Environmental impacts from ecotourism and outdoor recreation activities on terrestrial and aquatic ecosystems are well-reported in the literature, but less is known regarding the impacts of outdoor environmental education activities. Student activity during stream classes may cause substrate disruption and localized impacts on stream macroinvertebrates. We hypothesized that student activity would negatively impact macroinvertebrate community structure in three habitat types  within a site regularly used for stream classes while no impact from student activity would occur in the same three habitat types within an unused site. We addressed the hypothesis by sampling macroinvertebrates monthly for one year in the riffles, runs, and pools at the class site and the unused site within a fourth order stream in central Ohio. The results indicated reduced macroinvertebrate abundance and richness in the riffle at the class site during periods with student activity and no differences between site types during periods without student activity. No impacts of stream classes on macroinvertebrate communities were observed in runs or pools. The results suggest that environmental education organizations should avoid repetitive use of the same site for their stream classes to avoid reductions of macroinvertebrate abundance and taxa richness that can impact the students’ educational experience. Despite the benefits of ecotourism to host communities, tourists, and the local flora and fauna, researchers and industry professionals have observed environmental problems stemming from mass tourism2. Negative impacts of ecotourism at high-use sites have been documented globally across a wide range of terrestrial and aquatic ecosystems4. Notable examples in marine ecosystems include negative impacts on: (1) coral reefs in Brazil and the Philippines due to diving activities7; (2) shorebirds in Australia as a result of beachside driving8; and (3) bottlenose dolphin populations in New Zealand due to dolphin watching excursions9.The rising popularity of ecotourism has resulted in millions of tourists visiting natural areas across the globe annually10. Water-based recreational activities have been shown to cause physical habitat degradation, chemical pollution, and impacts to aquatic plants, vertebrates, and macroinvertebrates11. Specifically, macroinvertebrates are of interest to scientists because they are effective bioindicators of anthropogenic impacts and they play an integral role in the aquatic food web as detrivores and prey12. Experimental disturbances, instream recreational activities, and recreational stream crossings have been documented to negatively impact the population and community structure of freshwater macroinvertebrates. Experiments in Australia and Brazil that simulated instream trampling by hikers and bathers revealed changes in macroinvertebrate taxa composition and reductions in taxa richness and abundance14. Caires et al.15 observed an increase in drifting behavior in Baetis mayflies in a Utah river in response to instream hiking, but found no effect of instream hiking on macroinvertebrate abundance, taxa richness, and the abundance of the most common taxa. Wright & Li16 observed reductions in densities of caddisfly (Dicosmoecus gilvipes) larvae in Oregon due to recreational activities including streamside camping, fishing, goldpanning, swimming, and tubing. Laing17 noted decreased invertebrate abundance, insect abundance, Ephemeroptera Plecoptera Trichoptera (EPT) abundance, and taxa richness within tributaries of a scenic river in Nebraska due to canoers hiking through the water.Similarly, the impacts of recreational activities have been documented in numerous freshwater ecosystems20. Kidd et al.18 observed decreased water quality, taxa richness, percent EPT (excluding Hydropsychidae), and percent clingers and increased percentages of Chironomidae and Oligochaeta below stream crossings in Virginia. Conversely, Heth et al.19 found that taxa richness, EPT richness, Shannon diversity, and biotic index scores increased downstream of crossings in a Missouri stream in the summer and that no differences occurred in the winter. Holmquist et al.20 also observed increased invertebrate abundance, species richness, dominance, percent tolerant fauna, Chironomidae abundance, and Hilsenhoff biotic index scores below stream crossings in California streams in the late summer.Impacts on aquatic macroinvertebrates from recreational stream crossings have also been documented10. In contrast, environmental education activities are generally more structured and conducted as a regular component of the school curriculum via in-class lab activities, day-long field trips to nature centers and parks, and multi-day stays at resident outdoor education (ROE) centers. Such parks, nature centers, and ROE centers may host thousands of students over the course of an academic year22. Classes that focus on stream biomonitoring and/or involve stream exploration have become a popular component in many of these educational programs23. Aquatic activities and classes are typically conducted in the same body of water and often at the same site due to ease of access, safety, and/or tradition22. Consequently, organizations that serve large clienteles may unknowingly and inadvertently cause environmental disturbance in the same aquatic environment their activities and classes are intended to promote. However, empirical data to support this concern are presently limited.While effects of ecotourism and outdoor recreation on the environment have been well-studied, environmental education programs can impose similar impacts but have been far less studied. Ecotourism and outdoor recreation encompass activities in which participants engage by choice during their leisure time24 investigated the effects of ROE stream classes on aquatic macroinvertebrates in agricultural streams in central Ohio. Bossley & Smiley21 found that within a fourth order stream student instream activity did not alter short-term temporal trends in macroinvertebrate community structure in a riffle regularly used for ROE stream classes. Additionally, differences in macroinvertebrate community structure between the class riffle and a riffle unused by ROE stream classes were observed21. Bossley & Smiley22 also observed substrate rearrangement by students in ROE stream classes and found greater rock movement in the class riffle compared to an upstream riffle unaffected by student activity. Also, abundance, richness, EPT abundance, and clinger abundance were greater on rocks in the unused riffle than in the class riffle at the end of the six-week study22. These initial findings22 documenting spatial differences in macroinvertebrate community structure suggest the potential long-term impacts of student instream activity within this fourth order stream. In contrast, an investigation of macroinvertebrate community response to simulated substrate disturbance in riffles within headwater tributaries unused by ROE stream classes in central Ohio revealed no effect of trampling on macroinvertebrates24.As part of a series of four studies, Bossley & Smiley24 evaluating the impact of recreational activities and ROE classes in streams were mostly short term studies that encompassed one to three seasons. Additionally, previous studies evaluating the impacts of recreational activities and ROE classes in streams focused exclusively on riffles24 or conducted reach level assessments involving composited information from multiple habitat types18 and did not assess whether the anthropogenic impact on macroinvertebrates differed among habitat types. Habitat types in streams  differ in water depth, velocity, and substrate types27. It has also been well documented that macroinvertebrate abundance, diversity, and taxa composition also differ among habitat types within streams in North America, South America, Europe, Japan, Australia, and New Zealand33. Macroinvertebrates exhibit distinct preferences for substrate size and stability, and habitat types having greater substrate heterogeneity often exhibit greater macroinvertebrate biodiversity34. These documented differences in habitat characteristics and macroinvertebrate community structure among habitat types in streams suggest that the effects of disturbance on macroinvertebrates may differ among habitat types36.Within this manuscript we present the results of the fourth study that consists of a year-long investigation of trends in macroinvertebrate community structure across three habitat types  and four seasons between the class site and the unused site. Previous studies39 documented that macroinvertebrate responses to flooding and land use change differ among stream habitat types. Macroinvertebrate density, taxa richness, and taxa composition exhibited habitat specific responses to a spate in a second order stream in Japan30. Macroinvertebrate density decreased during the spate and then increased after the spate in riffles, but did not change during or after the spate in pools, glides, and backwaters30. Taxa richness decreased in pools after the spate, but did not change in riffles, glides, or backwaters30. Taxa composition changed in riffles and backwaters after the spate, but not in glides and pools30. Decreases in macroinvertebrate abundance after floods were the least in physically stable bedrock habitats and greatest in pools and runs in a river in Switzerland37. Percent EPT and percent Baetidae, Hydropyschidae, and Heptageniidae decreased in riffles after a spate, while percent EPT and percent Chironomidae increased in pools after a spate in a third order stream in Ohio38. Abundance and diversity of macroinvertebrates in riffles exhibited stronger relationships with physical and chemical variables associated with land use change than the abundance and diversity in pools and bank habitats in sand gravel streams in Georgia39. EPT taxa richness and Shannon diversity index in riffles were more sensitive indicators of land use change in riffles than in pools in headwater streams in Brazil33. However, whether the impacts of recreation and outdoor education activities on macroinvertebrates differ among riffles, runs, and pools is unknown.Previous studies23. Based on these observations, we hypothesized that ROE stream classes will negatively impact macroinvertebrate community structure within the riffle, run, and pool habitat types within the stream site regularly used by ROE stream classes during time periods when stream classes are in session. However, no impact will occur during time periods when stream classes are not in session. We addressed the hypothesis by conducting a year-long study that involved monthly sampling of aquatic macroinvertebrates in riffles, runs, and pools within the class site and an unused site within a fourth order stream. Additionally, to address the hypothesis we ensured that sampling was conducted during periods with student activity and periods without student activity.Prior observations of stream classes at Heartland Outdoor School (Heartland) documented that student instream activity involves physical disruption of the stream substrate throughout a stretch of stream encompassing riffle, run, and pool habitatsDuring the 12 month study, 21,911 macroinvertebrates and 56 taxa were captured within riffles  single factor effects of site type and student activity period within runs and pools were evident, these effects do not