MC density was higher in the fibrotic areas of lung tissue compared with spared alveolar areas or in controls. Female gender, low diffusion capacity for carbon monoxide, diffuse alveolar damage, and smoking were associated with a low MC density. MC densities of fibrotic areas had declined significantly in five subjects in whom both SLB in the stable phase and autopsy after an acute exacerbation of IPF had been performed. There were no correlations of MC densities with survival time or future acute exacerbations. The MC density in fibrotic areas was associated with several clinical parameters. An acute exacerbation of IPF was associated with a significant decline in MC counts. Further investigations will be needed to clarify the role of these cells in IPF and in the pathogenesis of acute exacerbation as this may help to identify some potential targets for medical treatment for this serious disease.Mast cells (MCs) are known to be involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), although their role in acute exacerbations of IPF has not been investigated. The aims of the study were to evaluate the numbers of MCs in fibrotic and non-fibrotic areas of lung tissue specimens of idiopathic pulmonary fibrosis (IPF) patients with or without an acute exacerbation of IPF, and to correlate the MC density with clinical parameters. MCs of IPF patients were quantified from surgical lung biopsy (SLB) specimens (The online version contains supplementary material available at 10.1007/s10753-021-01582-0. Idiopathic pulmonary fibrosis (IPF) is a severe interstitial lung disease of unknown cause, in which the phenomenon of acute exacerbation (AE) is responsible for significant mortality , 2. The Mast cells (MCs) are immune cells whose significance in pulmonary diseases has been best characterized in allergic asthma; the role of MCs in other respiratory diseases is less clear , 5. PeriA higher number of MCs have been observed in IPF compared with other interstitial lung diseases or normal controls , 9–15. MAlthough MCs appear to be involved in the pathogenesis of pulmonary fibrosis, the role of these cells in the development of AE-IPF has not been investigated. There were two aims of this study: (1) to evaluate the numbers of MCs in fibrotic and non-fibrotic areas of lung tissue specimens of IPF patients with and without an AE-IPF, and (2) to correlate the MC density with clinical parameters such as age, gender, pulmonary function test results, and smoking status. We also wanted to investigate whether the MC count was associated with the risk for mortality or the occurrence of AE-IPF.n = 21) were identified either by applying the current criteria for AE-IPF in those subjects in whom lung tissue material had not been obtained during the episode of AE-IPF (n = 13), or on the basis of the surgical lung biopsy material, i.e., diffuse alveolar damage (DAD) in parallel with usual interstitial pneumonia (UIP) indicative of AE-IPF (n = 8) [The study cohort consists of 47 IPF patients treated in Oulu University Hospital or Oulaskangas Hospital in Northern Finland between 1991 and 2019. All study subjects had undergone a surgical lung biopsy procedure for diagnostic purposes. The lung tissue specimens taken at autopsy were also studied from seven subjects out of 47. Non-diseased lung tissue material from 12 non-smokers who had undergone lung cancer surgery served as controls  results examined at a time near the biopsy date, antifibrotic drug use, medical treatment preceding AE-IPF in patients with DAD in a biopsy or autopsy specimen, and smoking data were collected systematically from the medical records. Patients with less than 5 pack-years of smoking history were regarded as non-smokers. Age and follow-up time were calculated by using the biopsy procedure date and death, transplantation or last follow-up date (11/8/2020). Death dates were collected from death certificates housed in the national registry of Statistics Finland.Formalin-fixed and paraffin-embedded lung specimens were cut into 3.5 μm sections, de-paraffinized in xylene and rehydrated in a descending ethanol series. The staining was performed by using Dako REAL EnVision Detection System . Microwave-stimulated antigen retrieval was performed in Tris–EDTA, pH9, and endogenous peroxidase was neutralized with peroxidase blocking solution (Dako). After the specimens had been incubated with a primary monoclonal mouse anti-human mast cell tryptase antibody  diluted 1:200 in antibody diluent (Dako) for 30 min at room temperature, a biotinylated secondary HRP rabbit/mouse antibody (Dako) was added. The color was developed with 3,3′-diaminobenzidine  and counterstaining was performed with hematoxylin. As a negative control, the primary antibody was replaced by mouse isotype control . The stained sections were digitized with Leica-Aperio AT2 .2 areas in fibrotic areas of lung tissue in IPF and in normal alveolar areas of control subjects. Due to the small amount of fibrotic areas in surgical lung biopsy samples of two subjects, five, and eight 0.16-mm2 areas were assessed instead of ten. In addition, three 0.16 square millimeter areas of non-fibrotic alveoli were assessed from specimens of 33 IPF patients, in whom there was enough preserved lung tissue to permit this analysis, i.e., in study subjects with IPF, the numbers of mast cells in both fibrotic and non-fibrotic areas were counted. The MC density of fibrotic areas was the value used when the correlations with clinical parameters were calculated. J.S. calculated the MCs from all lung tissue specimens. Two other investigators (S.L. and M.K.), calculated the mast cell densities of 20 different study subjects. In addition, MC densities of honeycombing areas, fibroblast foci and areas of dense fibrosis were calculated from 12 representative samples (S.L. and M.K.).Digitized lung tissue specimens were examined by using virtual microscopy software . The MCs were visualized maximally at 40 × magnification and were enumerated from ten randomly selected 0.16-mmt-test or paired samples t-test to compare means when appropriate. The intraclass correlation coefficient was determined to evaluate the interrater reliability of MC counts. Survival analysis was performed by using Kaplan–Meier curves, and risk for earlier death or earlier episode of AE-IPF was evaluated by using Cox regression model. Medians and quartiles of MC densities were utilized to determine the cutoff values for Kaplan–Meier and Cox regression analyses. We included MC densities of SLB samples, not autopsy samples, in the Kaplan–Meier and Cox regression analyses.IBM SPSS Statistics for Windows, Version 27.0  was used to perform statistical analysis, and Origin(Pro), Version 2019b , was utilized for preparing the graphs. Means and standard deviations were calculated for parameters that were normally distributed. Medians and interquartile ranges were determined for parameters that were not normally distributed. We used independent samples N = 1), autopsy (N = 5) or both in autopsy and lung biopsy specimens (N = 2).The characteristics of the study subjects are shown in Table 2 (SD 140) in the area of dense fibrosis, 530 MCs/mm2 (SD 110) in the honeycombing area, and 160 MCs/mm2 (SD 40) in fibroblast foci. Figure The MC densities in different types of lung tissue specimens are shown in Table 2 SD 40 in thep = 0.041) and current smokers had a lower MC density than ex- or non-smokers (p = 0.023). There was no association with forced vital capacity (FVC) and MC density, but in contrast, a high diffusion capacity for carbon monoxide (DLCO) was linked with a high MC density (p = 0.010). Patients with IPF had a higher MC density in their fibrotic lung tissue areas when compared with normal lung tissue derived from controls (p < 0.001), while the difference between non-fibrotic areas in IPF and controls was not statistically significant. We found no correlation of a future episode of AE-IPF with MC density in lung tissue biopsies taken in the stable phase of the disease.The MC densities in the fibrotic areas of lung tissue correlated with several clinical parameters, as shown in Table p < 0.001)  had a lower MC density than patients without AE at the time of biopsy  (1) Table , Fig. 2.p = 0.175). MC densities in tissue specimens obtained at diagnosis in the stable phase of the disease, without clinical or histological evidence of AE-IPF at the time of biopsy date, were not correlated with the time to the first future episode of AE-IPF .The correlation between the survival time and MC density was not statistically significant, although there was a slight trend towards a shorter survival and low MC density (319 MCs or less per square millimeter) ) with a high MC density , 13, 15,The discrepancies between the results mentioned above might be partly due to the differences in MC staining techniques: Andersson et al. found the association of a high MC density with low FEV1% predicted only with MCs double positive for both chymase and tryptase . ShimborIn addition to the differences in MC staining techniques, inconsistent results concerning MC densities and their correlations with clinical parameters might also be related to the methods used to calculate and choose the tissue areas for MC quantification. The determination of one MC density value for the whole area of tissue sample does not take into account the varying proportions of fibrosis and spared alveolar tissue in different tissue specimens, a phenomenon typically present in IPF where fibrotic areas of lung tissue alternate with less-affected parenchyma . ConsistWe could demonstrate a significant decline in the MC density of five patients who had undergone a lung biopsy for diagnostic purposes in the stable phase of disease and in whom an autopsy had been performed after death caused by an AE-IPF. No similar findings have been reported earlier, because there are very few studies involving multiple lung tissue specimens obtained from the same individual in different stages of pulmonary fibrosis.To our knowledge, there are no previously published studies investigating lung MC density in AE-IPF patients. Animal models have suggested MCs and their mediators have a role in the pathogenesis of acute respiratory distress syndrome (ARDS), a phenomenon in which DAD is present similarly to AE-IPF −30. HoweMCs are known to be activated by several different mechanisms, e.g., immune receptors, bacteria and their products, viruses, cytokines and inflammatory mediators, endogenic peptides, and physical stimuli . MCs areIn the study by Overed-Sayer et al., nintedanib, but not pirfenidone, inhibited fibroblast-mediated MC survival in vitro and this finding was confirmed in the rat bleomysin model . In our We were not able to find a statistically significant association of low MC density with short survival time, although a trend in this direction could be observed. It should be noted that we correlated survival time with MC density determined from patients’ SLB samples, of which only 3 were obtained during AE-IPF with a relatively low MC density. It was not reasonable to correlate MC cell densities of autopsy samples with survival time due to the lack of follow-up time of these patients. One may speculate that if we had had SLB specimens obtained slightly before death from a larger number of IPF patients, then it is possible that a correlation of low MC density and short survival time might have been found.Overall, we managed to gather a rather comprehensive lung biopsy material from IPF patients, namely lung tissue specimens from 47 patients with additional autopsy material from 7 patients; as far as we are aware, this is the largest histological material on IPF patients from which an MC quantification has been performed. In earlier studies, the study material has mainly included tissue specimens from fewer than 20 IPF patients , 13, 16,To conclude, the MC density had significantly declined in subjects with an AE-IPF indicating that these cells may play variable roles in different stages of the disease. Altogether, the role of inflammation, MCs and other immune cells should be studied further in order to gain a more profound understanding of the pathobiology of IPF as this may make it possible to identify new targets for medical treatment for this serious disease.Supplementary file1 (PDF 163 KB)Below is the link to the electronic supplementary material.


