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


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


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


Informed by the poststructuralist theory, this study investigates the case of Ming, a Chinese professor of English, about the impacts of his first language (L1) and second language (L2) learning experience, and the changes of social contexts on his L1 and L2 identities construction. It was found that being a learner of English as a Foreign Language (EFL), Ming’s identities development aligned with the poststructuralist theory, in which it is considered dynamic, fluid and conflicting. Ming negotiated and renegotiated his identities in various social contexts in China and the United States and finally gained acceptance into the L2 academic community. This study not only analyzes Ming’s experience with his language learning and identities, but also unravels that conflicts may be part of the process of identities construction, and encourages learners to be persistent and emotionally resilient, while using certain strategies to retain a stable L1 identity so that they can navigate through the negative encounters during the second language acquisition (SLA) process to sustain the development of their identities and L2 abilities. The relationships between social contexts, language and identities, and how they affect the learners’ second language acquisition (SLA) have been well documented in the literature. For instance, Traditional frameworks such as the acculturation model embraced the monolingual model, which unrealistically oversimplifies the process of L2 learning and disregarded the learners’ heritage language and culture. However, the exponential increase in mobility and globalization in the last two or three decades has resulted in greater diversity in various communities. English as a lingua franca also seems to gain its momentum, and many people from non-English speaking countries began to learn English. The monolingual model can no longer reflect the realities of the world today. As such, more recently, several researchers argued against these frameworks with the poststructuralist model, which captures the complexity of L2 learning and renders the learning outcome unpredictable. For example, Based on the previous literature, there seems to be a need for more research on the complexity of how L1 and L2 learning affect the learner’s identities construction. In addition, there is a paucity of studies on the development of the learner’s identities associated with changing social contexts, which can be operationally defined in this study as the different residence countries, school settings, communities, circles of friends and family of an English as a Foreign Language (EFL) learner.In order to better understand L1 and L2 identities construction along this line of research, the current study examines the case of Ming, a Chinese professor of English, about his L1 and L2 learning experience, L1 and L2 identities construction, and how language related experience and social contexts positively or negatively impact both identities.The theory of poststructuralism has been widely accepted in the academic community. Previous empirical studies substantiated the poststructuralist framework in learner identity and learner identity construction. Although English learners’ L2 identity construction may be independent of that of their official language in their home country, the process of their L2 identity construction may be difficult and complex. Therefore, a number of researchers conducted case studies investigating how L2 students negotiate their identities in an attempt to gain access to their L2 community. In more recent literature, scholars continue conducting similar studies, or updating their theories to gain a deeper insight into English learners’ L2 identity construction, especially how L2 learners negotiate their identities, the techniques they use and how they may mitigate such struggles. The research conducted on learner identity and its development supported the advocacy of multi-competencies and multilingualism. Furthermore, While previous studies unfolded the L2 learners’ dynamic negotiation of identities, L2 competence, and power positions in diverse contexts to gain legitimate membership in the L2 and international communities, the complexity of how L1 and L2 learning experience impact the construction of L1 and L2 identities, and how both identities are affected by social contexts, are yet to be fully explored. The current study aims to shed lights on these issues by conducting a case study on a highly proficient Chinese professor of English, Ming, who was born and raised in China but went to the United States for his MA and Ph.D. studies. The case study seeks answers to the following research questions:1)To what extent did Ming’s L1 and L2 learning experiences affect his L1 and L2 identities construction in different social contexts of China and the United States?2)How had Ming’s different social contexts in China and the United States impacted his L1 and L2 identities development?This study took a qualitative case study approach, with Ming as our focal participant. The case study approach was used in this research because of its many advantages. For example, Ming was born and raised in a southern province of China and excelled in his Chinese and English, from a young age. After graduating with a BA in English in China, he prepared for the TOEFL and the GRE on his own and achieved nearly perfect scores, with 110 on TOEFL (its scores range from 0 to 120) and 98th percentile on the GRE. He went to the United States and stayed there for 10 years to pursue his MA and Ph.D. degrees in English. Upon graduating from his Ph.D. program, he started teaching and conducting research at a university in China. His L2 learning experience and identities of L1 and L2 might have been impacted because of a change in social contexts from China to the United States in general. More specifically, his social contexts in this study encompass different countries, school settings, communities, the circle of friends and family, etc. Therefore, he was chosen for this case study. A summary of Ming’s profile is presented below.Two different data sources, namely, Ming’s written autobiographical narrative