indicate the influence of ROE stream classes. The effect of site type on the response variable is the same in both student activity periods and the effect of student activity period on the response variable is the same in both site types22 documenting the impact of ROE classes on macroinvertebrates in riffles. Notably, the previous results22 occurred within field studies conducted between April and May of 2014, which is after the sampling for this study was completed. This observation is important, because it suggests a multi-year impact of ROE stream classes on macroinvertebrates within the riffle of the class site in 2013 and 2014. The results within Alum Creek differ from an experimental simulation of student-induced trampling conducted in June of 2015 in the adjacent headwater tributaries. A one-time disturbance simulating the effect of trampling caused by ROE stream classes did not influence macroinvertebrate community structure in riffles of the tributaries24. These contrasting results are likely due to a combination of differences in the extent of site usage by ROE stream classes, substrate size, and stream size between the fourth order Alum Creek sitesthis study,22 and the first and second order tributary sites24. The contrasting results suggest that the ROE stream classes are more likely to affect macroinvertebrates in riffles that are more frequently visited by students, predominantly composed of cobble and other large substrate types, and located in fourth order streams. Conversely, ROE stream classes are less likely to affect macroinvertebrates in riffles that are less frequently visited by students, composed predominantly of gravel, and located in first or second order streams. Future research examining the influence of educational activities in streams needs to determine if the effect differs among streams with different substrate sizes and among different stream sizes.The findings of decreased macroinvertebrate abundance and taxa richness within riffles as a result of ROE stream classes concurs with results from previous studies17 that documented a reduction in macroinvertebrate abundance and taxa richness in riffles in response to recreational activities. Laing17 observed that macroinvertebrate abundance and taxa richness were reduced in riffles within high use tributaries compared to riffles of lesser used tributaries of the Niobrara Scenic River in Nebraska. However, these reductions were confined to summer months when the riffles were subjected to high usage by canoers, kayakers, and tubers while no difference between stream types was evident during periods of low usage in spring and early autumn17. Hardiman and Burgin13 found that within a riffle in a fourth order upland canyon stream in Australia macroinvertebrate abundance and taxa richness were lower in experimentally trampled quadrats than untrampled quadrats immediately after experimental trampling was conducted. However, macroinvertebrate abundance and taxa richness recovered within 15 days after trampling within the experimentally trampled quadrats. Similarly, Holmquist et al.20 documented downstream impacts of recreational stream crossings and observed an increase in macroinvertebrate abundance and taxa richness below stream crossings compared to above the crossings in second orders streams in the Yosemite National Park, California. Heth et al.19 also documented increased taxa richness and EPT taxa richness downstream of recreational stream crossings in a Missouri stream in the summer. Our results from riffles and those of Laing17, Hardiman and Burgin13, Holmquist et al.20, and Heth et al.19 suggest that recreational and educational activities on macroinvertebrates in streams may have both localized and downstream impacts that influence macroinvertebrate abundance and taxa richness in different ways.These results documenting an impact of ROE stream classes on macroinvertebrates in riffles are similar to the results of other studies24 that concluded that natural disturbances, among site differences, and temporal differences had a greater influence on macroinvertebrate community structure than recreational activities or ROE stream classes. Wright and Li16 documented that larval caddisfly density was lower in riffle and glide habitats in high use recreational sites compared to low use recreational sites in an Oregon stream before an 85 year event flood then did not differ between high and low use site types after the flood. Heth et al.19 found that macroinvertebrate communities within riffles in a Missouri River exhibited larger changes among sites than changes that occurred above and below stream crossings. Results from riffles in headwater tributaries within the Heartland property adjacent to Alum Creek24 documented that macroinvertebrate community structure was not influenced by experimental substrate disturbance, but differed among riffles within a stream and among time periods. The results from runs and pools combined with those of Wright and Li16, Heth et al.19, and Bossley and Smiley24 suggest that future research needs to identify the disturbance threshold that results in recreational and educational activities having a greater effect on macroinvertebrate communities than natural disturbances and/or spatio-temporal differences.The results from runs and pools are similar to those studies23. Stream classes achieve this goal by giving students an opportunity to capture and study the unique organisms that can be found in streams. The greater the variety and abundance of macroinvertebrates that live within a study reach, the greater the opportunity for discovery. Although some parks that regularly host recreationists have adopted the precautionary principle 13, we have observed that ROE organizations generally do not follow this practice. Based on the results of this study, we recommend that environmental education organizations maintain multiple sites within a stream designated for educational programming and alternate use between sites to avoid a reduction in macroinvertebrate abundance and taxa richness that might result from overuse of a single site.In conclusion, the effects of student activity resulting from outdoor education stream classes on macroinvertebrate abundance and taxa richness were observed in riffles but not on other response variables or in other habitat types  during this year long study. These findings carry important implications for environmental education organizations that conduct stream classes, especially for those who serve large clienteles and seasonally or annually conduct numerous visits to the same stream site. Conducted over the long-term, heavy visitation at a stream site can negatively impact the stream biota, which can compromise the educational quality of the experience for participants. An underlying goal of many environmental education programs and outdoor education excursions is to provide students with a positive association with nature42 with moderate sinuosity (=1.3), no constrictions, minimal to no canopy cover, and substrate composed primarily of coarse gravel (D50 = 38% coarse gravel substrate). The class site experiences more substrate disturbance and movement than the unused site due to the activity of ROE stream classes22. The unused site  is a 61 m reach that served as the control and has never been used for stream classes by Heartland or other ROE organizations. The unused site is located approximately 1.2 km upstream of the class site. The unused site is also characterized by forested riparian habitat with riparian widths >30.5 m and an agricultural field bordering the riparian habitat on the right bank. Alum Creek at the unused site is a C-4 riffle-pool stream42 with moderate sinuosity (=1.3), no constrictions, minimal to no canopy cover, and substrate composed primarily of coarse gravel (D50 = 36% coarse gravel substrate) with some large boulders.The study was conducted at two sites within the upper Alum Creek, which is a fourth-order stream located in Morrow County in central Ohio, USA. The class site  is a 61 m reach that has been regularly used since 2010 for stream classes taught by Heartland, which is an ROE center that provides overnight, multi-day, hands-on nature and environmental science programs for K–12 students. Heartland’s stream classes include measurements of select water chemistry variables, collection of live aquatic macroinvertebrates by dipnet, kick seine, and rock-picking, and on-site identification of captured organisms. During the collection of macroinvertebrates, students intentionally disturb the stream substrate by plowing into the gravel with their feet or flipping rocks over with their hands or feet. This process dislodges macroinvertebrates so they can be caught in nets. Students may also temporarily remove individual rocks from the water to pick off macroinvertebrates by hand. Macroinvertebrates captured by the students are released after identification. The class site is characterized by forested riparian habitat with riparian widths >30.5 m and bordered by an agricultural field on the right bank. Alum Creek at the class site is a C-4 riffle-pool stream3/s with an annual average daily discharge of 2.4 m3/s. Mean daily discharge on sampling days ranged from 0.12 to 3.9 m3/s with an average of 0.8 m3/s, which confirmed our sampling was conducted under base flow conditions.During the study water temperature, conductivity, total dissolved solids, turbidity, and nitrate concentrations were similar between the two sites Table . The pH 44. The experimental design is a modification of the BACI design in that we sampled a control site (unused site) and a treatment site (class site) before and after two time periods with student activity during which stream classes were regularly conducted in the class site. The first time period with student activity extended from mid-April to early June 2013 and the second time period with student activity occurred from late September through early November 2013. Approximately 3500 students participated in 166 stream classes at the class site during the study. Specifically, 89 stream classes were conducted in the first student activity period and 77 stream classes were held in the second student activity period. Thus, these two time periods are characterized by repetitive pulse disturbances. Three time periods with no student activity from ROE stream classes (recovery periods) also occurred before and after both student activity time periods. The first recovery period occurred from February through mid-April 2013. The second recovery period occurred from early June through late September 2013 and the third recovery period occurred from early November 2013 through January 2014.The study was conducted from February 7, 2013 to January 4, 2014 and used a modified BACI (Before-After-Control-Impact) designMacroinvertebrate sampling was conducted during the first week of each month from February 2013 to January 2014. Monthly macroinvertebrate sampling was conducted at the downstream class site first and then the upstream unused site. Three samples were collected each month from each of the three habitat types  in the class site and the unused site. Each month we collected three Surber samples  from randomly selected locations in the single riffle at each site. The substrate within the frame of the Surber sampler was disturbed and mixed with a hand trowel to a depth of 10 cm or to the underlying bedrock for 30 