Therefore, it is necessary to design high-performance gas sensors for toxic gas detection. In this sense, graphene-based materials are promising for use as toxic gas sensors. In addition to experimental investigations, first-principle methods have enabled graphene-based sensor design to progress by leaps and bounds. This review presents a detailed analysis of graphene-based toxic gas sensors by using first-principle methods. The modifications made to graphene, such as decorated, defective, and doped to improve the detection of NOx, SOx, and CO toxic gases are revised and analyzed. In general, graphene decorated with transition metals, defective graphene, and doped graphene have a higher sensibility toward the toxic gases than pristine graphene. This review shows the relevance of using first-principle studies for the design of novel and efficient toxic gas sensors. The theoretical results obtained to date can greatly help experimental groups to design novel and efficient graphene-based toxic gas sensors.Detecting and monitoring air-polluting gases such as carbon monoxide (CO), nitrogen oxides (NO The conversion of energy from one form to another many times affects the air composition in several ways. It is well-known that fossil fuels have been powering industrial development and the amenities of modern life that we enjoy. However, the combustion of fossil fuels contributes to a great extent to composition variations of the atmosphere, and this is mainly due to harmful gas emissions.x), and sulfur oxides (SOx), among others. In this context, health expenditures have increased due to air pollution, which is mainly associated with the rapid industrialization of many countries. Consequently, the disruption of ecological balance and serious public health issues caused by harmful gases are raising global concerns . Ad. Ad2 andgraphene . The comadsorbed . Finallyadsorbed . The comadsorbed .Many theoretical studies have been conducted on the use of doped graphene as toxic gas sensors. The results evidence that doped graphene sheets are good candidate materials as gas sensors. To experimentally confirm some of the above-mentioned theoretical predictions, various doped graphene materials have been synthesized and evaluated as toxic gas sensors ,107,108.x, and SOx toxic gases were revised and analyzed in detail. Based on this review, we concluded the following:(a)The interaction between toxic gases and pristine graphene is weak, which reduces the sensitivity and selectivity of pristine graphene toward the toxic gases.(b)The pristine graphene decorated with transition metals is a promising material for use in a toxic gas sensor. However, up to now these types of studies are still scarce; therefore, more theoretical studies on the sensitivity and selectivity of pristine graphene decorated with transition metals toward the toxic gases should be carried out.(c)It was observed that graphene with single-vacancy defects interacts stronger with the toxic gases compared to pristine graphene. Therefore, it is a promising material for use in toxic gas sensors. In addition to point defects, line or multivacancy defects should be investigated at the DFT level, to enrich graphene functionalities.(d)Bilayer and multilayer graphene exhibit higher different dimensionalities than single-layer graphene, which can increase the number of possible defect types, namely, point defects, line defects, and so on. At the theoretical level, more attention should be paid to understanding stable bilayer and multilayer graphene with randomly distributed defects.(e)A large number of theoretical studies have addressed the use of doped graphene as a toxic gas sensor. The evidence indicates that doped-graphene sheets are good candidate materials. However, up to date, DFT studies on the selectivity of doped graphene toward the toxic gases are limited. Therefore, more theoretical studies on the selectivity of doped graphene toward the toxic gases should be carried out. In addition, feasible approaches to facilitate the desorption of toxic gas on the doped graphene surface should be investigated.(f)The pyridinic-type N-doped graphene and doped vacancy-defected graphene are good materials for use in toxic gases sensors. However, more DFT-based studies on pyridinic-type N-doped graphene and doped vacancy-defected graphene as toxic gas sensors are needed.(g)The reasons for the difference of adsorption energy obtained by using different functionals  in the calculation methods should be compared and analyzed.(h)This review shows the importance of theoretical studies for the design of novel and efficient toxic gas sensors. The theoretical results obtained up to now can help and motivate experimental groups to design novel and efficient graphene-based toxic gas sensors.This review presents a detailed and critical analysis of current progress of graphene-based toxic gas sensors using first-principle methods. Following the development of graphene as a gas sensor, it has gained considerable interest from both a theoretical and a technological viewpoint. Therefore, modifications made to graphene to improve the detection of CO, NO


Presentation at academic conferences is an important marker of research productivity. However, not all accepted abstracts progress to full publication, and there is anecdotal evidence suggesting an imbalance in sex and ethnicity amongst presenters. There is a lack of data evaluating the outcome of prize presentation sessions at academic surgical conferences in the UK. This study aimed to analyse the outcomes and demographics from presentations at prize sessions at two prestigious UK surgical conferences.This retrospective observational study compared data on all Moynihan (Association of Surgeons of Great Britain and Ireland) and Patey  prize presentations from 2000 to 2020. The primary outcome was rate of publication. Secondary outcomes included demographic differences in sex and ethnicity, publication according to prize outcome, academic affiliation, time to publication, and journal impact factor.P = 0.019).Some 442 accepted abstracts were identified over the 21-year period, with 71.0% from the Moynihan sessions and 79.3% from the Patey sessions leading to full publications, with a median time to publication of 448 days (IQR 179–859) in journals with relatively high impact factors . Of the 442 prize presenters, 85 (19.2%) were female. The majority of the presenters were White males , followed by Asian males . However, there was a continuously increasing overall trend of female presenters from 2000 to 2020 (Publication rates from the two prize sessions were high, with presenters publishing in journals with high impact factors. There, however, was a disparity in sex and ethnicity amongst presenters. Academic surgical conferences provide a forum to showcase up-to-date surgical research through education, discussion and presentation of new work. Participation at conferences is an important marker of academic productivity. Although abstracts submitted to conferences are reviewed by a panel of scientific experts prior to acceptance, they are not subject to the same rigorous peer-review process of a journal publication. Whilst desirable, full publication is not always achievable and studies have reported that less than 50% of abstracts presented at surgical conferences progress to a full publication –4.There is anecdotal evidence to suggest that imbalances in sex and ethnicity exist amongst presenters at conferences –8, whichhttps://www.asgbi.org.uk) annual congress and the Surgical Research Society  annual meeting are two of the most prestigious academic surgical conferences in the UK and Republic of Ireland. The highest ranked abstracts submitted to these annual conferences are shortlisted for presentation at the Moynihan and Patey prize sessions, respectively, with one presenter being awarded each prize.The Association of Surgeons of Great Britain and Ireland  and NamSor Version 2 diasporaBatch (https://www.namsor.com/)” application programming interfaces (API), respectively. Ethnicity was coded into four main ethnic groups  as defined by the Office for National Statistics . The sex and ethnicity APIs are automated linguistic matching software programmes used routinely to identify sex and ethnicity and limit observer bias or discrimination [Sex  and ethnmination , 12. ThrRussell Group (https://russellgroup.ac.uk/) or Ivy League (https://ivyleague.com/) universities. Outside the UK and USA, according to the Times Higher Education World University Rankings 2021 (https://www.timeshighereducation.com/), the top three universities within the Republic of Ireland  and international universities ranked within the top 100 institutions worldwide were also considered prestigious. Presentations were categorised as published if a PubMed citable original article was identified in addition to the published abstract presentation that was authored by the presenter/first author and contained data presented at the conference. Time to publication was calculated in days from the date of conference presentation to the date of first online publication. Where article publications occurred prior to conference presentation the time to publication was recorded as 0 days. Journal impact factors were derived from the 2019 Journal Citation Reports published by Clarivate in 2020 . The journals where these studies were published were subclassified into surgical, medical, basic science/translational and miscellaneous based on their attributions on Scimago Journal and country rank (SJR).Shortlisted presentations were assigned as nominees or winners depending on the outcome at each conference. Academic affiliation was categorised according to whether the presenter/first author was linked to a university and if so, whether the university was considered prestigious. Prestigious universities were classed as either 2) and Mann–Whitney U tests were used to compare categorical and continuous variables as appropriate. Univariate regression analysis with the likelihood ratio test was used to compare time trends by year and presented using 3-year rolling averages. Comparisons were made between prizes (Moynihan and Patey), winners and nominees, and by sex and ethnicity. All analyses were conducted using Stata Statistical Software v16.1  with significance set at the 95% level and P < 0.05 considered significant.Data normality was assessed by visualising distribution plots, and descriptive statistics were used to report demographics including percentage frequencies, medians and interquartile ranges (IQR). Fisher’s exact, Chi-square . Amongst the presenters, 61.1% were White, followed by 29.0% Asian, 0.5% Black, and 9.5% Other/Arab. Of the 442 presenters, 316 (71.5%) had affiliations with a prestigious university, 83 (18.8%) with a non-prestigious university, and 43 (9.7%) did not specify a university affiliation. Throughout 2000 to 2020, there was an increasing proportion of presenters having an affiliation to a prestigious university  at the ASGBI Moynihan and 304 (68.8%) at the SRS Patey prize sessions. The general characteristics of the presenters are summarised in Table P = 0.057, Table P = 0.468). The 98 full-text publications generated from the Moynihan prize group were published in journals with a median impact factor of 5.33 (IQR 3.36–5.79), whereas the 241 publications from the Patey prize group were accepted in journals with a median impact factor of 4.55 .The majority of the research output presented at both Moynihan and Patey prize sessions led to publications , with 71.0% from the Moynihan and 79.3% from the Patey prize sessions resulting in full-text publications (p < 0.001). The journals where these studies were published were surgical (38.6%), medical (23.3%), basic science/translational (33.3%) and miscellaneous (4.7%), with differences found in the journal type between the Moynihan and Patey prize sessions (P = 0.017), which mirrors the types of research presented  and 30.1% were on clinical research. Between the two prize sessions, presentations from the Patey prize sessions were more likely to be laboratory-based, 79.9% vs 47.8% . Prize winners were more likely to publish in journals with a higher impact factor  .There was a significant disparity in sex amongst the Moynihan and Patey prize presenters, with 80.8% of the overall prize presenters being male Table . BetweenP = 0.019). Throughout the 21-year period, there was a steadily increasing trend of non-White presenters across both conferences  were White men, followed by Asian men 112, 25.3%), as shown in Fig. %, as sho7.8% wereP = 0.027) Table p = 0.058).Of the abstracts presented at both prize sessions, an overwhelming majority of the senior authors were male and White (88.0%) with a significant disparity noted in female senior authors ((14.0%, This study found that the abstracts presented at the ASGBI Moynihan and SRS Patey prize sessions resulted in a high rate of publication following presentation, and with an acceptable time to publication. Nearly 71% of abstracts from the ASGBI Moynihan and 79% from the SRS Patey prize sessions led to publications. The median time from presentation to publication was 448 days, reflecting the length of the peer review and publication process . PresentInterestingly, there was significant disparity in sex across both prize sessions, with male presenters forming the majority at 80%. This was consistent in both the Moynihan and Patey prize sessions. However, there was a steadily increasing trend of female presenters, rising from 15% in 2000 to 33% in 2020. Additionally, ethnicity subgroup analysis highlighted that the majority of presenters were White, followed by Asians. Across both prize sessions over the 21-year period, only one Black female, one Black male and 16 Asian female presenters were recorded out of a total of 442 presenters. The significant sex and ethnicity differences persisted even amongst senior authors. A focus should be placed on encouraging more women and ethnic minority groups to consider academic surgery as a potential career choice early on in their training. Additionally, educating medical students on study design, statistics and the range of possibilities that a career in academia can bring is paramount to fostering interest. Finally, embedding mentorship and flexibility in the different pathways into academic surgery might encourage future aspiring trainees to consider this as a career option.A Cochrane review  of over Academic surgery is historically perceived to be one of the more difficult career options for women , 15. A rAlthough the issue of ethnic diversity in surgery has received more coverage in the USA , 24, the.Analysis of the Moynihan and Patey prize sessions at the ASGBI and SRS conferences, respectively, may not be representative of the remaining sessions at the conferences. However, the two prestigious prize sessions are important in highlighting the best of surgical research. Additionally, prestigious prize sessions provide an opportunity to showcase the breadth and excellence of surgical research across the UK and Republic of Ireland. “Gender” API and ethnicity software programmes were used for the names of presenters and senior authors and could be subject to some erroneous findings. However, both systems have been validated in multiple studies , 12. ForPrestigious prize sessions highlight important and impactful academic research in surgery. Associated publication rates were high, with prize winners publishing in journals with high impact factors. The significant disparity in sex and ethnicity of both presenters and senior authors, across both prize sessions, reflects the current state of academic surgery. More needs to be done, to address this imbalance and encourage diversity and representation across the echelons of academic surgery.