and a semi-structured interview, were employed in this study. Both were done in English. Five guiding questions see  were genIn order to investigate Ming’s L1 and L2 learning experiences and identities construction, his narrative data were analyzed chronologically, based on three critical time periods. Each of the time periods is of significance to his L1 and/or L2 identities development. The first period occurred in the context of a high school and a university in China, where his L1 and L2 learning experiences as well as L1 and L2 identities construction began. The second period impacted his L1 and L2 identities development as his social contexts changed, when he first arrived in a mid-west city in the United States for his MA study. This is the period when he experienced the most struggles with his L1 and L2 identities. In contrast, the third period witnessed how Ming mitigated such conflicts and how he further developed both of his identities during his Ph.D. study in a large metropolitan city on the east coast of the United States. To deepen the analysis and triangulate the data, follow-up interview data were transcribed, coded, analyzed and presented based on the three time periods in the findings as follows: (1) L1 and L2 learning experiences and related language identities construction in China; (2) Initial L2 learning experiences in a mid-west city of the United States and language identities struggles; and (3) Reconciling L1 and L2 identities during the Ph.D. program in a large city of the east coast of America. Moreover, the poststructuralist theory was used to provide a deeper analysis of this case study. Member check was conducted with the participant to ensure accuracy and validity of data analysis.It is necessary to analyze Ming’s English learning experiences from three different time periods chronologically in order to capture his changing L1 and L2 identities. Ming’s profile and English learning are summarized in As Ming grew up in a multilingual environment with exposure to different dialects, he considered himself a multilingual Chinese. It seemed that at that time, his L2 identity was much weaker as compared to his L1 identity, since it was only “a foreign language” to him. Nevertheless, his L2 identity developed from a beginner of English in junior high school to an advanced learner at university:Excerpt 1 from narrativeAt the age of 11, I began to learn English as a foreign language in junior high school…I became an English major in college and studied the language, culture, and pedagogy of English. Apparently, I am a native speaker of Chinese… English is just a foreign language to me.”“…As an avid reader and skillful writer in his L1, Chinese, Ming believed that some of his L1 reading and writing skills were positively transferred to his acquisition of L2 English, which boosted his confidence in reading and writing in English and subsequently reinforced his L2 identity.Excerpt 2 from narrative“My solid foundation in Chinese literacy contributed a lot to my acquisition of English literacy later on which increased my confidence in the English language immensely.”When Ming arrived in a mid-west city of the United States for his MA study, he still considered himself an English learner:Excerpt 3 from narrative“I considered myself a lifetime English learner. In the MA program, I read on average 300 pages English materials per week, and delivered six to eight presentations and wrote four term papers  every semester, plus a thesis in the 4th semester. Meanwhile, I was watching English TV and interacted with Americans on a daily basis. the language environment changed to English. most of my professors and classmates were Americans.”Excerpt 4 from interviewmy English improved a lot. In the second semester of my MA program, I wrote a critical and insightful book review in English and published it in a refereed journal. Then I published another paper in my 3rd semester. At that time, none of the American students in my MA cohort published any papers.”“…He was developing his L2 identity in the United States. Meanwhile, he reached out to various Chinese and American discourse communities, and retained his L1 identity.Excerpt 5 from narrativeHowever, my roommate was a Chinese graduate student and I called my family once a week. In addition, I regularly interacted with people in the Chinese community. I was able to retain my L1 self in the United States.”“… However, such developments of his identity were not without struggles and conflicts.Even though he was able to gain acceptance into both the American and the Chinese communities in the United States, the communities themselves were separated from each other. He felt as if two people, namely, his L1 self and his L2 self, were trapped living within one body. Moreover, Ming also felt that his L1 identity threatened his L2 identity at times, and vice versa:Excerpt 6 from interview“When Americans were hostile toward my L1 self, it impeded my L2 learning, because I felt like I was not eligible to speak English. One day, I was doing grocery shopping at a local supermarket in that mid-west city. I was talking to my Korean friend about some random stuffs. All of a sudden, an American guy walked by and said to me “You are Chinese, why do you speak English? That’s my language!” I did not confront him or argue with him. Somehow I felt that he owned the English language and I did not have the right to use it. It was a horrible feeling.”Another hurdle that Ming needed to overcome was the unfamiliarity of American culture, for instance, the background knowledge of jokes. A lot of the time, when the professors said something funny in class, he did not know what was going on when everyone else was already laughing out loud.Excerpt 7 from interview“Sometimes, when talking to my American classmates, they were also joking too , but I did not get the joke and had no clue what they were talking about. Those were embarrassing moments.”He also encountered problems when interacting with Americans.Excerpt 8 from interview“Chinese people may say “have you eaten?” as a way to say “hello” to each other. Of course, I was not stupid enough to say that to greet my American friends. However, I was not used to saying “how is it going?” or “what’s up?” yet when I bumped into my friends in the street or on campus. Instead, I greeted them in the Chinese way by saying “where are you going?”