Standard convolutional filters usually capture unnecessary overlap of features resulting in a waste of computational cost. In this paper, we aim to solve this problem by proposing a novel Learned Depthwise Separable Convolution (LdsConv) operation that is smart but has a strong capacity for learning. It integrates the pruning technique into the design of convolutional filters, formulated as a generic convolutional unit that can be used as a direct replacement of convolutions without any adjustments of the architecture. To show the effectiveness of the proposed method, experiments are carried out using the state-of-the-art convolutional neural networks (CNNs), including ResNet, DenseNet, SE-ResNet and MobileNet, respectively. The results show that by simply replacing the original convolution with LdsConv in these CNNs, it can achieve a significantly improved accuracy while reducing computational cost. For the case of ResNet50, the FLOPs can be reduced by 40.9%, meanwhile the accuracy on the associated ImageNet increases. Convolutional neural networks (CNNs) have shown remarkable achievements in various vision tasks ,5,6,7,8.At present, the commonly used convolutions are Groupwise Convolution , DepthwiModel compression is considered as another reliable and economic method to improve the efficiency of the convolutional neural network, which can be roughly divided into three categories: (a) Connection pruning ,29; (b) Instead of directly pruning the whole model, we choose to integrate the pruning technique into the design of convolutional filters. In this way, the model can automatically learn to know which input features are most valuable for each single output, so that it enables to extract better features with fewer filters. To achieve this objective, we design a new type of convolutional filter—Learned Depthwise Separable Convolution (LdsConv), which can be directly plugged into existing standard architecture to reduce floating point of operations (FLOPs) and meanwhile improve the accuracy.To integrate the pruning methods, we develop the two-stage training framework to divide the training task into picking and combining. In the first stage, the LdsConv picks out the most valuable input features and applies more filters to them by pruning technique. In the second stage, the additional pointwise convolution combines the output of the first stage and produces the output features. The idea of division of labour and progressive working has been reflected in computer vision. For example, the two-stages detection framework  divides Our experiments show that by replacing the standard/depthwise convolution with the LdsConv in CNNs, it can improve the accuracy and reduce computational costs in the following models: ResNet , DenseNeWe integrate the weight pruning method into the depthwise separable convolutional filter and develop the two-stage training framework.We design an efficient convolution filter named Learned Depthwise Separable Convolution, which can be directly inserted into the existing CNNs. It can not only reduce and computational cost, but also improve the accuracy of the model.https://github.com/Eutenacity/LdsConv.We validate the effectiveness of the proposed LdsConv through extensive ablation studies. To facilitate further studies, our source code, as well as experiment results, will be available at Our main contributions are three-fold:Ever since the pioneering work on Alexnet  and VGG Model compression is another popular method to improve the efficiency of the convolutional neural network. Refs. ,29 removThe proposed LdsConv inserts the weight pruning process into the training. Therefore, the LdsConv embedded model is able to be trained from scratch without a pre-trained model. Different from  which onIn this section, we first introduce Depthwise Separable Convolution and LdsConv. Then we describe the details about the utilization of LdsConv. We also discuss implementation details and show how to replace Depthwise Separable Convolution with LdsConv.R, O, R filters to every input channel for each output. Thus, a standard convolution has the weight matrix with the size of H and W denote the height and the width of the filter. To reduce the computational cost, the depthwise separable convolution splits the standard convolution into two: a depthwise convolution for filtering, that only applies a single filter to the corresponding input channel for the output one, and a pointwise convolution for combing the outputs of the depthwise convolution and producing final output channels. The depthwise convolution is parameterized by the kernel of the size Consider a standard convolution that takes an Considering the strength of the depthwise separable convolution, it is highly desirable to design a more complex architecture to enhance the capability of the convolution so that the neural network can decide on which feature should be applied. In doing so, we need a novel convolution architecture, named Learned Depthwise Convolution (LdsConv). As shown in Initially, we adopt a group convolution which divides a standard convolution of size During the training process, we gradually screen out less important filters for each group. The importance of the filters is evaluated by the k with a range from 1 to 4 to represent that the number of remaining filters is k is presented, which both has a good balance of parameter and accuracy and fits all around dataset and network scale.It is important to consider and determine how many filters should be removed before the combining stage. Formally, we set a hyper-parameter In contrast to methods that prune weights in pre-trained models, our weight pruning process is plugged into the training procedure. Thus, we define the stage factor to determine the times of pruning. For a group filter weight M to ensure that filters belong to input features with a bigger number of possible remained filters would be penalized more strongly.To reduce the negative impact on the accuracy induced by pruning, we deliberately set the number of remaining filters of each input feature to be even avoid the case that most of remained filters extract information from only a small number of input features. As we know, it is hard to optimize the number of filters as they are non-differentiable. We thus define the coefficient of k. Furthermore, In each training iteration in picking stages, we first find the filters that have the highest probability to remain. Then, we check their input features to get the number of probably remaining filters of each input feature. Finally, we restrain these filters belong to input features having a big number of probably remaining filters. To this end, we use the following regularizer for a group filter weight R and output channel O of the original convolution and the pruning factor k. The initial value of the weight is set by the index information of the remaining filters. At the end of picking stages, we convert the sparsified model into a network with regular modules that can be efficiently deployed on devices without special hardware and software support. For this reason, we add additional pointwise convolutions to each LdsConv to build Depthwise Separable Convolution see . This opWe adopt the cosine shape learning rate schedule during training, which smoothly changes the learning rate, and usually improves the accuracy ,52,53. FIn addition to the use of LdsConv, we briefly describe how to replace standard convolutional filters and depthwise separable convolutional filters with LdsConv filters.When we try to replace a standard convolution with our proposed LdsConv, the most important hyper-parameter is the group cardinality In general, a pointwise convolution exists in each depthwise separable convolution. So, we do not need to worry about the problem mentioned above. In other words, we can simply replace the depthwise convolution with our proposed LdsConv. However, parameters and FLOPs may increase if we do not make any adjustments. Therefore, we suggest adding an additional convolution before or after the LdsConv to reduce the number of input or output channels of the LdsConv. The right part of In this section, we validate the effectiveness and efficiency of the proposed LdsConv. We first present ablation studies for image classification on Cifar . Then, wWe conduct a series of ablation studies to find the best situation to implement LdsConv filters and then check its robustness in different models.We use stochastic gradient descent (SGD) algorithm to train all the models. Specifically, we adopt Nesterov momentum with a momentum weight of 0.9 without dampening, and use a weight decay of k and stage factor s for the LdsConv.We do experiment with DenseNet-BC-100 architecture having a growth rate of 12  on the Ck to 2. The result shows that The first part of k, which vary from 1 to 4. In addition, we set the stage factor s to 4 which means all models have the same times of pruning. The results presented in the third part of k to 2 is a good choice which balances both the accuracy and the number of parameters. We can also reduce the pruning factor k to 1 or even integrate the additional pointwise convolution with the sequent convolution to reach a higher reduction to weights.We do experiment with several pruning factors To check the effectiveness of our balance loss function, we apply it to the models with varied pruning factors. The fourth part of s is set to 4 and the pruning factor k is set to 2 for all models. The result shows that the accuracy first rises up and then goes down. When To evaluate the effect of the group cardinality ResNet50  which isk = 2) without additional convolutions (AC) in the final part of To verify the function of each stage, we first explore the norm value of the picking results and then evaluate the effect of the additional convolution. The three panels of k to 2, the stage factor s to 4 and the balance loss function active. In DenseNet, we set its pruning cardinality To evaluate the effectiveness of the proposed LdsConv with the situation discussed in the above in different networks, we choose currently popular models as the baselines including ResNet , DenseNeIn a set of experiments, we test LdsConv filters on the ImageNet dataset.We use the SGD method to train all the models and adopt Nesterov momentum with a momentum weight of 0.9 without dampening using a weight decay of k to 2 and the stage factor s to 4. Except for DenseNet, we set the group cardinality In the experiments on ImageNet, we set the balance loss function active, the pruning factor We continue to use ResNet , DenseNeTo investigate the compressing ability of our proposed LdsConv, we adjust the bottleneck block with LdsConv in the ResNet to a extreme state as shown in To further verify the effectiveness of our approach, we do several experiments using three different networks, namely, ND , FLGC 4 and GDCoWe further apply the Grad-CAM  to modelFrom In this work, we propose a new type of convolution called LdsConv. We have compared our proposed convolutional filters with the original convolutional filters on various existing architectures. Experimental results show that our LdsConv is more efficient than existing convolutions in these models. We also have compared the LdsConv method with the FLOPs compression methods and similar motivated works. Results from our experiments show that the proposed method produces the overall best accuracy while still having competitive FLOPs.