For the first time, this study investigated the extraction of bioactive substances with different polarities from immature honey pomelo fruit (IPF), a by-product of pomelo planting processing that causes resource waste and environmental pollution, using novel continuous phase transition extraction technology (CPTE). The results showed that CPTE was suitable for extracting essential oil, naringin, and pectin in sequence according to their polarities. The naringin extraction process was optimized by the response surface methodology, resulting in an extract ratio up to 99.47%. Moreover, the pectin extracted from IPF by CPTE showed better quality compared to commercial counterparts, as evidenced by lower protein and ash contents and higher white value. Together, these results suggested that CPTE could be a promising technology to improve the application value of IPF. For instance, the extracted bioactive components can be utilized as nutraceutical food ingredients. The scientific insights from this study will contribute to the development of functional food ingredients and comprehensive utilization of farming by-products.The immature honey pomelo fruit (IPF) is a huge agro-industrial by-product generated during pomelo planting. Although IPF is rich in nutrients, more than 95% of IPF is discarded annually, which causes resource waste and a serious environmental problem. Here, we report a novel continuous phase transition extraction technology (CPTE) to improve the comprehensive utilization of IPF by sequentially generating high value products and solve pollution problems related to their disposal. First, essential oil was successively extracted by CPTE at a yield of 1.12 ± 0.36%, in which 43 species were identified. Second, naringin extraction parameters were optimized using the response surface methodology (RSM), resulting in a maximum extraction rate of 99.47 ± 0.15%. Finally, pectin was extracted at a yield of 20.23 ± 0.66%, which is similar to the contents of commercial pectin. In conclusion, this study suggested that IPF was an excellent potential substrate for the production of value-added components by CPTE. Citrus grandis (L.) Osbeck), a member of the citrus family, is widely cultivated in Southeast Asia and some other Asian countries [Pomelo  and were stored at room temperature. The composition of the IPF  was analyzed according to methods adapted from previous research ,19,20,21HPLC-grade acetonitrile and hexane were purchased from Fisher Scientific . n-butane (99.99%) was obtained from Shenzheng Shenyan Gas Co., Ltd. . Galacturonic acid and the naringin standard were purchased from Aladdin Co., Ltd. . Anhydrous methanol, phosphoric acid, and petroleum ether were available from Guangzhou Chemical Reagent Factory .IPF was mainly produced in April and May each year, so the IPF samples were collected in both April and May. Two samples were washed twice with tap water, then cut in half and dried in oven at 35 °C until its weight remained constant. The dried IPF samples were stored at room temperature for further analysis.The essential oil content was determined by the Soxhlet extraction method . The pecPrior to extraction, the IPF was ground into a 40 mesh powder. The essential oil was extracted under n-butane using CPTE equipment. The extraction was performed under 0.5 MPa for 60 min at 50 °C. The solvent was concentrated at 70 °C for 70 min.The extracted essential oil was then analyzed with a gas chromatography mass spectrometer (GC-MS)  equipped with a DB-5 capillary column  following a published method . The temIn order to improve the extraction rate of naringin, all extraction experiments were performed in accordance with a central composite design (CCD) with 3 factors and 3 levels based on preliminary experiments. These factors were extraction temperature, time, and raw material granularity (RMG), respectively. The factors and independent variables are listed in The remaining IPF after essential oil extraction was used as the starting material for naringin extraction. Anhydrous methanol was used extract naringin under 0.3 MPa. The solvent was concentrated at 75 °C. The extracted naringin and the naringin remaining in IPF dregs after extraction were analysis by HPLC, using the parameters described above. The naringin extraction rate was calculated using the following equation:The remaining IPF dregs after naringin extraction were used as the starting materials for pectin extraction. Extractions were conducted under continuous deionized water (Ph = 1.5) under 0.3 MPa at 90 °C for 60 min. The solvent was concentrated by vacuum evaporation at 90 °C for 60 min. Pectin concentrate was desalinated using a small electrodialysis desalination unit . Subsequently, 2 volumes of 95% ethanol were added to the sample and incubated for 4 h under 4 °C to precipitate pectin. The pectin was separated by high speed centrifugation, then washed three times with 95% ethanol followed by freeze-drying to obtain the final product. The pectin yield was calculated using the following equation:The extracted pectin was analyzed for the determination of the content of galacturonic acid, moisture content, ash content, degree of esterification (DE), protein content, and total sugar content, according to methods adapted from previous researches ,19,20,21t-test was performed to test the mean difference between two groups, whereas analysis of variance (ANOVA) followed by Duncan’s multiple range test was used for the comparison of differences among three or more groups. p < 0.05 was used as the threshold for determining significance.All assays were performed in triplicate, and the results are expressed as the mean ± standard deviation (SD). Student’s p < 0.05). This is in agreement with previous research showing that flavonoid content is the highest during the immature period [p < 0.05). As the naringin content in April was twice as high as in May, we selected the IPF collected in April for the further study, even though the pectin content was slightly lower.In order to determine which sample should be used for further analysis, the content  of naringin and pectin of IPF produced in April and May were quantified A. We foue period . HoweverThen we determined the levels of essential oil and other components in IPF harvested in April. As shown in Essential oil, as a volatile oil, is a general term for oily liquids in plants which have an aromatic odor. Many studies have shown that pomelo peel essential oil presents numerous biological activities, including antibacterial, anti-inflammatory, as well as swelling and pain relief effects ,27. The D-limonene is a common substance in citrus essential oils and a major contributor to citrus aromas. It is considered as a marker substance in essential oils, such as pomelo, orange peel, and bergamot , and is In a previous study, a microwave irradiation treatment was conducted to extract essential oil from pomelo, and it performed better than hydrodistillation in terms of the yield of essential oil (1.88 ± 0.06% yield) . In anotThe IPF residue after essential oil extraction was used as the starting material for naringin extraction, and the naringin extraction rates obtained under different conditions are listed in p = 0.0003 < 0.001) (p > 0.05), indicating that the error of the test is small, and the non-test factor has little influence on the result. The goodness of fit (R2 = 0.9646) illustrated that the model has a high degree of fit and reflects real experimental results.ANOVA analysis revealed that the polynomial model was statistically significant (< 0.001) . The p-vThe relationship between independent variables and their effects on the extraction rate of naringin were determined using a three-dimensional response surface curve generated by modeling .We found that all parameters tested had significant effects on the extraction process. Noticeably, ANOVA analysis revealed that the extraction time is the most important factor affecting the extraction rate, followed by temperature. As the temperature rose, the extraction rate of naringin also increased, particularly at the beginning of the extraction process. In addition, our results indicated that the solubility of naringin was greatly affected by temperature. Moreover, an increased temperature could reduce the viscosity of the solvent and improve solute vapor pressure , leadingThe biological activities of naringin have been shown in many studies. In the medical and food industries, naringin is the subject of significant demand as a pharmaceutical component and food additive. The traditional extraction methods for naringin still have the disadvantages of low purity, low extraction rate, and environmental pollution. According to our analysis using Design-Expert software, the optimum conditions for maximum naringin extraction rate were 76.4 °C for 3.7 h at 39.1 mesh RMG. The predicted extraction rate was 99.32%. We then verified this result by extracting the naringin under the optimum conditions and determining the corresponding extraction rate. For our implementation, the conditions were adjusted to 75 °C for 4 h extraction time at 40 mesh RMG. The test was performed in triplicate and the actual extraction rate was 99.47 ± 0.15%, which was very close to the predicted value for the optimal conditions.Next, we compared the extracted sample and commercially available standards by HPLC. As shown in After naringin extracting, the IPF dregs remaining were used as the starting material for further pectin extraction. We found that the IPF dregs contained 44.02 ± 1.30% pectin, which was 3.12 times higher than that of the original proportion. This indicated that the extraction of essential oil and naringin from the IPF during the process led to the increase of the percentage of pectin remaining in IPF dregs, which would promote the effective extraction of pectin .After the extraction process described earlier, pectin was obtained from IPF dregs with a yield of 20.23 ± 0.66%. Data in In this study, IPF has been used to produce value-added components  via a novel CPTE technology. The naringin extraction process was optimized using RSM, and the extract ratio of naringin was up to 99.47%. The quality of extracted IPE pectin, characterized by protein content, ash content, and white values, were much better than commercial pectin. Together, these results indicate that CPTE is an efficient and eco-friendly extraction technology that provides a significant advancement in the development and utilization of IPF and offers new insights pertaining to the comprehensive utilization of this agro-industrial by-product.