. Guess what? Some Americans responded “none of your business.””Apparently they were offended by what he said. Americans seem to have a strong sense of boundaries between each other. They value privacy and try to mind their own business. He stumbled over cultural issues like that quite a bit.On the other hand, after living in the United States for about a year, Ming began to encounter issues with some friends back in China. After he went to the United States for graduate study, he barely hand wrote Chinese anymore. As a result, his ability in Chinese writing decreased. Even though his everyday Chinese was not much affected, he sometimes had trouble expressing academic stuffs in Chinese.Excerpt 9 from narrative“One day, I was talking on the phone in Chinese to a friend in China and he asked me about the format of a research paper. I could not think of the Chinese words for “double spaced,” so I just said the English words, which he did not understand. He was offended and screamed at me: “Why do you have to show off your English? Do you think you are all that?”. Things like that happened to me a few times.”Even though it was not his intention to say English words to his friends in China, his L2 identity still posed a threat to his L1 self and caused miscommunication with his Chinese friends.After the master’s program, Ming moved to a large city on the east coast for his Ph.D. degree. There, his academic English further improved. He was serving as a research assistant and later a teaching assistant at university where he interacted on a daily basis with students and colleagues. During his doctoral study, he published quite a few papers in international refereed journals.He was also able to maintain his L1 identity, especially when his American undergraduate students and his American colleagues highly respected him and his L1 identity, and welcomed him into the academic community. Perhaps it was because the university was located in a metropolitan city with a quite diverse population, people tended to embrace diversity more, and incidents like the one in the supermarket did not happen to Ming again.Excerpt 10 from narrative“For some reason, some of my American undergraduate students and professors in the doctoral program preferred to call me by my Chinese name Ming rather than my English name Tom. I felt that they respected me for who I am and valued my Chinese cultural background.”His confidence in his L2 continued to grow as he published more journal articles and excelled in his doctoral study. He finally found a way to accept both his L1 and L2 identities, and became a multilingual, multicultural individual.Excerpt 11 from interview“In the Ph.D. program, I got straight A’s. I remembered a professor of the Ph.D. program was a well-known tough grader. He rarely gave A’s to his students and his tough feedback even made some doctoral students cry. I survived his class and got an A for active participation in class discussions and writing compelling term papers.”When people doubted his L2 identity, he seemed to be able to fall back on his L1 identity, which was more stable and comforting to him. His knowledge about World Englishes also helped.Excerpt 12 from interview“Sometimes people in my neighborhood picked on my Chinese accent and said that You don’t have a perfect American accent. How can you teach English then?”Ming was bothered by those negative comments at the beginning, but then as he read more literature on World Englishes, he knew well enough to tell those people that the monolingual model of English learning was problematic and unrealistic. In addition, as a lingua franca, English is being used world wide, instead of by people from English speaking countries only. Different varieties of Englishes are being redefined by researchers. His Chinese accent just shows that he was speaking a variety of English, which he was feeling comfortable with.Furthermore, Ming was inspired by some well-established scholars in his field, who originally came from Asia but were working in the United States. They shared similar cultural backgrounds and probably went through similar struggles to fight prejudice as well.Excerpt 13 from interview“In addition…there were quite a few Asian researchers…(who) were leaders in the field. I looked up to them as my role models and thought that my Chinese accent did not matter as long as I kept publishing papers and that should not be an obstacle to my career. Instead, my L1 self and culture were an asset in my research and teaching.”Finally, Ming no longer thought his L1 and L2 identities were conflicting. Instead, both were assets.Below is a summary table of Ming’s L1 and L2 identities construction in different social contexts, reflecting the intricate relationships among his language learning, social contexts and identities construction.Ming considered himself a life time English learner, and kept expanding his L2 identity in these three critical periods of his L1 and L2 learning. In the beginning, Ming’s L1 and L2 identities developed separately in China. Although his identity became a struggle when Ming first arrived in the mid-west of the United States for his MA study, during his Ph.D. study, his L1 and L2 identities converged and he was finally feeling comfortable with his identity as a multilingual. Ming was an additive bilingual. He moved to the United States after he graduated from university in China. By that time, his L1 identity was already fully developed. He managed to retain his L1 identity throughout his stay in the United States. Thus, his L2 identity was additive to his L1 self.The construction of Ming’s L1 and L2 identities can be explained by Norton’s poststructuralist theory. His identities were fluid, dynamic and changed depending on the contexts. For example, during Ming’s MA study, his L1 and L2 identities were dynamic and fluid. When he was in school, he may present his L2 self to his professors and peers. However, when he was at home or talking to his family in China over the phone, his L1 self dominated. The struggles and challenges Ming went through were not only due to cultural gaps between the east and the west, it was also because of the power relations. For instance, when Ming could not think of the Chinese