seconds.45. Specifically, the lower run was sampled in February 2013 and all other even-numbered months  throughout the study, while the upper run was sampled in March 2013 and all other odd-numbered months  throughout the study. Substrate mixing as part of the Surber sampling in runs was conducted in the same manner as that used for the Surber sampling in riffles.Each site included one run above the riffle and one run below the riffle. Thus, three Surber samples were collected from one run in each site during each month with sampling alternating between the lower and upper runs each month. Alternate sampling between lower and upper runs avoided the potential for compounding effects of investigator-induced disturbance on top of the student-induced disturbance45. The lower pools were sampled in February 2013 and subsequent even-numbered months, while the upper pools were sampled in March 2013 and subsequent odd-numbered months. Macroinvertebrates from pools were sampled with a stove pipe sampler  because the Surber sampler is not suitable for collecting macroinvertebrates within the deeper and slower flowing water typically found in pool habitats. Substrate within the stovepipe sampler was disturbed and mixed with a hand trowel to a depth of 10 cm or to the bedrock layer for 30 seconds. Following substrate disturbance the water column within the stovepipe sampler was strained by dipping a 500 μm hand net into the stovepipe sampler and dragging it through the water for 10 seconds. This straining process was conducted three times to maximize the likelihood of collecting all macroinvertebrates dislodged during the 30-second disturbance.In addition to runs, pools and areas of slow-moving water along the stream edge  were also located upstream and downstream of the riffle in each site. Three samples were collected from the pools in each site during each month. Alternate sampling of the lower and upper pools each month avoided the potential for investigator-induced disturbanceet al.46 and Voshell47. All macroinvertebrates, except Turbellaria, Collembola, Hirudinea, Acariformes, and Amphipoda were identified to family level using Merritt et al.46 and Voshell47. Taxa not identified to family level were identified using Merritt et al.46 and Voshell47 as follows: Turbellaria (class), Collembola (subclass), Hirudinea (subclass), Acariformes (super order), and Amphipoda (order). Organisms too small or damaged to identify to the specified taxonomic resolution stated above were excluded from statistical analyses. This taxonomic resolution was chosen because our initial analyses24 and others evaluating the impacts of recreational activities18 indicated that it is sufficient for the evaluation of the impacts of ROE stream classes.Contents of each sample were rinsed in a bucket of water, poured through a 500 μm hand net, transferred into a plastic bag, and preserved with 100% ethanol. The Surber net and hand net were also inspected for any clinging specimens, which were hand-picked and added to the bag prior to taking the next sample. In the laboratory, each sample was transferred into a plastic container and preserved in 100% ethanol for long-term storage until identification could be completed. No subsampling was conducted such that all macroinvertebrates from each sample were hand-picked using a 50x Wild Heerbrugg dissection microscope and stored in vials containing 95% ethanol. All specimens from each sample were later identified to class, subclass, super order, and family using Merritt 48; 4) evenness 49; 5) percent EPT ; 6) percent Chironomidae ; 7) Clinger taxa richness (number of clinger taxa in each sample); 8) Clinger abundance (number of clingers captured in each sample); 9) Trichoptera taxa richness (number of Trichoptera families in each sample); and 10) Trichoptera abundance (number of Trichoptera captured in each sample). The habit guild  of each insect taxa was compiled with family level information from Poff et al.50, Vieira et al.51, and Merritt et al.46 (Table 52 using the Sorensen (Bray Curtis) distance matrix and 500 iterations.Ten univariate community response variables were calculated for each sample from each habitat type  at each site during each month. Response variables included: 1) macroinvertebrate abundance (number of macroinvertebrates captured in each sample); 2) taxa richness (number of taxa represented in each sample); 3) Shannon diversity index41.We used linear mixed effect model analysis with site type  and student activity period (time periods with and without student activity) as fixed effects to determine if macroinvertebrate community response variables differed among site types and student activity periods. Linear mixed effect model analyses were conducted because they enable us to address the effect of pseudoreplication that occurs as a result of repeatedly sampling the same sites through time by incorporating a random effect into the model. The random effect used in each linear mixed effect model was that which was identified as the best random effect Tables –4 from p53. The Tukey Test 54 was used to determine differences among means if the linear mixed effect model analyses indicated that a significant effect occurred. Prior to conducting linear mixed effect model analyses we examined normal q-q plots created with the qqPlot function in the car package55, conducted the Shapiro-Wilk normality test using the shapiro.test function56, and conducted the Levene’s test for homogeneity of variance with the leveneTest function in the car package55 to determine if the response variables met the assumptions of normality and equal variance. Response variables that did not meet the assumptions were either log transformed or arcsine square root transformed prior to linear mixed effect model analyses (Tables 56 and a significance level of 0.05.Linear mixed effect model analyses and AIC tests were conducted with the lme function in the nlme packages Tables –4. UnivaSupplementary Information Tables A1-A6


PNPLA3 I148M variant and TM6SF2 E167K variant are recognized as the major genetic modifiers of nonalcoholic fatty liver disease (NAFLD). The present study sought to evaluate the potential additive effect of the two variants on the risk of NAFLD in Qingdao Han Population, China.PNPLA3 I148M variant and TM6SF2 E167K variant in a cohort of 512 unrelated NAFLD patients and 451 healthy controls by sequencing and polymerase chain reaction analysis. In addition, serum lipid profiles and liver enzymes were determined by standard clinical laboratory methods.We genotyped PNPLA3 148 locus G allele and 6.69% for TM6SF2 167 locus T allele. The PNPLA3 I148M variant was significantly associated with the risk of NAFLD in an additive model . And, our data suggested a strong link between the TM6SF2 E167K variant and the risk of NAFLD in a dominant model . In addition, the increasing of the number of risk alleles were associated with the risk of NAFLD .The minor allele frequencies were 45.48% for I148M and E167K variants in a manner of an additive effect could improve risk prediction for NAFLD in a Qingdao Han Population cohort.Combining the Register.gov: ChiCTR1800015426.Chinese Clinical Trial  Nonalcoholic fatty liver disease (NAFLD) is identified as a metabolic stress injury of the liver, as well as a burgeoning public health issue/economic burden with a high global prevalence of up to ~ 25% , 2. The PNPLA3 I148M variant and TM6SF2 E167K variant, are recognized as the major genetic modifiers of NAFLD [PNPLA3 I148M and TM6SF2 E167K variants may have an additive effect in the regulation of lipid metabolism [Notably, two ‘star gene’ variants, of NAFLD . More reof NAFLD . Interestabolism –14. HoweThe present study was conducted in accordance with the principles of the World Medical Association Declaration of Helsinki. All the subjects have signed written informed consent according to the study protocol approved by the Ethics Committee of Qingdao Municipal Hospital before participation .We recruited a total of 512 unrelated Qingdao Han NAFLD patients determined by liver ultrasonography (a fairly reliable and accurate noninvasive method with sensitivity of 84.8% and specificity of 93.6% ) and 451Blood samples were collected after 12-h fast for serological assays and genetic analysis. Serum concentration of albumin (ALB), fasting plasma glucose (FPG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), alkaline phosphatase (ALP), triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL), uric acid (UA) were obtained using standard methods in the Central Laboratory of Qingdao Municipal Hospital.PNPLA3 I148M variant and TM6SF2 E167K variant was performed by polymerase chain reaction (PCR) with the following primers. Primers for PNPLA3 I148M and TM6SF2 E167K were: 5′- AACTTCTCTCTCCTTTGCTTTCACA -3′ (forward), 5′- GGAGGGATAAGGCCACTGTAGA -3′(reverse); 5′- TGTCTCAGAACAAACAAACAAACAGA -3′ (forward), 5′- GTAGGGGATGGTGAGGAAGAAG -3′ (reverse). The PCR amplification profile was conducted as follows: 95 °C for 10 min, 40 cycles before denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min and elongation 30 s at 70 °C. The PNPLA3 and TM6SF2 genotypes were detected by ABI 3730XL  and calculated by Gene Mapper 4.1 software.Genomic DNA Purification Kit  was used to extract genomic DNA according to the manufacturer’s protocol from the noncoagulated blood samples. The genotyping of t test or paired samples t test. The Hardy-Weinberg equilibrium was measured using the χ2 test. Genotype and allele frequencies between healthy controls and NAFLD patients were analyzed by chi-square test. The DNA distributions between the two groups were determined by χ2 test and Fisher’s exact test where appropriate. The association between the PNPLA3 I148M and/or TM6SF2 E167K variants and NAFLD was evaluated by the odds ratio (OR) with 95% confidence interval (CI) and performed by logistic regression analysis. In addition, an additive model  for PLPLA3 gene and a dominant model  for TM6SF2 gene were assumed to assessed the potential additive effect of the two variants. Statistical significance was defined as P-value less than 0.05.SPSS 20.0 statistical software  was performed for statistical analysis. The baseline characteristics between healthy controls and NAFLD patients were showed as mean ± standard deviation, and the differences were examined using student’s P < 0.05).Demographic and clinical characteristics of healthy controls and NAFLD patients were presented in Table PNPLA3 I148M and TM6SF2 E167K were both in Hardy-Weinberg equilibrium in both two groups . The minor allele frequencies of the two single-nucleotide polymorphisms (SNPs) in the whole subjects were 45.48% for PNPLA3 I148M and 6.69% for TM6SF2 E167K, respectively. As listed in Table PNPLA3 I148M and TM6SF2 E167K reached significance . Moreover, Table PNPLA3 I148M variant was significantly associated with the risk of NAFLD in an additive model . Interestingly, it suggested a strong link between the TM6SF2 E167K variant and the risk of NAFLD in a dominant model .The genotypic distribution of PNPLA3 I148M had higher BMI, serum levels of ALT, AST, GGT, ALP, TG, TC, LDL and UA . The serum level of HDL did not reached significance (P = 0.967). However, only serum concentrations of some lipid profiles  showed inversely strong links between carriers of the T allele of TM6SF2 E167K and the noncarriers. The T allele of TM6SF2 E167K was associated with lower levels of serum TG