To broaden the operating range of photoreceptors, mammalian rod photoresponse recovery is accelerated mainly by two calcium sensor proteins: recoverin, modulating the lifetime of activated rhodopsin, and guanylate cyclase-activating proteins (GCAPs), regulating the cGMP synthesis. We found that decreasing rod intracellular Ca2+ concentration accelerates the flash response recovery and increases the basal PDE6 activity (βdark) maximally by ~ 30% when recording local electroretinography across the rod outer segment layer from GCAPs−/− recoverin−/− mice. Our modeling shows that a similar elevation in βdark can fully explain the observed acceleration of flash response recovery in low Ca2+. Additionally, a reduction of the free Ca2+ in GCAPs−/− recoverin−/− rods shifted the inhibition constants of competitive PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) against the thermally activated and light-activated forms of PDE6 to opposite directions, indicating a complex interaction between IBMX, PDE6, and calcium. The discovered regulation of PDE6 is a previously unknown mechanism in the Ca2+-mediated modulation of rod light sensitivity.Phosphodiesterase-6 (PDE6) is a key protein in the G-protein cascade converting photon information to bioelectrical signals in vertebrate photoreceptor cells. Here, we demonstrate that PDE6 is regulated by calcium, contrary to the common view that PDE1 is the unique PDE class whose activity is modulated by intracellular Ca These molecules hydrolyze cyclic AMP and cyclic GMP, second messenger molecules involved in a vast number of signaling pathways, including gene regulation, cell proliferation and differentiation, metabolism, immune function, memory, and visual transduction3. PDEs, together with the cAMP and cGMP synthetizing cyclases, regulate the intracellular cyclic nucleotide concentrations. Therefore, the modulation of the activity of different PDEs is among the key therapeutic targets in humans and animals5.Cyclic nucleotide phosphodiesterases (PDEs) regulate numerous processes essential in health and disease12-fold range6. This requires efficient light adaptation mechanisms in various levels of visual perception, which in the photoreceptor level are mostly mediated by controlling intracellular Ca2+ concentration. Here we investigate calcium-dependent modulation of PDE6, an enzyme that hydrolyzes cGMP and mediates the conversion of photon absorption to an electrical signal in the phototransduction cascade, taking place in the outer segments of vertebrate photoreceptor cells. In rod photoreceptors, photon-activated rhodopsin sequentially activates several G-proteins, transducins. The activated transducins bind to PDE6 molecules and dislocate the inhibitory PDE6γ-subunits covering the catalytic sites. This converts PDE6 molecules from their basally active form, where the enzyme activity is set by spontaneous thermal activations, to light-activated form, which hydrolyzes cGMP at nearly diffusion-limited rate7. PDE6 activation causes a rapid drop in the cGMP concentration, leading to the closure of cyclic nucleotide-gated (CNG) channels in the rod outer segment plasma membrane, reduction of the circulating “light-sensitive” current, and hyperpolarization of the cell .Our visual system can function in different illuminations covering a 10+/Ca2+K+ (NCKX) exchangers located in the outer segment plasma membrane leads to a rapid decrease in the cytoplasmic calcium concentration. With strong light stimuli, the intracellular calcium level can drop by more than tenfold in mouse rods10. The decrease in Ca2+ activates fast light adaptation mechanisms through calcium sensor proteins and accelerates the photoresponse recovery. Guanylate cyclase-activating proteins (GCAPs) augments the cGMP synthesis by the activation of guanylate cyclase11. Recoverin controls the rhodopsin kinase affinity to rhodopsin and modulates the lifetime of light-activated rhodopsin14 and possibly light-activated PDE616. Calmodulin can modulate the affinity of cGMP to CNG channels17, but this mechanism has been regarded as negligible for mammalian rod light adaptation18. Further, we, together with others, demonstrated that in addition to GCAPs and recoverin, there exists an unknown calcium-mediated light adaptation mechanism in mouse rods16.The closure of CNG channels upon light absorption diminishes the inflow of calcium into the photoreceptor cells, while the continuing extrusion of calcium by the Na−/− recoverin−/− background. Using the cGMP clamp paradigm19 and the modeling of dim flash responses, we found that a drop in calcium concentration can cause ~ 30% increase in the basal PDE6 activity. In addition, we found that the decrease in the extracellular calcium concentration modulates the inhibition efficacy of the non-selective competitive inhibitor, 3-isobutyl-1-methylxanthine (IBMX), for PDE6, and surprisingly, this modulation shifts the inhibition constants against the light-activated and basally activated forms of PDE6 to opposite directions. This suggests a complex interaction between IBMX, PDE6 subunits, and calcium.In this study, we recorded local electroretinography across the rod outer segment layer (LERG-OS) from isolated mouse retinas with microelectrodes. We demonstrate that lowering of the extracellular calcium concentration reduces the light-sensitivity of rods by accelerating the photoresponse recovery in GCAPsThe use and handling of the animals were in accordance with the Finnish Act on the Protection of Animals Used for Scientific and Educational Purposes (497/2013) and the Government Decree on the Protection of Animals Used for Scientific and Educational Purposes (564/2013). The study was carried out in compliance with the ARRIVE guidelines.−/− and recoverin−/− mice11 kindly provided by Dr. Jeannie Chen , were used in this study. The mice were in 12/12 h dark/light cycle, and they were let to dark-adapt overnight before the experiment. The mice were euthanized by CO2 inhalation and cervical dislocation. The eyes were enucleated, and a small incision was made along the equator of the eyes. The eyes were bisected by enlarging the incision with micro scissors, and the isolated eyecups were placed to cooled nutrition solution. One eyecup was stored at + 7 °C in the nutrition solution in a light-tight container to be used later on the same day. The retina was removed from the eyecup with forceps and micro scissors under a microscope, and the whole retina was placed in the recording chamber inside a light-protective Faraday cage. All procedures were performed under dim red light.GCAPs recoverin double knockout (DKO) mice of both sexes, derived from GCAPs19. The retina was placed on a filter paper photoreceptor side upwards. Local electroretinography was recorded across the rod outer segment layer (LERG-OS). The recording electrode (tip diameter 2–5 µm) was passed into the depth of ~ 25 µm in the retina, and the reference electrode , a cell-permeant competitive PDE6 inhibitor, was added to the nutrition solution in concentrations of 5, 10, 20, and 40 µM. All chemicals were purchased from Merck Group / Sigma-Aldrich .During the experiments, the nutrition solution filled the open chamber, and the laminar flow of the solution perfused the photoreceptor side of the retina at a constant rate (~ 3 ml/min). The composition of the nutrition solution was (mM): Na2+ concentration to a very low level. In the low Ca2+ solution, the total calcium concentration was adjusted to 100 µM , and free calcium concentration of the solution was set to ~ 20 nM with 3.4 mM EGTA, calculated with the “EGTA calculator”23. The pH of this solution was adjusted to 7.5 with NaOH.The calcium-dependent mechanisms in rod photoreceptors were manipulated by decreasing the CaThe recording chamber was placed on top of a heat exchanger, the temperature of which was controlled with a water circulating heating bath . All recordings were conducted at 37 ± 1 °C. The temperature in the recording chamber was continuously monitored with a calibrated thermistor . The perfusing solution was connected to the signal ground with a 4.7 µF capacitor to reduce high-frequency noise.max = 532 nm; Lumileds, Amsterdam, Netherlands). The homogeneity of the full field stimulus beam on the retina was verified with a camera-based beam profiler . 1 ms light flashes were used to stimulate the retina. The absolute light intensity incident on the retina was measured with a calibrated photodiode . The amounts of rhodopsin photoisomerizations in rods (R*rod−1 or R*rod−1 s−1) caused by the stimuli were calculated based on the rod outer segments dimensions , the LED emission spectrum, the photodiode spectral sensitivity curve, and the pigment template24 as described in25.Light stimulation was accomplished with a LED  controlled feedback from the recorded LERG-OS voltage signal to the light source was used in the cGMP clamp procedure to keep the recorded signal constant by adjusting the background light level after the introduction of PDE inhibitor IBMX to the retina. The closed-loop light control was accomplished digitally in LabVIEW software.c = 500 Hz (8-pole Bessel filter) and afterward digitally filtered with fc = 100 Hz.Data acquisition and LED controls were handled with a data acquisition card  and custom-made LabVIEW software. The recorded DC signals  were sampled at 1000 Hz with a voltage resolution of 15 nV. The signals were first low-pass filtered with a cut-off frequency of f−/− recoverin−/− mouse flash responses was investigated in 10 isolated retinas. The responses were recorded while perfusing the isolated retina with normal Ca2+ solution (containing 1 mM Ca2+) and then with low Ca2+ solution (containing ~ 20 nM free Ca2+). In 5 experiments, the low Ca2+ solution was changed back to normal Ca2+ solution and the flash responses were recorded again. Complete response families covering the whole operation range of rods were collected in each solution for all but one retina. Additionally, in 4 experiments, response families were collected only in low Ca2+ solution . One to twenty-one responses were averaged with each flash strength depending on the response amplitude and noise level. The derived parameter values  and in low Ca2+ solution (n = 7 retinas) as described in19. The cGMP clamp experiments in normal and low Ca2+ were conducted with separate retinas. All experiments lasted less than 2.5 h. The statistical significance of the calcium-induced inhibition constant modulation against light-activated PDE6 by calcium was tested using a two-tailed unpaired t-test with unequal variances. Each retina was considered as one observation. The basal PDE6 activity and the inhibition constant against basally activated PDE6 were determined by pooling the data from each retina and calculating the mean value and the 95% confidence limits or the standard error of regression for the parameters.The inhibition constants against light-activated and basally activated PDE6, and the basal PDE6 activity with cGMP clamp were determined in normal Ca19, while the methods for determining the phosphodiesterase inhibitor inhibition constants are published in26. Here we give a summary of the utilized phototransduction models and the core ideas behind the methods.The phototransduction models used in this study and the theoretical background of cGMP clamp have been described in detail in28.In the rod photoreceptor cytoplasm without any stimulating light, the rate of cGMP synthesis by guanylate cyclase, The PDE6 activity can be decreased by introducing a phosphodiesterase inhibitor to the rod cytoplasm. A competitive PDE6 inhibitor decreases the catalytic activity of both basally activated PDE6 and light-activated PDE6 with inhibition constants mentclass2pt{minim−1 s−1)19:PDE6 inhibitors reduce the rate by which the PDE6 enzyme hydrolyzes cGMP. When a competitive inhibitor of PDE6 is introduced into the photoreceptor cell, it will reduce the rate of light-induced cGMP hydrolysis according to an equation26:The