word for “double spaced,” he spoke English to his friend in China (who didn’t speak English), which made his friend angry. This is mainly because English is at the top of the language hierarchy, and the fact that Ming spoke English unintentionally put his friend at a disadvantage. It was after going through different changes in contexts and difficulties that Ming successfully developed his L2 identity.Other findings show Ming had much positive L1 transfer to his L2 literacy, hence he looked at his L1 identity positively. Such may be due to his social context (a high school in China) at the time that allowed him to construct his L1 identity with ease. Furthermore, it seemed that Ming’s L1 identity conflicted with his L2 self after he went to the United States for his MA study. This was due to his social contexts at the time. He was studying in an MA program in a mid-west city in the United States. The culture in the mid-west is relatively conservative, being less liberal than that in the west and east coasts. Even though Ming was well accepted by both the Chinese and the English communities at that mid-west university [this is consistent with Ming’s struggles in his L2 identity are consistent with the previous studies e.g., . SpecifiThis is similar to the research conducted by Moreover, Ming’s Ph.D. study and continued publications  helped him gain confidence in his English and L2 identity. The fact that his professors, and peers valued his diverse background helped to nurture his identity as “a multiple and a dynamic social construct” , p 56. Hheld dear” in his “heart.” He also expressed that his “favorite food is still Chinese dishes.” While existing studies promote multilingualism, and advocate against acculturation and monolingualism, it is essential to point out that although Ming was not fully acculturated, he became more inter-culturally sensitive, and embraced diversity. This implies that although English learners do not have to fully accept the L2 culture and lifestyle, it may be beneficial for them to have an open mind and to be more inter-culturally sensitive, so that they may interact with their L2 communities easily.Furthermore, Ming was raised in a multilingual environment with exposure to different dialects. Consequently, he thought of himself as a multilingual, which may have enabled him to maintain such an identity in the United States, even when his L1 and L2 identities conflicted with each other. Contrary to Ming’s autobiographical narrative and interview data seemed to align with the conceptual framework of the poststructuralist theory, described by Furthermore, it is equally important to recognize that not all discourse communities augment Ming’s struggles with his identities. Some  may nurture and fast-track his L2 identity construction and encourage both of his L1 and L2 identities to co-exist and thrive.Ming’s experience revealed important implications on English learners’ identity construction and sustainable development. First, there may be a relationship between students’ L1 and L2 identities and L2 learning. Various researchers studied the socio-contextual framework of L2 learning  of the learner identities construction. There may be others who may have different experiences in their English learning and their identities construction. For example, some may have moved to an English speaking country at a younger age, at an older age, or may be born in that country. Some learn L2 in an L2 speaking context whereas others may learn it in an L1 environment. Nevertheless, they are also multilinguals/bilinguals who may have a unique point of view on various pertaining topics. Therefore, it may be important for one to acknowledge, respect and embrace such diversity, instead of giving preference to one “type” of multilinguals/bilinguals. Findings of various studies  may offer empirical data to substantiate or challenge existing theories, or develop new theories and techniques to alleviate the challenges in students’ L2 identity construction, and contribute to the sustainable development of students’ L2 skills.For the above reasons, more empirical research (case studies and survey plus interviews) should be conducted to provide stakeholders with a more realistic and panoramic picture that reflects the complexities of L2 learners identities construction, the potential opportunities and the role of learner agency as a result of sustaining the development of their L2 identity and creating their multilingual identity.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Foreign Studies College, Hunan Normal University. The patients/participants provided their written informed consent to participate in this study.YX was responsible for the conceptualization, most of the methodology and editing. AZ contributed to the first draft of the manuscript. Both authors contributed to the data collection, revising, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We employ graphene microelectrodes fabricated on PET substrate in chronic in vivo lifetime. We demonstrate stable chronic recordings of cortical potentials in multimodal imaging/recording experiments using transparent graphene microelectrodes fabricated on PET substrate. On the other hand, graphene microelectrode arrays built on SU-8 substrate exhibit extensive crack formation across microelectrode sites and wires after one to two weeks, resulting in total failure of recording capability for chronic studies.Our results show that graphene microelectrodes fabricated on PET substrate work reliably after 30 days accelerated aging test performed at 87 °C, equivalent to 960 days in vivo experiments.PET shows superior reliability as a substrate for graphene microelectrode arrays for chronic Graphene is a unique neural interface material enabling cross-talk free integration of electrical and optical recording and stimulation techniques in the same experiment. To date, graphene-based microelectrode arrays have been demonstrated in various multi-modal acute experiments involving electrophysiological sensing or stimulation, optical imaging and optogenetics stimulation. Understanding chronic reliability of graphene-based transparent interfaces is very important to expand the use of this technology for long-term behavioral studies with animal models. I.in vivo experiments [Electrophysiology has