Coral reef islands provide precious living space and valuable ecological services for human beings, and its sustainability cannot be ignored under the pressure of human activities. Carrying capacity (CC) assessment has gradually become an important means to measure sustainability of islands. However, there is little comprehensive evaluation of the carrying capacity of coral reef islands, and traditional evaluation methods are difficult to express the social-ecological characteristics of coral reef islands. The present paper proposes a comprehensive assessment model for coral reef island carrying capacity (CORE-CC) which comprises dimensions of resources supply, environmental assimilative, ecosystem services, and socio-economic supporting. According to the characteristics of the coral reef islands, the core factors and indicators of each dimension are selected and the corresponding assessment index system of "pressure-support" is constructed. The assessment involves (1) identification of carrying dimensions and core factors, (2) pressure/support measurement and (3) assessment of carrying state. A case study is conducted in Zhaoshu Island of China, demonstrating the applicability of CORE-CC model and serving as a reference for adaptive management. Coral reef islands are formed by biological debris produced by the erosion, deposition and cementation of coral sediments. As a special type of islands, coral reef islands not only provides the carrier for human habitation and the fulcrum for protecting and utilizing the ocean, but also provides valuable ecosystem services, such as food provision, coastal protection, recreational activities and numerous cultural services4. However, due to the effects of local human activities, such as overfishing, land runoff, and pollution discharge, coral reefs’ sustainability is questioned around the world5. Coral reef systems are social-ecological systems where the interaction between humans and nature is particularly complex4. Human beings are the main body in the development and utilization of coral reef resources, and at the same time they bring threats and damage to coral reef ecosystems, but they are also the promoters of the protection and management of coral reef environment6. However, the management of the ecological environment should not only be passive restoration, active adaptive management to maintain the health of the ecosystem is more important7. The core task of adaptive management is to identify and analyze the basic resource advantages and stress factors of the regional ecosystem, and propose management models and countermeasures on this basis9. Currently, some scholars tried to achieve sustainable development of coral reef social-ecological system by revealing the interactions among socio-economic and ecological environment factors or addressing the stressors and the impact pathways, few of which have applied the carrying capacity theory in coral reef islands13. Carrying capacity is a scientific concept for measuring the relationship between human socio-economic activities and the natural environment, and an important tool for measuring and managing human sustainable development14. Meanwhile, the carrying capacity analysis method combines qualitative comparison and quantitative evaluation, which can reveal the stress factors and characteristics of the regional ecosystem to a certain extent, and can provide a basis for adaptive management15. Carrying capacity can be an effective perspective to measure the sustainability of coral reef islands16.Coral reefs are among the richest and most diverse ecosystem types found in the global ocean17. Accompanied with the development and enlarging range of human activities, the research on carrying capacity has changed from researches of biotic population growth law to the comprehensive carrying capacity covering natural resource endowments and human development demands18. The scopes of carrying capacity applications have been expanded, such as land resource carrying capacity20, water resource carrying capacity22, environmental carrying capacity24, ecological carrying capacity26, social carrying capacity28 and comprehensive carrying capacity31. The progression of carrying capacity has gradually attached importance to the influence of human activity factors on the carrying capacity19. The evaluation object has gradually shifted from a single resource and environmental element to the carrying capacity for multiple or comprehensive elements32.The concept of carrying capacity (CC) derives from a demographic focus on the sustainable growth of population or livestock farming, this concept demonstrates the relationship between a population (carrying object) and the environment (carrier)33, the researches on comprehensive assessment of CC are also applied to various fields of the ocean, such as Marine ecosystem, coastal zone and islands, etc37. However, as one of the most important marine ecosystems and type of islands, case studies on coral reef island carrying capacity are still limited to a single capacity, for instance, diving tourism carrying capacity or aquaculture carrying capacity40. The social-ecological system of coral reef islands is complex, we need to comprehensively assessment its carrying capacity to better reflect the entire system’s capacity.With the development of the marine economy, human activities, coastal development and pollution have gradually changed and destroyed the capacity of marine ecosystems41, ecological footprint method42, energy analysis44, and the comprehensive indicator system46. The system dynamics model can quantitatively analyse the intrinsic relationship between the structure and function of various complex systems; however, it is difficult to select the parameter indicator, and the large quantity of data limits the application of the model and the similitude to reality47. Both the ecological footprint method and the emergy analysis method simplify the complicated processes by an equivalent term which can neither tell considerable information relevant to the complexity of coupled social-ecological systems nor sufficiently feature a specific area48. Two common comprehensive indicator systems are as follows: one is to use a multi-dimensional indicator system, select indicators from the dimensions of resources, environment, ecology, socio-economic and assume ideal values for comprehensive evaluation; the other aims to build a PSR (Pressure-State-Response) or DPSIR (Driving-Force-Pressure-State-Impact-Response) model as a system layer or distinguish between pressure indicators and support indicators50.Fortunately, the assessment methods of ordinary islands provide a reference for the comprehensive assessment of coral reef islands CC. These methods mainly include the system dynamics model51, some limitations exist. Logic feedback frameworks based on PSR or DPSIR are usually applied in qualitative evaluation due to the difficulties in data collection and weight determination52. For the multi-dimensional indicator system method, most studies divide the carrying capacity system into a resource standard, environmental standard and human society standard, and calculate the indexes of each standard layer as a whole. However, the ultimate comprehensive research results are difficult to judge whether the examined carrying capacity is overloaded50. Recently, many scholars believe that it is necessary to analyze the relationship between the support of the carrier and the pressure carried by the carrier to reflect the carrying capacity, so that the state of the carrying capacity can be more accurately judged55. But the evaluation objects are mostly single elements, such as water resources53, ecological environment54, etc., which can only provide partial understanding of sustainability55. Therefore, we can combine the multi-dimensional indicator system method and pressure-support analysis method to evaluate the carrying capacity more comprehensively and accurately. Meanwhile, in view of the commonness of the islands and the uniqueness of the coral islands in the ecological dimension, the evaluation index system and methods for the coral reef island CC should be improved accordingly.Although the comprehensive indicator system method is more flexible and more in line with the actual situationTo address these shortcomings, this paper presents a comprehensive assessment model of coral reef island carrying capacity (CORE-CC), which is composed of four dimensions: resources supply, environmental assimilative, ecosystem services and socio-economic supporting. Comprehensively evaluates the carrying state according to the corresponding relationship between the pressure and support capacity of the same core indicators. Finally, a case study is conducted in a typical coral reef island of Zhaoshu, and serves as a reference for local adaptive management.56. Therefore, it is necessary to understand the conflict between the limited support capacity of the coral reef island system and the pressure caused by the increase in population and human activities58. In other words, it is essential to check whether population and human activities exceed the capacity provided by coral reef Island to achieve the mission of sustainable development. However, the pressure brought by human activities on coral reef islands is reflected in many aspects, such as resources extraction and utilization, pollutant discharge, ecological damage caused by excessive access to ecosystem services, etc. Correspondingly, the coral reef islands system provides supports of resource supply, environmental assimilation and ecosystem service to cope with pressures. Although human socio-economic development is the source of pressures, it can improve the structure and function of the regional ecological environment through economic investment and technological innovation to increase the carrying capacity. Thus the essence of coral reef islands carrying capacity (CORE-CC) is kind of comparison between the pressure caused by human activities and the support capacity of the coral reef islands system, covering the four dimensions of resource, environmental, ecological, and socio-economic  human presence and shaping of landscapes for long time periods, leading to the development of semi natural environment to urbanization with the changes of economy and society62; (c) economy dominated by tourism, services and fisheries63; (d) it has both terrestrial and marine ecosystems, as well as a unique coral reef ecosystem16. These characteristics lead to the identification of the core factors for each dimension of CORE-CC. In addition, the word "CORE" means that the model considers the core factors of each dimension.64. Among them, water is a precious source of life65, land resources provide a foothold for human survival66, and most coral reef islands rely on imports for food and fuel64. Considering the scarcity and importance of resources, ensuring the sustainability of water and land resources is the focus of this research67. Therefore, Land resources and water resources are used herein as the core factors to measure the resources supply capacity in CORE-CC.The resources supply dimension refers to the ability of the reef islands ecological environment to provide natural resources for human activities. Coral reef islands can provide humans with resources such as land, water, food, and a small amount of fuel56. However, coral reef islands have few large industries, and at the same time, due to the fast air circulation of the coral reef islands, makes air pollution is easy to reduce. Furthermore, scarce land resources are usually not used as pollutant discharge carriers, and the seawater around the island is the main receptor for pollution discharge68. Therefore, the water assimilation is used herein as the core factor to measure the environmental assimilative capacity.The environmental assimilation dimension refers to the migration of the reef islands environment and the self-purification function of transforming pollutants. The environmental assimilation of the coral reef islands should theoretically include the water assimilation, atmospheric assimilation and soil assimilation69. Coral reef islands have the characteristics of land-sea transition patches, and their ecosystem service capacity should be considered from both terrestrial ecosystem and marine ecosystem71. Among them, the marine is dominated by coral reef ecosystem services, which is also a unique feature of the CORE-CC.The ecosystem services dimension refers to the ability of ecosystems within a certain time and space to provide services such as ecological regulation, ecological support, and ecological culture54. Its capacity depends on the level of socio-economic development and the degree of support for the sustainable development of coral reef islands72. Therefore, the socio-economic supporting capacity is measured using socio-economic development and protective initiatives as the core factors.The socio-economic supporting dimension refers to the ability of human socio-economic to improve the resources supply capacity, environmental assimilative capacity and ecosystem services capacity of the coral reef islands. Human socio-economic's support behavior includes