ratio of amplification constants without and in the presence of inhibitor yields a linear equation that can be used to determine the inhibition constant for PDE6 inhibitors against light-activated PDE6, −/− mice, the guanylate cyclase-activating proteins have been knocked out, and the cGMP synthesis rate by guanylate cyclase, Introduction of PDE6 inhibitor reduces the basal PDE6 activity from For a competitive PDE6 inhibitor, Eq.  gives10\8). Thus, 26:The relative change in the circulating current in rods, and thus the extracellular voltage across the rod outer segment layer, is proportional to the relative change in the intracellular cGMP concentration to the power of the Hill’s coefficient for CNG channels 8. The change in the cGMP concentration after a flash stimulus can be described by the differential equation that takes into account the synthesis of cGMP by guanylate cyclase and the hydrolysis of cGMP by both basal and light-activated PDE6 activity,r(t) depends on the relative change in the cytoplasmic cGMP concentration in the following way:−/− recoverin−/− mice40. Additionally, cell-to-cell variations are averaged out in the mass potential ERG signal, which reduces the deviation between experiments.Additionally, the LERG-OS flash response waveform mentclasspt{minimatial Eq.  numericaA), the lifetimes of activated rhodopsin α), and the Hill’s coefficient for CNG channels  play only a minor role. This approximation holds when When modeling photoresponses, the values for several free parameters need to be estimated: The amplification constant of phototransduction (t in Eq. ), while t in Eq. ) and theion, Eq.  simplifi19. The analysis presumes that the modeled response is sub-saturated and that the basal PDE6 activity is lower than 6 s−1. Here, we use Eq.  retina, in normal Ca2+ conditions  and after the extracellular calcium concentration was dropped to ~ 20 nM (red traces). The low extracellular Ca2+ is expected to drive the intracellular calcium in rods well below that during rod saturating light (near 20 nM10). Hence, the low Ca2+ treatment can be expected to drive the intracellular calcium sensor proteins out of their physiological operation range and to uncover any remaining component of calcium-mediated light adaptation, as found in16. Despite the absence of GCAPs and recoverin, the lowering of Ca2+ was still able to accelerate flash response recovery without affecting the activation phase , we compared the early phase of flash responses recorded in normal and in low Ca2+ conditions by fitting the flash response model to the first 34 ms of the response leading edge . This r2+ conditions. The −2. Figure 2+ in19, where the optimal fit to population-averaged response families was achieved with rhodopsin and PDE6 lifetimes of 28 ms and 196 ms, respectively, and with an amplification constant of 18.9 s−2 . The comparison of these analyses suggests that neither rhodopsin lifetime, PDE6 lifetime, nor phototransduction amplification constant change significantly in DKO mouse rods due to the reduction in intracellular Ca2+ concentration.Figure 2+, rhodopsin lifetime was fixed to the value determined by modeling, 2+ and in low Ca2+ solution. For 5 out of the 10 retinas, control recordings were conducted in normal Ca2+ solution also after the low Ca2+ treatment. We observed no substantial differences between the parameter values obtained before the introduction and after the washout of low Ca2+ solution.To analyze the key parameters of the dataset in normal and low Cages (Eq. ) separatmentclass2pt{minim2+ shown in Fig. 47. In DKO mouse retinas, the proteins regulating guanylate cyclase activity, GCAPs, are knocked out, and therefore, no calcium-mediated modulation of guanylate cyclase activity should remain. Hence, a potential source for the low Ca2+ induced acceleration of flash response kinetics might be the increased basal PDE6 activity. To test this hypothesis, we conducted cGMP clamp experiments with DKO mouse retinas in normal and in low Ca2+ conditions.Since the acceleration of photoresponse shut-off in low CaThe cGMP clamp experiments were conducted by recording the LERG-OS signal while illuminating the retina with closed-loop PID-controlled background light, which received its feedback from the recorded LERG-OS signal. One example of cGMP clamp runs is shown in Fig. 2+ conditions. Figure 26. The inhibition constant against light-activated PDE6, 2+ without IBMX (red traces) and with 40 µM IBMX (blue traces). Figure 2+  and in low Ca2+ (red circles). The amplification constant decreases in both solutions as predicted by Eq. (2+ but only 7.6 ± 0.5 µM in low Ca2+ (n = 7). This difference was statistically significant (t(9) = 3.52, P = 0.007, two-tailed unpaired t-test with unequal variances).In order to determine the basal PDE6 activity using the cGMP clamp paradigm, we needed to know the potency of IBMX in inhibiting PDE6 in its light-activated and basally active form both in normal and in low Caons (Eq. ). Figured by Eq. . Surprisrmax. The LERG-OS saturation amplitude before the addition of the inhibitor, rmax,control, was determined from the plateau level of LERG-OS response to a strong stimulus before the cGMP clamp procedure. The growth of the saturation amplitude was determined from the maximal increase in the LERG-OS voltage after the turn-off of the background light in the cGMP clamp  and in amp Fig. .2+  and in low Ca2+ (red dots). The downregulation of cGMP increase induced by high IBMX concentrations can be seen as the nonlinear behavior in Fig. 2+ and 49.0 µM  in low Ca2+. As described earlier, the 2+ solution  and 5.7 ± 0.1 s−1 in low Ca2+ solution .cGMP clamp experiments were conducted on 9 retinas in normal Can in Eq. . The Hil2+ induced desensitization of responses and acceleration of response shut-off kinetics, we modeled dim flash responses with a phototransduction model . Figureentclass1pt{minima2+-dependent regulation of phototransduction in mammalian rods. Our results support two major conclusions: (1) The basal PDE6 activity is not constant as previously thought but depends on calcium concentration. Low levels of calcium, mimicking the behavior of intracellular calcium in strong lights, increase the basal PDE6 activity. (2) Calcium modulates the PDE6 inhibition constant of the non-specific PDE inhibitor IBMX, revealing an interaction between Ca2+ and PDE6.In this study, we investigated the role of PDE6 in Ca16. The phototransduction machinery of mammalian rods has two well-known negative feedback mechanisms mediated by the calcium sensor proteins GCAPs and recoverin13. Here we show that calcium can modulate rod flash responses even in GCAPs−/− recoverin−/− background. In our experimental approach, we lowered the extracellular free Ca2+ to around 20 nM. The Na+/Ca2+K+ exchange mechanism in the outer segment plasma membrane drives the intracellular Ca2+ concentration down towards the equilibrium value 2+ conditions, the recovery of GCAPs−/− recoverin−/− mouse flash responses accelerated, leading to a decrease both in the fractional amplitudes and integration times of dim flash responses.In a previous study, we demonstrated that fast light adaptation in mouse rods is completely mediated by calcium ions and that at least one unknown negative feedback mechanism must be involved in mouse rod phototransduction2+ might be caused by modulation of several mechanisms: shortening of the lifetime of light-activated rhodopsin or PDE6, dynamic enhancement of guanylate cyclase activity, or increase in the basal PDE6 activity8. Since we made our investigations on the GCAPs−/− recoverin−/− mice, the acceleration of response deactivation cannot be mediated by calcium-dependent modulation of guanylate cyclase by GCAPs11, lifetime of rhodopsin14, or PDE616 by recoverin. Additionally, switching between normal and low Ca2+ did not affect the dominant time constant of saturated flash response recovery, reflecting the lifetime of light-activated PDE644. As Ca2+ did not modulate the dominant time constant, 2+-induced acceleration of flash response kinetics was apparent in the recovery phase, while the early phase remained unchanged. Hence, we conclude that low Ca2+ did not affect phototransduction amplification. A third known calcium sensor protein, calmodulin, modulates the affinity of cGMP to CNG-channels in rods17. The 50. Lowering the extracellular Ca2+ to ~ 20 nM should decrease the intracellular Ca2+ well below this value and drive calmodulin modulation out of its functional range, and thus, dynamic calmodulin-mediated cGMP affinity modulation during flash responses is unlikely in low Ca2+ solution. The low Ca2+-induced steady-state change in the affinity of cGMP to CNG-channels should manifest as modulation of absolute response amplitudes but would affect neither flash response amplitudes normalized with the saturation amplitude . Rod phototransduction, with PDE6 as a key effector enzyme, is probably the best-known G-protein mediated signaling cascade in vertebrates, and PDE6 function and inhibition can be investigated electrophysiologically in live photoreceptors.Cyclic nucleotide phosphodiesterases have a central role in a multitude of cellular signaling mechanisms, and therefore, specific inhibition of different PDE isoforms would have a great functional impact and therapeutic utility  and that this interaction would be modified by Ca2+ without significant effect on the catalytic activity of PDE6. Antagonistic interaction between the γ-subunit and IBMX would manifest as apparently larger inhibition constants determined in the presence of γ-subunits compared to those determined in the absence of γ-subunits, i.e. in trypsin-activated PDE6 . In order to explain the low Ca2+-induced modulation of KI values, the ability of the γ-subunit to hinder PDE6 inhibition by IBMX should weaken in the light-activated state and intensify in the basally activated state in low Ca2+. However, clarification of this mechanism requires further studies.Most phosphodiesterases share highly conserved amino acid sequences in their catalytic domains, suggesting high similarity in the hydrolytic sites between different PDEs78. If the basal PDE6 activity was caused by the subunit containing the high-affinity transducin-binding site (without or with the binding of transducin) and the light-activated cGMP hydrolytic activity was produced by the subunit with the low-affinity transducin-binding site, the inhibition of these subunits by IBMX could differ. This might explain the different KI against basally and light-activated PDE6 in low Ca2+.Our finding may also relate to a recent result suggesting that substantial PDE6 activation requires the binding of two transducins to one PDE6 dimer, the binding of the first transducin to the high-affinity transducin-binding site causing negligible cGMP hydrolytic activity while binding of the second transducin to the low-affinity transducin-binding site brings about the full PDE6 activity