been one of the major tools for basic neuroscience research and clinical practice for decades. There have been vast technological advances in electrode technologies, allowing for high-temporal resolution recording of neuronal spikes and local field potentials generated by neuronal ensembles. Optical techniques such as multi-photon imaging has opened new avenues for high-spatial resolution mapping of neural activity from hundreds of neurons , 2. Comberiments –8. Grapheriments , high coeriments  and bioceriments , 12, makeriments .in vivo chronic animal experiments on mice with simultaneous 2-photon imaging and electrical recordings using the PET graphene electrode array and evaluate the stability of the electrode impedance and the recording noise over time.Transparent graphene arrays can be fabricated on different types of flexible polymer substrates, which exhibit optical transparency in wavelength ranges from 450 nm to 850 nm, commonly used in optical imaging and stimulation experiments. Different types of polymers including polyimide , SU-8 1, PET 5,, ParylenII.A.We fabricated 16-channel graphene microelectrode arrays with a 100 μm electrode size and 400 μm spacing on SU-8 and PET substrates –d. BesidB.s mimics the series resistance of the solution, the constant phase element (CPE) represents the Helmholtz double-layer capacitance (Cdl), the bounded Warburg element (Wb) simulates any diffusion processes occurring, and Rct is the charge-transfer resistance, which simulates any faradaic reactions [Both graphene microelectrodes fabricated on a PET substrate and SU-8 substrate were characterized using electrochemical impedance spectroscopy (EIS) in 0.01 M phosphate buffered saline (PBS) to study the graphene/electrolyte interface –d. Impedeactions . Represeq [dl. Computational values of Cdl (around 10 μF cm−2) are larger than computational values of Cq (around 1–4 μF cm−2) [dl is experimentally measured using Au electrodes [q is computed by fitting EIS results to the equivalent circuit model shown in ct and Wb values are both large, so the electrode behaves capacitively, which is consistent with well-known characteristics of graphene.Graphene gives rise to an additional quantum capacitance component represented by Cq , 21 in sμF cm−2) . Cdl is ectrodes , and Cq C.in vivo lifetime for implantable devices [ref). For our study, Tref is set at 37 °C, for that is the average body temperature of mice.Accelerated aging tests have been used to simulate  devices –27. The  devices . ΔT is ein vivo environments, we suspended the microelectrode arrays in a glass beaker and immersed them in 0.01 M PBS at pH 7.4, a buffer solution commonly used in accelerated aging tests due to the constant pH level [in vivo. Four graphene microelectrode arrays fabricated on a PET substrate and four graphene microelectrode arrays fabricated on an SU-8 substrate were used for a 30-day accelerated aging test. The impedance of working channels was tracked every three days for PET and every day for the SU-8 arrays which showed early failure.To simulate pH level . The setpH level , and theD.2) was made over the left hemisphere and the graphene microelectrode array was implanted to cover visual cortices. A glass imaging window was placed on top of the electrode array and further secured with vetbond (3 M) and dental acrylic. To protect the array while the mice were in the home cage, a custom 3D-printed case was employed. Electrophysiological data were recorded by the RHD2000 amplifier board and RHD2000 evaluation system (Intan Technologies), which has a large input impedance which allows us to achieve high SNR with our relatively high impedance electrodes. It is important to note that relatively high impedance of graphene electrodes necessitates recording systems with large input impedance to prevent signal attenuation. The data was processed using custom Matlab code.All procedures were in accordance with protocols approved by the UCSD Institutional Animal Care and Use Committee and the guidelines of the National Institutes of Health. Adult mice (cross between CaMKIIa-tTA (JAX 003010) and tetO-GCaMP6s (JAX 024742), 2 months old) were anesthetized with isoflurance (3% for induction and 1% for maintenance) and a circular piece of scalp was removed. A custom-built head-post was implanted to the exposed skull (1 mm posterior to lambda) with cyanoacrylate glue and cemented with dental acrylic . A stainless-steel screw  was implanted over right olfactory bulb as reference. A square craniotomy  exhibited visual PBS permeation between the PET substrate and SU-8 encapsulation layer . Liquid B.All four graphene microelectrode arrays fabricated on SU-8 substrate succumbed to crack failures prior to the conclusion of the 30-day accelerated aging test. The average impedance of SU-8 arrays over the 30-days is plotted along with the standard deviation in A summary of all the arrays detailing equivalent lifetime age and failure mechanisms is listed in Some studies in literature have suggested SU-8 as a reliable material for neural applications , 35, 36 C.in vivo studies. Therefore, for chronic in vivo experiments, we only used transparent graphene microelectrodes fabricated on PET substrates. To further evaluate the reliability of the transparent graphene microelectrode arrays in chronic imaging and recording experiments, we implanted the graphene microelectrodes over the visual cortex of transgenic mice. The transgenic mice express GCaMP6s [Based on the results of the accelerated aging tests, transparent graphene microelectrode arrays fabricated on a PET substrate were determined to be more reliable for chronic  GCaMP6s , which i GCaMP6s . Similar2) positioned 15 cm away from the right eye using Psychtoolbox [2. The neuron cell bodies are clearly identified due to the high transparency of the graphene electrodes and PET substrate (in vivo experiments combining electrophysiological recordings with 2-photon microscopy.In addition, we observed stimulus-induced responses in multiple ECoG channels, showing delayed on-responses and elevated signal power across wide frequency bands , 6e. Durhtoolbox . One of ubstrate . Our resIV.in vivo lifetime. On the other hand, graphene microelectrodes fabricated on SU-8 substrate exhibited failures due to different forms of crack formation across the electrodes or wires. We further demonstrated the reliability of graphene microelectrode arrays fabricated on PET in chronic 45-day in vivo studies in mice. After 45 days, graphene electrodes still exhibit low noise and impedance and can reliably record ECoG during calcium imaging. Our studies conclude that compared to other substrate materials, PET shows superior reliability and is therefore an ideal substrate of the graphene microelectrodes arrays for chronic in vivo studies, combining simultaneous electrophysiology and optical imaging.We performed systematic accelerated aging tests to investigate reliability of transparent graphene microelectrode arrays fabricated on PET and SU-8 substrates. Graphene microelectrodes fabricated on PET substrate show high reliability with all functioning channels after 30 days of accelerated aging or 2.63 years equivalent supp1-2979475