and TC .Furthermore, Table PNPLA3 148 locus, by coding the genotypes 0, 1 and 2 for CC, CG and GG; for TM6SF2 167 locus, by coding the genotypes 0 and 1 for CC and CT + TT, respectively). The group with 0 risk allele was regarded as the control group. Excitingly, the prevalence of NAFLD increased following with the increase of the number of risk alleles  showed an additive effect of the two variants, compared to individuals with 0 risk allele . Furthermore, our pilot study [PNPLA3 I148M and TM6SF2 E167K variants may increase hepatic lipid content by upregulating the expression of sterol regulatory element-binding transcription factor 1c and fatty acid synthase. However, to the best of our knowledge, all other previous studies have not reported the underlying molecular mechanism of this potential additive effect. These encouraging findings provide the potential perspective in new NAFLD classification and the probability of offering a new important tool for risk prediction of NAFLD in the coming years.NAFLD ranked as the most concerned chronic liver disease in the coming decades worldwide, and, nonalcoholic steatohepatitis is recognized as the second leading indication for liver transplantation in the USA –19. Notatabolism . More retabolism  of obeselleagues  demonstr studies , 22. Morot study  performePNPLA3 I148M and TM6SF2 E167K variants and serum concentrations of lipid profiles. Several studies showed inconsistent metabolic associations of NAFLD risk alleles. For instance, some studies demonstrated a strong link between TM6SF2 E167K variant and lower plasma levels of TC, TG and LDL [PNPLA3 I148M variant and plasma concentrations of lipid profiles was also inconsistent. [PNPLA3 I148M had higher serum levels of TG, TC and LDL . And, the serum concentrations of some lipid profiles  showed inversely strong links between carriers of the T allele of TM6SF2 E167K and the noncarriers. The T allele of TM6SF2 E167K was associated with lower concentrations of serum levels of TG and TC . These different findings may inasmuch as the population ethnicity, diagnostic method of NAFLD and demographic and clinical characteristics. Therefore, the relationship between the two ‘star gene’ variants and clinical features of NAFLD population needs further investigation.We additionally investigated the relations between  and LDL –26, but  and LDL , 27, 28.sistent. –31. Our This study has some limitations. First one is the perform of ultrasonography to determine NAFLD, instead of magnetic resonance imaging and/or liver biopsy. Notably, liver ultrasonography cannot provide reliable quantitative data . In addiPNPLA3 I148M and TM6SF2 E167K variants and the risk of NAFLD in a Qingdao Han Population cohort. More importantly, combining the I148M and E167K variants could improve risk prediction for NAFLD in a manner of an additive effect. This study may provide the potential perspective in new NAFLD classification and the identification of high risk NAFLD populations in the future.In conclusion, we confirmed the significant association between both the


The in vivo effects of Fab-6HD5 pre-administration were maintained over a long period of time for at least 10 days. Using flow cytometry analysis, we also found that Fab-6HD5 did not recognize the IgE Cε3 domain containing specific binding sites for FcεRIα. Furthermore, deletion-mapping studies revealed that Fab-6HD5 recognized conformational epitopes on the Cε2 domain of IgE. Given that the Cε2 domain plays a key role in stabilizing the interaction of IgE with FcRIα, our results suggest that the specific binding of Fab-6HD5 to the Cε2 domain prevents allergic reactions through destabilizing the preformed IgE-FcεRIα complex on rat mast cells. Although the present study was performed using animal models, these findings support the idea that a certain antibody directed against IgE CH domains may contribute to preventing allergic diseases through interacting with IgE-FcεRIα complex.Immunoglobulin E (IgE) plays a central role in the pathogenesis of Type I hypersensitivity through interaction with a high-affinity receptor (FcεRIα). For therapeutic applications, substantial attention has been focused recently on the blockade of the IgE interaction with FcεRIα. While exploring better options for preventing allergic diseases, we found that the Fab fragment of the rat anti-murine IgE antibody (Fab-6HD5) strongly inhibited passive cutaneous anaphylaxis (PCA)  In general, quality of life is often impaired in patients with asthma and allergic rhinitis. Immunoglobulin E (IgE), which was originally discovered in 1966 by Ishizaka et al.3, plays a pivotal role in allergic diseases, such as hay fever, food allergies, atopic dermatitis, asthma and anaphylaxis6. IgE binds to a high-affinity receptor (FcεRIα) on the surface of mast cells and basophils7. Cross-linking of IgE bound to FcεRIα with multivalent allergens results in the release of histamine and other chemical mediators that act on the surrounding tissues and the most common symptoms of Type I hypersensitivity10. Given that histamine plays a key role in inflammation, the use of antagonists targeting the histamine H1 and H4 receptor has been shown to be effective for the treatment of allergic diseases12. Allergen-specific immunotherapy through restoring immune tolerance to allergens has also been a widely used therapeutic approach13. Among potential treatments, anti-IgE therapy, which involves the neutralization of IgE by an anti-IgE antibody, appears to be the most promising strategy to treat allergic diseases to date. Indeed, omalizumab is the most widely used anti-IgE antibody drug18, and it leads to rapid and dramatic falls in free serum IgE levels as well as FcεRIα expression in mast cells20. Its clinical effects have been demonstrated in allergic diseases, such as asthma and chronic urticaria24. Because omalizumab consists of an intact, whole IgG molecule, its use in clinical trials showed several adverse effects, including two cases of anaphylaxis26. Undigested anti-IgE receptors and its F(ab’)2 induce a significant histamine release from mast cells, whereas the binding of Fab’ monomer fragments with receptors fails to do so27, which suggests that the Fab fragment of IgE reduces the risk of anaphylaxis. To analyze the anaphylactic reactions elicited by IgE antibodies, we previously produced rat monoclonal antibodies that react with murine IgE. In the present study, we focused on one of these antibodies (6HD5) and analyzed its mechanism of action for preventing allergic reactions.Allergic diseases, including asthma, are the most common chronic diseases, and their prevalence has increased worldwide in recent decadesin vivo effects on IgE-mediated anaphylactic reactions using a passive cutaneous anaphylaxis (PCA) assay. First, we injected serial dilutions of anti-dinitrophenyl (DNP) IgE (SPE-7)28 intradermally into rats. Twenty-four hours later, several dilutions of anti-IgE antibodies  were injected into the same sites. Following an extravasation assay with Evans blue and DNP-BSA, the results revealed that a minimal amount of Fab-6HD5 or 6HD5 (1.25 μg/ml) could inhibit the PCA reactions  was sufficient to completely inhibit the PCA reactions.To explore all avenues for preventing allergic diseases using anti-IgE therapy, we focused on a Fab fragment of a monoclonal anti-IgE antibody (Fab-6HD5) and investigated its To gain further insights into the role of Fab-6HD5 in allergic reactions, we next investigated how long the inhibitory effects of Fab-6HD5 on PCA reactions lasted. In the first group, SPE-7 was injected intradermally into rats on day 0. Then, serial dilutions of Fab-6HD5 were injected into the same sites on day 1. Two hours later, Evans blue and DNP-BSA were injected intravenously for the challenge. In the second group, an Evans blue extravasation assay was performed on day 10. The results demonstrated that an inhibition of PCA reactions was observed at a concentration of Fab-6HD5 ranging from 0.63 μg/ml in the day-1 group and 0.32 μg/ml in the day-10 group  activation. For this purpose, rat basophilic leukemia cells (RBL/2H3) were incubated overnight with DNP-IgE (SPE-7). Following sensitization, cells were incubated with serial dilutions of highly purified Fab-6HD5 for 2 hours at 37 °C. After washing the cells to remove excess IgE and Fab-6HD5, the cells were then stimulated with DNP-BSA to trigger degranulation. After stimulation, Syk activity and the percentage of β-hexosaminidase released were measured as described in the Methods section. Consistent with the results obtained using the in vivo PCA assay, our results demonstrated that Fab-6HD5 inhibits Syk activity and β-hexosaminidase release from RBL/2H3 cells in a dose-dependent manner in vitro . After washing, the cells were incubated with Fab-6HD5, Fab-HMK-12 or anti-Ig light chain (λ) antibodies, followed by PE-goat anti-rat IgG, and the samples were then subjected to FACS analysis. The results shown in Fig. + 6HD5+ and SPE-7+ λ + cells were 91.3% and 85.9%, respectively, whereas SPE-7+ HMK-12+ cells were 64.6%. Similar results were also obtained for another IgE isotype clone 142a (anti-TNP IgE), i.e., SPE-7+ 6HD5+ (86.3%), SPE-7+ κ + (95.1%) and SPE-7+ HMK-12+ (54.9%) cells. These results indicate that Fab-6HD5 interacts with preformed IgE-FcεRIα complex on the surface of mast cells.According to recent studies, several anti-IgE Fab fragments inhibit IgE-mediated serotonin release from mast cells+ 6HD5+ and SPE-7+ λ + cells were 90.2% and 92.3%, respectively, whereas SPE-7+ HMK-12+ cells were 4.05%. Likewise, similar results were obtained for 142a, i.e., SPE-7+ 6HD5+ (97.7%), SPE-7+ κ + (88.8%) and SPE-7+ HMK-12+ cells (1.09%). Taken together, these results suggest that Fab-6HD5 is not directed against the IgE Cε3 domain, which is involved in binding to FcεRIα34.To confirm these results, competition experiments were performed by premixing SPE-7 with Fab-6HD5, Fab-HMK-12, or anti-Ig light chain (λ) antibodies before incubation with RBL-2H3 cells   and binds to FcεRIα on mast cells and eosinophils with extraordinarily high affinityε2) Fig. . These mε2) Fig. . Under rTo further narrow down the binding epitopes on the Cε2 domain, we prepared several GST fusion molecules with Cε2 fragments Fig. . Western36. These findings support our previous observations that indicate that some anti-IgE antibodies, including 6HD5, can remove an IgE molecule already bound to FcεRIα on the surface of mast cells or basophilic leukemia cells and inhibit IgE-mediated systemic or local anaphylactic reactions37.It had been thought that the Cε2 domain is not involved in the interaction between IgE and FcεRIα. However, deletion of the Cε2 domain from the IgE H chain constant region increased the rate of dissociation of IgE from the receptor, which suggests that the IgE Cε2 domain plays a pivotal role in stabilizing the interaction of IgE with FcεRIαin vivo, as well as Syk activity and β-hexosaminidase release from rat basophilic cells in vitro. Flow cytometry analysis and deletion-mapping studies revealed that specific binding of Fab-6HD5 to the IgE Cε2 domain prevents allergic reactions over a long period of time through interacting with a preformed IgE-FcεRIα complex on the surface of rat mast cells. Given that the Cε2 domain plays a key role in stabilizing the interaction of IgE with FcεRIα, our findings suggest that pre-administration of a certain antibody directed against IgE CH