The COVID-19 pandemic provides a major opportunity to study fishing effort dynamics and to assess the response of the industry to standard and remedial actions. Knowing a fishing fleet’s capacity to compensate for effort reduction  allows differentiating governmental regulations by fleet, i.e., imposing stronger restrictions on the more resilient and weaker restrictions on the less resilient. In the present research, the response of the main fishing fleets of the Adriatic Sea to fishing hour reduction from 2015 to 2020 was measured. Fleet activity per gear type was inferred from monthly Automatic Identification System data. Pattern recognition techniques were applied to study the fishing effort trends and barycentres by gear. The beneficial effects of the lockdowns on Adriatic endangered, threatened and protected (ETP) species were also estimated. Finally, fleet effort series were examined through a stock assessment model to demonstrate that every Adriatic fishing fleet generally behaves like a stock subject to significant stress, which was particularly highlighted by the pandemic. Our findings lend support to the notion that the Adriatic fleets can be compared to predators with medium-high resilience and a generally strong impact on ETP species. In fact, on the one hand lockdowns introduced practical difficulties on board of vessels to applying social distances and, on the other hand, they caused a collapse of demand for seafood due to the closure of fish markets and restaurants and the reduction of direct sales, which in turn contributed to decrease fishing activities2. Both large and small-scale fisheries were affected, in different ways depending on the area4. Initially, the global reduction of fishing activity was due to the asynchronicity of the lockdown start time and fishing seasonality. Now, about two years into the pandemic, the time when this period began is becoming less and less relevant.Large-scale collectors of vessel data  have estimated that the 2020 March–April lockdowns, imposed by several governments, reduced fishing activities by about 10% in the world and by about 50% in Europe7 . To do this, fishing locations should be either extracted from the AIS records directly (when this information is available) or estimated from the sequences of coordinates embedded in these data (as in the case of the present experiment). Statistical and machine learning models are commonly used for these estimation tasksstress factors that reduce its abundance. Such factors include (Table 10). A spatial analysis was also performed, to identify the response of some fleets to the high stress level experienced due to the 2020 lockdowns and the sanitary restrictions. The fleets selected for this analysis were the large-scale fleets of the Adriatic Sea, the area with the highest regional bottom trawling footprint in the world11. In the Adriatic Sea, most of the fishing activity is carried out by four fleets targeting small pelagic and demersal fish species—pelagic pair trawlers, purse seiners, bottom otter trawlers and beam (or Rapido12) trawlers—which currently account for more than 70% of revenues13  species during the 2020 lockdownsstress factors, including the COVID-19 restrictions. The main traits analysed in each fleet were the speed of effort recovery (intrinsic growth rate) and the maximum number of fishing hours exceeding which regulations need to be introduced (carrying capacity).Our analysis measured specific fleet traits that depend on fishing method  and market capacity. These traits emerged from the similarity of the fleet effort dynamics to stock rebuilding dynamics. They summarise fleet responses to the The scenario emerging from our analysis is that the main Adriatic Sea fleets behave like apex predators towards their target stocks and that they show from medium to high resilience to effort reduction. Understanding and applying these findings has the potential to improve fishery and environmental management, minimise economic damage, maximise stock recovery and protect biodiversity. Notably, national supervisory bodies currently pursue stock rebuilding by mostly acting on the monthly fishing hours irrespective of fleet fishing method. Since different fleets suffer differently from impacts and have different recovery times, imposing stronger restrictions  on the more resilient and weaker restrictions on the less resilient would improve fishery management.: Did the lockdowns influence fishing activities in the Adriatic Sea and did these mirror broader patterns in the Italian seas?RQ1: Which gear types were most affected by the 2020 lockdowns in the Adriatic Sea?RQ2: Did the lockdowns change the potential impact of fisheries on ETP species?RQ3: Can an Adriatic Sea fleet’s response to effort reduction be modelled as that of an apex-predator stock with measurable resilience and carrying capacity?RQ4Altogether, our paper aims at answering the following specific research questions: This section describes methodological steps. Primary data sources location, volume, and properties are detailed in Section 23 (GFW). The complete census of Automatic Identification System (AIS) transmission data from the Astra Paging collector24 was used to conduct a detailed analysis of fishing activity in the Adriatic basin. The AIS data contain high reporting frequency information (every 5 minutes per vessel) on vessel position, speed over the ground, course and rate of turn. We used the data of the EU Fleet Register25 (EUFR) to make a consistency check and a quantitative evaluation of AIS data quality . Algorithmic details are given in Section We extracted coarse-scale patterns (0.5RQ1). We further explored the fishing activity patterns in the Adriatic Sea by calculating the barycentres of fishing activity of each gear over time using the AIS dataset .We used the GWF dataset to perform a large-scale analysis of fishing patterns at 0.526 (IUCN) information on the ETP species living at those sites allowed establishing the overlap between fishing activities and the species at risk, thus highlighting how their potential impact on such species changed during the pandemic . IUCN data are authoritative and reliable to assess whether an observed species is an ETP species28 and have been already used in pattern analyses, e.g., to assess potential habitat change due to climate change29. The IUCN assessments are carried out by experts and specialists who have sufficient knowledge and data of the species, and are members of authoritative scientific commissions30. The United Nations General Assembly has granted observer status to IUCN and thus we considered it a reliable data source. The number of ETP species inhabiting high-effort cells was used to identify the locations where the potential impact was most severe. Several ETP species living in small areas in the Adriatic Sea are subject to intense fishing effort and high traffic. In particular, the total fishing hours can be as many as 20  and the unexploited population size that the environment can naturally sustain . These two parameters allow estimating the maximum sustainable yield (MSY), i.e., the maximum amount of a stock’s biomass that can be taken without compromising the stocks’ ability to reconstitute it. A time series of catch statistics is a sequence of yields from the available stock biomass. Current data-limited stock assessment models can estimate the available annual stock biomass from the annual catch time series. They commonly rely on stock production models that correlate catch with biomass through r and k  stock-assessment model10 uses an abundance index catchabilityq, a coefficient that reflects fishery efficiency . AMSY uses the Schaefer stock dynamic equation to relate MSY, r and kq (the carrying capacity multiplied by catchability). If t is a monthly index), then r is its monthly growth and kq is the stock abundance in the absence of fishing pressure.The term  Section -SI. The  Section -SI. AMSYRQ4. We assumed each fleet  to be a stock, whose total monthly fishing hours were a measure of abundance stress factors that contribute to the reduction of the fleet’s fishing effort. If r represents the recovery speed of the fleet’s fishing effort, i.e., its response (including fishers’ reaction) to the combination of external factors that reduced its monthly fishing effort. Consequently, kq is the maximum number of hours the fleet can spend fishing before near saturation of its resources, i.e., the target stocks and the market. Beyond this number the fishery is no longer sustainable and regulations need to be introduced. According to this view, the catch MSY the maximum sustainable reduction of fishing hours that would not compromise the sustainability of the fishery with respect to its economic and natural resources . The time series kq and do not exceed the MSR.We used AMSY to study the Adriatic fleets’ response to regulations over time, particularly during 2020, and answer research question r and kq for the Adriatic fleets that employ OTB, PTM, TBB, and PS gears, and unfolded the differences between their growth rates and sustainable hours to produce novel information for fishery management. In this context, the reduction of fishing hours due to the pandemic was crucial to highlight data differences corresponding to the responses of the different fleets in 2020.In summary, we estimated This section describes the results of our analyses. Further numerical details that support this analysis are reported in Section excellent kappa agreement31 between AIS-estimated and EUFR-licensed gears over the five gear categories demonstrated the reliability of our classification algorithm. Additionally, EUFR data were well represented by the AIS data and vice versa, reflecting a reasonably good quality and consistency of the AIS data used in our analyses. Evaluation details are given in Sections The GFW data of March–April 2020 and 2019 indicate that, in the Italian seas, fishing during the lockdowns involved a wider area and only As a result of the fishing cell processing of the GFW data, although we detected effort reduction in all Italian seas, in March–April 2020 some locations were still characterised by a high fishing effort with thousands of hours Fig. -SI. ThisWe performed a spatio-temporal analysis to study the different fleets’ contributions to the overall fishing footprint, which demonstrated heterogeneous patterns. Indeed, PS fishery was little affected by the lockdown restrictions. The TBB fishery was strongly impacted by the EU lockdowns and made a slow recovery. The PTM fleet was moderately affected by the lockdowns and made a swift recovery in the whole Adriatic. The OTB fleet returned to pre-lockdown levels only at the end of 2020 and was strongly impacted by the lockdowns. Further details are given in Section t}∘ Fig. . The 202In February and March–May 2020 the high-effort fishing areas and those inhabited by ETP species showed a strong overlap. An evident effect of the 2020 EU lockdowns, detected both in the GFW and the AIS datasets, was a scattering and overall reduction of the overlap during this period Figs. b, 5. Anar-kq solutions  of the Schaefer dynamic for each fleet. This indicates that the fleet monthly effort time series fit well the Schaefer dynamics10, lending support to the hypothesis that fleet behaviour the can be assimilated to that of a fish stock. The fleets’ 32. One major finding was the synchronicity of PTM, OTB, and TBB fleet seasonality and the independent behaviour of the PS fleet. A detailed representation of the lockdown period and most of 2020 , produced several 32. In May, at the end of the 2020 lockdowns, the overall fishing effort reverted to pre-lockdown levels, albeit not for all fleets. In fact, the purse seine fleet was not severely affected, apart from a change in the general barycentre pattern and the pelagic pair trawling fleet was moderately impacted and experienced slightly longer barycentre shifts than the previous years. The beam trawling and bottom otter trawling fleets were the most affected; indeed, the lockdowns strongly reduced their effort in a period that was already less productive and changed their fishing range extension and patterns. These fleets operate in specific areas and they did not adjust their strategies and patterns. Their effort recovered slowly and reverted to pre-lockdown levels only at the end of 2020. The 2020 lockdowns were generally beneficial for ETP species, because fishing activities at the locations where their density was higher decreased substantially in March and April but also throughout 2020. Only the purse seine fleet continued an intense activity at the locations at higher risk of impact. The overlap of pelagic pair trawlers with ETP species shrank briefly and returned to pre-lockdown levels in May. The risk of impact from beam trawlers and bottom otter trawlers showed a considerable reduction. This finding predicts a potential population size increment of ETP species, to be checked against the next abundance surveys when available.In