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264146521	pes2o/s2orc	v3-fos-license	Network disruption via continuous batch removal: The case of Sicilian Mafia Network disruption is pivotal in understanding the robustness and vulnerability of complex networks, which is instrumental in devising strategies for infrastructure protection, epidemic control, cybersecurity, and combating crime. In this paper, with a particular focus on disrupting criminal networks, we proposed to impose a within-the-largest-connected-component constraint in a continuous batch removal disruption process. Through a series of experiments on a recently released Sicilian Mafia network, we revealed that the constraint would enhance degree-based methods while weakening betweenness-based approaches. Moreover, based on the findings from the experiments using various disruption strategies, we propose a structurally-filtered greedy disruption strategy that integrates the effectiveness of greedy-like methods with the efficiency of structural-metric-based approaches. The proposed strategy significantly outperforms the longstanding state-of-the-art method of betweenness centrality while maintaining the same time complexity. INTRODUCTION T He study of complex networks has emerged as a crucial interdisciplinary field, providing insights into systems as diverse as social interactions, biological processes, infrastructural designs, financial markets, transportation networks, and communication systems [1], [2], [3].Within this realm, understanding network disruption strategies is of paramount importance, particularly for mitigating the resilience of undesirable networks such as criminal organisations or disease-spreading pathways [4], [5].The task of disrupting a network can be formulated as the task of finding the smallest set of nodes that, if removed from the network, determine the largest decrease in the size of the largest connected component (LCC, for short) of the network.Unfortunately, previous studies show that such a problem is NP-hard [6], [7], [8]. Existing approaches to network disruption can generally be classified into three categories.The first category of approaches uses structural metrics, such as degree or betweenness centrality, to rank nodes and progressively delete them from the network according to their rank [9], [10], [11].The second category of methods starts by identifying nodes on the basis of their specific role within the network, such as those nodes that lie between predefined communities [12], the so-called articulation points whose removal would disconnect the network [13] or the nodes of high k-shell indices [14].These selected nodes are then further ranked according to metrics like degree or betweenness centrality.The third set of approaches for disrupting a network formulates the disruption process as a two-step procedure: the initial step focuses on selecting nodes to decycle the network, followed by choosing nodes to break the resulting tree structures [6], [15].Surprisingly, despite the efficacy of various newly developed techniques in specialised network contexts, betweenness centrality is still shown to be the most universally effective strategy for network disruption.[16]. In this work, we further investigate the disruption strategies in the context of criminal networks [17].Specifically, we focus on a Sicilian Mafia Network [18], [19].Unlike conventional criminal organisations, Mafia clans are characterised by unique structural attributes: they are composed of loosely coupled groups that can span across multiple generations [20].These groups, often referred to as "coscas", "families" or "clans", are bound by strong relational ties and reciprocal altruism.Mafia groups can have a profound influence over economic, social, and political sectors in some countries [21].Therefore, finding a way to effectively disrupt mafia clans is crucial not only for academic research but also serves as a practical guide for Law Enforcement Agencies (LEAs) in devising targeted countermeasures. Due to the inherent structural characteristics of criminal networks and the constraints of limited police resources, attacks against such networks are often not accomplished in one stroke.Mafia gang is strongly rooted in a specific social and professional environment, and such an environment acts as a protective shield over the gang leaders (also known as bosses) and its members.A boss lives in disguise and often enlists the help of people who are not affiliated with the gang (e.g.doctors and nurses who provide medical treatments to the boss without revealing her/his identity to the police).Efforts to catch mafia bosses therefore take many years and they start with people who support the activities of a mafia gang without being part of the gang; police forces try to catch the boss by investigating and arresting people who are "closer and closer" to the boss.Police proceeding ends with the arrest of the boss along with a circle of accomplices and collaborators and, consequently, a peculiarity of the process of dismantling a criminal network is that the network is not attacked one node at a time, but the investigations simultaneously target groups of nodes. arXiv:2310.09987v1 [cs.SI] 16 Oct 2023 Therefore, we view criminal network disruption as a continuous batch removal process [18].In this framework, a specified number of target nodes are removed at each iterative step.This approach extends traditional strategies, which typically assume the removal of only a single node at a time.Also, given the constraints of limited police resources, it may be necessary to concentrate efforts in a specific location.As such, we also examine the impact of various disruption strategies constrained to the LCC, rather than targeting the whole network.We evaluated seven distinct disruption strategies within the context of continuous batch removal on the mafia network: degree centrality [9], betweenness centrality [22], collective influence [11], CoreHD [15], APTA [13], as well as variations of degree and betweenness centrality that incorporate secondary ranking criteria for instances of tied rankings.We discover that betweenness centrality and APTA emerge as the most effective strategies when there are no constraints limited to the LCC.On the other hand, degree centrality performs exceptionally well within the LCC constraint, especially when the batch size is small. In pursuit of a strategy that could surpass traditional approaches, we initially adopted a greedy algorithm.In each iteration, our algorithm identifies and removes the batch of nodes resulting in the greatest reduction of the LCC size.As anticipated, the greedy strategy significantly outperforms classical methods, particularly when the batch size exceeds one.However, the greedy approach is computationally infeasible for large networks or large batch sizes.To address this issue, we optimise the greedy algorithm by narrowing the search space.Our study suggests that a large proportion of nodes targeted by the greedy method also rank high in both betweenness and degree centrality, and selected nodes are often articulation points.Thus, we introduce a novel approach, called Structurally-Filtered Greedy Approach (SF-GRD), which restricts the search space to nodes that are highly ranked in these metrics.The results show that SF-GRD achieves a disruption effect comparable to the greedy algorithm while maintaining the same time complexity as betweenness centrality. To summarise, our contribution in this work is manifold: • We propose a generalised robustness metric that is more suitable for effective disruption strategies in a continuous batch removal setting; • Focusing on the disruption of the mafia network and aiming to mimic actual police raid operations, we propose to apply the constraint of within-LCC in a continuous batch removal setting; • We elucidate the disparate impacts of the within-LCC constraint on degree centrality and betweenness centrality; • After analysing the result of a naive greedy approach, we propose a Structurally-Filtered Greedy approach that not only outperforms traditional disruption strategies but also maintains the same polynomial time complexity as betweenness centrality. The remainder of this paper is organised as follows: Section 2 summarises current approaches in dismantling criminal networks; Section 3 explores and proposes metrics for assessing network disruption processes; Section 4 presents our case study dataset, along with requisite prepro-cessing steps and initial insights; Section 5 outlines classical disruption strategies, introduces the within-LCC constraint, and provides a comprehensive discussion of the results; Section 6 describes the Structurally-Filtered Greedy approach (SF-GRD), demonstrating its effectiveness and efficiency; and finally, Section 7 offers concluding remarks. RELATED LITERATURE Criminal and covert networks are flexible organisations that can quickly adapt their organisation and behaviour to survive.However, empirical research on the dynamics of criminal networks is still limited [18], [19], [23], [24] due to the lack of accurate and complete datasets.Simulation studies have shown that removing actors in the most central positions in the criminal network is one of the most efficient strategies [23]. The main limitation of existing node removal processes stems from the fact that these processes simulate the arrest of one individual at a time.In contrast, law enforcement often arrests both the leader of the organisation (referred to as the boss) along with several accomplices who helped the boss escape (e.g., by protecting her/his anonymity). We also point out that attempts to remove one or more nodes from a criminal network generally do not lead to the dismantling of the organisation, but only its weakening.Gangs are able to reorganise quickly by, for instance, electing new leaders, changing the communication procedures as well as the rules for recruiting new members.Paradoxically, a criminal organisation can become stronger and more cohesive after its reorganisation [25], [26], [27]. In this paper, we focus on the size of the largest connected component (LCC) of the graph(s) associated with a mafia gang: indeed, an LCC that covers almost all the nodes of a criminal gang allows all its members to communicate and coordinate to carry out illicit tasks; a significant reduction in the LCC severely inhibits the ability of gang members to communicate and, consequently, severely damages the criminal power of the gang.The activities required to rebuild the LCC are costly and dangerous because, for example, the gang must elect new members with the aim of connecting separated sub-communities.Such an activity is hard to implement in the short term because the potential mediators need to be trusted individuals from all the involved sub-communities.Furthermore, the police have to deal with smaller sub-communities, it is easier to thwart gang reconstitution attempts.In this study, we propose strategies to reduce the size of the LCC and, from a technical point of view, our problem is similar to some well-known problems in graph theory concerning the decomposition of graphs into connected components.More specifically, a vertex cut of a graph G = ⟨V, E⟩ is a subset of nodes V ′ of V such that the graph G ′ obtained by deleting nodes in V ′ from G has at least k (non-empty and pairwise disconnected) components [8], [28], [29].The vertex k-cut problem requires constructing a vertex k-cut of minimum cardinality, if it exists, and, as shown by Cornaz et al. [8], such a problem is NP-hard for any k ≥ 3. The k-cut problem consists in finding the smallest set of edges E ′ to remove from G to produce at least k connected components [30], [31].Cornaz et al. [8] solved the k-VC problem using a general-purpose integer linear programming solver; more recently, Zhou et al. developed a fast local search algorithm to solve it [32]. Our work advances the state of the art by proposing an algorithm to find the nodes whose removal gives the largest reduction in the LCC.Our approach is much more accurate than current best betweenness-based methods and maintains the same computational complexity.Since criminal networks extracted from court documents typically contain around a hundred nodes, the betweenness computation can be performed efficiently, making our approach suitable for supporting law enforcement investigations. WORKS In the analysis of network behaviour under different disruption scenarios, particularly when evaluating robustness or vulnerability, it is crucial to employ measures that encapsulate the specifics and impact of the disruptions.This is essential across a range of fields, such as controlling the spread of diseases, investigating the propagation of information in social networks and understanding the structural integrity of criminal networks.Two main measures have been proposed to quantify the effectiveness of disruptions, each with its own merits and applicability. One-Time Disruption The first category of disruption measures we consider is called one-time significant disruption [6], [9]. The objective is to minimise the number of nodes that need to be removed to reduce the size of the largest connected component (LCC) below a certain threshold \|V \| * = q\|V \|, where V is the set of nodes in the network, and q ∈ (0, 1).More formally, we understand these metrics as the solution of the following optimization problem: It defines the smallest set of nodes that, when removed, results in an LCC smaller than the given threshold, quantifying the effectiveness of a one-time disruptive strategy. Continuous Disruption The second category of disruption measures used in our study are robustness measure and they evaluate how the disruption process evolves as nodes are progressively removed from the network [16], [33]. We define the robustness R as the average of the normalised sizes of the LCC after each node's removal, until all nodes are removed, that is: where N is the total number of nodes, i is the current node to remove, and L(i) is the size of the LCC after removing i. The lower bound is reached in a star graph and when the first removed node is the center of the star; while the upper bound is taken at a complete graph (LCC is viewed as 0 when all nodes are removed in the calculation). The robustness measure not only provides a means to evaluate the inherent resilience of a network structure to node removal, but it also serves as a quantitative tool to compare the effectiveness of different disruption strategies. Robustness of partial removal Although the original robustness measure tracks the entire disruption, it is very likely we care more about the effectiveness of only removing a subset of network nodes.Many effective disruption strategies would rank the nodes according to their structural attributes, and the removal of a small percentage of top-ranked nodes would significantly break the network into very small components, making the removal of the remaining nodes insignificant. For example, when applying a betweenness-degree disruption strategy on a real-world criminal network [18], the network has been significantly broken after 20% of nodes are removed, -LCC size has dropped to 7, about just five percent of the original LCC size.And, the network becomes completely disrupted when 40% of nodes are eliminated. Therefore, we propose a novel robustness measure for partial removal, denoted R@r, which aims to capture the effects of removing a small percentage of nodes in the network.Furthermore, we also generalise the removal process to support batch removal, where at each step, more than one node can be removed.Batch removal process is also a better reflection of the police raid operation in the real world, as combating organised crime often requires continuous actions, and the effectiveness (the number of nodes removed) that can be achieved by each operation is related to the deployable police force. Specifically, with a batch size b, the robustness measure R@r is defined as the average of the normalised sizes of the LCC for each removal step, up to r percent of total nodes removed.Mathematically, it is given by: where N is the total number of nodes, S is the number of steps until a fraction r of total nodes are removed, and L(k) is the size of the LCC after step k.Clearly, when the percentage r equals 1, and the batch size b equals 1, it degrades to the original robustness measure R.This measure provides a focused view of the initial stages of the disruption process, which are often the most critical, allowing us to better evaluate the performance of different disruption strategies. DATASET DESCRIPTION In this section, we introduce our case-study dataset, the preprocessing steps we performed as well as some basic characteristics of the dataset. The dataset was derived from the pre-trial detention order issued by the Court of Messina's preliminary investigation judge on March 14, 2007, which was towards the end of the major anti-mafia operation referred to as the A red circle around a node signifies leadership roles within the network, including positions such as bosses and executives.Additionally, the size of a node is proportional to its degree, and the width of a link is proportional to its weight. "Montagna Operation".The particular investigation was a prominent operation focused on two mafia clans, i.e., the "Mistretta" family and the "Batanesi" clan.From 2003 to 2007, these families were found to have infiltrated several economic activities, including major infrastructure works, through a cartel of entrepreneurs close to the Sicilian Mafia. The pre-trial detention order reported the composition of both the Mistretta family and the Batanesi clan; judicial proceedings followed a chronological narrative scheme which detailed the illicit activities pursued by the members of the Mistretta clan before the imprisonment of a boss (denoted as Boss X to preserve anonymity).The Boss X has been selected by the most influential mafia syndicates to settle the conflicts between the Mistretta and the Batanesi families.The conflicts were unleashed from the extortion imposed on local entrepreneurs in the construction of the highway that connects the city of Messina to Palermo. Two graphs were originally constructed from the court order [34]: A meeting network and a phone call network (both networks are available at https://doi.org/10.5281/zenodo.3938818). The meeting network represents physical encounters, while the phone calls network depicts telecommunication interactions.Meetings involving suspected individuals can be broadly classified as follows: (a) meetings that aim to define the organisational structure of the mafia syndicate along with its relationships with entrepreneurs and other mafia syndicates operating in surrounding areas.Boss X always attended these meetings and has always been accompanied by at least two trusted men.(b) Meetings involving people who occupied the lowest levels of the mafia syndicate hierarchy; the purpose of these meetings was, in general, to design illicit activities and usually, only two people were involved in these meetings.For each one, the date of the meeting, the place, and the participants were recorded.Participants in a meeting were identified by a unique code.The procedure to build the Meetings network was as follows: (a) Each person who participated in at least one meeting corresponds to a node in the network; (b) two subjects in the Meetings network are connected by an edge if both of them attended at least one meeting; (c) edge weights reflect the number of meetings that two individuals jointly attended.The phone call network is built in a similar manner. Although the meeting and phone-call graphs individually offer valuable insights into the social dynamics among the subjects under investigation [19], they each capture only a subset of the interactions.Remarkably, these two graphs share about 50% of their nodes, indicating that they are not isolated spheres of activity but rather interlinked facets of a more complex organisation.To harness the full range of interaction types, we propose to unify these separate but overlapping networks into a single, comprehensive network: distinct edges from the two networks are added to the new network, and when edges appear in both the meeting and phone-call networks, their weights are summed up as the new weight for the edge in the integrated network.This consolidated graph, which we name the Unified Graph, allows for a more accurate and comprehensive understanding of the underlying criminal organisation, covering both formal and casual interactions among individuals.After carefully examining the dataset, we introduced three data-preprocessing steps to address some data issues in the original edge lists: • Retaining the larger weights.We detected inconsistent edge weights in the original edge list data, 29 cases in the meeting list, and 22 cases in the phone-call list, respectively.To deal with weight inconsistency, we chose to keep the larger value that represents the strength of the connection between two individuals.• Keeping only the largest connected components.All three networks contain several tiny cliques and individual nodes that are disconnected from the LCC.We chose to exclude them from each network and focus on the core component of the criminal organisation.• Removing self-loops.We removed self-loops from the data as they are not meaningful in the context. After performing the preprocessing steps above, we obtained three key criminal networks: the Meeting Graph, the Phone-Call Graph, and the Unified Graph.Their features are summarised in Table 1.Initially, the Meeting-DC and Phone-Call-DC graphs consist of 95 and 94 nodes, which decrease to 86 and 85 nodes, respectively, in their connected versions.This suggests that the connected versions are comprised of the major components of the original graphs, capturing the core interactions, which is also evidenced by the marginal change in edge count. Moving on to the specific graphs, the Meeting Graph has a higher average degree (5.63) and clustering coefficient (0.70) compared to the Phone-Call Graph, which has values of 2.66 and 0.10, respectively.This suggests that meetings involve more densely connected individuals, whereas phone calls occur in a more dispersed manner.After effectively merging them into the Unified Graph, the average degree (4.78) and clustering coefficient (0.46) fall between those of the Meeting and Phone-Call graphs. The visualisation of the Unified Graph is given in Figure 1.As human capital is another important dimension to consider in criminal networks [35], we take advantage of the accompanying node feature information and categorise individuals based on both their familial and leadership roles.The details of this classification are in Table 2. Lastly, when we compare the Unified Graph to randomly generated graphs with the same number of nodes and average degree, we observe significant differences.The Unified Graph has a much higher clustering coefficient (0.46 compared to 0.03) and also a smaller average path length (3.11 compared to 3.29).These features indicate a tightlyknit community structure with efficient pathways for information or influence to flow.These are key considerations for understanding the complexities inherent in criminal activities and for designing effective intervention strategies. DISRUPTION STRATEGIES In this section, we describe strategies for efficiently disrupting a network.We recall that the problem of finding the smallest subset S ⊆ N of nodes of a network G = ⟨N, E⟩ that, when removed from G, yields a graph G ′ which largest connected component has size at most a fixed threshold C is NP-complete [6].A brute force solution would consider every possible subset of the nodes, resulting in a factorial time complexity. To efficiently tackle the network disruption problem, we need to design approximate strategies that select the most promising nodes according to some metric (typically a measure of centrality) and progressively remove those nodes from the network. In the following, we first introduce five mainstream disruption strategies and then discuss how we modify and apply them to our study. Disruption strategies The first three attack strategies are derived from wellstudied centrality metrics and they rank nodes according to a pre-defined metric and progressively remove nodes according to the ranking.The other two attack strategies consider specific sub-structures within a graph (e.g. the 2cores or the connected components of that graph) to determine which nodes to remove. • Degree centrality (DEG) [9].This attack simply ranks nodes according to their importance measured by the degree centrality.The degree centrality of a given node i is calculated by C D (i) = j∈V a ij , where A = (a ij ) is the adjacency matrix.We can consider two variants of the DEG strategy: the first is to compute degree centrality only once on the original graph; the second is to recompute degree centrality after each removal, thus accounting for dynamic changes in the perturbation process.We choose the recalculated degree approach to maximise its effectiveness in a continuous batch removal setting.That is, a number of nodes with the highest degrees are removed at each step until a predetermined total percentage of nodes are removed. • Betweenness centrality (BTW) [22].Betweenness centrality measures nodes' importance by measuring the number of times a node appears in the shortest path between all pairs of nodes and, thus, it can be regarded as a global centrality metric as it requires the full knowledge of the graph topology.The betweenness centrality of a node i is defined as: where σ(s, t \| i) is the number of shortest paths between s and t passing through node i, and σ(s, t) is the number of all shortest paths between s and t.As for the DEG strategy, we adopt the recalculated betweenness centrality to remove the top-ranked nodes at each step. • Collective Influence (CI) [11].Collective influence is a recently proposed centrality metric which takes into account the degree of nodes within a given radius when evaluating a node's impact on network disruption; intuitively, a node close to more high-degree nodes will have a higher collective influence score.Specifically, the collective influence CI(i) of a node i is computed as follows: where k i is the degree of node i, B(i, ℓ) is the ball of radius ℓ centered on node i.We define B(i, ℓ) as the set of nodes x at a distance at most ℓ from i.Here the distance of two nodes is defined as the length of the shortest path joining them, and δB(i, ℓ) is the frontier of the ball, that is, the set of nodes at a distance ℓ from i. Essentially, j represents nodes that are l-hop neighbours of i.In our experiment, l is set to 1, and the ranking is recalculated after each removal. • CoreHD [15].By prioritising the decycling of a network, CoreHD focuses on the 2-core [36] when selecting a node to remove -more specifically, remove the highest-degree node from the 2-core at each step and, if more than one node has the same highest degree, one of them is randomly chosen.When 2-core no longer exists, the highestdegree node in the remaining tree-like graph is removed. Fig. 2: A snapshot of a graph during the BTW-DEG disruption process.After removing 28 nodes, the original graph has fragmented into cliques of different sizes: one 5-clique, four 3-cliques, and eight 2-cliques, with the remaining nodes being isolated.Since betweenness centrality can no longer discriminate between the nodes, degree centrality will be used to select a node from the 5-clique. In our batch-removal setting, CoreHD is extended to support multiple node removals at each step. • Articulation Point Targeted Attack (APTA) [13].An Articulation Point (AP) is any node in a graph whose removal increases the number of connected components.The removal of an AP node will split a connected component into multiple smaller connected components.APs can be identified in linear time using Tarjan's Algorithm [37]. APTA is a greedy AP attack that removes the most destructive AP (that is, the one causing the largest decrease in LCC size) at each step.APTA is similar to CoreHD in that they both limit the pool of targeted nodes.However, unlike from the above procedures, the "ranking" of AP nodes is no longer given by a structural metric, but is determined with the goal of minimising the size of LCC.We further modify the APTA attack so it can support multiple AP node removal.In cases where APs don't exist, nodes of the highest degree or nodes identified by another designated structural metric will be removed instead. Equality and Localisation in Disruption Strategies Building upon the five popular disruption strategies outlined above, there are still opportunities to enhance the efficiency of these approaches.Two specific enhancements can be considered to improve these strategies, namely: 1) Introduction of a secondary ranking criterion. Our first observation is that nodes could have the same centrality value, and in such a case, we would not be able to identify the next target node.An effective strategy is to combine a secondary criterion for node removal with our primary criterion: for example, we could select nodes based on their betweenness, and if two or more nodes have the same betweenness, we would favour nodes with the highest degree.The resulting strategy is called BTW-DEG (see Figure 2 for more details).The use of a secondary criterion is ; also useful in non-centrality-based node removal approaches (i.e.CoreHD and APTA): if Core or AP do not exist, nodes can still be selected according to the secondary criterion. 2) Selection within the LCC.Second, by narrowing the selection of nodes to those within the LCC rather than targeting nodes across the entire graph, we can create a more focused and efficient disruption process.Since the objective is to achieve the minimum robustness score (Equation 3), focusing on dismantling the LCC could enhance the effectiveness of the disruption, especially when only one node is removed at each step.Furthermore, this approach resonates with real-world scenarios, such as a police raid operation targeting a specific location, making it a practical and meaningful strategy for network disruption. The enhancements discussed, such as the incorporation of secondary ranking criteria and emphasising the LCC, serve to refine our understanding and application of network disruption strategies.These refinements not only optimise existing strategies but also open avenues for customised approaches tailored to specific disruption objectives or network structures.As we transition into the experimental analysis, we will explore how these enhancements impact the effectiveness of each disruption strategy in the targeted criminal network. The dynamic batch removal disruption process is outlined in Algorithm 1.All code and experiments are available at https://github.com/UTS-CASLab/Disrupt-Criminal-Network. Result and Discussion In the experiment, we implement the five classic disruption strategies, plus two strategies that utilise a secondary centrality, i.e., DEG-BTW, and BTW-DEG, on the Unified Graph.The disruption is done in a dynamic manner until a percentage of nodes are removed, that is to say, re-ranking nodes after each removal step.Furthermore, we also present the situation where the constraint of disrupting only the LCC is applied.The detailed result is shown in Table 3. First, in the standard setting where target nodes are chosen from the entire graph, BTW, BTW-DEG, and APTA are found to be the most effective strategies across batch sizes, followed by DEG and DEG-BTW.In contrast, CI and CoreHD demonstrate inferior performance in network disruption.Although CI is designed to provide a more comprehensive view of a node's influence by considering its extended neighbourhood and is proposed as an improvement over DEG, it actually fails to identify nodes that are pivotal for maintaining overall network connectivity and falls behind DEG.Take the star graph as an example, CI assigns zeros to all nodes, including the centre of the star, whereas DEG ranks the centre node as the most important one.When it comes to CoreHD, the approach could be effective if the primary goal is to break cycles within the network.However, nodes in cycles may not be the nodes that, if removed, would cause the network to fragment Next, let us see how the constraint of within-LCC influences the performance of different strategies with different batch sizes.When batch size equals 1, imposing the constraint of within-LCC consistently improves the performance for all disruption strategies, especially for degreebased approaches such as DEG, DEG-BTW, CI and CoreHD.The rationale behind this improvement is straightforward: the primary goal of the disruption process is to minimise the size of the LCC at each step.Thus, concentrating the disruption efforts on the LCC is intuitively beneficial. When the batch size increases to 2 and beyond, the effects of the within-LCC constraint begin to diverge depending on the type of strategy employed.For degreebased approaches like DEG, DEG-BTW, CI, and CoreHD, the within-LCC constraint consistently enhances performance across various batch sizes (the only exception is at R@40% with a batch size of 4 due to an increased number of connected components of comparable sizes at a later stage).This is likely because degree centrality is inherently a local measure that quantifies a node's immediate influence based on its direct connections.When the scope of the algorithm is narrowed to the LCC, this local effect is magnified.The LCC, being the most connected and structurally significant part of the network, often contains high-degree nodes that are crucial for maintaining the network's largest connected structure.Therefore, focusing on the LCC enables these degree-based methods to target the most impactful nodes more efficiently, further optimising the disruption process. In contrast, for betweenness-based approaches like BTW and BTW-DEG, the application of the within-LCC constraint results in worse performance when the batch size is larger than one.The reason behind this is that betweenness centrality relies on global information, and narrowing the focus to the LCC might neglect key bridging nodes that exist outside of it.Overall, our results suggest that when the batch size is relatively small (batch size ranging from 1 to 3), the best disruption strategies are found to be those having within-LCC constraints.Also, degree-based approaches benefit more from a focus on LCC. Lastly, the result also sheds light on the impact of incorporating a secondary centrality measure.Generally, DEG-BTW outperforms DEG, and BTW-DEG is more effective than BTW.There are instances where the strategies with and without secondary centrality measures yield identical outcomes, especially when a small percentage of nodes are targeted to be removed, as indicated by R@20%.This suggests that in the initial phases, the top-ranked nodes are usually structurally distinct, making the primary centrality measure sufficient for effective node removal.However, as the percentage increases, the benefit of including a secondary centrality becomes more evident.For instance, we observed a decline in the number of identical outcomes between single-metric and dual-metric strategies-from 9 pairs at R@20% to just 4 pairs at R@40%.This indicates that as more nodes are targeted for removal, a dual-metric approach to node ranking becomes increasingly beneficial. It's noteworthy that degree centrality, due to its computational efficiency, emerges as the most commonly employed secondary ranking criterion.It also plays a pivotal role in CoreHD, as core only identifies a candidate set of nodes, and it is within this set that degree centrality is employed to finalise the rankings.Moreover, in the absence of articulation points or a core, degree centrality becomes the fallback option for ranking the nodes in APTA and CoreHD. DIRECT OPTIMISATION ALGORITHMS The above experiment revealed the effectiveness of introducing a secondary centrality metric as well as including the constraint within the LCC.The best disruption strategies are found to be APTA (when b = 1), DEG-based strategies (when b = 2 or 3), and BTW-based strategies (when b = 4). A natural continuation is, of course, to find another disruption strategy that outperforms them.Inspired by the outstanding performance of the greedy approach APTA, and also due to the relatively small size of the graph we manage in our analysis, we propose to first apply a naive greedy disruption strategy on all nodes, not only on the articulation points.Then, based on the findings from the experiment, we propose a novel greedy disruption strategy that also takes advantage of crucial structural features. A Naive Greedy Disruption Approach In order to conduct a continuous batch removal, at each step, the naive greedy disruption approach (GRD) goes over all possible combinations of all nodes and removes nodes leading to the largest drop in LCC size.The experiment is done on the Unified Graph and the result is reported in Table 4.The improvement of GRD is evident, especially when the batch size is larger than one.When the batch size equals one, the performance of GRD and APTA are quite similar.This can be attributed to the fact that most individual nodes that lead to the largest drop in LCC size are also articulation points.However, as the batch size increases, the advantages of GRD become more pronounced.This suggests that although the greedy approach aims to maximise the disruption effect only at each individual step, it does achieve an overall better performance compared to the best structural-metric-based approaches. One would expect that the within-LCC constraint should limit the effectiveness of a greedy approach due to a reduced search space.Certainly, there is no difference with b equal to one since a greedy algorithm would naturally target a node within the LCC to achieve a reduction in its size.The difference between with or without the constraint within-LCC is still not significant when b equals 2. As the batch size exceeds two, the performance of the standard GRD significantly outpaces that of the with-in LCC GRD.This implies that, with larger batch sizes, the freedom to remove nodes from multiple components becomes increasingly important for the overall effectiveness of GRD, as opposed to removing all nodes of the batch size within the LCC. Structurally Filtered Greedy Disruption Obviously, GRD's significant improvement in performance comes at the expense of efficiency: the time complexity of looping over the combination of n nodes alone is O(N n ), which is clearly infeasible even for moderate values of n.Therefore, we further analyse the rankings and characteristics of three key structural metrics for those nodes found by GRD.To meet the continuous batch removal process, these metrics are recalculated after each removal step.The three metrics, namely, betweenness centrality, degree centrality and articulation points, are selected because they have proven to be the most effective structural-based metrics to disrupt the criminal network.The detailed ranking TABLE 6: Detailed node categories using different disruption approaches, highlighting the first three steps for b = 3. Nodes marked with 'F' belong to mafia families, while 'L' denotes a leadership role. DEG-BTW GRD SF-GRD Step Step 3 N12 (F), N11 (L), N29 N43, N29, N69 N48 (F), N45 (F), N51 (F) information is shown in Figure 3.It is clear that a large portion of the nodes found in GRD also have high rankings in betweenness centrality and degree centrality, and the majority of them are identified as articulation points.This finding inspired us to create a structurally filtered greedy disruption strategy (in short, SF-GRD).The SF-GRD algorithm is designed to enhance the efficiency of the naive greedy disruption approach by constraining the search space to nodes that exhibit specific structural characteristics.Specifically, SF-GRD targets a union of nodes that either rank highly in betweenness centrality, excel in degree centrality, or are identified as articulation points.Moreover, to maintain a constant-size search space, we limit our selection to the top-ranking articulation points, sorted by their degree centrality.The SF-GRD algorithm is formally described in Algorithm 2. It determines the target nodes to be removed in a single disruption step.For a continuous batch removal process, the function select_nodes_to_remove in Algorithm 1 can be replaced by the SF-GRD function. The performance outcomes for SF-GRD, GRD, and the top structure-metric-based methods are summarised in Table 5.Overall, SF-GRD excels over structure-metric-based strategies and achieves comparable effectiveness to that of GRD.Like GRD, SF-GRD also benefits from relaxing the within-LCC constraint when the batch size is larger than one.In order to gain a deeper understanding of the disruption process, we take the case of R@20% and b = 3 as an example and plot how each step of removal affects the size of the LCC, when different disruption strategies are applied (Figure 4).Under this setting, SF-GRD performs closely to GRD, and both of them outperform DEG-BTW by a large margin.After the removal of 27 nodes, all three approaches have reached a similar disruption effect, with the size of LCC dropping to around 5 and 6.That also explains why we should focus more on the initial disruption steps. Furthermore, we examine the roles of the removed nodes identified by those approaches.We focus on the first three removal steps with a batch size equal to 3 and report the nodes removed together with their roles -whether they are from the two mafia clans or they take a leadership role.We can see that nodes associated with both mafia families and leadership roles (marked with F &L) are universally targeted by all three disruption strategies within the initial two steps.In fact, 5 out of the 6 nodes removed in these early stages possess such dual roles.Nodes that are important to hold the network together, also hold important roles in the organisation.Our finding aligns with the intuition that we should first take out the leaders in order to disrupt the network.Certainly, the importance of certain nodes might diminish along with the decomposition of the network, which explains why GRD targets the nodes without specific roles at the third step.In a disrupted network, nodes previously less influential may take on new significance, while the influence of originally key nodes may wane.Lastly, we discuss the time complexity of the proposed SF-GRD approach and compare it with GRD and BTW.At each step, SF-GRD involves first the calculation and ranking of three structural metrics, which are the betweenness centrality, the degree centrality, and the degree ranked articulation points.Among them, the dominating computational cost comes from the betweenness centrality, which is O(V E + V 2 log V ) using the Brandes algorithm [38].Then, in the greedy search loop, the total number of combinations is t ′ b , and the calculation of the size of the LCC is (V + E).Here, t ′ is the size of the search space, equal to 3 times t, where t is the number of nodes selected for each of the three structural metrics (see Algorithm 2).Therefore, the time complexity for SF-GRD is O(V E +V 2 log V + t ′ b •(V +E)).Since t ′ and b are usually set to be small integers (in our experiment, t ′ is set to be 15, and b is capped at 4), the time complexity for large graph would degrade to that of the betweenness centrality, i.e., O(V E + V 2 log V ). Time Complexity Analysis In comparison, GRD is looping over all combinations of node tuples from the entire graph, leading to a prohibitive time complexity of O V b • (V + E) .The comparison of the time complexity and the actual running time is given in Table 7.We used a machine with an Intel Xeon 6238R CPU at a base frequency of 2.2 Hz to run the experiment.We observe that SF-GRD is substantially more efficient than GRD while maintaining only a slightly higher computational cost compared to BTW.Specifically, with b = 4, SF-GRD is more than four orders of magnitude faster than GRD. In short, through a structurally-filtered scope, SF-GRD has brought down the combinatorial complexity of GRD to a polynomial time complexity while still achieving a comparable performance.SF-GRD successfully combines the best of both worlds: the effectiveness of GRD-like methods and the efficiency of structural-metric methods. CONCLUSION We first introduced a generalised robustness metric suitable for partial and continuous batch removal processes.We then evaluated several disruption strategies on a Sicilian Mafia network and proposed a within-LCC constraint which mimics the police raid operations.We revealed that this constraint would limit the effectiveness of the betweennessbased approaches but would enhance the performance of degree-based ones with a moderate batch size. Furthermore, we proposed a structurally-filtered greedy disruption strategy based on the structural characteristics of the nodes removed by a naive greedy approach.We found that the new algorithm significantly outperformed all classic disruption strategies while maintaining the same time complexity as the betweenness centrality approach.In addition to the applications of our proposed approaches in various complex network scenarios, a particularly interesting direction for future research involves exploring how other structural characteristics can provide valuable insights into network disruption.Considerations may extend to metrics like the graphlet degree [39], [40], the clustering coefficient [41], and the quadrangle coefficient [42]." Fig. 1 : Fig.1: The Unified Graph.Different colours represent different clans within the mafia network.A red circle around a node signifies leadership roles within the network, including positions such as bosses and executives.Additionally, the size of a node is proportional to its degree, and the width of a link is proportional to its weight. Fig. 3 :Algorithm 2 : 2 G 4 targetNodes ← [] ; 5 search 6 for 8 G 10 if lcc size current <= min lcc size then 11 min lcc size ← lcc size current ; 12 targetNodes ← node tuple ; 13 return Fig.3: Structural characteristics of the nodes removed by GRD.A large percentage of nodes, across different batch sizes, rank highly in the betweenness centrality and degree centrality, and are also articulation points. Fig. 4 : Fig. 4: A detailed disruption process.With the batch size setting to 3, 27 nodes are removed in 9 steps. TABLE 1 : Network description and comparison.The table lists six network metrics for seven different networks.The original disconnected graphs for meetings and phone calls are labelled as Meeting-DC (1 st column) and Phone-Call-DC (2 nd column), respectively.Their union forms the Unified-DC network (3 rd column).After isolating the largest connected components, we obtain the Meeting Graph (4 th column), Phone-Call Graph (5 th column), and Unified Graph (6 th column).The final column presents averaged statistics for 1,000 random graphs, each having the same number of nodes and edges as the Unified Graph. TABLE 2 : Classification of nodes based on family and leadership roles TABLE 3 : Dynamic disruption of various batch sizes, measured in R@20% and R@40%.Seven disruption strategies are included, performed both on the entire graph and on the LCC (referred to as Standard and W-LCC, respectively).The lowest R-values for each column are put in bold, and the lowest values across two columns are asterisked. TABLE 4 : Comparison of best-performing strategies at each batch size b with the Naive Greedy Disruption Approach (GRD), measured in R@20% and R@40%.The lowest Rvalues are highlighted in bold font. TABLE 5 : Performance comparison of the best performing structure-metric-based approach, GRD, and SF-GRD, measured in R@20% and R@40%.The number of highestranking nodes t is set to be 5 in the experiment. TABLE 7 : Time complexity and actual experimental time comparison.N is the number of nodes in a graph, E is the number of edges, b is the batch size, and t ′ is the size of the search space, set to be 15 in the experiment.	2023-10-17T06:42:50.377Z	2023-10-16T00:00:00.000	{ "year": 2024, "sha1": "50fb0941eb892cd6ac915708a37e2e1a4d500301", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "50fb0941eb892cd6ac915708a37e2e1a4d500301", "s2fieldsofstudy": [ "Computer Science", "Law", "Political Science" ], "extfieldsofstudy": [ "Medicine", "Computer Science" ] }
270244251	pes2o/s2orc	v3-fos-license	OD-YOLO: Robust Small Object Detection Model in Remote Sensing Image with a Novel Multi-Scale Feature Fusion As remote sensing technology has advanced, the use of satellites and similar technologies has become increasingly prevalent in daily life. Now, it plays a crucial role in hydrology, agriculture, and geography. Nevertheless, because of the distinct qualities of remote sensing, including expansive scenes and small, densely packed targets, there are many challenges in detecting remote sensing objects. Those challenges lead to insufficient accuracy in remote sensing object detection. Consequently, developing a new model is essential to enhance the identification capabilities for objects in remote sensing imagery. To solve these constraints, we have designed the OD-YOLO approach that uses multi-scale feature fusion to improve the performance of the YOLOv8n model in small target detection. Firstly, traditional convolutions have poor recognition capabilities for certain geometric shapes. Therefore, in this paper, we introduce the Detection Refinement Module (DRmodule) into the backbone architecture. This module utilizes Deformable Convolutional Networks and the Hybrid Attention Transformer to strengthen the model’s capability for feature extraction from geometric shapes and blurred objects effectively. Meanwhile, based on the Feature Pyramid Network of YOLO, at the head of the model framework, this paper enhances the detection capability by introducing a Dynamic Head to strengthen the fusion of different scales features in the feature pyramid. Additionally, to address the issue of detecting small objects in remote sensing images, this paper specifically designs the OIoU loss function to finely describe the difference between the detection box and the true box, further enhancing model performance. Experiments on the VisDrone dataset show that OD-YOLO surpasses the compared models by at least 5.2% in mAP50 and 4.4% in mAP75, and experiments on the Foggy Cityscapes dataset demonstrated that OD-YOLO improved mAP by 6.5%, demonstrating outstanding results in tasks related to remote sensing images and adverse weather object detection. This work not only advances the research in remote sensing image analysis, but also provides effective technical support for the practical deployment of future remote sensing applications. Introduction Due to the ongoing advancements in remote sensing technologies, the utilization of Remote Sensing Images (RSIs) has expanded significantly, including in the detection of lake surface water [1], applications in grain production [2] and landslide research [3].In recent years, the capabilities of neural network models have continuously improved.And, computer vision technology has advanced significantly, resulting in object detection methods that are vastly different from those of the past.The models of deep learning can extract deeper features from remote sensing images, showing great potential for applications in the recognition of remote sensing image targets [4]. However, the characteristics of remote sensing images also lead to several problems as follows: Firstly, remote sensing images include satellite images and images taken by unmanned aerial vehicles (UAVs), which differ significantly from ground-level images.What is more, remote sensing images exhibit rich diversity, capturing subjects across a variety of scales, perspectives, and forms.This diversity makes object detection more challenging.Secondly, remote sensing images have very large differences in object size, and these images cover both small and large targets.This difference in scale increases the complexity of the object detection task.Further, remote sensing images have a complicated background condition, which introduces a new consideration of environmental factors for object detection.The textures and colors of the objects in the images can closely resemble those of the background and complicate their differentiation.Therefore, it raises the level of challenge in detecting.Simultaneously, images captured through remote sensing often suffer from diminished object details and insufficient information, posing challenges to precise object detection.Finally, the weather has a great influence on remote sensing images; for example, there is a big difference between rainy days and sunny days in terms of the ability to capture objects, and bad weather may lead to blurred images or noise.The combination of these factors compounds the challenges associated with detecting targets in RSI. This paper introduces the OD-YOLO model framework in response to the challenging conditions of remote sensing images, aiming to enhance its object detection capabilities within remote sensing imagery tasks.We have designed a DRmodule, which enhances feature extraction for geometric structures.According to the Feature Pyramid Network (FPN) of YOLOv8 [5], this paper introduces the Dynamic Head module, which effectively improves the feature fusion capability across layers in the FPN.This paper proposed a new OIoU loss function.The OIoU can more accurately describe the localization of the targets in images.These improve the performance of the model, which shows an improvement of about 5.2% in mAP50 and 4.4% in mAP75 compared to recentadvanced methods. The main contributions of this paper are as follows: 1. In this paper, we propose the OD-YOLO framework for target detection in remote sensing scene captured by unmanned aerial vehicles. 2. An enhanced feature extraction Detection Refinement module (DRmodule) and OIoU loss function are employed to improve the model's capacity to gather features from small objects and detect them. 3. Experiments with a remote sensing object detection dataset prove that the OD-YOLO effectively boosts the performance in detect objects in remote sensing scenes. The rest of the paper is organized as follows: Section 2 offers an overview of related work in the field.Section 3 provides a thorough explanation of the method we propose.The results from comparative and ablation studies are detailed in Section 4. Section 5 explores the disadvantages of our approach and offers ideas for improvements in the future.Finally, Section 6 provides a summary of the findings and conclusions drawn from this research. Related Work 2.1. YOLO Model As deep learning grows, CNN has made great achievements, and YOLO [6] is one of the most popular models in CNNs.The YOLO model boasts an extensive array of applications in industry due to its simple structure, lightweight network and excellent detection results.Zhukov et al. [7] used the YOLO model with an attention mechanism and achieved better results in rail lines defect detection.Zhao et al. [8] have improved the YOLOv5 by making it lightweight to enable the rapid detection of sewer defects.Chen et al. [9] used the YOLO model to achieve good results in suspicious object detection in millimeter wave images.Ding et al. [10] improved the YOLOv5 model's capacity for global feature fusion by integrating the EPMS module.Tang et al. [11] improved the object detection ability of the YOLO model in complex traffic roads by introducing an RMA module.Wu et al. [12] proposed the SOD-YOLO model to achieve outstanding capacity in detecting mini-goal in the high-voltage transmission lines.Yuan et al. [13] effectively improved the precision in detect-ing defects of PCB by using the LW-YOLO model.Song et al. [14] proposed MED-YOLO to improve the model's recognition ability in complex traffic scenes.Du et al. [15] developed the DSW-YOLO model for strawberry fruit recognition and successfully implemented it in a strawberry-picking robot, achieving relatively good results.Wan et al. [16] proposed the LGP-YOLO, which has led to considerable advancements in identifying surface defects on light guide plates. In other methods, the ability of YOLO model's feature extraction is mainly improved through the attention mechanism, which essentially assists the model to accurately pinpoint and identify the target objects.But mostly, these models ignore the neck structure feature structure.In this paper, we raise the capacity of the feature fusion of the FPN in the framework.To achieve this, our method uses the Dynamic Head [17] to improve the overall recognition precision. Remote Sensing Object Detection Over the past few years, as objects detection technology has continually evolved, significant progress has been made in object detection in remote sensing imagery.However, many problems persist in detecting small targets, which are difficult to identify.Du et al. [18] effectively tackled the issue of losing crucial feature information of small-scale targets during multiple downsampling by combining feature integration across multiple scales and integrating an attention module.Liang et al. [19] proposed an object identification approach based on a novel cloud collaboration and restructured convolutional architecture, which improves the model's capacity in detecting remote sensing scenes in real time.Wu et al. [20] introduced the GNAM module, which combines several attention mechanisms to create a global normalized attention weight.This helps better utilize valuable information in the input feature channels and spatial dimensions, improving the effectiveness of the model in identifying remote sensing targets.Liu et al. [21] integrated a data augmentation algorithm with a highly efficient subspace attention module, while also fine-tuning the quantity of detection heads and refining the loss function.In a training period, Mai et al. [22] employed a dynamic dual-domain alignment (DDA) approach, which addresses possible mismatches in spatial and feature domains throughout the learning phase.Zhang et al. [23] introduced Drone-YOLO, which improves the capability of the model in understanding different scales features by utilizing the APFN [24] structure and RepVGG [25]. This paper proposes the DRmodule to tackle the difficulties of identifying small and diverse-shaped objects in remote sensing imagery.This module effectively lowers the likelihood of missing small objects and improves the model's capabilities in identifying targets across remote sensing images. Small Object Detection In traditional machine learning tasks, detecting small objects has always been a challenging task because the features of small object images are not distinct, making them difficult for models to recognize.Attention mechanisms and improved loss functions are often used to promote the model's effectiveness in the tasks of detecting small target.Mo et al. [26] incorporated multi-attention into YOLOv5 to yield improved outcomes in detecting small objects on airport runways.Yang et al. [27] merged an enhanced channel attention mechanism with a better version of E-ELAN [28] to introduce an upgraded YOLOv7 model, which is designed to identify small spots on grape leaves.Aibibu et al. [29] combined the strengths of various networks to improve the detection performance of small target vehicles.Liu et al. [30] utilized dynamic snake convolution [31] and introduced WISE-IoU [32] to boost the model's effectiveness in detecting small traffic-related objects.Wang et al. [33] proposed a joint attribute soft-sharing and contextual local method to improve the model's efficacy in re-identifying pedestrians. In this paper, the OIoU loss function is used, which is a more precise and stable metric tailored to small target detection.This approach enhances the precision and resilience in detecting objects. Proposed Method This paper uses the YOLOv8n model as a foundational architecture and presents the OD-YOLO framework.We propose the DRmodule in the OD-YOLO framework, specifically designed to identify small objects in remote sensing images, aiming to improve the model's feature extraction performance.Additionally, we incorporate the OIoU loss to boost the model's ability to semantically express detection boxes of small objects.Our model also integrates a Dynamic Head to strengthen the feature fusion ability in the feature pyramid.OD-YOLO enhances feature representation, leading to improved recognition accuracy in identifying objects within remote sensing imagery tasks. As shown in Figure 1, three components are in the framework of the OD-YOLO: Backbone, Neck and Head.Before being fed into the network, the input image's resolution will be adjusted to 640 × 640.Mosaic data augmentation stitches together four images using random cropping, brightness adjustments, and flipping.Feature Pyramid Network (FPN) [34] and Path Aggregation Network (PANet) [35] are used in OD-YOLO's neck, and finally, the OD-YOLO uses Dynamic Head to boost feature fusion capability.At last, the model outputs detection boxes, confidence scores, and categories in the form of a Decoupled Head. Detection Refinement Module Objects in remote sensing images may be relatively small.It is challenging for YOLO to detect samll targets because of the lack of specific information.To address these challenges, we designed a module called the Detection Refinement module (DRmodule), which is placed in the Backbone of the YOLO module to further enhance detection performance.The DRmodule enhances multi-scale expressive abilities and improves performance in low-resolution settings for detecting small targets in remote sensing images by integrating DCNv2 [36,37] and the Hybrid Attention Transformer [38].As shown in Figure 2, the DRmodule uses DCNv2 to derive geometric features from the input features.Then, a Hybrid Attention Transformer (HAT) is applied for feature extraction before using DCNv2 again to extract deeper features.These two sets of features are concatenated together.By fusing features across scales and adapting to deformations, the DRmodule enhances the effectiveness of the model in handling complicated situations in the images of remote sensing, thus improving the precision and reliability of target detection. Deformable Convolutional Networks In traditional CNN, convolution kernels have a very fixed structure, which results in poor feature extraction capability for certain geometric structures.DCNv2 [36,37] is an enhanced convolution technique capable of adaptively modifying the convolution kernel shape to more effectively accommodate the target's deformation or multi-scale features.The scale of targets in remote sensing imagery can differ substantially, and DCNv2 can better capture features of targets at different scales.Objects in remote sensing images may undergo unstructured deformations, such as tilting or morphing.DCNv2 can handle these situations better by adapting the shape of its convolution kernels, thus boosting the precision of object detection. For a DCNv2, in a 3 × 3 convolution, we assume that the relative positions of the convolutionally extracted features are as shown Equation (1). In DCNv2, each position of the convolution feature extraction has a position offset ∆p and a weight coefficient ∆m, learned from the preceding feature map.Therefore, the final feature output map is as shown Equation (2). Here, p 0 denotes the value at each location on the output feature map y, ∆m n is a decimal between [0, 1] used to represent the weight of that position.p n represents the point in the input feature map.