The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C‐terminal fragments of the protein TDP‐43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS‐PRM) can generate a high‐resolution map of pathological TDP‐43 peptide ratios to form the basis for quantitation of abnormal C‐terminal TDP‐43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p‐TDP‐43, p‐tau, alpha‐synuclein, and beta‐amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full‐length and truncated (disease‐specific) TDP‐43 peptides. This informed the synthesis of heavy isotope‐labeled peptides for absolute quantification of TDP‐43 by MS‐PRM across 16 ALS, 8 Parkinson’s, 8 Alzheimer’s disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C‐terminal fragments in ALS‐TDP urea fractions. Subsequent MS analysis resolved specific TDP‐43 N‐ and C‐terminal peptides, including a novel N‐terminal truncation site‐specific peptide. Absolute quantification of peptides by MS‐PRM showed an increased C:N‐terminal TDP‐43 peptide ratio in ALS‐TDP brain compared to normal and disease controls. A C:N‐terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6–100) and specificity of 100% (CI 68–100), and from Parkinson’s and Alzheimer’s disease with a sensitivity of 93% (CI 70–100) and specificity of 100% (CI 68–100). N‐terminal truncation site‐specific peptides were increased in ALS in line with C‐terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N‐terminal ratio but coexistent limbic TDP‐43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP‐43 fragments in human brain, which could form the basis for an antibody‐free assay. Our methodology has the potential to help clarify if specific pathological TDP‐43 peptide signatures are associated with primary or secondary TDP‐43 proteinopathies. The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C‐terminal fragments of the protein TDP‐43. Here, we used a high‐resolution map of TDP‐43 peptides to form the basis for absolute quantitation of abnormal C‐terminal TDP‐43 fragment enrichment. The C:N‐terminal peptide ratio is increased in ALS brains and N‐terminal truncation‐specific peptides confirm the pathological cleavage of TDP‐43 in ALS and surprisingly AD. TDP‐ as CTFs , 5. Alth as CTFs , 7, biociofluids . Studiesiofluids . Limitatiofluids , 12, 13.post mortem tissue. The detection of additional N‐terminal truncation site‐specific peptides was used to quantify the process of pathological cleavage of TDP‐43 into N‐terminal truncated CTFs. This method specifically discriminated ALS from other neurodegenerative diseases such as Parkinson’s disease (PD) and AD. Interestingly, by this method we were able to identify some cases of AD with coexistent LATE neuropathological change (LATE‐NC); however, further work is required to establish whether our method is suitable for the identification of disease‐specific “TDP‐43 strain signatures” associated with the spectrum of primary and secondary TDP‐43 proteinopathies.We aimed to develop an antibody‐independent method to biochemically detect CTFs by mass spectrometry to enable the absolute quantification of pathology. Detecting specific C‐ and N‐terminal TDP‐43 peptides allowed us to measure the pathological accumulation of C‐terminal TDP‐43 relative to its N‐terminal end in urea fractions extracted from TDP‐43 pathology positive ALS 22.1Primary motor cortex (BA4) or, if unavailable, dorsolateral prefrontal cortex (BA9/46) and spinal cords from individuals diagnosed with ALS, PD or AD, and from normal controls (CTL) were obtained from the Oxford Brain Bank. Donors had provided written informed consent for brain donation and the use of the material and clinical information for research purposes under Research Ethics Committee approval .2.2Post mortem delay was calculated in hours from time of death to fixation and the age at death was recorded. Genetic testing was only conducted in the presence of a clear family history.For several ALS cases severity of physical symptoms was classified as per the revised ALS Functional Rating Scale (ALSFRS‐R) with a maximum of 48 points. Here, lower values represent a more severe disease stage  and the 2.3All cases were formally assessed for microscopically visible protein aggregates using standard criteria used in international brain banking (PMC3266529). In brief, formalin‐fixed paraffin‐embedded sections (six microns) were stained with the following primary antibodies : phosphorylated TDP‐43 (pTDP‐43) that targets pS409/410 ; Anti‐ß‐Amyloid ; AT8 ; and Purified Mouse Anti‐alpha‐Synuclein . All were developed with the DAKO Envision Kit and 3,3‐diaminobenzidine tetrahydrochloride (DAB) as chromogen. Sections were examined using an Olympus BX50 microscope with an attached Olympus digital camera (for photography).2.4Fractionation of tissue was performed as described by Neumann and colleagues , with th2.5The urea fractions were diluted in LDS sample buffer (Thermo Fisher) supplemented with reducing agents (Thermo Fisher) and boiled for 5 min. Proteins were separated on 10% NuPAGE pre‐cast Bis‐Tris gels (Thermo Fisher), transferred using an iBlot2 apparatus (Thermo Fisher). Membranes were Ponceau‐stained, then blocked using protein free Blocking buffer (Thermo Fisher), and probed overnight at 4°C with a polyclonal TDP‐43 C‐terminus antibody recognizing TDP‐43 at epitopes from amino acid 260 onwards  or phosphorylated TDP‐43 at S409/410  and 2 h at room temperature with secondary antibody rabbit HRP. Antibody