terms A second aim was to assess puberty‐related effects on RS functional connectivity, and most interestingly, whether this was also a better predictor of RS connectivity than chronological age. First, a simple GAMM model to visualize effect of sex and age on PDS development was tested with the following model Results showed that PDS score was described by a main effect of sex and spline effect of age. That is, pubertal development manifested differently for boys and girls and increased with age  for each RS connection. ICCs were computed by estimating a null model Equation 8 with ma33.1r) for each connection of interest per time point. Most regions were positively functionally connected, with the strongest positive connections between the amygdala–hippocampus and subcallosal cortex–frontal medial cortex. Negative connectivity was observed between dorsal and ventral cortical regions, such as the dorsal ACC–subcallosal cortex, and dorsal ACC–frontal medial cortex.Figure 3.2We assessed age‐related changes in RS connectivity using GAMM .For boys, subcortical–cortical connectivity between the nucleus accumbens–frontal medial cortex and putamen–frontal medial cortex related negatively to PDS scores, with increasing PDS significantly decreasing RS connectivity (similarly to what was observed for age)  and caudate–dorsal ACC (p = .02) showed a significant effect of PDS over and above age at baseline.For girls, subcortical–cortical connectivity between the hippocampus–dorsal ACC , caudate–dorsal ACC , and putamen–frontal medial cortex  was significantly negatively related to PDS scores, with increasing PDS related to decreased RS connectivity . Given that prior studies testing subcortical reactivity suggested that puberty may be a driving factor for nonlinear age patterns . These findings fit well with prior cross‐sectional reports (Fareri et al., Together, these findings may be interpreted as change in local versus distributed networks in which specific functional networks both strengthen and weaken across adolescence. Previous work also showed that structural networks become more segregated across development (Baum, Ciric, Roalf, Betzel, et al., 4.2Previous studies focusing on functional reactivity have often suggested that pubertal development may advance or enhance growth trajectories of brain development, although most studies up to now focused on brain structural development (e.g., Herting & Sowell, Until now, a large body of work on pubertal effects on neural development is based on animal studies. Recent animal evidence suggests that puberty may be a critical driver in reward‐circuitry development. For instance, animal work has observed a reduction in medial PFC volume and synapses in postpubertal rats, and neuronal losses during pubertal onset (Walker et al., Note that when testing sex differences, we observed only few differences in functional connectivity between boys and girls. That is, amygdala–hippocampus connectivity, a connection most prominently influenced by sex, was stronger for boys than for girls, and hippocampal–ventral ACC connectivity was stronger for girls than for boys. Previous research on sex differences has mostly been done in adults (e.g., Alarcón, Cservenka, Rudolph, Fair, & Nagel, 4.3r = .94; Gabard‐Durnam et al., RS connectivity is inherently susceptible to effects of motion, which can have a marked influence on developmental findings. In the current study, we have taken steps to account for such possible confounds (see Satterthwaite et al., A second limitation of our analyses may be that we used a set of anatomical ROIs based on the Harvard–Oxford atlas. Using anatomical ROIs versus a functional brain atlas (such as the areal atlas of Power et al., 5The current study used RS functional connectivity in a large longitudinal sample to understand developmental changes in connectivity between and within subcortical and medial prefrontal regions. These findings have implications for future research: they confirm patterns of subcortical–cortical connectivity changes, and advance insights by suggesting an important role for pubertal development in the development of subcortical–cortical functional connectivity. This may be an important starting point for further understanding of hormonal effects operating on functional connectivity development, and the link with real‐life reward‐driven behaviors.The authors declare no conflict of interest.