∆p n denotes the offset learned from the earlier feature map.DCNv2 dynamically adapts to each feature extraction position by introducing a deviation that enables dynamic feature extraction.It also incorporates a weight, allowing for varied feature expression based on each position's unique characteristics.It can capture detailed information about the boundaries and complex shapes of target objects more accurately, especially useful for detecting objects that change shape.This approach effectively describes the geometric shapes of targets in remote sensing image recognition and boosts OD-YOLO's capability to extract features from small objects. Hybrid Attention Transformer Remote sensing object detection tasks face challenges such as highly complex backgrounds and multi-scale objects, which often lead to limitations in the recognition accuracy and generalization capabilities of traditional models.By using HAT's advanced features, including its robust global and local information processing abilities and acute sensitivity to complex backgrounds, the model significantly enhances its capability to detect objects of different scales. As shown in Figure 3, to derive shallow features from the image, HAT initially applies a convolution process, and it employs several RHAGs and a 3 × 3 convolution process for deriving deep feature.After that, the convolution layer and a Pixel Shuffle layer [39] is used for rebuilding the resolution, and then another 3 × 3 convolution layer is used to produce the ultimate image.The RHAG has several HAB layers; an OCAB layer and a 3 × 3 convolutional layer with a residual connection are used to achieve better reconstruction effects.This module comprises two principal elements: Window-based Self-Attention [40] and Channel Attention [41].First, the input features are normalized, then processed using the window-based self-attention mechanism.This mechanism segments the features maps into local windows, and every window will calculate self-attention to capture the association information of local areas.Next, through the channel attention, more global features are introduced to calculate channel attention weights.This attention module utilizes global insights to weight the features, thus activating more pixels.In HAT, the OCAB module is additionally proposed, as shown in Figure 4. Compared to the CAB module, it introduces an overlapping cross-attention layer to establish cross-connections between windows in window self-attention, enhancing the network's representation ability.As shown in Figure 5, the specific computation procedure resembles that of the W-SMA module [40], but in calculating the attention mechanism, zero-padding is applied to the original image during the computation of K/V, allowing for learning from the content in another window through a larger window.When a feature map is input into this layer, it is divided into several M * M windows, which serve as the Query in the attention mechanism.At the same time, the original feature map undergoes zero-padding controlled by the parameter γ.The padded feature map is divided into M 0 * M 0 feature maps, which serve as the Key/Value.The attention algorithm is as shown in Equation (3).The design of this module enables better utilization of the pixel information within a window for queries, thereby improving the model's performance. Here, d stands for the dimension of Q and K T , and B stands for the encoding of the position.The computation of Q, K, V is as described above.The calculation of attention is refered to in the literature [42].Through incorporating the HAT module into the architecture of the OD-YOLO model, we boost the model's capacity to process fine features in remote sensing images and markedly promote the model's adaptability to the unique spatial variability of remote sensing images through HAT's channel and window self-attention mechanisms.This improvement boosts the precision in the detection of the remote sense and strengthens the model's reliability against complex backgrounds and varied target sizes. Dynamic Head A major challenge in detecting objects in remote sensing is handling target detection across multiple scales and against complex backgrounds.Due to the difficulties, traditional object detection methods often struggle to accurately identify small targets or targets within complex backgrounds.Therefore, we employed Dynamic Head [17] to promote the performance on the small targets detection. In traditional Feature Pyramid Networks [34], F ∈ R L×H×W×C represents the features across all levels of the feature pyramid, where L represents the number of layers, and H, W, C represent the height, width, and channels.In the Dynamic Head, we define S = W × H so the tensor in the Feature Pyramid Network is reshaped into F ∈ R L×S×C .Scale-aware, Spatial-aware, and Task-aware attention mechanisms are incorporated in the Dynamic Head.Their structures are illustrated in Figure 6. Scale-aware Attention dynamically integrates features from various scales by depending on the significance, adjusting them according to their semantic importance.This attention mechanism can effectively improve performance issues in object detection caused by differences in object scales.The specific calculation equation is as shown in Equation (4). Here, f (•) is a convolutional layer close to 1 × 1, and δ is an activation function, with the specific expression being δ = max(0, x+1 2 ), F represents the input feature map.Spatial-aware Attention, designed with reference to DCN [36,37], is an attention mechanism in spatiality, which enables sparse sampling of features at different levels.It can unify features of different positions and levels.The specific calculation equation is shown in Equation (5). Here, L stands for the level of the FPN, w is a module similar to convolution for multidimensional feature sampling used for feature collection, and F represents the input feature.c represents the sampled channel.K represents the area for sparse feature sampling, while ∆p k and ∆m k , respectively, represent the position's offset and the position's weight, both of which are learned from the input feature map.Task-aware Attention is a dynamic, task-sensitive attention mechanism that dynamically turns off and on the features of certain channels in different tasks.The specific calculation equation is shown in Equation (6). Here, F c stands for the features in the channel c , F is the input feature map.OD-YOLO uses Dynamic Head as its detection head to greatly improve its ability to recognize targets from remote sensing images.This is particularly effective for small targets and complex backgrounds.Dynamic Head's special attention mechanism offers better feature details, making the model more adaptable to changes in size and more focused on important areas.This boosts the detection quality.The enhancements not only improve accuracy on remote sensing images, but also make the model better at identifying targets in complex settings. OIoU Loss Function In the tasks of detecting objects, the difference between the model's predictions and the true target is described by the loss function.It plays a crucial role as the model learns to be accurate.During the process of remote sensing target detection, the objects detected are typically small in size; therefore, accurately describing the detection boxes is quite challenging.When dealing with small objects, traditional loss functions might have some issues.Since small objects usually occupy fewer pixels in an image, traditional loss functions are quite sensitive to the pixel shift between predicted and true target boxes, which can lead to unstable results.Therefore, we propose OIoU loss function to precisely detect boxes.Its calculation process mainly includes the following parts: Angle Loss We use δ to represent the size of angle loss in OIoU.In this paper, B signifies the box predicted, and B GT signifies the real detection box, as shown in Figure 8.The definition of δ is shown in Equation (7). where C h is the height disparity between the predicted and true detection boxes, and γ represents the Euclidean distance between the centers of the two detection boxes.Throughout the training phase, if α ≤ π 4 minimize α first; if not, focus on reducing β.Equation (7) describes the angular difference between the two detection boxes.If α is π 2 or 0 during training, the angle loss is 0. In the convergence process, if α ≤ π 4 , minimizing α will be prioritized; otherwise, minimizing β will be prioritized. Distance and Shape Loss In OIoU, we define the distance loss as ϵ.As shown in Figure 9, B GT represents the real box, and B represents the predicted box.B c and B GT c .represent the centers of the two detection boxes, respectively.The calculation equation for ϵ is shown in Equations ( 8) and (9). Here, the expressions of ρ x and ρ y are in Equation (9). Equations ( 8) and ( 9) show the discrepancy in the distance of the midpoints of the predicted and actual boxes.These equations take into account the Euclidean distance in both the x and y directions and use an exponential function to control the sensitivity of the loss.Here, µ is an adjustment parameter used to balance the impact of angle loss on distance loss.The goal of these equations is to bring the center of the predicted box as close as possible to the center of the real box, thus reducing the model's error in target localization. B B GT C W C H . This shows how distance and shape loss is calculated between the predicted and real object boxes. We also define the shape loss as ζ, with the specific calculation equation shown in Equations ( 10) and (11). Here, (w, h) and (w GT , h GT ) stands for the width and height of the predicted and actual boxes.The parameter range of θ is [2,6], signifying the level of focus on shape loss, and it is set to 4 in the OD-YOLO.The expressions for W w and W h are shown in Equation (11). Equations ( 10) and (11) measure the shape discrepancy between the predicted box and the real box.Through calculating the relative error in position of two boxes, and applying an exponential function to adjust the sensitivity of the loss, it adjusts the emphasis on shape loss.The goal of shape loss is to predict when true boxes are more closely aligned, improving detection accuracy. In OIoU, there is another part of the distance loss that more accurately describes the variance in shape and distance in the predicted box and the real box.Here, we use η to represent it, with the specific calculation process shown as Equation (12). Here, σ denotes the Euclidean distance of two points.C w and C h indicate the minimum bounding rectangle's width and height for the predicted and real bounding boxes.b and b GT represent the centers of the two boxes, while (w, h) and (w GT , h GT ) stand the width and height of the predicted and real boxes.Equation ( 12) introduces a comprehensive distance loss, which scrutinizes the variations in shape and center position more closely between the predicted boxes and the actual ones.It calculates the square of the discrepancies in the shape of the boxes and center points between the predicted and real boxes.Then it is divided by the width and height of the minimum bounding rectangle that encloses both the predicted and actual boxes.It measures the differences in size and position between the two boxes.This design aims to more accurately measure and optimize the differences between the predicted and real boxes, further boosting the model's effectiveness in detecting objects within remote sensing imagery. OIoU In OIoU, we use IoU to describe the difference between predicted and real detection boxes.Figure 10a shows the intersection between the detection boxes, and Figure 10b shows their union.We use A to represent the predicted detection box, and B to represent the real detection box.The calculation equation for IoU is shown in Equation (13). In summary, the OIoU loss function is calculated as in Equation ( 14). By introducing the OIoU loss function, the OD-YOLO model can more accurately detect small objects in remote sensing images and also improve its ability to recognize objects against complex backgrounds.Experimental results show that OD-YOLO outperforms several advanced models on the VisDrone dataset, with significant improvements in both mAP50 and mAP75 metrics.This demonstrates the OIoU loss function's efficiency and superiority in tasks involving object detection in remote sensing imagery. Experiment 4.1. Experiment Details In this paper, the image resolution is set to 640 × 640.We choose the Adam optimizer to fine-tune the parameters, setting an initial learning rate of 0.01 and a weight decay of 0.0005.All computational tasks, including training and testing, are executed on an NVIDIA RTX 4060 Laptop GPU, utilizing Pytorch 2.0.1 and CUDA 11.8.To efficiently manage local memory resources, the BatchSize is established at 8. We use Mosaic data augmentation [44] in a training period and merge the four images together. Dataset Visdrone We chose the challenging VisDrone-2019 dataset [45], which has 8599 images to evaluate our model.These images were taken by drones at various positions and altitudes.In this dataset, there are lots of photos with small detection targets, dynamic blur, and obstructions.These challenges help evaluate the model's effectiveness.The dataset is by default divided into 6471 training images and 548 validation images, totaling about 540k detection boxes.The rest being used as the test set, which is not used for training or evaluation. Foggy Cityscapes We chose a challenging dataset for adverse weather conditions: Foggy Cityscapes [46].This dataset is based on Cityscapes and each image has been fogged with coefficients of 0.01, 0.02, and 0.005, providing valuable reference for object detection in adverse weather.The dataset consists of a total of 8925 training images and 1500 validation images. Evaluation Metrics This paper mainly uses mAP50 and mAP75 as the key indicators, and their calculation process is as follows.First, we need to calculate the average precision (AP).In mAP50, we consider a detection correct if the IoU between the predicted box and the ground truth box is greater than 50%.In mAP75, we consider a detection correct if the IoU is greater than 75%.After calculating the AP for each category, we take the average of the AP values for all categories to obtain the final mAP.The specific computational equation is as shown in Equation (15). Experimental Results We performed a comparison analysis of our proposed framework against alternative methods, with the results presented in Table 1.OD-YOLO achieves 36.16% on mAP50 and 21.6% on mAP75.When compared with YOLOv8n, YOLOv5s, Cascade-RCNN, RefineDet, and M2S, it shows an improvement of 5.2%, 9.9%, 4.2%, 7.3%, and 6.4% on mAP50, and 4.4%, 7.4%, 6.6%, 7.5%, and 7.4% on mAP75.These results show that OD-YOLO not only detects more objects accurately on the VisDrone dataset for remote sensing images, but it is also better at detecting objects of varying scales and complexities.Especially at high IoU thresholds, OD-YOLO still maintains high detection accuracy.This means it locates and recognizes targets very accurately.It shows OD-YOLO's strengths and potential in detecting targets.Table 2 details the comparison between the OD-YOLO model and the YOLOv8n model in detecting different types of targets on the VisDrone dataset.It lists the average precision (AP) for 10 categories, as well as the mAP at IoU thresholds of 0.5 and 0.75.Thorough comparing the AP, mAP50 and mAP75 of YOLOv8n and OD-YOLO in each category, it is clear that OD-YOLO performs better in almost all categories.Specifically, OD-YOLO not only shows a clear improvement in precision when dealing with small target recognition tasks but also demonstrates higher detection accuracy at higher IoU thresholds.This enhancement validates the efficacy and promise of the OD-YOLO model for applications in remote sensing image analysis.Figure 11 showcases the detection outcomes for five representative images using YOLOv8n and OD-YOLO.From the illustrations, it is evident that OD-YOLO surpasses YOLOv8n in several key areas: it excels in identifying smaller objects, operates more effectively in dimly lit environments, and demonstrates superior performance in navigating complex roadways.OD-YOLO showcases heightened confidence in its target detections, with marked improvements in recognizing distant small targets and those obscured by shadows.Moreover, in intricate scenes, OD-YOLO achieves greater accuracy in classifying various categories and boasts a higher success rate in target identification.This enhanced capability is particularly pronounced in the model's handling of small object recognition, where OD-YOLO consistently outperforms its predecessor.The advancements made by OD-YOLO can be attributed to its refined algorithms that better interpret the nuances of remote sensing imagery, including the intricate play of light and shadow, as well as the model's adeptness at processing the detailed textures and shapes indicative of small and distant objects.This increased accuracy not only improves the reliability of the detections, but also minimizes the instances of false positives and negatives, crucial for applications requiring high precision. In order to evaluate the effectiveness of the model, this study plots the Precision-Recall curve.Each point on the curve represents the precision and recall at a specific threshold.The shape of the curve reflects the classifier's performance at different thresholds.Generally, we aim for a high precision while maintaining a high recall, to ensure that all positive samples are identified as much as possible while minimizing false positives.Figure 12 shows the PR curve for YOLOv8n, and Figure 13 shows the PR curve for OD-YOLO.In the figures, it is evident that OD-YOLO achieves higher precision at the same recall level.This means the OD-YOLO model can provide more accurate predictions while maintaining a high recall rate.The Precision-Recall curve of OD-YOLO is smoother, indicating that the predictions from the OD-YOLO model are more stable and consistent.A confusion matrix is a commonly used tool for evaluating classifiers in classification problems.It shows how well the classifier performs by organizing the prediction results into a matrix based on the true and predicted categories.This visually shows how the classifier does with different categories.Figure 14 represents the confusion matrix for YOLOv8n, and Figure 15 represents the confusion matrix for OD-YOLO.In the confusion matrix, the horizontal axis shows the true values, and the vertical axis shows the predicted values.By looking at the confusion matrices of both models, OD-YOLO has higher accuracy and lower error rates for category, showing that it is better at correctly predicting multiple categories.Compared to YOLOv8n, OD-YOLO has fewer mix-ups and is more accurate in distinguishing samples of different categories.Overall, OD-YOLO's confusion matrix is better than YOLOv8n's, with more true positives, lower misclassification rates, higher precision, and fewer cases of mixing up categories.In this paper, we use Grad-CAM [51] technology to visualize features.We chose the C2f layer before the 128 × 128 × 128 Dynamic Head of Figure 1.This layer integrates features from several smaller scales within the backbone, demonstrating the backbone's ability to extract features for small objects.In the visualized heatmap, the deeper the color, the greater the contribution to the outcome.As shown in Figure 16, OD-YOLO demonstrates a significant improvement in the capability to derive features from small targets compared to YOLOv8n.These feature extraction capabilities come from the DRmodule's superior ability to extract small-scale features, and OIoU helps the model better locate the position and shape of the objects.To better verify the role of OIoU in small object detection, we used YOLOv8n as the training model and conducted experiments on the Visdrone dataset for comparison.In the standard YOLOv8n, the CIoU loss function is used.We conducted comparison experiments by replacing the loss function.As show in Table 3, the experiments showed that OIoU has a significant advantage in small object detection compared to other loss functions.It improved the AP metric by up to 1.4%, mAP50 by up to 1.8%, and mAP75 by up to 1.4%.This indicates that OIoU has a clear advantage in small object detection.This advantage comes from OIoU's more accurate consideration of the angle and distance differences between the predicted box and the ground truth box, resulting in more accurate bounding box positioning.To evaluate the performance of the OD-YOLO model in complex weather conditions, we chose the challenging Foggy Cityscapes dataset for comparison experiments.It is important to note that the Foggy Cityscapes dataset is derived from the Cityscapes dataset, and its labels also come from Cityscapes.However, because the Cityscapes dataset is suitable for both object detection and panoptic segmentation tasks, some class settings are not suitable for evaluating object detection.Therefore, we adopted the mainstream method for evaluating the model on Foggy Cityscapes: training with all classes together and selecting the average Precision (AP) of eight classes (person, rider, car, truck, bus, train, motorcycle, bicycle) and the mean AP of these eight classes for comparison. Table 4 shows the performance of OD-YOLO and other models on the Foggy Cityscapes dataset.From the table, we can see that OD-YOLO has a significant advantage compared to other models.Compared to YOLOv8n, YOLOv5n, SIGMA, and DeFRCN, OD-YOLO shows a notable improvement in each category, with an overall mAP increase of 5.5%, 8%, 8%, and 20.4%, respectively, for the eight categories.Compared to MILA, although there is a slight decrease in the car, bus, and train categories, the overall mAP increased by 2.6%.The experimental results indicate that OD-YOLO significantly enhances feature extraction capability for object recognition tasks under adverse weather conditions.Figure 17 shows a comparison of typical prediction results between YOLOv8n and OD-YOLO on the Foggy Cityscapes dataset.In the first image, YOLOv8n mistakenly detects a traffic sign as a person, while OD-YOLO better detects the person in dense fog conditions.In the second image, OD-YOLO accurately detects a train and a small, occluded car.In the third image, OD-YOLO successfully detects a car that is partially occluded by a nearby car and also accurately detects a person in dense fog conditions.In the fourth image, OD-YOLO accurately detects a nearby car covered in dense fog.In the fifth image, OD-YOLO avoids YOLOv8n's mistake of identifying a traffic sign as a car and successfully detects a motorcycle.This demonstrates that OD-YOLO has better object detection performance under adverse weather conditions compared to YOLOv8n.The improvement in OD-YOLO's detection capability under adverse weather conditions comes from the enhanced modules within OD-YOLO.These modules better extract the less obvious features of objects in adverse weather, particularly the DRmodule's ability to extract blurred features.Such enhancements allow the model to meet higher accuracy requirements for object recognition in adverse weather conditions. The Result of Ablation Study To evaluate the effectiveness of each module and investigate their impact on the accuracy of the algorithm, we conducted a variety of ablation experiments.Starting with YOLOv8n as the baseline, we incrementally added improvements.According to the ablation experiment data in Table 5, incorporating the DRmodule into the backbone network improved the mAP50 and mAP75 by 1.3% and 0.9%, respectively, compared to the original network.This demonstrates that the DRmodule significantly enhances the model's ability to capability to derive geometric shape features, boosting its performance.Adding the Dynamic Head module on top of the DRmodule further increased mAP50 and mAP75 by 3.1% and 2.6% indicated that Dynamic Head greatly improves the feature fusion ability in the feature pyramid of the YOLO model, achieving excellent results in object detection.Finally, including the OIoU loss function in the model resulted in an additional increase of 0.8% in mAP50 and 0.5% in mAP75, showing that OIoU accurately describes the prediction accuracy of small object detection, thereby enhancing model performance. GFLOPs represent the number of floating-point operations required by the model, indicating its computational complexity.FPS represents the number of frames that can be detected per second.From the ablation experiments, OD-YOLO increased the computational complexity by 3.7 GFLOPs compared to YOLOv8n.Although the FPS decreased from 256.4 to 134.3, maintaining 134.3 FPS on devices with limited computing power still allows for very smooth object detection tasks.This drop in frame rate is completely justified. Discussion While the model demonstrates superior performance in small object detection within remote sensing images, there are several limitations and avenues for future work that merit attention.One of the primary limitations of the OD-YOLO model lies in its computational efficiency and resource requirements.The integration of complex modules like the DRmodule and the feature fusion detection head, while beneficial for accuracy, significantly increases the computational load.This can pose challenges for real-time applications or when deploying on hardware with limited processing capabilities.Secondly, object image recognition in remote sensing can be affected by objective factors such as weather.Although in this experiment, OD-YOLO performed very well under adverse weather conditions, there is still a need to explore and address object detection in extreme conditions. Looking ahead, there are several promising directions for enhancing the OD-YOLO model.First, optimizing the model architecture to reduce computational demands while maintaining or even improving accuracy would make OD-YOLO more practical for a broader array of applications, including those requiring real-time processing.Investigating lightweight versions of the model that do not compromise significantly on performance could be particularly beneficial.For example, using lightweight methods such as Ghost-Module [59] to replace certain structures in the model can improve the model's real-time performance while maintaining accuracy.Secondly, we will explore more data processing techniques.For example, in extreme weather conditions, we can use advanced image processing techniques such as dehazing [60].In the future, we will focus more on researching image processing methods for remote sensing image object recognition, facilitating object detection under extreme conditions.Thirdly, to achieve better detection results, we will fine-tune our model to achieve optimal performance. In conclusion, while the OD-YOLO model represents a significant step forward in remote sensing image analysis, continuous efforts in addressing its limitations and exploring future directions are essential for advancing the field and meeting the evolving demands of practical applications. Conclusions In this paper, we introduce the OD-YOLO model, designed specifically for the task of recognizing targets in remote sensing images.This model incorporates three key components: the DRmodule, which boosts feature fusion; a detection head that improves the feature pyramid structure; and the OIoU loss function, tailored to enhance recognition of small targets.Our experiments conducted on the VisDrone and Foggy Cityscapes dataset demonstrate that OD-YOLO outperforms existing models in detecting small targets and bad weather condition.Moving forward, we aim to further refine the model's performance, striving to develop more effective target recognition approaches for remote sensing imagery. Figure 1 . Figure 1.The framework of OD-YOLO.Four DRmodules are placed in the backbone for feature extraction.In the neck, features across scales are fused.Finally, detection targets are output using three dynamic heads of different scales. Figure 2 . Figure 2. The structure of DRmodule.It integrates Deformable Convolutional Networks and Hybrid Attention Transformer for enhanced geometric feature extraction in remote sensing imagery. Figure 3 . Figure 3. Diagram of the Hybrid Attention Transformer (HAT) in the DRmodule, showing how it extracts basic features, processes them with attention groups for detailed analysis, and then rebuilds the image to better capture object details for detection. Figure 4 . Figure 4.The Overlapping Channel Attention Block (OCAB) in the HAT, showing how it uses attention across different areas and channels to better detecting targets. Figure 6 . Figure 6.The Dynamic Head incorporates three attention mechanisms: Scale-aware, Spatial-aware, and Task-aware attentions.This diagram shows the process of dynamically adjusting feature emphasis on different scales, spatial regions, and task-specific features to enhance object detection performance. Figure 7 . Figure 7.The structure of Dynamic Head, showing how it combines attention mechanisms to decide on object classes and their locations in the image. Figure 8 . Figure 8. Diagram showing how angle loss is calculated between the predicted and real object boxes. Figure 10 . Figure 10.Illustration of the Intersection over Union (IoU) calculation for object detection.(a) Intersection; (b) Union. Figure 11 . Figure 11.The detecting result comparison between YOLOv8n and OD-YOLO.(a) Original images.(b) The result of YOLOv8n.(c) The result of OD-YOLO. Figure 16 . Figure 16.The figure displays a comparison of heatmaps between YOLOv8n and OD-YOLO.(a) is the original picture, (b) is the heatmap of YOLOv8n, and (c) is the heatmap of OD-YOLO.It can be observed in the figure that the colors representing small targets in OD-YOLO are deeper, indicating that OD-YOLO has a stronger capability for feature extraction. Figure 17 . Figure 17.The detecting result comparison in Foggy Cityscapes dataset between YOLOv8n and OD-YOLO.(a) Original images; (b) The result of YOLOv8n; (c) The result of OD-YOLO. Table 1 . The Comparsion of various state-of-the-art models on VisDrone dataset. Table 2 . The result of different classes on VisDrone dataset. Table 3 . The Comparsion of various loss function on VisDrone dataset. Table 4 . The Comparsion of various state-of-the-art models on Foggy Cityscapes dataset. Table 5 . The result of ablation study on VisDrone dataset.	2024-06-05T15:15:35.705Z	2024-06-01T00:00:00.000	{ "year": 2024, "sha1": "5ef3e04550e1cced8a5ab2daffee9c341e4e8fd8", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1424-8220/24/11/3596/pdf?version=1717387717", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a5bb148da53ce71427880bdce535394eba97a48b", "s2fieldsofstudy": [ "Environmental Science", "Computer Science", "Engineering", "Geography" ], "extfieldsofstudy": [ "Medicine" ] }
228899906	pes2o/s2orc	v3-fos-license	FPGA-Based On-Board Hyperspectral Imaging Compression: Benchmarking Performance and Energy Efﬁciency against GPU Implementations : Remote-sensing platforms, such as Unmanned Aerial Vehicles, are characterized by limited power budget and low-bandwidth downlinks. Therefore, handling hyperspectral data in this context can jeopardize the operational time of the system. FPGAs have been traditionally regarded as the most power-efﬁcient computing platforms. However, there is little experimental evidence to support this claim, which is especially critical since the actual behavior of the solutions based on reconﬁgurable technology is highly dependent on the type of application. In this work, a highly optimized implementation of an FPGA accelerator of the novel HyperLCA algorithm has been developed and thoughtfully analyzed in terms of performance and power efﬁciency. In this regard, a modiﬁcation of the aforementioned lossy compression solution has also been proposed to be efﬁciently executed into FPGA devices using ﬁxed-point arithmetic. Single and multi-core versions of the reconﬁgurable computing platforms are compared with three GPU-based implementations of the algorithm on as many NVIDIA computing boards: Jetson Nano, Jetson TX2 and Jetson Xavier NX. Results show that the single-core version of our FPGA-based solution fulﬁls the real-time requirements of a real-life hyperspectral application using a mid-range Xilinx Zynq-7000 SoC chip (XC7Z020-CLG484). Performance levels of the custom hardware accelerator are above the ﬁgures obtained by the Jetson Nano and TX2 boards, and power efﬁciency is higher for smaller sizes of the image block to be processed. To close the performance gap between our proposal and the Jetson Xavier NX, a multi-core version is proposed. The results demonstrate that a solution based on the use of various instances of the FPGA hardware compressor core achieves similar levels of performance than the state-of-the-art GPU, with better efﬁciency in terms of processed frames by watt. Introduction Hyperspectral technology has experienced a steady surge in popularity in recent decades.Among the main reasons that have given hyperspectral imaging greater visibility is the richness of spectral information collected by this kind of sensor.This feature has positioned hyperspectral analysis techniques as the mainstream solution for the analysis of land areas and the identification and discrimination of visually similar surface materials.As a consequence, this technology has acquired increasing relevance, being widely used for a variety of applications, such as precision agriculture, environmental monitoring, geology, urban surveillance and homeland security, among others.Nevertheless, hyperspectral image processing is accompanied by the management of large amounts of data, which affects, on one hand, its real-time performance and, on the other hand, the requirements of the on-board storage resources.Additionally, the latest technological advances are promoting to market hyperspectral cameras with higher spectral and spatial resolutions.All of this makes the efficient data handling, from an on-board processing, communication, and storage point of view, even more challenging [1,2]. Traditionally, images sensed by spaceborne Earth-observation missions are not on-board processed.The main rationale behind this is the limited on-board power capacity that forces the use of low-power devices, which are normally not as highly performing as their commercial counterparts [3][4][5][6][7][8].In this regard, images are subsequently downloaded to the Earth surface where they are off-line processed on high-performance computing systems based on Central Processing Units (CPUs), Graphics Processing Units (GPUs), Field-Programmable Gate Arrays (FPGAs), or heterogeneous architectures.In the case of airborne capturing platforms, images are normally on-board stored and hence the end user could not access them until the flight mission is over [9].Additionally, unmanned aerial vehicles (UAVs) have gained momentum in recent years.They have become a very popular solution for inspection, surveillance and monitoring since they represent a lower-cost approach with a more flexible revisit time than the aforementioned Earth-observation platforms.In this context, hyperspectral image management is addressed similarly to how it is done in airborne platforms, although a lot of efforts have been made recently to transmit the images to the ground segment as soon as they are captured [10,11]. Regrettably, the data transmission from the aforementioned remote-sensing observation platforms introduces important delays.They are mainly related to the transference of large volumes of data and the limited communication bandwidths between the source and the final target, which has also kept relatively stable over the years [6,12,13].Consequently, it reveals a bottleneck in the downlink systems that can seriously affect to the effective performance of real-time or nearly real-time applications.However, the steadily growing data-rate of the latest-generation sensors makes it compulsory to reach higher compression ratios and to carry out a real-time compression performance in order to prevent the unnecessary accumulation of high amounts of uncompressed data on-board and to facilitate efficient data transfers [14]. In this scenario of limited communication bandwidths and increasing data volumes, it is becoming necessary to move from lossless or near-lossless compression approaches to lossy compression techniques.Despite most of the state-of-the-art lossless compressors bringing a quite satisfactory rate-distortion performance, the former approaches provide very moderate compression ratios of about 2∼3:1 [15,16], which presently are not sufficient to handle the high input data-rate of the newest-generation sensors.For this reason, a research effort targeting lossy compression is currently being made [17][18][19][20][21]. Due to the limited on-board computational capabilities of remote-sensing hyperspectral acquisition systems, low-complexity compression schemes stand as the most practical solution for such restricted environments [21][22][23][24].Nevertheless, most of the state-of-the-art lossy compressors are generalizations of existing 2D images or video compression algorithms [25].For this reason, they are normally characterized by high computational burden, intensive memory requirements and a non-scalable nature.These features prevent their use in power-constrained applications with limited hardware resources, such as on-board compression [26,27]. In this context, the Lossy Compression Algorithm for Hyperspectral Image Systems (HyperLCA) [28] was developed as a novel hardware-friendly lossy compressor for hyperspectral images.HyperLCA was introduced as a low-complexity alternative that provides a good compression performance at high compression ratios with a reasonable computational burden.Additionally, this algorithm permits compressing blocks of image pixels independently which promotes, on the one hand, the reduction of the data to be managed at once besides the hardware resources to be allocated.On the other hand, the HyperLCA algorithm becomes a very competitive solution for most applications based on pushbroom/whiskbroom scanners, paving the way for real-time compression performance.The flexibility and the high level of parallelism intrinsic to the HyperLCA algorithm has been previously evaluated in earlier publications.In particular, its suitability for real-time performance in applications characterized by high data-rates with restrictions in computational resources due to power, weight or space was tested in [29]. In this work, we focus on a use case where a visible-near-infrared (VNIR) hyperspectral pushbroom scanner is mounted onto a UAV.In particular, we have analyzed the performance of FPGAs for the lossy compression of hyperspectral images against low-power GPUs (LPGPUs) in order to establish the benefits and barriers of using each one of these hardware devices for the on-board compression task.Specifically, we have implemented the HyperLCA algorithm in a Xilinx Zynq-7000 programmable System on Chip (SoC).We have selected this SoC because it can be found in low-cost, low-weight and compact-size development boards, such as MicroZed TM and PYNQ boards.Although UAVs have been consolidated as trending aerial observation platforms, their acquisition costs are still not accessible for many end customers, not only those who want to purchase them but also those who lease their services.For this reason, we must also aim to solve the economic implications that comes along with these devices.On this basis, in this work we have focused on the on-board computing platform, searching for a less expensive alternative that, in exchange, cannot offer the same level of both performance and functionality than other costly commercial products.Additionally, it is important to note that while experiments carried out in this work are oriented to the current necessities imposed by an application based on drones, all drawn conclusions can be extrapolated to other fields in which remotely sensed hyperspectral images have to be compressed in real time, such as spaceborne missions that employs next-generation space-grade FPGAs. The rest of this paper is organized as follows.Section 2 gives outlines of the majority issues around the operations involved in the HyperLCA algorithm.In addition, it also includes a detailed explanation about the implementation model developed for the execution of the HyperLCA compressor on the selected FPGA SoC.Section 3 analyzes the obtained experimental results in terms of both quality of the compression process and hardware implementation points of view.Section 4 discusses the strengths and limitations of the proposed solution and includes a comparison evaluation of the obtained results with those achieved by a selection of LPGPUs embedded systems, following the implementation model shown in [29].Finally, Section 5 draws the main conclusions of this work. HyperLCA Algorithm The HyperLCA algorithm is a novel lossy compressor for hyperspectral images especially designed for applications based on pushbroom/whiskbroom sensors.Concretely, this transform-based solution can independently compress blocks of image pixels regardless of any spatial alignment between pixels.Consequently, it facilitates the parallelization of the entire compression process and makes it relatively simple to stream the compression of hyperspectral frames collected by a pushbroom or whiskbroom sensor.Additionally, the HyperLCA compressor also allows fixing a desired minimal compression ratio and guaranties that at least this minimum will be achieved for each block of image pixels.This makes it possible to know in advance the maximum data rate that will be attained after the acquisition and compression processes in order to efficiently manage the data transfers and/or storage.Additionally, the HyperLCA compressor provides quite satisfactory rate-distortion results for higher compression ratios than those achievable by lossless compression approaches.Furthermore, the HyperLCA algorithm preserves the most characteristic spectral features of image pixels that are potentially more useful for ulterior hyperspectral analysis techniques, such as target detection, spectral unmixing, change detection, anomaly detection, among others [30][31][32][33][34]. Figure 1 shows a graphic representation of the main computing stages involved by the HyperLCA compressor.First, the Initialization stage configures the same parameters (p max ) employing the input parameters CR, N bits and BS.Since the HyperLCA compressor follows an unmixing-like strategy, the p max most representative pixels within a block of image pixels to be independently processed are compressed with the highest precision.Then, other image pixels are reconstructed using their scalar projections, V, over the previously selected pixels.Therefore, p max is estimated according to the minimum desired compression ratio to be reached, CR, the number of hyperspectral pixels within each image block, BS and the number of bits used for representing the values of V vectors.Second, these p max characteristic pixels are selected using orthogonal projection techniques in the HyperLCA Transform stage.It results in a spectral uncorrelated version of the original hyperspectral data.Thirdly, the outputs of the HyperLCA Transform stage are adapted in the following HyperLCA Preprocessing stage for being later more efficiently codified in the fourth and last HyperLCA Entropy Coding stage.In the following lines, the operations involved in these four algorithm stages are further analyzed in order to give a glance of the decisions taken for the acceleration of the HyperLCA algorithm in hardware devices based on FPGAs. General Notation Before starting the algorithm description, it is necessary to introduce the general notation followed thorough the rest of this manuscript.In this sense, HI = {F i , i = 1, ..., nr} is a sequence of nr scanned cross-track lines of pixels acquired by a pushbroom sensor, F i , comprised by nc pixels with nb spectral bands.Pixels within HI are grouped in blocks of BS pixels, M k = r j , j = 1, ..., BS , being normally BS equal to nc, or multiple of it, and k spans from 1 to nr•nc BS .μ represents the average pixel of each image block, M k .Therefore, C is defined as the centralized version of M k after being subtracted μ from all image pixels.E = {e n , n = 1, ..., p max } saves the p max most different hyperspectral pixels extracted from each M k block.V = {v n , n = 1, ..., p max } comprises p max vectors of BS elements where each v n vector corresponds to the projection of the BS pixels within M k onto the corresponding n extracted pixel, e n .Q = {q n , n = 1, ..., p max } and U = {u n , n = 1, ..., p max } save p max pixels of nb bands that are orthogonal among them. HyperLCA Initialization The HyperLCA compressor must first determine the number of pixels, e n , and projection vectors, v n , (p max ) to be extracted from each image block, M k , according to the input parameters CR, N bits and BS, as shown in Equation (1).In it, DR refers to the number of bits per pixel per band used for representing the hyperspectral image elements to be compressed.As can be deduced, the number of selected pixels, p max , directly determines the maximum compression ratio to be reached with the selected algorithm configuration.Furthermore, bigger p max results in better reconstructed images but lower compression ratios. HyperLCA Transform The HyperLCA compressor is a transform-based algorithm that employs a modified version of the well-known Gram-Schmidt orthogonalization method to obtain a compressed and uncorrelated image.In this regard, the HyperLCA Transform stage oversees carrying out the spectral transform.For this reason, it is the algorithm stage that involves the most demanding computing operations and thus, which are more prone to be accelerated in parallel dedicated hardware devices. The HyperLCA Transform is described in detail in Algorithm 1.As can be seen, the HyperLCA Transform stage receives as inputs the hyperspectral image block to be compressed, M k , and the number of hyperspectral pixels to be extracted, p max .As outputs, the HyperLCA Transform states the set of the p max most different hyperspectral pixels, E, and their corresponding projection vectors, V.For doing so, the hyperspectral image block, M k , is first centralized by subtracting the average pixel, μ, from all pixels within M k , which results in the centralized version of the data, C, in line 3. Second, the p max most characteristic pixels are sequentially extracted from lines 4 to 15.In this iterative process, the brightness of each image pixel is calculated in lines 5 to 7. It is defined as the dot product of each image pixel with itself (see line 6).The extracted pixels, e n , are those pixels within the original image block, M k , with the highest brightness in each iteration, n (see line 8 and 9).Afterwards, the orthogonal vectors q n and u n are accordingly defined as shown in lines 10 and 11, respectively.u n is employed to estimate the projection of each image pixel over the direction spanned by the selected pixel, e n , deriving in the projection vector, v n in line 12.Finally, this information is subtracted from C in line 13. Accordingly, C retains in next iterations the spectral information that cannot be represented by the already selected pixels e n .For this reason, once the p max pixels have been extracted, C contains the spectral information that will be not recovered after the decompression process.Hence, it is represented of the losses introduced by the HyperLCA compressor.Consequently, the remaining information in C could be used to establish extra stopping conditions based on quality metrics, such as the Signal-to-Noise Ratio (SNR) or the Maximum Absolute Difference (MAD).In this work, these extra stopping conditions have been discarded, and thus, a fixed number of p max iterations is executed for all image blocks, M k .Algorithm 1 HyperLCA Transform.Extracted pixels: e n = r j max ; 10: Inputs: M q n = c j max ; 11: Projection vector: {Additional stopping condition checking.}15: end for HyperLCA Preprocessing To ensure an efficient entropy coding of the HyperLCA Transform outputs, they must be first adapted in the HyperLCA Preprocessing stage.This transformation process is performed in two steps: Scaling V Vectors V vectors represent the projection of each pixel within M k over the direction spanned by each orthogonal vector, u n , in each iteration.Therefore, potential values of each element of V vectors are in the range of (−1, 1].However, the later Entropy-coding stage works exclusively with integers.Consequently, elements within V must be scaled to fully exploit the dynamic range offered by the input parameter N bits , as shown in Equation (2).After doing so, the scaled Vvectors are rounded up to the closest integer values. Error Mapping The codification stage also exploits the redundancies within the data in the spectral domain to assign the shortest word length to the most common values.With it, the compression ratio achieved by the HyperLCA Transform could be even increased.To this end, the output vectors of the HyperLCA Transform ( μ, E and scaled V) are lossless pre-processed in the Error Mapping stage.To do so, the prediction error mapper described in the Consultative Committee for Space Data Systems (CCSDS) Blue Books is employed [35].In this regard, each output vector, ( μ, e n and scaled v n ) is individually processed and transformed to be exclusively composed of positive integer values closer to zero. HyperLCA Entropy Coding The Entropy Coding is the last stage of the HyperLCA compressor.It follows a lossless entropy-coding strategy based on the Golomb-Rice algorithm [36].As in the Error Mapping stage, each single output vector is independently coded.For doing so, the compression parameter, M, is estimated as the average value of the targeted vector.Afterwards, each of its elements is divided by M in order to obtain the results of the division, the quotient (q) and the remainder (r).On one hand, the quotient, q, is codified using unary code.On the other hand, the remainder, r, could be coded using b = log 2 (M) + 1 bits if M is power of 2. Nevertheless, M can actually be any positive integer.For this reason, the remainder, r is coded as plain binary using b − 1 bits for r values smaller than 2 b − M, otherwise it is coded as r + 2 b − M using b bits. HyperLCA Data Types and Precision Evaluation Most of the compression performance achieved by the HyperLCA algorithm is obtained in the HyperLCA Transform stage, originally designed to use floating-point precision.However, FPGA devices are, in general, more efficient dealing with integer operations.Additionally, the execution of floating-point operations in different devices may produce slightly different results.For this reason, the performance of the HyperLCA algorithm using integer arithmetic was largely drawn in [37] in order to adapt it for being more suitable for this kind of device.In particular, it was used the fixed-point concept in a custom way employing integer arithmetic and bits shifting [38].In this previous work, two different versions of the HyperLCA Transform were considered employing 32 and 16 bits, respectively, for representing the image values stored in the centralized version of the hyperspectral frame to be processed, C. It is important to note that the aforementioned proposed versions were developed for working with hyperspectral images whose element values could be represented with up to 16 bits per pixel per band as maximum.In this context, the quality of the compression results is very similar between the 32-bits fixed point and the single precision floating-point versions.Nevertheless, the compression performance obtained by the 16-bit version is not as competitive as the other two solutions.It is shown that this 16-bit approach provides its best performance for very high compression ratios, small BS and images packaged using less than 16 bits per pixel per band.This makes the 16-bits version into a very interesting option for applications with limited hardware resources, especially the availability of in-chip memory, one of the weaknesses of FPGAs. On this basis, we have made some performance-enhancing improvements to the 16-bit version to obtain better compression results.In this context, we have assumed that the available capturing system codes hyperspectral images with a maximum of 12 bits, which is also the most common scenario in remote-sensing applications [39,40] and in the one outlined in this manuscript.Table 1 summarizes the number of bits used by the two algorithm versions introduced in [37], known as I32 and I16, and the one described in this work, referred as to I12, for some algorithm variables.It should be pointed out that when integer arithmetic is used, V vectors are directly represented using integer data types and do not need to be scaled and rounded to integer values. As described in Section 2.1.3,the information presents in matrix C decreases with every iteration, n, though some specific values may increase in some quite unlikely situations.For this reason, initial values of C were divided by 2 in the previous 16-bit version in order to avoid overflowing, what directly decreases the precision in one bit.In the new version proposed in this work, this fact is discarded since we have 2 bits extra for these improbable situations.As can be noticed from Table 1, image C is stored employing 16 bits in the new I12 version as well as in the I16 version.However, it is assumed that images values are coded employing 12 bits as maximum instead of 16 bits.It permits having 2 extra bits for representing the integer part of the fixed-point values of C elements.Consequently, image precision is not altered as in previous 16-bit version for dealing with possible overflowing scenarios.Additionally, this new version also allows having 2 bits for representing the decimal part of the fixed-point values of matrix C, which is not possible in the I16 version. FPGA Implementation of the HyperLCA Algorithm An FPGA (Field-Programmable Gate Array) can be seen as a whiteboard for designing specialized hardware accelerators (HWacc), by composition of predefined memory and logic blocks that are available in the platform.Therefore, a HWacc is a set of architectural FPGA resources, connected and configured to carry out a specific task.Each vendor proposes its own reconfigurable platform, instantiating a particular mix of such resources, around a particular interconnection architecture. FPGAs provide flexibility to designers, since the silicon adapts to the solution, instead of fitting the solution to the computing platform as is the case of GPU-based solutions.On top of that, FPGA-based implementations can take advantage of the fine-grain parallelism of their architecture (operation level) as well as task-level concurrency.In this paper, the HyperLCA lossy compressor has been implemented onto a heterogeneous Zynq-7000 SoC (System-on-a-Chip) from Xilinx that combines a Processor System (PS), based on a dual core ARM processor, and a Programmable Logic (PL), based on a Artix-7/Kintex-7 FPGA architecture. The development process followed a productive methodology based on High-Level Synthesis (HLS).This methodology focuses the effort on the design and verification of the HWacc, as well as the exploration of the solution space that helps to speed up the search for value-added solutions.The starting point of a methodology based on HLS is a high-abstraction model of the functionality to be deployed on the FPGA, usually described by means of high-level programming languages, such as C or C++.Then, HLS tools can generate the corresponding RTL (Register Transfer Level) implementation, functionally equivalent to the C or C++ model [41,42]. Productivity is the strongest point of HLS technology, and one of the main reasons why hardware architects and engineers have been recently attracted to it.However, HLS technology (more specifically, the tools that implement the synthesis process) has some weaknesses.For example, despite the fact that the designer can describe a modular and hierarchical implementation of a HWacc (i.e., grouping behavior via functions or procedures), all sub-modules are orchestrated by a global clock due to the way the translation to RTL from C is done.Another example is the rigid semantic when specifying dataflow architectures, allowing a reduced number of alternatives.This prevents the designer from obtaining optimal solutions for certain algorithms and problems [43,44], as was the case of the HyperLCA compressor.Therefore, to overcome the limitations of current HLS tools, a hybrid solution that combines modules developed using VHDL and HLS-synthesized C or C++ blocks has been selected.The result allows the achievement of the maximum performance.Otherwise, it could not be possible to realize either the synchronization mechanisms or the parallel processing proposed in this work.On top of that, this approach makes it also possible to optimize the necessary resources because of the use of custom producer-consumer data exchange patterns that are not supported by HLS tools. The four main stages of the HyperLCA compressor, described in Section 2.1 and shown in Figure 1, have been restructured in order to better adapt to devices with a high degree of fine-grain parallelism such as FPGAs.Thus, the operations involved in these parts of the HyperLCA algorithm have been grouped in two main stages: HyperLCA Transform and HyperLCA Coder.These new stages can run in parallel, which improves the performance. The Initialization stage (Section 2.1.2),where the calculation of the p max parameter is done, is considered only at design time.This is because several hardware components, such as internal memories or FIFOs, must be configured with the appropriate size.In this sense, it is worth mentioning that the p max value (Equation ( 1)) depends on other parameters also known at design time (the minimum desired compression ratio (CR), block size (BS) and the number of bits (N bits ) used for scaling the projection vectors, V, and, therefore, can be fixed for the HWacc. HyperLCA Transform HyperLCA Transform stage performs the operations of the HyperLCA transform itself (described in Section 2.1.3),and also the computation of the average pixel and the scaling of V vector.These are the most computational demanding operations of the algorithm and, together with the fact that they are highly parallelizable by nature, are good candidates for acceleration. Figure 2 shows an overview of the hardware implementation of the HyperLCA Transform stage.Avg_Cent, Brightness and Proj_Sub modules have been modeled and implemented using the aforementioned HLS tools, while the memory buffers and custom logic that integrates and orchestrates all the components in the design have been instantiated and implemented using VHDL language, respectively.This HWacc has a single entry corresponding to a hyperspectral block (M k ) that will be compressed, while the output is composed of three elements (which differs to the output of Algorithm 1) in order to achieve a high level of parallelism.It must also be mentioned that the output of the HyperLCA Transform stage feeds the input of the HyperLCA Coder stage that performs the error mapping and entropy coding in parallel.Thus, the centroid, μ, is obtained as depicted in Algorithm 1, while the p max most different hyperspectral pixels, E, are not directly obtained.In this regard, the HWacc provides the indexes of such pixels, j max , in each loop iteration of Algorithm 1 (outer loop), while the HyperLCA Coder is the responsible to obtaining each hyperspectral pixel, e n , from the external memory, in which is stored the hyperspectral image, to build the vector of most different hyperspectral pixels, E. Finally, the projection of pixels within each image block, V, is provided by the HWacc in a batch mode, i.e., each loop iteration (outer loop of Algorithm 1) obtains a projection (v n ) that forms part of the V vector. The architecture of the HLCA Transform HWacc can be divided into two main modules: Avg_Cent, which corresponds to lines 2 and 3 of Algorithm 1, and Loop_Iter (the main loop body, lines from 5 to 14).These modules are connected by a bridge buffer (BBuffer), whose depth is only 32 words, and also share a larger buffer (SBuffer) with capacity to store a complete hyperspectral block.The size of the SBuffer depends on the BS algorithm parameter and its depth is determined at design time.The role of this SBuffer is to avoid the costly access to external memory, such it is the case of double data rate (DDR) memory in the Zynq-7000 SoCs. The implementation of the SBuffer is based on a first-in, first-out (FIFO) memory that is written and read by different producers (i.e., Avg and Brightness) and consumers (i.e., Cent and Projection) of the original hyperspectral block and the intermediate results (transformations).Since there are more than one producer and consumer for the SBuffer, a dedicated synchronization and control access logic has been developed in VHDL (not illustrated in Figure 2).The use of a FIFO contributes to reduce the on-chip memory resources in the FPGA fabric, being its use feasible because of the linear pattern access of the producers and consumers.However, this type of solution would not have been possible with HLS because the semantic of stream-based communication between stages in a dataflow limits the number of producers and consumer to one.Also, it is not possible, with the used HLS technology, to exploit inter-loop parallelism as is done in the proposed solution.Notice that there is a data dependency between iterations in Algorithm 1 (centralized block, C) and, therefore, the HLS tool infers that the next iteration must wait for the previous one to finish, resulting in fact in sequential computation.However, a deeper analysis of the behavior of the algorithm shows that the computation of the brightest pixel for iteration n + 1 can be performed as it has received the output of the subtraction stage, which will be still processing iteration n.The Avg_Cent module has been developed using HLS technology and contains two sub-modules, Avg and Cent, that implement lines 2 and 3 of Algorithm 1, respectively.Avg This sub-module computes the centroid or average pixel, μ, of the original hyperspectral block, M k , following line 2 of Algorithm 1, and stores it in CBuffer, a buffer that shares with Cent sub-module.During this operation, Avg forwards a copy of the centroid to the HyperLCA Coder via a dedicated port (orange arrow).At the same time, the Avg sub-module writes all the pixels of the M k into the SBuffer.A copy of the original hyperspectral block, M k , will be available once Avg finishes, ready to be consumed as a stream by Cent, which reduces the latency. Centroid ( ) MB Index (j max ) Figure 3 shows in detail the functioning of the Avg stage.The main problem in this stage is the way the hyperspectral data is stored.In our case, the hyperspectral block, M k , is ordered by the bands that make up a hyperspectral pixel.However, to obtain the centroid, μ, the hyperspectral block must be read by bands (in-width reading) instead of by pixels (in-depth reading).We introduce an optimization that handles the data as it is received (in-depth), avoiding the reordering of the data to maintain a stream-like processing.This optimization consists of an accumulate vector, whose depth is equal to the number of bands that stores partial results of the summation for each band, i.e., the first position of this vector contains the partial results of the first band, the second position the partial results of the second band and so on.The use of this vector removes the loop-carry dependency in the HLS loop that models the behavior of the Avg sub-module, saving processing cycles.The increase in resources is minimal, which is justified by the gain in performance.Cent This sub-module reads the original hyperspectral block, M k , from the SBuffer to centralize it (C).This operation consists of subtracting the average pixel, calculated in the previous stage, from each hyperspectral pixel of the block (line 3 of Algorithm 1). Figure 4 shows this process, highlighting the elements that are involved in the centralization of the first hyperspectral pixel.Thus, the Cent block reads the centroid, μ, which is stored in the CBuffer, as many times hyperspectral pixels have the original block (i.e., BS times in the example illustrated in Figure 4).Therefore, CBuffer is an addressable buffer that permanently stores the centroid of the current hyperspectral block that is being processed.The result of this stage is written into the BBuffer FIFO, which makes unnecessary an additional copy of the centralized image, C. As soon as the centralized components of the hyperspectral pixels are computed, the data is ready at the input of the Loop_Iter module and, therefore, it can start to perform its operations without waiting for the block to be completely centralized.The Loop_Iter module instantiates the Brightness and Proj_Sub sub-modules which have been designed and implemented using HLS technology.Both modules are connected by two FIFO components (uVectorB and qVectorB) using customized VHDL code to link them.Unlike Avg_Cent module, Loop_Iter module is executed several times (specifically p max times, line 4 of Algorithm 1) for each hyperspectral block that is being processed. Brightness This sub-module starts as soon as there is data in the BBuffer.In this sense, Brightness sub-module works in parallel with the rest of the system; the input of the Brightness module is the output of Cent module in the first iteration, i.e., the centralized image, C, while the input for all other iterations is the output of Subtraction sub-module that corresponds to the image for being subtracted, depicted as X for the sake of clarity (see brown arrows in Figure 2). Brightness sub-module has been optimized to achieve a dataflow behavior that takes the same time regardless of the location of the brightest hyperspectral pixel.Figure 5 shows how the orthogonal projection vectors q n and u n are obtained by the three sub-modules in Brightness.First, the Get Brightness sub-module reads in order the hyperspectral pixel of the block from the BBuffer (C or X, it depends on the loop iteration) and calculates its brightness (b j ) as specified in line 6 of Algorithm 1. Get Brightness also makes a copy of the hyperspectral pixel in an internal buffer (actual_pixel) and in SBuffer.Thus, actual_pixel contains the current hyperspectral pixel whose brightness is being calculated, while SBuffer will contain a copy of the hyperspectral block with transformations (line 6 and assignment in line 13 of Algorithm 1). Once the brightness of the current hyperspectral pixel is calculated, the Update Brightness sub-module will update the internal vector brightness_pixel if the current brightness is greater than the previous one.Regardless of such condition, the module will empty the content of actual_pixel in order to keep the dataflow with the Get Brightness sub-module.The operations of both sub-modules are performed until all hyperspectral pixels of the block are processed (inner loop, lines 5 to 7 of Algorithm 1). The reason to use a vector to store the brightest pixel instead of a FIFO is because the HLS tool would stall the dataflow otherwise. Finally, the orthogonal projection vectors q n and u n are accordingly obtained from the brightest pixel (lines 10 and 11 of Algorithm 1) by the module Build quVectors.Both are written in separate FIFOs: qVectorB and uVectorB, respectively.Furthermore, the contents of these FIFOs are copied in qVector and uVector arrays in order to get a double space memory that does not deadlock the system