domains may provide better options for preventing Type I hypersensitivity reactions.We have demonstrated that a small amount of anti-IgE antibody Fab-6HD5 strongly inhibits PCA reactions 2) at 37 °C.Female Sprague-Dawley rats were purchased from SLC Inc., Japan. The animals were maintained under specific pathogen-free (SPF) conditions at the animal facility of Juntendo University. To perform the experiments under the same conditions but without bias, all SPF animals were allowed to acclimate for 7 days before they were used in experiments. All animal studies were performed according to the National Institute of Health Guidelines for Care and Use of Laboratory Animals. The experimental protocol was approved by the Animal Experimentation Committee of Juntendo University . The rat basophilic leukemia cells (RBL-2H3) were generous gifts from Dr. C. Ra . RHL-2H3 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% FCS and maintained in a humidified incubator  or anti-TNP-IgE (142a). Twenty-four hours later, 1 μg/ml of Fab-6HD5, 6HD5, Fab-HMK-12, Fab-HMK-12, rat IgG and anti-κ antibody were injected into the same sites. Two hours after the second series of injections, the rats were injected intravenously with 1 ml of mixture comprised of equal quantities of 0.5% Evans blue dye in saline and 1 mg/ml of DNP-BSA or TNP-BSA. The reactions (the diameters of the blue spots) were measured 30 min later. The experiments were repeated three times. All experiments were performed in triplicate by investigators  who were blinded to the measurements of the PCA reactions.PCA reactions were performed as previously describedSeveral sites of freshly shaved skin of 2 rats for each group were injected intradermally with 1 μg/ml of SPE-7 on day 0.In the first group, serial dilutions of Fab-6HD5 were injected into the same site on day 1. Two hours later, the rats were injected intravenously with 1 ml of mixture comprised of equal quantities of 0.5% Evans blue dye in saline and DNP-BSA (1 mg/ml).In the second group, an Evans blue extravasation assay was performed on day 10. The reactions (the diameters of the blue spots) were measured 30 min later.37. The rat IgG anti-murine κ antibody and phycoerythrin (PE)-labeled goat anti-rat IgG were purchased from BioLegend  and Jackson ImmunoResearch  respectively. Fab fragments of 6HD5 and HMK12 were prepared by Immune-Biological Laboratories Co. Ltd . Highly purified Fab fragments of 6HD5 were prepared using Ficin, a cysteine protease isolated from fig latex. Briefly, 6HD5 (1 mg/ml) was digested with 0.5 ml of the immobilized Ficin resin slurry (Thermo scientific prod. # 44881) twice in the presence of 25 mM cysteine. The resulting Fab-6HD5 fragments were used for in vitro blocking assays of Syk phosphorylation or β-hexosaminidase release.SPE-7 (anti-DNP murine IgE antibody) and 141a (anti-TNP murine IgE antibody) were purchased from Sigma-Aldrich Co. LLC (USA) and Pharmingen (USA), respectively. The rat monoclonal antibodies 6HD5 and HMK-12 (specific for murine IgE) have been described previouslyThe 30 molecules of DNP (70 kDa molecular weight) from bovine serum albumin were a generous gift from Dr. Z. Ovary . TNP-BSA and Evans blue dye were obtained from Wako Pure Chemical Industries .Anti-IgE antibodies bound to RBL-2H3 cells were detected by flow cytometry analysis.5 RBL-2H3 cells were incubated with 1 μg/ml of FITC-Labeled SPE-7 (anti-DNP IgE) or 141a (anti-TNP IgE) for 30 min at 4 °C. The cells were washed twice with PBS and incubated with 1 μg/ml of Fab-6HD5, Fab-HMK-12 or anti-Ig light chain antibodies for 30 min at 4 °C. After the washes, the cells were incubated with 10 ng/ml of PE-goat anti-rat IgG for 30 min at 4 °C. The cells were then washed twice with PBS and analyzed using a FACSCalibur . For competition experiments, 1 μg/ml of FITC-Labeled SPE-7 or 141a was premixed with 1 μg/ml of Fab-6HD5, Fab-HMK-12 or anti-Ig light chain antibodies. After incubation for 30 min at room temperature, the mixture was added to 5 × 105 RBL-2H3 cells and incubated for 30 min at 4 °C. The cells were washed twice with PBS and incubated with 10 ng/ml of PE-goat anti-rat IgG for 30 min at 4 °C. The cells were then washed twice with PBS and analyzed using a FACSCalibur For the experiment, 5 × 104 cells/well). The adherent cells were incubated with 1 μg/ml of mouse anti DNP-IgE (SPE-7) overnight at 37 °C. Following sensitization, the cells were washed and incubated with a serial dilution of highly purified Fab-6HD5 for 2 hours at 37 °C. The cells were washed three times with HEPES-buffered Tyrode’s solution, pH 7.4 (HEPES-Tyrode’s solution) to remove excess IgE and anti-IgE. Then, the cells were incubated with 100 ng/ml of DNP-BSA in 0.1% BSA-HEPES-Tyrode’s solution for 1 hour at 37 °C to trigger degranulation. After stimulation, aliquots (10 μl) of the supernatant from each well were incubated with 40 μl of substrate solution, p-nitrophenyl-N-acetyl-beta-D-glucosamine in 0.1 M citrate buffer, pH 4.5 (1.3 mg/ml) for 1 hour at 37 °C. The color formed by hydrolysis of the substrate to produce p-nitrophenolate was measured at 405 nm on a microplate reader . The total cellular content of β-hexosaminidase was measured in 1% TritonX-100 cell lysates.RBL-2H3 cells were seeded in 96-well flat bottom plates  for indicated periods and cell extracts were subjected to Western blotting. Proteins were detected with anti-phosphorylated Syk , anti-Syk (#2712), and anti-Tpt1  antibodies followed by HRP-conjugated anti-rabbit  or anti-mouse (#7076) antibody.RBL-2H3 cells were incubated with anti-TNP IgE (0.5 μg/ml)  for 1 h. Highly purified 6HD5-Fab (2 μg/ml) or control IgG2a  was added and further incubated overnight. After washing, cells were detached with 0.05% Trypsin-EDTA, collected, washed and resuspended at 2 × 10Total RNA was isolated from the mouse IgE-producing hybridoma SPE-7 and reverse-transcribed using oligo dT primers. The coding gene for the IgE H chain constant region (Cε1-4) and truncated regions were synthesized from the cDNA and subcloned into pGEX-6P-3 . The PCR primer sets and primer sequence information are listed as Table Samples of GST-IgE H chain constant fusion molecules (Cε1-4) were run on 12.5% of SDS-PAGE under reducing conditions, transferred to a PVDF membrane (Millipore corp.) and probed with Fab-6HD5 followed by peroxidase-conjugated goat anti-rat IgG, F(ab’)2 fragment specific . Signals were developed using Pierce ECL Plus Western Blotting Substrate (Thermo 32132). Immunoreactivity was detected with an Imagequant LAS 4000 . To remove the first antibody, the membranes were soaked in stripping buffer  at 65 °C for 30 min with occasional shaking. After washing the membranes three times with 0.05% Tween-PBS and blocking them with BlockAce , the membranes were re-probed with anti-GST-tag pAb  followed by HRP-conjugated anti-rabbit IgG . The ECL reaction and acquisition of chemiluminescent signals from immunoblots were performed as described above. For Syk phosphorylation assay, blots were probed with respective antibodies, anti-phosphorylated Syk, anti-Syk, and anti-Tpt1 followed by HRP-conjugated anti-rabbit or anti-mouse antibody.β-hexosaminidase release assay were analyzed by an unpaired t test using GraphPad PRISM to identify significant differences. P values < 0.05 were considered significant.Data generated from the Supplementary Information


Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates that are inherently unstable, making it difficult to study. Here we developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. A subset of recursive sites were validated by RT-PCR and sequencing. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, we observe that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of  Drosophila melanogaster cells using data from sequencing of nascent RNA captured within minutes after transcription. We used these methods to identify hundreds of previously unknown sites of recursive splicing, occurring commonly in fly introns longer than 40kb and often in genes involved in morphogenesis and development. We observed that recursive splicing is associated with increased splicing accuracy of long introns, which are otherwise often spliced inaccurately, potentially explaining its widespread occurrence in long fly introns.The splicing of RNA transcripts is an essential step in the production of mature mRNA molecules, involving removal of intron sequences and joining of flanking exon sequences. Introns are usually removed as a single unit in a two-step catalytic reaction. However, a small subset of introns in flies are removed via splicing of multiple distinct consecutive segments in a process known as recursive splicing. This pathway was thought to be quite rare since intermediates of recursive splicing are seldom detected. In this study, we developed three new computational approaches to identify sequence reads, read pairs and patterns of read accumulation indicative of recursive splicing in  Drosophila melanogaster genome are known to undergo recursive splicing, in which two or more adjacent sections of an intron are excised in separate splicing reactions, each producing a distinct lariat , (2), (2)D. mDrosophila as well and have been used in gene prediction in fly genomes.We calculated the strength of splice sites using a maximum entropy model as implemented in maxEntScan  using 9 Gene Ontology enrichment analyses were performed using a custom script to avoid significant gene ontology terms with overlapping gene sets. Specifically, the script used the Flybase gene ontology annotation downloaded from the Gene Ontology Consortium website  and searhttps://github.com/jpaggi/recursive.Source code for our pipeline to identify recursive splicing sites is available at S1 Fig(A) RNA-seq pre-processing steps to convert reads into an array of read densities: (1) summing the read coverage for each base-pair (top) and (2) replacing the read counts in annotated repeat regions and 100 flanking nt with median read density in 900 nt flanking regions (bottom) (B) MCMC algorithm infers probability that each position in intron is a recursive splice site, where upon entering each round with a previously accepted state, this state is perturbed to propose a new state and the new state is either accepted or rejected. The procedure is performed over 107 rounds, with sampling every 50 rounds to obtain a probability that each base pair is a recursive site. (C) Sequence information is used in conjunction with MCMC-inferred probabilities to predict recursive sites.