research and development of more resources, pollution control, environmental construction, ecological protection and restoration construction, etcAs a result, it leads to differences in the economic structure and functional orientation of different coral reef islands, the types and quality of factors that CORE-CC focuses on in each dimension are also different. Therefore, it is more meaningful to select the core factors of each dimension of CORE-CC according to the common characteristics of coral reef islands. The coral reef islands are common characterized by: (a) insularity, i.e. small size, limited water resources and heavily dependent on imports problemsAccording to the analysis conducted above, the core factors of each dimension of CORE-CC are determined Table . In the In summary, the following measurable indicators of each core factor are collected. Since CORE-CC is expressed into a comparison of the safe thresholds and the pressure generated by human demand, a set of methodologies for indicators estimation are described from the ends of pressure and support. For the core factors of resources and environment, the supply of resources and the capacity of environment are taken as the support, while the actual demand of resources and the discharge of pollutants in the social economic system are taken as the pressure. For the core factors of ecosystem and socio-economic, the actual ecological services provided by the ecosystem and the actual economic input provided by the socio-economic system are taken as the support, while the expected value of a specific standard or environmental quality improvement requirements is taken as the pressure.Ci is the comprehensive ecological suitability score of the i-th evaluation unit, i = 1,2,3…m; Cij is the suitability grade score for the j-th evaluation factor of the i-th evaluation unit, j = 1,2,3…n; Wj is the weight of the j-th factor. The results of evaluation unit are classified into four levels: forbidden, limited, less suitable, or suitable. Among them, the area land of less suitable and suitable is accounting for the suitable construction land resource.This factor is measured by indicator of suitable construction land resource. Land resources have functions of production, carrying capacity, providing raw materials, saving and value-added, etc. The CORE-CC focuses on the spatial carrying capacity function of land as the basic activity place of human beings and building base, and the analysis of its supply capacity mainly focuses on construction land. The pressure end of this indicator is the developed land use area of coral reef islands, and the support end is the area suitable for construction land. The amount of land resources suitable for construction refers to the amount of land resources whose natural conditions and socio-economic conditions are suitable for construction purposes and will not adversely affect the ecological environment after development. The calculation is mainly obtained through the ecological suitability assessment of construction land. The assessment of the ecological suitability of construction land is the process of assessing the suitability of the land for various construction uses from the perspective of ecological environmental protection. The main factors affecting the suitability of the land are selected . Based on the GIS tool, a multi-factors weighted scoring method is used to calculate the appropriate construction land resources. The comprehensive ecological suitability score is obtained by using the following equations:WAR is the total available water resource, WG is available ground water, WT is water transferred in, WRe is reclaimed wastewater, and WD is desalinated sea water.This factor is measured by indicator of the available water resource that refers to the maximum amount of water resources that can be developed and used within a certain time and space without prejudice to environmental systems. The pressure end of this indicator is the annual water consumption of the total population, while the support end is the annual total available water resources, which is composed of available surface water, available ground water, trans-boundary water and wastewater reclamation. The calculation equation is as follows:73. In this paper, inorganic nitrogen (IN) and inorganic phosphorus (IP) as the main pollutant indicators. A DHI MIKE3 software was used to establish a coupling model of hydrodynamics and water quality in the offshore waters of the coral reef islands. The movement and water quality of pollutants in the offshore waters were simulated, and the response field results reflecting the relationship between the pollutant concentration and the emission sources in the near waters were obtained. In addition, the classic linear optimization method is used to obtain the maximum total pollution load of each pollution source under the premise of meeting specific water quality standards, which is used as the water assimilation capacity74. It is expressed as,i is the sea area number, j is the point source number, i, i, i, j, j, AC is the seawater assimilative capacity.According to the characteristics of the treatment of coral reef island pollutants, the water assimilation refers to the ability of the seawater around the coral reef island to keep the water body clean through the migration of water mass and degradation of pollutant concentration. The pressure end of this indicator is calculated according to the annual emissions from point sources , while the support end is estimated by the water assimilative capacity proposed in the total maximum daily load (TMDL) process, to achieve compliance with the pollutant standards75. Therefore, the LAI can approximately represent the ecological service function. For a region, the sum of average LAI for all types of land cover (Sum LAI) is often used to reflect the level of ecosystem services76. The pressure end of this indicator is the expected value of the Sum LAI to meet the functional orientation and development planning of the coral reef islands, while the support end is the actual measured value. The equation is as follows:LS is the sum LAI value, i is the land cover type, Li is the averaged LAI value of i-th land cover type, Ai is the area of the i-th land cover type, Ais is the sum area of land covers. The types and proportions of land cover can be obtained through remote sensing image supervision and classification combined with field investigation. The averaged LAI for each type of biome/land covers employ the empirical values78 listed in Table This factor is measured by indicator of Sum LAI. Leaf area index (LAI) is one of the most widely used parameters in ecological science, which is used to describe the ecological health of natural ecosystems. Most of the ecological service functions of terrestrial natural ecosystems originate from metabolic processes of plants or related processes, including photosynthesis, transpiration, and material circulation, and the leaf area plays a decisive role to a large extententclass1pt{minima79. Among which, Hard corals are reef-building and when they die the skeleton endures and creates a base for other corals to grow on. They are considered as the most important component of coral reefs because they build durable structures that provide habitat for countless coral reef organisms80. Furthermore, if the coral reef has a high biodiversity of plants and animals that perform different functions, the buffer capacity is usually higher81. Thus, this factor is measured by indicators of hard corals coverage, species number hard corals, coral reef fish density, and species number of coral reef fishes for assessment. Referring to the coral reef health status standard set by Zufikar82 and Sun83, the pressure end of these indicators is the demand value that can maintain the health of coral reef ecosystem, and the supporting end is the actual value of these indicators. These indicators are available through field surveys and remote sensing. Among them, the calculation formulas of hard corals coverage and coral reef fish density are as follows:HCC)n is the number of hard corals on each survey line, and M is the total number of points on each line.Hard corals coverage (Df)N is the total number of reef fish on the sample line; L is the total length of the sample line (m); W is the width of one side of the sample line (m).Coral reef fish density  has equal opportunities for the factors that restrict the CORE-CC, so the same weight is used for assessment. For dimension index assessment, we define the surplus ratio of the dimension index as:Di is the surplus ratio of the i-th dimension, ni is the number of core indicators of the i-th dimension index, dij is the same as above. For Di > 0, it implies the surplus state. For Di = 0, it implies the full- loaded state. Whereas for Di < 0, it implies the over-loaded state.According to the theory of regional carrying capacity vector assessment proposed by Yu and MaoD is the surplus ratio of the CORE-CC, m is the number of dimensions . For D > 0, the coral reef island is fully capable of carrying the pressure induced by human interference, whereas for D < 0, the coral reef island is over-loaded and negative to carry the pressure.For the CORE-CC assessment, combining vector of each dimension index to get a surplus ratio of CORE-CC index, which can be described as:2. It is formed by the accumulation of white coral skeletal and shell sands on a reef platform, and is about 4.4 m above sea level. There are lots of hard disk phosphorous lime soil on the island, and the vegetation is mostly Scaevola sericea. ZSI community belongs to Sang City, Hair Province, China. The residents are generally composed of fishermen, government workers and civil servants who have settled down for a long time or visited for a short time. With the gradual opening of the island's tourism policy, its tourism industry is booming and the number of tourists is increasing. Since the official establishment of Sansha municipal government in 2012, the living conditions and environment of the local residents in ZSI have been significantly improved, and the infrastructure construction of road facilities, power grid, sewer and so on is complete. With the development and utilization of coral reef islands strengthened, the pressure on the ecological environment of the ZSI is also increasing. Through the comprehensive carrying capacity assessment to provide a reference for the sustainable development of ZSI.Zhaoshu Island (ZSI) is one of the main coral reef islands of the Xisha Islands in the South China Sea. Its geographical coordinates are 16°59′N, 112°16′E, and an area of about 0.22 kmAccording to the assessment method described above, an assessment index system for the carrying capacity of the coral reefs of ZSI was established, and the carrying stage of ZSI in 2017 was evaluated and analyzed. The data of each indicator are obtained through field surveys, local government statistical yearbooks, interviews with local government staff, historical research literature, and remote sensing monitoring.The surplus ratio of each core indicator is calculated by Eq. , and the3/a and tends to have a net surplus of 44%. Although the construction intensity of civil buildings continues to increase, the suitable construction land resource remains 0.13 km2 with a 38% surplus. The seawater assimilation capacity indicator has the highest surplus degree, with a surplus ratio of 87%. This shows that ZSI's offshore water body (1 km buffer zone) has sufficient assimilation capacity to carry the discharge of IN and IP pollutants, and can ensure that the water quality meets the national optimal water quality standard.On the whole, 4 of the 11 core indicators of ZSI’s CORE-CC in 2017 were overloaded. The Sum LAI, Hard corals coverage, and coral reef fish density in the ecosystem service capacity dimension are overloaded by 4%, 145%, and 67%, respectively, indicating that both land and marine ecosystems cannot provide stable and adequate ecosystem services. Among them, the marine ecosystem is particularly overloaded, with two indicators of the highest degree of overload. This indicates that ZSI's coral reef ecosystem service capacity has been severely degraded by climate change and human activities. The R&D expenditure indicator in the social support capacity dimension is 60% overloaded, indicating that under the situation of economic development, the investment in research on ZSI is relatively lagging. As a practice of the national ecological civilization reform, the ‘Ecological Island’ project implemented by the State Oceanic Administration has a positive impact on the ecological environment of ZSI, making the environmental protection investment indicator appear surplus stage. The core indicators of the resource supply dimension are all surplus. Thanks to the establishment and operation of desalination plant, the available water resource of ZSI is 105 mBased on the analysis of the core indicators, a vector of carrying capacity surplus ratio model is used for the comprehensive assessment of CORE-CC to determine the comprehensive carrying stage and degree of each dimension and the entire coral reef islands system Fig. . In gene87, so we are not