this paper, we have unfolded several aspects of the influence of the 2020 lockdowns on the Adriatic Sea fisheries, which were found to reflect the coarse-scale effects detected in all the Italian seas. Our findings indicate that the lockdowns slowed down a fast and generalised spread and increase of fishing activity that could severely affect the already heavily exploited Adriatic Sea stocks33. Notably, the bluefin tuna farming industry has been flourishing in the Croatian islands since 200034 and its production volume doubled between 2014 and 201835. In this industry, part of the feed consists of local small pelagic fish36, whose demand was probably stable also during the lockdowns. As regards vessel presence in the Adriatic, the slight reduction of registered EU vessels in 2020 with respect to 2019  indicates inertia in recovering fishing hours from one month to the next, due to the fact that the effects of the stress factors spanned several months. These effects include (i) the progressive relaxation of restrictions, (ii) the long rebuilding time and seasonal availability of target stocks, and (iii) the entry-exit scheme of fishing vessels. A fleet’s intrinsic growth rate summarises its capacity and effectiveness to adapt to these effects, which also depend on the fleet size, whereas the kq trait summarises the capacity of the resources exploited by the fleet to sustain its fishing activity and estimates a maximum sustainable number of fishing hours for each fleet; this information has the potential to be directly used in management strategies. The kq trait principally correlates with target stock availability but also embeds the capacity of the supply chain to manage the catch. If the supply chain were unable to receive and sell the catch, the fishing activity would need to be reduced. This effect is typically negligible in the current greedy global economic system, but the pandemic has magnified its importance. It is also worth noting that a side effect of inadequate supply chain capacity is a higher level of food waste37. Thus, balancing supply and demand in the seafood chain by including market constrains into fishery management would also enhance environmental sustainability.Our data indicate that the 2020 lockdowns occurred during a seasonal fishing effort reduction involving all fleets except the purse seiners. The charts illustrating the relative effort reduction Fig. a,b indic38. The lockdowns have affected both the supply chain (via market closure) and vessel activity (via the sanitary restrictions and the forced stop). Since these aspects are correlated, they have generated a vicious circle that has acted as a complex additional stress factor on the Adriatic fleets. The r and kq traits calculated in the present study account for reconfiguration of the supply chain change and the fishery response to it in an implicit manner, as they summarise a fleet’s response to all the stress factors occurring in a specific area and fishing context. A comprehensive management strategy would need to measure the efficiency and response of a supply chain to the loss of fishing hours and resources in an explicit way and could integrate this information with our findings42.The pandemic has highlighted the very low resilience of the seafood system to market closure and its differentiated impact across countries and social levelsIn summary, the COVID-19 pandemic has offered the opportunity to observe and quantify some key intrinsic traits of Adriatic Sea fisheries. Our findings, have the potential to help decision-makers produce monthly harvesting control rules to (i) cut the number of fishing hours per fleet (which should be well below the maximum sustainable reduction); (ii) take into consideration fleet resilience by imposing stronger restrictions on fast-recovering fleets (pelagic pair trawlers and purse seiners) and less harsh restrictions on slow-recovering fleets (bottom otter and beam trawlers), and (iii) optimise harvesting control rules based on fishing seasonality, fleet size, and fishery volumes. These control rules should also take into account the spatial overlap between areas of intense fishing activity and the geographic distribution of ETP species if they are to enhance ecosystem sustainability. Harvesting control rules conceived in this way would go a considerable way towards fishery sustainability viewed in terms of ecosystem protection, control of the economic pressure, stock rebuilding, lower food waste and sustained resource availability.r and kq traits and (iii) produce helpful information to management organisations. The proposed machine learning model for gear classification is flexible enough to be used with other AIS data and areas. The type of spatial analysis described can be directly re-used with other areas to extract spatio-temporal trends and patterns and evaluate the overlap between fishing activity and ETP species presence. It is also general enough to process the activity and gear classifications estimated by other algorithms, e.g., the GFW global scale data, and quickly produce estimations for new areas.The presented approach can be used for other marine areas and fleets beyond the Adriatic, e.g., to all Mediterranean Sea fleets, because the type of analysis presented is mostly independent of the specific application case. For example, the AMSY model can process new fleet data to (i) verify that these follow Schaefer dynamics, (ii) estimate Supplementary Information.


The objective of this study was to explore whether soluble programmed death ligand 1 (sPD-L1) is a potential prognostic biomarker in patients with non-small cell lung cancer (NSCLC).A comprehensive search of electronic databases was carried out. Original studies with inclusion of sPD-L1, progression-free survival, and overall survival in NSCLC were eligible. The primary endpoints were overall survival and progression-free survival. Hazard ratios (HRs) and 95% confidence intervals (CIs) were applied for data analysis.P < 0.001) and progression-free survival . A subgroup analysis revealed that high levels of sPD-L1 were correlated with poor overall survival in patients treated with immunotherapy .Eight studies involving 710 patients with NSCLC were included in the analysis. A pooled data analysis revealed that high levels of sPD-L1 were correlated with poorer overall survival (HR = 2.34; 95% CI = 1.82–3.00; This pooled analysis of published data suggests that sPD-L1 may serve as a readily available biomarker for survival in NSCLC patients treated with ICI based treatment. Prospective studies with well-designed standard assessment methods should be conducted to validate the prognostic role of sPD-L1 in NSCLC.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021283177. Lung carcinoma is the most aggressive cancer worldwide . ApproxiPD-L1 is expressed in different types of cancer ; membranPD-L1 can be divided into membrane-bound PD-L1 and sPD-L1 , and theThis systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) . The proThe inclusion criteria were as follows: (a) patients were with NSCLC, (b) the sPD-L1 levels were analyzed in serum or plasma, (c) the study was reported in a full research publication in English, (d) the relationship with human survival outcomes  was determined, and (e) the number of included cases was not less than 20. The exclusion criteria were as follows : (a) comTwo independent reviewers (GL and ZZ) extracted information by reviewing the eligible studies. The extracted data were as follows: the first author, the year of publication, the country where the study originated from, cancer type, sample size, age, study design, cutoff value of sPD-L1, Eastern Cooperative Oncology Group performance status, smoking status, lines of ICI treatment, tissue PD-L1 tumor proportion score, follow-up time, survival outcomes with regard to high/low sPD-L1 levels, and the relationship between clinicopathologic features and sPD-L1 concentrations.The quality of the included studies was evaluated according to the Newcastle–Ottawa Quality Assessment Scale (NOS) . The scoI2  and 95% confidence interval (CI). Review Manage (5.4 version) (Cochrane Centre) was used. Furthermore, random-effect model was applied. Heterogeneity was evaluated using I2 , 33. SubI2 . STATA sA total of 2,382 studies were retrieved from the database search. The study selection process is illustrated in P < 0.001). Moreover, there was no significant heterogeneity among the studies  (P < 0.001), with no heterogeneity    using thP < 0.001). Moreover, there was no significant heterogeneity among the studies  (P < 0.001) (Six studies (496 patients) reported the outcomes of patients receiving ICIs , 43–46.  = 0.43) , 43–46 uA sensitivity analysis was performed to determine the stability of the findings . The anaMoreover, in patients with NSCLC receiving ICIs, the characteristic of patients from the included studies were provided in P = 0.40)   Figure 4P < 0.001) and PFS  in patients with NSCLC. Moreover, the level of sPD-L1 may be considered as a prognostic marker for patients with NSCLC who received immunotherapy—poorer . This was consistent with previous studies by Khan et al. (P < 0.001). Okuma et al. (vs. 7 ng/ml). He et al. . This is consistent with a previous study showing that the sPD-L1 levels can be a prognostic marker in patients with melanoma receiving ICIs (Six studies (496 patients) , 43–46 ring ICIs . Moreoveing ICIs .An open question that remains to be answered is what makes sPD-L1 a suitable prognostic marker for cancer. The biology rationale is not clear to us. A potential hypothesis is that the inhibition of sPD-L1 can result in a similar function to other checkpoint inhibitors, thereby achieving a checkpoint inhibitor effect. Some studies reported that the inhibition of sPD-L1 restricting tumor growth showed a similar mechanism to that of anti-PD-L1 in mAb-injected mice , 55. A fThe relationship between the PD-L1 expression of the tissue and the level of sPD-L1 is not fully understood. As described in the study of Mazzachi et al. , there wEGFR mutations. However, sPD-L1 concentrations were associated with live metastasis (P = 0.015). Mazzachi et al.  suggested no significant difference in survival outcomes   . This isThis study had some limitations. First, some of the included studies were retrospective studies, and there may have been a selection bias or publication bias, as positive results are more easily published in journals compared with negative results. Second, in regard to heterogeneity, all analyses used the random-effect model . In the In conclusion, our meta-analysis indicates that sPD-L1 has a prognostic role in patients with NSCLC. Moreover, low sPD-L1 levels may be a prognostic factor in patients receiving immunotherapy. A high expression of sPD-L1 was correlated significantly with worse OS and PFS. Prospective studies with well-designed and standard assessment methods  should bThe original contributions presented in the study are included in the article/GL and ZZ contributed to the study design and manuscript drafting. GL and F-MK contributed to editing and proving. All authors contributed to the article and approved the submitted version.This study was supported by the Natural Science Foundation of Shenzhen (no. JCYJ20170307095828424). China NSF8187110989 as well as Shenzhen Science and Technology Commission (Grant No: KQTD20180411185028798).