(TIF)Click here for additional data file.S2 Fig(A) The probability derived from the sawtooth MCMC model of a site being a recursive site for the final set of recursive sites (light orange), all sites with minimal support from any method (dark orange), and random sites placed down in the same introns (grey). (B) The sawtooth score (see light orange), all sites with minimal support from any method (dark orange), and random sites place down in the same introns (grey). (C) Number of recursive sites (left) and high-confidence sites (right) identified by one of multiple identification pipelines, with the majority of recursive sites identified by both junction reads and sawtooth scores, as well as present in the Duff et al. dataset. (D) The gene expression levels of genes with recursive introns  relative to the junction spanning read support for each recursive intron , showing the varying power to identify recursive sites with the sawtooth recursive method (orange), junction-spanning reads alone (blue), or both methods (black). (E) The cumulative distribution of distances between the recursive site identified with the sawtooth recursive method and the best matching recursive motif (orange) and random sites placed down in the same introns (grey) are significantly different.core see  for the (PDF)Click here for additional data file.S3 FigA) and intronic 3' splice sites (B), for recursively spliced introns from three genes: Ten-m (left), Luna (middle), and Tet (right). PCR was performed on first-strand cDNA from nascent RNA from Drosophila S2 cells isolated after 5 minutes of labeling with 4sU and bands were visualized on a 1.5% agarose gel with a 50bp ladder. Schematics indicate the junctions across which the amplicons were designed. Junction boundaries were confirmed by sequencing for lanes 3, 5, 17, 18, and 19 in (A). (C) PCR products across recursive segments and intronic 3' splice sites from first-strand cDNA from steady-state RNA for the Ten-m recursively spliced intron. The junction boundary was confirmed by sequencing for lane 4. (D) Representative sequence traces confirming the junction boundaries for two novel 5'-RS recursive splicing events (top and middle) and one RS-3' event (bottom). Peaks delineate specific nucleotides, including A (green), C (blue), G (black), and U . For each of these events, the full band was sequenced , however only 30nt around the junction is shown here for visualization purposes.PCR products from recursive segments anchored to intronic 5' splice sites ((TIF)Click here for additional data file.S4 Figtop), medium-confidence recursive sites (middle) and high-confidence recursive sites (bottom).Sequence logo for all intronic AG|GT sites ((PDF)Click here for additional data file.S5 Fig(A) Distribution of lengths of recursive segments  for medium-confidence recursive segments (yellow) and high-confidence recursive segments (gold). (B) Recursive segment length distributions  for introns with varying numbers of recursive sites (x-axis). (C) The number of splice junction reads (y-axis) spanning a 5' splice site and recursive site (blue), two recursive sites (gold), and a recursive site and 3' splice site (yellow) across the labeling periods (x-axis). (D) Junction reads used to estimate splicing half-lives for recursive segments (red lines), centered on 3' recursive sites (red dots) for each segment. Incomplete splicing is estimated from intron-exon junction reads (pink bars). Completed splicing is estimated from a sum across split-junction reads between the 5' splice site and recursive site (light blue bars), two recursive sites (orange bars), a recursive site and the 3' splice site (yellow bars), and the 5' splice site and 3' splice site . Each segment’s splicing is informed by different types of junction reads dependent on the position in the intron, as drawn for an intron with three recursive segments. (E) Splicing half-lives (y-axis) for recursive segments with varying positions across the intron (x-axis), where on average, all segments in an intron tend to be spliced out at similar rates. (F) The distribution of mean life-times (y-axis) for recursively spliced introns  relative to non-recursive introns chosen to match the length of the recursive introns (grey).(PDF)Click here for additional data file.S6 Figy-axis) across both 3' splice sites (orange) and 5' splice sites (blue) for recursive sites (right) and non-recursive introns matched for intron length (left). Significance is indicated such that **: P < 0.01 and ***: P < 0.001, with a Mann-Whitney U test.Distribution of splice site strengths Click here for additional data file.S1 TableColumn 1 –intron: Coordinates of intron containing recursive site, with chr:start-end:strand.Column 2 –gene: FlyBase gene symbol for parent gene.Column 3 –TPM: Gene expression values calculated using kallisto (TPMs).Column 4 –completed_splicing_junction_reads: Number of junction reads supporting completed splicing across the entire intron.Column 5 –recursive_site: Coordinate for the recursive site.Column 6 –method: Method used for identification of the recursive site, where “junction” indicates site identified by either RachetJunction or RachetPair, “sawtooth” indicates site identified by RachetScan, and “both” indicates site identified by both methods.Column 7 –in_duff: Flag indicating the recursive site was identified in the Duff et al. study.Column 8 –high_confidence: Flag indicating the recursive site was identified as a high-confidence site (1) or a medium-confidence site (0).Column 9 –junction_reads: Comma-separated list of the number of junction reads (5'-RS) supporting the recursive site in each timepoint (combined across replicates) .Column 10 –spanning_read_pairs: Comma-separated list of number of spanning read-pairs supporting the recursive site in each timepoint (combined across replicates) .Column 11 –sawtooth_score: Sawtooth score for the recursive site, as defined in the Methods.Column 12 –mcmc_probability: Probability of this site being a recursive site, as derived from the MCMC sampling procedure integral to the RachetScan method.Column 13 –recursive_index: Recursive index for the recursive site, as defined in the Methods.Column 14 –motif: Sequence found around the recursive site.Column 15 –motif_score. Motif score for the recursive site, as defined in the Methods.Column 16 –downstream_reads: Number of splice junction reads originating from the 3' end of the exon.Column 17 –intron_body_reads: Number of reads in the body of the intron.(TXT)Click here for additional data file.S2 TableColumn 1 –intron coordinates: Coordinates of intron containing recursive site, with chr:start-end:strand.Column 2 –junction type: Type of junction the lariat spans Column 3 –junction coordinates: Coordinates of the junction that the lariat spansColumn 4 –branchpoint: Coordinate of the branchpoint identified by the lariat spanning readColumn 5 –sequence: Sequence overlapping the branch point (the putative branch point nucleophiles is at base 11)Column 6 –timepoint: labeling period in which the lariat read was identifiedColumn 7 –read ID: Identifier of the lariat spanning read.(XLSX)Click here for additional data file.S3 TableColumn 1 –PRIMER PAIR: Name of primer pair usedColumn 2—Name: Name of individual primer Column 3—Sequence: Sequence of individual primerColumn 4 –Expected amplicon size: Expected amplicon size if recursive segment has not yet been spliced out. Molecules where the recursive segment has been spliced will not be amplified with these primer combinations.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 TableColumn 1 –intron: Coordinates of intron containing recursive site, with chr:start-end:strand.Column 2 –gene: FlyBase gene symbol for parent gene.Column 3 –recursive_site: Coordinate for the recursive site .Column 4 –segment_type: Indicator whether the segment is spliced to a recursive site. (“segment”) or to the 3’ splice site .Column 5 –segment_len: Length of the segment (nucleotides).Column 6 –segment_num: Position of the segment relative to other segments in the intron.Column 7 –three_length: Length of the region from the recursive site (or 3' splice site) to the polyA site of the transcript (nucleotides).Columns 8–10 –ie_count_[timepoint]: count of the intron-exon junction reads for each of the labeling periods (summed across three replicates per labeling period)–for recursive sites, this overlaps the recursive site, while for the final segment this overlaps the 3' splice site.Columns 11–13 –ee_count_[timepoint]: count of the exon-exon junction reads for each of the labeling periods (summed across three replicates per labeling periods)–this includes junctions deriving from recursive intermediates, as outlined in the Methods.Column 14 –halflife: Half-life of the recursive segment computed using the junction dynamics approach described in Pai et al. 2017 (min).Column 15 –txn_to_three: Time to transcribe the remainder of the intron from the recursive site to the 3’ splice site (min).(TXT)Click here for additional data file.


In this study, we assess the effect of the lake size on the accuracy of a threshold-based classification of ground-fast and floating lake ice from Sentinel-1 Synthetic Aperture Radar (SAR) imagery. For that purpose, two new methods (flood-fill and watershed method) are introduced and the results between the three classification approaches are compared regarding different lake size classes for a study area covering most of the Yamal Peninsula in Western Siberia. The focus is on April, the stage of maximum lake ice thickness, for the years 2016 and 2017. The results indicate that the largest lakes are likely most prone to errors by the threshold classification. The newly introduced methods seem to improve classification results. The results also show differences in fractions of ground-fast lake ice between 2016 and 2017, which might reflect differences in temperatures between the winters with severe impact on wildlife and freshwater fish resources in the region. Patterns of low backscatter responsible for the classification errors in the centre of the lakes were investigated and compared to the optical Sentinel-2 imagery of late-winter. Strong similarities between some patterns in the optical and SAR data were identified. They might be zones of thin ice, but further research is required for clarification of this phenomenon and its causes. Several studies have successfully demonstrated the ability of using C-Band SAR data to distinguish between areas of ground-fast and floating lake ice of thermokarst lakes  and the ice-water boundary   and Sentinel-1B (launched in April 2016) are part of the EU’s Copernicus Program and carry an identical SAR instrument, the C-SAR. The two satellites share the same polar, sun-synchronous orbit, separated by 180lane ESA . C-SAR clane ESA . The incThe default operating mode is IW  with VV and vertical-horizontal (VH) polarization over land, which satisfies most service requirements ESA . EW modeFor 2016, three adjacent scenes acquired on 2 April, covering nearly the whole Yamal Peninsula were selected. For 2017, two adjacent scenes of the same orbit acquired on 9 April were used. In addition to the scenes used for the classification, several EW HH-polarized scenes from February to June 2016 and 2017 were used for a visual interpretation of specific patterns that cause differences between the results obtained with the different methods.2.3.Sentinel-2 is the second pair of earth observation satellites of the EU’s Copernicus program. Sentinel-2A was launched into orbit in June 2015 and carries a multispectral imager, simply called MultiSpectral Instrument (MSI), which acquires images in 12 spectral bands  were obtained for that purpose. At high latitudes, a high percentage of cloud cover in scenes is very common, leaving only a few useful acquisitions throughout the year.In optical images from summer, when the lakes are free of ice, the outlines and also the shallow shelves of many arctic lakes are often clearly visible. Since the freezing depths vary from year to year, the images can only give hints where ground-fast ice might have formed and where most likely not. The shallow shelf regions, where ground-fast ice usually forms in the winter, are often seen as brighter rings around the lake margins in optical summer images  dataset of the National Geomatics Center of