surprised that ZSI’s ecosystem service carrying state are overloaded. As an administrative division newly established in 2012, ZSI experienced a lot of reclamation activities and excessive pollution caused by intensive shipping in the initial stage of construction, which may lead to the degradation of coral reef ecosystem services. Although the ecological restoration of ZSI has been ongoing, at least it has not yet returned to the ideal state.The CORE-CC model proposed in this paper indicates the new connotation of coral reef islands comprehensive carrying capacity from the four dimensions of resources, environment, ecosystem and socio-economic. Moreover, the core indicators are selected according to the characteristics of each dimension, which is conducive to quickly determining the carrying status. The results of the comprehensive assessment highlight the overall pressures on the coral reef island, which can help local decision-making and provide information for the coral reef island’s sustainable development. The case study of ZSI showed that the model can be applied to local contexts and that is potentially useful in practice as a guide for planning. There is still a large surplus in the dimension of resource supply and environmental assimilation of ZSI's carrying capacity, but this is only because ZSI is in the primary stage of construction and the optimistic situation brought by the small population and industrial scale. At the same time, if the assessment standard of environmental assimilation capacity is changed to coral healthy water quality limit, its carrying capacity will be greatly reduced. Therefore, although the carrying capacity of resources and environmental is in surplus state, it does not mean that resources can be squandered and pollution can be discharged at will. Due to the common interference of human activities and natural changes, factors that threaten ecological resilience and health set the tone for the regional decline in coral reef ecosystem serviceIn terms of socio-economic supporting, due to the lack of significant economic benefits and good investment allocation of ZSI, the positive feedback of ecological environment is hindered. At present, ZSI focuses on the physical carrier of human survival, and gradually implements basic functions in a human-controlled manner. In the realization process of functional zones under the guidance of policies, due to the open source of production materials and construction capital, the CORE-CC of ZSI has a relatively large elastic range, and the autonomous carrying capacity is relatively weak. Maintaining a good foundation of care conditions and establishing appropriate regulations and policies for risk control are important prerequisites for enhancing the self-sufficiency of coral reef island resources and ensuring their sustainability. In terms of marine eco-civilization practice proposed by 18th CPC National Congress, timely release of priority measures to ease supply pressure, strengthen ecological restoration, and promote the optimal allocation of resources and services to ensure the sustainable development of ZSI.88, especially when the value range of the pressure end and the support end are close. Therefore, we recommend setting a certain amount of uncertainty buffer (such as 10%) as warning zone near the critical point, which means that when the result is greater than 0 and less than 10%, the carrying capacity is considered to be in a warning state. This allows society time to react to early warning signs approaching the critical point and the subsequent possible overload risk89, instead of entangled in the uncertainty of the carrying capacity state. Another type of uncertainty relates to indicator weights. Weights can indirectly integrate the views of stakeholders90 or experts' opinions on local policy priorities91, and are often used as measures of perceived importance of the subgroup to the system92. We used equal weights due to a lack of knowledge of the study area. However, in practice, the importance of various carrying capacity factors to coral reef islands with different economic structures, stakeholders and development policies may not be the same94. In this regard, we encourage development and use of weights for localised assessments to maintain the usefulness of the model in different types of coral reef islands.The comparative analysis of pressure-support indicators may help to understand the concept and mechanism of carrying capacity, but it also introduces the uncertainty of the quantification of carrying capacity. The CORE-CC model essentially takes the pressure-support index comparison result d see Eq.  equal toWe strive to have a comprehensive and versatile model for coral reef islands, but the core factors and indicators selected in this article may not cover all of them. The indicators of each dimension may be more abundant in future research, such as increasing energy supply and fishery supply. Considering the difficulties encountered in assessing the correct match between pressure and support and establishing standard thresholds, the representation of ecosystem service capacity needs to be further improved. In view of these problems, it is necessary to further explore ways to enrich the CORE-CC framework, propose an acceptable indicators and standard thresholds list, and dynamically monitor the resource consumption and ecological environment status related to social development.According to the sustainable development needs of coral reef islands and the essence of the concept of carrying capacity, this paper proposes a conceptual model of coral reef island carrying capacity (CORE-CC) from the perspective of pressure-support. The model includes four dimensions: resource supply, environmental assimilation, ecosystem services and socioeconomic supporting. According to the characteristics of the coral reef islands, the core factors and indicators of each dimension are selected and the corresponding assessment index system of "pressure-support" is constructed. Using the pressure/support comparative analysis method, the comprehensive assessment of carrying capacity was conducted with ZSI as the case study. The results show that the ZSI’s CORE-CC in 2017 still maintained a carrying capacity surplus ratio of 5%. Among them, the resource supply capacity and environmental assimilative capacity are in a good carrying state, and the surplus ratios were 41% and 87%, respectively. The ecosystem service capacity and social support capacity are both overloaded, with overload degrees of 36% and 20%, respectively. This is also the key factors that lead to ZSI’s CORE-CC nearing full load, and it must be targeted for improvement.Case studies show that the CORE-CC model is suitable for measuring the carrying capacity of coral reef islands. The measurement results not only show the carrying states of each dimension, but also provide a data basis for resource balance, total amount control and ecological construction. This will help provide a support for decision-making on the scale, structure, and layout of coral reef islands development.


Biliary metastasis of colorectal cancer is a manifestation of metastatic liver carcinoma, and is often difficult to differentiate from cholangiocarcinoma. Further, lower bile duct metastasis of colorectal cancer is rare. We report the case of a 74-year-old woman who underwent pylorus-preserving pancreatoduodenectomy for lower bile duct metastasis of rectal cancer.The patient had undergone laparoscopic low anterior resection for rectal cancer (pT3N0M0 stage IIA) 6 years ago, laparoscopic anterior liver resection for liver metastasis (Couinaud segment V) 3 years ago, and left and caudal lobectomy with extrahepatic bile duct resection for left intrahepatic bile duct metastasis 6 months ago. A follow-up examination showed a 15 mm mass in the common bile duct, for which she underwent pylorus-preserving pancreatoduodenectomy. Histological and immunohistological examination of the specimens revealed similar cytokeratin (CK) expression patterns, which were negative for CK7 and positive for CK20. Therefore, the definitive diagnosis was metastasis from rectal cancer.In summary, we encountered a case of lower bile duct metastasis from rectal cancer, which is often difficult to differentiate from cholangiocarcinoma. In such patients, CK7 and CK20 expression patterns are important in differentiating the two. The mechanism of metastasis in this case was considered to be through cancer cell implantation from lymphatic spread, or through distant metastasis of the primary cancer. Biliary metastasis of colorectal cancer is a manifestation of metastatic liver carcinoma . IntrabiA 68-year-old woman, who had a history of hypertension, hyperlipidemia, and diabetes mellitus, underwent laparoscopic low anterior resection (LLAR) for treatment of her rectal cancer in December 2011. Histological examination revealed a well differentiated tubular adenocarcinoma, pT3N0M0 stage II A according to the Union for International Cancer Control (UICC) TNM classification system  scan revealed liver metastasis in Couinaud segment V Fig. . A diagnTwo years after undergoing hepatectomy, the follow-up CT scan revealed dilation of the left intrahepatic bile duct and a high-density mass in the common trunk of segment II and segment III (B23). Magnetic resonance cholangiopancreatography (MRCP) and endoscopic retrograde cholangiopancreatography (ERCP) also revealed a mass in B23 Fig. . The bioIn October 2017, a follow-up CT scan revealed a 15 mm mass with enhancement in the early phase in the common bile duct, and lymphadenopathy at 8a. MRCP also revealed a 15 × 6 mm low intensity mass at the end of the common bile duct, and a slightly high diffusion-weighted imaging (DWI) but no corresponding low signal on the apparent diffusion coefficient (ADC) map  expression patterns of the specimens, which were negative for CK7 and positive for CK20 Fig. , indicatBiliary metastasis of colorectal cancer is quite common and has a good prognosis. A characteristic imaging finding for biliary metastasis is dilation of the intrahepatic bile ducts, and this often makes it difficult to differentiate intrahepatic cholangiocarcinoma from metastatic liver carcinoma. Immunohistological staining is useful for this differentiation. CK7 negative and CK20 positive expression pattern has a 93% positive predictive value for colorectal metastatic cancer. The pattern in our patient had only a 4% positive predictive value for cholangiocarcinoma [In this case, based on the immunohistological findings, we determined that the intrahepatic bile duct cancer in April 2017 and the lower bile duct cancer in October 2017 were metastases of the rectal cancer in 2011. Only a few cases of lower bile duct metastasis from colorectal cancer have been reported –7. Sano As mentioned above, our case likely followed the same process of metastasis. Considering the rarity of bile duct metastasis of colorectal cancer, it is quite unlikely that bile duct metastasis occurred twice in this case. Our histological examination revealed that the surgical margin was preserved and that there was no vascular, lymphatic, or neuronal invasion. We considered that the mechanism of metastasis involved cancer cell implantation. Implantation was considered to occur at the injured bile duct during the ERCP, or at the site of the resection stump during the left and caudal lobectomy with extrahepatic bile duct resection. However, it is uncertain whether seeding by biopsy was involved.Our case showed some differences from former reports. There was isolated LN 8a metastasis in our case. Considering the mechanism of LN metastasis, there are two possibilities: hematogenous metastasis from primary rectal cancer or lymphatic invasion from former metastases. If hematogenous metastasis is the cause, the metastasis is not a local disease, but a systemic one; hence, chemotherapy may be needed in some cases. In contrast, if lymphatic invasion is the cause, the metastasis is a local disease and may need complete excision. In this case, we considered the mechanism of LN metastasis to be lymphatic invasion from either left intrahepatic bile duct metastasis of rectal cancer or lower bile duct metastasis of rectal cancer. In general, adjuvant chemotherapy after hepatectomy for bile duct metastasis of colorectal cancer, or even liver metastasis of colorectal cancer, does not have sufficient evidence of prognostic improvement. Our institution’s policy, therefore, is not to perform adjuvant chemotherapy after complete resection for distant metastasis.We have been following up on the patient for over 2 years without chemotherapy, and there has been no sign of metastasis and recurrence. If any evidence occurs, she may be considered for chemotherapy. Further studies in more patients are needed to evaluate whether chemotherapy will prevent recurrence or not.In conclusion, we managed a case of lower bile duct metastasis of rectal cancer in which CK7 and CK20 expression patterns were useful to confirm whether it was liver metastasis or cholangiocarcinoma. The mechanism of metastasis was considered to be either cancer cell implantation with lymphatic invasion or distal metastasis of the primary cancer.


Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification. Ribosome biogenesis is a complex process with multifaceted steps involving rRNA synthesis, ribosomal protein expression and subsequent transport into the nucleus, rRNA modifications and folding, as well as protein assembly ,3,4,5,6.snoRNAs are essential small RNA molecules that have been identified to regulate and guide the posttranscriptional chemical modifications of rRNA, namely 2′O-methylation and pseudouridylation ,21,22. TThe sequences of the C (RUGAUGA) and D (CUGA) boxes as well as the H (ANANNA) and ACA box are used to differentiate between the two main snoRNA classes ,25. AddiHID2) snoRNA leads to developmental leaf polarity and overall slow-growth defects [The formation of snoRNPs contributes not only to the function of the particle but also to the nucleolar import, and it helps to stabilize the snoRNA . Modific defects  similar  defects ,3,32,33.SnoRNAs are discussed to be largely localized in the nucleolus and to function in rRNA modification, but recent studies expanded this functional repertoire. It was demonstrated that snoRNAs dually act in the regulation of rRNA modification and in pre-mRNA splicing as exemplified for SNORD27 in HeLa cells . MoreoveArabidopsis thaliana [HID2 mutants displayed no change in 2´-ribose methylation at position G2620, leading to the conclusion that other snoRNAs might fulfill this action as backup partners.In plants, U3, U14, and U49 were identified early based on similarity to the snoRNAs in yeast or vertebrates ,39,40. Hthaliana , the numthaliana ,26,43. Tthaliana . Howeverthaliana . HoweverA. thaliana were complemented by the analysis of the RNA family in rice [The global studies of the snoRNA content in  in rice ,46. This in rice . Analyzi in rice ,49.Recently, a link between snoRNA and miRNA function has been suggested based on the identification of dicistronic genes encoding precursors processed to snoRNA and miRNA molecules . MoreoveA. thaliana cell suspension culture was fractionated into the nuclear depleted cytoplasm and nucleus. Ethidium bromide staining revealed two dominant pre-rRNA precursors, 35S and 27SB, in the nuclear fraction  was subjected to denaturing gels. RNA molecules smaller than 200 nt were excised and used for next generation sequencing. In total, 12 million reads obtained by sequencing the RNA of the total fraction as well as 7 or 15 million reads of the nuclear fractions were mapped to the genome of thaliana b. The tothaliana b, althou; TAIR10 ) was ratA. thaliana proteins. However, in contrast to tRNAs for serine, tRNAs for leucine integration are only medium abundant and tRNAs for valine are among the tRNAs with lowest abundance. In turn, while tryptophan and cysteine are low-abundance amino acids, a high tRNA content was identified for both. Thus, while the gene copy number appears to correlate with the amino acid abundance, a correlation between tRNA abundance and amino acid occurrence could not be observed. Considering that the theoretical amino acid content based on and the amino acid occurrence in all annotated proteins correlates with the determined amino acid content in a cell [For tRNAs, it has been discussed that their copy number correlates with amino acid occurrence in the proteome . In heren a cell , this obn a cell  or reguln a cell ,63.The general coverage of the rRNA was comparable for the three nuclear samples, while the coverage is not homogenous throughout the rRNA transcript d. We assHID2 mutants uncovered a pre-rRNA degradation mode in the nucleus, degrading the 3′ end of the 27SB pre-rRNA [A large number of reads was obtained for the isolated small RNAs mapped to the 5′ external transcribed spacer (ETS) and the mature 18S rRNA. A certain variation of reads mapping to the 5′ and the central region (bp 5600–5800) of the 18S rRNA was observed, for which a higher read coverage was observed in the nuclear or cell lysate fraction. In contrast, a large number of reads observed for the isolated RNAs from the total lysate fraction were mapped to the 5′ region of the 25S rRNA, while reads for this region were less frequent when the RNA of the nuclear fractions was analyzed. Considering the two facts that RNAs migrating at sizes smaller than 200 nt were excised from the gel and that rRNA depletion was performed, reads covering the rRNA originated from specific or unspecific rRNA breakdown. Thus, the observed difference of the coverage of the 5′ region of 25S could result from a higher stability of this rRNA in pre-ribosomal complexes dominating the rRNA pool in the nucleus when compared to mature ribosomes with high abundance in the cytoplasm. Alternatively, the analysis of pre-rRNA ,65, whicA. thaliana or Oryza sativa [The identification of snRNAs was largely comparable between nuclear fractions and the total lysate a. Furthea sativa ,52 have a sativa  and the a sativa  was analp-value is considered. In general, we followed the RFAM-based classification with one exception: the U3-like snoRNAs. While prediction suggested an H/ACA-type snoRNA, this family is generally described as C/D box snoRNA. In addition, for the newly found snoRNAs, it was searched for complementary sites on the rRNA  staining . After 15 min incubation on ice, NP-40 was added , followed by thorough mixing; 3 mL of homogenate was loaded onto 1 mL of 10% sucrose in HNB buffer and centrifuged at 4 °C at 2.150 g for 10 min. The supernatant consisted of cytoplasmic extract, which was used for RNA purification [RNA isolation—RNA isolation from different plant tissues was performed as established ,57. For fication ,57.RNA sequencing—RNA libraries of total cell extract and nucleus were size separated on polyacrylamide gels. RNAs smaller than 200 nucleotides were extracted. RNA was prepared for next generation sequencing by GenXPro , including rRNA depletion. Stranded single-end reads of 100 nucleotides were created on Ilumina NextSeq 500 and controlled for their quality by FASTQC (www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were mapped to the genome of A. thaliana (TAIR10 [ (TAIR10 ) with Ne  and rRNin snOPY  or publiin snOPY ) were usDetection of new snoRNAs in A. thaliana—Detection of new snoRNAs . Region (TAIR10  to ident (TAIR10 . “Contig (TAIR10  and RNAs (TAIR10 .Fluorescence in situ hybridization (FISH)—RNA-FISH was performed in A. thaliana roots as described [2PO4, and 8.4 mM Na2HPO4). The root tips were arranged onto a glass slide, covered, and gently squashed, and the glass slides with coverslip was immersed in liquid nitrogen for 5 s. The cover slip was removed from the squashed root cells, and tissues were air dried  and subsequently immersed in 70% ethanol on a coplin jar . Before hybridization, slides were removed and ethanol was evaporated. Roots were washed twice with wash buffer . Cy-3 labeled probes were mixed  in 100 µL hybridization buffer , added to roots, and incubated in a humid chamber box . Cover slips were removed, and roots were washed twice with 200 µL wash buffer and incubated in wash buffer . The nuclei were stained with 100 µL of DAPI solution . After washing twice with 300 mM NaCl and 30 mM sodium citrate, pH 7.0, samples were incubated for 2 min with 100 µL of Anti-fade GLOX buffer  followed by incubation in 100 µL of Anti-fade GLOX buffer containing enzymes . A coverslip was placed and sealed with nail varnish. For imaging, Zeiss LSM780 Confocal Laser scanning microscope was used in which Cy3, and DAPI were excited at 543 nm and 405 nm respectively while the emission signal was recorded at 570 nm and 460 nm, respectively. All images were prepared using Image J [escribed . The sno Image J .Northern blotting and hybridization—For northern blotting, 5 µg each of cytoplasmic and nuclei RNA was separated alongside low-range ssRNA marker on 12% denaturing PAGE containing 7 M urea in TBE . RNA was mixed with equal volume of 2× formamide-loading buffer , heat denatured , and placed on ice before loading. After electrophoresis, gels were stained with ethidium bromide and electro-blotted onto a HvBond Nylon membrane . RNA was UV cross-linked with the membrane; 50-pmol oligonucleotides  in a 20 µL reaction using polynucleotide kinase according to manufacturer’s instructions  and subsequently added onto the membrane pre-hybridized with 50 mL of hybridization buffer  and 0.05 mg/mL MB-grade DNA  and was shaken overnight at 37 °C. The membranes were washed with low and high stringency buffer containing 6X SSC and 2X SSC plus 0.1% SDS, respectively. Radioactivity was imaged using Typhoon scanner  and quantified with Image J [leotides  were 5′  Image J .