and allows Proj_Sub sub-module to read several times (concretely BS times) the orthogonal projection vectors q n and u n to obtain the projected image vector, v n , and transform the current hyperspectral block.This module also returns the index of the brightest pixel, j max , so that the HyperLCA Coder stage reads the original pixel from the external memory, such as DDR, where the hyperspectral image is stored in order to build the compressed bitstream.Proj_Sub Although this sub-module is represented by separate Projection and Subtraction boxes in Figure 2, it must be mentioned that both perform their computations in parallel.The Proj_Sub sub-module reads just once the hyperspectral block that was written in SBuffer by the Brightness sub-module (X). Figure 6 shows an example of Projection and Subtraction stages.First, each hyperspectral pixel of the block is read by the Projection sub-module to obtain the projected image vector according to line 12 of Algorithm 1.At the same time, the hyperspectral pixel is written in PSBuffer, which can store two hyperspectral pixels.Two is the number of pixels because the Subtraction stage begins right after the first projection on the first hyperspectral pixel is ready, i.e., the executions of Projection and Subtraction are shifted by one pixel.Figure 6 shows such behavior.While pixel r 1 is being consumed by Subtraction, pixel r 2 is being written in PSBuffer.During the projection of the second hyperspectral pixel (r 2 ), the subtraction of the first one (r 1 ) can be performed since all the input operands, included the projection v n , are available, following the expression in line 13 of Algorithm 1. The output of the Projection sub-module is the projected image vector, v n , which is forwarded to the HyperLCA Coder accelerator (through the Projection port) and to the Subtration sub-module (via the PBuffer FIFO).At the same time, the output of the Subtraction stage feeds the Loop_Iter block (see purple arrow, labeled as X, in Figure 2) with the pixels of the transformed block in the i th iteration.It means that Brightness stage can start the next iteration without waiting to get the complete image subtracted.Thus, the initialization interval between loop-iterations is reduced as much as possible because the Brightness starts when the first subtracted data is ready.The FPGA-based solution described above highlights how the operations are performed in parallel to make the most out of such technology.Also, it has been spotted specific synchronization scenarios that are not possible to design using solely HLS.due to the current communication semantics supported by the synthesis tools.For example, is not possible for current synthesis tools to perform an analysis of the data and control dependencies such as the one done in this work.However, a hybrid solution based on HLS and hand-written VHDL code to glue the RTL models synthesized from C/C++ models, brings to life an efficient dataflow (see Figure 7).In this regard, the use of optimal sized FIFOs to interconnect the modules is key.For example, while the Brightness sub-module is filling the SBuffer, the Projection sub-module is draining it, and at the same time this sub-module supplies to the Subtraction sub-module with the same data read from SBuffer.Finally, Subtraction sub-module feeds back the Brightness sub-module through the BBuffer FIFO.The Brightness sub-module fills, in turn, the SBuffer, with the same data, closing the circle; this loop is repeated p max times. Furthermore, the initialization interval between image blocks has been reduced.The task performed by the Avg sub-module for block k + 1 (see Figure 7) can be scheduled at the same time that the Projection and Subtraction sub-modules are computing their outputs for block k, and right after the completion of the Brightness sub-module for block k.This is possible since the glue logic discards the output of the Subtraction sub-module during the last iteration.This logic ensures that the BBuffer is filled with the output from the Avg sub-module that feeds the first execution of Brightness for the first iteration of block k + 1, resulting in an overlapped execution of the computation for blocks k and k + 1.Unfortunately, despite the optimizations introduced in the dataflow architecture, the HWacc is not able to reach the performance target as shown by the experimental results (see Section 3).The value in the column labeled as 1 PE (Processing Element) is clearly below the standard frame rates provided by contemporary commercial hyperspectral cameras.However, the number of FPGA resources required by the one-PE version of the HWacc is very low (see Section 3.2) which makes it possible to implement further parallelism strategies to speed up the compression process.Thus, three options open up for solving the performance problem. Avg First, task-level parallelism approach is possible by means of the use of several instances of the HWacc working concurrently.Second, increase the intra-HWacc parallelism using multiple operators, computing several pixel bands at the same time.Thirdly, a combination of the two previous approached.Independently of the strategy chosen, the limiting factor is the version of the Xilinx Zynq-7000 programmable SoC, that would have enough resources to support the design.In Section 4, a detailed analysis of several single-HWacc (with variations in the number of PEs) and multi-HWacc versions of the design is drawn. So far, it has been described the inner architecture of a HWacc that only performs a computational operation over a single band component of a hyperspectral pixel.However, it can be modified to increase the number of bands that are processed in parallel.Thus, the HWacc of HyperLCA compressor turns from a single-PE to multiple PEs.This fact opens two new challenges.First challenge is to increase the width of the input and output ports of the modules, in accordance with the number of bands that would be processed in parallel.It must be mentioned that it is technologically possible because HLS-based solutions allow designers to build their own data types.For example, if a band component of a hyperspectral pixel is represented by an unsigned integer of 16-bits, we could define our own data type consisting of an unsigned integer of 160-bits packing ten bands of a hyperspectral pixel (see Figure 8).The second challenge has to do with the strategy to process the data in parallel.In this regard, a solution based on the map-reduce programming model has been followed.Figure 8 shows an example of the improvements applied to the Cent stage, following the above-mentioned optimizations.The input of this stage is the hyperspectral block, M k , and the average pixel, μ, which are read in blocks of N-bands.The example assumes that the block is composed of ten bands and uses an user data type, specifically an unsigned int of 160-bits (10 bands by 16-bits to represent each band).Then, both blocks are broken down into the individual components that feed the PEs in an orderly fashion.This process is also known as map phase in the map-reduce programming model [45].It must be mentioned that the design needs as many PEs as number of divisions made in the block. Once the PEs have performed the assigned computational operation, the reduce phase of the map-reduce model is executed.For Cent sub-module, this stage consists of gathering in a block the partial results produced by each one of the PEs.Thus, a new block of N-bands is built, which in turn is part of the centralized block (C), which is the output of Cent sub-module. Coder The HyperLCA Coder is the second of the HWacc developed, responsible for the error mapping and entropy-coding stages of the HyperLCA compression algorithm (Figure 1).The coder module teams up with the transform module to perform in parallel the CCSDS prediction error mapping [35] and the Golomb-Rice [36] entropy-coding algorithms as the different vectors are received from the HyperLCA Transform block. The HyperLCA transform block generates the centroid, μ, extracted indexes of pixels, j max , and projection vectors, v n , for an input hyperspectral block, M k .These arrays are consumed as they are received, reducing the need for large intermediate buffers.To minimize the necessary bandwidth to the memory that stores the hyperspectral image, only the indexes of the selected pixels in each iteration of the transform algorithm (line 8 of Algorithm 1) are provided to the coder (MB_index port). The operation of the transform and coder blocks overlaps in time.Since the coder takes approximately half of the time the transform needs to generate each vector (see Section 3.2) for the maximum number of PEs (i.e., the maximum performance achieved), a contention situation is not taking place, reducing the pressure over the FIFOs that connect both blocks and, therefore, requiring less space for these communication channels. Figure 9 sketches the internal structure of the HyperLCA Coder that has been modeled entirely using Vivado HLS.It is a dataflow architecture comprising three steps.During the first step, the prediction mapping and entropy coding of all the input vectors are performed by the coding command generator.The result of this step is a sequence of commands that are subsequently interpreted by the compressed bitstream generator.The generation of the bitstream was extracted from the entropy-coding original functionality, which enabled a more efficient implementation of the latter, which could be re-written as a perfect loop and, therefore, Vivado HLS was able to generate a pipelined datapath with the minimum initiation internal (II = 1). DDR HLCA Transform The generation of the compressed bitstream is simple.This module is continuously reading the cmd_queue FIFO for a new command to be processed.A command contains the operation (unary of binary coding) as well as the word (quotient or reminder, see Section 2.1.5)to be coded, and the number of bits to generate.Unary and binary coding functions simply iterate over the word to be coded and produces a sequence if bits which corresponds to the compressed form of the hyperspectral block. Finally, the third step packs the compressed bitstream in words and written to memory.For this implementation, the width of the memory word is 64 bits, the maximum allowed by the AXI Master interface port for the Zynq-7020 SoC.The bitstream packer module instantiates a small buffer (64 words) that is flushed to DDR memory once it has been filled.This way, average memory access cycles per word is optimized by means of the use of burst requests. As mentioned above, the HyperLCA Transform block feeds the coder with the indexes of the extracted pixels e n , which correspond to the highest brightness as the hyperspectral block is processed.Hence, it is necessary to retrieve the nb spectral bands from memory before the coder could start generating the compressed stream for the pixel vector.This is the role played by the pixel reader module.As in the case of the bitstream packer step, the pixel reader makes it use of a local buffer and issue burst requests to read the bands in the minimum number of cycles. While the computing complexity of the coder module is low, the real challenge when it comes to the implementation of its architecture is to write a C++ HLS model that is consistent through the whole design, verification and implementation processes.To achieve this goal, it has been provided the communication channels with extra semantic so as to keep the different stages sync, despite the fact the model of computation of the architecture falls into the category of GALS (Globally Asynchronous, Locally Synchronous) systems.Side-channel information is embedded in the cmd_queue and compressed_stream FIFOs that connects the different stages of the coder.This information is used by the different modules to reset their internal states or stop and resume their operation (i.e., special commands to be interpreted by the bitstream generator or a flush signal as input to the packing module).This way, it is possible to integrate under a single HLS design all the functionality of the coder, which simplifies and speeds up the design process.On top of that, this strategy allowed avoiding the need to tailor the C++ HLS specification to make it usable in different steps of the design process.For example, it is common to write variations of the model depending on whether functional (C simulation) or RTL verification (co-simulation) is taking place due to the fact that the former is based on an untimed model and the latter introduces timing requirements [46,47]. Designing for parallelism is key to obtain the maximum performance.The dataflow architecture of the coder ensures an inter-module level of concurrency.However, the design must be balanced as to the latency of the different stages in the dataflow.Otherwise, the final result could be jeopardized.As mentioned before, decoupling the generation of the compressed bitstream from the entropy-coding logic, led to a more efficient implementation of the latter by the HLS synthesis tool.Also, this change helped to redistribute the computing effort, achieving a more balanced implementation. In the first stage of the dataflow (coding command generator) a simple logic that controls the encoding of each input vector plus a header is implemented.It is an iterative process that performs the error mapping and error coding over the centroid, and p_max times over the extracted pixels and projections vectors.The bulk of this process is, thus, the encoding algorithm.The encoding is delegated in another module that implements an internal dataflow itself.In this way, it is possible to reduce the interval between two encoding operations.As can be seen in Figure 9, the prediction mapping and entropy-coding sub-modules communicates through a ping-pong buffer for the mapped vector. To conclude this section, it is worth mentioning a couple of optimizations carried out related to the operations pow and log, which are used by the prediction mapping and entropy-coding algorithms.This type of arithmetic operation is costly to implement in FPGAs since the generated hardware is based on tables that consume on-chip resources (mainly BRAM memories), and an iterative processes that boosts latency.Since the base used in this application is 2, it can be largely simplified.Thus, we can substitute the pow and log operations by a logical shift instruction and the GCC compiler __builtin_clz(x) built-in function, respectively.This change is part of the refactoring process of the reference code implementation (golden model) that is almost mandatory at the beginning of any HLS project.The __builtin_clz(x) function is synthesizable and counts the leading zeros of the integer x.Therefore, the computation of the lowest power of 2 higher than M, performed during the entropy coding, is redefined as follows: Reference Hyperspectral Data In this section, we introduce the hyperspectral imagery used in this work to evaluate the performance of the proposed computing approach using reconfigurable logic devices.This data set is composed of 4 hyperspectral images that were also employed in [29], where the HyperLCA algorithm was implemented in low-power consumption embedded GPUs.We have kept the same data set in order to compare in Section 3 the performance of the developed FPGA-based solution with the results obtained by the GPU accelerators. In particular, the test bench was sensed by the acquisition system extensively analyzed in [48].This aerial platform mounts a Specim FX10 pushbroom hyperspectral camera on a DJI Matrice 600 drone.The image sensor covers the range of the electromagnetic spectrum between 400 and 1000 nm using 1024 spatial pixels per scanned cross-track line and 224 spectral bands.Nevertheless, the hyperspectral images used in the experiments only retain the spectral information of 180 spectral bands.Concretely, the first 10 spectral bands and the last 34 bands have been discarded due to the low spectral response of the hyperspectral sensor at these wavelengths. The data sets were collected over some vineyard areas in the center of Gran Canaria island (Canary Islands, Spain) and in particular, in a village called Tejeda, during two different flight campaigns.Figure 10 and Figure 11 display some Google Maps pictures of the scanned terrain in both flight missions, whose exact coordinates are 27°59 35.6 N 15°36 25.6 W (green point in Figure 10) and 27°59 15.2 N 15°35 51.9 W (red point in Figure 11), respectively.Both flight campaigns were performed at a height of 45 m over the ground, which results in a ground sampling distance in line and across line of approximately 3 cm.The former was carried out with a drone speed of 4.5 m/s and the camera frame rate set to 150 frames per second (FPS).In particular, this flight mission consisted of 12 waypoints that led to a total of 6 swathes, but only one was used for the experiments, which has been highlighted in green in Figure 10.Two portions of 1024 hyperspectral frames with all their 1024 hyperspectral pixels were selected from this swath to generate two of the data sets that compose the test bench.A closer view of these selected areas can be also seen in Figure 10.These images are false RGB representations extracted from the acquired hyperspectral data. The latter was carried out with a drone speed of 6 m/s and the camera frame rate set to 200 FPS.The entire flight mission consisted of 5 swathes, but only one was used for the experiments in this work, which has been highlighted in red in Figure 11.From this swath, two smaller portions of 1024 hyperspectral frames were cut out for simulations.A closer view of these selected areas is also displayed in Figure 11.Once again, they are false RGB representations extracted from the acquired hyperspectral data.For more details about the flight campaigns, we encourage the reader to see [48]. Evaluation of the HyperLCA Compression Performance The goodness of the I12 version of the HyperLCA algorithm proposed in this work has been evaluated and compared with previous I32 and I16 versions of the algorithm presented in [37] and the single precision floating-point (F32) implementation presented in [29].For doing so, the hyperspectral imagery described in Section 2.3 has been compressed/decompressed using different settings of the HyperLCA compressor input parameters. In this context, the information lost after the lossy compression process has been analyzed using three different quality metrics.Concretely, the Signal-to-Noise Ratio (SNR), the Root Mean Squared Error Additionally, deviations in the values of the three quality metrics employed in this work between I32, I12 and F32 versions are almost negligible, with the advantage of halving the memory space required for storing C. Second, it can be also concluded that the HyperLCA lossy compressor is able to compress the hyperspectral data with high compression ratios and without introducing significant spectral information losses. Evaluation of the HyperLCA Hardware Accelerator Section 2.1 describes the FPGA-based implementation of the HyperLCA compressor as defined in Algorithm 1.The architecture of the HWacc is divided into two blocks, Transform and Coder that run in parallel following a producer-consumer approach.Therefore, for the performance analysis of the whole solution, the slowest block is the one determining the productivity of the proposed architecture. The HyperLCA Transform block bears most of the complexity and computational burden of the compression process.For that reason, several optimizations (see Section 2.1.3)have been applied during its design in order to achieve a high degree of parallelism and, thus, reduce the latency.One of the most important improvements is the realization of the map-reduce programming model, to enable an architecture with multiple PEs (see Figure 8) working concurrently on several bands.The experiments carried out over the different alternatives for the HyperLCA Transform block are intended to evaluate how the performance and resource usage of the FPGA-based solution scales, as the number of PEs instantiated by the architecture grows up. The configuration of the input parameters has been set as follows: CR parameter has been set to 12, 16 and 20; the BS parameter has been set to 1024, 512 and 256 and; N bits parameter gets the values 12 and 8.The value of p max is obtained at design time from these parameters following Equation ( 1), and are listed in the last column of Table 3. Concerning the sizing of the various memory elements present in the architecture, it is determined by two parameters: the number of PEs to instantiate (parallel processing of hyperspectral bands) and the size of the image block to be compressed.It is worth mentioning that in this version of the HWacc, the number of PEs must be a divisor of the number of hyperspectral bands in order to simplify the design of the datapath logic. First, the data width of the architecture must be defined.Such parameter is obtained multiplying the number of PEs by the size of the data type used to represent a pixel band.In the version of the HWacc under evaluation, the I12 alternative has been selected, due to its good behavior (comparable to the I32 version as discussed in Section 2.1.6)and the resource savings it brings.For the I12 version, a band is represented with an unsigned short int which turns into 16-bit words in memory.On the contrary, choosing the I32 version, the demand for memory resources and internal buffers (such as the SBuffer) would double, because an unsigned int data type is used in the model definition.Thus, if the HWacc only instantiates a PE, the data width will be 16-bits, whereas if the HWacc instantiates 12 PEs (i.e., the HWacc performs 12 operations over the set of bands in parallel), the data width will be 192-bits.Second, the depth of the SBuffer must be calculated following Equation (6), where BS is the block size, nb the number of bands (in our case is fixed to 180), and N PEs is the number of processing elements. SBu f f erDepth min Being optimal as to the use of BRAM blocks within the FPGA fabric is compulsory since this resource is highly demanded as the number of PEs increases (seen in Table 4).On top of that, keeping the use of resources under control brings along some benefits such as helping the synthesis tool to generate a better datapath and, therefore, to obtain an implementation with a shorter critical path or contain the consumption of energy.Table 3 lists the memory requirements demanded by SBuffer for the 108 different configurations of the HyperLCA Transform block evaluated in this work.The SBuffer component is, by far, the largest memory instantiated by the architecture and it is implemented as a FIFO.SBuffer component has been generated with the FIFO generator tool provided by Xilinx which only allows depths that are power of two.Therefore, from a technical point of view, it is not possible to match the minimum required space of SBuffer with the obtained from the vendor tools.To mitigate the waste of memory space derived from such constraint of the tool, the SBuffer has been broken into two concatenated FIFOs (i.e., the output of the first FIFO is the input of the second) but keeping the facade of a single FIFO to the rest of the system.For example, the minimum depth of SBuffer for PE = 1 and BS = 256 is 46,080.With a single FIFO, the smallest depth with enough capacity generated by Xilinx tools would be 65,536.Therefore, the unused memory space represents approximately 30% of the overall resources for SBuffer.However, by using the double FIFO approach, one FIFO of 32,768 words plus another one of 16384 would be use.Only ≈ 6% of the assigned resources to SBuffer would be misused. To evaluate the HyperLCA Transform hardware accelerator, the proposed HWacc architecture has been implemented using the Vivado Design suite.This toolchain is provided by Xilinx and features a HLS tool (Vivado HLS) devoted to optimize the developing process of IP (Intellectual Property) components FPGA-based solutions for their own devices.The first implemented prototype instantiated one HWacc targeting the XC7Z020-CLG484 version of the Xilinx Zynq-7000 SoC.This FPGA has been selected because of its low-cost, low-weight and high flexibility, features that make it an interesting device to be integrated in aerial platforms, such as drones.The aim of this first prototype is to evaluate the capability of a mid-range reconfigurable FPGAs such as the XC7Z020 chip, for a specific application such as the HyperLCA compression algorithm.Hence, and due to the amount of resources available on the target device, the maximum possible number of PEs for the single-HWacc prototype is 12. Table 4 summarizes the resources required, which have been extracted from post-synthesis reports, for each of the 108 versions of the HWacc that process different block sizes of 180-band hyperspectral images of the data set (see Section 2.3).Several conclusions can be derived from these figures.In first place, the amount of digital signal processors (DSPs), flipflops (FFs) and look-up-tables (LUTs) resources increases with the number of PEs but are similar for different values of the BS parameter.On the contrary, the demand of BRAMs depends directly on BS and increases slightly with PE for a given block size.The PE = 10 version needs a special remark.Such version represents an anomaly in the linear behavior of the resource demand.Even with the use of a double FIFO approach, as explained before, the total capacity of the BRAM used to instantiate SBuffer is clearly oversized for that datawidth to assure that a hyperspectral block and its transformations (M k and C, respectively) could be stored in-circuit.Second, in addition to the resources needed by the HWacc of the HyperLCA Transform, it is necessary to take into account those corresponding to the other components in the system such as the Coder or the DMA (Direct Memory Access) to move the hyperspectral data (M k ) from/to DDR to/from the hardware accelerators.These extra components will make use of the remaining resources (specially LUTs, which is the most demanded as can be seen in Table 4), establishing a maximum of 12 for the number of PEs. Table 5 shows the post-synthesis results for the HyperLCA Coder block.The resources demanded by the coder does not depend on the BS parameter or the number of PEs.It is important to mention that the majority of the BRAM, FFs and LUTs resources are assigned to the two AXI-Memory interfaces that the HLS tool generates for the Pixel Reader and the Bitstream Packer modules (see Figure 9).Table 6 shows the throughput, expressed as the maximum frame rate, for a specific configuration of the HWacc using two clock frequencies: 100 MHz and 150 MHz (Table A1 extends this information by adding the number of cycles to compute a hyperspectral block).Columns labeled as PE denote the number of processing elements instantiated by the HWacc, which in combination with the input parameters (N bits , BS, CR and p max ), show the average number of hyperspectral blocks that the HWacc is able to compress per second (FPS).The maximum frame rate has been normalized to 1024 hyperspectral pixels per block, which is the size of the frame delivered by the acquisition system.It is worth noting that these results include the transform and coding steps, which are performed in parallel.Table 7 shows the coding times (in clock cycles) compared to the time needed by the transform step with the best configuration possible (i.e., PE = 12).The HyperLCA Coder takes roughly 50% less time on average and, since the relation between both hardware components is a dataflow architecture, the latency of the whole process corresponds to the maximum; that is, the delay of the Transform step. One key factor is the minimum frame rate that must be supported for the targeted application.Ideally, such threshold would correspond to the maximum frame rate provided by the employed hyperspectral sensor (i.e., 330 FPS).However, the experimental validation of the camera set-up in the drone (Section 2.3), tells us that frame rates between 150 and 200 are enough to obtain hyperspectral images with the desired quality, given the speed and altitude of the flights.Therefore, a threshold value of 200 FPS is established as the minimum throughput to validate the viability of the HyperLCA hardware core.In Table 7, it has been highlighted (bold type-faced cells) the configurations that would be valid given this minimum.Thus, it can be observed that the PE = 12 version, for both clock frequencies, and the PE = 10 version at 150 MHz reach the minimum frame rate, even for the most demanding scenario (N bits = 8, BS = 1024 and CR = 12). In turn, by using lower values for N bits the compression rate is reduced due to the higher number of V vectors extracted by the HyperLCA Transform.This means that more computations must be performed to compress a hyperspectral block.It must be mentioned that the PE = 10 version meets the FPS target in all the scenarios but the most demanding one when the clock frequency is set to 100 MHz.As for the PE = 10 version, the PE = 6 version does not reach the minimum FPS in a few scenarios (concentrated in N bits = 8 and BS = 1024), but in can be a viable solution given the actual needs of the application set-up.In addition to the performance results listed in Table 6, Figure 12 graphically shows the speed-up gained by the FPGA-based implementation as the number of PEs increases.The values have been normalized, using the average time for the PE = 1 version as the baseline.Several conclusions can be drawn from this figure.First, it is observable that the PE = 12 version of the HWacc performs ×7 times (N bits = 12) to ×7.6 times (N bits = 8) faster than PE = 1 version.This configuration is the one that guarantees the fastest compression results.Second, the speed-up gain is nearly linear for PE = 2 and PE = 4 versions, whereas the scalability of the accelerator drops as the number of PEs is higher (see Figure 12a,b).This behavior is seen for both N bits = 8 and N bits = 12 configurations (see Figure 12c,d).However, for higher values of BS and CR the shape of the curve shows a better trend though not reaching the desired linear speed-up. Discussion In this paper, we present a detailed description of an FPGA-based implementation of the HyperLCA algorithm, a new lossy transform-based compressor.As fully discussed in Section 3.2, the proposed HWacc meets the requirements imposed by the targeted application in terms of the frame rate range employed to capture quality hyperspectral images.In this section, we would like to also provide a comprehensive analysis of the suitability of the FPGA-based HyperLCA compressor implementation and a comparison with the results obtained by an embedded System-on-Module (SoM) based on a GPU that has been recently published [29]. Concretely, María Díaz et al. introduce in [29] three implementation models of the HyperLCA compressor.In this previous work, the parallelism inherent to the HyperLCA algorithm was exploited beyond the thread-level concurrency of the GPU programming model taking advantage of the CUDA steams.In particular, the third approach, referred to as Parallel Model 3 in [29], achieves the highest speed-up, especially for bigger image blocks (BS = 1024).This implementation model bets for pipelining the data transfers between the host and the device and the kernel executions for the different image blocks (M k ).To do this, such proposal exploits the benefits of the concurrent kernel execution through the management of CUDA streams.Unlike the FPGA-based implementation model proposed in this paper, only the HyperLCA Transform stage is accelerated in the GPU.In this case, the codification stage is also pipelined with the HyperLCA Transform stage but executed on the Host using another parallel CPU process.Table 2 collects the quality results of the compression process issued by the aforementioned GPU-based implementation model in terms of SNR, MAD and RMSE (F32 version).For more details, we encourage the reader to see [29]. To compare the performance of the GPU-based implementation model and the FPGA solution presented here, we are going to use two assessment metrics: the maximum number of frames compressed in a second (FPS) and the power efficiency in terms of FPS per watt.The latter figure of merit is of great importance given the target application, since it is critical to maximize the battery life of the drone.Additionally, the GPU-based model introduced in [29] has been executed in three different NVIDIA Jetson embedded computing devices: Jetson Nano, Jetson TX2 and the most recent supercomputer Jetson Xavier NX. These modules have been selected for the reasonable computational power provided at a relatively low power consumption.Table 8 summarizes the most relevant technical characteristics of these embedded computing boards.As can be seen, the Jetson Nano module integrates the less advanced, oldest generation of the three GPU architectures, instantiating the fewer execution units or CUDA cores as well.On the contrary, Jetson Xavier NX represents one of the latest NVIDIA power-efficient products, which offers more than 10X the performance of its widely adopted predecessor, Jetson TX2. 9 collects the performance results obtained for the three GPU-based implementations of the HyperLCA and the most powerful implementation of the HWacc in a Zynq-7020 SoC (PE = 12).For each implementation, it is specified the clock frequency and power budget.Several algorithm parameters have been tested over 180-band input images.In addition, Figure 13 displays the obtained FPS according to different configurations of the input image block size (BS).For the sake of simplicity, we only represent results for N bits = 8 since the behavior is similar for N bits = 12 as the reader can see in Table 9. From the performance point of view, the most competitive FPGA results, compared to the fastest GPU implementations on Jetson Nano and Jetson TX2, are for the smallest input block size (BS = 256), resulting very similar to BS = 512 and even better compared to Jetson Nano.For the largest block size (BS = 1024) the performance of the solutions based on GPUs, is higher because of how the parallelism is inferred in both architectures.On the one hand, the FPGA architecture can process 12 bands at a time, overlapping the computation of several groups of pixels due to the internal pipeline architecture.Therefore, processing time linearly increases as the number of pixels in the image block is higher.On the other hand, GPUs can process all the pixels in an image block in parallel, regardless the size of the block.Thus, processing time is nearly constant, independently of the value of parameter BS.However, this assumption does not hold when it comes to reality, since it has to be taken in consideration the time required for memory transfers, kernel launches and data dependencies.In this context, the time spent to setting up and launching the instructions to execute a kernel or perform a memory transfer must be also taken into account.As analyzed in [29], the time used in transferring image blocks of 256 pixels is negligible in relation to the overhead of launching the memory transfers and the additional required logic.However, the time required for transferring image blocks of 1024 pixels is comparable with the overhead of initializing the copy.Consequently, the bigger the size of the block, the better performance obtained by GPU-based implementation.For this reason, the trend of the FPGA performance function (see blue line in Figure 13) decreases as BS increases, while the opposite behavior is shown for the GPU-based model regardless of the desired CR.This pattern is also present when analyzing the results for the Jetson Xavier NX.However, in this case, the GPU clearly outperforms the maximum number of FPS achieved by the FPGA for all algorithm settings.Nevertheless, it should be noticed by the reader that the Jetson Xavier NX represents one of the latest, most advanced NVIDIA single-board computers whereas the Xilinx Zynq-7020 SoC that mounts the ZedBoard (i.e., XC7Z020-CLG484) is a mid-range FPGA several technological generations behind the Jetson Xavier GPU.Despite the fact that there are more powerful FPGA devices currently on the market, one of the main objectives of this work is to assess the feasibility of the reconfigurable logic technology for high-performance embedded applications such as HyperLCA under real-file conditions.At the same time, it is also a goal of this work to explore the minimum requirements of an FPGA-based computing platform that is able to fulfil the performance demands and constraints of the hyperspectral application under study.Thus, the selected version of the Xilinx Zynq-7020 SoC meets the demand for all algorithm configurations, at a lower cost.Going deeper in the analysis of the results, Figure 14 plots the power efficiency for each targeted device, measured as the FPS achieved divided by the average power budget.The picture shows how the efficiency varies in relation to the size of the input image blocks (BS).Jetson boards are designed with a high-efficient Power Management Integrated Circuit that handles voltage regulators, and a power tree to optimize power efficiency.According to [49][50][51], the typical power budgets of the selected boards amount to 10 W, 15 W and 10 W for the Jetson Nano, Jetson TX2 and Jetson Xavier NX modules, respectively.In the case of the XC7Z020-CLG484 FPGA, the estimated power consumption after Place & Route stage in Vivado toolchain goes up to 3.74 W at 150 MHz.Based on the trend lines shown in Figure 14, it can be concluded that the FPGA-based platform is by far more efficient in terms of power consumption that the Jetson Nano and TX2 NVIDIA boards, for all algorithm configurations.As in the case of the performance analysis (Figure 13), the power efficiency of the FPGA slightly decreases with higher BS values while GPU-based implementations present an opposite behavior.As a result, the FPGA-based solution remains a more power-efficient approach for the smallest image block size (BS = 256) and shows similar figures for BS = 512 and higher CR.Nevertheless, for BS = 1024, Jetson Xavier NX clearly outperforms the proposed FPGA-based solution.The reasons that explain this behavior root in the fact that GPU-embedded platforms have been able to significantly increase their performance while maintaining or even reducing the power demand.The combination of architectural improvements and better IC manufacturing processes have paved the way to an scenario where embedded-GPU platforms are gaining ground and can be seen as competitors of FPGAs concerning power efficiency. Although the initial FPGA solution has proved to be sufficient, given the real-life requirements of the targeted application, we have ported the proposed design to a larger FPGA device.The objective is two-fold.First, to evaluate if it is possible to reach the same level of performance (in terms of FPS) than that obtained by the Jetson Xavier NX implementation with current FPGA technology.Second, to study how FPGA power efficiency evolves as the complexity of the design increases, and compare it to the results obtained by the Jetson Xavier NX. Thus, a multi-HWacc version of the design was developed using the XC7Z100-FFV1156-1 FPGA, one of the biggest Xilinx Zynq-7000 SoCs, as the target platform.The new FPGA allows up to 5 instances of the HyperLCA component working in parallel.The selected baseline scenario is the configuration where the single-core FPGA design obtained the worst results compared to the Jetson Xavier NX (i.e., BS = 1024 and Nbits = 8 for all CR values).Synthesis and implementation was carried out by means of Vivado toolchain using the Flow_AreaOptimized_high and Power_ExploreArea strategies, respectively.As can be seen in Figure 15a, the performance of the multi-HWacc version grows almost linearly with the number of instances.There is a loss due to the concurrent access to memory in order to get the hyperspectral frames and the necessary synchronization of the process, which is the responsibility of the software.The new multi-core FPGA-based HyperLCA computing platform can reach the same level of performance for the maximum number of instances.As to the efficiency in terms of energy consumption per frame processed by the collection of HWaccs, with just three instances the FPGA is comparable to the GPU (above the Jetson Xavier NX results for CR = 16 and CR = 20) and better for four and five instances of the HWacc. Conclusions The suitability of the HyperLCA algorithm for being executed using integer arithmetic was already examined in further detail in previous state-of-the-art publications.Nonetheless, in this work we have contributed to its optimization providing a performance-enhancing alternative that has brought about a substantial performance improvement along with a significant reduction in hardware resources, especially aimed at overcoming the scarcity of in-chip memory, one of the weakness of FPGAs. In this context, the aforementioned modified version of the HyperLCA lossy compressor has been implemented onto an heterogeneous Zynq-7000 SoC in pursuit of accelerating its performance and thus, complying with the requirements imposed by a UAV-based sensing platform that mounts a hyperspectral sensor, which is characterized by a high frame rate.The adopted solution combines modules using VHDL and synthesized HLS models, bringing to life an efficient dataflow that fulfils the real-life requirements of the targeted application.On this basis, the designed HWaccs are able to reach frame rates of compressed hyperspectral image blocks higher than 330 FPS, setting the baseline scenario in 200 FPS, using a small number of FPGA resources and low power consumption. Additionally, we also provide a comprehensive comparison in terms of energy efficiency and performance between the FPGA-based implementation developed in this work and a state-of-the-art GPU-based model of the algorithm on three low-power NVIDIA computing boards, namely Jetson Nano, Jetson TX2 and Jetson Xavier NX. Conclusions drawn from the discussion show that although the FPGA-based platform is by far more efficient in terms of power consumption than the oldest-generation NVIDIA boards, such as the Jetson Nano and the Jetson TX2, the newest embedded-GPU platforms, such as the Jetson Xavier NX, are gaining ground and can be seen as competitors of FPGAs concerning power efficiency. On account of that, we have also introduced a multi-HWacc version of the developed FPGA-based approach in order to analyze its evolution in terms of performance and power consumption when the number of accelerators increases in a larger FPGA.Results conclude that the new multi-core FPGA-based version can reach the same level of performance as the most efficient embedded GPU systems.Also, looking at the energy consumption, FPGA performance per watt is comparable from just three instances of the HWaccs. Finally, we would like to conclude that although the work described in this manuscript has been focused on a UAV-based application, it can be easily extrapolated to other work in the space domain.In this regard, FPGAs have been established as the mainstream solution for on-board remote-sensing applications due to their smaller power consumption and above all, the accessibility to radiation-tolerant FPGAs [6].That is why the FPGA-based model proposed in this manuscript efficiently implements all HyperLCA compression stages in the programmable logic (PL) of the SoC, that is the FPGA.Hence, it can easily be adapted to be performed on other space-grade certified FPGAs. Figure 1 . Figure 1.Data flow among the different computing stages of the HyperLCA compressor. Figure 2 . Figure 2. Overview of the HyperLCA Transform hardware accelerator.Light blue and white boxes represent modules implemented using HLS.Light red boxes and FIFOs represent glue logic and memory elements designed and instantiated using VHDL language. Figure 6 . Figure 6.Example of Projection and Subtraction stages. Figure 7 . Figure 7. Dataflow of HyperLCA Transform hardware accelerator.Overlapping of operations within inter-loops and inter-blocks. Figure 8 . Figure 8. Map-reduce programming model and data packaging on Cent stage. Figure 9 . Figure 9. Overview of the HyperLCA Coder hardware accelerator. Listing 1 : FPGA implementation of costly arithmetic operations during entropy coding.// O r i g i n a l code b = l o g 2 (M) + 1 ; d i f f e r e n c e = pow( 2 , b ) − M; //FPGA o p t i m i z a t i o n b = ( 3 2 − _ _ b u i l t i n _ c l z (M) ) ; d i f f e r e n c e = (1 < <b ) − M; Figure 10 . Figure 10.Google Maps pictures of the vineyard areas sensed during the first flight campaign.False RGB representations of the hyperspectral images employed in the experiments. Figure 11 . Figure 11.Google Maps pictures of the vineyard areas sensed during the second flight campaign.False RGB representations of the hyperspectral images employed in the experiments. Figure 12 . Figure 12.Speed-up obtained for multiple PEs compared to the 1 PE version of the HyperLCA HW compressor.(a) Speed-up N bits = 8; (b) Speed-up N bits = 12; (c) Speed-up curve N bits = 8; (d) Speed-up curve N bits = 12. Figure 13 . Figure 13.Comparison of the speed-up obtained in the compression process, in terms of FPS and the input parameter BS, reached by a Xilinx Zynq-7020 programmable SoC following the design-flow proposed in this work versus the GPU-based implementation model described in [29] performed onto some NVIDIA power-efficient embedded computing devices, such as Jetson Nano, Jetson TX2 and Jetson Xavier NX.(a) FPS N bits = 12, CR = 12; (b) FPS N bits = 12, CR = 16; (c) FPS N bits = 12, CR = 20. Figure 14 . Figure 14.Comparison of the energy efficiency in the compression process, in terms of the ratio between obtained FPS and power consumption and the input parameter BS, reached by a Xilinx Zynq-7020 programmable SoC following the design-flow proposed in this work versus the GPU-based implementation model described in [29] performed onto some NVIDIA power-efficient embedded computing devices, such as Jetson Nano, Jetson TX2 and Jetson Xavier NX.(a) FPS N bits = 12, CR = 12; (b) FPS N bits = 12, CR = 16; (c) FPS N bits = 12, CR = 20. Figure 15 . Figure 15.Evolution of the performance (a) and energy efficiency (b) of a multi-core version of the FPGA-based HyperLCA computing platform.Comparison with the GPU-based implementation model described in [29] performed onto NVIDIA Jetson Xavier NX (N bits = 8, BS = 1024, using a Xilinx Zynq-7000 XC7Z100-FFV1156-1). Table 1 . Number of bits used for the integer and decimal parts used to represent the variables involved in the HyperLCA Transform.Three versions of the algorithm were developed to use integer arithmetic (I32, I16, I12). Table 3 . Design-time configurations parameters of the HyperLCA transform hardware block for hyperspectral images with 180 bands. Table 4 . Post-Synthesis results for the different versions of the HyperLCA Transform for a Xilinx Zynq-7020 programmable SoC and image block up to 180 bands. Table 5 . Post-Synthesis results for the HyperLCA Coder block for a Xilinx Zynq-7020 programmable SoC and pixel size up to 180 bands. Table 6 . Maximum frame rate obtained using the FPGA-based solution on a Xilinx ZynQ-7020 programmable SoC for hyperspectral images with 180 bands. Table 7 . Comparison of the computation effort made by the Transform and Coding stages. Table 8 . Most relevant characteristics of the NVIDIA modules Jetson Nano, Jetson TX2 and Jetson Xavier NX. Table 9 . [29]mum frame rates obtained by the proposed FPGA implementation and the GPU-based model introduced in[29]for the NVIDIA boards Jetson Nano, Jetson TX2 and Jetson Xavier NX.	2020-11-19T09:14:17.365Z	2020-11-13T00:00:00.000	{ "year": 2020, "sha1": "355b0d5ca7891933f64800b1c0a50d2d1ec0aaa6", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2072-4292/12/22/3741/pdf?version=1605505121", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "eb3eb521f4fbe209871f3d26f54dac3dd3d74a5a", "s2fieldsofstudy": [ "Environmental Science", "Computer Science", "Engineering" ], "extfieldsofstudy": [ "Computer Science" ] }
207930193	pes2o/s2orc	v3-fos-license	Federated Learning for Edge Networks: Resource Optimization and Incentive Mechanism Recent years have witnessed a rapid proliferation of smart Internet of Things (IoT) devices. IoT devices with intelligence require the use of effective machine learning paradigms. Federated learning can be a promising solution for enabling IoT-based smart applications. In this paper, we present the primary design aspects for enabling federated learning at network edge. We model the incentive-based interaction between a global server and participating devices for federated learning via a Stackelberg game to motivate the participation of the devices in the federated learning process. We present several open research challenges with their possible solutions. Finally, we provide an outlook on future research. I. INTRODUCTION Recently, edge computing has gained significant interest due to its ability of extending cloud computing utilities and services to the network edge with low-latency. Numerous Internet of Things (IoT) applications such as augmented reality, autonomous driving, forest fire surveillance, industry 4.0, smart health-care, among others, require edge processing with low latency [1]. In such applications, the involved IoT end devices have stringent computational resource constraints. One way to provide those IoT edge devices with on-demand computing resources is by using a remote cloud. However, the inherent delay pertaining to end-to-end communications with a cloud server can lead to intolerable latency. Therefore, edge computing is a promising solution to enable latencysensitive IoT applications by providing low-latency on-demand computing resources [2]. On the other hand, the data generated by end IoT devices offers an opportunity of using machine learning schemes to enable intelligent applications. Therefore, it is indispensable to use of machine learning at the edge to enable various smart applications. Traditional machine learning use centralized training data at a data center which requires migrating of data from a massive number of geographically distributed smart IoT devices to a centralized location for training [3]. Storing user data at a centralized location of a third party raises serious privacy concerns. To cope with the limitation of not preserving the users' privacy in centralized learning, it is important to introduce distributed, edge-deployed learning algorithms such as federated learning [4]. Federated learning allows privacy preservation by avoiding use of centralized training [4]. An overview of how federated learning can enable IoT-based smart applications is presented in Fig. 1. Depending on how the global learning model is being operated, we can distinguish two categories of federated learning: Cloud-based federated learning and edge-based federated learning [5]. Edge-based federated learning involves a set of devices within close vicinity and computation of global learning model at edge server. On the other hand, a cloud-based federated learning model involves the computation of a global learning model at a cloud for IoT devices that are geographically distributed over a large area. Hereinafter, we consider only edge-based federated learning because of the prime role that it will play in tomorrow's wireless and IoT networks [1]. To benefit from the deployment of federated learning, it is important to address few technical challenges that include local device computational and communication resources optimization. In addition, there is a need for effective incentive mechanisms to motivate the participation of users in the learning of a global federated learning model. Several recent works have considered machine learning in enabling IoT-based smart applications [6]- [9]. The works presented in [6], [7] mostly rely on centralized machine learning solutions which can have limitations in terms of scalability as well as privacypreservation. In [8], the authors studied a federated learning framework to provide efficient resource management at the network edge. The work in [8] presented building blocks, different neural network schemes, and key enablers of machine learning at network edge. However, the works in [8] and [9] do not discuss the important challenges pertaining to incentive design and network optimization under edge-based federated learning. Our key contributions include: • We present the key design challenges and opportunities for implementation of federated learning in edge networks. • To the best of our knowledge, this is the first work to review resource optimization and incentive mechanism for federated learning over edge networks. A. Resource Optimization Optimization of communication and computation resources is absolutely necessary to enable the main phases of federated learning, such as local computation, communication, and global computation. Computation resources can be either those of a local device or of an edge server, whereas communication resources are mainly radio resources of the access network. In the local computation phase, every selected device performs a local model update using its dataset in an iterative manner. The allocation of local device computational resources strongly depends on the device energy consumption, local learning time, and local learning accuracy. In addition, the heterogeneity of the local dataset sizes significantly affects the allocation of local computational resources. Device energy consumption and local learning time are strongly dependent on the CPU capability of the edge device. Increasing the device CPU frequency yields an increase in energy consumption and a decrease in learning time. Similarly, the local computational latency increases for a fixed frequency with an increase in local learning accuracy. Therefore, it is evident that here is a need to study the tradeoff between computation energy consumption, computational latency, learning time, and learning accuracy. On the other hand, the access network and core network resources must be allocated optimally in the communication phase [10]. B. Learning Algorithm Design Federated learning involves the usage of local and global computation resources in addition to communication resources. Several machine learning techniques, such as long short-term memory, convolutional neural network, support vector machines, and Naive Bayes schemes can be used at each local device [3]. To enable federated learning, numerous optimization schemes, such as federated averaging (FedAvg) and FedProx can be used to train non-convex federated learning models [11]. FedProx is the modified version of FedAvg and it counts for statistical heterogeneity among users. FedAvg is based on running stochastic gradient descent (SGD) on a set of smart devices with statistical homogeneity to yield local model weights. Subsequently, an averaging of the local weights is performed at the edge computing server located at BS. FedProx has similar steps as FedAvg, but the difference lies in local device minimizing of objective function that considers the objective function of FedAvg with an additional proximal term which limits the impact of local device data non-independent and identically distributed (non-IID) on the global learning model. FedAvg does not guarantee theoretical convergence, while FedProx shows theoretical convergence. In FedAvg and FedProx, all the devices are weighted equally in global federated learning model computation without considering fairness among devices. However, there exist significant variations in different devices nature (i.e., hardware variability). To address such fairness issues, a so-called q-FedAvg algorithm has been recently proposed. The idea of q-FedAvg is to give higher weights to the devices with poor performance by modifying the objective function of the typical FedAvg algorithm. To introduce potential fairness and reduce training accuracy variance, the local devices having a high empirical loss (local loss function) are emphasized by setting large values of q in the q-FedAvg. Specifically, the value of q determines the amount of fairness, greater that value of q more will be the fairness and vice versa. On the other hand, an adaptive control scheme has been proposed regarding the adaptation of global aggregation frequency for federated learning [5]. Moreover, the adaptive control scheme offers a desirable tradeoff between global model aggregation and local model update to minimize the loss function with resource budget constraint. All of the above discussed methods are used for a single task global federated learning model. We can use a multi-task learning model for multiple tasks, whose data is distributed among multiple edge nodes in a federated learning setting. In [12], federated multi-task learning (FML) has been proposed while considering fault tolerance and joint optimization of both communication and computational resources. C. Incentive Mechanism Design In addition to resource optimization and learning algorithm design, a set of devices involved in the training of a global federated learning model must be given proper incentives to ensure the trustworthiness of their participation in federated learning. Incentives are possible in different forms, such as user-defined utility and money-based rewards. Several frameworks such as game theory, matching theory, and auction theory can be used in the design of incentive mechanisms for federated learning [13], [14]. For instance, consider an incentive mechanism based on game theory in which an edge server and and edge users act as a set of players. The edge server announces a reward as an incentive to the participating nodes while maximizing its benefits in terms of improving global federated learning model accuracy. On the other hand, the edge users try to maximize their individual utilities to improve their benefit. In this regard, utility can, for example, be defined as the improvement of local learning model accuracy within the allowed communication time during the training process. An improvement in the local learning model accuracy of the end-user increases its incentive from the edge server and vice versa. This process of incentive-based sharing of model parameters is continued until convergence to some global model accuracy level. III. INCENTIVE BASED FEDERATED LEARNING OVER EDGE NETWORKS A. System Model Consider a multi-user system comprised of a BS and a set of user devices with non-IID and heterogeneous data sizes. Enabling federated learning over such edge networks involves the use of local device computational resources, cloud computational resources, and communication resources that must be optimally exploited. In a typical federated learning environment, the participating user equipment (UE) have to iterate over their local data with possibly non-IID and unbalanced nature, to train a global model. However, UEs are generally reluctant to participate in federated learning due to limited computing resources and limited communication resources [10]. Thus, enabling federated learning requires some careful design considerations that include: • First of all to motivate UEs for participation, it is necessary to model the economic interaction between the BS and the UEs. Within each global iteration, the BS can offer a reward rate (e.g., $/iterations) to the UEs for their selection of the optimal local iteration strategy (i.e., CPU-frequency cycle) that can minimize the overall energy consumption of federated learning, with a minimal learning time. • The set of resource-constrained UEs involved in federated learning has numerous heterogeneous parameters: Computational capacity, training data size, and channel conditions. This heterogeneity of UEs significantly affects the local learning model computation time for a certain fixed local model accuracy level. To compute the local learning model within fixed allowed time for resource-constrained UEs with heterogeneous parameters, the local learning model accuracy will be different for different UEs. Therefore, it is necessary to tackle the challenge of heterogeneous local learning model accuracy of the participating UEs for synchronous federated learning. B. Stackelberg Game Solution The BS employs an incentive mechanism for motivating the UEs to participate in training of a global federated learning model. However, heterogeneous UEs have different computational and communication costs needed to train a global model. Therefore, they expect different reward rates to perform optimally in a federated learning setting. On the other hand, the BS seeks to minimize the learning time while maximizing the accuracy level of the learning model. Thus, this complex interaction between the BS and the UEs can be naturally cast as a Stackelberg game with one leader (BS) and multiple followers (UEs). Here, for the offered reward, the BS aims at maximizing its utility that is modeled as a function of key federated learning performance metrics such as the number of communication rounds needed to reach a desirable global federated learning model accuracy level. Correspondingly, the UEs will respond to the offered reward by the BS and choose their local strategy (i.e., the selection of CPU-frequency cycle for local computation) to maximize their own benefits. Evaluating the responses from the UEs, the BS will adjust its reward rate, and the process repeats until a desired accuracy level is obtained. To this end, the BS must carefully design an incentive mechanism to influence available UEs for training the global model. In the proposed framework, the sequence of interactions between the BS and the UEs to reach a Stackelberg equilibrium is as follows: • At the beginning, each rational UE in federated learning submits its best response (i.e., optimal CPU-frequency) to the BS for the offered reward rate, to maximize its local utility function. Specifically, each UE considers the viability of the offered reward rate for their incurred computational and communication costs in federated learning. • Next, the BS evaluates these responses, thereafter, updates and broadcasts its offered reward rate to the UEs, to maximize its own utility function (i.e., minimizing the overall energy consumption and the learning time) for the learning problem. • To this end, with the optimal offered reward, the UEs will correspondingly tune their strategy and update response that solves their individual utility maximization problem. Hereafter, the iterative process continues in each round of interaction between the BS and UEs. • In summary, we follow the best response dynamic algorithm to achieve the Stackelberg equilibrium. For this, with the first-order condition, we first find a unique Nash equilibrium at the lower-level problem (among UEs), and, then, use a backward induction method to solve the upperlevel problem (the BS's problem). C. Performance Evaluation In this section, we evaluate the performance of our proposed incentive-based federated learning model. We consider three participating UEs having different channel conditions explicitly, and having equal local data size. At each UE, we define the mean square error of the learning problem, i.e., the local relative accuracy metric, as θ. Further, the utility model for UEs is chosen as a concave function in terms of local relative accuracy θ and offered reward from the BS. In Fig. 3a, the impact of the offered reward rate r on the relative accuracy θ for three UEs is shown. Note that, a smaller value of θ means higher accuracy. An increase in the offered reward rate will motivate UEs to iterate more within one global iteration, resulting in a lower value of θ, which is intuitive. The heterogeneous responses of UEs is the result of individual computational limitations, local data size, and communication channel conditions. The impact of the communication channel conditions on local relative accuracy for a randomly chosen UE, with defined computational characteristics and local data size is illustrated in Fig. 3 and Fig. 4. For clarity, we use a normalized communication time to quantify the adversity of channel conditions. Here, a unit value for the normalized communication time signifies poor channel conditions. As the normalized communication time increases, we observe that the UEs prefer to iterate more locally to avoid expensive communication costs. Fig. 4 presents the relationship between the offered reward rate and local relative accuracy over the communication cost at a particular UE. The heatmap plot reveals the optimal response behavior for the UEs to maximize the utility function at the given channel conditions. To this 5HZDUGUDWHU &RPPXQLFDWLRQFRVW 5HODWLYHDFFXUDF\ Fig. 4: Impact of offered reward rate, communication cost versus local relative accuracy. end, we observe heterogeneity in responses of the participating UEs, under different wireless network conditions and due to their local strategies, for the offered incentive to perform federated learning. Thus, it is crucial to have an appropriate incentive design to align responses of the participating UEs for improving the performance of the federated learning model. A. Resource Optimization for Blockchain based Federated Learning An attacker might attack the centralized server involved in federated learning in order to alter global model parameters. In addition, a malicious user might alter federated learning parameters during the communication phase. To cope with such security and robustness issues, blockchain based federated learning (BFL) can be used. BFL does not require central coordination in the learning of the global model that results in enhanced robust operation. In BFL, all the users send their local model parameters to their associated miners, which are responsible for sharing local model updates through a distributed ledger. Finally, local model updates of all the devices involved in learning are sent back by miners to their associated devices for the local models aggregation. Although BFL provides benefits of security and robustness, there exist significant challenge of computational and communication resources optimization to reach a consensus among all miners. Static miners can be implemented at the BS, whereas wireless mobile miners can be implemented using unmanned aerial vehicles (UAVs). However, UAVs based mobile miners pose more serious resource allocation challenges than static miners at the BS. B. Context-Aware Federated Learning How does one enable more specialized federated learning according to users contextual information? Context-awareness is the ability of a devices/system to sense, understand, and adopt its surrounding environment. To enable intelligent context-aware applications, federated learning is a viable solution. For instance, consider keyboard search suggestion in smartphones in which the use of federated learning is a promising solution. In such type of design, we must consider context-awareness for enhanced performance. Unique globally shared federated learning model must be used separately for regions with different languages to enable more effective operation. Therefore, the location of the global model must be considered near that region (i.e., micro data center) rather than a central cloud. C. Mobility-Aware Federated Learning How does one enable seamless communication of smart mobile devices with an edge server during the learning phase of a global federated learning model? A seamless connectivity of the devices with a centralized server during the training phase must be maintained. Mobility of devices must be considered during the device selection phase of federated learning protocol. Deep learning-based mobility prediction schemes can be used to ensure the connectivity of devices during the training phase of a globally shared global model. V. CONCLUSIONS AND FUTURE RECOMMENDATIONS In this paper, we have presented the key design aspects, incentive mechanism, and open research challenges, for enabling federated learning in edge networks. Finally, we present several recommendations for future research: • Generally, federated learning involves training of a global federated learning model via an exchange of learning model updates between a centralized server and geographically distributed devices. However, wireless devices will have heterogeneous energy and processing power (CPU-cycles/sec) capabilities. Moreover, some of the devices might have noisy local datasets. Therefore, there is a need for novel federated learning protocols that will provide criteria for the selection of a set of local devices having sufficient resources. The selection criteria of the devices must include long-lasting backup power, sufficient memory, accurate data, and higher processing power. • A set of densely populated devices involved in federated learning might not be able to have real-time access to the edge server located at the BS due to a lack of communication resources. To cope with this challenge, one can develop new federated learning protocols based on socially-aware device-to-device (D2D) communication. Socially-aware D2D communication has an advantage of reusing the occupied bandwidth by other users while protecting them by keeping the interference level below the maximum allowed limit. Initially, multiple clusters based on social relationships and the distance between devices should be created. Then, a cluster head is selected for every cluster based on its highest social relationship with other devices. Within every cluster, a sub-global federated learning model is trained iteratively by exchanging the learning model parameters between the cluster head and its associated devices. Then, the sub-global	2019-11-06T03:29:04.000Z	2019-11-06T00:00:00.000	{ "year": 2019, "sha1": "2a3d09bbdfe21418ce75d6973f71028fa9192b89", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1911.05642", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "2a3d09bbdfe21418ce75d6973f71028fa9192b89", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