signal was detected with ECL reagent  using a ChemiDoc MP Imaging System (Bio‐Rad). For interpretation of the immunoblots the ratios of the intensities of the CTFs over full‐length TDP‐43 were compared.2.6Total protein amounts were visualized by silver staining using a ProteoSilver Silver Stain kit (Merck) as per the manufacturer’s protocol and using solutions provided in the kit. Briefly, after electrophoresis, NuPAGE Bis‐tris gels were fixed overnight at room temperature in fixing solution (10% acetic acid (Millipore) and 50% Ethanol (VWR) in MilliQ water). The gels were consecutively washed for 10 min in 30% Ethanol, MilliQ water, sensitizer solution, and silver solution. Staining was visualized by incubating with developer solution for 30–70 s, the reaction was stopped by addition of ProteoSilver Stop solution and washing with MilliQ water for 15 min.2.7Urea fractions were thawed on ice and used for digestion. For in‐gel digestion gel bands were excised with a scalpel after electrophoresis and gel pieces rinsed in 200 μl wash solution (50% methanol and 5% acetic acid in MilliQ water). Two‐hundred microliters of acetonitrile was added to dehydrate the gel pieces prior to reduction with 5 mM DTT and alkylation with 20 mM IAA. After dehydration samples were first rehydrated in 200 μl of 100 mM ammonium bicarbonate and then, in 30 µl of 200 ng/µl chymotrypsin (Promega) in 50 mM ammonium bicarbonate and incubated overnight. Fifty microliters of 50 mM ammonium bicarbonate buffer was added to the samples, 50 µl of extraction buffer 1 (50% acetonitrile and 5% formic acid in MilliQ water) and 50 µl of extraction buffer 2 (85% acetonitrile and 5% formic acid in MilliQ water) were added and supernatant with eluted peptides collected. For in‐solution digestion samples were reduced with 5 mM of dithiothreitol (Alfa Alsar) followed by alkylation with 20 mM IAA. Samples were subsequently precipitated by methanol–chloroform extraction. Pellets were fully resuspended by addition of 6 M urea buffer (Merck) and brief sonication. Samples were reduced to a final concentration of 1 M Urea by adding 50 mM TEAB buffer. The samples were incubated with 2 μg trypsin/chymotrypsin (Promega) at 37°C/25°C on a shaker for 14–16 h. The reaction was stopped by addition of formic acid (Millipore). Samples were desalted using SOLA SPE columns (Thermo Fisher). All in‐solution and in‐gel digested samples were dried in a speed‐vac (Thermo Scientific) and pellets were resuspended in 2% acetonitrile with 0.1% formic acid diluted in MilliQ water.2.8Peptides from in‐solution and in‐gel digestion were analyzed by nano ultra‐high‐performance liquid chromatography (nano‐UPLC) tandem mass spectrometry (MS/MS) using a Dionex Ultimate 3000 nano‐UPLC, (Thermo Fisher) coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher). Data were acquired in data‐dependent mode with a resolution of 120,000 full width half maximum at m/z 200 in the survey scan (375–1500 m/z) and with EASY‐IC using the reagent ion source (202 m/z) for internal calibration. MS/MS spectra were acquired after precursor isolation in the quadrupole with an isolation window of 1.2 Th, dynamic precursor selection (top speed mode) with a 3‐s fixed duty cycle time, and 60‐s dynamic precursor exclusion. Isolated precursor ions were fragmented by CID with a Normalized Collision Energy of 35%. Parallelization was enabled and MS/MS spectra were acquired in the linear ion trap for up to 250 ms with an ion target of 4000 in rapid scan mode. Raw MS data were analyzed using Progenesis QI for Proteomics v3.0 (Nonlinear Dynamics). MS/MS spectra were searched against the UniProt Homo Sapiens Reference proteome (retrieved 15/11/2016) using PEAKS with precursor mass tolerance of 10 ppm and a fragment ion tolerance of 0.5 Da. Carbamidomethylation of Cysteines was defined as a fixed modification, deamidation of Asparagine and Glutamine, and oxidation of Methionine as variable modifications. Peptides scoring ≥20 and FDR <1% were imported into Progenesis QIP.2.9As previously described, in‐gel digestion of low molecular weight bands (20–28 kDa) of ALS urea fractions were used to identify the truncation site‐specific TDP‐43 peptides . These p2.10For the measurement of endogenous (light) TDP‐43 peptides by mass spectrometry with parallel reaction monitoring (MS‐PRM), C‐ and N‐terminal and truncation site‐specific peptides detected from the discovery experiments and a previously reported sequence  were use2.1110 ratio (light:heavy peptide)) were compared across diagnostic groups. The C:N‐terminal peptide ratio was calculated by dividing non‐log absolute abundances of the light peptides. All significant differences were tested comparing ALS to all other diagnostic groups using one‐way ANOVA with Dunnett’s multiple comparison test. p‐values <0.05 were considered as statistically significant. Standard measures of diagnostic test validity were performed on non‐log abundances of the light peptides. Sensitivity, specificity, and predictive values accompanied by their confidence interval (CI) stated at the 95% level were calculated for varying peptide cut‐off levels. The optimal cut‐off level for dichotomizing values was selected as the situation maximizing the Youden index (For interpretation of the PRM results absolute abundances of the light peptides (logen index . The rec33.1Post mortem time from death to fixation was the same between groups (p = 0.6). Clinical characteristics of patients are given in Table All clinical diagnoses were neuropathologically confirmed and the characteristic neuropathology of each diagnostic group used for the final MS‐PRM analysis is shown in Figure 3.2First, we used LC–MS/MS analysis to detect unique peptides for the absolute quantification of TDP‐43 Figure . In‐soluFor absolute quantification of endogenous (light) peptides by MS‐PRM the corresponding heavy isotope‐labeled analogs of a list of tryptic and chymotryptic TDP‐43 peptides were generated Table  and test3.3p = 0.04) and an increased level of CTF‐25 in ALS compared to CTL, PD, and AD  in accordance with the enrichment of CTFs in ALS brain tissue   protein fractions from the same tissue cohort of 16 ALS, 8 PD, 8 AD, and 8 CTL used for PRM were performed at equal volumes to verify the recovery of protein prior to digestion and to confirm depletion of abundant