C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). There are three main disease mechanisms: loss of function of C9ORF72 protein, gain of function from the accumulation of sense and antisense (GGGGCC)n in RNA, and from the production of toxic dipeptides repeat proteins (DPRs) by non-AUG initiated translation. While many of the downstream mechanisms have been identified, the specific pathogenic pathway is still unclear. In this article, we provide an overview on the currently available literature and propose several hypotheses: (1) The pathogenesis of C9orf72-associated ALS/FTD, which cannot be explained by a single mechanism, involves a dual mechanism of both loss and gain of function. (2) The loss of function and gain of function can cause TDP-43 aggregation and damage nucleocytoplasmic transport. (3) Neurodegeneration can be caused by an accumulation of toxic substances in neurons themselves. In addition, we suggest that microglia may cause neurodegeneration by releasing inflammatory factors to neurons. Finally, we summarize several of the most promising treatment strategies.The expanded GGGGCC hexanucleotide repeat in the non-coding region of the  C9orf72 gene is an important genetic cause for ALS/FTD, accounting for roughly 40% of familial ALS patients, 25% of familial FTD patients and as high as 88% in familial ALS/FTD patients  is a chronic progressive and fatal neurodegenerative disease caused by the degeneration of upper and lower motor neurons. It is characterized by progressive muscle weakness and atrophy, eventually leading to respiratory failure. Approximately 5–50% of ALS patients have clinical symptoms of frontotemporal dementia (FTD) . FTD is patients . ALS andC9orf72 gene (chromosome 9 open reading frame 72 gene) is located on the short arm of chromosome 9 and is transcribed into three major transcripts , which produce the two protein isoforms [C9ORF72-S (C9-S) and C9ORF72-L (C9-L)]. The GGGGCC hexanucleotide repeat mutation is located on intron 1 between exons 1a and 1b  loss-of-function mechanism: decreased transcription of the C9orf72 coding region that leads to reduced production of the C9ORF72 protein, also known as C9ORF72 haploinsufficiency; (2) gain-of-function mechanism: sense and antisense (GGGGCC)n RNA can sequester or alter the function of RNA-binding proteins and other proteins; (3) By non-AUG initiated translation: sense and antisense (GGGGCC)n RNA produce five toxic dipeptides repeat proteins (DPRs) (The pathogenesis of s (DPRs) . Based oC9orf72 expansion carrier study, V1 and V2  in the frontal cortex and cerebellum were reduced compared to the control group, with no reduction in V3 observed  that activates Rab-GTPases . FurtherC9orf72 knockout cells, FIS1 and C9orf72 were identified as a synthetic lethal interaction , C9orf72 haploinsufficiency may increase glutamate receptors on the surface of iMNs, leading to excitotoxicity and impaired clearance of DPRs, leading to neurotoxicity, ultimately resulting in neurodegeneration . Compared with C9450C mice, C9450CC9orf72+/− and C9450CC9orf72–/–mouse showed more severe learning, memory and motor deficits , are widely distributed throughout the CNS, supporting the involvement of gain-of-function mechanisms . AdditioDMPK gene and is a typical RNA toxicity disease. DM1 is believed to be caused by RNA transcripts (RNA foci) isolating crucial RNA-binding proteins , leading to cause widespread splicing abnormalities (The first reported RNA foci were in Myotonic dystrophy type 1 (DM1) patients, which is caused by a CTG repeat expansion of the malities .C9orf72-associated FTD/ALS patients thus promoting research into possible RNA toxicity mechanisms. RNA foci are formed by the transcription of (GGGGCC)n repeats in the sense or antisense direction. Sense RNA foci exist in most brain regions, such as cortical regions, granule cells of the hippocampal dentate gyrus, and cerebellum. However, antisense RNA foci appear to be most common in cerebellar Purkinje cells and spinal motor neurons (n and antisense (CCCCGG)n into zebrafish embryos can independently induce a motor axonopathy in the absence of DPRs n repeats can form both G-quadruplexes (G-Qs) and hairpins 36 causing eye degeneration and death, while poly(GA)36 and poly(PA)36 had no effect (100 and poly(GR)100 cause a significant decrease in survival rate (50 mice and (PR)100 mice have exhibited neurodegeneration and behavioral deficits, which gradually aggravated over time, suggesting age-dependent neurodegeneration 50 have been found to interact with RBP’s as well as proteins with low complexity sequence domains (LCD), thus damaging the assembly, dynamics, and functions of membraneless organelles   that arginine-containing DPRs function through are largely unknown. Through proteomic analysis, poly(GR)mplexes) . Accordimplexes) . Under smplexes) . Throughmplexes) . Poly (Gmplexes) . Poly (Pmplexes) . Heterocmplexes) .in situ neuronal which can recruit 26S proteasomes and impair proteasome function, consequently abnormal protein homeostasis  also show toxicity in heterologous cells, primary neuronal cultures and mice. Poly(GA) aggregates form twisted ribbons eostasis .C9orf72-associated ALS/FTD , and are regulated by nuclear transport receptors knowns as transport factors (TFs), most of which are in the karyopherin family, such as importins that facilitate entry into the nucleus and exportins that facilitate exit from the nucleus. In addition, this process involves the Ras-related nuclear protein (Ran)GTP-RanGDP cycle, in which the small GTPase Ran regulates cargo across the nuclear membrane through its own nucleotide state, and RanGAPs, RanBPs, and RCC1 play an important role in the nucleotide transformation of Ran . Thereforansport .in vivo  can sequester nucleocytoplasmic transport proteins into inclusion, such as RanGAP1 and Pom121  and TFs . The NPCThe loss of C9-S, formation of RNA foci, or the accumulation of DPRs might promote cytoplasmic mislocalization of TDP-43. Cytoplasmic accumulation of TDP-43 exacerbates nucleocytoplasmic transport defects in a positive feedback loop and at the same time enhances levels of DPRs, thus leading to a vicious circle . TherefoC9orf72-associated ALS/FTD has no effective treatment. The only two pharmacological treatments approved by the USA Food and Drug Administration for ALS treatment are riluzole and edaravone. Here, we introduce several of the most promising novel treatment strategies below.To date, C9orf72-associated toxicity acquisition mechanisms, such as the use of antisense oligonucleotides (ASO) or RNA interference (RNAi). ASO is a short DNA/RNA sequence complementary to the target gene mRNA. Through the RNase H-dependent pathway, an ASO combines with a complementary target mRNA, which activates RNaseH leading to mRNA degradation and thus inhibiting the production of a given protein. Alternatively, through steric blocking it can prevent RNA splicing/processing without degradation. Both mechanisms can reduce protein expression of target genes. However, the pathogenesis of C9orf72-associated ALS/FTD, which cannot be explained by a single mechanism, involves a dual mechanism of both loss and gain of function. Therefore, an important goal of ASOs targeting C9orf72 is to reduce the expression of (GGGGCC)n mutations while maintaining the expression of normal alleles. Some studies have shown that ASOs can target (GGGGCC)n without lowering the mRNA level of C9orf72 mRNA, significantly reducing the number of RNA foci and the percentage of cells containing RNA foci in each cell in induced pluripotent stem cells (iPSCs) from C9orf72 ALS patients . Another method of gene silencing is through the use of an RNAi strategy. RNAi works when a double-stranded DNA homologous to an endogenous mRNA is introduced into the cell, the mRNA is degraded. RNAi will be described below in gene therapy.Gene silencing is a good way to deal with patients . At the nic mice . AlthougC9orf72-associated ALS/FTD. For example, TMPyP4 and KPT-276 can restore nucleocytoplasmic trafficking. Tauroursodeoxycholic acid (TUDCA) and salubrinal can decrease ER stress  in the brain and spinal cord is required. Adeno-associated viruses (AAVs) are a promising method of gene therapy for neurological diseases. Different serotypes of AAVs have different tissue tropism and transduction efficiencies. For example, after intravenous administration, AAV9 has the ability to cross the blood–brain barrier and tends to transduce glial cells while AVV6 preferentially transduces neurons . Evidencnscripts . HoweverHTT gene, as it has high specificity for CAG repeats of mutant HTT genes but no effect on normal genes. Excitingly, two companies, Sangamo Therapeutics and Pfizer, are currently collaborating on the use of ZFP-TF technology to treat C9orf72C9-associated ALS/FTD.Recently, Sangamo Therapeutics designed allele-specific zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat in Huntington’s disease . Once a C9orf72 repeat expansion in ALS and FTD, uncovering the mystery of C9orf72C9-associated ALS/FTD will help us find effective therapeutic targets, thereby having a significant influence on patients suffering from these debilitating neurodegenerative diseases.In view of the important position of QY wrote the manuscript. BJ and LS made suggestions and edited the manuscript. All authors read and approved the final manuscript.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.