3556073	pes2o/s2orc	v3-fos-license	Deciphering general characteristics of residues constituting allosteric communication paths Considering all the PDB annotated allosteric proteins (from ASD - AlloSteric Database) belonging to four different classes (kinases, nuclear receptors, peptidases and transcription factors), this work has attempted to decipher certain consistent patterns present in the residues constituting the allosteric communication sub-system (ACSS). The thermal fluctuations of hydrophobic residues in ACSSs were found to be significantly higher than those present in the non-ACSS part of the same proteins, while polar residues showed the opposite trend. The basic residues and hydroxyl residues were found to be slightly more predominant than the acidic residues and amide residues in ACSSs, hydrophobic residues were found extremely frequently in kinase ACSSs. Despite having different sequences and different lengths of ACSS, they were found to be structurally quite similar to each other - suggesting a preferred structural template for communication. ACSS structures recorded low RMSD and high Akaike Information Criterion(AIC) scores among themselves. While the ACSS networks for all the groups of allosteric proteins showed low degree centrality and closeness centrality, the betweenness centrality magnitudes revealed nonuniform behavior. Though cliques and communities could be identified within the ACSS, maximal-common-subgraph considering all the ACSS could not be generated, primarily due to the diversity in the dataset. Barring one particular case, the entire ACSS for any class of allosteric proteins did not demonstrate"small world"behavior, though the sub-graphs of the ACSSs, in certain cases, were found to form small-world networks. Introduction: Starting from Monod−Wyman−Changeux(1) and Koshland−Némethy−Filmer(2) models, investigations of allosteric regulation of protein function have over half-acentury long, rich and multifaceted history. There are so many excellent reviews that have attempted to capture the essence of various aspects of research on this topic that even a cursory enlisting of them will run for pages. The most recent ones include (3)(4)(5)(6)(7)(8). To summarize these efforts, one can merely observe that while a lot has been unearthed about the physico-chemical nature of allosteric signal transduction, the various modes through which the long-distant communication is achieved, the structural details of cooperativity revealed during this process, there are still significant aspects of allosteric regulation, especially in the context of generalized characterization of the process, that need to be better understood. The present work reports a few generalized findings about the allosteric communication. enzymes (27). Similarly, while the necessary role of the symmetrical multimeric proteins have been discussed in one report(28), roles of monomeric proteins or that of multimeric proteins apparently without symmetry in achieving allosteric communications have been reported also (29,30). Taking notice of these differences, we chose to look beyond the symmetry aspects, be it crystallographic or selfsimilar(31), but by resorting to a collection of related approaches to decipher some consistent patterns embedded latently in structural, biophysical and topological nature of allosteric communication sub-structures(ACSS). We focused on the analysis of mobilities of residues forming ACSS. We compared protein fluctuations derived from crystallographic Debye-Waller B-factors of experimentally solved crystal structures with those obtained from the root mean square fluctuations (RMSF) profile from computational modelling. Materials : The curated database ASD(Allosteric Database)(32) was used to retrieve protein structures with information about the identified allosteric communication paths. In some cases we spotted differences in the description of protein structures provided by the ASD and the PDB. These cases were not considered for the study. Retaining the typification scheme provided by the ASD, the finally selected set of 30 proteins that were further divided in four groups: the kinases, the nuclear receptors, the peptidases and the transcription factors. The PDB IDs of these 30 proteins are: 1CZA, 1DKU, 1E0T, 1PFK, 1S9I, 1SQ5, 2BTZ, 2JJX, 2OI2, 2VTT, 2XRW, 3BQC, 3EQC, 3F9M, 3MK6, 4AW0 (-as the kinase group of allosteric proteins); 1IE9, 1XNX, 2AX6 3S79 (-group of nuclear receptors); 1SC3, 2QL9, 4AF8 (-the peptidases); and 1JYE, 1Q5Y, 1R1U, 1XXA, 2HH7, 2HSG, 3GZ5 (-the transcription factors). Methodology: Resorting to a coarse-grained representation of residues, and a reduced amino acid alphabet is more likely to lead to generalized ideas from the investigation of the ACSS of 30 proteins. A mere two-letter hydrophobic-polar classification of the residues would have been too broad to reveal the complexity of the problem. Thus we resorted to a scheme(33, 34) which has been found to be extremely successful in protein structure prediction studies (35,36). Here the 20 amino acids are expressed with a reduced 8-letter alphabet scheme; that is: GLU and ASP -as acidic, ARG, LYS and HIS -as basic, GLN and ASN -as amides, SER and THR -as hydroxyls, TRP, TYR, PHE, MET, LEU, ILE and VAL -as hydrophobic, and GLY and ALA -small residues. PRO and CYS are special among the 20 amino acids for obvious reasons; because of their special status, each one of them are placed as singleton groups. This coarse-grained description was used to study both population characteristics of the ACSS constituents and to undertake the network-based investigations of ACSS. Because the central theme of this study was to measure the extent by which the residues involved in allosteric communication differ from all remaining residues, various tests were conducted throughout the study to compare residues constituting ACSS with non-ACSS ones. Atoms of the residues not identified (viz., color-coded) by the ASD as part of allosteric communication paths, were considered to be non-ACSS residues and atoms. We were interested to know whether the sub-structures of the allosteric communication paths have structural similarities among themselves, or not; so i.e. the kinase allosteric communication paths will be characterized by a certain set of canonical parameters, while the nuclear receptor's allosteric communication paths will be different by certain (structural) degrees, etc. However, we realized that a mere structural superposition of the ACSS, will fall short of our goal because the substructures in question are characterized by different sequence compositions, different lengths, and additionally may contain various gaps and insertions. Because neglecting certain parts of these sub-structures would have caused loss of valuable information, we used THESEUS 2.0 software(37) that superposes multiple protein structures without throwing away the gaps in them. For network-based studies, Python's NetworkX was used as the graphing library, while matplotlib was used for image generation. Because statistical tests are necessary to categorically establish the general traits in the allosteric proteins and yet, because the present study considers a limited set of allosteric proteins as belonging to different four classes two non-parametric tests (Wilcoxon signed-rank test and Friedman's non-parametric test)(38-40) were employed to ascertain the traits of the obtained results. In investigating the "small world network" characteristics, methodologies elaborated in (41) were implemented by us; details about the theoretical basis of the methodology, thus, can be found there. To gather the answer to the question of whether or not the ACSSs are SWNs or not, at multiple resolutions, we studied the problem by generating the Erdös-Rényi (E-R) random graph at three probabilities: 0.3, 0.5 and 0.7. The computational modelling is a key to solving many fundamental problems of molecular biology. Prediction of protein structures and interactions(42) as well as structural transformations taking places during unfolding, folding and aggregation processes have been studied by computer simulations at different levels of resolution and timescales (43-52). For more efficient simulations one uses coarse-grained (CG) models which reduce the complexity of each amino acid by representing it by a single node or group of pseudo atoms (42, [53][54][55]. In our work, the CABS-flex method (56) is used for predicting protein fluctuations. CABS-flex employs a coarse grained CABS model (54) -efficient and versatile tool for modelling protein structure, dynamics and interactions (57)(58)(59)(60). In CABS model one uses coarse-grained representation for a protein in which four atoms per residue are retained. The four atoms include Cα, Cβ, center of mass of the rest of side chain and the center of the virtual Cα-Cα bond. A high resolution lattice model with realistic interactions between atoms is implemented at which Cα atoms of the polypeptide chain are confined to the simple cubic lattice model with the lattice spacing of 0.61Å. Large number (800) of the possible virtual Cα-Cα bonds orientations keeps the protein conformation space flexible and ensures overcoming lattice anisotropy problems. Unlike the alpha Carbon atoms, which are restricted to the lattice, the remaining atoms in CABS model are off-lattice. Conformations obtained by CABS-flex simulations further can be reconstructed to physically sound atomistic systems using coarse-grained to atomistic mapping methods (57,61).The interactions between atoms are described by a realistic knowledge-based potential, while protein-solvent interactions are approximated using implicit solvent model. More detailed description of the CABS force field can be found elsewhere (54,62). Discrete conformation space, coarse-grained representation and solvent treated in implicit fashion involved in CABS model greatly reduce the tremendous number of degrees of freedom and free energy landscape roughness. Such simplifications seem to be a reasonable compromise compared to more computationally demanding (and in many cases prohibitive due to the huge system size involved) explicit-solvent all-atom modelling. It is worth noting that despite rather drastic above mentioned simplifications and approximations involved in CABS modelling, the results on structure prediction and dynamics from CABS model simulations reasonably agree with both more sophisticated all-atom MD simulations as well as experimental results (57,60,(63)(64)(65). To address the question whether B-factors from protein data bank file are consistent with mean square fluctuations of atoms from simulations, we perform near-native simulations for three conceptually different proteins, 1Q5Y from transcription factors group, 1SC3 from the peptidases and 2JXX from the kinase group of allosteric proteins. 3.1: The thermal fluctuation of residues in allosteric communication paths: Mass distribution in proteins is known to be inhomogeneous (66); thus, it is not realistic to expect that protein atoms populating diverse spatial zones of a protein will be restricted by equal degrees in their allowed structural thermal fluctuations. Alongside the dynamics needed to ensure the propagation of the structural signal through protein, the ACSS residues possess their inherent thermal fluctuational dynamic. The construction of the residual-interaction networks depends on the value of the cutoff distance, that may be different than the commonly used value of 6.5 Å (67). To quantify the extent of fluctuations of the ACSS residues, versus fluctuations of non-ACSS residues, we extracted B-factors from the coordinate files of the protein structures in protein data bank (PDB) (68) for all 30 proteins. Results found from this investigation, however, are far from simple. While the (naive) expectation was to observe that the residues in ACSS have more inherent fluctuations, obtained results demonstrated that the thermal fluctuations are not only residue-specific but also specific to the type of allosteric proteins considered; Table 1 contains the details of this investigation. The results shown in Table 1 reveal certain interesting findings. For example, the hydrophobic residues (barring PHE) in ACSS have been found to represent large thermal fluctuations, significantly larger than the non-ACSS hydrophobic residues. The global averages of B-factors for residues VAL, LEU, ILE and MET in ACSS's have shown a consistent difference (of ~+9.0) in comparison to the non-ACSS ones for the same set of proteins. On the other hand, the polar residues belonging to ACSS have smaller B-factors in comparison to the same non-ACSS residues, barring the case of GLU which has the same B-factors in both cases. However, the differences in B-factors of the polar residues in ACSS and non-ACSS regions are not found to be as high as that observed for the hydrophobic residues. Differences of B-factors in PRO, CYS and GLY were not considered because they occurred in very low number cases (≤ 3). To assess whether and by what extent the B-factors of different families of allosteric proteins differ from each other, we subjected the mean values to Friedman's nonparametric test (alternatively referred to as 'non-parametric randomized block analysis of variance')(38, 39). We chose to employ Friedman's test because, ANOVA requires the assumptions of a normal distribution and equal variances (of the residuals) to hold, none of which is found to be existing in our case (viz., that in Table 1), while Friedman test is free from the aforementioned restrictions. The null hypothesis for the test was that the B-factors of the four types of ACSS are the same across repeated measures. Result obtained from the test categorically demonstrates that there indeed exists a substantial difference in the B-factors of these four classes of ACSSs. Results obtained from B-factors of four types of ACSS was Freidman X 2 = 20.4 > 16.266 (P value at 0.001, with 3 degrees of freedom), whereby the null hypothesis was rejected comprehensively. To ascertain the degree to which the B-factors of ACSS residues in each of the four classes of allosteric proteins differ from the B-factors of the non-ACSS residues, each of the classes were subjected to Wilcoxon signed rank test (36), which is a nonparametric analogue of paired t-test for correlated samples, without assuming that the population is normally distributed. The null hypothesis for each of the comparisons was that the median difference between pairs of observations is zero. Result obtained from the tests revealed that the B-factors of ACSS residues in each of the classes differed significantly than the B-factors of the non-ACSS residues. For the kinase class of allosteric proteins we found, W kinase =87 >> 23 ([W(α=0.01,17)=23); for the peptidase class W peptidase =8 > 2 ([W(α=0.05,7)=2] (we note that W is not defined in 0.01 at degrees of freedom 7 (though W(0.01,8)=0), whereby, the critical value comparison is being reported at the weaker 0.05 level); for the Nuclear Receptors, W NR =15 > 5 ( [W(α=0.01,11)=5]); and for the transcription factors, W TF = 6 > 5 ([W(α=0.01,11)=5]). Thus, the null hypothesis was rejected in each of the four cases with extremely high confidence. Composition of the ACSS population: Allosteric signalling achieved at the structural level show certain differences for various proteins (69)(70)(71). Thus, we expect to observe differences in composition of ACSS residues for four different classes of proteins. We found that basic residues are more frequent in ACSS than the acidic ones. To demonstrate this prevalence let us take a closer look at the composition of ACSSs for kinases: the acidic residues were found in 12/133 cases, while the basic residues occurred in 32/133 cases. For the class of transcription factors the basic residues in ACSSs occurred in 13/35 cases, whereas the acidic residues occurred in 5/35 cases. The hydrophobic residues were found to occur in ACSSs of kinases with significant frequency (59/133 cases), but were be notably small in ACSS of transcription factors (2/35) and in peptidases (1/11). Hydroxyl residues were found to be more common in ACSSs than the amide residues, for kinase ACSS: amide residues 6/133, and hydroxyl residues 14/133 cases. PRO and CYS populations although are extremely small in ACSS, show that CYS occurs slightly more frequently than PRO. The small amino acids (GLY and ALA) were found in very small frequency in ACSSs, while TRP was not found as part of any of the ACSSs. 3.3: Structural Superimposition of Multiple Allosteric Communication Paths: Results obtained from the structural superimposition of multiple ACSSs demonstrated clearly that the allosteric communication paths, for any type of allosteric protein, match closely each other in their structures. Upon superimposing the PDB-coordinates of ACSSs of all proteins for each of the four classes using 'Theseus' software we obtained results shown in detail in Supplementary Material:1. Here we report the two most prominent results, a: RMSD for the superposition, and b: the Akaike Information Criterion (AIC). AIC proposed by Akaike (72) has become commonly used tool for statistical comparison of multiple theoretical models characterized by different numbers of parameters. Because the RMSD of two superposed structures indicates their divergence from one another a small value is interpreted as a good superposition. In contrast, the higher magnitude of AIC indicates better superposition. We found that the ACSS paths, despite belonging to different proteins and corresponding to sequences of varying lengths, consistently demonstrated lower RMSD values and significantly higher AIC values in comparison to non-ACSSs parts of the structures (see the details in the Supplementary Material:1). 3.4.1: Centrality of ACSS: The process of allosteric signal communication is directional, but the richness of the constructs available to study networks becomes apparent by using non-directional graph-theoretical framework. Thus, instead of asking 'what is the route of allosteric signal propagation for a specific protein?', which is already provided by ADB, we asked questions like: 'how robust the ACSSs are, compared to non-ACSS parts of the proteins?', or , 'how does the fluctuation of one arbitrarily-chosen residue influence the spread of allosteric signal through ACSS?', or , 'how probable is it that allosteric communication occurs through a randomly chosen shortest path between two residues belonging to ACSS?', etc. To answer these and similar questions of general nature, we started our investigation by studying the centrality aspects of the ACSS network. The centrality metrics quantify the relative importance of a protein residue (viz. the vertex) or a residue-toresidue communication path (viz., an edge) in the network description of ACSS. There are many centrality measures, we chose to concentrate upon three fundamental measures outlined in Freeman's classic works (73,74), namely: degree centrality, betweenness centrality and closeness centrality. Because our ACSS networks are unweighted, we did not calculate the Katz centrality. Also, though eigenvector centrality (75,76) and Gould index present refined knowledge of network centrality, they don't basically differ from degree centrality, and were neglected in the present work. Degree centrality reveals the local characteristics of the ACSS graph while the other two measures, (betweenness and closeness centralities), tend to reflect upon the global network structure, because they rely on counting shortest paths. Degree Centrality: Degree centrality for any protein residue in an ACSS network is calculated in a straightforward way, by counting the number of residue-residue communication links connecting that residue (implementing the classical definition (73) to the context of ACSS). Degree centrality of any ACSS residue provides an idea about the local structure around that residue, by measuring the number of other residues connected to it. We note that degree centrality is a local measure that does not provide any information about the network's global structure. We have found that the average degree centrality of ACSS residues, irrespective of the type of allosteric proteins, is lower than the average degree centrality of the non-ACSS residues. This result, alongside that obtained from the other centrality measures are presented in Table-2. Maintaining equality in the size (i.e. the number of residues considered) in every quartet of networks (one ACSS network and three non-ACSS networks from the same protein), the average degree centrality was calculated for every group of ACSS and non-ACSS residues; which in turn, was calculated after calculating the degree centrality of each residue belonging to ACSS and non-ACSS fragments. These residue-specific details of degree centrality for every protein considered, for ACSS and similar size non-ACSS fragments, are provided in Supplementary Material-2. We note that the average degree centrality of ACSS fragments consistently show lower values than similar non-ACSS fragments. The kinase and the transcription factor ACSSs have similarly low average values of degree centrality. The nuclear receptors have them significantly higher, while the peptidases ACSSs have the highest average values of degree centrality. We note also that the typical differences between average degree centrality of ACSS and non-ACSS fragments are: ~0.10 for kinases, ~0.8 for nuclear receptors, ~0.9 for peptidases, and ~0.13 for transcription factors. More importantly, the consistent observation of lower average degree centrality of ACSS fragments irrespective of the type of allosteric proteins suggests that it is a general feature. In terms of the network theory the degree centrality is the diagonal element of adjacency matrix that corresponds to the sum of all off-diagonal elements in a given row/column. In an alternative way, one may as well describe the degree centrality as the number of paths of unit length coming out from a given vertex. Degree centrality shows the ability of a given vertex to influence or to be influenced by the local structure around it. It can be considered as a marker (or even predictor) of immediate effects of propagating through a network. Thus, the consistently lower values of average degree centrality observed in ACSS fragments suggests that nature attempts to shield them from perturbations which may destabilize allosteric communication. In terms of attempts to shield the ACSS from perturbations influencing residual interaction network, degree centrality demonstrates similarity with the concept of eigenvector centrality, the caveat being that the later provides a metric to assess direct and indirect influences taking place over a long time-interval while the former measures immediate effects of a perturbation solely. The global centrality measures: While the degree centrality provides a measure to assess the possibility of immediate involvement of a residue in influencing the signal communication in residue interaction network of a protein, the concepts of closeness centrality and betweenness centrality provide ideas of how the global topology of the network influences the signal propagation. Closeness centrality of any connected graph measures how "close" a vertex is to other vertices in a network; this is computed by summing up the lengths of the shortest paths between that vertex and other vertices in the network. Closeness of a vertex, thus, can be interpreted as a predictor of how long it may take for that vertex to communicate with all other vertices. In the framework of protein residue connectivity network, the residues with low closeness score can be identified as ones that are separated by short distances from other residues. It can be expected that they receive the structural signal (i.e. instantaneous fluctuation or perturbation) faster, being well-positioned to receive this information early. We indeed found that the average closeness centrality of the ACSS network is lower in comparison to the non-ACSS fragments, for all the types of allosteric proteins. However, the difference between the extent of average closeness centrality between ACSS and non-ACSS fragments was found to vary over a larger scale than what was observed for average degree centrality. For graphs of equal sizes, the difference between the average closeness centrality of ACSS and non-ACSS fragments was found to be: ~0.04 for peptidases, ~0.05 for nuclear receptors, ~0.11 for kinases, and ~0.22, for transcription factors (see Table-2). We also found that while ACSSs of transcription factors and kinases have small values of average closeness centrality, suggesting fast propagation of allosteric signals, nuclear receptors, and peptidases are significantly less capable of rapid transmission of the signal in the allosteric communication. Based on values of closeness centrality of ACCS fragments, peptidases are expected to be twice slower in propagating allosteric signals in comparison to transcription factors and kinases. To assess the extent to which the centrality measures differ for four different classes of proteins, we subjected them to Friedman non-parametric test. The null hypothesis was that the centrality measures across the four different classes are the same under repeated measures. Since the value of the test statistic Q=13.67 > 12.838 (p-value), the null hypothesis was rejected comprehensively. The betweenness centrality provides more idea about the global network structure; for every vertex of the network the betweenness centrality specifies the fraction of the shortest paths (geodesics) that pass through that vertex. In this sense, such measure assesses the influence that a given vertex (residue) has over the transmission of a structural signal. A residue with large betweenness centrality score can be expected to have a large influence on the allosteric signal propagating through the ACSS network. Results obtained by us shown inTable-2, are found to be less clear to interpretation. While the average betweenness centrality of ACSS networks in peptidases is found to be higher in comparison to non-ACSS fragments, the similar data for kinases, nuclear receptors, and transcription factors show opposite behaviour. These results imply that for three out of four considered classes of allosteric proteins the residues constituting the ACSS net do not fall in the geodesics which communicate the allosteric signal. -Though it seems unexpected at the first, a close scrutiny at the definition of betweenness centrality reveals that this is not only possible but can probably be expected. The implicit assumptions in linking the signal transmission with betweenness centrality are, first, that the information propagation through the network takes place along the geodesics, and second, that the information is an undivided chunk. While such characterizations can be useful in many cases of network science, the determinism of a biological network may include certain provisions for error handling and fault tolerance mechanisms developed through evolution. Thus, propagation of information (the structural signal in the case of allostery) across ACSS although deterministic may not follow the simplistic geodesic-based route. A geodesic, i.e. the shortest path, is identified as the path through which the two given vertices are connected by the smallest number of intermediate vertices. The mean of the shortest path lengths reflects the expected distance between two connected vertices. While it has been reported that residues constituting the ligand binding sites of proteins have low average of their shortest path lengths (77), the ACSS networks studied in the present work tends to suggest that the allosteric signal communication may not take place along the shortest path. Second, as the various recent works of allostery tend to suggest, the structural fluctuation-based signal propagating across protein, may not always be treated as an undivided entity (78)(79)(80). Thus, the average betweenness centrality studies for ACSSs need further investigations based on availability of larger datasets in the future. Cliques and Communities in ACSS: Cliques are the complete subgraphs, where every vertex is connected to every other vertex. A clique is considered maximal only if it is not found to be a subgraph of some other clique. Communities are identified through partitioning the set of vertices, whereby each vertex is made a member of one and only one community. Because of their higher order connectivity, the cliques detected in protein structures are considered to indicate regions of higher cohesion (in some cases, rigid modules). Do the ACSSs embody certain common characteristics in their connectivities which can be revealed through the cliques and communities? To answer this question, we subjected the ACSSs of each of the four classes of proteins to investigation, which implemented (81) and (82) algorithms. We found that indeed the ACSS modules can be partitioned into cliques and communities (83,84). These results are presented in Fig.-1 Maximum common subgraphs to describe the ACSS: A maximal common subgraph of a set of graphs is the common subgraph having the maximum number of edges. Many attempts have been made for the last two decades to apply this methodology in protein science (85)(86)(87). Finding the maximal common subgraph is a NP-complete problem (88). To solve this difficult problem a backtrack search algorithm proposed by McGregor(89) and a clique detection algorithm of Koch (88), are traditionally used. However, for our ACSSs, some of which are quite large in size, neither McGregor's nor Koch's algorithm was found to be applicable; primarily because of the huge computational costs incurred by the exponential growth of intermediary graphs of varying sizes. Thus, upon generating the subgraphs for each of ACSSs (using Python's NetworkX), we had to resort to the brute-force method to identify the maximum common subgraph for each of the ACSS classes. In some cases, the number of cliques was found to be large; e.g. for 2BTZ (see Supplementary Mat.-3), while in some other cases only one clique was found (e.g. for 2VTT (Res78:hydrophobic-Res76:basic-Res71:basic); or for 2XRW (Res198:hydrophobic-Res230:basic-Res231:hydrophobic)). Fig-2 presents five maximum common subgraphs composed of 3 vertices and 3 edges, obtained from five subsets (each comprised of 5 to 7 proteins) of kinase ACSSs. The fact that different combination of subsets of kinase ACSSs were found to represent different maximum common subgraphs, point out, why, finding a general maximum common subgraph (composed of more than two nodes and one connecting edge) for the entire set of kinase ACSSs was found to be impossible for this dataset. 3.7: How frequently do the allosteric communication paths form small world network? Investigating whether in general the ACSS residues belonging to the four different classes of allosteric proteins constitute 'small world' networks (SWN) or not is important; because SWNs are more robust to perturbations, and may reflect an evolutionary advantage of such an architecture (90,91). There are numerous previous works which talk about SWN (92) and the relevance of SWN in investigating the protein structural networks (93)(94)(95) and protein-protein interaction networks (96). The SWN (92), constitute a compromise between the regular and the random networks, because on one hand they are characterized by large extent of local clustering of nodes, like in regular networks, and on the other hand they embody smaller path lengths between nodes, something that is distinctive for random networks. Because of the ability to combine these two disparate properties, not surprisingly, it has been shown that networks demonstrating the 'small-world' characteristics tend to describe systems that are characterized by dynamic properties different from those demonstrated by equivalent random or regular networks (92,(96)(97)(98)(99)(100). Greene and Higman (95) have shown that protein structure networks which demonstrate both the long-range and short-range interactions exhibit a SWN character; however, the ones demonstrating only the long-range interactions cease to remain SWNs. Additionally, a study of a benchmarked set of 15 pairs of ACSSs with effector and substrate both present in at least one of the two structures demonstrated that the clusters possessing at least one substrate or an effector molecule exhibited SWN characteristics (101). This inspired us to perform a thorough investigation of the entire set of ACSSs for each of the four classes of allosteric communication paths. We have found that whether ACSSs exhibit SWN nature or not -is a complex problem; while the complete ACSS of a protein may not always demonstrate SWN characteristics, many sub-graphs of non-trivial lengths of the same ACSS reveal SWN character. To elucidate, the ACSSs of all kinases, all peptidases, all transcription factors, and three nuclear receptors (1IE9, 2AX6, 3S79), were found to not demonstrate SWN characteristics. In fact, because residues constituting the ACSS tend typically to not cluster in a particular region of protein structure but are distributed over entire structure, (except of 1XNX), all the ACSSs (mentioned above) were found to be represented by unconnected graphs. Similar disconnected nature of the graphs was revealed by the non-ACSSs fragments. To get a better understanding of the topology of networks, the unconnected graphs of ACSSs and non-ACSSs fragments were first segmented into a set of sub-graphs of non-trivial lengths (≥ 3 nodes); then, shortest paths in the respective subgraphs were calculated. The graph fragmentation was performed using Python's NetworkX library (with 'matplotlib' library used for image generation). Structural graphs of all ACSS or non-ACSS fragments were found to be fragmented into more than one sub-graph in almost every case (except of nuclear receptor protein 1XNX). To ascertain the nature of clustering characteristics the mean clustering coefficient as well as the mean shortest paths for each of these sets of subgraphs representing ACSS or non-ACSS were calculated (see Table-3). The SWN character was observed only for ACSS of 1XNX (Fig.-3). However, the subgraphs of ACSSs with non-trivial number of nodes (namely ≥ 3) demonstrate the SWN character, for 6 cases, with the details provided in Table-4. Changing the cut-off distance from 6.5Å to another value (e.g. 10Å or larger) doesn't change the fundamental behaviour, because, the residues involved in ACSS will be still scattered over the entire protein structure. CABS-flex simulations With the help of CABS modelling, we computed the values of root mean square fluctuations RMSF, shown as red curves (on right Y-axis) on middle plots in Figs. 4a, 4b and 4c. The values of B-factors are shown as black curves (on left Y-axis). Although quantitative comparison between B-factors and RMSFs is not possible due to different temperatures and environmental factors used in simulations and experiments, qualitatively the data agree. The most fluctuating protein residues during near-native state simulations result in a series of peaks in RMSF profile (red curve, right X-axis) which correlate with experimentally measured B-factor values (black curve, left Y-axis). Upper and bottom snapshots in Fig. 4 correspond to protein representation coloured by crystallographic B-factor values from PDB and by RMSF values from CABS-flex simulation, respectively. 1Q5Y consists of two alpha-helices and four beta strands. First two peaks in RMSF profile (Fig. 4a) correspond to regions connecting the first alpha helix with its neighbouring beta-strands. The third peak is associated with beta hairpin loop connecting the second and third beta strands. Finally, the fourth and fifth peaks correspond to the regions connecting second helix with its neighbouring beta-strands. The results from B-factor profile are slightly different. Both alpha-helices have high temperature factors values. As follows from B-factor and RMSF profiles (Fig. 4a) each of two broad peaks corresponding to H1 and H2 on B-factor profile splits into two peaks on RMSF profile. This phenomenon possibly occurs as a result of competition between the energy gain and the entropy loss of different secondary structural elements upon temperature increase (102). At low crystallization temperature complexity of structural variations of alpha-helix ensemble of microstates is comparable to the one from less structured connecting loop ensemble of microstates. However, as temperature T increases less structured connecting regions gain more entropy compared to structured alpha helix, as a consequence at room temperature residues from connecting loops are more destabilized having larger RMSF values than those forming alpha-helices. In other words, as T increases, alpha helix destabilizes to a smaller extent, compared to its connecting loops. However overall, protein fluctuations from RMSF profile can be mapped to B-factor values and agree well with experimentally measured X-ray B-factors Fig. 4b and Fig. 4c show RMSF and B-factor profiles as well as corresponding snapshots for two different proteins, 1SC3 and 2JXX. RMSF and B-factor profiles have multiple peaks which are interpreted as a sign of least stable parts of proteins studied. Similar to 1Q5Y protein, we observe that connecting loops as well as C-and N-terminals are the most fluctuating parts of protein. The overall trend is that the mobility of atoms obtained from simulations are in good agreement with the crystallographic B-factors. 4.Conclusion: The aim of the present work was to decipher some general patterns of residues forming the ACSS of 30 allosteric proteins, and compare them with non-ACSS residues in the same proteins. Our aim was to report the general quantifiable differences between these two (aforementioned) sets of residues and not to study the general mechanism of allosteric communication. By performing the CABS-based simulations of proteins around their native conformations we demonstrated that protein fluctuations depicted by RMSF profiles can be mapped to B-factors and show satisfactory degree of agreement with experimental data. Our results may benefit the protein engineering community and those studying the general mechanism of allosteric communication or in general, long-distance communication in proteins. The knowledge of the topological invariants of communication paths and the biophysical, biochemical and structural patterns may help in a better understanding of allostery. As many recent papers (103)(104)(105)(106)(107) have pointed out, the long-distance communication features within proteins involve several types of non-linear characteristics that may often be dependent on transient fluctuations, making it difficult to arrive at a generalized dynamic picture. However a generalized static picture of the long-distance communication route can be obtained, which may help to better understand such communication schemes, especially those related to allostery. The present work attempted to report such generalized findings. While certain yet-unknown (to the best of our knowledge) patterns regarding the thermal fluctuation profile of ACSS atoms, the structural and topological nature of the ACSS have come to light, incongruities of our findings regarding the extent of betweenness centrality in ACSS network and their small-world nature indicates the need for more focused studies directed at these issues, which in turn, may shed new light on allosteric signal communication. For example, proteins, in general, are fractal objects with known characteristics of trapping energy (108)(109)(110)(111). Do the findings on betweenness and on small-world network nature reported in this work indicate the possibility of energy traps in ACSSs? -We plan to probe into many such questions in future. Table 2 Legend: The three types of major centrality measures calculated on the ACSS and non-ACSS graphs of the same size. Peptidases No SWN was observed even among the subgraphs of residues constituting ACSSs of peptidases. Transcription Factors No SWN was observed among the subgraphs of residues constituting ACSSs of transcription factors.	2018-02-27T23:21:21.000Z	2017-02-03T00:00:00.000	{ "year": 2019, "sha1": "531b414baa619d8a1b2da5dafb00f32b38c5c62b", "oa_license": "elsevier-specific: oa user license", "oa_url": "https://doi.org/10.1016/j.bpj.2016.11.2698", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "531b414baa619d8a1b2da5dafb00f32b38c5c62b", "s2fieldsofstudy": [ "Biology", "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Chemistry", "Biology", "Physics" ] }
16652331	pes2o/s2orc	v3-fos-license	Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene. Introduction Inflammation, specifically proinflammatory prostanoids, greatly contribute to shape tumor microenvironment and to neoplastic progression [1]. Bacterial lypopolisaccharide (LPS) induces epithelial and immune cells to release mediators such as prostaglandins, which are synthesized by cyclooxygenase-2 (COX-2). COX-2 is directly involved in the development of chronic inflammation, including inflammatory bowel disease [2,3]. COX-2 is an immediateearly response gene induced locally by LPS and pro-inflammatory cytokines [4,5]. COX-2 expression levels are upregulated in a significant fraction of colorectal adenomas and adenocarcinomas [6][7][8]. COX-2 products induce changes in cell adhesion properties enhancing the metastatic potential. High levels of COX-2 are associated with poor prognosis [9,10]. Regular use of COX inhibitors greatly reduces the relative risk of developing colon cancer and contrasts cancer stemness [11,12]. COX-2 expression is regulated by epigenetic mechanisms. For example, COX-2 gene is frequently methylated and silenced in a subset of colorectal tumors (CIMP+ tumors) [13]. Also, in chronic inflammation induced by Helicobacter pylori (Hp) infection, COX-2 is modulated by DNA methylation [14]. At this stage, mechanisms underlying LPS-induced COX-2 gene modulation and programming have been only partly addressed. However, epigenetic changes in host cells induced by LPS have been recently investigated [15][16][17][18][19][20]. We have previously shown that Hp induces specific epigenetic events at COX-2 and iNOS promoter regions in the first phases of exposure of gastric cells to Hp [18,19]. Furthermore, in intestinal epithelial cells LPS activates IL-8 gene expression by inducing rapid histone modifications in the IL-8 promoter chromatin [17]. In this work, we investigated whether LPS exposure affects epigenetic signatures at COX-2 gene in intestinal epithelial cells. We show that LPS induces cycles of methylation in the DNA and in histone H3 around the COX-2 promoter. Specifically, methylation cycles of H3 lysine 27, a repressive mark associated with transcription inhibition, are induced by LPS and carried out by the H3K27 demethylase JMJD3 to activate COX-2 gene in intestinal epithelial cells. Databases Gene sequences were retrieved by the Ensembl database: COX-2, accession number ENST00000367468. Quantitative ChIP analysis ChIP analysis was performed as previously described [24] using the EpiQuikTM chromatin immunoprecipitation kit from Epigentek Group Inc. (Brooklyn, NY). Specifically, 0.5 × 10 6 cells treated or untreated were grown to 80%-90% confluency on a 100 mm plate, then trypsinized, collected and washed with PBS. The cells were cross-linked in fresh culture medium with 1% formaldehyde and blocked with 1.25 M Glycine solution. After washing with PBS (2x) the samples were immunoprecipitated with the antibodies according the manufacturers' instructions. Antibodies used for Protein-DNA immunoprecipitation were: anti-H3-Acetyl (Upstate cat#06-599), anti di-methyl-H3K9 (Upstate cat#07-214), anti tri-methyl-H3K27 (Upstate cat#07-449, Biotechonology, Dundee; UK) anti-di-methyl-H3K4 (Abcam Inc. cat#Ab7766), anti-JMJD3 (ABGENT, cat#AP1022a, San Diego; CA), anti-EZH2 (Cell Signaling TECHNOLGY, cat#AP1022a, Danvers, MA) and normal mouse IgG as a negative control antibody. DNA samples were subjected to quantitative PCR analyses, using SYBR 1 Green Taq (Qiagen) in a Chromo4 Real Time thermocycler (BIORAD). Amplification of the COX-2 promoter was performed using the primers: PCXF 5'-AAGGGGAGAGGAGGGAAAAATTTGTG-3' (position from nucleotides -89 to -114) and PCXR, 5'-GAGGCGCTGCTGAGGAGTT CCTG-3' (position from nucleotides +44 to +66). The quantitative PCR conditions were: 95°C for 15 min followed by 40 cycles of 95°C for 15 sec, 62°C for 30 sec, and 72°C for 30 sec. The signal obtained by precipitation with the control IgG was subtracted from the signals obtained with the specific antibodies. Results are expressed as percentage of the input, and calculations take into account the values of at least three independent experiments. Gene silencing by RNA interference HT-29 cell lines were plated at a density of 50% per 6-well plates 24 hrs before transfections. Cells were transfected with JMJD3 siRNA (Qiagen, Hs_jmjd3_2, SI00449827, target sequence: cagcaggaatgccaaggtgaa) or AllStars negative Control siRNA (Qiagen, 1027281) at a final concentration of 5nM using Lipofectamine 1 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were treated with LPS (50 ng/ml), then harvested at the indicated time points and subjected to mRNA and ChIP analyses as described above. DNA methylation analysis by MALDI-TOF Sodium bisulfite treatment. Genomic DNA was isolated with DNeasy extraction kit (Qiagen) according to the manufacturer instructions. Sodium bisulfite conversion was performed using EZ DNA Methylation Kit (Zymo Research, CA USA). One μg for each sample of genomic DNA, eluted in 30 μL of H 2 O, was used for bisulfite treatment. Amplicons used for methylation analysis were obtained from 50 ng of bisulfite treated genomic DNA. Statistical Analysis Statistical significance between groups was assessed by Student's t test. Data are expressed as means Å ± standard deviation (SD). All experiments were repeated at least three times. A p value < 0,01 and 0,05 was considered to be statistically significant. LPS induces COX-2 gene in HT-29 intestinal cells To investigate COX-2 promoter (Fig 1A) regulation, we first determined the changes in COX-2 mRNA levels induced by LPS in HT-29 human intestinal epithelial cells. HT-29 cells, primed with IFN-γ were treated with LPS, then COX-2 mRNA levels were measured by real-time PCR at different time points (Fig 1B). COX-2 mRNA levels markedly increased in response to LPS, peaking at 1 hour after stimulation with 50 ng/ml LPS ( Fig 1B) and then gradually decreased over time. We noticed two peaks of COX-2 mRNA levels at 1h and 6h of LPS treatment, suggesting that LPS induces cyclic transcriptional events to activate COX-2 expression in HT-29 cells. LPS-dependent histone modifications at COX-2 promoter Histone methylation and acetylation at the COX-2 promoter chromatin following LPS stimulation were analyzed by ChIP assays using anti-acetylated H3, dimethylated H3K4 (H3K4me2), dimethylated H3K9 (H3K9me2) and trimethylated H3K27 (H3K27me3) antibodies (Fig 2A). We found that the chromatin activation marks, H3K4me2 and H3 acetyl, increased significantly at COX-2 promoter chromatin 1 hour after LPS stimulation returning to basal levels at 12-24 hours time points (Fig 2A). Similarly to mRNA accumulation, there are two peaks, at 1 and 6 h after LPS exposure, of H3K4me2 levels (compare Figs 1B and 2A). Conversely, the repressive chromatin marks, H3K9me2 and H3K27me3 displayed an opposite pattern of cyclical changes upon LPS. Specifically, H3K27me3 levels were high in untreated HT-29 cells, decreased at 1 h, partially restored at 2 h and again decreased at 6 h time points after LPS treatment. At 12-24 hours H3K27me3 levels were similar in stimulated and unstimulated cells (Fig 2A). H3K9me2 levels displayed a single cycle, because they decreased early (1h LPS) and returned to the levels of unstimulated cells thereafter. These data suggest that H3K27 methylation changes may be relevant for LPS induction of COX-2 gene and that they may be dependent on changes in the levels or the recruitment of H3K27 methylase and demethylase enzymes (namely, EZH2 and JMJD3) upon LPS stimulation. To define more precisely this point, we analysed first the concentration of these two enzymes following LPS stimulation. Fig 2B shows that EZH2 expression was significantly and rapidly (1 hour) reduced by LPS, while JMJD3 levels were significantly induced by LPS and remained high up to 6 h of LPS stimulation. Second, we analyzed the recruitment of EZH2 and JMJD3 at the COX-2 promoter upon LPS stimulation. Fig 2C shows that JMJD3 recruitment at COX-2 promoter displayed the same cyclical changes of the activation marks (Fig 2A, left panels). As expected, the pattern observed in the recruitment of JMJD3 was reciprocal to that of its natural substrate, H3K27me3 (Fig 2C left panel and Fig 2A right panels). Collectively, LPS induced at COX-2 promoter a cyclical loss and gain of repressive and activation markers, respectively. These oscillatory cycles of epigenetic markers faithfully reflect COX-2 mRNA accumulation in response to LPS. JMJD3 depletion inhibits COX-2 induction by LPS To investigate the role of JMJD3 demethylase in LPS-induced COX-2 activation, we performed a JMJD3 knock-down experiment. HT-29 cells were transfected with JMJD3 siRNA and JMJD3 and COX-2 mRNA levels were measured by qRT-PCR. In LPS-untreated siRNA-transfected cells, JMJD3 expression decreased more than 70% and did not increase upon LPS treatment (Fig 3). COX-2 induction by LPS was completely inhibited in JMJD3-depleted cells Chromatin from controls and from HT-29 cells treated with LPS for 1, 2, 6, 12, 24 hours, was immunoprecipitated with the indicated antibodies. Each experiment was repeated at least three times, and the data are presented as percentages of input DNA (mean ± SD). , p < 0.01; ,p<0,05; (B) Total RNA was extracted at the indicated time points after treatment with LPS and subjected to qRT-PCR ( Fig 3A). ChIP analysis shows that reduction of cellular JMJD3 inhibits the recruitment of the enzyme and the demethylation of H3K27 (Fig 3). Collectively, these data demonstrate that JMJD3 and H3K27 demethylation is required for COX-2 induction by LPS. Dynamic changes of CpGs methylation at the COX-2 promoter Histone H3 methylation changes can be associated with DNA methylation patterns [26]. Specifically, we have previously described that rapid transient DNA methylation changes at the COX-2 promoter are associated with transcriptional activation and precede histone modifications in gastric cells exposed to Helicobacter pylori [18]. Along the same line, here, we investigated the DNA methylation dynamics at COX-2 promoter in LPS-treated HT-29 cells. Twenty-two CpG sites located in the COX-2 promoter region were analyzed by MALDI-TOF MS technique (Sequenom), on both lower and upper strands (Fig 4). DNA methylation was evaluated during the first 2 h of LPS exposure at intervals of 10 minutes in order to be able to detect possible transient changes. Fig 4 shows cycles of methylation/demethylation with a 40 min period upon LPS exposure. CpGs upstream or around the transcription start site (TSS) were methylated and demethylated in a strand specific fashion (-176 and +25 from TSS). Conversely, the CpGs in the body of the gene were methylated-demethylated symmetrically on both strands. It is worth noting that some CpGs were methylated-demethylated only during the first cycle (40 min), while others (+108/115, +124/127, +132, +139/145, +171, +198/208, +217, +225/231) displayed one or more methylation cycles with a 40 min interval between the peaks (Fig 4). Collectively, these results show very similar oscillatory patterns of DNA and histone methylation changes induced by LPS. The timing of these cycles suggest that DNA methylation changes precede or are synchronous with histone H3K27 demethylation events induced by LPS and carried out by JMJD3. It remains to be seen whether the two events are causally linked. Discussion In the present study, we describe the chromatin and DNA methylation changes occurring at the promoter of COX-2 gene in intestinal epithelial cells as direct consequence of LPS exposure. LPS-dependent COX-2 gene activation was marked by transient cyclic epigenetic events including increase of H3 acetylation and H3K4me2 activation marks, decrease of H3K9me2 and H3K27me3 repressive marks and early cyclic DNA methylation/demethylation events at specific CpG sites of COX-2 gene-regulatory region. Most importantly, we found that JMJD3 demethylase was enriched at COX-2 promoter in concomitance with depletion of H3K27me3 levels and displacement of EZH2, a H3K27 methylase. Finally, the essential role of JMJD3 was assessed by siRNA experiments. Cycles of histone H3K27 methylation induced by LPS at the COX-2 promoter Ours and others' recent studies demonstrate that bacteria, or their components such as LPS, are able to induce changes in histone modifications in exposed cells, thereby altering the host's analyses of histone demethylases JMJD3 and methyltransferases EZH2. The mRNA levels were normalized to G6PD levels and expressed relative to control cells (CTRL). (C) ChIP experiments were performed with anti-JMJD3 and anti-EZH2 antibodies. COX-2 promoter DNA sequences recovered after the indicated treatments were quantified by real-time PCR. Each experiment was repeated at least three times, and the data are presented as percentages of input DNA (mean ± SD). , p < 0.01; **,p<0,05. doi:10.1371/journal.pone.0156671.g002 Cyclical Chromatin Changes during LPS-Dependent COX-2 Activation transcriptional program and possibly affecting the host innate immune response [15][16][17][18][19][20]. However, the majority of the previous studies were carried out in immune cells (neutrophils, monocytes, macrophages and dendritic cells) [27] while it is not well known the response to LPS of epithelial cells, which represent the first barrier against microbes. Moreover, most of studies addressing LPS-induced epigenetic modifications at COX-2 gene were focused on histone phosphorylation and acetylation dynamics and few on histone and DNA methylation [15,16,28,29]. In an early study [30] it was reported that LPS induced IL-12 production by a rapid and specific nucleosome re-organization at IL-12 promoter region in murine macrophages. Transient changes in H3 acetylation and H3K4, H3K9 and H3K27 methylation in IL-8 gene promoter were induced by LPS and pretreatment of HT-29 colon cancer cells with deacetylase inhibitors amplified LPS-induction of IL-8 [17]. A more precise analysis overtime of histone H3K27 methylation at the COX-2 promoter reveals at least 2 cycles of methylation involving H3K27. JMJD3 demethylase seems an important mediator of LPS induced H3-K27 methylation cycles, because depletion of this enzyme severely impairs LPS induction of COX-2 and abolishes H3K27 methylation cycles (Fig 3). However, recruitment of the demethylase (JMJD3) and the loss of the methyltransferase (EZH2) at the COX-2 promoter is steady and progressive (Fig 2B), suggesting that not the concentration but the activation of JMJD3 is cyclical. We propose that methylation cycles of H3K4 and H3K27 follow reciprocal patterns (Fig 2A). Methylation of H3K4 transiently halts demethylation of H3K27 and this allows the ordered recruitment of transcription initiation factors. These events impact also on RNA accumulation, because we observed a cycle also at COX-2 RNA levels following LPS challenge. We wish to stress that we could detect these cycles (RNA included) because the cells have been synchronized in two steps and are responsive to LPS stimulus. With the time 6-12 hours after the initial LPS challenge, transcription stochastically de-synchronizes and the histone methylationdemethylation cycles are not detectable (Fig 2). Finally, the simultaneous presence of H3K9 and H3K27 methylation marks at COX-2 gene promoter region has been previously suggested to contribute to the maintenance of constitutive heterochromatin and more stable gene silencing [31]. However, our data show that LPS stimulation is able to induce rapid and simultaneous loss of both repressive marks at COX-2 promoter. Cycles of CpG methylation induced by LPS at the COX-2 gene The most striking finding presented here is the temporal association between histone H3K27 and H3K4 methylation cycles with methylation cycles of selected CpGs in COX-2 gene. There are similar examples of cyclical DNA methylation in genes induced by estrogens [32,33] or cyclical histone H3 K9 and K4 methylation in genes induced by retinoic acid [34]. This is the first example of temporal correlation between histone and DNA methylation. Although we have not clarified the mechanistic link between the two methylation events, we note some specific signatures of the CpGs undergoing to methylation cycles around the TSS of COX-2 gene upon LPS challenge. The CpGs in the promoter region (-176 and +25) were transiently methylated in the minus strand only, while the CpGs in the body of the gene (+108 to +225) underwent periodic methylation on both strands (Fig 4), similarly to CpGs in estrogen responsive genes [32,33]. Moreover, some CpGs displayed a single methylation cycle at 40 min of LPS exposure, while others were periodically methylated in two methylation-transcription cycles, or in three cycles (+135 and + 217), with a time interval period of 40 minutes between each cycle (Fig 4). We hypothesize that these epigenetic changes define a specific transcription territory in or around the gene and its regulatory regions. For example, these transient and differential methylation marks may differentially label the regulatory regions and the gene body. Overall, we believe that the analysis of the epigenetic changes induced by LPS to activate COX-2 provides a specific temporal window on the early phases of transcription. Moreover, because aberrant methylation of COX-2 is frequent in colon cancer [13], it will be very important to investigate whether transient cyclical methylation occurring during transcription cycles may become inaccurate and distorted thus potentially contributing to aberrant methylation changes at COX-2 gene promoter during neoplastic progression.	2018-04-03T03:04:18.292Z	2016-06-02T00:00:00.000	{ "year": 2016, "sha1": "f72ce38dbf2ed87434219294e750c9f2631155d9", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0156671&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "f72ce38dbf2ed87434219294e750c9f2631155d9", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