proteins after sequential fractionation Figure . The uren tissue . It is n) Figure .3.410 ratio (light peptide:heavy peptide), Figure p = 0.001)  . To investigate if peptide abundances reflect the pathological increase of CTFs in insoluble protein fractions from ALS motor and prefrontal cortex tissue, we calculated the C:N‐terminal peptide ratio. The C:N‐terminal peptide ratio was increased in ALS compared to all other diagnostic groups   Figure . In cont) Figure , but una) Figure . This suTo determine the potential of using C‐ and N‐terminal peptide quantification as a diagnostic tool to discriminate ALS from other diseases, receiver operating characteristic (ROC) analysis was used Figure . A N‐ter3.5p = 0.0008 and p < 0.0001, respectively) . Only when calculating the Truncation 1: N‐terminal peptide ratio an increase in ALS compared to AD was observed (p = 0.04) caused by the respective higher N‐terminal peptide abundance in AD.To specifically quantify N‐terminal truncation of TDP‐43, to further proof the pathological truncation of TDP‐43 in smaller CTFs, the truncation site‐specific peptides were measured within the insoluble protein fraction from motor and prefrontal cortex tissue. Quantification of truncation site‐specific peptide 1 (Truncation 1) showed an increase in ALS motor and prefrontal cortex urea fractions compared to PD and CTL  Figure . SurprisROC analysis showed that a Truncation 1 peptide abundance >0.005 yielded the optimal discrimination between ALS and PD, with a sensitivity of 100% (CI 67.56%–100.0%) and 100% specificity (CI 56.55%–100.0%) Figure . Applyinp = 0.001 and p = 0.01, respectively) , PD, and CTL (p < 0.0001 and p = 0.0001). No significant difference between ALS and AD was observed when calculating the Truncation 2: N‐terminal peptide ratio (p = 0.79). According to this, ROC analysis demonstrated a Truncation 2 peptide abundance >0.004 yielded the optimal discrimination between ALS and PD with a sensitivity of 100% (CI 70–100) and 100% specificity (CI 18–100)  showed a significant increase in ALS motor and prefrontal cortex urea fractions compared to PD and CTL  Figure , but Tru) Figure . ApplyinIn summary, quantification of the TDP‐43 peptide signature in motor and prefrontal cortex urea fractions showed that in ALS the C:N‐terminal peptide ratio and truncation site‐specific peptides were increased in the presence of TDP‐43 neuropathology. In contrast, in AD motor cortex where TDP‐43 neuropathology is microscopically absent the C:N‐terminal ratio was normal, but truncation site‐specific peptides were similarly increased Figure .3.6To investigate, if an increase of C:N‐terminal peptide ratio and truncation peptides also represent pTDP‐43 pathology we immunoblotted motor cortex urea fractions of ALS and AD cases with the highest MS‐PRM values compared to PD and CTL cases. We observed the highest immunoreaction for pTDP‐43 at 45 and 25 kDa in ALS followed by AD with LATE‐NC, while pTDP‐43 was absent in PD and CTLs with low MS‐PRM values Figure . As expe3.7Due to the biochemical evidence of pathological TDP‐43 truncation in AD motor cortex samples using highly sensitive PRM, we sought to investigate if this finding represents the coexistence of LATE‐NC in significant cases of AD. Therefore, we re‐examined our cohort for microscopic evidence of pTDP‐43 pathology. We found evidence of LATE‐NC in six out of eight cases of AD, but not in PD, ALS, or controls confirming the sensitivity of our assay. The principal anatomical distribution of pTDP‐43 in LATE‐NC was different from that typically found in ALS, mainly involving the amygdala, entorhinal cortex, and hippocampus, but not the motor cortex Figure .3.8p = 0.0001) , PD, or AD (both p = 0.9)   Figure . This su) Figure . When ca) Figure . Truncat4Post mortem validation of TDP‐43 pathology currently relies on qualitative rather than absolute quantitative data . Professor Benedikt Kessler, Dr. Mattéa Finelli, and Mr Connor Scott have nothing to report.Conception and design of the study: RF, EG, BMK, KT, and MRT; Acquisition and analysis of the data: EF, PDC, MJF, CS, OA, and RF; Drafting a significant portion of the manuscript or figures: EF, PDC, MJF, CS, BMK, RF, EG, KT, and MRT; Review and editing: RF, BMK, EG, KT, and MRT; Funding acquisition: MRT, EF, BMK, and PDC; Resources: RF, BMK, OA, and MRT; Supervision: RF, EG, KT, and MRT. All authors critically reviewed the manuscript and approved its submission.Supplementary MaterialFIGURE S1 Neuropathology of the study cohort. (A) ALS‐TDP with granular‐, skein‐ and neuritic pTDP‐43 aggregates in the motor cortex (layer three) and nucleus hypoglossus (inset). (B) PD with alpha‐synuclein‐positive Lewy bodies, granular deposits and Lewy neurites inthe substantia nigra (inset: pigmented dopaminergic neuron with Lewy body H&E). (C and D) classical AD with a neuritic beta‐amyloid‐positive plaque (C) and neurofibrillary tangles in the granule cells of the hippocampus (D) . Scale bar 20 μmFIGURE S2 Silver stains of insoluble and soluble protein fractions. Representative silver stains demonstrate the total protein amount of (A) 0.5 μl of the soluble (TX) protein fractions. (B) 1 μl of the insoluble (urea) protein fractions extracted from cortex and spinal cord post mortem tissue of CTL, ALS, PD and AD patients. Depletion of abundant proteins is demonstrated in urea fractionsFIGURE S3 Skyline export of light and heavy peptides for PRM analysis. Representative Skyline exports shown with Savitzky‐Golay  retrieved from a pooled ALS cortex urea fraction sample. The top row represents light peptides and the bottom row heavy isotope‐labelled peptides, where different coloured graphs represent fragment ions. (A) N‐terminal chymotryptic light and heavy peptide. (B) C‐terminal chymotryptic light and heavy peptide. (C) Truncation 1 chymotryptic light and heavy peptide. (D) Truncation 2 chymotryptic light and heavy peptideTABLE S1 Detailed characteristics of post mortem tissueTABLE S2 Detection of TDP‐43 peptides from in‐solution trypsin digestions of cortex urea fractions from ALS and CTL casesTABLE S3 Detection of semi‐specific truncation site‐specific TDP‐43 peptides from chymotrypsin in‐gel digestion of cortex urea fractionsTABLE S4 List of TDP‐43 peptides for heavy isotope‐labelled peptide productionClick here for additional data file.