The SARS-CoV-2 pandemic has caused unpredictable mortality and economic losses globally. With no approved drug for the treatment, the accurate diagnosis of COVID-19 becomes essential. RNA based test takes several hours and require extensive human intervention for RNA extraction and RT-PCR, but it is preferred over the antibody-based detection as the latter does not detect the early stage infections.•RT-PCR based COVID-19 diagnosis is vulnerable to the false-negative results due to inaccurate sample isolation or RNA extraction.•RNA pool of multiple samples from an individual improves the chances of detection of SARS-CoV-2 by RT-PCR.•Inclusion of specific marker genes would ensure the right RNA source from the desired cell.The RT-PCR being a gold standard of COVID-19 diagnosis offers highly standardized detection of the SARS-CoV-2 RNA, still vulnerable for false-negative diagnosis due to absence of infected cells in the sample or inaccurate RNA extraction. Hence there is a need to develop alternative protocols and methods for the accurate COVID-19 diagnosis. Here we propose two additional steps in RT-PCR based COVID-19 diagnosis to minimize false-negative detection. The first step involves collection of four samples from an individual. Each sample should be collected from nasopharyngeal and oropharyngeal regions on day 01, mixed together followed by RNA extraction and then repeating the same exercise on day 03. The RNA extracted on day 01 and day 03 must be pooled together to be used in the RT-PCR. Second, we propose the inclusion of the control marker genes specific to nasal goblet cell, type-II pneumocyte and absorptive enterocytes to ensure the specificity of the RNA source. Overall, these additional steps in the proposed method would increase the chances of SARS-CoV-2 detection in the infected population and would limit the false-negative diagnosis of COVID-19 and hence the spread of this disease.  Specification tablest, 2021 there have been 94,963,847 confirmed cases of COVID-19, including 2,050,857 deaths reported to WHO so far (https://covid19.who.int/). An individual with COVID-19 shows symptoms of influenza, such as dry cough, severe headache, fever, and tiredness. In individuals infected with SARS-CoV-2, the critically ill condition may lead to organ function damage, such as acute kidney injury, liver dysfunction, cardiac injury and acute respiratory distress syndrome, which can result in a long-term overall decrease in lung function. Some of the critical cases may also lead to death. The severe conditions and mortalities have been observed more in older patients or patients with a weak immune system Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19) in humans. With the relatively larger genome size of around 30 kb compared to the other RNA viruses, the SARS-CoV-2 genome codes for four types of proteins: spike (S), membrane (M), nucleocapsid (N), and envelope (E) So far there is no new drug developed and approved for the treatment of COVID-19, but there are treatment options adopted worldwide to treat COVID-19 patients. Among those, the convalescent plasma (CP) therapy and drug repurposing offer the potential options of treatment in absence of an approved new drug to treat SARS-CoV-2 infection The convalescent plasma (CP) is the plasma-derived from COVID-19 patients who have successfully overcome the infection. In the past, the CP had been used for the treatment of other deadly diseases caused by viruses such as Severe Acute Respiratory Syndrome (SARS) and Spanish flu, as the plasma provides neutralizing antibodies to the patient against infectious agents. Emil von Behring, a well-known scientist from Germany received the noble prize in 1901 for treating diphtheria using the CP. Although the results of the treatments using the CP have been of mixed types, as there is always a challenge of getting the desirable and right titer of the neutralizing antibody prior to giving the plasma to COVID-19 patients. On the drug development side, there is no new drug developed and approved yet for the treatment, and in such a situation the drug repurposing that uses an existing approved drug for the treatment of another disease may offer the solution Recently, the attempts have been also made using the microfluidic platform for the detection of COVID-19. A microfluidic-based platform allows the diagnosis of a clinical fluid sample via microchannel which offers faster and high sensitivity detection Although the antibody-based tests offer a shorter turnaround time and high specificity, these tests fail to detect the early stage infection in an individual as it takes a few days to get the antibody response after infection. Therefore the RT-PCR test has been the gold standard so far considering the speed, sensitivity and accuracy of the test along with successful detection of early-stage infection. Although, RT-PCR is the most commonly used test for the SARS-CoV-2 diagnosis, there is a huge scope of improving the accuracy and sensitivity of the COVID-19 diagnosis. When inaccurately done, it may lead to two situations. A false-positive diagnosis, which labels a normal individual as SARS-CoV-2 positive or a false negative diagnosis that fails to identify an infected individual. The false-positive individual may undergo unnecessary psychological stress with long quarantine and social isolation; a false negative individual may spread infection around and contribute to the severity of the pandemic For a diagnostic test to be efficient, the very first step is the collection of samples from the patient. The Centre for Disease Control (CDC) has recommended the collection of upper respiratory specimens, preferably from nasopharyngeal (NP) or oropharyngeal (OP) region for the RT-PCR test. As of June 2020, the CDC has also allowed nasal swabs to be taken by the patient or health worker and used as a valid specimen for testing when NP swabs are not available. The present method of sample collection involves one-time collection from either the NP or OP region of a potentially infected individual. This method does not ensure getting the virus-containing cells or free virus in the samples even if an individual is infected. In the case of low virus titers in NP or OP regions, there are chances of missing out on the infected cells and hence the SARS-CoV-2 RNA. In addition, there may be cases when collected samples have no cells at all, irrespective of an individual's infection status. SARS-CoV-2 can be found in all or any of these specimens of NP, OP and nasal swabs. Testing on each specimen may be necessary depending on the variability of patient condition or a need for re-testing after a negative result, but testing multiple samples from the same individual in RT-PCR would be time taking and expensive. To maximize the chances of getting the virus or infected cells in the samples collected, and to minimize the false negative diagnosis, we propose a 4-swab method. In this method on day-1, the samples must be collected from the NP and OP regions and then pooled together in the Virus Transport Media (VTM) followed by RNA extraction to prepare the RNA pool-A. On day-3, the samples must be collected again from the NP and OP regions, pooled together in the VTM followed by RNA extraction to prepare the RNA pool-B. The RNA pool-A and RNA pool-B must be mixed together, and the mixed RNA can be used as the template source for the RT-PCR. This RNA pool would also contain SARS-CoV-2 RNA if the individual to whom the samples belong, is infected. The pooling of samples from NP and OP regions and then mixing the RNA samples from day-1 and day-3 would enhance the chances of SARS-CoV-2 RNA detection, even if the sampling method has missed the virus at one of the two sites or attempts. There has been evidence that the chances of detection of the virus from an infected person are 66% in the case of nasal samples, and 33% in the case of pharyngeal samples. Considering these events as mutually exclusive, the probabilities of detection would be higher when more than one sample is collected from NP and OP regions and pooled together. Even though this method would require sample collection twice from the same patient, it can be easily implemented on the patients admitted in hospitals, or people undergoing quarantine. Also, using the pooled RNA as template source would bring down the frequency of false-negative diagnosis.Once the RNA is isolated and pooled, the presence of SARS-CoV-2 RNA can be tested using the RT-PCR. In RT-PCR, the SARS-CoV-2 RNA, if present in the RNA pool is reverse transcribed to make the complementary DNA (cDNA), which is then amplified into numerous copies using specific primers. Different regions of viral RNA such as E gene and RNA dependent RNA polymerase (RdRp) can be amplified with specific primers in real-time to confirm the presence of SARS-CoV-2 RNA in the patient samples.To avoid the false-negative diagnosis, different kits have included specific internal controls such as external RNA added at the time of RNA extraction and then tested during the RT-PCR to ensure that the negative diagnosis is not due to RNA degradation. Also, multiple kits use RNAse-P as an internal control to ensure the stability of RNA as well as the source of the RNA isolated, but it is not universally included in all the diagnostic kits, currently being used for the COVID-19 diagnosis. Also, RNAse-P confirms the presence of cellular RNA but since it is expressed in multiple cell types, it does not qualify to be a signature marker of a specific cell potentially infected with the SARS-CoV-2. Therefore, the inclusion of cell-specific controls becomes significant to minimize false negative COVID-19 diagnosis in RT-PCR based methods.To confirm the presence of cells in the samples collected, ideally, the sample should contain the RNA from the goblet secretory cells of the nasal passages, type-II pneumocyte of lungs and absorptive enterocytes of the intestine, the cell types which are known to get infected by SARS-CoV-2. At least one out of these 3 genes should get amplified in RT-PCR irrespective of the presence of SARS-CoV-2 RNA in that sample or infection status of that individual. No amplification of even 1 out of these 3 genes would suggest the absence of those specific cells in the sample or inaccurate RNA extraction. Samples showing amplification of any of these 3 genes and no amplification of viral genes should be considered as true negative. Even after the inclusion of these genes as internal controls, there is still a possibility that an individual being infected is not releasing enough of the virus into NP or OP regions to be picked up and detected, but the chances would be minimal due to multiple samples and using the RNA pool for the RT-PCR test as described earlier.Using pooled RNA for RT-PCR, and inclusion of specific marker genes from goblet nasal secretory cells, type-II pneumocytes of lungs and absorptive enterocytes of the intestine will not only minimize the frequency of false-negative detection, it would also help in limiting the spread of the virus by identifying and containing such patients in the long run. Overall by reducing the false-negative diagnosis of COVID-19 significantly, this method will contribute to limit the further spread of the current SARS-CoV-2 pandemic Author has no conflict of interest in regards to the information shared in the manuscript.