103480964	pes2o/s2orc	v3-fos-license	An efficient iodide ion chemosensor and a rewritable dual-channel security display material based on an ion responsive supramolecular gel By introducing multi-self-assembly driving forces, coordination binding sites and signal groups into the same molecule, a well designed functional gelator G1 was synthesized. The gelator G1 could form a stable Pb-coordinated supramolecular metallogel (PbG) accompanied with aggregation-induced fluorescence emission (AIE). PbG shows the reversible selective fluorescent response for I under a gel– gel state. The detection limit of PbG for I is 1.0 10 7 M. The AIE fluorescence of PbG could be reversibly switched “on–off–on” under gel–gel states via alternatively adding I and Pb water solution into PbG. Other anions could not induce similar stimuli-response for PbG. Interestingly, when a writing brush dipped in I water solution was used to write on the xerogel film of PbG, the film did not show any color changes. However under UV at 365 nm, a clear dark writing image appeared. This dark writing could be erased by brushing Pb on the film. More interestingly, when the PbG film containing the invisible I writing was exposed to iodine vapor, a clear brown writing appeared on the film. However, when this film was placed under the room atmosphere for one minute, the brown writing gradually disappeared. Therefore, the PbG film could act as not only a convenient reversible I detection test kit, but also an erasable dual-channel security display material. However, although a signicant amount of effort has been devoted to the development of metal ions coordinated supramolecular gels, 40 it is still a big challenge to design and synthesize supramolecular gels that can optically sense a given chemical stimulus with a specic selectivity. In view of this, as a part of our research interest in supramolecular chemistry, [41][42][43][44][45] we attempted to control the stimuli-response properties of supramolecular gels through the competitive coordination between supramolecular gelators, metal ions and guest compounds. Herein, we designed and synthesized a gelator G1 based on multi-assembly driving forces, 46-49 uorescent signal groups 50 and coordination binding sites. 51,52 The gelator G1 could form a stable supramolecular organogel in various solvents at very low critical gelation concentrations (CGCs). Aer the addition of Pb 2+ to the G1 ethanol organogel (OG), OG could form a stable Pb 2+ -coordinated supramolecular metallogel PbG accompanied by the pale blue aggregationinduced uorescence emission (AIE). [53][54][55][56] The AIE of PbG could be reversibly controlled by iodide anions with a specic selectivity in gel-gel states. PbG could act as not only a convenient reversible I À detection test kit, but also an erasable dual-channel security display material. It is worth mentioning that the security display materials have become of increasing importance. 57,58 Results and discussion First, we carefully investigated the gelation properties of G1. As shown in Table S1, † the gelator G1 could form a stable supramolecular organogel in various solvents at very low critical gelation concentrations. Among these solvents, the gelator G1 showed the lowest CGC (0.40%, wt/v%, 10 mg mL À1 ¼ 1%) and the highest gel-sol transition temperature (78 C) in ethanol. The G1-based supramolecular organogel OG in ethanol is more stable than the gel in other solutions. Therefore, we investigated the inuence of metal ions on the G1 organogel in ethanol. Interestingly, aer the addition of Pb 2+ to the G1 ethanol organogel (OG), OG could form a stable Pb 2+ -coordinated supramolecular metallogel PbG. As shown in Fig. 1, PbG has no uorescence emission in hot ethanol solution (T > T gel ). However, when the temperature of this hot ethanol solution dropped below the T gel of PbG, the emission intensity at 340 nm showed an evident increase and reached a steady state, which indicated that the uorescence emission of metallogel PbG was aggregation-induced emission (AIE). The anion response capability of the supramolecular metallogel PbG was primarily investigated by adding various anions in water solutions (AcO À , HSO 4 À , H 2 PO 4 À , F À , Cl À , Br À , I À , N 3 À , SCN À , ClO 4 À , CN À , CO 3 2À , S 2À and SO 4 2À , 1 mol L À1 ) to PbG. As shown in Fig. 2, when adding water solutions of various anions to the small amount of metallogel PbG on a spot plate, only I À could quench the uorescence of PbG, while other anions could not. These results indicated that PbG could selectively sense I À , which was attributed to I À competitively binding with Pb 2+ . Moreover, the I À response properties of PbG were investigated by uorescence titrations. As shown in Fig. 3, with the addition of I À into PbG, the spectra showed evident red shis, which was attributed to the coordination of I À with Pb 2+ . In PbG, Pb 2+ coordinated with the gelator through the acylhydrazone moiety. When I À was added into PbG, Pb 2+ coordinated with I À and the acylhydrazone moiety was released, which induced the uorescence spectra of the gel to undergo the red shis. In addition, the emission intensity at 474 nm decreased with increasing the concentration of I À . The detection limit of the uorescence spectra changes, which was calculated on the basis of 3d/S, 60 was 2.037 Â 10 À6 M (Fig. S4 †) for I À anion. Interestingly, aer the addition of Pb 2+ into the I À containing PbG, the uorescence of PbG recovered, which was attributed to the Pb 2+ coordination with G1 again. These properties make PbG act as an I À and Pb 2+ controlled "on-off-on" uorescent switch. By alternate addition of I À and Pb 2+ , the switching could be performed reversibly at least for three cycles with a small uorescent efficiency loss (Fig. 4). In order to facilitate the use of the metallogel PbG, the I À detection lm based on PbG was prepared by pouring the heated ethanol solution of PbG onto a clean glass surface and then drying in air. The PbG lm was white under natural light and showed a blue uorescence emission under UV at 365 nm. When a writing brush dipped in I À water solution was used to write on the lm, the lm did not show any color changes. However, under UV at 365 nm, a clear dark writing image appeared (Fig. 5). This dark writing image could be erased by brushing Pb 2+ on the lm. More interestingly, when the PbG lm containing the invisible I À writing was exposed to iodine vapor, a clear brown writing appeared on the lm. However, when the lm was put under the room atmosphere for one minute, the brown writing disappeared gradually. Therefore, the PbG lm could act as not only a convenient reversible I À detection test kit, but also an erasable dual-channel security display material. In order to investigate the self-assembly and stimuliresponse mechanism of PbG, a series of experiments was carried out. First, in the concentration dependent 1 H NMR of G1 (Fig. 6), the -NH-(H b ) and -N]CH-(H a ) resonance signals showed signicant downeld shis as the concentration of G1 rose. These results revealed that in the gelation process, the -NH-(H b ) and -N]CH-(H a ) groups formed hydrogen bonds with the -C]O groups on the adjacent gelators. On the other hand, with a gradual increase in concentration, the 1 H NMR signal of phenyl protons (H c , H d , H e , and H f ) showed an evident upeld shi, indicating that the p-p stacking interactions between the phenyl groups involved in the gelation process. 61 Therefore, the gelator G1 self-assembled to supramolecular organogel OG by the hydrogen bonds, p-p stacking as well as the vdW existing in the long alkyl chains. The formation mechanism of supramolecular metallogel was also investigated by 1 H NMR titrations, as shown in Fig. 7. With the addition of Pb 2+ , the -NH-(H b ) group on the gelator showed signicant downeld shis, which indicated that the gelator coordinated with Pb 2+ via the acylhydrazone moiety. In addition, in the IR spectra (Fig. S5 †) the stretching vibrations of -C]O and -C]Nof G1 showed obviously shis from 3455 and 1650 cm À1 to 3583 and 1588 cm À1 , respectively. These phenomena indicated that in PbG, Pb 2+ coordinated with the nitrogen and oxygen atoms on the acylhydrazone group. This presumed self-assembly and coordination mechanism was also supported by the T gel of OG and PbG. For instance, as shown in Fig. 8, under the same condition, the T gel of OG was signicantly higher than that of PbG. The large differences of Fig. 3 Fluorescence spectra of PbG (1%, in ethanol, G1-Pb 2+ ¼ 1 : 1) with increasing concentration of I À (using 1 mol L À1 TBA in water solution as the I À sources), l ex ¼ 340 nm. Fig. 4 Fluorescent "on-off-on" cycles of PbG, controlled by the alternative addition of I À and Pb 2+ , l ex ¼ 340 nm. Fig. 5 Writing, erasing and coloration of a natural light invisible image on a PbG supramolecular gel film (obtained from 1% ethanol metallogel, PbG, G1-Pb 2+ ¼ 1 : 1. Writing: write in I À water solution; erasing: brush Pb 2+ water solution; coloration: expose the PbG film into the iodine vapor ca. 5 s). The photographs were taken at room temperature under room light and exposed to a 365 nm UV light. T gel between OG and PbG were ascribed to the breakage of intermolecular hydrogen bonds between -N]C-H on one gelator and -C]O on the other one in OG, which was caused by the coordination of Pb 2+ with the gelator G1. Moreover, the Pb 2+ coordination process reduced the distance of p-p stacking between the phenyls, which enhanced the aggregation induced emission of PbG. This proposed mechanism was also supported by the XRD patterns (Fig. S6 †). The XRD patterns of OG, PbG and the PbG treated with I À showed the peaks at 2q ¼ 18.62-27.70 , corresponding to the d spacing 3.5Å, 3.45Å, and 3.8Å, respectively. As shown in Scheme 2 and Fig. S6, † in OG, the peaks at 21.28 and the d spacing 3.5Å were attributed to the p-p stacking, which existed in the phenyl groups of OG. While, in PbG, the d spacing changed to 3.45Å, which was attributed to the Pb 2+ coordination process reducing the distance of p-p stacking between the phenyls. Meanwhile, the d spacing 3.8Å was attributed to the interlamellar spacing between the supramolecular chains. In addition, aer the formation of PbG, the peaks of OG (at 2q ¼ 21.28, 23.22, 23.90, 25.30 ) disappeared and these peaks reappeared aer PbG was treated with I À . These phenomena conrmed that Pb 2+ coordinated with OG and induced a change in the XRD pattern of OG, while the addition of I À into PbG induced the competitive coordination of I À with Pb 2+ and led to the recovery of XRD peaks. According to 1 H NMR, IR and XRD, the ion response mechanism of OG and PbG could be presumed as in Scheme 2. To get further insight into the morphological features of the supramolecular organogel OG, metallogel PbG and PbG treated with I À , SEM studies were carried out with their xerogels, respectively. As shown in Fig. S7, † the SEM images of OG showed an overlapped rugate layer structure. The metallogel PbG also showed overlapped rugate layer structures and the aggregation structure of layer was compacted. However, aer I À was added into the PbG xerogel, the micro states experienced obvious changes. There were a large number of micro cavities formed in the xerogel of PbG. These micro cavities provided the PbG xerogel with the properties for the adsorption of iodine vapor. Therefore, the mechanism of iodine vapor-caused color change could be attributed to the iodine vapor adsorption into these micro cavities. Conclusions In summary, a novel supramolecular gelator G1 has been designed and synthesized. The gelator G1 could form a stable supramolecular metallogel PbG. Through the competitive coordination of Pb 2+ and I À with the gelator G1, the aggregation-induced emission of the supramolecular metallogel PbG was controlled as "on-off-on". More interestingly, aer Pb 2+ competitive coordinated with I À , there were a large number of micro cavities formed in the PbG xerogel, which enabled the PbG xerogel to absorb the iodine vapor and show a brown color. PbG could act as not only a convenient high selective and sensitive I À detection test kit, but also an erasable dual-channel secret documentation medium. Scheme 2 Chemical structure of the G1 and the presumed self assembly and reversible stimuli-response mechanism.	2019-04-09T13:04:01.521Z	2017-08-02T00:00:00.000	{ "year": 2017, "sha1": "3a350b0ef7a1eaa75015824090e4335f1d664e3a", "oa_license": "CCBY", "oa_url": "https://pubs.rsc.org/en/content/articlepdf/2017/ra/c7ra06238a", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "d1b1037c1979ed504a8c98f8e92d95a189990a7f", "s2fieldsofstudy": [ "Materials Science", "Chemistry" ], "extfieldsofstudy": [ "Materials Science" ] }
236275343	pes2o/s2orc	v3-fos-license	Survey of airborne organic compounds in residential communities near a natural gas compressor station: Response to community concern Introduction: Natural gas compressor stations are located throughout the country and are used to maintain gas flow and ensure continuous distribution through the pipeline network. Compressor stations emit many air contaminants including volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs). While the serious health effects associated with the inhalation of elevated pollutant levels are clear, the relationship between proximity to natural gas compressor stations and residential health effects is not well understood. Community members living near a natural gas compressor station in Eastern Ohio expressed concerns regarding their air quality; therefore, the objective of this study was to assess exposure to airborne organics in residential air near the compressor station. Methods: Our team conducted a 24-hour air sampling campaign to assess outdoor and indoor air contaminant levels at 4 homes near the Williams Salem Compressor Station in Jefferson County, Ohio. Air quality was assessed using two techniques: 1) summa canisters to quantify VOC concentrations and 2) passive air samplers to evaluate a broader panel of VOCs and SVOCs. Results: Among the three homes situated < 2 km from the compressor station, indoor benzene levels were 2–17 times greater than the Ohio Environmental Protection Agency (EPA) indoor standard due to vapor intrusion. Multiple other VOCs, including ethylbenzene, 1,2,4-trimethylbenzene, 1,2 dichloroethane, 1,3 butadiene, chloroform, and naphthalene also exceeded state standards for indoor concentrations. Several SVOCs were also detected inside and outside participants’ homes, including benzene and naphthalene derivatives. Conclusion: Our results validate the community members’ concerns and necessitate a more comprehensive epidemiological investigation into the exposures associated with natural gas compressor stations and methods to mitigate elevated exposures. Alarming levels of VOCS were detected inside of homes. Further research is needed to determine the source of VOC exposure and potential health effects. Introduction Natural gas production and use has increased in the United States over the past decade (Administration, U.S.E.I. 2020). Natural gas is extracted through conventional and unconventional methods such as hydraulic fracturing or "fracking". Once extracted, natural gas is transported from the production site to storage facilities and consumers across the nation by an elaborate system of more than 300,000 miles of pipeline and more than 1,400 compressor stations across the country (Administration, U.S.E.I. 2020). Compressor stations, also referred to as pumping stations, are located along the natural gas pipeline network to increase pressure to maintain gas flow and ensure continuous transportation of the natural gas through the pipeline. Depending on specific factors, such as the diameter of the pipeline and topography, compressor stations are typically located every 50 to 100 miles. Compressor stations have been identified as a major source of air pollutants (nitrogen dioxide, carbon monoxide, volatile organic compounds (VOCs), semi-volatile organic compounds (SVOCs), formaldehyde, and particulate matter) with levels often exceeding regulated standards (Macey et al., 2014). The health hazards of elevated VOC exposure are well documented (Lee et al., 2010;Pappas et al., 2000;Agency, U.S.E.P. 2000;Registry, A.f.T.S.a.D. 2014, Filley et al., 2004Loh et al., 2007) with adverse health effects including headaches, nausea (Lee et al., 2010), fatigue (Norbäck et al., 2017), skin and eye irritation (Agency, U.S.E.P. 2017), respiratory issues (Pappas et al., 2000), and central nervous system damage (Filley et al., 2004). However, there is limited information about exposure to air contaminants and resulting health effects in residents living near natural gas compressor stations. One recent ecological study evaluated county-level VOC emissions data across the US and identified that higher levels of total VOCs were reported around compressor stations (Hendryx and Luo, 2020). The emissions were also associated with greater age-adjusted mortality rates (Hendryx and Luo, 2020). A study in New York identified compressor stations as posing a significant public health risk due to toxic air emissions including VOCs (Russo and Carpenter, 2019). Even at low levels of exposure, benzene, a VOC and known carcinogen, has been repeatedly linked with increased risk of developing blood disorders including nonlymphocytic leukemia, chronic non-lymphocytic leukemia and chronic lymphocytic leukemia (Society, 2016). Other VOCs such as 1,3 butadiene and ethylbenzene are associated with adverse health outcomes including respiratory effects, eye irritation, dizziness and fatigue (Agency, U.S.E.P. 2000;Registry, A.f.T.S.a.D. 2014). In addition, exposure to SVOCs, such as polycyclic aromatic hydrocarbons (PAHs), can pose high risks to acute and chronic health effects including impaired lung function, eye irritation, nausea, DNA, kidney, lung and liver damages, and cancers (Council, 2014). Several PAHs have often been detected near fracking-related activities, including compressor stations (Council, 2014;Luek and Gonsior, 2017). For example, naphthalene is one of the probable carcinogenic PAHs emitted at compressor stations. Exposure to naphthalene can lead to adverse health effects on immune system, reproductive, brain and nervous systems (Council, 2014;Registry, A.f.T.S.a.D. 2008). Residents of Jefferson County in Eastern Ohio reached out to our research team with concerns about a nearby natural gas compressor station, the Williams Salem compressor station. These community members reported unsettling odors inside and outside of their homes and were concerned about the health impacts of the air quality. One resident remarked that on days in which the odor was very strong they would notice symptoms such as a burning in his/their nose and throat and debilitating headaches (Haberley, 2020). Another community member described eye irritation, breathing issues, nasal and throat dryness, fatigue, and excruciating headaches (Haberley, 2020, Haberley, 2020, Haberley, 2020. Our team responded to these concerns by developing a community-academic partnership with the objective of determining the concentration of VOCs and SVOCs on these properties. Study Design: In August 2020 our team conducted a 24-hour air sampling campaign to assess outdoor and indoor airborne organics at 4 homes near the Williams Salem Compressor Station in Jefferson County. Air quality was assessed using two techniques: 1) summa canisters to quantify VOC concentrations and 2) passive air samplers to evaluate a broader panel of VOCs and SVOCs. Both air sampling devices were hung on a shepherd's hook outside each home by our field team, away from known emission sources (i.e., vehicles). A second set of samplers was concurrently deployed inside each home on a table in the main room. During the sampling period residents were asked to continue their routine household activities. Samplers were deployed by a member of the field team between 7:00 AM and 10:00 AM on August 5, 2020. Samplers were collected in the same order as deployed on the morning of August 6, 2020 such that the total sampling time was 24 hours at each site. All samples were collected on the same day to ensure similar weather patterns across sites. Written consent was provided by residents prior to participation Approval for this study was received by the Institutional Review Board at the University of Kentucky. Summa Canisters: EPA recommended methods were used to measure VOCs, methane, and formaldehyde using summa canisters. An air flow controller was attached to each canister to monitor the sampling flow over the assessment period. Flow controllers were calibrated to a primary standard prior to each deployment. All canisters were batch certified by ALS Environmental before sample collection. Collected samples were immediately transported to ALS Environmental (Cincinnati, Ohio) for analysis of 62 VOCs, formaldehyde, and methane. VOCs were analyzed by EPA Method TO-15, methane was measured following NIOSH and OSHA methods using gas chromatography, and formaldehyde concentrations were assessed by OSHA Method 1007. The detection limits are detailed in the Supplemental Information Table 1. Twenty-four-hour average concentrations were calculated for all detected gases. Individual results from the summa canister sampling were shared with each participant followed by a phone call to discuss the results and any questions that arose. FreshAir Clips: FreshAir clips are passive samplers developed to assess exposure to VOCs and SVOCs. The design of these samplers has been previously described (Doherty et al., 2020). Briefly, airborne pollutants were captured using thin-film of polydimethylsiloxane (PDMS) which were custom fabricated as PDMS sorbent bars. PDMS absorbs compounds with a wide range of air-octanol portioning coefficients, including a broad array of pesticides, PAHs, VOCs, polybrominated diphenyl ethers (PBDEs), and phthalates (Lin et al., 2020;Koelmel et al., 2020). PDMS sorbent bars were housed in a PTFE chamber which was mounted in a magnetic clip. In this study, each PTFE chamber contained four pre-cleaned PDMS sorbent bars. PTFE chambers were wrapped with pre-baked aluminum foil and individually stored in air-tight 60 mL glass jars. These samplers were prepared by our team at Yale University and shipped overnight to the field team on the day prior to sample collection. The shipment included samplers for deployment with participants (n=8), field blanks (n=2), and transport blanks (n=6). Immediately prior to deployment at participants' home, a member of the field team mounted the PTFE chamber in a magnetic clip. At the end of the 24-h exposure assessment period, samplers were collected by a member of the field study team. The PTFE chamber was removed from each magnetic clip, wrapped with pre-baked aluminum foil, and individually stored in air-tight glass jar. All samplers were sent via cold-chain overnight shipment back to Yale for chemical analysis. Once received, PDMS sorbent bars were removed from each PTFE chamber and placed them back to air-tight glass storage vials using stainless steel forceps. Samples were stored at −20°C prior to analysis. PDMS sorbent bars samples were analyzed using a thermal desorption unit (TDU, Gerstel, Linthicum, MD, USA) coupled with a Q-Exactive Orbitrap mass spectrometer (Thermo-Fisher, Waltham, MA, USA). During analysis, thermally extracted analytes were first cryo-focused, then transferred to the GC column (TG-5SILMS, 20m x 0.25mm x 0.25 μm). Full scan electron ionization (EI) mass spectra (m/z 53.4 -800) was recorded at an acquisition rate of 4Hz, at 60,000 resolution. The detailed method has been previously described (Koelmel et al., 2020). For this study, QCs and blanks (laboratory, field, and transport) were analyzed every 5 samples. Raw mass spectral data were analyzed using TraceFinder 4.1 (Thermo). A seven-point calibration curve (0 to 1000 pg/μL) was developed for each compound, then applied for quantification for a total of 71 compounds. All coefficients of determination (R 2 ) were greater than 0.95. Peak integration was checked manually prior to data export. After chro-matographic peak-picking, blank feature filtering (BFF) was performed using field and transport blanks to remove compounds with high levels of background contaminations 35 . To compare indoor and outdoor airborne contaminant levels detected using the FreshAir clips, we conducted a fold change analysis using the mean concentration measured for each compound. Questionnaire: Each participant completed a brief questionnaire about their home characteristics during the sampling period, such as the use of tobacco inside the home, the operation of an air conditioner, propane heater and/or gas stove, and windows open or closed. Additionally, participants indicated if any products which emit odors/aerosols were used near the sampling devices such as air fresheners, candles, hair spray, perfume, paint, markers, disinfectants, adhesives, caulks, photographic solutions, kerosene, gasoline, fuel oil, farming equipment and/or lawn mowers. Results Three of the homes (Homes A-C) were within 2 km of the compressor station and the fourth home was approximately 16 km away (Home D) ( Table 1). All homes were located northeast of the Williams Salem Compressor Station. During the sampling period the temperature ranged from 62°F to 82°F, the average wind speed was approximately 6 miles per hour and the wind direction was northwest. This data was captured by the Weather Forecast Office (WFO) located in Wheeling, West Virginia, approximately 30 miles from the participants' homes. Homes A -C were built between the 1930s and the 1960s. Home D is newer and was built in the 2000s. Home B was the only site to report that the windows were open during the sampling timeframe. A household member at Home C smoked inside during the sampling period. Numerous VOCs (n 25) were detected in Homes A, C and D using the summa canisters (Table 2). Seven VOCs were detected at levels that exceeded the state EPA indoor air standard due to vapor intrusion (Ohio EPA, 2021). Indoor benzene levels at Homes A, C, and D were 2 to 17 times greater than the Ohio EPA indoor standard (3.1 μg/m 3 ) (Ohio EPA, 2021); a maximum concentration of 55.2 μg/m 3 was measured at Home A. At Home A, indoor levels of 1,2,4-trimethylbenzene were 13 times greater than the state standard and levels of ethylbenzene were 5 times higher than the standard. Additionally, levels of chloroform and naphthalene were elevated (2.64 μg/m 3 and 2.20 μg/m 3 , respectively) inside Home A. At Home C, the indoor levels of 1,3 butadiene and 1,2 dichloroethane both exceeded the indoor standards with concentrations of 7.50 μg/m 3 and 2.4 μg/m 3 , respectively. VOCs, as measured using the summa canisters, were not detected at any of the outdoor sampling locations. Seventeen airborne contaminants were detected using the FreshAir Clip (Fig. 1A) and included a nitroaromatic, isophorone, a haloether, seven PAHs, a phthalate, a chlorinated hydrocarbon, three organo-chlorine pesticides, a pyrethroid, and an organophosphate ester flame retardant. Like the summa canisters, indoor exposure levels collected with the FreshAir Clip were increased compared to outdoor samples. However, unlike the summa canisters with which VOCs were below above detection limits outdoors (Table S1), quantifiable chemical exposures were measured outdoors using the FreshAir clips. Using these passive air samplers, similar profiles of airborne contaminants were measured inside and outside each home, including PAHs and nitro-aromatics (Fig. 1B). Indoor exposures were dominated by nitrobenzene, 2-methylnaphthalene, bis(2-chloro-1-methylethyl) ether, 2,4-dinitrotoluene, diethyl phthalate, and triphenyl phosphate (Fig. 1C). Four compounds were found to have significantly elevated levels outdoors (all PAHs) compared to indoor samples across all homes (Fig. 1C). It is important to note that during this exposure assessment we asked residents to continue their typical routines. At Home B, windows were kept open for the 24-hour sampling period. Despite being less than 2 km from the compressor station, indoor levels of all VOCs evaluated using summa canisters were below detection limits (Table 2). Indoor concentrations of airborne contaminants measured with the FreshAir Clips at Home B were also lower than other homes tested (Fig. 1B). Discussion In our study, we detected 25 VOCs and 17 SVOCs inside the homes located near the Williams Salem Compressor Station in Jefferson County, Ohio. Of the VOCs detected, indoor air levels of benzene, 1,2,4-trimethylbenzene, 1,2-dichloroethane, 1,3-butadiene, chloroform, ethylbenzene, and naphthalene were particularly concerning, exceeding the state indoor standard. Detected SVOCs included benzene and naphthalene derivatives. Exposure to elevated concentrations of VOCs is associated with the symptoms reported by the community members, such as headaches, fatigue, respiratory issues, and nasal irritation (Pappas et al., 2000;Norbäck et al., 2017;Agency, U.S.E.P., 2017). Long-term exposure to the measured SVOCs is unclear. Furthermore, previous research has described associations among indoor exposure to VOCs at concentrations, comparable to those detected in our study, and health outcomes (Norback et al., 2017;Wickliffe et al., 2021). According to the Agency of Toxic Substance and Disease Registry (ATSDR), benzene, toluene, ethylbenzene, and xylene (BTEX) are frequently emitted from natural gas compressor stations (Services, U.S.D.o.H.a.H., 2016). We observed levels of 1,2,4trimethylbenzene and benzene that were more than ten times greater than the standard. Our results align with a previous exploratory study in New York which similarly described high levels of benzene, an established carcinogen (Society, 2016), near fracking activity and compressor stations (Macey et al., 2014). Low levels of ambient benzene exposure have been associated with hematologic malignancies such as T-cell lymphoma, follicular lymphoma, and myeloid dysplastic syndromes (Teras et al., 2019). Exposure to ambient benzene at levels similar to what was observed in our study have been associated with congenital disorders such as spina bifida in the offspring of exposed mothers (Lupo et al., 2010). There is no known threshold for benzene related health effects (Organization, W.H., 2010); therefore, reducing benzene levels to as low as possible is optimal for human health. (Russo and Carpenter, 2019). Acute inhalation exposure to 1,3-butadiene can lead to symptoms such as nasal and eye irritation, fatigue, headaches, and vertigo (Organization, W.H., 2012). Chronic exposure to the carcinogen has been linked with cardiovascular effects (Penn and Snyder, 2007), leukemia (Macaluso et al., 2021), and several other cancers (Organization, W.H. 2012). According to the US EPA, naphthalene has been classified as a possible human carcinogen. Health effects associated with exposure include cataracts, retinal damage, headache, nausea, gastrointestinal issues, and fatigue (US EPA 2020). Using county-level data, a recent study described associations between mortality rates and VOC emissions from compressor stations (Hendryx and Luo, 2020). While it is known that compressor stations emit VOCs and the literature is clear on the serious dangers associated with the inhalation of elevated VOC levels, the emission profiles of SVOCs and the human health impacts associated with these exposures from compressor stations is limited. In addition to environmental measures, further examination of the individual health impacts of exposure to these emissions would be insightful. While outdoor contaminant levels were found using the passive air sampling technique, detected outdoor levels were lower compared to the concentrations measured indoors, even in the house where windows were kept open during assessment period. Following a similar trend, the summa canisters did not detect VOCs outside; VOC concentrations were only detected when sampling indoors. While more comprehensive sampling is necessary to determine the pathway of exposure, we hypothesize that the elevated indoor contaminant levels may be due to vapor intrusion, because measurements from assessed homes constantly showed elevated indoor levels of commonly found petroleum-related vapor intrusion contaminants (i.e., benzene) regardless of the age of buildings, and personal activities conducted during sampling. Furthermore, elevated levels indoors may be attributable to the poor ventilation. According to American Society of Heating, Refrigerating and Air-Conditioning Engineers Standard 62.2 (The American Society of Heating, R.a.A.-C.E., 2020), residential buildings typically receive 0.35 air changes per hour, which may result in accumulation of outdoor pollutants in the indoor space (Agency, U.S.E.P., 2020). To further explore this hypothesis, indoor air sampling should be conducted at varying levels throughout the home and ground water, soil, and/or sub-slab vapor data should be collected. Toluene/benzene (T/B) ratios have bene established to represent vehicle emissions. The indoor air T/B ratio at home D, located the furthest from the compressor station, was approximately 3:1 which may indicate vehicular emission as the source of exposure (Miller et al., 2011). More data is needed to investigate this pathway of exposure. As people spend most of their time inside (Owen et al., 2010), indoor air quality is a critical aspect of public health. The undetectable or near undetectable indoor levels of VOCs and SVOCs in Home B may be due to the open windows during the sampling period. This finding demonstrates the potential effectiveness of enhanced ventilation in reducing exposure (The American Society of Heating, R.a. A.-C.E., 2020, Agency, U.S.E.P., 2020, Godish and Spengler, 1996). Opening windows is feasible in mild to warm weather; however, extreme temperatures require an alternate solution to improve indoor air quality. Despite compliance with EPA regulations (Haberley, 2020), our study detected elevated levels of airborne organics homes near the Williams Salem Compressor Station. Annual emission inventories assume a constant release of emission; however, monitoring studies have demonstrated that emissions are episodic (Brown et al., 2015), released from scheduled or accidental "blowdowns". These events occur when there is a buildup of pressure within the compressor and the gas is vented or flared directly into the air to depressurize and prevent a potential explosion. Since air emissions from compressor stations release transient high concentration plumes that are not captured by annual average concentration, future studies should conduct campaigns that allow for more comprehensive characterization of emissions. Due to the rapid rise of natural gas and fracking technology coupled with the lack of transparency involved in the processes, many questions surround the potential public health impacts (Adgate et al., 2014, Fisher et al., 2018. Previous research demonstrates that residents living near fracking operations are more likely to experience stress and depression (Casey et al., 2018). Specifically, in Jefferson County, community members have also expressed concern surrounding their quality of life (Haberley, 2020). Future research should explore the impact of compressor stations on a variety of health outcomes including psychosocial factors. In this study, the community members reported detecting odors and experiencing health effects since the addition of the nearby Williams Salem compressor station. We did not collect source data close to the compressor station since our exploratory study was designed to address the residents' concerns by measuring VOC/SVOC concentrations at their homes. While many of the chemicals that we detected indoors naturally occur in petroleum and are associated with natural gas production, there are additional sources of indoor VOC/ SVOC exposure such as paints, cleaners, furniture polish, detergents, pesticides, solvents, and tobacco smoke (Agency, U.S.E.P., 2017). To account for these potential exposures, we administered a questionnaire to each resident inquiring about the use of VOC/SVOC emitting products during the sampling timeframe. Future research would benefit from having a trained environmental technician inspect each home for additional sources of VOC/ SVOC emissions. While this study did not capture source data and therefore cannot link the high levels of indoor VOC/SVOCs to the compressor station, the results validate the community members' concerns and call for a more extensive epidemiological investigation. Additionally, while our study was prompted by antidotal reports of health effects, we did not capture health symptoms in this study. Our findings highlight the need for future epidemiologic research to thoroughly investigate these claims. Many previous studies have utilized aggregate exposure data (Russo and Carpenter, 2019, Macey et al., 2014, Hendryx and Luo, 2020, Koehler et al., 2018, limiting the ability to characterize specific individuallevel exposures stemming from natural gas compressor stations. Future research should aim to fill this research gap by directly quantifying contaminant exposures at individual households. In this study, our comprehensive sampling methods, the summa canister and the passive Fresh Air sampler, enabled us to detect a broader range of airborne contaminants to characterize the range of emissions from compressor stations. infrastructure will continue to increase the number of homes located near compressor stations. Therefore, understanding the public health impacts associated with compressor stations needs to be a research priority. Conclusions An exploratory 24-hour air sampling campaign identified concerning levels of VOCs and SVOCs inside the homes of residents living near a natural gas compressor station. The results validate the community members' concerns and necessitate a more comprehensive epidemiological investigation into the exposures associated with natural gas compressor stations. Supplementary Material Refer to Web version on PubMed Central for supplementary material.	2021-07-26T00:05:40.602Z	2021-06-13T00:00:00.000	{ "year": 2021, "sha1": "f050ecb140b196b5b05f15bcfdf7a84d4939c416", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.envadv.2021.100076", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "194fb44950adb603c1fdac0f1e0db5219e94064e", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Medicine", "Environmental Science" ] }
1326058	pes2o/s2orc	v3-fos-license	Growing Finely-Discriminating Taxonomies from Seeds of Varying Quality and Size Concept taxonomies offer a powerful means for organizing knowledge, but this organization must allow for many overlapping and fine-grained perspectives if a general-purpose taxonomy is to reflect concepts as they are actually employed and reasoned about in everyday usage. We present here a means of bootstrapping finely-discriminating tax-onomies from a variety of different starting points, or seeds, that are acquired from three different sources: WordNet, ConceptNet and the web at large. Introduction Taxonomies provide a natural and intuitive means of organizing information, from the biological taxonomies of the Linnaean system to the layout of supermarkets and bookstores to the organizational structure of companies. Taxonomies also provide the structural backbone for ontologies in computer science, from common-sense ontologies like Cyc (Lenat and Guha, 1990) and SUMO (Niles and Pease, 2001) to lexical ontologies like WordNet (Miller et al., 1990). Each of these uses is based on the same root-branch-leaf metaphor: the broadest terms with the widest scope occupy the highest positions of a taxonomy, near the root, while specific terms with the most local concerns are located lower in the hierarchy, nearest the leaves. The more interior nodes that a taxonomy possesses, the finer the conceptual distinctions and the more gradated the similarity judgments it can make (e.g., Budanitsky and Hirst, 2006). General-purpose computational taxonomies are called upon to perform both coarse-grained and fine-grained judgments. In NLP, for instance, the semantics of "eat" requires just enough knowledge to discriminate foods like tofu and cheese from non-foods like wool and steel, while specific applications in the domain of cooking and recipes (e.g., Hammond's (1986) CHEF) require enough discrimination to know that tofu can be replaced with clotted cheese in many recipes because each is a soft, white and bland food. So while much depends on the domain of usage, it remains an open question as to how many nodes a good taxonomy should possess. Princeton WordNet, for instance, strives for as many nodes as there are word senses in English, yet it also contains a substantial number of composite nodes that are lexicalized not as single words, but as complex phrases. Print dictionaries intended for human consumption aim for some economy of structure, and typically do not include the meaning of phrases that can be understood as straightforward compositions of the meaning of their parts (Hanks, 2004). But WordNet also serves another purpose, as a lexical knowledgebase for computers, not humans, a context in which concerns about space seem quaint. When space is not a issue, there seems no good reason to exclude nodes from a concept taxonomy merely for being composites of other ideas; the real test of entry is whether a given node adds value to a taxonomy, by increasing its level of internal organization through the systematic dissection of overly broad categories into finer, more intuitive and manageable clusters. In this paper we describe a means by which finely-discriminating taxonomies can be grown from a variety of different knowledge seeds. These taxonomies comprise composite categories that can be lexicalized as phrases of the form "ADJ NOUN", such as Sharp-Instrument, which represents the set of all instruments that are typically considered sharp, such as knives, scissors, chisels and can-openers. While WordNet already contains an equivalent category, named Edge-Tool, which it defines with the gloss "any cutting tool with a sharp cutting edge", it provides no structural basis for inferring that any member of this category can be considered sharp. For the most part, if two ideas (word senses) belong to the same semantic category X in WordNet, the most we can infer is that both possess the trivial property X-ness. Our goal here is to construct taxonomies whose form makes explicit the actual properties that accrue from membership in a category. Past work on related approaches to taxonomy creation are discussed in section 2, while section 3 describes the different knowledge seeds that serve as the starting point for our bootstrapping process. In section 4 we describe the bootstrapping process in more detail; such processes are prone to noise, so we also discuss how the acquired categorizations are validated and filtered after each bootstrapping cycle. An evaluation of the key ideas is then presented in section 5, to determine which seed yields the highest quality taxonomy once bootstrapping is completed. The paper then concludes with some final remarks in section 6. Related Work Simple pattern-matching techniques can be surprisingly effective for the extraction of lexico-semantic relations from text when those relations are expressed using relatively stable and unambiguous syntagmatic patterns (Ahlswede and Evens, 1988). For instance, the work of Hearst (1992) typifies this surgical approach to relation extraction, in which a system fishes in a large text for particular word sequences that strongly suggest a semantic relationship such as hypernymy or, in the case of Charniak and Berland (1999), the part-whole relation. Such efforts offer high precision but can exhibit low recall on moderate-sized corpora, and extract just a tiny (but very useful) subset of the semantic content of a text. The KnowItAll system of Etzioni et al. (2004) employs the same generic patterns as Hearst (e.g., "NPs such as NP1, NP2, …"), and more besides, to extract a whole range of facts that can be exploited for web-based question-answering. Cimiano and Wenderoth (2007) also use a range of Hearst-like patterns to find text sequences in web-text that are indicative of the lexico-semantic properties of words; in particular, these authors use phrases like "to * a new NOUN" and "the purpose of NOUN is to " to identify the formal (isa), agentive (made by) and telic (used for) roles of nouns. Snow, Jurafsky and Ng (2004) use supervised learning techniques to acquire those syntagmatic patterns that prove most useful for extracting hypernym relations from text. They train their system using pairs of WordNet terms that exemplify the hypernym relation; these are used to identify specific sentences in corpora that are most likely to express the relation in lexical terms. A binary classifier is then trained on lexico-syntactic features that are extracted from a dependency-structure parse of these sentences. Kashyap et al., (2005) experiment with a bootstrapping approach to growing concept taxonomies in the medical domain. A gold standard taxonomy provides terms that are used to retrieve documents which are then hierarchically clustered; cohesiveness measures are used to yield a taxonomy of terms that can then further drive the retrieval and clustering cycle. Kozareva et al. (2008) use a bootstrapping approach that extends the fixed-pattern approach of Hearst (1992) in two intriguing ways. First, they use a doubly-anchored retrieval pattern of the form "NOUNcat such as NOUNexample and " to ground the retrieval relative to a known example of hypernymy, so that any values extracted for the wildcard * are likely to be coordinate terms of NOUNexample and even more likely to be good examples of NOUNcat. Secondly, they construct a graph of terms that co-occur within this pattern to determine which terms are supported by others, and by how much. These authors also use two kinds of bootstrapping: the first variation, dubbed reckless, uses the candidates extracted from the double-anchored pattern (via ) as exemplars (NOUNexample) for successive retrieval cycles; the second variation first checks whether a candidate is sufficiently supported to be used as an exemplar in future retrieval cycles. The approach we describe here is most similar to that of Kozareva et al. (2008). We too use a double-anchored pattern, but place the anchors in different places to obtain the query patterns "ADJcat NOUNcat such as " and "ADJcat * such as NOUNexample". As a result, we obtain a finelydiscriminating taxonomy based on categories that are explicitly annotated with the properties (ADJcat) that they bequeath to their members. These categories have an obvious descriptive and organizational utility, but of a kind that one is unlikely to find in conventional resources like WordNet and Wikipedia. Kozareva et al. (2008) test their approach on relatively simple and objective categories like states, countries (both closed sets), singers and fish (both open, the former more so than the latter), but not on complex categories in which members are tied both to a general category, like food, and to a stereotypical property, like sweet (Veale and Hao, 2007). By validating membership in these complex categories using WordNet-based heuristics, we can hang these categories and members on specific WordNet senses, and thus enrich WordNet with this additional taxonomic structure. Seeds for Taxonomic Growth A fine-grained taxonomy can be viewed as a set of triples Tijk = <Ci, Dj, Pk>, where Ci denotes a child of the parent term Pk that possesses the discriminating property Dj; in effect, each such triple expresses that Ci is a specialization of the complex taxonym Dj-Pk. Thus, the belief that cola is a carbonated-drink is expressed by the triple <cola, carbonated, drink>. From this triple we can identify other categorizations of cola (such as treat and refreshment) via the web query "carbonated * such as cola", or we can identify other similarly fizzy drinks via the query "carbonated drinks such as ". So this web-based bootstrapping of fine-grained category hierarchies requires that we already possess a collection of fine-grained distinctions of a relatively high-quality. We now consider three different starting points for this bootstrapping process, as extracted from three different resources: Word-Net, ConceptNet and the web at large. WordNet The noun-sense taxonomy of WordNet makes a number of fine-grained distinctions that prove useful in clustering entities into smaller and more natural groupings. For instance, WordNet differentiates {feline, felid} into the sub-categories {true_cat, cat} and {big_cat, cat}, the former serving to group domesticated cats with other cats of a similar size, the latter serving to cluster cats that are larger, wilder and more exotic. However, such fine-grained distinctions are the exception rather than the norm in WordNet, and not one of the 60+ words of the form Xess in WordNet that denote a person (such as huntress, waitress, Jewess, etc.) express the defining property female in explicit taxonomic terms. Nonetheless, the free-text glosses associated with WordNet sense-entries often do state the kind of distinctions we would wish to find expressed as explicit taxonyms. A shallow parse of these glosses thus yields a sizable number of fine-grained distinctions, such as <lioness, female, lion>, <espresso, strong, coffee> and both <messiah, awaited, king> and <messiah, expected, deliverer>. ConceptNet Despite its taxonomic organization, WordNet owes much to the centralized and authority-preserving craft of traditional lexicography. Con-ceptNet (Liu and Singh, 2004), in contrast, is a far less authoritative knowledge-source, one that owes more to the workings of the WWW than to conventional print dictionaries. Comprising factoids culled from the template-structured contributions of thousands of web users, ConceptNet expresses many relationships that accurately reflect a public, common-sense view on a given topic (from vampires to dentists) and many more that are simply bizarre or ill-formed. Looking to the relation that interests us here, the IsA relation, ConceptNet tells us that an espresso is a strong coffee (correctly, like WordNet) but that a bagel is a Jewish word (confusing use with mention). Likewise, we find that expressionism is an artistic style (correct, though WordNet deems it an artistic movement) but that an explosion is a suicide attack (confusing formal and telic roles). Since we cannot trust the content of ConceptNet directly, lest we bootstrap from a highly unreliable starting point, we use WordNet as a simple filter. While the concise form of ConceptNet contains over 30,000 IsA propositions, we consider as our seed collection only those that define a noun concept (such as "espresso") in terms of a binary compound (e.g., "strong coffee") where the head of the latter (e.g., "coffee") denotes a WordNet hypernym of some sense of the former. This yields triples such as <Wyoming, great, state>, <wreck, serious, accident> and <wolf, wild, animal>. Veale and Hao (2007) also use the observations of web-users to acquire common perceptions of oft-mentioned ideas, but do so by harvesting simile expressions of the form "as ADJ as a NOUN" directly from the web. Their approach hinges on the fact that similes exploit stereotypes to draw out the salient properties of a target, thereby allowing rich descriptions of those stereotypes to be easily acquired, e.g., that snowflakes are pure and unique, acrobats are agile and nimble, knifes are sharp and dangerous, viruses are malicious and infectious, and so on. However, because they find that almost 15% of their web-harvested sim-iles are ironic (e.g., "as subtle as a rock", "as bulletproof as a sponge-cake", etc.), they filter irony from these associations by hand, to yield a sizable database of stereotypical attributions that describes over 6000 noun concepts in terms of over 2000 adjectival properties. However, because Veale and Hao's data directly maps stereotypical properties to simile vehicles, it does not provide a parent category for these vehicles. Thus, the seed triples derived from this data are only partially instantiated; for instance, we obtain <surgeon, skilful, ?>, <virus, malicious, ?> and <dog, loyal, ?>. This does not prove to be a serious impediment, however, as the missing field of each triple is quickly identified during the first cycle of bootstrapping. Overview of Seed Resources Neither of these three seeds is an entirely useful knowledge-base in its own right. The WordNetbased seed is clearly a representation of convenience, since it contains only those properties that can be acquired from the glosses that happen to be amenable to a simple shallow-parse. The ConceptNet seed is likewise a small collection of low-hanging fruit, made smaller still by the use of WordNet as a coarse but very necessary noisefilter. And while the simile-derived distinctions obtained from Veale and Hao paint a richly detailed picture of the most frequent objects of comparison, this seed offers no coverage for the majority of concepts that are insufficiently noteworthy to be found in web similes. A quantitative comparison of all three seeds is provided in Table 1 We can see that WordNet-derived seed is clearly the largest and apparently the most comprehensive knowledge-source of the three: it contains the most terms (concepts), the most features (discriminating properties of those concepts), and the most triples (which situate those concepts in parent categories that are further specialized by these discriminating features). But size is only weakly suggestive of quality, and as we shall see in the next section, even such dramatic differences in scale can disappear after several cycles of bootstrapping. In section 5 we will then consider which of these seeds yields the highest quality taxonomies after bootstrapping has been applied. Bootstrapping from Seeds The seeds of the previous section each represent a different starting collection of triples. It is the goal of the bootstrapping process to grow these collections of triples, to capture more of the terms -and more of the distinctions -that a taxonomy is expected to know about. The expansion set of a triple Tijk = <Ci, Dj, Pk> is the set of triples that can be acquired from the web using the following query expansions ( is a search wildcard): 1. "Dj * such as Ci" 2. "Dj Pk such as " In the first query, a noun is sought to yield another categorization of Ci, while in the second, other members of the fine-grained category Dj-Pk are sought to accompany Ci. In parsing the text snippets returned by these queries, we also exploit text sequences that match the following patterns: 3. " and Dj Pk such as " 4. " and Dj * such as Ci" These last two patterns allow us to learn new discriminating features by noting how these discriminators are combined to reinforce each other in some ad-hoc category formulations. For instance, the phrase "cold and refreshing beverages such as lemonade" allows us to acquire the triples <lemonade, cold, beverage> and <lemonade, refreshing, beverage>. This pattern is necessary if the bootstrapping process is to expand beyond the limited vocabulary of discriminating features (Dj) found in the original seed collections of triples. We denote the mapping from a triple T to the set of additional triples that can be acquired from the web using the above queries/patterns as expand(T'). We currently implement this function using the Google search API. Our experiences with each query suggest that 200 snippets is a good search range for the first query, while 50 is usually more than adequate for the second. We can now denote the knowledge that is acquired when starting from a given seed collection S after t cycles of bootstrapping as Kt S . Thus, Web queries, and the small snippets of text that they return, offer just a keyhole view of language as it is used in real documents. Unsurprisingly, the new triples acquired from the web via expand(T') are likely to be very noisy indeed. Following Kozareva et al. (2008), we can either indulge in reckless bootstrapping, which ignores the question of noise until all bootstrapping is finished, or we can apply a noise filter after each incremental step. The latter approach has the additional advantage of keeping the search-space as small as possible, which is a major consideration when bootstrapping from sizable seeds. We use a simple WordNet-based filter called near-miss: a new triple <Ci, Dj, Pk> is accepted if WordNet contains a sense of Ci that is a descendant of some sense of Pk (a hit), or a sense of Ci that is a descendant of the direct hypernym of some sense of Pk (a near-miss). This allows the bootstrapping process to acquire structures that are not simply a decorated version of the basic WordNet taxonomy, but to acquire hierarchical relations whose undifferentiated forms are not in WordNet (yet are largely compatible with WordNet). This nonreckless bootstrapping process can be expressed as follows: T ∈ filter near−miss  expand T ' } Figure 1 and figure 2 below illustrate the rate of growth of triple-sets from each of our three seeds. Referring again to table 1, we note that while the ConceptNet collection is by far the smallest of the three seeds -more that 7 times smaller than the simile-derived seed, and almost 40 times smaller than the WordNet seed -this difference is size shrinks considerably over the course of five bootstrapping cycles. The WordNet nearmiss filter ensures that the large body of triples grown from each seed are broadly sound, and that we are not simply generating comparable quantities of nonsense in each case. An Example Consider cola, for which the simile seed has one triple: <cola, refreshing, beverage>. After a single cycle of bootstrapping, we find that cola can now be described as an effervescent beverage, a sweet beverage, a nonalcoholic beverage and more. After a second cycle, we find it described as a sugary food, a fizzy drink and a dark mixer. After a third cycle, it is found to be a sensitive beverage, an everyday beverage and a common drink. After a fourth cycle, it is also found to be an irritating food and an unhealthy drink. After the fifth cycle, it is found to be a stimulating drink, a toxic food and a corrosive substance. In all, the single cola triple in the simile seed yields 14 triples after 1 cycle, 43 triples after 2 cycles, 72 after 3 cycles, 93 after 4 cycles, and 102 after 5 cycles. During these bootstrapping cycles, the description refreshing beverage additionally becomes associated with the terms champagne, lemonade and beer. Empirical Evaluation The WordNet near-miss filter thus ensures that the parent field (Pk) of every triple contains a value that is sensible for the given child concept (Ci), but does not ensure that the discriminating property (Dj) in each triple is equally sensible and apropos. To see whether the bootstrapping process is simply padding the seed taxonomy with large quantities of noise, or whether the acquired Dj values do indeed mark out the implicit essence of the Ci terms they describe, we need an evaluation framework that can quantify the ontological usefulness of these Dj values. For this, we use the experimental setup of Almuhareb and Poesio (2005), who use information extraction from the web to acquire attribute values for different terms/concepts, and who then compare the taxonomy that can be induced by clustering these values with the taxonomic backbone of Word-Net. Almuhareb and Poesio first created a balanced set of 402 nouns from 21 different semantic classes in WordNet. They then acquired attested attribute values for these nouns (such as hot for coffee, red for car, etc.) using the query "(a\|an\| the) * Ci (is\|was)" to find corresponding Dj values for each Ci. Unlike our work, these authors did not seek to acquire hypernyms for each Ci during this search, and did not try to link the acquired attribute values to a particular branching point (Pk) in the taxonomy (they did, however, seek matching attributes for these values, such as Temperature for hot, but that aspect is not relevant here). They acquired 94,989 attribute values in all for the 402 test nouns. These values were then used as features of the corresponding nouns in a clustering experiment, using the CLUTO system of Karypis (2002). By using attribute values as a basis for partitioning the set of 402 nouns into 21 different categories, Almuhareb and Poesio attempted to reconstruct the original 21 WordNet categories from which the nouns were drawn. The more accurate the match to the original WordNet clustering, the more these attribute values can be seen (and used) as a representation of conceptual structure. In their first attempt, they achieved just a 56.7% clustering accuracy against the original human-assigned categories of WordNet. But after using a noise-filter to remove almost half of the web-harvested attribute values, they achieve a higher cluster accuracy of 62.7%. More specifically, Poesio and Almuhareb achieve a cluster purity of 0.627 and a cluster entropy of 0.338 using 51,345 features to describe and cluster the 402 nouns. 1 We replicate the above experiments using the same 402 nouns, and assess the clustering accur acy (again using WordNet as a goldstandard) after each bootstrapping cycle. Recall that we use only the D j fields of each triple as features for the clustering process, so the comparison with the WordNet goldstandard is still a fair one. Once again, the goal is to determine how much like the humancrafted WordNet taxonomy is the tax onomy that is clustered automatically from the discriminating words D j only. The clustering ac curacy for all three seeds are shown in Tables 2, 3 and 4. Both the WordNet and ConceptNet seeds achieve comparable accuracies of 68% and 67% respectively after 5 cycles of bootstrapping, which compares well with the accuracy of 62.7% achieved by Poesio and Almuhareb. However, the simile seed clearly yields the best accuracy of 84.3%, which also exceeds the accuracy of 66.4% achieved by Poesio and Almuhareb when using both values and attributes (such as Temperature, Color, etc.) for clustering, or the accuracy of 70.9% they achieve when using attributes alone. Furthermore, bootstrapping from the simile seed yields higher cluster accuracy on the 402noun data-set than Veale and Hao (2008) themselves achieve with their simile data on the same test-set (69.85%). But most striking of all is the concision of the representations that are acquired using bootstrapping. The simile seed yields a high cluster accuracy using a pool of just 2,614 fine discriminators, while Poesio and Almuhareb use 51,345 features even after their feature-set has been filtered for noise. Though starting from different initial scales, each seed converges toward a feature-set that is roughly twenty times smaller than that used by Poesio and Almuhareb. Conclusions These experiments reveal that seed knowledge of different authoritativeness, quality and size will tend to converge toward roughly the same number of finely discriminating properties and toward much the same coverage after 5 or so cycles of bootstrapping. Nonetheless, quality wins out, and the simile-derived seed knowledge shows itself to be a clearly superior basis for reasoning about the structure and organization of conceptual categories. Bootstrapping from the simile seed yields a slightly smaller set of discriminating features than bootstrapping from the WordNet seed, one that is many times smaller than the Poesio and Almuhareb feature set. What matters is that they are the right features to discriminate with. There appears to be a number of reasons for this significant difference in quality. For one, Veale and Hao (2007) show that similes express highly stereotypical beliefs that strongly influence the affective disposition of a term/concept; negatively perceived concepts are commonly used to exemplify negative properties in similes, while positively perceived concepts are widely used to exemplify positive properties. Veale and Hao (2008) go on to argue that similes offer a very concise snapshot of those widely-held beliefs that are the cornerstone of everyday reason- Bootstrapping Cycle Purity ing, and which should thus be the corner-stone of any general-purpose taxonomy. In addition, beliefs expressed via the "as D j as C i " form of similes appear to lend themselves to re-expression via the "D j P k such as C i " form; in each case, a concept C i is held up as an exemplar of a salient property D j . Since the "such as" bootstrapping pattern seeks out expressions of prototypicality on the web, a simile-derived seed set is likely the best starting point for this search. All three seeds appear to suffer the same coverage limitations, topping out at about 82% of the words in the Poesio and Almuhareb data-set. Indeed, after 5 bootstrapping cycles, all three seeds give rise to taxonomies that overlap on 328 words from the 402-noun test-set, accounting for 81.59% of the test-set. In effect then, bootstrapping stumbles over the same core of hard words in each case, no matter the seed that is used. As such, the problem of coverage lies not in the seed collection, but in the queries used to perform the bootstrapping. The same coverage limitations will thus apply to other bootstrapping approaches to knowledge acquisition, such as Kozareva et al. (2008), which rely on much the same stock patterns. So while bootstrapping may not be a general solution for acquiring all aspects of a general-purpose taxonomy, it is clearly useful in acquiring large swathes of such a taxonomy if given a sufficiently high-quality seed to start from.	2014-07-01T00:00:00.000Z	2009-03-30T00:00:00.000	{ "year": 2009, "sha1": "6bb1b0deed2bbf0b30ee437caab52edea2b3916d", "oa_license": null, "oa_url": "https://dl.acm.org/doi/pdf/10.5555/1609067.1609160", "oa_status": "BRONZE", "pdf_src": "ACL", "pdf_hash": "a8276b86612df84a650371dfb80798b995643744", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Computer Science" ] }