China (NGCC) was used to extract the lakes from the SAR imagery. Its full derivation is described in Bartsch et al. . The GLC3.3.1.Wet snow or liquid water on top of the lake ice layer results in backscatter similar to ground-fast lake ice. Only frozen days need to be therefore considered. Daily maximum air temperatures recorded at the weather station Marre-sale  were used as an indicator if melting processes took place during acquisitions. On 2 April 2016, the maximum air temperature was −5.4 The Sentinel Application Platform (SNAP) toolbox of the European Space Agency (ESA) was used for pre-processing of all SAR images. First, thermal noise and border noise were removed. Thermal noise can be seen as radiometric disturbances at the edges of the subswaths used by the TOPSAR system. Those disturbances were found to be significantly more striking for cross-polarized data  and wide deeper central parts (darker blue or green colour) could clearly be distinguished in the Sentinel-2 summer imagery. One hundred metre distance buffer zones were calculated from the outlines inward for each of the 20 lakes to represent the shelf areas, where the potential occurrence of ground-fast ice is assumed. Centroids were calculated for each of the 20 lakes, and circles with 1 km diameter around these centroids were computed to represent the deeper areas, where only the occurrence of floating ice is expected. The percentages of ground-fast and floating ice detected by the three methods were calculated for each of the shelf- and central zones.Results have been also compared to a similar dataset derived from ENVISAT ASAR Wide Swath (WS) mode data . BetweeThe watershed method detected significantly larger amounts of ground-fast ice pixels for lakes smaller than 1.600 kmThe fractions detected above 12.800 km studied ).As can be seen in In a visual inspection of the classification results and the original SAR scenes, 3 major types of features that cause significant differences between the results of the different classification methods for large lakes could be identified. Some of the lakes show wide areas in the assumed floating ice region, where backscatter is significantly lower than in the surroundings and in other lakes. These wide patches are thought to cause large classification errors, especially with the threshold method. An example of this phenomenon can be seen in Another line of low backscatter, that appears to be a crack within the ice, can be seen in These ‘dots’ are especially interesting, as they clearly stand out against the surrounding much brighter pixels. Further visual investigations of SAR time series for lake Neyto showed that these patterns change throughout the winter season, becoming more apparent and larger with time, and also differ strongly when compared between the two years  the lake5.The similarity between the threshold and flood-fill method may not be surprising for the smallest lake size classes, as pixels of low backscatter are thought to be connected to each other anyway, but for the remaining lakes smaller than 12.800 kmThe watershed method is thought to classify smaller areas of floating lake ice in central parts of small lakes incorrectly as ground-fast lake ice. This might be caused by the small number of central lake pixels that would belong to the floating ice class. If all of the few central pixels show comparably low backscatter values below −10.0 dB, the whole lake is classified as ground-fast.For the larger classes (12.800 kmIn the following, two visual examples shall illustrate some points discussed above. Winter 2017 was generally colder than average, including some periods of extreme cold. The minimum temperature recorded at the weather station Marre-sale was −39.0 The used lake mask may introduce classification errors. It is static and from a different year than the Sentinel data. It has been shown by Trofaier et al.  that theThe lower ground-fast ice values of the ASAR WS product may result from the coarser spatial resolution. More mixed pixels are expected to occur along the lake rims. The higher ground-fast ice values for larger lakes point to the uncertainties due to different backscatter responses of ice in the lake centres.The differences for the larger lakes underline the importance of analysing possible classification errors for the largest lakes. Three feature types that could be largely responsible for the classification errors with the threshold method were identified. For the line-type features, cracks might indeed be responsible for the observed low backscatter, which appears to be the case in a comparison to the optical data in The comparison to optical data clearly indicates strong similarities between the dot-like patterns of low backscatter and the presumably snow-free areas of lake Neyto in 2016 seen in the optical acquisition in Walter et al.  note, thLarge circular patterns in lake ice, which can be identified with optical satellite data, have been also reported for Lake Baikal as well as Hovsgol (Kouraev et al. The occurrence of locally thin ice in central Yamal was also observed by Nenets. For example, reindeer herders reported in the summer 2017, that once they were crossing the large lake Yambuto  in mid-MMuch more research is needed to evaluate the origin of the mentioned patterns. The resemblance of the patterns in the SAR images with the presumably snow-free areas in 6.Within this study, the role of lake size and the effects of local phenomena on discriminating ground-fast from floating ice on Arctic lakes on and near the Yamal Peninsula using Sentinel-1 SAR imagery of April 2016 and April 2017 was assessed. Arctic lakes are large emitters of carbon dioxide and methane, which is also released in the winter. Mapping the extent of floating and ground-fast ice accurately can be useful for emission studies. Moreover, it is important for understanding sublake permafrost dynamics and lake habitat changes. With the flood-fill and the watershed method, two new methods relying on image processing techniques were introduced. The two methods were designed to separate shallow shelf regions from central parts of the lakes in the SAR imagery because the threshold classification showed partly large assumed errors in the central parts. The results of the new methods were compared to the results of the threshold classification for different lake size classes. The comparison indicates, that the new methods are likely capable of reducing errors observed in the threshold classification for the largest lakes significantly, but no total assessment could be performed because no ground truth data were available. The classifications also show considerable differences between the two years, which might reflect the colder winter of 2017 when compared to 2016. Additionally, three types of features that are thought to cause the differences between the threshold and the other classification approaches were identified. Cracks in the lake ice are thought to be one of them. Other sources are rather wide patches of low backscatter in the SAR images, that could not be related to any phenomenon yet. The third type is dot-like patterns of low backscatter. A time series analysis revealed that these patterns vary over time and tend to intensify in late winter. The presence of locally thin ice could be confirmed by reports from reindeer herders. The patterns also vary between the two years and showed large similarities with optical Sentinel-2 images acquired when the lakes were partly free of snow. It is not clear what these patterns are caused by and only speculations about their origin could be given in this study. However, the patterns seem to be a very interesting subject for future studies.


The as-prepared P(dCz2-co-dTC2) displayed a high ΔT (57.0%) and multichromic behaviors ranging from yellowish green, greenish gray, gray to purplish gray in different redox states. Five organic electrochromic devices (ECDs) were built using dCz- and dTC-containing homopolymers and copolymers as anodic materials, and polythiophene) (PProdot-Me2) as the cathodic material. The P(dCz2-co-dTC2)/PProdot-Me2 ECD presented remarkable electrochromic behaviors from the bleached to colored states. Moreover, P(dCz2-co-dTC2)/PProdot-Me2 ECD displayed a high optical contrast , short switching time (ca. 0.3 s), high coloration efficiency (528.8 cm2 C−1) at 580 nm, and high redox cycling stability.A series of carbazole-based polymers (PdCz, P(dCz2- The ΔT of P(CDPN-co-EDOT) was 36% and 43% in visible zone and near-IR region, respectively. Polythiophene and its derivatives are widely studied owing to their interesting characteristics such as narrow band gaps and desirable stability in air [T of these PEDOT derivatives were 44–63% at ca. 590 nm [Over the past numerous years, several inorganic and organic electrochromic materials have been extensively studied for use in the rear-view mirrors of vehicles, displays, helmet visors, and windows of buildings . In thespolymers , and widpolymers , polytripolymers , polyindpolymers , polycarpolymers , polypyrpolymers , polythipolymers , polyselpolymers , poly) . The P, P(dCz2-co-dTC2), P(dCz1-co-dTC2)) with different dCz/dTC feed molar ratios were electrosynthesized to study their potential applications in ECDs. 2,6-di(9H-carbazol-9-yl)pyridine unit comprises two carbazole groups linked by a pyridine core, such donor-acceptor-donor (D-A-D) configuration is efficient for narrowing the band gap of polymers. Moreover, two carbazole groups in each repeat unit of polymer backbone facilitate the formation of radical cations and dication upon oxidation. In addition, the chemical structure of 3,6-di(2-thienyl)carbazole group displays that a carbazole unit inserts between two thiophene units, PdTC is easier to oxidize due to the donor capability of the carbazole. Poly has been shown to have a short conjugation length. The incorporation of thiophenes at 3,6-positions of carbazole unit would extend this conjugation length, leading to low oxidation potentials. Furthermore, five ECDs consisted of PdCz, P(dCz2-co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), or PdTC as the anodic material, and PProdot-Me2 as the cathodic material were fabricated and their spectroelectrochemical characteristics, transmittance-time profiles, and long-term electrochemical stability were studied in detail.In the present study, two homopolymers (PdCz and PdTC) and three copolymers pyridine (dCz), 3,6-di(2-thienyl)carbazole (dTC), and Prodot-Me methods ,25. Elec33:53:14 .co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC films were carried out in an 0.2 M LiClO4/acetonitrile (ACN)/dichloromethane (DCM)  solution, and the feed ratio of species for anodic polymer films were listed in 3) for 3 cycles. The Ag/AgNO3 electrode was calibrated using ferrocene. PProdot-Me2 film was electrodeposited potentiostatically at 1.0 V (vs. Ag/AgNO3). Polymeric thicknesses at the electrode surfaces obtained from an Alpha-Step profilometer  were about 180–300 nm.The electrosynthesis of PdCz, P(dCz2-co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), or PdTC film as the anodic layer and PProdot-Me2 film as the cathodic layer. The active areas of anode and cathode were 1.5 cm2. The ECDs were built by arranging the reduced and oxidized polymeric films to face each other, and they were isolated by a PMMA/PC/ACN/LiClO4 composite electrolyte.ECDs were constructed using PdCz, P, and the path length of cell