Rationale: circular RNAs (circRNAs) have been demonstrated to play a crucial role in cancer progression. KIAA1429, a key component of the m6A methyltransferase complex, has recently been reported to promote hepatocellular carcinoma (HCC) progression by regulating the m6A methylation. The aim of present study is to investigate the role of circular RNAs in KIAA1429-mediated HCC progression.Methods: RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (m6A-seq) were utilized to identify KIAA1429-regulated circRNAs. The effects of circDLC1 on proliferation and metastasis of hepatoma cells were examined in vitro and in vivo. RT-qPCR was used to measure the expression of circDLC1 in HCC tissues and hepatoma cells. RNA FISH, RIP assays and biotin-labeled RNA pull-down were used to investigate the downstream effector of circDLC1. The downstream targets of circDLC1 were identified using RNA-seq.Results: Our data demonstrated that circDLC1 was downregulated in HCC tissues and closely relevant to favorable prognosis. Overexpression of circDLC1 inhibited the proliferation and motility of hepatoma cells in vitro and in vivo, while silencing of circDLC1 played the opposite role. Mechanistic investigations revealed that circDLC1 could bind to RNA-binding protein HuR, which subsequently reduced the interaction between HuR and MMP1 mRNAs, and thus inhibited the expression of MMP1, ultimately contributing to inhibition of HCC progression.Conclusion: Our work suggests that circDLC1, a downstream target of KIAA1429, is a promising prognostic marker for HCC patients, and the circDLC1-HuR-MMP1 axis may serve as a potential therapeutic target for HCC treatment. Liver cancer is one of the most common malignant tumors and the fourth leading cause of cancer-related death worldwide N6-Methyladenosine (m6A) modification, methylation of adenosine at the N6 position, is a widespread and abundant modification in messenger RNA (mRNA) and non-coding RNAs (ncRNAs) in eukaryotes Circular RNAs (circRNAs), known as covalently closed and single-stranded transcripts, have been proved to exist in many species In this study, we identified circDLC1  as a KIAA1429-regulated circRNA, which was closely associated with favorable prognosis. In addition, circDLC1 was found to interact with RNA-binding protein HuR and block the interaction between HuR and MMP1 mRNAs. Our study identified a novel circRNA that was regulated by m6A modification and provided new insights into the pathogenesis of HCC.All HCC and paired adjacent normal tissue samples (n = 110) were obtained from deidentified patients who had undergone surgical resection in West China Hospital . The protocols of this study were conducted in accordance with the Ethical Review Committees of Sichuan University. According to the policies of the committee, the written informed consent was provided from each patient.2. Actinomycin D was purchased from Sigma-Aldrich.Huh-7, Hep3B, HepG2 and SK-Hep1 cell lines were purchased from the Shanghai Cell Bank Type Culture Collection Committee . SNU449 cell line was obtained from ATCC . Short tandem repeat (STR) analyses were performed by third-party biology services with all cell lines to characterize the identities of the cells . All cells were cultured in DMEM  containing 10% FBS (HyClone) at 37 °C and 5% COin situ Hybridization Kit (RiboBio Biotechnology), according to the manufacturer's instruction. Images were acquired by the A1RþMP Confocal Laser Microscope System (Nikon).Cy3-labled probes against 18S and U6 were designed and synthesized by RiboBio (RiboBio Biotechnology). Cy3-labelled circDLC1 probes . In brief, magnetic beads were incubated with 5 μg of antibody against HuR  or DHX9  and normal IgG  for 30 min at 25 °C. The cell lysate and treated magnetic beads were incubated overnight at 4 °C. After 6 times washing, proteinase K digestion buffer was used to digest the RNA-protein complexes for 45 min at 55 °C. The enriched RNAs were extracted by phenol: chloroform: isoamyl alcohol (125 : 24 : 1). Then, the purified RNAs were subsequently analyzed by RT-qPCR to assess the enrichment of target RNAs to the target proteins.0.3% formaldehyde was used to cross-link and glycine solution was used to quench the cells (Millipore). Cross-Linked RNA immunoprecipitation assays were performed according to the manufacturer's protocol of Magna RIP0.3% formaldehyde was used to cross-link and glycine solution was used to quench the cells (Millipore). Sangon Biotech synthesized the biotin-labelled circDLC1 probes and control probes . Biotin-5 cells were injected subcutaneously into the right axilla of mice. Tumor volume was measured by a caliper weekly and calculated as length × width2 × 0.52. For the liver orthotopic-implanted models, each liver of mice was injected with 1 × 106 cells. After 6 weeks, mice were sacrificed, and the livers were visualized using the IVIS@ Lumina II system . All tumor samples were embedded in paraffin and confirmed by hematoxylin and eosin staining. Furthermore, 2 × 106 cells were injected into mice to established the tail intravenous injection models through the tail vein. After 6 weeks, mice were sacrificed, and the lungs were visualized using the IVIS@ Lumina II system .All animal experiments were authorized by the Institutional Animal Care and Use Committee of Sichuan University. In this study, the BALB/c nude mice were all male and 6 weeks old, which were purchased from BEIJING HFK BIOSCIENCE . All mice were fed under standard pathogen-free conditions. All surgical procedures were performed with sodium pentobarbital anesthesia. 5 × 10t test, ×2 test, the Wilcoxon signed-rank test, was conducted as appropriate. Kaplan-Meier method was applied to measure the survival curves, and the log-rank test was used to assess the differences. Correlations were assessed by Pearson correlation analysis. P values less than 0.05 indicated statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001.All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software). The Student p<0.05). Among these differentially expressed circRNAs, 33 were upregulated and 40 were downregulated in shKIAA1429 cells compared with shCtrl cells  and control cells (shCtrl). We detected 73 distinct circRNAs with 2-fold change (P < 0.0001) and RFS (P = 0.0003)  by RT-qPCR. The expression of circDLC1 was much lower in HCC tissues than paired adjacent normal tissues Figure A. FurtheOn the other hand, the expression of KIAA1429 in paired HCC and adjacent normal tissue samples (n = 40) was examined by RT-qPCR and IHC. Notably, the expression of KIAA1429 was obviously upregulated in HCC tissues compared to paired adjacent normal tissues Figure C. CollecNext, we examined the physical circular structure of circDLC1. CircDLC1 is derived from exons 14, 15 and 16 of the DLC1 gene , and the result of Sanger sequencing proved the presence of back-splicing junction Figure A. Also, Previous study revealed that the long flanking introns with inverted complementary sequences usually facilitated the biogenesis of circRNAs from their internal exons Previous studies have demonstrated that DHX9 could bind to the flanking inverted complementary sequences of its target RNAs to inhibit the pairing of these sequences Six human hepatoma cell lines were chosen to measure the expression of circDLC1 by RT-qPCR. Among them, the lowest expression of circDLC1 was observed in HCC-LM9, moderate expression of circDLC1 was observed in SNU 449, HepG2 and Hep3B, and the highest expression of circDLC1 was observed in Huh7 and SK-Hep1 A. For fuin vivo, circDLC1 knockdown SK-Hep1 cells and circDLC1-overexpressing HCC-LM9 cells were subcutaneously injected into nude mice. We observed that tumor volume was remarkably increased when circDLC1 was knocked down, whereas overexpression of circDLC1 showed an inverse result  staining validated the number of metastatic foci was also increased both in liver and lung. However, the liver and lung metastasis model exhibited opposite results when circDLC1 was overexpressed  RBP family and is responsible for the stabilization and (or) translation of many target mRNAs, which typically bear U-rich 3 UTRs Next, we investigated the mechanism of MMP1 downregulation mediated by HuR knockdown. We found that HuR silencing decreased MMP1 mRNA levels by enhancing its degradation, because silencing HuR lowered the half-life of MMP1 mRNA significantly Figure H. MoreovA previous study has demonstrated that MMP1 inhibited cell proliferation and invasion in HCC Mounting evidence reveal that m6A modification plays a critical role in different human cancers Multiple studies have reported that the alternative splicing of RNA was regulated by m6A modification Previous studies demonstrated that HuR is responsible for the stabilization and (or) translation of many target mRNAs, which typically bear U-rich 3' UTRs MMP1, a member of the zinc-dependent endopeptidase family, has been demonstrated to be closely relevant to migration and invasion in a number of cancers In summary, our study identified circDLC1 as a novel downstream effector of KIAA1429-mediated m6A modification and confirmed the critical role of m6A modification in regulating the biogenesis of circRNAs. Furthermore, we demonstrated that circDLC1 suppresses the growth and metastasis of HCC via the HuR-MMP1 axis. Our findings shed new light on the underlying mechanisms of HCC progression and suggest that circDLC1 may serve as a prognostic biomarker and potential therapeutic target of HCC patients.Supplementary figures and tables.Click here for additional data file.