239621898	pes2o/s2orc	v3-fos-license	Biomechanical Effects of Polyetheretherketone and Titanium Rods in Transforaminal Lumbar Interbody Fusion: A Finite Element Analysis Background: Currently, the comprehensive biomechanical evaluation of polyetheretherketone (PEEK) rods in transforaminal lumbar interbody fusion (TLIF) is limited. The purpose of this study was to compare the biomechanical differences between titanium alloy (Ti) rods and PEEK rods in TLIF. Methods: L3-5 lumbar models were developed using the �nite element method. Four surgical models of TLIF were constructed by simulating different fusion methods and rods: cage fusion with Ti rods, cage fusion with PEEK rods, bone graft alone with Ti rods, and bone graft alone with PEEK rods. The range of motion (ROM) and stress distribution of the surgical and adjacent segments were then compared. Results: Compared to the Ti rods, the PEEK rods increased the ROM by 0.7–20% at the L4/5 segment and decreased the ROM by 0.8–15.1% at the L3/4 segment. The disc stresses at the L3/4 level were similar among the surgical models (0.79–1.80 MPa). The peak stresses of the screws, rods, and bone-screw interfaces in the PEEK rod models were 0–1.2 times, 1.6–4.4 times, and 0–1.4 times lower than those of the Ti rod models, respectively. PEEK rods increased the average strain of the bone graft by 0.5–61.6% and the stresses of the cage by 0.9–44.1% and endplates by 2.1–52.9%. Conclusion: In TLIF, PEEK rods played a positive role in restoring the ROM. They also increased the strain of the bone graft, stresses of the endplates and cages, and the risk of rod fracture and reduced the stress of the screw-rod system. Bone grafts alone combined with PEEK rods had acceptable biomechanical behavior in TLIF. Introduction Transforaminal lumbar interbody fusion (TLIF) is performed in clinics for the treatment of lumbar degenerative diseases and is favored by spine surgeons [1][2][3].In contrast to posterior lumbar interbody fusion, the unilateral transforaminal approach is used in TLIF to decrease the risk of nerve root and dural injury and to prevent the destruction of the normal structure of the spine [4][5][6].Studies on clinical results, radiography, and biomechanics have shown that patients who undergo TLIF have relatively good clinical outcomes [2,[7][8][9].However, spinal fusion alone cannot provide su cient strength and stability for the surgical segment, which may lead to spinal instability or even surgical failure [6,10].Therefore, a supplemental posterior pedicle screw xation system was introduced to improve the biomechanical stability of the spine and surgical success rates [11,12]. Currently, titanium alloy (Ti) is the preferred material for pedicle-based screw xation systems because of its excellent strength, fatigue resistance, and biocompatibility [13,14].The superior construct stiffness of the Ti xation system ensures spine stability and promotes solid fusion [11,15,16].However, as a stress shielding effect and abnormal motion are caused by excessive rigidity of the xation system, it is di cult to avoid adjacent segment disease (ASD) and instrumentation failure [17][18][19].Some studies have reported that the reoperation rate due to ASD was at least 20% within 10 years [20].In TLIF with traditional rigid xation, related ASD and screw loosening events have also been reported [21,22]. Therefore, researchers look forward to nding new substitute materials to address this issue. Novel polyetheretherketone (PEEK) rods, whose elastic modulus is close to that of bones, are used in clinical practice as an alternative to Ti rods in an effort to reduce the incidence of adverse events [13,15,23].The biocompatibility, toxicity, and imaging characteristics of PEEK rods are better than those of Ti rods, which are the basis of their being potential clinical substitutes [24,25].Importantly, studies have revealed that PEEK rods can not only provide su cient strength and stability but also reduce stress shielding, which may contribute to overcoming ASD and instrumentation failure [19,26,27].However, most of these studies did not consider interbody fusion when constructing models of semi-rigid xation with PEEK rods [18,[28][29][30].Overall, there is a shortage of a comprehensive biomechanics evaluation of PEEK rods in TLIF. The purpose of this study was to compare the biomechanical differences between Ti rods and PEEK rods in TLIF, which may provide some theoretical evidence for their use in clinical treatment. Study design and setting This was a nite element (FE) analysis study, which aimed to compare the biomechanical differences between Ti rods and PEEK rods in TLIF, performed in a laboratory setting. Intact FE model Computed tomography (CT) data of the L3-5 vertebrae were obtained from a healthy male (29 years old, 176 cm height, 60 kg weight, with no history of spine-related diseases, and trauma).This study was approved by the Ethics Committee of our hospital, and informed consent was obtained from the volunteer.The procedure for lumbar model reconstruction was similar to that used in previous studies [31].Thin-layer (0.625 mm) CT data were saved in DICOM format and imported into Mimics (Materialise Inc., Leuven, Belgium) to generate a surface model.The solid model was constructed using 3-Matic software (Materialise Inc.) and meshed using HyperMesh (Altair Engineering, Inc., Troy, Michigan, USA).The de nition of material properties, model assembly, and analysis were performed using Abaqus (Hibbitt, Karlsson, and Sorensen, Inc., Providence, Rhode Island, USA). Consistent with a previous study on the lumbar FE model, the 1 mm thick cortical layers and the 0.5 mm thick endplates covered the surface of the vertebral body (Fig. 1a-c) [31].The nucleus pulposus (NP) was simulated as an incompressible uid element, which accounted for 40% of the intervertebral disc volume (Fig. 1d) [32].The annulus brosus (AF) was constructed using a heterogeneous ber-reinforced composite consisting of annulus bers and a ground substance (Fig. 1e-f) [31,33].The ligaments were modeled as tension truss elements, including the anterior longitudinal, posterior longitudinal, avum, supraspinous, interspinous, intertransverse, and capsular ligaments (Table 1).The facet articular cartilage with a thickness of 0.2 mm was tied to the bone, and the friction coe cient between articular surfaces was 0.1 [8].A convergence analysis was performed by ensuring that the strain energy error was less than 5% [27]. Surgical FE models In this study, the intact model was modi ed and four surgical models were constructed: TLIF with a cage and two Ti rods (Cage + Ti rods), TLIF with a cage and two PEEK rods (Cage + PEEK rods); TLIF with autogenous bone graft alone and two Ti rods (bone graft alone + Ti rods); and TLIF with autogenous bone graft and two PEEK rods (bone graft alone + PEEK rods). To simulate the process of decompression and fusion, left L4-5 facetectomy was performed, and then the entire NP, left posterior part of the AF, and capsular and avum ligaments were removed in all the surgical models (Fig. 2a-b) [34].A banana -shaped PEEK cage was placed on the anterior part of the L4-L5 intervertebral space (height, 9.5 mm; length, 32 mm; width, 10 mm; and surface area, 1.86 cm 2 ; BKMeditech USA, Las Vegas, NV, USA) (Fig. 2c) [35].Cancellous bone was implanted in the inner and outer spaces of the cage to ll the intervertebral space.To remove the overlap between the cage and the endplates, a "Boolean calculation" was performed.Cage-bone graft interfaces were assigned a "tie" constraint, and cage-endplate interfaces were assigned a friction coe cient of 0.2 [34].In the bone graft alone models, cancellous bone was used to ll in the L4/5 space.It was assumed that the surface of the bone graft was rigidly connected to the vertebral surface to simulate complete fusion. In terms of posterior xation, the pedicle screw-based xation system consisted of four screws (diameter, 6.5 mm; length, 45 mm) and two connecting rods.The screw was made of Ti-6Al-4V and inserted into the vertebra.The connecting rods were categorized as Ti-6Al-4V rods and PEEK rods.The rods were cylindrical with a diameter of 5.5 mm and length of 58 mm (Table 2).The bone-implant and screw-rod interfaces were de ned in the "tie" contact condition. Loading conditions This study focused on the ranges of motion (ROMs) in all models under the loading conditions of exion, extension, lateral bending, and rotation, as well as the maximum von Mises stress of the adjacent disc, endplates, cages, screw-rod system, and bone graft in the surgical model.The ROM was calculated based on a previous study [34].By xing the inferior surface of the L5 vertebrae to limit its degree of freedom, 400 N of vertical preload and 8 Nm of bending moments were applied on the L3 superior surface simultaneously to simulate physiological load and motion in coronal, sagittal, and axial planes, respectively.Additionally, the ROMs of the L3/4 and L4/5 segments were compared with those of previous studies to validate the intact model [34,36,37]. Model validation In this study, an intact spinal model of L3-5 was constructed and validated.Under extension, exion, lateral bending, and axial rotation, the ROM was 2.84°, 3.96°, 2.36°, and 1.90° in the L4/5 segment, and 3.26°, 3.69°, 2.55°, and 2.45° in the L3/4 segment, respectively (Fig. 3).Under similar load conditions, the agreement of the ROMs between those of the current study and previous studies, including those tested on cadavers and FE studies, were acceptable, indicating that the current model could be used for further studies. PEEK rods versus Ti rods After validating the effectiveness of the intact model, four surgical models were constructed.Substantially, compared with the intact model, the ROMs of the L4/5 segment for all surgical models decreased (0.58°-1.62°), while the ROMs of the L3/4 segment increased (3.23°-6.20°)(Fig. 4a-b). Whether in cage fusion or bone graft alone fusion, the ROMs of the L4/5 segment in the PEEK rods xation models were greater than those in the Ti rods xation models (0.83°-1.62° vs. 0.58°-1.57°).Correspondingly, the ROMs at the L3/4 segment were lower in the PEEK rod xation models than in the Ti rod xation models (3.23°-6.17°vs. 3.60°-6.20°). The peak stresses of the cage in the PEEK rods xation models were 7.2-30.4MPa, which were greater than those of the Ti rods xation models (6.6-25.3MPa) under all loading conditions (Fig. 5a).The peak stresses of the disc at the L3/4 segment for all surgical models increased compared to those of the intact model (0.79-1.80 MPa vs. 0.64-1.46MPa), while the disc stresses of the PEEK rod xation models were similar to those of the Ti rod xation models (0.79-1.79 MPa vs. 0.80-1.80MPa) (Fig. 5b).Furthermore, the peak stresses of the endplates were 8.83-61.44MPa in the models with PEEK rods, and were 7.96-50.82MPa in the models with Ti rods (Fig. 5c).Compared to Ti rod xation models, PEEK rod xation models could reduce the peak stresses of screws (37.59-65.72MPa vs. 39.08-122.96MPa) (Fig. 5d).The largest stresses experienced by the PEEK rods were 7.36-37.40MPa, which were much lower than those experienced by the Ti rods (20.84-124.06MPa) (Fig. 5e).Furthermore, the peak stresses at the bone-screw interfaces in the PEEK rod groups were lower than those in the Ti rod groups (21.10-44.80MPa vs. 26.47-105.48MPa) (Fig. 5f). It was found that the average strain of bone graft in the PEEK rod xation models was larger than that in the Ti rod xation models (570.67-9570.85µE vs. 481.42-7433.68µE) (Fig. 6a-c).In addition, the average strain of the outer bone graft around the cage was much greater than that of the inner bone graft in the cage (1544.30-7519.96µE vs. 481.42-1298.92µE). Cage fusion versus bone graft alone fusion In the cage fusion models, the ROMs of the L4/5 and L3/4 segments were 0.58°-1.49°and 3.51°-6.20°,respectively.In the bone graft alone fusion models, the ROMs were 0.74°-1.62°and 3.23°-6.09°at the L4/5 and L3/4 segments, respectively.The peak stresses of the endplates were much lower in the bone graft alone models than in the cage fusion models (7.96-18.69MPa vs. 18.34-61.44MPa).The adjacent disc stresses were similar between bone graft alone models and cage fusion models (0.79-1.79 MPa vs. 0.79-1.80MPa).Compared to bone graft alone fusion models, cage fusion models could reduce the peak stresses of screws (37. Discussion As a novel "compliant" material, PEEK rods are available as substitutes for traditional Ti rods [15].Although some studies have demonstrated that PEEK rods have excellent performance, there is still a lack of su cient biomechanical evidence to support the use of PEEK rods in TLIF.In our study, we found that PEEK rods in TLIF played a positive role in restoring normal motion and reducing stress shielding. However, the PEEK rods also simultaneously increased the risk of rod fracture, endplates collapse, and cage failure. The L4/5 segment was chosen for fusion and xation because it has the highest clinical incidence (58.3%), which is determined by its anatomical structure and physiological function [38].It is generally accepted that the aim of a lumbar surgery, including TLIF, is to provide stability after decompression, essentially sacri cing part of the ROMs for symptom improvement.In our study, the ROMs of the L4/5 segment for all surgical models were decreased by 41.1-69.5% compared to that of the intact model.Compared to the Ti rod rigid xation models, the PEEK rod semi-rigid xation models increased the ROM at the L4/5 segment by 0.7-20%.It was suggested that PEEK rods could not only stabilize the movement of the spine to a great extent but also allow for more ROMs than that of Ti rods.In cadaveric biomechanical testing, Gornet et al. also showed that there was no signi cant difference in stability between PEEK rods and Ti rods [14].Yeager et al. concluded that the stability of PEEKs rod was similar to that of Ti rods in vitro [26].Moreover, we found that the change in the ROMs caused by PEEK rods during lateral bending and rotation was greater than that created during exion and extension.We inferred that the axial stiffness provided by PEEK rods was similar to that of Ti rods, but the bending stiffness of PEEK rod models was lower than that of Ti rod models.It is known that axial stiffness affects exion and extension, whereas lateral bending stiffness affects rotation and lateral bending [26].Therefore, axial stiffness is mainly provided by the anterior column, while the posterior internal xation is important to bending stiffness [23,26]. ASD is a common long-term complication of lumbar fusion.Abnormal motion (quality and quantity) and intervertebral disc pressure (IDP) of adjacent segments are closely related to ASD [39].In this study, we found that, compared to the intact model, the ROMs and intradiscal peak stresses of the L3/4 segment of all the surgical models increased by 31.8-68.0%and 18.5-39.0%,respectively.Cunningham et al. found that spinal instrumentation increased proximal IDP by as much as 45% in in vitro biomechanical testing [40].Jin et al. showed that although both PEEK rods and Ti rods increased the inter-segmental rotation and IDP in the upper adjacent segments, the PEEK rods generated fewer changes than those of titanium rods [41].Similarly, Nikkhoo et al. demonstrated that there was no signi cant difference in the ROM of adjacent segments between the PEEK rod models and the intact models, and PEEK rods reduced the disc height loss, uid loss, and disc stresses of adjacent segments under cyclic loading compared with those of Ti rods [30].Additionally, during exion, the increase in disc stresses at L3/4 was highest (29.7-39.0%),and this was similar to that reported by Hsieh et al. (50%) [23].In fact, the structural stiffness provided by both rigid and semi-rigid xation systems is signi cantly greater than that of the normal spinal unit, which further changes the ROM and IDP of adjacent segments [13,41].The ROM increases at the adjacent level may be derived from the non-physiological center of motion, which is a compensatory change to xed segments [29,42].However, this adverse compensation makes the spine's originally complex but regular coupling movement to become irregular, resulting in facet hypertrophy, an IDP change, and altered biomechanics [42].Remarkably, PEEK rods reduce the ASD incidence owing to their low structural stiffness and physiological load sharing [14,17].Furthermore, the biomechanical effect of the rigidity of the rod on the adjacent segments seems to be more important than the amount of fusion mass [41].Athanasakopoulos et al. reported a retrospective clinical study comprising 52 patients with posterior lumbar internal xation using PEEK rods, and no ASD was observed after a mean follow-up period of 3 years [43].However, although the disc stresses of the PEEK rod models at the adjacent level were better than those of the Ti rod models, the difference was not signi cant in our study.Generally, there is still a lack of high-quality clinical studies to verify the effect of PEEK rods on ASD. The concept of exible xation is re ected not only in the partial restrictions on the ROM but also in reasonable load sharing [18].Some researchers believe that low back pain is caused by abnormal load transfer rather than an abnormal ROM [17,29].As expected, the PEEK rods showed superior performance in terms of stress distribution.In our study, the peak stresses of screws, rods, and bone-screw interfaces in PEEK rod models were 0-1.2 times, 1.6-4.4times, and 0-1.4 times lower than those of Ti rod models, respectively.Fan et al. showed that PEEK rods decreased the stress of pedicle screws by 12.0-36.7%and rods by 2.5-5.6 times compared to cases where Ti rods were used; however, the ratio of peak stress to yield stress of PEEK rods (10.2-15.7%)was greater than that of Ti rods (5.1-11.1%)[27].The ratio in our study was 7.4% for PEEK rods and 2.8-16.5% for Ti rods, which also suggested that the fracture risk of PEEK rods was high.Theoretically, PEEK rods with low elastic modulus can reduce the structural stiffness and transfer loads to the anterior column.This could unload the stress of screws and bone-screw interfaces and is especially important for patients with osteoporosis [44].Ahn et al. showed that the PEEK rod system transmitted 27.5% of the axial compressive load, while this ratio was 66.7% in the Ti rod system [18].Gornet et al. proved that the PEEK rod loads were at least 6% less than the titanium rod loads under all loading conditions [14]. In addition, we found that PEEK rods increased the average strain of the bone graft by 0.5-61.6%and stresses of the cage by 0.9-44.1% and those of endplates by 2.1-52.9%.As noted earlier, this was because the PEEK rods transferred load to the anterior column.Furthermore, we found that the strain of the bone graft in the periphery of the cage was greater than that in the interior of the cage, suggesting that fusion may begin from the periphery.However, the bone graft of the anterior column experienced stress, which stimulated its growth and fusion according to Wolff's law.Wang et al. found that the PEEK rod group had better fusion than the Ti rod group after posterior bone graft fusion and internal xation in canines [45].However, PEEK rods simultaneously increased the risk of cage failure and endplate collapse.It is well known that cages are important than posterior xation in maintaining spinal stability because the anterior column bears approximately 75% of the load [23]. The application of the cage is to overcome issues related to intervertebral space reduction caused by autogenous bone due to its weak strength [46].However, given the high elastic modulus of the cage and the small contact area, which should be > 30%, events of cage subsidence and endplate collapse are still worthy of careful evaluation [47].In our study, we found that compared to cage fusion, bone graft alone fusion increased the ROMs of the xed segment, reduced the peak stresses of the endplates, and increased the average bone graft strain.However, bone grafting alone also increased the peak stresses of screws, rods, and bone-screw interfaces.In a clinical study involving 23 patients who underwent TLIF with autologous bone graft fusion alone, Sleem et al. found that autologous bone grafts alone could also achieve satisfactory clinical outcomes [48].Interestingly, it was noticed that bone graft alone combined with PEEK rods had superior biomechanical characteristics, especially in terms of improving the bone graft strain, endplate stress, and rod stress.However, whether the model could provide su cient stability and strength remains to be further studied because there is still no recognized ROM and load sharing. This study has some limitations.First, muscles were not considered in the model.Undeniably, the role of muscles is complex and important.Second, our data were obtained from a healthy, young man.The human lumbar spine of each individual is unique and is also affected by the age, presence of disease, and other factors.Third, we only analyzed the biomechanical behavior of PEEK rods after rigid fusion without considering fusion failure.Fourth, we simpli ed the loading pattern, material properties, and interaction of the implants and the spine, but the actual situation was severely complicated. Conclusion In TLIF, PEEK rods could not only signi cantly restrain the ROM of surgical segments, but also allow more exibility compared to those of Ti rods.Simultaneously, PEEK rods decreased the stresses of the screws and bone-screw interfaces and increased the strain of the bone graft and stresses on the endplates and cages.Although the PEEK rods reduced the stresses of the rods, the risk of rod fracture increased.The effect of PEEK rods on adjacent discs was similar to that of Ti rods.Moreover, bone graft alone fusion was superior to cage fusion in providing the ROM, increasing bone graft strain, and reducing endplate stresses, but it also increased the stresses of the posterior xation system.Theoretically, bone graft alone combined with PEEK rods have acceptable biomechanical features in TLIF.Further studies are required to con rm the nding in the future. 1 Abbreviations nite element Figure 2 The Figure 2 Figure 3 Comparison Figure 3 Table 1 De nition of materials properties in the nite element models. Table 2 De nition of materials properties and dimensions of the implants.	2021-09-27T19:28:14.445Z	2021-08-12T00:00:00.000	{ "year": 2021, "sha1": "82886454de3a72fe6c7d8d416abee9668c5052ea", "oa_license": "CCBY", "oa_url": "https://doi.org/10.21203/rs.3.rs-795573/v1", "oa_status": "GREEN", "pdf_src": "ScienceParsePlus", "pdf_hash": "cdc093a38740ca2c57cf532950cb2d5b1ae96361", "s2fieldsofstudy": [ "Engineering", "Medicine" ], "extfieldsofstudy": [ "Materials Science" ] }
33183054	pes2o/s2orc	v3-fos-license	Factors Associated with the Risk of Articular Surface Perforation during Anchor Placement for Arthroscopic Acetabular Labral Repair Background The purpose of this study was to evaluate factors associated with the risk of articular surface perforation during anchor placement for arthroscopic acetabular labral repair using follow-up computed tomographic arthrography (CTA). Methods Forty-six patients (29 males and 17 females) underwent arthroscopic labral repair using 142 suture anchors (55 large anchors and 87 small anchors). The patients were followed with CTA 1 year postoperatively. Anchor position was assessed by the insertion angle and the distance of the suture anchor tip from the articular cartilage. The incidence of malposition of suture anchors was assessed in follow-up CTA. The location and incidence of malposition were compared between two groups divided according to the diameter of suture anchor. Results The mean insertion angle and distance were significantly different between the groups. Of the 142 anchors, 15 (11%) were placed in the cartilage-bone transitional zone. Articular involvement was most common at the 3 o'clock position of the suture anchor (six out of 33 anchors, 18.2%). Both the insertion angle and distance showed small values in the articular involvement group. Conclusions The radiographic analysis of the placement of suture anchors after arthroscopic labral refixation based on follow-up CTA demonstrates that articular involvement of anchors is related to the location on the acetabular rim (clock position) and anchor diameter. Clinics in Orthopedic Surgery • Vol. 9, No. 4, 2017 • www.ecios.org Several anatomical studies have attempted to identify the safe angle for anchor placement in different regions of the acetabulum or to modify the portals to create a better angle for anchor placement. [11][12][13][14] The purpose of this study was to evaluate factors associated with the risk of articular surface perforation during anchor placement for arthroscopic acetabular labral repair using follow-up computed tomographic arthrography (CTA). METHODS The design and protocol of this retrospective study were approved by the Institutional Review Board of Chung-Ang University Hospital (IRB No. C2016133-1876) and performed in accordance with the principles of the Declaration of Helsinki. Informed consent was waived. We performed arthroscopic labral repair on 200 patients between January 2011 and December 2014. Of those, 46 patients (29 males and 17 females) who underwent 1-year follow-up CTA were enrolled in our study. Arthroscopic labral repair was performed using 142 anchor sutures in the 46 patients. The patients were divided into two (a large anchor group and a small anchor group) according to the diameter of the suture anchor used. Fifty-five anchors (larger than 2.3 mm) were used in the large anchor group and 87 anchors (smaller than 1.4 mm) were used in the small anchor group. After conducting a radiological analysis of postoperative CTA results, the patients were also subdivided into an articular involvement group and a non-involvement group. Surgical Technique One senior surgeon (YCH) repaired 46 acetabular labral tears in 46 hips with femoroplasty in 21 hips and acetabuloplasty in 18 hips. Hip arthroscopy was performed with the patient positioned in the supine position on a standard fracture table. Traction was applied with slight extension and adduction of the hip joint using sufficient force to open the joint approximately 1 cm. Two or three portals (anterolateral, anterior, and/or posterolateral) were placed for treatment of femoroacetabular impingement including arthroscopic labral repair. After assessing the amount of bone overhang, the pincer lesion was resected, or rim trimming was accomplished using a 5.5mm round burr. The amount of bone resected depended on the extent of overhang. After resecting the pincer lesion, the single loop technique was used in a small labrum (less than 6 mm in height). In a large labrum (more than 6 mm in height), the labral base refixation technique was used. Arthro-Pierce (Smith & Nephew, Andover, MA, USA) was used to pierce the labrum, and a stiff, nonabsorbable suture was passed through the base of the labrum. PEEK standard anchors ( (Fig. 1). These were secured with a straight drill guide. Then, using a suture passer, a suture limb was passed through a small portion of the labral substance. An arthroscopic sliding knot was made and passed down through the cannula to secure the repair. Two to four anchors were placed at the superior (12 o' clock), anterosuperior (1-2 o' clock), and anterior (3 o' clock) positions. Anchors fixed in other locations were excluded from this study. Next, traction was released and the hip was flexed 45°, allowing passage of the camera along the femoral head-neck junction into the peripheral space. The femoral head-neck junction and lateral epiphyseal vessels were visualized during this maneuver. Instruments were placed into the peripheral compartment through the anterolateral or distal lateral accessory portal. After confirming a cam lesion, which was defined by local abnormalities and dynamic examination, bumpectomy was performed to restore the femoral head-neck offset by transforming the convex lesion into a concave surface. 15) The operation was completed with joint lavage and injection of a local anesthetic. Radiological Evaluation Standardized CTA was performed preoperatively in all patients, and follow-up CTA was recommended 1 year after surgery. All measurements were performed with a picture archiving and communication system (M view; Infinitt, Seoul, Korea). Coronal, axial, and sagittal reconstruction was created ( Fig. 2A). Two musculoskeletal radiologists independently measured the radiographic parameters. The interobserver reliability showed a high level of agreement (0.87) on separate measurements of the lateral center edge angle (LCEA) and the calculated correlation. LCEA values were measured on coronal images (Fig. 2B). Using sagittal images, we classified anchor locations as superior (12 o' clock), anterosuperior (1-2 o' clock), or anterior (3 o' clock). The location of each anchor was determined on the coronal and axial computed tomography (CT) scout images (Fig. 2C). The insertion angle was measured by extending A B C Clinics in Orthopedic Surgery • Vol. 9, No. 4, 2017 • www.ecios.org a line 15 mm from the insertion point to the acetabular cartilage as described by Lertwanich et al. 12) and drawing another line from the insertion point through the anchor (Fig. 3A). The minimum distance between the anchor/ drill hole and the articular surface of the acetabulum was then measured (Fig. 3B). The angle of anchor insertion was measured at the superior and anterosuperior positions on the coronal images, and the distance between the anchor/drill hole and the articular surface of the acetabulum for the superior and anterosuperior anchors was also measured. Anchors in the anterior position had the same measurements taken on the axial images. The articular surface was considered to be involved in cases in which the suture anchor was located in the cartilagebone transitional zone on sagittal images (white arrow in Fig. 3C), and such cases were assigned to the articular involvement group (Fig. 3C). Statistical Analysis Both musculoskeletal radiologists evaluated the CTA findings and consensus was achieved for any discrepancies. We assessed inter-and intraobserver reliability of the measured parameters, including the LCEA, insertion angle, and distance using Kappa coefficients. Reliability was classified as follows: 0, poor; 0-0.20, slight; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, substantial; and greater than 0.80, almost perfect agreement. 16) The Student t-test was used to detect any differences in the insertion angle and distance between the articular involvement group and the non-involvement group. The chi-square test was used to compare categorical variables such as gender and suture anchor type. IBM SPSS ver. 22.0 (IBM Corp., Armonk, NY, USA) was used for all analyses and a significant difference was defined as p < 0.05. All data is presented as the mean and standard deviation. RESULTS The study subjects included 29 males and 17 females with a mean age of 32.7 ± 9.5 years and a mean body mass index of 23.0 ± 4.2 kg/m 2 . The mean LCEA angle was 29.6° ± 5.3°. Of a total of 142 anchors, 55 were PEEK standard anchors and 87 were all-suture anchors. Twenty-eight anchors were located at the 12 o' clock position, 81 at the 1-2 o' clock positions, and 33 at the 3 o' clock position. Fifteen anchors (10.6%) were placed in the cartilage-bone transitional zone (Table 1). On the radiological analysis of the 142 anchors, the mean LCEA angles of the PEEK standard anchor group (large anchor group) and the all-suture anchor group (small anchor group) were 29.1° ± 6.8° and 29.9° ± 4.0°, respectively (p = 0.372). The mean insertion angles of the PEEK standard anchor group and all-suture anchor group at the 12 o' clock position were 20.7° ± 2.8° and 25.0° ± 5.6°, respectively (p = 0.022). The mean angle of insertion at the 3 o' clock position was 17.6° ± 4.6° for the PEEK standard anchor group and 26.2° ± 7.4° for the all-suture anchor group (p = 0.002). There was no significant difference in the insertion angle at the 1-2 o' clock positions between the two groups (p = 0.757) ( Table 2). The mean distance to the articular cartilage at the 12 o' clock position was 5.8 ±1.1 mm for the PEEK standard anchor group and Values are presented as number (%), mean ± standard deviation (range), or mean ± standard deviation. The mean distance to articular cartilage at the 1-2 o' clock position was 5.9 ± 1.8 mm for the PEEK standard anchor group and 6.6 ± 1.9 mm for the all-suture anchor group (p = 0.038). However, the distance between the two groups at the 3 o' clock position was not statistically significantly different (p = 0.158) ( Table 3). On the subgroup analysis of the articular involvement group and non-involvement group, there was no notable difference in age (p = 0.809), sex (p = 0.158), body mass index (p = 0.708), and LCEA (p = 0.638). However, the anchor location (p = 0.023), anchor type (p = 0.025), insertion angle (p = 0.001), and distance (p < 0.001) were significantly different between the two groups. Articular involvement was noted in six out of 33 anchors (18.2%) located at the 3 o' clock position and in nine out of 81 anchors (11.1%) located at the 1-2 o' clock position. However, articular involvement was not observed for 28 anchors located at the 12 o' clock position. The incidence of articular involvement was higher in the PEEK standard anchor group (10 out of 55, 18.2%) than in the all-suture anchor group (5 out of 87, 5.7%). Both insertion angle and distance showed small values in the articular involvement group (Table 4). DISCUSSION The goal of any successful acetabular labral repair procedure is stable fixation without complications. Several ana-tomic and cadaver studies have attempted to suggest safe angles for acetabular labral repair. 11) However, there has been no report regarding the prevalence of malposition of anchors during labral repair. In this study, using CTA, we observed that 15 of 142 anchors (11%) were placed in the cartilage-bone transitional zone. Anchors placed at the 3 o' clock position were more frequently located in the cartilage-bone transitional zone. Larger-diameter (≥ 2.3 mm) PEEK standard anchors were more frequently located in the cartilage-bone transitional zone than were smallerdiameter (≤ 1.4 mm) all-suture anchors. Several anatomic studies have reported different safety angles according to acetabular location. In particular, the 3 o' clock position in the acetabulum has been described as a high-risk area for cartilage damage. In this study, suture anchors at the 3 o'clock position had the highest likelihood of being located in the cartilage-bone transitional zone (18.2%, 6/33 anchors). Hernandez and McGrath 11) performed an anatomic study of safe suture angle with three different suture anchor systems using nine human cadavers. They found that the maximal angle of insertion at the anterior rim location was the narrowest among three acetabular locations studied. Foster et al. 17) reported similar findings in a study on six cadavers. They found a smaller distance from the articular surface when using a more anterior position, and showed a very small distance at 4 o' clock. Our findings correspond well with their results. We found that suture anchors at the anterior (3 o' clock) position had a significantly lower insertion angle compared with those at the superior (12 o' clock) and anterosuperior (1-2 o' clock) positions. Therefore, when placing anchors at the 3 o' clock position, care should be taken to avoid articular injury during arthroscopic procedures. Theoretically, large-diameter suture anchors are more prone to penetrate the cartilage or the cartilage-bone transitional zone. We found that larger-diameter (≥ 2.3 mm) PEEK standard anchors were indeed more frequently located in the cartilage-bone transitional zone than were smaller-diameter (≤ 1.4 mm) sutures. Hernandez and McGrath 11) reported similar findings. They found that 2.4-mm anchors were inserted at a significantly greater maximal angle than were 3.0-mm anchors (p = 0.04). They also noted especially small widths at the level of the labral insertion (less than 3 mm) at the anterior location, and suggested using drills of < 3.0 mm for anchor insertion if the labral attachment was used as the starting point. They suggested that using an anchor larger than 3.0 mm would be more likely to cause articular damage. To prevent articular damage during placement of suture anchors, several methods can be considered. First, small-diameter suture anchors could be used to minimize cartilage damage. Second, a change in portal placement can increase safety. Stanton and Banffy 18) evaluated the distance from the articular surface according to portal placement using six cadavers. They analyzed the three common portals: distal anterolateral accessory (DALA), mid-anterior (MA), and anterolateral (AL) portals, and reported that the DALA portal produced a larger distance from the articular surface and was safer than were the other portals. At a depth of 15 mm, the average distance from the articular surface in the anterior position (MA portal), the superior position (AL portal), and the anterior position (DALA portal) was 5.26, 3.41, and 8.01 mm, respectively. Third, trimming of the acetabular rim can produce a safer angle. Hernandez and McGrath 11) measured the width of the acetabular bone at the point of labral insertion as well as at depths of 5, 10, 15, and 20 mm from the labral insertion point at the 12 o' clock, 1:30, and 3 o'clock positions in nine human cadavers. They found the 3 o' clock position to be significantly wider at deeper (15 and 20 mm) points. Suture anchor placement done at the 3 o' clock position is associated with a narrow insertion angle and small articular distance. We recommend widening of the safety angle with trimming of the acetabular rim and use of an accessory portal such as the DALA portal, especially for suture anchor placement done at the 3 o' clock position. In all-suture type anchors, the use of a curved guide, flexible drill, and flexible suture anchor inserter may provide more precise placement in the acetabular rim at the 3 o' clock position. Nho et al. 13) performed a cadaver study that compared suture anchor placement in the acetabular rim using straight versus curved drill guides, and measured the insertion angle and distance of the suture anchor during labral refixation. The curved suture anchor guide significantly increased the insertion angle (p = 0.009) and the distance from the articular cartilage to the anchor (p = 0.003). In addition, the use of a curved guide, flexible drill, and flexible suture anchor inserter could allow for placement of suture anchors without changing anchor placement position in the acetabular rim. 13,17) This study had several limitations. First, it was a retrospective small case series. Second, we did not analyze the clinical results and complications according to anchor type. All-suture type anchors have a lower probability of articular involvement and a wider margin of safety angle. However, there are doubts about their strength and durability. Therefore, further research on the clinical and imaging results according to anchor type is necessary. Finally, there is a concern about radiation dose in studies using CT. However, in our study, the radiation dose ranged from 2.55 to 7.75 mSv, which is not higher than that given during acute abdominal CT (usually from 5 to 8 mSv) and is lower than that used for obstetric-gynecological pelvic CT (10 to 16 mSv). [19][20][21] The radiographic analysis of the placement of suture anchors after arthroscopic labral refixation using followup CTA demonstrates that articular involvement is related to the location on the acetabular rim (clock position) and diameter of the suture anchor.	2018-04-03T06:19:58.806Z	2017-11-10T00:00:00.000	{ "year": 2017, "sha1": "2468c47bae8afbf03c2d145593aed302676ca76c", "oa_license": "CCBYNC", "oa_url": "https://doi.org/10.4055/cios.2017.9.4.405", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "2468c47bae8afbf03c2d145593aed302676ca76c", "s2fieldsofstudy": [ "Engineering", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
209444997	pes2o/s2orc	v3-fos-license	Experimentally Calibrated Kinetic Monte Carlo Model Reproduces Organic Solar Cell Current-Voltage Curve Kinetic Monte Carlo (KMC) simulations are a powerful tool to study the dynamics of charge carriers in organic photovoltaics. However, the key characteristic of any photovoltaic device, its current-voltage ($J$-$V$) curve under solar illumination, has proven challenging to simulate using KMC. The main challenges arise from the presence of injecting contacts and the importance of charge recombination when the internal electric field is low, i.e., close to open-circuit conditions. In this work, an experimentally calibrated KMC model is presented that can fully predict the $J$-$V$ curve of a disordered organic solar cell. It is shown that it is crucial to make experimentally justified assumptions on the injection barriers, the blend morphology, and the kinetics of the charge transfer state involved in geminate and nongeminate recombination. All of these properties are independently calibrated using charge extraction, electron microscopy, and transient absorption measurements, respectively. Clear evidence is provided that the conclusions drawn from microscopic and transient KMC modeling are indeed relevant for real operating organic solar cell devices. organic solar cell. It is shown that it is crucial to make experimentally justified assumptions on the injection barriers, the blend morphology, and the kinetics of the charge transfer state involved in geminate and nongeminate recombination. All of these properties are independently calibrated using charge extraction, electron microscopy, and transient absorption measurements, respectively. Clear evidence is provided that the conclusions drawn from microscopic and transient KMC modeling are indeed relevant for real operating organic solar cell devices. Introduction Kinetic Monte Carlo (KMC) simulations have successfully been used to model the charge carrier dynamics in organic photovoltaics (OPVs) on the ps to µs time scale. For instance, it was shown that in thin-film OPV devices, thermalization in the disorder-broadened density of states (DOS) does not complete before charges are extracted. [1][2][3][4] The conclusions from these studies are drawn from the fitting of time-resolved experiments performed under certain bias conditions such as short circuit or open circuit. Other authors used KMC modeling to focus on the process of charge recombination and its dependence on the morphology in slabs of material, i.e., in absence of contacts. [5][6][7][8][9] However, it is still an open question to which extent nonequilibrium phenomena and other aspects that are not accounted for in macroscopic simulations such as quasi-equilibrium drift-diffusion (DD) models, govern the steady-state operation of complete OPV devices. To answer the question, it would be highly desirable to have a microscopic model that is also able to describe the current-voltage (J-V ) curve, particularly the open-circuit voltage (V OC ) and the fill factor. Modeling J-V curves with KMC has so far proven nearly impossible. One of the main challenges is the presence of two injecting contacts. While it may be acceptable to consider the contacts as simple sinks for electrons and holes in transient extraction experiments (performed at V V OC ), this simplification does not work for situations closer to V OC . When the internal field is low, contacts inject many charge carriers into the active layer. This high carrier density is demanding from the computational point of view and challenging to correctly account for. Even though a few concepts exist how contacts can be implemented in KMC, literature studies have so far failed to fully describe J-V data of real devices or are based on assumptions that are not justified experimentally. [10][11][12] Besides computational challenges, the injected charge density also sets the boundary conditions for the recombination of photogenerated carriers. [13] Charge recombination generally becomes more important when going from short circuit to open circuit because transport will slow down. Indeed, the competition between charge extraction and recombination has been demonstrated to be the main determinant of the device fill factor. [14][15][16] For a device model to be reliable it must therefore capture the hopping transport characteristics and the recombination kinetics at the same time. Even though the mechanisms of charge recombination are highly disputed, it is commonly accepted that the morphology plays a key role. [6,17,18] For instance, it is well documented that aggregated donor or acceptor domains may lower the recombination rate. [19][20][21][22] However, although the morphology of many donor/acceptor blends is well characterized by electron microscopy and other techniques, the nanostructure is often neglected in KMC and an effective medium is assumed instead. [23] Here, we present a KMC model that successfully predicts device J-V curves while simultaneously accounting for nonequilibrium hopping transport and recombination dynamics. We show that this is only possible when correct assumptions are made on the injection barriers, the morphology of the active layer, and the charge recombination rate. All these properties are calibrated by independent experimental techniques such as charge extraction, electron microscopy and transient absorption. We are thereby introducing a device model that works on a multitude of length and time scales. As such it will be useful for future investigations on the interplay between elementary processes and device characteristics of organic solar cells and other optoelectronic devices. Material System The aim of this work is to develop and experimentally calibrate a KMC model that fits both transient experiments and device J-V curves. Our material system for experimental calibration is TQ1:PC 71 BM, [24] an archetypal polymer/fullerene blend. The reason for choosing TQ1:PC 71 BM is that for this specific system a clear picture of the carrier dynamics has emerged from time-resolved measurements and previous modeling, which is summarized in a recent review article. [23] Hence, many of the parameters for the KMC model are already known; in particular, it has been shown that the charge extraction in thin devices with an active-layer thickness ≈ 100 nm is strongly affected by nonequilibrium effects. Figure 1 shows KMC Describes Device Current-Voltage Curve The KMC model, which is extended and experimentally calibrated in this work to fully describe OPV devices, has been introduced previously. [ Table 1. Open symbols are simulations with the same parameter set, but assuming too low injection barriers of 0.1 eV (triangles) or only an effective medium without PC 71 BM aggregates (squares). hopping rate ν ij from site i to site j separated by a distance r ij is given by where ν 0 is the attempt-to-hop frequency, α the inverse localization length, ∆E ij = E j − E i the energy difference between the sites, and kT the thermal energy. Hopping is assumed to take place in a Gaussian DOS, where E is the single particle energy, E 0 the mean energy, and σ the width of the Gaussian DOS or the energetic disorder. We note that without of loss of generality, also other energy distributions could be assumed in the model, such as an exponential DOS. From previous studies, however, it is known that a Gaussian DOS gives the most appropriate description for the present TQ1:PC 71 BM system both when describing transient and steadystate experiments. [23,25] In this work, only hopping between nearest neighbors on a regular, six-fold coordinated lattice was considered. In this configuration, the localization length α is unimportant; the first exponential term of Equation (1) was implicitly included in ν 0 , that is, the rate of downward nearest-neighbor hops. The core working principle of a KMC model is to simulate the time evolution of a system As mentioned above, the presence of injecting contacts causes computational challenges. Charge injection is mediated by the injection barriers, i.e., the energy offset between the contact Fermi level and the respective molecular orbital of the semiconductor. Especially for low barriers, carriers may oscillate multiple times across the contact interface before injection/extraction finally takes place. We mitigated this 'small barrier' problem by only allowing for a transfer if the number of charges next to the contact interface deviates from its equilibrium value. The transfer is modeled as hopping event with an attempt frequency ν 0,cont of the same order as for the transport of the faster carrier (here: electrons) in the semiconductor. This ensures that charge collection is not limited by the contacts. Both the cathode and anode were considered nonselective; hence, possible losses due to diffusion of carriers into the 'wrong' contact are implicitly accounted for. An advantage of KMC simulations is that no explicit assumptions about the formalism of charge recombination need to be made. Recombination of free charges involves the formation of a CT pair as intermediate. Exciton formation is explicitly allowed, but requires overcoming the relevant energy level offset between the TQ1 and PC 71 BM; as such, it can be interpreted as the inverse of charge separation, i.e., the splitting of (CT) excitons into free electrons and holes. As discussed in more detail below, it is then the inverse lifetime of the CT state that determines the recombination rate and must be calibrated experimentally. The filled circles in Figure 1a show that after the calibration discussed below, the KMC model fits the J-V curve of the TQ1:PC 71 BM solar cell well within experimental accuracy and matches both the device V OC and fill factor. Table 1 lists the key parameters used for the simulations. We note that these values are not the result of a fitting routine but come from independent characterizations. The hopping parameters were chosen in such a way that they represent earlier experiments, such as time-resolved electric-field-induced second harmonic generation (TREFISH) [4] and temperature-dependent space-charge-limited currents (SCLC), [4,26] but at the same time allow efficient calculations. This was done by assuming a single disorder for electrons and holes (σ e = σ h ≡ σ) and adjusting the attempt frequencies ν 0 such that the macroscopic transport characteristics of TQ1:PC 71 BM, e.g., the contrast between electron and hole mobility, are still captured (see Supporting Information for details). Figure 1b shows that also with the symmetrized hopping parameters, relaxation in the DOS is far from being complete when photogenerated carriers are extracted. This is true for both short-circuit and open-circuit conditions, which indicates that nonequilibrium effects may affect charge extraction along the entire J-V curve. A detailed discussion of how the nonequilibrium effects influence the individual performance parameters will be the topic of another publication. The main result of this study is that a KMC model that can describe full J-V characteristics requires an appropriate description and calibration of the injection barriers and the morphology in the active layer. If wrong or too simple assumptions are made on these properties, our otherwise well validated KMC model can no longer describe the device (Figure 1a, open symbols). Because this mainly concerns V OC and the fill factor, these observations are closely related to the charge recombination. In the following sections we will therefore focus on the factors that determine the shape of the J-V curves in the fourth quadrant, that is the injection barrier height, the blend morphology, and the recombination rate. Calibration of Injection Barriers The injection barriers set the carrier density in the device around the built-in voltage. To get a realistic estimate of the barrier height, we compare the results of charge extraction experiments in the dark with device simulations. As KMC calculations are computationally too expensive for this approach, we used a DD model instead. [27,28] This is justified because the charges treated here were not photogenerated, but injected from the contacts, so that the complexities of exciton/charge separation are bypassed. Furthermore, charges are injected from thermalized reservoirs (contacts), so that it is reasonable to describe them by a quasi-equilibrium mobility. The mobility values were estimated by inserting the hopping parameters in Table 1 in the mobility functional by Pasveer et al. [29] Charge recombination is assumed to be strictly bimolecular with the steady-state recombination coefficient (6 × 10 −18 m 3 s −1 ) taken from experimental studies on TQ1:PC 71 BM. [30,31] Figure 2a illustrates the effect of the injection barrier height on the average carrier density. Here, we chose devices with an active-layer thickness of 150 nm; only at these larger thicknesses a 'bulk' region is established, which makes the comparison with charge-extraction experiments more reliable. [32,33] Note that especially at higher densities the carrier profiles are not perfectly symmetric, which is due to the imbalanced electron and hole transport. [28,34] The experiments to be simulated are charge extraction by linearly increasing voltage (CE-LIV) and bias-assisted charge extraction (BACE). In both techniques, the device is held at a certain pre-bias (V pre ) until a steady state is reached; the charges in the device are then extracted by applying a triangular (CELIV) or rectangular (BACE) voltage pulse. The dark carrier density is calculated from the transient current J(t) via where q is the elementary charge, d the active-layer thickness, J 0 the displacement current measured at V pre = 0, and t f the time at which charge extraction is completed. Note that in the form of Equation (3), the carrier density represents the average of electrons and holes, as pointed out by Hawks et al. [35] Figure 2b shows that CELIV and BACE give a consistent picture of the carrier density as a function of voltage. At V pre = 0.9 V, which approximately corresponds to open-circuit conditions under 1-sun illumination, n dark is about 1 × 10 22 m −3 . This is the same order of magnitude as for the photogenerated carrier density and indicates the importance of injected carriers for charge recombination. As can be seen, the best description of the dark carrier density and its voltage dependence is obtained for a barrier height of 0.2 eV; with this value, the KMC model reproduces the experimental J-V curve (see Figure 1). We note that the discrepancy between CELIV/BACE and DD simulation at voltages well below the built-in voltage is merely due to experimental limitations. In this regime, most carriers are situated in the thin space-charge regions close to the contacts, which makes them only partly visible to charge-extraction experiments. [14,36] If instead too small injection barriers are selected as input for the KMC model, it can no longer describe both V OC and the fill factor. The open triangles in Figure 1 illustrate this for a barrier height of 0.1 eV. Although this is not pursued further in this work, we would like to stress that this finding shows that defining a contact as 'Ohmic', in the sense that it does not limit injection and extraction in a particular experiment, is insufficient. Here, injection barriers of 0.1 and 0.2 eV both give rise to 'Ohmic' injection, implying bulk-limited transport under forward bias, but these barriers are not equivalent in terms of the resulting photovoltaic behavior. Another interesting observation is that, as one would expect, lowering the injection barriers from 0.2 to 0.1 eV leads to an increase in V OC . But at the same time the fill factor becomes reduced, so that the overall power conversion efficiency stays roughly the same. Hence, we can deduce from our KMC simulations that reducing the injection barriers does not per se lead to a better performing solar cell device. Closer examination of this aspect, however, requires more extensive parameter studies, which are beyond the scope of the present paper and will be the subject of future work. Morphology Governs Charge Recombination In our previous KMC studies the photoactive blend was assumed as an effective hopping medium without any morphological features. [1,3,4] This zero-order approximation is reasonable when describing experiments on the ps-µs timescale where charge recombination is insignificant. However, we find that the effective-medium approach fails to fully describe the device J-V curve (Figure 1, open squares). In order to obtain a more realistic picture of the morphology, we performed transmission electron microscopy (TEM). Figure 3a blends are commonly attributed to fullerene domains because of their higher density. However, this assignment is not unambiguous; the different intensities could also be caused by phase contrast due to local crystallinity differences. For comparison, we investigated the same sample in scanning transmission electron microscopy (STEM) mode using a high-angle annular dark field (HAADF) detector. [37,38] In the Supporting Information we show that HAADF-STEM reveals very similar structures as in Figure 3a, but of inverted contrast. This clearly confirms that the clusters seen in TEM are PC 71 BM aggregates, in agreement with earlier work on similar blend systems. [39] The main effect of aggregation is to reduce the energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) compared to the amorphous material. This creates an energy cascade with a driving force for carriers to move from the (molecularly mixed) amorphous regions towards the (material-pure) aggregates and will affect the way how charges separate and recombine. [19][20][21][22] We implemented the aggregates in the KMC model as 7 × 7 inclusions in a 10 × 10 unit cell describing the mixed donor/acceptor phase (Figure 3a, inset). Inclusions were assumed to consist of pure PC 71 BM with a 0.2 eV lower-lying LUMO compared to the mixed phase; all other properties were left unchanged to keep the number of unknown parameters at a minimum. We did not consider pure TQ1 domains, as our TEM experiments do not provide any evidence for them. This is reasonable, since TQ1 is a relatively amorphous polymer that has no strong tendency to form aggregates, in particular in blends with excess fullerene. [40] Note that the aggregate size in the KMC model is smaller than what is suggested from the electron microscopy images. This was done to keep the simulation box computationally tractable while still getting reasonable statistics. The size of the inclusions and the unit cell were chosen such that the donor/acceptor ratio of the blend is maintained. A detailed examination of the structure size on the device performance is beyond the scope of this work; however, first tests indicate that the actual size of the aggregates is much less important than their presence. Likewise, a 0.1 eV lower-lying LUMO for the aggregate phase did not make any relevant difference as compared to the used 0.2 eV. Only with the inclusions in the effective hopping medium we were able to match the fill factor of the experimental devices. Figure 3b shows that this is due to a reduction of the charge recombination. Importantly, the presence of aggregates simultaneously reduces the yields of geminate and nongeminate recombination. This confirms earlier suggestions that the generation and recombination of free charges are coupled via the ability of CT pairs to separate. [41,42] In other words, the possibility for carriers (here: electrons) to lower their energy by moving to the aggregates will not only increase the charge separation yield, but also reduce the nongeminate recombination. This is a clear hint that the different ability to form aggregates/phase-pure domains may explain why different OPV materials show so different recombination rates compared to the Langevin model. In the context of this work, Table 1 but varied decay rate of CT states into the ground state. Inset: Illustration of the relevant states and transitions for charge recombination. (b) Measurements with various initial carrier densities by varying the pump fluency from 2.5 × 10 16 to 6 × 10 17 photons · m −2 and simulations for a fixed decay rate of k CT = 3 × 10 7 s −1 . Experimental data from Andersson et al. [43] however, it means that it is the kinetics of the CT states, i.e., how they dissociate and (re-)associate, that must be calibrated experimentally. Calibration of the Recombination Rate The inset in Figure 4 illustrates the kinetic model of charge recombination that has emerged from literature. [2,19,30,41,42] As we discuss to some detail in the Supporting Information, recombination in TQ1:PC 71 BM is not limited by the rate k enc at which free carriers meet to form an interfacial CT complex. This implies that the probability for the CT pair to dissociate is much higher than to decay to the ground state (k d k CT ). It has been shown that in such a situation an equilibrium between CT states and free charge carriers is established. [2,41] The position of the equilibrium is determined by the rate k CT , which is the relevant parameter in the KMC model to calibrate the recombination. In order to do so, we use the results of transient absorption (TA) experiments. TA is a pump-probe technique that optically tracks a carrier population created by a short light pulse over time. As the experiment is carried out under flat-band conditions, the measured decay solely reflects the recombination kinetics. Figure 4a shows the TA decay of a TQ1:PC 71 BM device for a pump fluency of 4 × 10 16 photons · m −2 taken from literature. [43] The traces are attempts to describe the experiment with our KMC model. One can clearly see that the (inverse) CT state lifetime is the crucial parameter for the decay dynamics. The best fit on short time scales is obtained for k CT = 3 × 10 7 s −1 . Figure 4b demonstrates that with the calibrated value for k CT , we are able to reasonably describe transient absorption data for a range of initial carrier densities. On longer time scales, however, the fit between TA experiment and KMC model is not as good. The reason for this are the symmetrized transport parameters we use for computational effectiveness. As discussed in the Supporting Information, the disorder σ and attempt-to-hop frequency ν 0 are largely interchangeable, i.e., increasing the one parameter can be compensated by decreasing the other and vice versa. This interchangeability allows us to use the values given in Table 1, which keep the KMC calculations manageable, while still reproducing the measured quasi-steady state mobilities. Nevertheless, using symmetrized transport parameters remains a simplification, so that some of the details necessary to describe the full TA traces are lost. In Figure S2 we show that a better fit can be obtained when the 'real', non-symmetrized values for σ and ν 0 are used in the simulation. However, significant differences between the parameter sets are only noticeable at very high initial carrier densities (∼ 10 24 m −3 ) and on the time scale of µs and beyond. At those times, most of the carriers have already been extracted, as can be seen from the histograms in Figure 3b and from previous experiments. [1,4,23] Hence, the use of the simplified transport parameters is well justified when describing a solar cell under standard operating conditions. Conclusions We were taken at an acceleration voltage of 300 kV in a FEI Titan 80-300. Discussion of Symmetrized Hopping Parameters A well-known problem of kinetic Monte Carlo (KMC) simulations of hopping transport in energetically disordered media is that calculation times explode when the normalized disorderσ = σ/kT becomes larger than 3-4. An additional problem arises in bipolar systems if the attempt to hop frequencies ν 0 of the two charge species (electrons and holes) differ by more than roughly an order of magnitude. In this case, the hopping events of the species with the highest ν 0 outnumber those of the slower species. This leads to poor statistics for the slower species that can only be cured by increasing the total number of hops considered in the simulation, i.e. by increasing the total calculation time. Unfortunately, the previously determined hopping parameters for the TQ1:PCBM system studied here cause both problems to arise. For holes in TQ1 we found in Refs. S1-S5 σ h ≈ 50-100 meV and ν 0,h ≈ 0.1-1 × 10 10 s −1 and for electrons in PCBM σ e ≈ 120 meV and ν 0,e ≈ 1×10 13 s −1 . In order to keep calculation times to acceptable levels (days), we had to use partially symmetrized hopping and disorder parameters. In this, we could make use of the previously observed interchangeability of the disorder and the attempt-to-hop frequency, [S2] where increases in one of the two parameters can be largely compensated by a simultaneous increase in the other parameter. Bearing the above in mind, we used σ e = σ h = 75 meV for both electrons and holes, and ν 0,e = 1×10 11 s −1 and ν 0,h = 1 × 10 10 s −1 , keeping the corresponding steady state electron mobility significantly larger than that of the holes. The other parameter that has (a minor) influence on the hopping process in the used model is the nearest neighbor distance a NN = 1.8 nm that we fixed to the value that we obtained from temperature-dependent charge transport studies. [S5] A consequence of the symmetrized hopping parameters is that the other rates in the simulation, specifically the recombination rate of the CT exciton, become relative to these values. This explains why the used CT rate k CT = 3 × 10 7 s −1 is two to three times lower than the typical values used before. [S1,S3] S2 Charge Recombination in TQ1:PC 71 BM The three relevant transitions for the recombination of photogenerated charges are the separation rate of CT states (k d ), the encounter rate of free electrons and holes (k enc ), and the decay rate of CT states into the ground state (k CT ). Importantly, the encounter complex formed by two independent carriers has been identified as the same CT state as involved in charge separation. [S6,S7] Only if the rate at which CT states recombine were much faster than the rate at which they dissociate (k CT k d ), the recombination would be encounter-limited and Langevin theory applicable. In practice, however, virtually all OPV blends show recombination rates that are substantially reduced compared to the Langevin model. The apparent steady-state recombination rate constant of 6 × 10 −18 m 3 /s we assume Section 2.3 in the main text for TQ1:PC 71 BM implies a reduction of 2 orders of magnitude. Such large reduction factors cannot be explained by geometrical confinement, i.e., the fact that electrons and holes are attributed to different material phases and only meet at the heterointerface. [S8] We must therefore assume that recombination in TQ1:PC 71 BM is not encounter-limited. This is reasonable considering the following: although the details of charge separation are not yet well understood, the rate k d will depend on the ability of the two involved carriers to move away from each other. Still being a hopping process, this will happen with a rate on the order of 10 10 to 10 13 s −1 . Because this is much faster than the rate at which the CT state decays into the ground state, i.e., its inverse lifetime (k CT = 10 7 to 10 8 s −1 ), there is enough time to establish an equilibrium between CT states and free electrons and holes. [S1, S9] In other words, the carriers forming the CT state have multiple attempts to escape from their mutual Coulomb attraction before they ultimately recombine. One can easily see that it is then the rate k CT which determines the position of the equilibrium; decreasing k CT will shift the equilibrium more towards dissociation, effectively decreasing the charge recombination rate detectable experimentally. S3 Figure S1: Transmission electron micrograph in HAADF-STEM mode of the same TQ1:PC 71 BM blend film as discussed in the main text. The HAADF signal in STEM originates from electrons that have inelastically scattered to high angles when transmitted through the sample. The signal intensity is higher if the electrons scatter against molecules of higher average atomic number (Z-number). This is likely not relevant here, since the TQ1 monomer and the PC 71 BM molecule have a similar average Z-number. The signal intensity is also increased for regions of higher density or thickness. Since the film thickness is rather uniform, the bright regions in the HAADF-STEM image show areas of higher density. This correlates well with what is known about fullerenes and their aggregates. [S10-S12] For the electrons σ e = 120 meV and ν 0,e = 1 × 10 13 s −1 was assumed and for the holes σ h = 83 meV and ν 0,h = 5 × 10 9 s −1 , according to previous experimental work. [S3] As mentioned in the text above, using these parameters required us to assume a slightly higher CT rate of k CT = 8 × 10 7 s −1 .	2019-12-20T21:32:50.000Z	2019-12-20T00:00:00.000	{ "year": 2019, "sha1": "d4819325ed4cb46668e472f54e059e638c62d5f9", "oa_license": "CCBY", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/solr.202000029", "oa_status": "HYBRID", "pdf_src": "Arxiv", "pdf_hash": "87f5e12bb47995c37d7ffc275e605095bb98912f", "s2fieldsofstudy": [ "Physics", "Engineering", "Materials Science" ], "extfieldsofstudy": [ "Materials Science", "Physics" ] }