was 1 cm.The as-prepared polymer films and ECDs were characterized using a CHI627D electrochemical analyzer  and an Agilent Cary 60 UV-Visible spectrophotometer . The system of electrochemical experiments was implemented in a three-constituent cell. The working electrode was an ITO coated glass plate, the counter electrode was a platinum wire, and an Ag/AgNO4/ACN/DCM solution in the potential range from 0.0 to 1.4 V. The scan rate was 100 mV s−1. When the number of CV cycles increased, the current density of redox peaks increased with an increasing scanning number, implying the growth of polymer films on ITO surfaces [3), respectively, can be ascribed to the presence of radical cation and dication in a dCz unit. The CV curves of PdTC show an oxidation and a reduction peaks at 1.05 and 0.37 V, respectively, as shown in co-dTC1), P(dCz2-co-dTC2), and P(dCz1-co-dTC2) appeared at 1.25, 1.19 and 1.15 V, respectively, and the reduction peaks of P(dCz2-co-dTC1), P(dCz2-co-dTC2), and P(dCz1-co-dTC2) located at 0.50, 0.43 and 0.39 V, respectively, as shown in co-dTC1), P(dCz2-co-dTC2), and P(dCz1-co-dTC2) films. The polymer films were further characterized using FT-IR, co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC films. The characteristic peaks of PdCz are shown in −1 indicates the doping of PdCz film with the ClO4−. The characteristic peaks at around 1600 cm−1 represent the aromatic C=C stretching vibration. The band at 1220 cm−1 is related to C–C formation. The peak at around 1450 cm−1 can be ascribed to the C–N stretching of the carbazole unit [surfaces . As dispole unit . −1 as shown in −1 could be attributed to the presence of dTC in P(dCz2-co-dTC1), P(dCz2-co-dTC2), and P(dCz1-co-dTC2) films, implying dCz- and dTC-containing copolymer films were successfully synthesized. The polymerization schemes of PdCz, P(dCz-co-dTC), and PdTC are shown in There was no conspicuous characteristic peak of PdCz at ca. 790 cmco-dTC2) film was immersed into clean electrolyte solution, and then run in new CV curves. The as-prepared P(dCz2-co-dTC2) film was swept at 10, 50, 100, 150, and 200 mV s−1 in 0.2 M LiClO4/ACN/DCM using CV. As shown in co-dTC2) film showed a single quasi-reversible redox process and the anodic and cathodic peak current densities increased linearly with increasing scan rate (inset in co-dTC2) film was not a diffusion-controlled process [The first rinse P, P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC electrodes in an ACN/DCM solution containing 0.2 M LiClO4. PdCz does not show distinct absorption peaks at 0.0 V but two new peaks appear at 430 and 785 nm gradually upon increasing of the applied potential, which can be attributed to the growth of charge carrier bands [co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC electrodes display absorption peaks at 406, 410, 405, and 402 nm at 0.0 V, respectively, which can be assigned to the π-π* transition of P(dCz2-co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC electrodes in their reduced state. When potentials in the oxidative direction were applied, the π-π* transition bands of P(dCz2-co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC electrodes waned and new charge carrier bands (550 nm and 860 nm for P(dCz2-co-dTC1); 545 nm and 820 nm for P(dCz2-co-dTC2); 548 nm and 830 nm for P(dCz1-co-dTC2); 549 nm and 815 nm for PdTC) were seen at the longer wavelengths zone. The optical band gap (Eg) values of PdCz, P(dCz2-co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC electrodes were 3.01, 2.43, 2.48, 2.42, and 2.45 eV, respectively.co-dTC1) film was yellowish green, gray, and purplish grey at 0.0, 0.6, and 1.1 V, respectively, whereas P(dCz2-co-dTC2) film was yellowish green, greenish gray, gray, and purplish gray at 0.0, 0.6, 0.8, and 1.1 V, respectively, and P(dCz1-co-dTC2) film was yellow, gray, and dark purplish-grey at 0.0, 0.4, and 1.1 V, respectively. The colorimetric values , CIE  chromaticity values  and diagrams of the five polymer films at various potentials are listed in PdCz displayed multichromic behaviors with a light gray color in its neutral state (0.0 V), an iron grey color (0.9 V) and a dark gray color (1.2 V) during stepwise oxidation. The colors of PdTC electrode were light yellowish-gray, light gray, and iron grey at 0.0, 0.7, and 1.3 V, respectively. The dCz- and dTC-containing copolymer electrodes displayed different multichromic behaviors with PdCz and PdTC electrodes. P(dCz2-co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC films were monitored by potential stepping between neutral (0.0 V) and oxidized states (+1.1 V) with a residence time of 10 s. The ΔT values of PdCz, P(dCz2-co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC films were 19.7% at 790 nm, 55.7% at 790 nm, 57.0% at 784 nm, 50.6% at 790 nm, and 48.0% at 790 nm, respectively. The ΔT of P(dCz2-co-dTC1), P(dCz2-co-dTC2), and P(dCz1-co-dTC2) films were larger than those of PdCz and PdTC films in 0.2 M LiClO4/ACN/DCM solution, indicating copolymers contain both dCz and dTC units increased ΔT significantly. The τc of electrodes (a), (b), (c), (d), and (e) were 4.0 s, 2.7 s, 3.5 s, 4.2 s, and 4.3 s at 3rd cycles, respectively, and the τb of electrodes (a), (b), (c), (d), and (e) were 0.8 s, 2.7 s, 1.8 s, 5.6 s, and 6.0 s at 3rd cycles, respectively. Different τc and τb in each set of samples may be attributed to the contact area between electrolytes and electrodes.As displayed in Tox and Tred are the transmittances in the oxidized and the reduced state, respectively.ΔOD can be determined using the formula ,(1)ΔOD=η) is another important parameter for practical application of polymer films in ECDs and it can be determined using the equation [Qd implicates the amount of injected charges per unit electrode area. The η values of PdCz, P(dCz2-co-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC films are 81.4 cm2 C−1 at 790 nm, 99.3 cm2 C−1 at 790 nm, 248.4 cm2 C−1 at 784 nm, 145.3 cm2 C−1 at 790 nm, and 164.9 cm2 C−1 at 790 nm, respectively (co-dTC2) film presents the highest η among the five polymer films.The coloration efficiency (equation : (2)η=ΔOectively . P/PProdot-Me2, and PdTC/PProdot-Me2 ECDs at different applied potentials. 2 ECD did not show distinct absorption peaks at wavelength ranging from 350 to 450 nm at 0.0 V, P(dCz2-co-dTC2)/PProdot-Me2 and PdTC/PProdot-Me2 ECDs displayed distinct absorption peaks at 410 and 402 nm at 0.0 V, which could be assigned as the absorption peaks of P(dCz2-co-dTC2) and PdTC films at 0.0 V, respectively. The absorption peaks of P(dCz2-co-dTC2) and PdTC films fade with the increasing applied potential, and new absorption bands were emerged gradually at around 580 nm. This can be attributed to the stepwise reduction of PProdot-Me2 film. The colors of PdCz/PProdot-Me2 ECD are silver gray, gray, and purplish-gray at 0.0, 1.0, and 1.3 V, respectively. P(dCz2-co-dTC2)/PProdot-Me2 and PdTC/PProdot-Me2 ECDs reveal four kinds of colors from bleached to colored states. The colors of P(dCz2-co-dTC2)/PProdot-Me2 ECD are yellowish green, iron gray, dark blue, and purplish-blue at −0.8, 0.8, 1.2, and 1.5 V, respectively. PdTC/PProdot-Me2 ECD reveals yellowish-green, greenish-gray, grayish-blue, and purplish-blue at −0.8, 0.0, 0.8, and 1.7 V, respectively. The electrochromic photographs, colorimetric values , CIE chromaticity values  and diagrams of PdCz/PProdot-Me2, P(dCz2-co-dTC2)/PProdot-Me2, and PdTC/PProdot-Me2 ECDs at various applied potentials are listed in PdCz/PProdot-Me2 and P(dCz2-co-dTC2)/PProdot-Me2 ECDs were monitored by potential stepping between bleached and colored states with a residence time of 10 s. The ΔT, ΔOD, switching time, and η of PdCz/PProdot-Me2, P(dCz2-co-dTC1)/PProdot-Me2, P(dCz2-co-dTC2)/PProdot-Me2, P(dCz1-co-dTC2)/PProdot-Me2 and PdTC/PProdot-Me2 ECDs are listed in co-dTC2)/PProdot-Me2 ECD displays the highest ΔT, and P(dCz2-co-dTC1)/PProdot-Me2 and P(dCz2-co-dTC2)/PProdot-Me2 ECDs display higher ΔT and ΔOD than those of PdCz/PProdot-Me2 and PdTC/PProdot-Me2 ECDs, indicating that the use of copolymers (P(dCz2-co-dTC1) and P(dCz2-co-dTC2)) as the electrode materials brings about a higher ΔT at around 580 nm than those of homopolymers (PdCz and PdTC). T with reported ECDs, P(dCz2-co-dTC2)/PProdot-Me2 ECD shows higher ΔT than those reported for P(Bmco)/PEDOT [2 [co-bth)/PEDOT [2 [co-ProDOT)/triple-layer PEDOT-PSS ECDs [τc and τb of five ECDs in As displayed in o)/PEDOT , P(dNcbpo)/PEDOT , P/PEDOT , PtCz/PPPEDOT [2 , and P/PProdot-Me2, P(dCz2-co-dTC2)/PProdot-Me2, P(dCz1-co-dTC2)/PProdot-Me2 and PdTC/PProdot-Me2 ECDs were 507.0, 562.4, 528.8, 438.9 and 440.4 cm2 C−1, respectively. P(dCz2-co-dTC1) film with a dCz/dTC = 2/1 feed molar ratio presented the highest η among the five ECDs. η with reported ECDs: P(dCz2-co-dTC2)/PProdot-Me2 ECD showed higher η than those reported for p(dNcbph-co-bth)/PEDOT [2 [co-ProDOT)/triple-layer PEDOT-PSS ECDs [co-dTC2)/PProdot-Me2 ECD showed lower η than that reported for P(tnCz1-bTp2)/PProdot-Me2 ECD [The h)/PEDOT , PtCz/PPPEDOT [2 , and P/PProdot-Me2, and PdTC/PProdot-Me2 ECDs was detected at bleached and colored states by applying the voltage for 1 s for each 100 s interval [co-dTC2)/PProdot-Me2 ECD showed less transmittance change at the colored state than those of PdCz/PProdot-Me2 and PdTC/PProdot-Me2 ECDs, disclosing that P(dCz2-co-dTC2)/PProdot-Me2 ECD exhibited a satisfactory open-circuit memory effect.The open-circuit memory effect of PdCz/PProdot-Meinterval ,41. Figu2, P(dCz2-co-dTC2)/PProdot-Me2, and PdTC/PProdot-Me2 ECDs was monitored using CV at the 1st, 500th and 1000th cycles [2, P(dCz2-co-dTC2)/PProdot-Me2, and PdTC/PProdot-Me2 ECDs, respectively. P(dCz2-co-dTC2)/PProdot-Me2 ECD showed better long-term electrochemical stability than those of PdCz/PProdot-Me2 and PdTC/PProdot-Me2 ECDs, disclosing the redox stability of copolymerized P(dCz2-co-dTC2) film was higher than those of homopolymerized PdCz and PdTC films and ECD employed a copolymer (P(dCz2-co-dTC2)) as anodic polymer film gave rise to a better long-term electrochemical stability than those of homopolymers (PdCz and PdTC).The long-term electrochemical stability of PdCz/PProdot-Meh cycles . From thco-dTC1), P(dCz2-co-dTC2), P(dCz1-co-dTC2), and PdTC) were prepared using electrochemical copolymerization. Our experimental studies display that dCz- and dTC-containing copolymer electrodes showed multichromic behaviors and the electrochromic switching properties and coloration efficiency of the copolymers could be adjusted by various feed monomer molar ratios. P(dCz2-co-dTC1) film was yellowish green, gray, and purplish grey at 0.0, 0.6, and 1.1 V, respectively, whereas P(dCz2-co-dTC2) film was yellowish green, greenish gray, gray, and purplish gray at 0.0, 0.6, 0.8, and 1.1 V, respectively. P(dCz2-co-dTC2) film displayed a high ΔT (57.0 %) and a high η (248.4 cm2 C−1) at 784 nm in 0.2 M LiClO4/ACN/DCM solution. Five ECDs based on dCz- and dTC-containing anodic polymer electrodes and PProdot-Me2 cathodic polymer electrodes were built and electrochromic properties were characterized. P(dCz2-co-dTC2)/PProdot-Me2 ECD exhibited a high ΔT (45.8 % at 580 nm) and high long-term electrochemical stability, whereas P(dCz2-co-dTC1)/PProdot-Me2 ECD exhibited high η (562.4 cm2 C−1 at 580 nm) and a rapid switching time (≤0.3 s). In view of the above studies, dCz- and dTC-containing copolymers are amenable for use in ECDs.Five ECDs’ polymer electrodes (PdCz, P(dCz2-