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study focused on spatial and temporal variation in carabid beetle communities and environmental factors, and the association between them at spatial and temporal scales, based on data collected over 5 years from nine study sites on three mountains. We found that carabid beetle communities exhibited significant temporal variation, and that the patterns of temporal variation differ between mountains. Temporal variation in communities was suggested to occur in response to variations in the local climate. Our results suggest that temporal surveys of communities and climates at local scales are important for predicting temporal changes in communities. Such investigations are expected to reveal an additional fraction of variation in communities, and to provide information on previously overlooked underlying processes, especially with respect to global community patterns and changes in wider spatial scales.Spatial and temporal variation in ecological environments may result in spatial and temporal variation in communities. Temporal studies of biodiversity are essential for forecasting future changes in community structure and ecosystem function. Therefore, determining the mechanisms that drive temporal change in communities remains an important and interesting challenge in ecology. We quantified spatial and temporal variations in carabid beetle communities and site-specific environmental factors for 5 years at nine study sites on three mountains in the Baekdudaegan Mountain Range, Korea. Carabid beetle communities exhibited significant temporal variation, which was larger than spatial variations between and within mountains. Environmental factors mostly varied between sites within mountains. Community variation was only weakly associated with environmental factors at wide scales, i.e., between sites on three mountains, but was strongly associated at narrow spatial scales, i.e., between sites within one mountain. Our results indicate that temporal variation in communities occurs in response to variations in the local climate, and that the patterns of temporal variation differ between mountains. Thus, temporal surveys of insect communities and climates at local scales are important for predicting temporal changes in the communities. Spatial and temporal variation in ecological environments may reflect variability in spatial and temporal variation in communities . SpatialCarabid beetles are found in most terrestrial habitats, and their distribution often correlates with environmental variables ,13. ThesThe aims of this study were (1) to quantify the relative contribution of spatial and temporal variations in communities and environmental factors to total variation within an area, and (2) to examine the association between spatial and temporal variation in communities with environmental factors at different spatial and temporal scales. To this end, we examined two levels of spatial variation (between and within mountains) and one level of temporal variation (between years), to quantify and characterize the relative contribution of temporal and spatial variations to total variation in the beetle communities. We performed variance partitioning of measures of community composition and site-specific environmental factors, and examined covariation between them. We found large temporal variation in carabid communities but not in environments, and variation in community was not explained by variation in environmental factors at a wide geographical scale . Based on these results, we suggest that site-specific temporal variation in beetle communities and environmental factors should receive more attention when predicting temporal changes in beetle communities.Quercus spp.) provide the dominant cover at all study sites, with Pinus densiflora (Korean red pine), Pseudostellaria palibiniana, and Dioscorea quinqueloba present as understory vegetation. The study sites vary in litter layer development and understory vegetation  in the Baekdudaegan Mountain Range, Korea. The mountains are separated from each other by 55–84 km. The Baekdudaegan Mountain Range is a watershed crestline 1400–1500 km long, running through most of the length of the Korean Peninsula, from Mt. Paektusan in the north to Mt. Jirisan in the south . Oak tregetation .Changes in environmental variables at each study site were measured using HOBO data loggers . These loggers recorded air temperature, air humidity, light intensity, and soil temperature hourly from June 2010 to September 2015. Each logger was attached to a tree trunk 120 cm above the ground. Soil temperature was measured 5 cm underground. The data were downloaded directly from each logger every month. This system cannot measure precipitation; generally, this is difficult to measure hourly, although it is an important environmental parameter in predicting carabid beetle communities. Air humidity (relative humidity) was expected to be closely related to precipitation and was used as a surrogate.Pterostichus, three Synuchus, and one Dolichus; these species were regarded as morphospecies. The data of two samplings in a year were pooled, and constituted the sample for that site, month, and year, resulting in 45 data points across the nine sites over 5 years.To quantify community composition and its variation at each study site, carabid beetles were collected twice a year (July and August) for 5 consecutive years 2011–2015) using pitfall traps  containing an attractant (powder of silkworm pupae) [ using piThe sampling scheme (200 traps for 24 h) differs from that used in other studies ,16, whicTo summarize the environmental variables, we first calculated daily means from the data collected at hourly intervals at each site. Then, 12 environmental factors were calculated for each collection: summer mean air temperature, winter mean air temperature, summer maximum mean air temperature, winter highest mean air temperature, summer minimum mean air temperature, winter lowest mean air temperature, summer mean air humidity, winter mean air humidity, summer mean illumination (insolation), winter mean illumination, summer mean soil temperature, and winter mean soil temperature. Winter variables were calculated from the data collected in the December to February period before the first month of sampling. Summer variables were calculated from the data collected in the period from June to the date of the second sampling in the year. Summer and winter environments were mainly examined because, for carabid beetles in this region, summer conditions may be important for reproduction and population growth, and winter conditions may be important for survival during hibernation . These eTo quantify spatial and temporal variation in community composition, Sørensen’s indices were calculated between three sites on one mountain, based on data pooled for 5 years , and between sites on three mountains, based on data pooled for three sites and 5 years . Sørensen’s indices from 5 years of data obtained from within each site  and from each mountain  were also calculated, the latter of which was based on the data pooled for three sites on one mountain. In addition, Shannon’s diversity indices  for the To summarize and visualize variation in community composition in relation to environmental factors, we performed canonical correspondence analysis (CCA) based on 45 data points of carabid beetle community composition and the 12 environmental factors. CCA models based on the data from a single mountain were reported in our previous studies ,18, and To quantify the relative contributions of spatial and temporal variation to total variation in environmental factors and communities, we constructed generalized linear models (GLMs) with each of the 12 environmental factors and five measures of community composition  as dependent variables, and year, mountain, and site (nested within mountain) as independent variables. Identity link and normal distribution were assumed for metric data, and log-link and Poisson distributions were used for count data. Variation partitioning ,25 was uEnvironmental factors varied between mountains and between sites within a mountain. At all study sites during the study period, the monthly means of air temperature, soil temperature, and air humidity were highest in August and lowest in December or January .Mountain, site, and year contributed to variations in environmental factors . Summer Synuchus , Carabus (2357), and Pterostichus (1567) were dominant, followed by Pristosia (555).We collected 7607 individual carabid beetles, belonging to 33 species in 13 genera . At the The 45 community data points were segregated along two CCA axes . VariatiThe patterns of temporal change in community composition differed between mountains b–d. CommDiversity indices revealed that temporal variation in communities over 5 years was as large as spatial variation between sites within mountains and between mountains : temporaMountain, site, and year were responsible for the variation in the carabid communities . DiversiWe quantified spatial and temporal variation in carabid beetle communities and their background environments in the Baekdudaegan mountains in Korea over a 5-year continuous survey period. We found that temporal variation in communities was predominant and mostly larger than spatial variation between sites on a mountain and between mountains. For environmental factors, spatial variation between sites within a mountain represented the largest proportion of explained variation, followed by temporal variation, which accounted for the second largest proportion of explained variation. These results indicate that temporal change constitutes a significant component of variation in communities and environments but affects these components to different degrees. Because the geographical scale of this study was relatively narrow (Baekdudaegan Mountain Range in the Korean Peninsula), and the temporal scale was limited to 5 years, surveys performed at wider geographical and longer temporal scales may change the relative contributions of spatial and temporal variation, which may warrant further study.Which factor (or factors) was responsible for the observed temporal variation in carabid beetle communities? Insufficient survey size can randomly bias the observed composition of a community  and may introduce apparent temporal fluctuation of communities. However, our results indicate this may not be the case. When community samples were pooled for 5 years within sites, α-diversity increased and spatial β-diversity across sites within a mountain decreased, whereas this tendency was not observed in temporal β-diversity . TemporaA change in the local environment may cause temporal variation in communities. Our previous studies indicated that spatial and temporal variation in carabid beetle communities occurred in response to changes in local environmental factors ,18. HoweIt is also possible that environmental factors not investigated in this study may influence carabid beetle communities. The phenology of carabid beetles can be related to their life cycles: spring breeder species reproduce in the spring and overwinter as adults, and the autumn breeder species reproduce in autumn and overwinter as larvae . Thus, pEcological processes other than responses to environmental factors may also result in temporal variation in communities. Ecological and reproductive interaction between species can influence the population dynamics of species. Resource competition between species exerts a negative effect by affecting access to resources ,30. SpecOur results indicate that carabid beetle communities exhibit significant temporal variation. This highlights the importance of conducting temporal surveys of communities at local scales. Such investigations are expected to reveal an additional fraction of variation in communities, and to provide information on the underlying processes that have been overlooked, especially in studies of global community patterns and changes at wider spatial scales. Our results also suggest that detailed observation of local climates is necessary for predicting temporal change in communities. Such predictions may prove difficult if data are obtained at wider geographical scales. When responses of local communities to local environments differ between sites, statistical modeling of community responses to environmental factors may be difficult at wider geographical scales. This study calls for novel schemes for the statistical modeling of community changes that takes into account temporal variation.