119120907	pes2o/s2orc	v3-fos-license	The Lagrange-Poincar\'e equations for interacting Yang-Mills and scalar fields A special case of the Lagrange-Poincar\'e equations for the gauge field interacting with a scalar field is obtained. For description of the dynamics on the configuration space, the adapted coordinates are used. After neglecting the group variables the obtained equations describe the evolution on the gauge orbit space of the principal fiber bundle which is related to the system under the consideration. Introduction The behavior of systems with symmetry is determined by internal dynamics, which is often hidden, which presents significant difficulties in the case of the usual description of evolution. In the theory of reduction for mechanical systems with symmetry, this problem is solved using the Lagrange-Poincaré equations. Due to symmetry, the configuration space of mechanical systems can be regarded as the total space of the principal fiber bundle associated with the system. The Lagrange-Poincare equations are given by two equations: the "horizontal" equation which belongs to the kernel of the 1-form connection (naturally emergent in such systems) and the "vertical" equation related to the motion along the orbit of the principal fiber bundle. In case of the projection onto the base manifold (the orbit space of the principal fiber bundle), the horizontal equation describes the internal dynamics of the system. This dynamics is determined by the mechanical system that arises from the original system as a result of the reduction. In mechanics, the interrelation between the original system and the reduced one is well studied due to Marsden-Weinstein reduction theory. [1,2] But internal dynamics is also the main object of research in gauge theories -infinitedimensional dynamic systems that are invariant with respect to the action of a group of gauge transformations. Here, the true configuration space (the configuration space of physically observable variables) is the orbit space of the action of the gauge group. The main problem for these systems is that it is not possible to describe the local dynamics on the gauge orbit space in terms of the gauge-invariant variables. It is currently unknown how to do this in satisfactory way. The generally accepted method of describing local dynamics in orbit space is to use a special coordinate system in the principal fiber bundle. The coordinates of such a system are known by the name of the adapted coordinates [3][4][5][6] and are defined using local sections of the bundle. The sections are given by local surfaces (submanifolds) in the space of gauge fields. The local surfaces themselves are determined by equations that cannot be explicitly resolved, so parametric representations of the surfaces cannot be obtained. As a result, when introducing coordinates into the principal bundle, we are forced to deal with constrained variables (or dependent variables) as coordinates in this approach. In spite of this, the approach is widely used, for example, when quantizing gauge fields by the path integral method. [7][8][9] Studies of the classical evolution of gauge fields with the use of adapted coordinates for local descriptions of the dynamics have practically not been conducted. In this paper our goal is to obtain the Lagrange-Poincare equations for the gauge system formed from the Yang-Mills field interacting with the scalar field. We are based on our works [10,11] where we have considerd the mechanical system of two particals given on the product manifold consisting of the Riemannian manifold and the manifold represented by the vector space. It was assumed that the system under consideration is invariant with respect to the group action. The resulting reduced mechanical system was given on the corresponding associated bundle which serves as the base space of the principal bundle related to the system. The geometry of this special mechanical system is analogous to the gauge system we consider in the present article. So it can be regarded as the model system for our problem. The paper will be organized as follows. Section 2 is an introduction to our paper, where we recall our previous work from arXiv, where the mechanical system of two interacting particles was investigated. In Section 3 we explain how the adapted coordinates can be determined for the gauge interacting sistem formed from the Yang-Mills field and a scalar field. These coordinates correspond to the coordinates in the mechanical system. This provides the basis for using the Lagrange-Poincaré equations obtained earlier for the mechanical system, in deriving analogous equations for the gauge system under the study. In Section 3, we derive such equations for the gauge system using functional expressions for the terms of the Lagrange -Poincaré equations obtained earlier for the mechanical system. Details of derivations of the Lagrange-Poincaré equations are considered in Appendix. Mechanical system of two interacting particles In our previous works [10,11], we considered a special finite-dimensional mechanical system with the following Lagrangian: The configuration space of this system is the product manifold P × V . It was assumed that P is a smooth finite-dimensional Riemannian manifold (without the boundary) and V is a finite-dimensional vector space. So, (Q A , f n ), A = 1, . . . , N P and n = 1, . . . , N V , are the coordinates of a point (p, v) ∈ P × V in some local chart. Also, it was assumed that a smooth isometric free and proper action of the compact group Lie G on P × V was given. We dealt with the right action on P × V : (p, v)g = (pg, g −1 v). In coordinates, this action is written as follows: HereD n m (g) ≡ D n m (g −1 ), and by D n m (g) we denote the matrix of the finitedimensional representation of the group G acting on the vector space V . For our metric the Killing vector fields In the following, we will also use the condensed notation for indices: A ≡ (A, p). So, for example, the components of the Killing vector fields will be written as KÃ µ = (K A µ , K p µ ). From the general theory [1] it is known that in our case P × V can be regarded as a total space of the principal fiber bundle Due to this fact it is possible to express the coordinates (Q A , f n ) of the point (p, v) in terms of the the principal fiber bundle coordinates. The method of performing this for the typical principal bundle P(M, G) is wellknown [7,[12][13][14][15][16]. In approach close to ours was considered in [9] for the abelian gauge theory. It consists of using the local sectionsσ i of our bundle, π ′ ·σ i = id. But to defineσ i , it is necessary to use the sections σ i of the principal fiber bundle P(M, G): where a(p) is the group element defined by p = σ i (x)a(p). The adapted coordinates on P(M, G) are defined by means of the choice of the special local sections σ i . The sections are determined by the local submanifold Σ i of P, given by the equation {χ α (Q) = 0, α = 1, . . . , N G }. The coordinates of the points on the local submanifold Σ i will be denoted by Q * A , they are such that {χ α (Q * ) = 0}. That is, Q * A are dependent coordinates. In other words, the special section σ i is defined as the map We note that there exists a local isomorphism between trivial principal bundle Σ i × G → Σ i and P(M, G): [5,6,9] which allows us to introduce a local coordinates on P(M, G). In coordinates we have: where Q * B are the coordinates of a point given on the local surface Σ i and a α -the coordinates of an arbitrary group element a. This element carries the point, taken on Σ i , to the point p ∈ P which has the coordinates Q A . An inverse map ϕ −1 i , has the following coordinate representation: , a α (Q)). Here the group coordinates a α (Q) of a point p are the coordinates of the group element which connects, by means of its action on p, the surface Σ i and the point p ∈ P. These group coordinates are given by the solutions of the following equation: The coodinates Q * B are defined by the equation In the same way as for the principal bundle P(M, G), there exist a local isomorphisms of the principal fiber bundle P(P × G V, G) and the trivial principal bundlesΣ i × G →Σ i , where now the local surfacesΣ i are the images of the sectionsσ i . In this case we have the following coordinate functions of the charts: Here Q A and f m are the coordinates of a point (p, v) ∈ P × V , Q * A (Q) is given by (4) andf n (Q) = D n m (a(Q)) f m , a(Q) is defined by (3), and we have used the following property:D n m (a −1 ) ≡ D n m (a). The coordinates Q * A , representing a point given on a local surface Σ i , satisfy the constraints: χ(Q * ) = 0. The coordinate functionφ i mapsΣ i × G → π −1 (Ũ i ): ϕ i : (Q * B ,f n , a α ) → (F A (Q * , a),D m n (a)f n ). Thus, we have determined the special local bundle coordinates (Q * A ,f n , a α ), also called the adapted coordinates, in the principal fiber bundle π : P ×V → P × G V . The replacement of the coordinate basis (∂/∂Q B , ∂/∂a α ) for a new basis (∂/∂Q * A , ∂/∂f m , ∂/∂a α ) is performed as follows: -the matrix which is inverse to the Faddeev-Popov matrix: a) e is the unity element of the group. Thus, the metric (2) of the original manifold P × V in a new coordinate basis is given bỹ where G CD (Q * ) ≡ G CD (F (Q * , e)): B is the projection operator on the tangent plane to the submanifold Σ. It is given by (6) is the metric on G-orbit through the point (p, v): In our works [10,11], the Lagrange-Poincaré equations equations were derived using the so-called the horizontal lift basis on the total space of the principal fiber bundle. The new basis consists of the horizonal and vertical vector fields and can be determined by using the "mechanical connection" which exists [1] in case of the reduction of mechanical systems with a symmetry. In the principal fiber bundle P(P × G V, G), in coordinates (Q * A ,f n , a α ), the connectionω =ω α ⊗ λ α ({λ α } is the basis in the Lie algebra of the group Lie G) is given by the following expression: , the connection can be rewritten aŝ or using the condensed notations of indices likê In coordinates (Q * A ,f n , a α ), the horizontal lift basis (H A , H p , L α ) is given by the vector fields and also by the left-invariant vector field L α = v ν α (a) ∂ ∂a ν which is obtained from the Killing vector field K α (Q). Note that L α commutes with the horizontal vector fields H A and H p . In the definition of H M , new components of the projection operator were used. They are The operator NÃ B satisfy the following property: In a new coordinate basis (H A , H p , L α ), the metric tensor (6) is represented asǦ The components of the "horizontal metric"G H AB depending on (Q * A ,f m ) are defined as follows: In the coordinate basis (H A , H p , L α ), the original Lagrangian L has the following representation: where the new time-dependent variables ω A , ω p and ω α , which are associated with velocities, are given by The Lagrangian (9) was used in [10,11] for derivation of the Lagrange-Poincaré equations. This was done using the Poincaré variational principle. The following equations were obtained: (Here the condensed notation for indices is used: according to which the sum over the repeated indexR means the summation over R and r.) In these equations p σ = γ ασ ρ α ǫ ω ǫ , the curvature tensor F α SP of the connection A α P is given by and F α pm are defined in a similar way. The covariant derivative D R (d κσ (Q * ,f )) are given by The Christoffel symbols HΓR BM , HΓR qB and HΓR pq for the horizontal (degenerate) metricG H RT are defined by means of the equalities: And HΓ qBT and HΓ pqT have an analogous definitions. Taking into account the following properties: and the invariance of the potential V (Q * ,f ) under the action of the group G, this means that NR F V ,R = V ,F , we can rewrite the Lagrange-Poincaré equations in the following way: These equations will be used for derivation of the Lagrange-Poincaré equations in gauge theories. Adapted coordinates in configuration space of the gauge system with interaction Our aim is to extend the methods we have used for the finite-dimensional mechanical system with symmetry to the gauge system which describe the dynamics of Yang-Mills field interacting with the scalar field. The standard relativistically invariant Lagrangian for this system is singular (we can not determine the Hamiltonian using the Legendre transformation) in contrast to what we had for the model mechanical system. The problem is related to presence of the redundent variable A a 0 in the Lagrangian. Therefore, by setting A a 0 = 0 in the Lagrangian we obtain the Lagrangian which is free of this problem. Note, that the same can be performed by suitable gauge transformation So, the Lagrangian (Lagrange density) we will consider is the following Here k αβ = c τ µα c µ τ β is the Cartan-Killing metric on the group G, V 0 is some gauge-invarint potential. g 0 is a coupling constant. 2 The covariant derivative ∇ i is defined as follows: , whereJ α are the generators of the representationD n m (a) which acts (on the right) in the vector space V :f n =D n m (a)f m ,D n m (Φ(g, h)) =D m p (h)D p n (g). The generators satisfy the following commutation relation [J α ,J β ] =c γ αβJ γ , where the structure constantsc γ αβ = −c γ αβ . The Lagrangian (14) is invariant under time-independent gauge transformations of the gauge potentials and scalar fields: : The obtained Lagrangian looks as if it represents the motion of two "particle" in the product space P × V in the potential One of the space, P, is an infinite-dimensional Riemannian manifold. The gauge fields A a i can be regarded as points of this manifold. And the other space, V , is the space of functions with the values in the vector space V. Also, we are given an action of the group, the group of the gauge transformations, on the product space. This is analogous to what we have in reduction problem for dynamical system with symmetry in mechanics, which was considered in the previous section. Here we are interested in description of internal dynamics given on the gauge orbit space. The reduction theory for the gauge-invariant dynamical systems follows from the result obtained in [3-6, 17, 18], and in other works, where the geometric approach to the gauge fields was developed. First of all, the gauge fields A a µ (x) are regarded as coordinate representations of connections defined on the principal fiber bundle P (M, G) over the compact manifold M. 3 Then, in order to have a smooth free and proper action of the gauge group on the space of connections P, one must consider the irreducible connections. (The isotropy subgroup of these connections coincides with Z (G), the center of gauge group G .) The gauge transformation group must be groupG = G /Z (G). Moreover, the connections and the gauge transformation functions must belong to classes of Sobolev functions H k and H k+1 , respectively, with k ≥ 3 [3,5]. Only in this case one leads to the principal fiber bundle defined by π : P → P/G = M . The function space V of the matter fields f b (x, t) consists of the sections of the associated bundle Γ(P × G V). (These sections also must be from H k .) In our case, P ×V is the original configuration space of the gauge system with the Lgrangian (14), and the gauge orbit space P × G V , the base of the principal fiber bundle π ′ : P × V → (P × V )/G = P ×G V , is the configuration space of the physically observable quantities. From the quadratic part of the Lagrangian (14) it follows that the Riemannian metric of the original configuration space is flat. It can be presented as follows: is the metric on P and the metric on V is In these formulae we have used the extended notation for indices by which A (α,i,x) ≡ A αi (x) and f (m,x) ≡ f m (x). Note that the use of such notations helps in the generalization of formulas obtained in the finite-dimensional case to the corresponding formulas in field theories. From the gauge invariance of the Lagrangian (and the metric) it follows that the Killing vectors of the original metric are where components of this vector field are given by is a partial derivative with respect to x i ), and . We can determine the coordinates in the principal bundle for the gauge system under study just the same way as was done for a mechanical system with symmetry in a finite-dimensional space. ?The local sections Σ of the principal fiber bundle P(M ,G ), which are necessary for determination of the bundle coordinates in the total space P × V of the bundle π ′ , will be defined by means of the Coulomb gauge condition (or the Coulomb gauge): ∂ i A αi = 0. The gauge potentials that will satisfy this equation (dependent variables) will be denoted by A * i α . Note that dependent variables are typically used when quantizing gauge fields [6-9, 22, 23]. As was shown in previous section, for transition from the original coordinate (A α i , f a ) given on P × V to the adapted coordinates (A * i α ,f b , a µ ) of the principal fiber bundle it is requiered that the group coordinates a α (A) of the "point" A should be known. In mechanics, they are obtained as a solution of the equation (3): χ β (F A (Q, a −1 (Q))) = 0. For the Coulomb gauge, this equation is as follows: Then, the coordinates Q * of the corresponding point on a submanifold Σ are determined by the corresponding group transformation: In gauge theories, we have the following gauge transformation: With the obtained a α (x), f a is expressed in terms off a as follows: a α (x)). To obtain a new coordinate representation of the original Riemannian metric, we must transform the coordinate vector fields. The "vector fields" transformation formula is a strightforward generalization of the corresponding formula from the finite-dimensional case: where we have denoted byF the matrix which is inverse to the matrix F (µ,k,u) (α,i,x) defined as follows F satisfies the relation: Also, we have δ δf (n,x) = D m n (a(x)) δ δf m (x) . In formula (15), by N (ν,p,v) (µ,k,u) , which is equal to (β,j,y) , we have denoted the projection operator onto the subspace which is orthogonal to the component of the Killing vector field K (α,y) which is related to P. The projection operator N (m,y) For the Coulomb gauge, we have Therefore, the matrix Φ (restricted to the gauge surface) is equal to An inverse matrix Φ −1 can be determined by the equation That is, it is the Green function for the Faddeev-Popov operator: (The boundary conditions of this operator depend on a concrete choice of a base manifold M.) By a second group of variables, the Green function Φ −1 satisfies the following equation: Notice that in the formula (15), the matrix Φ −1 , as well as the other terms of the projector N, is given on the gauge surface Σ. In our principal bundle, the orbit metric d (µ,x)(ν,y) is determined by using the Killing vectors K (α,y) : That is, = γ µν (x, y) + γ ′ µν (x, y) An "inverse matrix" to the "matrix" d (µ,x)(ν,y) is defined by the following equation: . In explicit form this equation is written as follows: Thus, d (ν,y)(σ,z) is the Green function of the operator given by the expression in square brackets. It is assumed that a certain boundary condition for the equation is chosen. The Green function d (ν,y)(σ,z) and the Killing vectors are the main elements with by which the "Coulomb connection" (or "mechanical connection") is determined:ω =ω α ⊗ λ α in the principal fiber bundle P(P ×G V , G ): where the components of the connection are given by The following transformation of the coordinate basis in our principal bundle is connected with the replacement of the basis vector fields by the horizontal ones. This can be done with the help of horizontal projection operators, which are determined by the connection we have just defined. All this is similar to what we did in the finite-dimensional case. Therefore, we will not follow all the steps that ultimately must lead to the Lagrange-Poincaré equations in the functional space of gauge fields. Instead, for this purpose we will use the finite-dimensional equations (11), (12) and (13). The Lagrange-Poincaré equations in gauge theories The equations that we derive in this article are a special case of the Lagrange -Poincaré equations. In this article, we restrict ourselves to a particular case of the Lagrange -Poincaré equations. They can be obtained from finitedimensional equations if we assume that the expression under the projector N B A in the first horizontal equation (11) is zero. In addition, we neglect those terms of the first equations that explicitly depend on Killing vectors. Then, from our assumption and the structure of the second horizontal equation (12), it follows that the terms of the second equation with the projector N r B are equal to zero. Thus, we will deal with the following equations: Since the Riemannian metric of the original manifold of gauge fields is flat, G AB = δ AB must be used as a metric in these finite-dimensional equations. In addition, this fact must be taken into account when calculating the terms of equations are made with using the Killing relation. In this regard, we first transform the terms of the equations so that later it was possible to replace them by the appropriate functional expressions. Therms of equations with Christoffel symbols HΓ , curvatures F α and D m d κσ will be expressed using the Killing vectors, the components of the mechanical connection and the metric on the orbit. Further we will list the obtained representations for these terms. Christoffel symbols for the horizontal metric . . Note that before starting the transition to the functional representation in the Christoffel symbols, the partial derivatives of the connections are replaced using the following formulas: Another equivalent representation of derivatives are To obtain a functional representation for the members of the equations, one needs to treat the indices of variables as if they were compact notations of extended indices. Recall that our basic variables are ω A ≡Q * A , ω n ≡ḟ n . So, we have and similarly for ω n (t): ω n (t) → d dtf (n,x) (t) ≡ḟ n (x, t). To obtain functional representations for the terms of the equations, we made the following replacements: To get expressions in the right parts of the formulas, you need to take sum over repeated generalized indices. Sum over continuous indices means corresponding integration. There are also the appropriate functional representations for the connections. A But in our final formulas we do not use them, despite the fact that this can be done as it does not lead to the simplification of the already rather complex expressions. The results of our calculations -functional representations of the terms of the equations are presented in Appendix. Concluding remarks The obtained equations are represented a rather complex expressions. A possible simplification of these equations can be obtained by projection of the equations onto the orbit space of the principal bundle.This is achieved by setting the group variable to zero. This suggests that the full equations apparently describe the dynamics of the system in the excited state.A direct consequence of these equations are the equations for the relative equilibria of the dynamical system under consideration. These equations can easily be derived from the result of those equations that are obtained in the work. It should also be noted that the role played by the equations of the mechanical system. Without these equations, it would be difficult to understand in complex expression the nature and origin of individual terms of the equation for the gauge system. It remains unclear whether it is possible to somehow simplify the resulting equation based on some kind of symmetry. It is also not clear whether the equation for relative equilibria can be used to study the problem related to symmetry breaking.	2018-12-31T09:31:16.000Z	2018-12-31T00:00:00.000	{ "year": 2018, "sha1": "77348b126c3c054efcaade185c105263dc0312f2", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "77348b126c3c054efcaade185c105263dc0312f2", "s2fieldsofstudy": [ "Physics", "Mathematics" ], "extfieldsofstudy": [ "Physics", "Mathematics" ] }
1076530	pes2o/s2orc	v3-fos-license	Spontaneous Sleep-Like Brain State Alternations and Breathing Characteristics in Urethane Anesthetized Mice Brain state alternations resembling those of sleep spontaneously occur in rats under urethane anesthesia and they are closely linked with sleep-like respiratory changes. Although rats are a common model for both sleep and respiratory physiology, we sought to determine if similar brain state and respiratory changes occur in mice under urethane. We made local field potential recordings from the hippocampus and measured respiratory activity by means of EMG recordings in intercostal, genioglossus, and abdominal muscles. Similar to results in adult rats, urethane anesthetized mice displayed quasi-periodic spontaneous forebrain state alternations between deactivated patterns resembling slow wave sleep (SWS) and activated patterns resembling rapid eye movement (REM) sleep. These alternations were associated with an increase in breathing rate, respiratory variability, a depression of inspiratory related activity in genioglossus muscle and an increase in expiratory-related abdominal muscle activity when comparing deactivated (SWS-like) to activated (REM-like) states. These results demonstrate that urethane anesthesia consistently induces sleep-like brain state alternations and correlated changes in respiratory activity across different rodent species. They open up the powerful possibility of utilizing transgenic mouse technology for the advancement and translation of knowledge regarding sleep cycle alternations and their impact on respiration. Introduction Sleep is a fundamental circadian behavior that has fascinated scientists for centuries. Although its functional role is still enigmatic, sleep has a powerful influence on behavior, mental health, cognition, and different physiological functions in vertebrates [1][2][3][4]. Sleep is not a unitary process. It involves systematic transitions between different levels of brain activity. The initiation of sleep is characterized by the gradual development of high-amplitude low-frequency activity in the forebrain and lowered muscle tone (non-REM or slow wave sleep: SWS) that eventually progresses into a paradoxical brain state characterized by low voltage fast frequency activity in the cortex and hippocampal theta activity accompanied by a generalized muscle atonia and rapid eye movements (REM). The major anatomical brain structures that act as key players in the determination of the sleep/wake cycles have been identified (mesopontine nuclei, tuberomammillary bodies, locus coeruleus, raphe nucleus, hypothalamus) but the precise sequence of events that lead to brain state alternations and the mechanistic understanding of the process are still under debate [1,5]. The lack of an appropriate experimental model for sleep (other than sleep itself) is likely responsible for the difficult progress in this field. Murine natural sleep is characterized by a very irregular and unpredictable sequence of events. Sleep cycles have been reported to be between 20 seconds and 13 minutes in length, with irregular occurrence of REM states during sleep that can last mere seconds to a few minutes [6][7][8][9]. Respiratory activity during sleep is characterized by a drop in ventilation and respiratory muscle activity during SWS followed by an increase in respiratory frequency and variability preferentially during REM sleep, although ventilatory control and respiratory characteristics can change considerably amongst different mouse strains [10][11][12]. The development of transgenic mouse lines provides further experimental power for sleep studies since different neuronal populations can be labelled, selectively activated, and permanently or reversibly silenced. This general approach may provide insight into the functional role and dynamics of structures that are involved in brain state alternations and in the modulation of respiratory networks, as well as provide new working hypotheses on disease physiology and enable mechanistic hypotheses to be tested at both cellular and network levels. Recently, urethane anesthesia has been demonstrated to be a useful model of sleep dynamics in rats, where spontaneous brain state alternations at neocortical and hippocampal sites replicate, in a reproducible and predictable fashion, the spontaneous alternation between a SWS state and a REM state occurring in natural sleep [13]. In addition, it has also been demonstrated that under urethane, breathing shows changes correlated to these same brain state alternations that correspond to many of the typical respiratory features occurring during natural sleep and its stages [14]. Here we investigated the possibility that similar changes in forebrain activity and respiration occur in adult mice anesthetized with urethane. We show that, similar to rats, mice under urethane show spontaneous brain state alternations and correlated respiratory changes that also mimic sleep patterns. This confirms the potential of urethane anesthesia as a model for sleep across different rodent species and opens up a novel model system for determining mechanistic processes at a molecular and genetic level for sleep-like brain state changes and their physiological correlates. Materials and Methods Animal handling and experimental protocols were approved by the Biosciences and Health Science Animal Policy and Welfare Committees of the University of Alberta according to the guidelines established by the Canadian Council on Animal Care. Acute (urethane-anesthetized) mice preparation Thirteen adult male mice (7 CD1 mice and 6 C57BL/6 mice; 25-60g; mean=40.2±4.5g; no statistical difference in weight between the 2 strains, CD1= 47.4±6.3; C57BL/6 33.0±5.1; p=0.11) were used for acute experiments in this study. Mice were initially anesthetized with isoflurane (2% in 100% O 2 ) while the femoral vein was cannulated and urethane (~1.5 g/kg body weight) was gradually delivered i.v. to induce anesthesia. In some cases, when the intravenous route became blocked or proved difficult, urethane was administered or supplemented intraperitoneally to a similar final dosage. Additional doses of anesthesia (at 0.2g/ml concentration in 0.01ml amounts) were delivered as necessary to maintain a surgical plane of anesthesia which was verified on occasion during experiments by lack of reaction to hindpad pressure or other noxious stimuli such as tail pinches. We ensured, as shown previously for rats [13], that these stimulations did not alter the rhythmicity or phase of subsequent brain state alternations. Additional saline (0.1ml) was delivered subcutaneously every two hours in order to maintain hydration levels. A servo-controlled heating pad was set at 36-37±1°C to maintain body temperature (model 50-7220, Harvard Apparatus) and rectal temperature was continuously recorded during a subset of experiments (n=4). Bipolar EMG wire electrodes made with multi strand, Tefloncoated, stainless steel wires (Cooner Wire, Chatsworth, CA) were inserted into the genioglossus (GG), oblique abdominal (ABD), and intercostal muscles (INT) to measure breathing characteristics. For GG EMG placement, a skin incision was performed under the chin to expose the digastric muscle which lies above the mylohyoid and genioglossus muscles. Insulated wires with a 1mm length exposure were then inserted through the genioglossus muscle at 1-2mm distance from each other. For ABD EMG placement, a skin incision was performed on the right side of the abdomen, just below the rib cage and bipolar electrodes were placed in the oblique ABD muscle at 1-2mm distance from each other. Skin incisions were then sutured back to avoid drying of the muscles. For INT EMG placement, a skin incision was performed on the right side of the chest and the pectoral muscle was displaced in order to expose the intercostal muscles. Bipolar electrodes were placed in the intercostal muscles at 1-2mm distance from each other. Skin was then sutured back together. Bipolar signals were amplified at 10,000X gain and filtered between 100 and 20kHz using a differential amplifier (model 1700, AM-Systems Inc, Carlsborg, WA). Mice were then positioned on a stereotaxic frame and bipolar electrodes made with Teflon-coated stainless steel wires (~1mm vertical stagger; AM-Systems Inc.) were implanted in the hippocampal formation (HPC) according to the following coordinates relative to bregma: anterior-posterior (AP): -2.0; mediolateral (ML): ±1.5; dorsoventral (DV): -1.5 to -2.0mm. Local field potential (LFP) signals were amplified across contacts at 1000X gain and filtered between 0.1 and 10kHz bandwidth using the same differential amplifiers as for EMG. Hippocampal electrodes were lowered in the DV axis just below the position where a marked increase in multiunit discharge was detected via an audio amplifier (model 3300, AM-systems, Inc) and a prominent theta rhythm could be observed in the LFP. This corresponded to a relative position of both poles of the electrode across the pyramidal layer of CA1. Electrodes were then fixed to the skull by jeweler's screws and dental acrylic. Mice were allowed to stabilize for an hour before recordings took place. All recordings were made in a small, quiet, and artificially lit room and were always made while mice were positioned (using indwelling ear bars) in the stereotaxic apparatus which was oriented away from the centre of the room on a vibration proof table. They were spontaneously breathing room air. Thus, they had extremely limited (and virtually unchanging) external visual, auditory, somatosensory, and olfactory stimulation. All signals were sampled at 2 kHz following automatic antialiasing filtering using a PowerLab 16/30 data acquisition system (A D Instruments Inc., Colorado Springs, CO). At the end of the experiment, mice were euthanized by urethane overdose. Data analysis During urethane anesthesia, REM-like and SWS-like states were identified by performing spectrographic analysis on moving 6s intervals and calculating the percentage of total power at specific frequencies in LabChart Pro7 software. In this set of experiments, we used hippocampal LFPs in order to clearly identify the activated (i.e., REM-like) state by the expression of activity in the theta frequency range (3-4Hz) and the deactivated state (e.g. SWS-like) by the expression of the slow oscillation (~1Hz). Since spontaneous brain state alternations can be observed simultaneously from both cortical and hippocampal recording sites in rats, measurements in either site provide an accurate identification of forebrain state [13][14][15]. Indeed, we have found in these earlier studies that hippocampal theta provides a more sensitive index of forebrain activation. Since hippocampal activated (REM-like) states are characterized by a 3-4Hz theta rhythm, this state was defined when the power in the HPC recordings at the 3-4 Hz interval was ≥ 40% of total power for 3 consecutive 6 s intervals (FFT size 16K, Cosine Bell data window with 25% overlap). Conversely, a deactivated (SWS-like) state was defined when hippocampal power in the 0.2-1.2 Hz bandwidth was ≥40% of total power for 3 consecutive 6 s intervals. Periods that did not meet these two conditions (when power in the 3-4 Hz and 0.2-1.2 Hz bandwidths was < 40% of total power) were identified as transition states. Transition states have also been characterized during both urethane anesthesia and natural sleep in rodents as demonstrating transient (less than 0.5s in duration) neocortical oscillatory (7-15 Hz) spindle events, consistent with activity during stage 2 nREM sleep [13]. The rhythmic interaction of forebrain state alternations and fluctuations in body temperature, previously shown to be linked in urethane-anesthetized rats [16], were assessed in mice with continuous recordings of temperature (n=4). For this purpose, brain state was characterized by the log ratio of power in the theta and slow oscillation ranges from hippocampal LFP recordings. Using custom Matlab code (V 5.3, The MathWorks, Natick, MA), both power and temperature signals were down sampled to 10Hz, detrended and normalized to have peak to peak amplitudes of 1. Both autocorrelation and cross correlation functions were assessed on these processed signals, again in Matlab. The average period was calculated from the time of the first positive peak at non zero lag in the autocorrelation function and the lag across signals was similarly calculated from the time of the first positive peak closest to the zero lag in the cross correlation function. Phase angles were calculated by dividing the time lag across signals by the period of the alternations and multiplying by 360. Raw respiratory muscle EMG activity was rectified and integrated (time constant decay 0.08s); baseline and peak amplitude was calculated with the peak analysis application in LabChart7 Pro software. Respiratory period was obtained by measuring the time between two consecutive peak inspiratory events. In order to measure respiratory variability we obtained the coefficient of variation of the period by calculating the standard deviation of the respiratory period divided by its mean for each state across experiments. Sighs were identified by the presence of an augmented respiratory effort (>50% increase in integrated GG EMG peak amplitude in comparison to a regular breath) [17][18][19][20]. Single values were averaged according to the state and 1-tail paired t tests (for measurements across different states) and 2tail unpaired t tests (for comparisons across the two mice strains) were used to determine significance of results. Data values are reported as mean ± standard error of the mean. Brain state alternations in mice under urethane anesthesia Similar to what has been previously demonstrated in adult rats [13][14][15], 10 of 13 mice under urethane anesthesia displayed clear spontaneous brain-state alternations (Figure 1). In the 3 remaining mice, two were excluded for poor quality LFP signals that made automated determination of brain state difficult (although brain state alternations were apparent visually) and one did not display any systematic alternations despite good quality recordings. In total, we analyzed 24.33 hours of spontaneous recordings from 10 mice (5 CD1 and 5 C57BL/6). There were no significant differences in the various parameters extracted from any EEG and respiratory signal between the two strains (p>0.05 in all t-test between strains), therefore we report data both for individual strains and pooled data ( Table 1). Brain activity displayed quasi-rhythmic transitions between an activated (REM-like) state and a deactivated (SWS-like) state. The REM-like state was characterized by high power in the theta frequency bandwidth (range: 3.5-4.5 Hz) with a corresponding increase in power through the gamma band (25-40Hz), consistent with the expression of gamma activity during both natural sleep and during anesthesia in previous studies [21,22]. The SWS-like state was characterized by high power slow oscillation (SO) bandwidth (range: 0.2-1.2 Hz). In between these two states, a transition period was identified which exhibited intermediate electrographic characteristics (<40% power in both theta and SO frequency bands). The average duration (period) of state alternations was 13.90±1.38 min (CD1= 11.22±2.11min, n=5; C57BL/6= 16.15±1.32min, n=5; p=0.08; table 1), again comparable to that shown in rats [13]. During these alternations, the average time spent in the activated (REM-like) phase was 3.85±0. 48 Once mice developed spontaneous brain state alternations, the state changes persisted for several hours, further supporting the hypothesis that the alternations are intrinsic to the anesthetic effect induced by urethane and not generated by a gradual waning of the anesthetic level. Indeed, as previously confirmed in rats [13], we ensured that mice were always at a surgical plane of anesthesia, regardless of state. Although variable in timing across animals, the fluctuations in power and the period at which they alternated tended to be consistent within the same animal with occasional transient shortenings. Since our conditions were always similar across animals, it is unlikely that this variability was due to environmental conditions. In a subgroup of mice (n=4), we simultaneously measured core body temperature in order to evaluate any coupling between this measure and brain state alternations ( Figure 2). As previously shown in rats [16], rhythmic coupling was observed in all cases (albeit in 1 case, the coupling was weaker and transient across samples). This was observed from the similar period of fluctuations of power and temperature as shown by autocorrelation functions and by a parallel rhythmicity in the cross correlation function. Cross correlation functions between power ratios describing forebrain state alternations and body temperature showed that temperature peaks lagged behind those of power at an average of 3.68 ± 0.62 minutes (corresponding to an average Raleigh phase angle of 69.7°). Interestingly, this coupling was strongest for experiments in which the period of state alternations was extended. Indeed, in the above data, the average period of state alternations was 19.15±0.37 minutes. In a further subgroup of six mice (4 C57BL/6 and 2 CD1mice), we also assessed the influence of additional doses of anesthesia on cyclic state alternations (Figure 3). In four animals, supplemental doses of anesthesia (2-4mg -0.01-2ml) did not affect the duration nor the periodicity of brain state When active, GG EMG was consistently rhythmic and in phase with inspiration ( Figure 4). In 8 out of 10 mice, the integrated amplitude of GG EMG activity was significantly modulated by state. Integrated peak amplitude of GG EMG during REM-like states was reduced on average to 82.3±4.0% (p=6.6x10 -3 ) of SWS-like state values and to 88.5±3.2% (p=0.04) of transition state values (n=8). In one of the remaining mice the amplitude of GG EMG activity remained consistent across brain states and was rhythmically related to inspiration whereas in another mouse we could not detect any respiratory related signal. These changes are consistent with those observed in rats during both natural sleep and under urethane anesthesia [14,28,29]. ABD EMG activity was either absent (n=3) or had expiratoryrelated rhythmicity (n=4). In these latter mice, ABD activity displayed state dependent modulation, with integrated peak activity in REM-like state that was increased by 22.1±8.2% (p=0.02) as compared to transition states and increased by 59.6±25.6% (p=0.03) as compared to SWS-like states ( Figure 3). This was again consistent with previous work performed in adult rats, where, although variable in occurrence, expiratory ABD EMG activity displayed a prominent association with state in both urethane anesthesia and natural sleep [14]. In contrast to the above changes described for spontaneous state alternations, we additionally found that there was a marked difference in EMG activity elicited by a noxious stimulus delivered to the pad or tail which concomitantly induced a transient forebrain activation of hippocampal EEG. In both GG and INT traces, the delivery of this activating stimulus was associated with a modest increase in muscle tone. This suggests that brain activation achieved by noxious stimuli is not identical to that elicited spontaneously. Urethane anesthesia Urethane anesthesia has been widely used for a variety of physiological, and especially neurophysiological, experimental paradigms. Despite a relatively common usage, its exact mechanism of anesthetic action is still unknown. Urethane is considered to be a non-typical general anesthetic due to its only moderate effects on both excitatory and inhibitory neurotransmission [30] in contrast to other commonly used veterinary/scientific anesthetics (isoflurane, propofol, ketamine, and barbiturates), which typically display a primary action of promoting inhibitory GABAergic neurotransmission, reducing excitatory glutamaterigic transmission, or both [31][32][33][34]. One of urethane's most prominent cellular effects is the hyperpolarization of cortical neurons via potentiation of a resting potassium conductance [35]. Interestingly, modulation of potassium conductances that are active in the sub-threshold range (albeit different varieties) has been proposed to be important for producing a "sleepless" Drosophila mutant [36] and in the neurophysiological actions of adenosine, a classic and naturally occurring somnogenic agent [37]. This leads to the speculation that modulation of intrinsic membrane excitability as opposed to actions on either excitatory or inhibitory synaptic transmission may be a common mechanism for the maintenance of unconsciousness during both urethane anesthesia and natural sleep. Although our group was not the first to describe spontaneous brain state alternations under urethane anesthesia, a common and continuing mis-interpretation is that the activated state is indicative of a reduced (wake-like) level of anesthesia [38][39][40][41]. We previously, and conclusively, demonstrated that this is not the case with urethane-anesthetized rats [13]. Across brain state alternations, there was no change in the level of responsiveness to a painful stimulus nor was there an increase in tonic skeletal muscle tone when transitioning to activated states. Indeed spontaneous activated states under urethane, as with natural REM, were often characterized by a reduction in tonic skeletal muscle tone [13]. This is in contrast to the effects of delivering noxious stimuli to urethane anesthetized animals that can also activate the forebrain but that increase skeletal (as well as GG and INT EMG respiratory) muscle tone. In the present study, severe tail or hind paw pressure could activate both forebrain state and GG/INT EMG , but spontaneous changes into activated states showed the opposite profile on GG EMG activity and had no effects on INT EMG . Thus, given the host of other physiological variables that also parallel changes across the sleep cycle including temperature and ventilation [14,16], as well as the influence of pharmacological and brainstem manipulations known to influence sleep states [13] we conclude that the spontaneous alternations present under urethane most closely resemble the natural sleep cycle between REM and SWS. In a previous study [16] we discovered that thermal manipulations, similar to natural sleep, had a significant impact on brain state in urethane-anesthetized rats and could be an entraining influence upon their alternations. We also showed that despite this influence, brain state alternations and temperature fluctuations could be shown to be mutually independent, although they are likely coupled biological oscillators. In the present study, we took great care to ensure that any external fluctuations of heating were minimized by using a homeostatic system without marked feedback cycling. Even with the use of this system, like with our previous results in rats, we were able to demonstrate a significant and strong sleep-like coupling of brain state alternations to core temperature fluctuations in a majority of experiments. Our present results show that spontaneous transitions are not simply a species-specific reaction of rats to urethane anesthesia but that they also occur in mice. Similar to rats, the timing of brain state alternations is comparable to spontaneous uninterrupted SWS/REM cycles occurring in natural sleep of mice [7,9]. Furthermore, although our sampling was limited (n= 5 in each case), we did not observe any significant differences in neurophysiological or respiratory measures between the two different mouse strains we used; the outbred CD1 and inbred C57BL/6 strain. These results contrast with the reported changes in ventilatory behavior across different inbred mice strains [10,[42][43][44], although it has been shown that possible strain differences may be hampered by the use of urethane anesthesia [42]. We are not aware of any direct comparison of ventilator parameters between C57BL/6 and CD1 mice strains, however differences in neuroanatomy, behavior and pharmacological properties have been reported [45][46][47][48]. The current results suggest that mice under urethane anesthesia present a stereotyped alternation of brain state and respiratory behavior that makes differences between CD1 and C57BL/6 strains indistinguishable. State dependent modulation of breathing in urethane anesthetized mice. Similar to what we reported in adult rats [14], we observed obvious state dependent changes in several respiratory parameters in mice. In contrast to our previous results in rats, transition states (characterized by weak power in the hippocampus at both 4Hz and 1Hz frequencies) displayed respiratory characteristics more similar to the SWS-like states than REM-like states. This phenomenon may be due to differences in the intrinsic properties of the neuronal networks that control respiratory parameters in mice. Nonetheless, in mice, respiratory rate and respiratory variability were significantly increased during REM-like states compared to SWS-like and transition states. These changes reproduced what happens in natural sleep of both humans [23][24][25][26][27] and rodents [10,[12][13][14]49]. Sigh rate was higher in REM-like state versus SWS-like state, similar to what we observed in urethane anesthetized rats and what is observed in natural sleep in humans [25][26][27] and in rats [14]. Interestingly, very few studies have analyzed sigh frequency in mice across sleep states and they reported a reduction in sigh/hr during REM state compared to SWS [12,50]. This discrepancy between human and rat natural sleep data and our urethane anesthetized data in mice may be due to the very limited time mice spend in REM epoch (≤ 2min) [7,8] which makes sigh rate calculations extremely challenging or impossible in addition to the fact that sighs can often occur at the transitions between SWS and REM states [14]. When the activity of different respiratory muscles was examined, including INT, GG, and ABD, we detected systematic differences across states as well. For INT muscle activity there was no significant change across states, similar to our previous results in urethane anesthetized rats [14] and naturally sleeping rats [49,51], where both diaphragm and INT muscles do not show significant and consistent changes in EMG activity across states. However, reduced activity in external INT muscle is sometimes observed in humans during REM compared to SWS states [52]. The activity of the GG muscle was consistently reduced in REM-like epochs compared to SWS-like and transitions states, similar to what happens in natural sleep of humans and rodents, where both tonic and respiratory modulated activity of the GG muscle progressively declines as sleep progresses from wakefulness to SWS and REM sleep [14,28,29,[53][54][55]. We are not aware of studies on GG EMG activity in unanesthetized mice across sleep/wake cycles, but, as demonstrated by activity of the correlated masseter muscle in mice and humans [56,57], we are quite confident that our data on GG EMG activity in urethane anesthetized mice replicates the state dependent modulation that is present in humans, rats, and very likely in mice. These results further support the opportunity of using urethane anesthesia for the study of mechanisms governing state dependent modulation of GG activity in mice as well. Similar to our results in both natural sleep and under urethane anesthesia [14], we observed occasional increases in expiratory-related muscle activity at the level of ABD muscles. These results suggest that in the activated states of urethane anesthetized mice, expiratory related activity may also be recruited in order to favor ventilation when inspiratory activity is more irregular and variable. These results support the hypothesis that under urethane anesthesia, state dependent modulation of respiratory muscles occurs in a fashion that reproduces what happens in natural sleep, therefore allowing for mechanistic analysis of respiratory muscle control in an anesthetized preparations that display spontaneous and predictable changes that replicate phenomena occurring during natural sleep. In conclusion, the observation that urethane anesthesia recapitulates natural sleep-like alternations in mice as well as in rats, opens the possibility of applying this model of sleep to study cellular and network mechanisms in different transgenic mice that display aberrant brain activity, sleep pattern and/or respiratory disturbances, such as mouse models for respiratory disorders (congenital central hypoventilation syndrome, Prader Willi syndrome) or transgenic mice that display either constitutive or inducible activation or silencing of specific neurons involved in brain state alternations or in respiratory control across states. Author Contributions Conceived and designed the experiments: SP CTD. Performed the experiments: SP. Analyzed the data: SP CTD. Contributed reagents/materials/analysis tools: SP SG CTD. Wrote the manuscript: SP SG CTD.	2016-05-04T20:20:58.661Z	2013-07-30T00:00:00.000	{ "year": 2013, "sha1": "68d1c105ce061da086a32f41b040bace88c70101", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0070411&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "68d1c105ce061da086a32f41b040bace88c70101", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
251935140	pes2o/s2orc	v3-fos-license	STAT3 mutations in “gray-zone” cases of T-cell large granular lymphocytic leukemia associated with autoimmune rheumatic diseases A persistently increased T-cell large granular lymphocyte (T-LGL) count in the blood of more than 2 × 109/L for at least 6 months is necessary for a reliable diagnosis of T-LGL leukemia. In cases with LGL counts of approximately 0.5–2 × 109/L, a diagnosis of T-LGL leukemia can be made if clonal rearrangement of T-cell receptor (TCR) genes is present and if the patient shows typical manifestations of T-LGL leukemia, such as cytopenia, splenomegaly, or concomitant autoimmune disease. However, in cases with LGL counts of less than 0.5 × 109/L, the diagnosis of T-LGL leukemia is questionable (termed as “gray-zone” cases). Although mutations in signal transducer and activator of transcription 3 (STAT3) gene are the molecular hallmark of T-LGL leukemia, their diagnostic value in the “gray-zone” cases of T-LGL leukemia has not been evaluated – our study has been aimed to examine the prevalence of STAT3 mutations in these cases. Herein, we describe 25 patients with autoimmune rheumatic diseases, neutropenia, clonal rearrangement of TCR genes, and circulating LGL count of less than 0.5 × 109/L. Splenomegaly was observed in 19 (76%) patients. Mutations in the STAT3 were detected in 56% of patients using next-generation sequencing. Importantly, in 3 patients, no involvement of the blood and bone marrow by malignant LGLs was noted, but examination of splenic tissue revealed infiltration by clonal cytotoxic T-lymphocytes within the red pulp, with greater prominence in the cords. We suggest using the term “splenic variant of T-LGL leukemia” for such cases. Introduction Large granular lymphocytic (LGL) leukemia is characterized by the expansion of lymphocytes with abundant cytoplasm containing variably sized azurophilic granules and a reniform or round nucleus with mature chromatin (1). Approximately 85% of LGL leukemia patients exhibit T-cells with a mature post-thymic phenotype (1). T-cell LGL (T-LGL) leukemia has an indolent clinical course and involves the peripheral blood, bone marrow, and spleen (2). A typical manifestation of T-LGL leukemia includes neutropenia, splenomegaly, and LGL lymphocytosis, which are detected in up to 84%, 50%, and 52% of patients, respectively (3). Moreover, up to 40% of patients with T-LGL leukemia have concomitant autoimmune disorders, of which rheumatoid arthritis (RA) is the most common (1,4,5). Historically, a definitive diagnosis of LGL leukemia is made if the LGL count in the peripheral blood is more than 2 × 10 9 /L (normal range, < 0.3 × 10 9 /L) over a period of 6 months, with no clearly identified cause (3,6). Although this definition of T-LGL leukemia remained unchanged in the 2016 revision of the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, cases wherein the LGL count is less than 2 × 10 9 /L may be suggestive of this diagnosis (2). While a lower threshold of the absolute LGL count in the peripheral blood for T-LGL leukemia has not been defined by the WHO, the threshold of 0.5 × 10 9 /L is now generally accepted (5,7). In cases with LGL counts of approximately 0.5-2 × 10 9 /L, a diagnosis of T-LGL leukemia can be made if the LGLs are clonal and if the patient shows typical manifestations of T-LGL leukemia, such as cytopenia, splenomegaly, or concomitant autoimmune disorder (5,(7)(8)(9)(10). Infiltration of bone marrow by clonal T-LGLs and/or characteristic histopathological findings of LGL leukemia in the bone marrow with typical manifestations of T-LGL leukemia also confirm the diagnosis of T-LGL leukemia (8,11,12). However, cases with circulating LGL counts of less than 0.4-0.5 × 10 9 /L (termed as "gray-zone" cases) present a specific diagnostic challenge because of the difficulty in making a differential diagnosis with reactive T-LGL proliferation. Although T-cell clonality is necessary to distinguish T-LGL leukemia from reactive T-LGL proliferation, clonality does not equate to malignancy and is not enough to classify T-LGL proliferation as leukemia (13,14). Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway is a molecular hallmark of T-LGL leukemia (5). Activating point mutations in the STAT3 gene are found in up to 72% of T-LGL leukemia patients (15-18). Thus, detection of STAT3 mutations may serve as a valid reason for classifying the condition in diagnostically challenging cases as T-cell LGL leukemia (15). However, their prevalence in "gray-zone" cases has not been evaluated. In the present study, STAT3 gene mutations were evaluated using next-generation sequencing (NGS) in a cohort of 25 patients with autoimmune rheumatic diseases (ARDs), clinical presentation typical for T-LGL leukemia, clonal rearrangement of the T-cell receptor (TCR) gene, and absolute circulating LGL count of less than 0.5 × 10 9 /L. Patients and methods The inclusion criteria were as follows: patients aged > 18 years with diagnosed ARD and absolute neutrophil counts of < 1.5 × 10 9 /L. From 2008 to 2021, 106 patients admitted to V.A. Nasonova Research Institute of Rheumatology met the inclusion criteria and were included in the primary analysis. A flowchart of the patient selection process is shown in Figure 1. Peripheral blood smears were available for 83 patients. Manual differential tests were performed using Wright-Giemsastained peripheral blood smears to quantify the LGL percentage and calculate the absolute LGL counts. The circulating LGL count was ≤ 0.5 × 10 9 /L in 39 patients. In 25 of the 39 patients, T-cell clonality testing revealed clonal populations. These 25 patients were included in the final analysis. The clinical data of patients were collected, including age of onset of neutropenia or splenomegaly, sex, rheumatologic diagnosis, and bone marrow differential counts. Splenic tissue specimens (from 5 patients) and bone marrow trephine biopsies (from 15 patients) had previously been fixed in 10% formalin, routinely processed, embedded in paraffin, and stained with hematoxylin and eosin. Screening for STAT3 mutations was performed on samples in which a monoclonal TCR gene rearrangement pattern was detectable. For samples with a polyclonal TCR gene rearrangement pattern, STAT3 mutation screening was conducted if a monoclonal TCR gene rearrangement pattern was detected in other tissue samples from the same patient. For example, if a monoclonal TCR gene rearrangement was detected in the bone marrow but was absent in the blood sample, screening for STAT3 mutations was performed in both the bone marrow and blood samples. Ethics statement This study was approved by the Ethics Committee of the V.A. Nasonova Research Institute of Rheumatology (protocol #10 on the 20-01-2022). All patients gave written consent for collection, analysis of specimens and for publication of their data as results of the study. Evaluation of T-cell clonality T-cell clonality was examined using genomic DNA extracted from blood (25 patients), bone marrow (16 patients), and spleen tissue (5 patients) samples. T-cell clonality was evaluated based on the rearrangements of TCR-γ (Vγ-Jγ) and TCRβ (Vβ-Jβ, Dβ-Jβ) in all patients. T-cell clonality assays were performed according to the BIOMED-2 standardized protocol (19). Polymerase chain reaction (PCR) amplification was conducted using an automated DNA Engine Thermal Cycler (Bio-Rad, Hercules, United States), and fragments were detected using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, United States). The data were analyzed using GeneMapper software version 4.0 (Applied Biosystems). Detection of a weak clonal signal in the context of polyclonal signals was interpreted as ambiguous clonality. False positive results (pseudo-monoclonality) were excluded by identical dominant PCR products in repeated PCR analyses of the same sample and/or identical dominant PCR products in different test samples (blood, bone marrow, or spleen samples) from the same patient. Evaluation of STAT3 mutations STAT3 mutations were examined using genomic DNA extracted from specimens of peripheral blood (24 patients), bone marrow (15 patients), and the spleen (2 patients). Mutations in exons 19-21 of the STAT3 gene were identified by NGS. Appropriate DNA regions were amplified using primers for exons 19-20 (product length 502 bp) and exon 21 (product length 522 bp), as described previously (15). Statistical analysis Descriptive statistics are presented as numbers and percentages for categorical data and as medians and ranges for continuous data. A two-sample test for equality of proportions with continuity correction was used for statistical analysis. A p-value of < 0.05 was considered statistically significant. Results The clinical, laboratory, and pathologic characteristics of the 25 patients included in the final analysis are shown in Table 1. Overall, 22 patients had RA, 2 had systemic lupus erythematosus (SLE), and 1 had primary Sjogren's syndrome (SS). Six patients had SS in combination with other rheumatic diseases (secondary SS): 4 with RA and 2 with SLE. The median age of the patients at the time of neutropenia detection and/or splenomegaly was 58 years (range, 35-71 years). The female-to-male ratio was 3.2:1. Splenomegaly was observed in 19 (76%) patients, among whom 5 underwent splenectomy for diagnostic or therapeutic purposes. The absolute lymphocyte counts in the blood ranged from 0.24 to 1.782 × 10 9 /L, with a median of 0.96 × 10 9 /L. Bone marrow aspirate differential counts were available for 23 patients. In all cases, there were no signs of myelodysplasia. The proportion of lymphocytes in the bone marrow was between 5.2% and 41.6% (median 17.8%) of all nucleated cells, but only 4 (17%) patients had bone marrow lymphocytosis, defined as exceeding the upper limit of lymphocytes of > 23.8% of nucleated bone marrow cells. Interstitial/intrasinusoidal distribution of cytotoxic T-cells in the bone marrow was detected in 10 (67%) of the 15 patients examined. Examination of splenic tissue revealed lymphoid infiltration of both splenic cords and sinusoids within the red pulp, with greater prominence in the cords. The white pulp was preserved and contained prominent germinal centers in 3 patients and was atrophied in 2 patients. Immunohistochemical staining revealed that the lymphocytes infiltrating the red pulp were CD3 + /TIA1 + in all patients and CD16 + in 4 patients. Beta-F1 and TCR-γ positivity was observed in 3 and 1 patient, respectively. In another patient, approximately one-third of the T-cells expressed Beta-F1, and another third of the T cells expressed TCR-γ. In addition, approximately one-third of the T-cell population did not express TCR. Lymphocytes were CD8 + /CD4 − in three TCRαβ + cases and CD8 − /CD4 − in the remaining cases. Clonal (or ambiguous clonal) rearrangement of TCR genes in the peripheral blood was detected in 19 of the 25 patients: TCR-γ gene in 7 (28%) of the 25 patients and TCR-β gene in 18 (72%) of the 25 patients. Out of 16 bone marrow samples examined, clonal (or ambiguous clonal) rearrangement of TCR genes was detected in 13 cases (in 3 of these patients, it was not detected in the blood): TCR-γ gene in 7 (44%) of 16 patients and TCR-β gene in 13 (87%) of 15 patients (in one case this study was not performed). Clonal TCR-β gene rearrangements were detected more often than TCR-γ gene rearrangements both in the peripheral blood (72 vs. 28%, p = 0.004678) and bone marrow samples (87 vs. 44%, p = 0.03399). Clonal rearrangements of TCR genes in the spleen samples were detected in all 5 patients examined: TCR-γ gene in 4 of 5 patients and TCR-β gene in 3 of 4 patients (in one case this study was not performed). Overall, a clonal (or ambiguous clonal) rearrangement of TCR genes was detected in the blood and/or bone marrow samples in 22 (88%) of the 25 patients. In the 3 remaining patients, it was found only in the spleen samples. Point mutations in the STAT3 gene were identified in 14 (56%) of the 25 patients examined, including D661Y (5 patients), Y640F (4 patients), S614R (2 patients) and N647I, Y657_K658insY, K658R, N664T, and E616G in one patient each. Double mutation in the STAT3 gene was observed in 2 patients. STAT3 mutations were detected in 11 (46%) of 24 blood samples, 9 (60%) of 15 bone marrow samples, and in both the spleen samples (100%). In one bone marrow sample, STAT3 mutation was detected in the absence of a clonal rearrangement of the TCR genes. Discussion Clonal proliferation of T-LGLs has been detected in various clinical conditions, including autoimmune disorders, myeloid or lymphoid clonal hematologic malignancies, pure red cell aplasia, aplastic anemia, and paroxysmal nocturnal hemoglobinuria (29)(30)(31)(32)(33). In addition, small clonal proliferations of T-LGLs have been observed after allogeneic stem cell transplantation (34,35) and solid organ transplantation (36-38) as well as in patients with HIV (39) or cytomegalovirus infection (40). It is likely that some of these cases may represent an intensive immune response to antigen exposure more than would be expected for physiological T-cell clones, rather than true T-LGL leukemia. Although the etiology and pathogenesis of T-LGL leukemia remains unclear, the triggering event underlying T-LGL leukemia is believed to be related to chronic antigenic stimulation initially leading to a poly-or oligoclonal expansion of LGLs (41, 42). The proliferation of LGLs is supported by several cytokines, including interleukin (IL)-6, IL-12, and IL-15 (43)(44)(45)(46)(47). The activating point mutations of STAT3 in these antigen-driven LGLs can cause aberrant STAT3 signaling and, in combination with other factors, ultimately lead to the monoclonal expansion of LGLs (42,48). Monoclonal expansions of CD8 + T-lymphocytes have also been detected in healthy individuals (49-52). Bigouret et al. suggested that T-LGL clones exist in a significant number of healthy individuals and clinical symptoms, such as neutropenia and autoimmune diseases, can occur when T-LGL clone encounters an antigen and begins producing large amounts of effector molecules (49). The diagnosis of T-LGL leukemia in cases with small clonal populations in blood has been debated. The term "Tcell clonopathy of undetermined significance" was coined to emphasize the indolent clinical course of T-LGL leukemia in many patients and to eliminate the stigma associated with the term "leukemia." The term "T-cell clonopathy of undetermined significance" has been used regardless of the peripheral blood LGL count, clinical symptoms, and hematological abnormalities observed in patients (53,54). Subsequently, the term "T-cell clone of undetermined significance" (TCUS) was proposed for patients with a small clonal population of T-LGL and no diagnostic features of T-LGL leukemia (51,55). However, the diagnostic threshold of circulating LGLs to distinguish T-LGL leukemia from TCUS has not been defined, and the researchers proposed a threshold range from 500 cells/µL (51) to 2,000 cells/µL (55). In addition, the immunophenotypic signature of TCUS closely resembles that of T-LGL leukemia (51), and the immunohistochemical pattern of bone marrow in TCUS has not been studied yet. Activating point mutations in STAT3 are not pathognomonic for T-LGL leukemia and can be detected in various hematological and non-hematological malignancies (56). In addition, Kim et al. recently found somatic STAT3 mutations in CD8 + T-cells of healthy blood donors carrying the human T-cell leukemia virus type 2 (57). However, in an appropriate clinical context, STAT3 mutations are a molecular marker that is highly specific for T-LGL leukemia (15)(16)(17)(18)56). Nevertheless, the significance of STAT3 mutations in distinguishing T-LGL leukemia from TCUS is not clear. Similar to the MYD88L265P mutation, which alone is not an absolute diagnostic marker for Waldenstrom's macroglobulinemia and is found in 50-80% of patients with IgM monoclonal gammopathy of uncertain significance (58), STAT3 mutations alone are unlikely to allow a reliable diagnosis of T-LGL leukemia when clonal T-LGL proliferation is found. Monoclonal B-cell lymphocytosis (MBL) is a hematological condition characterized by the presence of a monoclonal population of B-lymphocytes with a count of less than 5 × 10 9 /L in the peripheral blood and without other features of a B-cell lymphoproliferative disorder, such as cytopenia, organomegaly, lymphadenopathy, or extramedullary involvement (59). The genomic aberrations characteristic of chronic lymphocytic leukemia (CLL) can be found in cases of MBL with an immunophenotype similar to that observed in CLL (60). Drawing an analogy between TCUS/T-LGL leukemia and MBL/CLL, it seems logical that the distinction between T-LGL leukemia and TCUS should be based on the blood LGL count and/or the presence of signs of T-LGL leukemia, such as cytopenia and/or splenomegaly. Although all patients in our cohort had absolute LGL counts in the peripheral blood below the currently accepted threshold of 0.5 × 10 9 /L, the typical manifestations of T-LGL leukemia, including neutropenia and splenomegaly, allowed us to classify these cases as T-LGL leukemia rather than TCUS. Patients with STAT3 mutations develop RA and neutropenia more often than patients without these mutations (15,16,61). This likely explains the higher frequency of STAT3 mutations in our cohort than in T-LGL leukemia patients in two recently reported large studies: 56% vs. 36%-40% (61, 62). The majority (88%) of patients in our cohort had RA. Manifestations of T-LGL leukemia in the setting of RA are sometimes clinically indistinguishable from Felty's syndrome (FS), a rare subset of seropositive RA complicated by neutropenia and often splenomegaly. The detection of a clonal rearrangement of TCR genes and/or an expanded T-LGL population (≥ 2 × 10 9 /L) supports the diagnosis of T-LGL leukemia but not of FS (63-66). As reported previously, PCR-based TCR gene rearrangement analysis according to the BIOMED-2 protocol followed by GeneScan analysis made the detection of clonal T-cell populations possible with a sensitivity of 5% (19, 67). Due to the low clonal expansion of T-LGLs in "gray-zone" cases, detection of a clonal peak may be impossible or questionable, making diagnosis much more difficult. Immunohistochemical examination of bone marrow biopsies is recommended in "gray-zone" T-LGL leukemia cases (7), but due to the expansion of reactive cytotoxic T-lymphocytes in patients with RA, this study cannot reliably distinguish T-LGL leukemia in the setting of RA from FS (64,66). Because TCR-γ gene rearrangement occurs in both αβTCR + and γδTCR + T-cells, it is the best target for analysis of T-cell clonality (19, 67). On the other hand, the TCR-β gene has more expansive combinatorial diversity in contrast to the TCR-γ gene, making the TCR-β gene the preferred target for identifying a true clonal process in αβTCR + proliferation. In our patient cohort, a clonal rearrangement of the TCR-β gene was detected significantly more often than monoclonal rearrangements of the TCR-γ gene both in the peripheral blood and bone marrow. According to the BIOMED-2 group recommendations (68, 69), TCR-γ and TCR-β gene rearrangements were analyzed simultaneously in our work. Blood and bone marrow examination provided complementary information and allowed the detection of T-cell clonality in 22 (88%) of the 25 patients. Three patients in our cohort presented a particular diagnostic challenge. These patients had seropositive RA, severe neutropenia, and massive splenomegaly, which, in the absence of evidence for clonal proliferation of LGLs in the blood and bone marrow, allowed an initial misdiagnosis of FS. Morphological examination of spleen samples showed infiltration of cords and sinusoids of the red pulp by cytotoxic T-lymphocytes with more marked involvement within the splenic cords, which is characteristic of spleen involvement by T-LGL leukemia (70). A monoclonal TCR gene rearrangement was detected in the spleen in all the 3 patients, and a STAT3 mutation was present in 1 of them. These patients presented an unusual variant of T-LGL leukemia, in which tumor cells are localized in the spleen, while very few or no tumor cells in the blood and bone marrow prevented the diagnosis of T-LGL leukemia. Because the term "leukemia" is used when there is extensive peripheral blood and bone marrow involvement, we suggest the term "splenic variant T-LGL leukemia" for such cases. In conclusion, we have described a cohort of patients with various ARDs and expansion of T-LGLs of less than 0.5 × 10 9 /L in the peripheral blood. Such cases present a diagnostic challenge because of the low clone size and require both TCR-γ and TCR-β gene rearrangement analysis in the blood and bone marrow. Although cases with a very small clonal population of T-LGLs remain the subject of terminological debate (T-LGL leukemia versus TCUS), clinical manifestations such as neutropenia and splenomegaly have allowed us to classify these cases as T-LGL leukemia. STAT3 mutations were detected by NGS in peripheral blood, bone marrow, and spleen in a total of 56% of patients in our cohort, providing an argument for considering these cases as atypical aleukemic presentation of T-LGL leukemia. In diagnostically unclear cases of neutropenia and/or splenomegaly in patients with ARDs, the detection of STAT3 mutations by NGS can indicate the diagnosis of T-LGL leukemia. Data availability statement The data presented in this study are deposited in the SRA repository, accession number: PRJNA867539. Ethics statement The studies involving human participants were reviewed and approved by the V.A. Nasonova Research Institute of Rheumatology (protocol #10 on the 20-01-2022). The patients/participants provided their written informed consent to participate in this study. Author contributions VG: data collection, analysis and interpretation, literature search, study design, and writing -review and editing the manuscript. YS, BB, NK, NR, and AS: data collection, analysis and interpretation, literature search, editing, and reviewing of the manuscript. All authors contributed to the manuscript and approved the submitted version.	2022-08-31T13:38:29.881Z	2022-08-31T00:00:00.000	{ "year": 2022, "sha1": "3a72bbc93771e3c0505e100b9001fb761527e792", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Frontier", "pdf_hash": "3a72bbc93771e3c0505e100b9001fb761527e792", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [] }
20558307	pes2o/s2orc	v3-fos-license	A Preliminary Investigation of the Interactions between the Brood Parasite Chalcoela iphitalis and Its Polistine Wasp Hosts The life history of Chalcoela iphitalis—a common brood parasite of social wasps—has been described in previous literature, but critical information regarding oviposition behavior and possible differential host parasitism remain cryptic. Here we report on infestation levels of this moth in field populations of paper wasps in Polistes and Mischocyttarus, as well as the oviposition behavior of the moths under a laboratory setting. We found evidence for differential parasitism between paper wasp genera in the field, with almost 50% nest infestation in P. bellicosus and no occurrences of moth infestation in M. mexicanus. Laboratory results revealed that oviposition occurs only at night and is stimulated by contact with the wasp nest or adult wasps. In this setting, eggs were laid largely on the substrate above or adjacent to the host nest, but not on the nest itself. Introduction The nests of eusocial paper wasps often support a diverse array of insect parasites and symbionts [1]. Understanding these insects with which paper wasps have co-evolved can ultimately help us understand the paper wasp ecology. The sooty-winged Chalcoela moth, Chalcoela iphitalis (Walker), is a brood parasite in the family Crambidae [2] which attacks Polistine wasps, including at least ten species of Polistes and one species of Mischocyttarus in the US and Central America [3]. However, evidence from a ten-year study indicated that C. iphitalis did not show a host preference between P. fuscatus and P. dominula [4]. Moth larvae feed on pupal or pre-pupal wasps before spinning silken cocoons containing layers of air pockets within wasp brood cells. Moth silk is not chewed through or removed by the wasps, and newly laid eggs and early instar wasp larvae can sometimes be observed on top of moth silk [3]. C. iphitalis overwinter inside the abandoned wasp nest and are typically bivoltine, with an adult emergence in the spring as well as late summer when nests containing brood are present. Infestations can be commonly observed, with up to 73% of nests parasitized in a population of one wasp species [5]. Parasitism of a population of Polistes exclamans in Texas by C. iphitalis has been detailed by Strassmann [5] over the course of three years. The average number of mature cells infested per nest ranged from 19-34% and varied significantly between years. Infestations usually peaked around July or August, and C. iphitalis may avoid ovipositing in previously infested nests. Each successful C. iphitalis larva kills one wasp pupa, but holes between cells were observed, indicating that some moth larvae may be feeding on multiple pupae. The ability of C. iphitalis to destroy a large percentage of wasp pupae in a small period of time may have a large impact on worker replacement at the nest. Strassmann and Thomas [6] conducted a principal component analysis on a population of P. exclamans and found that nest decline was correlated with C. iphitalis infestation. A similar study concluded that heavy infestations of C. iphitalis can be a primary cause of colony failure. Moth diapause inside of old nests may be a cause for the rarity of nest re-use by paper wasps [7]. Paper wasp nests attach to a variety of substrates that include natural vegetation and manmade structures, and nest location is thought to be an important aspect related to moth invasion [1]. In one study, approximately 60% of the Polistes nests located on manmade structures in Illinois were parasitized by C. iphitalis, whereas only 20% of nests built on trees or shrubs were parasitized, suggesting that nests in vegetation may be less preferred or difficult for moths to locate [8]. Strassmann [5] notes that this moth lays eggs in wasp nests at night. Strassmann [5] and West-Eberhard [9] also described the parasite alarm reaction of adult wasps when encountering a moth, during which wasps will react violently by biting or stinging the area where the moth had been and subsequently alarming other wasps through vibrations and wing flipping on the nest. Wasps continuing the alarm behavior will sometimes leave the nest and walk over the substrate for up to 10 h after initial detection of the moth [5]. Mechanisms by which the moth parasite is able to bypass the host defense is still unknown. The objective of this study was to determine parasitism rates in wasp species found in the Baton Rouge, Louisiana area, factors related to the parasitism, as well as oviposition behavior in the laboratory. To achieve this, we examined the behavior of both host and parasite in the laboratory, as well as the occurrence of C. iphitalis in field populations of Polistine wasps in southern Louisiana. Field Study Southern Louisiana study sites encompassed areas in Baton Rouge and St. Gabriel including Bluebonnet swamp, Burden Research Station, LSU Reproductive Biology Center, and LSU main campus. Areas were systematically searched for Polistine nests throughout the spring and summer of 2016. Because air temperature is a major factor affecting wasp activity [10], searches were carried out mostly on warm and sunny days when wasps were active, with search increments of 3-4 h each day. Areas for visual search included sheltered sites such as palm fronds, picnic shelters, eaves of buildings, as well as low shrub vegetation. Observations of wasps flying directly into a location sometimes led to the discovery of a nest in that manner. Upon discovery, each colony was identified to species and visited weekly for the duration of the season through colony decline. Presence of C. iphitalis infestation as well as percentage of cells infested were recorded. We used total cell number of nests that reached the pupal stage to calculate the percentage of cells with webbing (infested) for each species. Only nests that reached the pupal stage were included in our calculations due to the fact that C. iphitalis feeds only on pupal or pre-pupal wasp stages [3]. Laboratory Study In July of 2016, two moth-infested wasp nests (P. fuscatus and P. bellicosus) which had been abandoned by the wasps were collected in the field. The nests were brought back to the laboratory and placed together in the bottom of a 12.7 × 10.16 cm cylindrical clear plastic container, whereupon 29 C. iphitalis adults eclosed within two days. Two active Polistes dorsalis nests without evidence of previous C. iphitalis infestation were also collected from the field and glued to the top of identical separate plastic containers as noted above; however, these containers were inverted such that the lid served as the bottom of the cage. Both nests were of similar size, and one nest (A) consisted of two adult females while the other nest (B) consisted of four adult females. All brood stages (eggs, larvae, and pupae) were present in both nests, which were maintained in the laboratory and given access to honey, water, and wax worms daily under a natural photoperiod. The two moth-infested wasp nests and two active P. dorsalis nests were collected at Burden Research Station in Baton Rouge, LA, USA. To release moths from their cage to one of the two active P. dorsalis nests, container lids were removed and containers were placed together. This allowed the 29 moths in the lower cage to fly freely to the upper cage housing an active P. dorsalis nest for an immediate observation of 30 min. Two daytime (temperature = 23.1 • C and luminosity = 59) and two night-time (temperature = 21.9 • C and luminosity = 0) observations (28 and 29 July, data obtained from Onset HOBO ® UA-002 Pendant Temperature Light Data Logger) were conducted on each wasp nest. After the first 30-min daytime observation concluded, any moths remaining in the wasp cage area were placed back into their own cage, which was then placed underneath the next wasp nest for a 30-min observation. The behavior and interactions between wasp and moth were recorded. Because red light is not visible to most insects but provides light for observation by humans [11], a red light was utilized during night-time trials. After both 30-min night-time observations terminated, cages were separated and seven adult moths (from the 29 total) were placed in each wasp cage. Moths were then allowed further oviposition for 10 additional hours before being removed the next morning. This created a standard number of moths and oviposition time in each wasp cage. Removal of adult moths from wasp cages was facilitated by physical stimulation from a small paintbrush which encouraged downward flight into the moth cage. After each 10.5 h period, number of moth eggs and egg locations (wasp nest surface or cage surface including top, wall, or bottom) were noted in each wasp cage. In total, moths were permitted to oviposit in each wasp cage for 22 h (including daytime and night-time observational periods). Fourteen days after trials ended, two late instar wasp larvae were removed from each nest for examination of moth parasitism. Moth larval silk was noted in cells as evidence. A two-way ANOVA procedure (SAS ® 9.4, ©2016 Proc Mixed, SAS Institute Inc., Cary, NC, USA) was utilized to analyze differences between moth egg positioning (either top, wall, or bottom of cage) and egg number in both wasp cages. Polistes dorsalis Behavior under Laboratory Conditions Moths quickly encountered adult wasps at the nest (n = 9 observations, five in nest A and four in nest B) during night-time trials only, whereupon some wasps would display parasite alarm behavior with jerking movements and wing flipping [3]. The degree to which parasite alarm behavior was displayed varied greatly among individual wasps; some females left the nest in pursuit of the moth or continued this behavioral pattern for the remainder of the 30-min observational period (n = 3), while other individuals spent very little or no time (n = 5) performing the same behaviors after contact with moths. One wasp in nest A left the nest in pursuit of contacting moths three times, spending ≈40 s before returning to the nest each time; no other wasps left their nest during trials. In the first nest (A) containing two adults, one female exhibited a much higher degree of alarm than the other. The less-alarmed female raised her wings often during contact with the more-alarmed female, but spent most of her time walking slowly around the nest. She was also observed grooming and cell checking during this time. The more alarmed female walked jerkily around the nest, flipped her wings, and was not observed grooming or cell checking for the full 30-min trials. Abdominal wagging was observed once on the face of the nest by the more alarmed female. In the second nest (B), one out of four females displayed the alarm behavior as previously described, however the behavior was displayed only for 20 min after initial contact with the moth before the wasp returned to resting behavior. Two of the remaining three females were recently eclosed (<24 h) and reacted to the alarmed female by moving away from her and toward the top of the nest. The fourth female raised her wings and made antennal contact with the alarmed female but did not exhibit the same degree of alarm. Chalcoela iphitalis Behavior under Laboratory Conditions No wasp-moth interactions or moth flights were observed during daytime trials. Moths remained motionless on the walls and floor of their cage with antennae tucked back. During night-time trials, moths typically flew and landed within eight centimeters of the wasp nest with antennae fully extended and oscillating (Figure 1). In total, 14 moth-wasp or moth-nest contacts were observed. Moths and wasps made antennal contact during nine of these incidents, six of which resulted in moth oviposition within seconds of the encounter on the walls, ceiling, and floor of the cage, but never on the nest itself. When moths made antennal contact with an adult wasp, avoidance behavior was displayed as the moth tucked antennae back and ran rapidly in the opposite direction. Eggs were laid singly; often when an ovipositing moth would contact an existing egg on the cage surface with the tip of her abdomen, another egg would be deposited against it. This resulted in egg clumps of seven or less. Ovipositing females could be seen moving the tip of their abdomen from side to side as they walked along the surface of the cage. In two instances, moths hovered near the nest and made contact with the nest itself or an adult wasp before landing nearby and beginning oviposition. Moths were rarely (n = 1) observed walking on a nest surface. After completion of trials, one wasp cage (A) contained 136 C. iphitalis eggs, with 55 after the first 10.5-h period and 81 after the second, while the other cage (B) contained 16 eggs, 10 after the first period and 6 after the second. There was a significant difference between position of eggs in the cages (F(2,12) = 10.84, p < 0.002), with the majority of eggs located on the walls and ceiling closest to the nest and few or no eggs on the bottom (Figure 2). Fourteen days after trials ended, all wasp pupae on each nest had eclosed, leaving only a few late instar larvae for us to examine. No evidence of moth silk was present by this time. Upon removal of a late instar larva from nest A, two C. iphitalis larvae (each ~4 mm in length) were found in the bottom of that cell, and the wasp larva showed signs of predation due to feeding, including a sclerotized, discolored, and deformed posterior end (Figure 3). Removal of a late-instar wasp larva from nest B revealed two larger (~7 mm) C. iphitalis larvae which moved into an adjacent cell through a hole in the cell wall. The adjacent wasp larvae showed no visible sign of predation. Moth silk was first observed in the nests four weeks after trials started, indicating a 4-week developmental time for C. iphitalis in the laboratory at a temperature of 22 °C. In total, 14 moth-wasp or moth-nest contacts were observed. Moths and wasps made antennal contact during nine of these incidents, six of which resulted in moth oviposition within seconds of the encounter on the walls, ceiling, and floor of the cage, but never on the nest itself. When moths made antennal contact with an adult wasp, avoidance behavior was displayed as the moth tucked antennae back and ran rapidly in the opposite direction. Eggs were laid singly; often when an ovipositing moth would contact an existing egg on the cage surface with the tip of her abdomen, another egg would be deposited against it. This resulted in egg clumps of seven or less. Ovipositing females could be seen moving the tip of their abdomen from side to side as they walked along the surface of the cage. In two instances, moths hovered near the nest and made contact with the nest itself or an adult wasp before landing nearby and beginning oviposition. Moths were rarely (n = 1) observed walking on a nest surface. After completion of trials, one wasp cage (A) contained 136 C. iphitalis eggs, with 55 after the first 10.5-h period and 81 after the second, while the other cage (B) contained 16 eggs, 10 after the first period and 6 after the second. There was a significant difference between position of eggs in the cages (F (2,12) = 10.84, p < 0.002), with the majority of eggs located on the walls and ceiling closest to the nest and few or no eggs on the bottom (Figure 2). Fourteen days after trials ended, all wasp pupae on each nest had eclosed, leaving only a few late instar larvae for us to examine. No evidence of moth silk was present by this time. Upon removal of a late instar larva from nest A, two C. iphitalis larvae (each~4 mm in length) were found in the bottom of that cell, and the wasp larva showed signs of predation due to feeding, including a sclerotized, discolored, and deformed posterior end (Figure 3). Removal of a late-instar wasp larva from nest B revealed two larger (~7 mm) C. iphitalis larvae which moved into an adjacent cell through a hole in the cell wall. The adjacent wasp larvae showed no visible sign of predation. Moth silk was first observed in the nests four weeks after trials started, indicating a 4-week developmental time for C. iphitalis in the laboratory at a temperature of 22 • C. Discussion Although Chalcoela iphitalis attacks many different paper wasp species [3], our data suggest that the moth attacks Polistes bellicosus most often in this area, especially as compared with Mischocyttarus mexicanus. The only available host record for C. iphitalis from the genus Mischocyttarus is in M. Discussion Although Chalcoela iphitalis attacks many different paper wasp species [3], our data suggest that the moth attacks Polistes bellicosus most often in this area, especially as compared with Mischocyttarus mexicanus. The only available host record for C. iphitalis from the genus Mischocyttarus is in M. Discussion Although Chalcoela iphitalis attacks many different paper wasp species [3], our data suggest that the moth attacks Polistes bellicosus most often in this area, especially as compared with Mischocyttarus mexicanus. The only available host record for C. iphitalis from the genus Mischocyttarus is in M. basimacula, a species occurring in Central America [3]. In our study, Polistes bellicosus was the most commonly observed species and had the highest C. iphitalis infestation rate, while Mischocyttarus mexicanus was second most commonly observed species and had no infestations. This lack of infested M. mexicanus nests is of great interest, and several possibilities exist: enhanced behavioral and chemical defenses against the moth, insufficient host quality, more concealed nesting locations [8], or morphology of the two proximal abdominal segments, which are more elongate and stalk-like in Mischocyttarus than in Polistes. We speculate that this elongate petiole may add to the body flexibility for use of Van der Vecht's gland-a sternal gland which secretes repellant compounds that protect the nest against ants, yellowjackets, and house flies [12]. Van der Vecht's gland secretions have yet to be tested for repellency against parasitic Lepidoptera. It is plausible that the allomone produced by this gland in Mischocyttarus-which is typically applied to the nest petiole-prevents moth larvae from traveling down the petiole and into the comb in the same manner in which it prevents ants from doing so [13]. C. iphitalis has been noted to have no known host specificity within the Polistes genus [1]; however, a closely related species (C. pegasalis) with a similar life history to that of C. iphitalis was shown to have differential parasitism rates among Polistes species in Jamaica [14]. We found that Polistes species in our study were not attacked equally as often, and the percentage of infested cells also differed between species. However, the most frequently attacked species did not have the highest percentage of infested cells. This discrepancy may be due to the variation in the average number of cells built by each species [15]. If moths lay similar quantities of eggs at each nest, nests with fewer cells should have a higher percentage of total cells infested, which was indeed the case with P. metricus. A tradeoff between parasite vigilance behavior and foraging or brood care by adult female wasps was suggested as a possible explanation to differential parasitism among Polistes species [14]. However, oviposition of Chalcoela moths occurs mainly at night when wasps are not foraging [5], suggesting that other possible factors are at play. In the laboratory, we found that individual adult wasps exhibited a varying degree of alarm behavior after the detection of an adult moth. The wasp exhibiting the highest degree of alarm was also preforming abdominal wagging-a behavior observed more often in queens than in workers [16]. Avoidance of an alarmed individual by callow individuals in another instance was also observed, suggesting that alarm behavior may be correlated with social rank of the individual, as has been shown with aggression behavior [17]. Laboratory experiments also demonstrated that Chalcoela iphitalis oviposit on substrates surrounding the host nest rather than on the nest itself. This makes the substrate which the nest is built upon an important factor in avoiding predation by C. iphitalis. A substrate which has a large surface area (such as S. palmetto leaves or manmade structures) may be more conducive for moth oviposition, while a narrower substrate (such as twigs or small tree branches) may inhibit oviposition by the moth. In this study, we saw no infestation of nests built on twigs or branches in the field, supporting similar observations made by Reed and Vinson [8]. Antennal contact by a female moth with either an adult wasp or the wasp nest itself appeared to be a prerequisite for oviposition in the laboratory, but moths avoided walking onto or next to the nest. Antennal contact most likely provides host checking and oviposition stimulation, while avoidance behavior serves a function of remaining at a safe distance from defending wasps while ovipositing. Although the ectoparasitic moth larvae typically attack only pupae and prepupae [5], last instar wasp larvae were attacked in the laboratory. This could be due to the confined setting and lack of available pupae once all wasp pupae had matured. Field observations supported the basis of an exclusion of pre-pupal nests from the study, in that infestation of a pre-pupal nest was not observed. It is presumable that upon hatching, caterpillars located the nest pedicel and traveled into the nest, although this was not directly observed. Neonates likely go undetected by adult wasps due to their small size. Our results provide the first report of oviposition behavior of C. iphitalis and suggest differential host parasitism in field populations. Future studies focusing on behavioral differences of individual wasps during episodes of parasite invasion may provide the basis for differential alarm behavior among individuals within the colony, and provide further explanation for the differential parasitism by Chalcoela moths.	2017-11-27T16:36:43.994Z	2017-08-24T00:00:00.000	{ "year": 2017, "sha1": "f976b6dc6524f44e123b65188d60afbded8ebdf1", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2075-4450/8/3/89/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "f976b6dc6524f44e123b65188d60afbded8ebdf1", "s2fieldsofstudy": [ "Biology", "Environmental Science" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
220063588	pes2o/s2orc	v3-fos-license	Development of quantification software for evaluating body composition contents and its clinical application in sarcopenic obesity In sarcopenic obesity, the importance of evaluating muscle and fat mass is unquestionable. There exist diverse quantification methods for assessing muscle and fat mass by imaging techniques; thus these methods must be standardized for clinical practice. This study developed a quantification software for the body composition imaging using abdominal magnetic resonance (MR) images and compared the difference between sarcopenic obesity and healthy controls for clinical application. Thirty patients with sarcopenic obesity and 30 healthy controls participated. The quantification software was developed based on an ImageJ multiplatform and the processing steps are as follows: execution, setting, confirmation, and extraction. The variation in the muscle area (MA), subcutaneous fat area (SA), and visceral fat area (VA) was analyzed with an independent two sample T-test. There were significant differences in SA (p < 0.001) and VA (p = 0.011), whereas there was no difference in MA (p = 0.421). Regarding the ratios, there were significant differences in MA/SA (p < 0.001), MA/VA (p = 0.002), and MA/(SA + VA) (p < 0.001). Overall, intraclass correlation coefficients were higher than 0.9, indicating excellent reliability. This study developed customized sarcopenia-software for assessing body composition using abdominal MR images. The clinical findings demonstrate that the quantitative body composition areas and ratios can assist in the differential diagnosis of sarcopenic obesity or sarcopenia. The term 'sarcopenic obesity' has been proposed to identify obesity with low skeletal muscle function and mass 1 . The current definitions of sarcopenic obesity combine sarcopenia [which was registered in the International Classification of Diseases (ICD-10-CM) in 2016] 2 as defined through variable criteria, with the presence of obesity either defined as body mass index (BMI) or adiposity levels [3][4][5][6] . To date, there has been growing interest in sarcopenic obesity or sarcopenia. Several studies have reported that sarcopenia is closely associated with obesity 7,8 , physical disorders, a decline in quality of life 9 , metabolic complications, disease incidence 10,11 , and in particular, the treatment effects of cancer patients 12,13 . These studies indicate that more patients within the obese population have a weakened musculoskeletal system or increased fat mass in all age groups, and the risks and prevalence of sarcopenia increased with diseases such as liver fibrosis, obesity and metabolic syndrome 7,8,11 . However, current studies regarding the quantitative assessment of sarcopenia and/or sarcopenic obesity remain insufficient as diagnostic criteria and measurement techniques for muscle mass (or muscle loss) have not yet been established. Furthermore, it is unclear whether the specific mechanism of sarcopenic pathological responses involved in the decrement of muscle mass is aging 14,15 . Therefore, there is an unmet need for the establishment of a standardized method to assess muscle and fat mass. Scientific RepoRtS \| (2020) 10:10452 \| https://doi.org/10.1038/s41598-020-67461-0 www.nature.com/scientificreports/ Recent studies regarding diagnostic methods for sarcopenia or sarcopenic obesity have focused on the use of methods for the quantitative evaluation of body composition. Body composition imaging typically refers to the quantification of body fat and muscle mass, with evaluation methods including anthropometry, bioelectrical impedance analysis (BIA), and medical imaging [16][17][18] . Among these, the medical imaging techniques are regarded as the gold-standard in order to assess whole-body and specific regional muscle and fat mass. Medical imaging methods have included dual-energy X-ray absorptiometry (DEXA), computed tomography (CT), and magnetic resonance imaging (MRI). MRI, specifically shows great promise for quantifying the soft tissues including muscle, fat, nerve and ligaments and no ionizing radiation for patients [19][20][21] . There are only a few whole-body MRI studies focusing on quantification of actual body composition 22,23 . However, the application of whole-body MRI is restricted in clinical settings as the manual assessment of whole-body organ and tissue mass is time consuming. Several studies have investigated the use of single slices to estimate whole-body composition as an alternative method. Quantified composition on individual slices obtained at the lumbar spine showed a strong correlation with total visceral fat, subcutaneous fat and muscle mass [24][25][26][27] . Adipose tissue areas 5-10 cm above L4-L5 showed the strongest correlation with total visceral fat volume, whereas there was no association with subcutaneous fat areas. The area ~ 5 cm above L4-L5 was established as a predictor of total body-muscle volume 28 . In both genders, a single MRI scan at the level of the third lumbar spine (L3) is the best compromise as a site to assess total volume of visceral fat, subcutaneous fat and muscle 24 . Therefore, the use of single slice and automatic quantification software with a rapid processing time is useful for clinical implementation. Currently, there are few studies focusing on the quantification of body composition in sarcopenic obesity or sarcopenia. For this study, we developed a semi-automatic quantification software for body composition imaging using abdominal MR images and compared the differences between sarcopenic obesity and healthy controls for clinical application. Results Patient characteristics. The averaged enzyme levels in both groups are shown in Table 1. The serum biochemistry showed significant differences between the two groups as follows: aspartate aminotransferase (AST, p < 0.001), alanine aminotransferase (ALT, p < 0.001), fasting glucose (p = 0.014) and triglycerides (TG, p < 0.001). However, there was no significant difference in γ-glutamyl transpeptidase (GGT, p = 0.249) or alkaline phosphatase (ALP, p = 0.170). Compared with those of healthy controls, these changes in serum levels can closely suggest the changes in the metabolic status such as those in sarcopenia and obesity. Measurements of body composition contents using developed software. Five anonymized MRIs with the same slice at the L3 location ( Fig. 1) were selected and provided to six reviewers. Each reviewer independently analyzed the major composition contents in sarcopenic obesity (i.e., muscle, subcutaneous fat, and visceral fat). Table 2 lists the processes for qualitative and quantitative analyses. Figure 2 demonstrates the graphic user interface (GUI) of the developed software and an example image for major composition contents. The mean time for quantifying muscle and fat areas was 20.84 min per image (range 19.56-21.84 min) for the original software and 3.24 min per image (range 2.86-3.36 min) for the developed software. The total processing time using the developed software was reduced by 6.43 times compared with the original software (84% reduced time: Fig. 3). Differentiation of muscle and fat mass in sarcopenic obesity. MR image data in 30 patients with sarcopenic obesity and 30 healthy controls were analyzed with the developed software. The averaged areas (A) and ratio of muscle (M), subcutaneous fat (S), and visceral fat (V) in two groups are summarized in Table 3. There were significant differences in the subcutaneous fat area (SA); (p < 0.001; Fig. 4B) and the visceral fat area (VA); (p = 0.011; Fig. 4C), whereas no significant difference was found in the muscle area (MA); (p = 0.421; Fig. 4A). In the ratios, there were significant differences in MA/SA (p < 0.001; Fig. 4D), MA/VA (p = 0.002; Fig. 4E), and MA/ Table 1. General characteristics in sarcopenic obesity and healthy control groups. The difference between normal control and sarcopenia disease groups was analyzed by the independent two sample T-test. www.nature.com/scientificreports/ (SA + VA); (p < 0.001; Fig. 4F). Therefore, the ratios derived from muscle and fat areas are expected to be more powerful indexes for distinguishing sarcopenic obesity to healthy control. Inter-rater agreement. The interobserver variability in muscle and fat areas between 2 observers is summarized in Table 4. There was no significant difference between the averaged muscle and fat areas of the 2 observers. Overall, intraclass correlation coefficients (ICCs) were higher than 0.9, indicating an excellent reliability. The ICCs (range 0.952-0.994) were 0.977 for the measurements in sarcopenic obesity patients and 0.971 for the healthy control group, respectively. Therefore, the overall muscle and fat measurements of both observers showed an excellent agreement (p < 0.001). Discussion This study developed semi-automated software for assessing body composition on the basis of ImageJ. The study analyzed the difference between people with sarcopenic obesity and a healthy control group by measuring body composition areas from retrospective MRI datasets. In our study, abdominal MR images with 3-dimensional T1 high-resolution isotropic volume excitation (THRIVE) pulse sequence demonstrated good discrimination in diagnosing sarcopenic obesity patients (as SA and VA; p < 0.05). Moreover, the ratios of muscle and fat areas with better discrimination are MA/SA, MA/VA and MA/(SA + VA) (p ≤ 0.002) compared to areas. Therefore, our findings demonstrate that the areas & ratios of muscle and fat composition quantified at the single slice level of L3 can be useful for diagnosing sarcopenic obesity. This study investigated the reproducibility of inter-observer assessment. The muscle and fat areas measured by two observers were excellent with regard to inter-rater agreement (> 0.9), indicating reproducibility. Therefore, the sarcopenia software-based body composition measurements can be reproduced in clinical abdominal MR images. Compared with the original ImageJ program, the processing steps of developed software for quantification were simplified from 11 to 5 processes. Consequently, total processing times were greatly reduced to approximately 3 min per subject as shown in Fig. 2. Thus, our semi-automatic sarcopenia software with a rapid processing time would be beneficial for clinical implementation. Also, the advantage of the ImageJ program is that it is an open source platform based on the Java programming language. It provides high scalability through a Java plug-in and macro functionality. Further study is required, to test our software and other segmentation software such as the medical imaging interaction toolkit (MITK) and the imaging interaction toolkit (ITK). With regard to our study design, this retrospective study used inclusive criteria on the basis of BMI 29 and blood biochemistry from electrical health records (EHR) for enrollment of sarcopenic obesity patients. This retrospective enrollment may be considered a potential bias or variation. The potential risk factors in the selection of the patients (age, sarcopenic severity, gender and so on), the clinical conditions (initial management, drug type or dosage, treatment and etc.) and imaging setting (type of scanners/pulse sequences) or any combinations with the sources may represent bias. In the present study, the evaluation method for overweight obesity included the Korean standard BMI (> 25.0 kg/m 2 ) in conjunction with elevated serum enzyme alanine aminotransferase (ALT) levels. The BMIs and ALT levels in the sarcopenic obesity group were higher than those in the healthy control group. Image-based fat quantification is well reflected in the differences in the subcutaneous and visceral fat in the patient group, but not in the MA. A recent study reported that T2 (or T2-corrected) Dixon MRI and MR spectroscopy (MRS) techniques can provide reliable quantification of fat composition using proton density fat fraction (PDFF) and proton density water fraction (PDWF) while minimizing MR-specific effects 30 . Thus, this finding may be considered a good indicator for assessing the severity of obesity. Further cross-validation studies are essential in order to confirm the muscle/fat composition of a large cohort population together with other imaging methods. This study had several limitations. First, our study dealt with middle aged subjects in both groups. Several studies reported that aging affects muscle mass and maximum muscular strength including individual differences 10,11 . In addition to the muscle/fat assessment, the volumetric muscle measurement and muscle composition must be quantitatively investigated as these factors can differ greatly between individuals. This depends on many confounding factors such as physical activity, smoking and nutrition 31,32 . However, in this study, there was no consideration for aging, physical activity, smoking and nutrition as potential variables, which could influence the evaluation of the muscle mass and muscular strength. Further correlative studies are essential in order to clarify the physiological responses and how those potential variables affect sarcopenic obesity. Moreover, we suggest a standardized study protocol/design with prospective, large-scale cohort investigations according to age group (20-60 years and above). Second, BMI assessment is the simplest method for assessing obesity. However, it is limited to evaluate actual body composition because the values are indirect indices based on body weight, height, and waist circumference (i.e., not the actual muscle and fat mass). The BMI cut-offs as criteria of overweight and obesity are different from those in the World Health Organization (WHO) Expert committee (25.0-29.9 kg/m 2 ), the Western Pacific Regional Office (WPRO) of WHO (> 23.0 kg/m 2 ), the Asia Pacific region (> 25.0 kg/m 2 ) and other various countries 16 . Thus, a standardized index is required in order to accurately measure the actual amount of muscle and fat mass in patients with obesity or sarcopenia. To overcome this issue, we believe that our imaging-based quantification software could be a solution and could provide accurate muscle and fat information to physicians. Third, there is the issue of selecting a single slice at the L3 level for body composition measurement. Selecting a single slice instead of a whole-body MRI analysis may hold true in a cross-sectional study design. However, weight change estimates with a single slice cannot replace whole body assessments 24,33 . Thus, a single slice MR analysis should be carefully used to assess weight change in patients with sarcopenia. Also, MRI estimates of skeletal muscle mass are on mass rather than on tissue composition. The current issue of fat infiltration into muscle should be focused on actual muscle mass measurement. When compared with MRI, CT estimates include attenuation which provides further information of clinical relevance 34 . A solution could be a multivoxel MRS protocol for fat infiltration 35 . Fourth, most of the issues regarding sarcopenia Table 4. Inter-rater variability in muscle and fat measurements. MA muscle area, SA subcutaneous fat area, VA visceral fat area, ICC intraclass correlation coefficient, CI confident interval. Areas quantified from each observer are presented as means ± SD. *The differences between two observers in area measurements were assessed by the independent two sample T-test. † The intra-rater reliability between two observers was assessed by the intraclass correlation (ICC) test. www.nature.com/scientificreports/ relate to the skeletal muscles of the arms and legs (i.e., peripheral skeletal mass). This is again associated with the sequelae of sarcopenia, e.g., frailty. Thus, future studies must clarify the association between skeletal muscle estimates at L3 and the thigh. In conclusion, this study developed a customized sarcopenia-software for assessment of body composition using abdominal MRI images. Our software has the advantages of use in an open source platform and a rapid quantification time for clinical application. The clinical findings demonstrate that the quantitative body composition data such as areas and ratios can assist in the differential diagnosis of obesity and in determining the ratios of muscle and fat. These could be considered as imaging biomarkers for sarcopenic obesity in clinical practice. Subjects and method Ethics statement. We conducted a retrospective study, which was approved by the institutional review board (IRB) of Wonkwang University Hospital. Written informed consent was exempted by the approval of Wonkwang University Hospital IRB committee due to the use of anonymous archival data including MRI data and the use of electronic health records for the application of the developed software. This study was conducted in accordance with the Helsinki Declaration and Good Clinical Practice. Study population. In compliance with the legal guidelines on safety and IRB bioethics, a total of 60 subjects consisting of 30 obese patients with suspected sarcopenia (mean age 47.3 ± 19.4 years.) and 30 healthy controls (mean age 54.6 ± 17.2 years.) were enrolled in this study from January 2014 to April 2019 36 . The individuals complained of fatigue and inactivity, and they appeared to be weaker than their maximum muscular strength. The Korean standard BMI (kg/m 2 ) was used as the selection criteria 29 . Subjects were defined with the following BMI values in conjunction with serum enzyme levels 37 Measurement of body compositions on 3rd lumbar spine MRI. To measure body compositions, this study chose a single slice analysis instead of a whole body MRI analysis with a cross sectional study design 24,33 . The L3 level image was selected as the position for quantitative analysis (Fig. 1). Not only are visceral and subcutaneous fat visible in this position, but also the seven major muscles (the psoas, erector spinae, quadratus lumborum, transversus abdominus, external and internal obliques, and rectus abdominus) can be identified. Moreover, this L3 level includes the spine, intestines, kidneys, and liver. Hence, it is a key position for observing various anatomical areas 24,28 , it is considered the most suitable position at which to analyze the relationship between various conditions and diseases including sarcopenia, aging, obesity, and osteoporosis 38 . Software environment and software algorithm. In order to quantitatively analyze the muscle and fat mass in the patients with sarcopenic obesity, the software was developed on the open source ImageJ multiplatform software (ver.1.51t, Java 1.8.0_191 64bits), developed by the National Institutes of Health (NIH) 39 . To use the software, the Java standard edition (SE) Runtime Environment is required to be installed in advance. Table 2 lists the overall processes for qualitative and quantitative analyses on the original ImageJ software and the developed sarcopenia-specialized software. The main processes are divided into four steps (execution, setting, confirmation and extraction), and the existing 11 analysis processes in the original software are simplified into five processes in the developed software. Data processing and quantification of MR images. The procedures for MRI data processing are comprised of four steps as follows: execution, setting, confirmation and extraction. MRI data are opened from the developed sarcopenia-software in this study and analysis tools specific to the quantification of body composition are implemented as shown in Fig. 2A. A L3 level image was chosen from the axial MR images in each patient to identify the ROIs of muscle, subcutaneous fat, and visceral fat. For the pre-processing of the MRI data, the selected MR image is set in the Window. The Leveling and Threshold values as shown in Fig. 2B (F1 function key button), and their values are applied to the MRI data. After the setting, the ROIs (as shown in Fig. 2C) corresponding to the muscle, subcutaneous fat and visceral fat are roughly drawn on the MR image using the drawing tools on menu bar ( Fig. 2A). www.nature.com/scientificreports/ (4) The extraction step After the quantification of the body composition is completed for the confirmed ROIs from a physician (K.H.Y), the results are extracted into several file formats such as TIFF, PNG, JPG and BMP files. These files are used for the color-labeling of the ROI images, the ROI files for the confirmed ROIs, and the CSV files for the quantification data. To compare the original software and developed software, five anonymized MRI images at the same slice of L3 location were selected and provided to six reviewers. Each reviewer independently analyzed the major composition contents in sarcopenia (i.e., muscle, subcutaneous fat, and visceral fat). They had no knowledge of the clinical outcome or access to the readings of the other reviewers. To assess the inter-observer variability of the measurements, both radiologists independently assessed the L3 images. The overall measurements for each patient were calculated as a mean and standard deviation of the areas. Statistical analysis. The abdominal muscle and fat contents were compared with two independent groups using the statistical package for the social sciences program (SPSS ver. 20, Chicago, Illinois). The variation in muscle and fat contents was evaluated with an independent two sample T-test. Inter-rater agreement and reliability were estimated by calculating the intra-class correlation coefficient (ICC); (and a 95% confidence interval [CI]) between the muscle and fat areas for the same subject on the same system. The ICC values were considered as the basis to evaluate the level of reliability using the following guideline 40 : values less than 0.5 are indicative of poor reliability, values between 0.5 and 0.75 indicate moderate reliability, values between 0.75 and 0.9 indicate good reliability, and values greater than 0.90 indicate excellent reliability. Two-sided p values less than 0.05 were considered to indicate statistical significance in all tests.	2020-06-26T14:49:02.784Z	2020-06-26T00:00:00.000	{ "year": 2020, "sha1": "725a2c376149a721fa371efae41fb32a835ca765", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41598-020-67461-0.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "2d4afa4f5e98197e888936f6e769f8c645fad7f8", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
13407453	pes2o/s2orc	v3-fos-license	Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9 Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with 2H- and 31P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors. Introduction The increasing resistance of pathogens against conventional antibiotics has become a major concern for treatment of infectious diseases. Therefore, development of new classes of therapeutic agents is urgently required. One group of molecules that has attracted interest are cationic antimicrobial peptides. Unlike conventional antibiotics that bind to a target in the membrane or cytosol and act specifically on biochemical pathways, most antimicrobial peptides kill cells by permeabilizing the membrane through peptide-lipid interactions. Many studies have been conducted with antimicrobial peptides, and various membrane-destabilization mechanisms were proposed such as the ''barrel-stave'' and the ''carpet'' models. The former postulates that the peptide acts by perturbing the barrier function of membranes through transmembrane pore formation [1]. In the ''carpet'' model, the peptide acts in a detergent-like manner, covering the membrane surface until a threshold concentration is reached leading to membrane pore formation and finally to membrane disruption [2]. Other specific mechanisms have also been suggested, including phase separation due to specific peptide-lipid interactions [3,4] and detergent-like solubilization of the membrane [5]. All these mechanisms are not mutually exclusive and an individual peptide may induce different mechanisms of membrane damage depending on the membrane lipid composition [6,7]. Frog skin is by far the most important source of antimicrobial peptides, with more than half of the peptides described to date isolated from South American Hylidae and European, Asian or North American Ranidae [8]. Among these peptides, dermaseptins B and S, secreted in the skin of the South American tree frogs Phyllomedusa bicolor and P. sauvagei respectively, form a family of amphipathic, a-helical, closely related antimicrobial peptides with broad-spectrum microbicidal activities at micromolar concentrations against Gram-positive and Gram-negative bacteria, fungi, yeasts and protozoa [9]. One of these peptides, dermaseptin S9 (Drs S9), a cationic peptide, does not resemble any antimicrobial peptide identified to date, but is more similar to synthetic peptides that were originally designed as transmembrane mimetic model peptides [10][11][12], having a hydrophobic core sequence flanked at both termini by several positively charged residues. Usually, cationic antimicrobial peptides are unstructured in buffer, but fold into a-helical structure in the presence of lipids. Consistent with this, previous structural studies showed that Drs S9 folds into an ahelical structure in TFE/water mixtures, while the NMR spectrum in water shows very broad signals indicative of aggregation in aqueous solution [13]. Subsequently, it was shown that Drs S9 slowly assembles into amyloid-like fibrils at 500 mM in aqueous solution and that the antimicrobial and chemotactic activities of Drs S9 are modulated by its propensity to form fibrils [14]. Indeed, Drs S9 exhibited an antimicrobial activity against all Gramnegative bacteria strains tested at day 0 and day 3 whereas the peptide exhibited little or no antibacterial activity after 7 days of incubation. Surface plasmon resonance experiments indicated that Drs S9 binds to anionic or zwitterionic phospholipid vesicles [13]. This Drs S9/lipid interaction may be important for the antimicrobial activity of the peptide, for example because membranes may modulate the conformation or the fibril forming propensity of the peptide, and/or because the peptide may interfere with the permeability properties of the membrane. Despite this potential biological importance, to our knowledge these two studies [13,14] are the only ones so far on the interaction between membranes and Drs S9. To gain further insight into the nature of the initial steps of Drs S9-membrane interactions, including the potential importance of lipid composition, we performed a biophysical study of Drs S9 in lipid bilayers of different compositions. In particular, we used model membranes of the zwitterionic lipid phosphatidylcholine (PC) and the anionic lipid phosphatidylglycerol (PG) in a 7:3 ratio to mimic the lipid composition of microorganism plasma membranes and we used bilayers composed of pure PC and PG to analyze the importance of electrostatic interactions. To further understand the importance of amyloid-like fibril formation in relation to membrane-structure and membrane-damage induced by Drs S9, we also studied and compared the effect of addition of the peptide to preformed membrane vesicles versus intimate incorporation into the membranes via cosolubilization of peptide and lipid in organic solvent. Preparation of MLVs and LUVs Lipid films were made by dissolving the desired lipids in chloroform (for DOPC, POPC and DMPC) or chloroform/ methanol 3:1 (for DOPG, POPG and DMPG). The solvent was evaporated under dry nitrogen gas. The resulting films were then kept under vacuum desiccator for at least 30 minutes. Films were then rehydrated with appropriate buffer (10 mM Tris-HCl, 100 mM NaCl, pH 7.4 for the calcein leakage assay, DSC and NMR experiments and 10 mM sodium phosphate buffer, pH 7.4 for the CD experiments) at a temperature above the transition temperature of the lipids for 30 minutes. The lipid suspensions were frozen in liquid nitrogen and thawed in a 40uC water bath 3 times to obtain homogeneous multilamellar vesicles (MLVs). Large unilamellar vesicles (LUVs) with a mean diameter of 200 nm were prepared from the MLVs using the following protocol: the lipid suspensions were subjected to 10 freeze-thaw cycles, and passed 19 times through a mini-extruder (Avanti Polar Lipids, Alabaster, USA) equipped with a 200 nm polycarbonate membrane. The phospholipid content of lipid stock solutions and vesicle preparations was determined as inorganic phosphate according to Rouser [15]. Calcein-containing LUVs were made using the same protocol, except for the following adaptations. The buffer for hydration of the lipid films was replaced by a solution containing 70 mM calcein and 10 mM Tris-HCl (pH 7.4). Free calcein was separated from the calcein-filled LUVs using size-exclusion chromatography (Sephadex G50-fine) and elution with 10 mM Tris-HCl, 100 mM NaCl (pH 7.4). Peptide preparation When Drs S9 was incorporated into the vesicles, lipid and freshly peptide were solubilized in respectively CHCl 3 /CH 3 OH and TFE and then mixed together. The organic solvents were removed under high vacuum and the residual lipid/peptide film was hydrated in the appropriate buffer (pH 7.4). For experiments where Drs S9 was added to lipid vesicles, the preformed vesicles were slurried with a 1 mM peptide solution in milliQ water. A peptide:lipid molar ratio of 1:20 was used in all experiments. Thioflavin assays The kinetics of Drs S9 fibril formation were measured using the fluorescence intensity increase upon binding of the fluorescent dye Thioflavin T (ThT) to fibrils. A plate reader (Fluostar Optima, Bmg Labtech) and standard 96-well flat-bottom black microtiter plates in combination with a 440 nm excitation filter and a 485 nm emission filter were used. The ThT assay was started by adding 10 mL of a 2 mM Drs S9 (100 mM peptide) in aqueous solution to 190 mL of a mixture of 10 mM ThT, LUVs (2 mM lipids; peptide:lipid ratio 1:10) and 10 mM Tris/HCl, 100 mM NaCl (pH 7.4). The microtiter plate was shaken for 10 seconds directly after addition of all components, but not during the measurement. The ThT assay was performed 2 times, each in duplicate. The results presented here are the average of the different experiments; errors bars indicated the statistical dispersions. Electron microscopy Peptides and LUVs were incubated under the same conditions as in the Thioflavin T assay. Aliquots (25 mL) of this mixture were adsorbed onto glow-discharged carbon-coated 300-mesh copper grids for 2 min. Grids were then blotted and dried. Grids were negatively stained for 45 s on 2% uranyl acetate, blotted and dried. Grids were examined using a Jeol 2100 electron microscope operating at 200 kV. Calcein permeability assay in large unilamellar vesicles A plate reader (Fluostar Optima, Bmg Labtech) was used to perform calcein leakage experiments in standard 96-well transparent microtiter plates. Measurements were conducted on calcein-loaded DOPC, DOPC/DOPG 7:3 and DOPG LUVs. The Drs S9 was added to a mixture of calcein-containing LUVs in 10 mM Tris-HCl, 100 mM NaCl, pH 7.4 buffer. The final concentrations were 20 mM for lipids and 1 mM for peptide (peptide:lipid ratio of 1:20). Directly after addition of all components, the microtiter plate was shaken for 10 s using the shaking function of the plate reader. The plate was not shaken during the measurement. Fluorescence was measured from the bottom, every minute, using a 485 nm excitation filter and a 520 nm emission filter. The temperature was approximately 28 uC 6 3 uC. The maximum leakage at the end of each measurement was determined via addition of 1 mL of 10% Triton-X100 to a final concentration of 0.05% (v/v). The release of fluorescent dye was normalized according to the following equation: In this equation, L T is the fraction of dye released (normalized membrane leakage), F T is the measured fluorescence intensity, and F 0 and F 100 are the fluorescence intensities at time zero and after addition of Triton-X100, respectively. The calcein leakage experiment was performed 4 times, each in triplicate, on different days. The results presented here are the average of the different experiments, 6 standard deviation. Monolayer experiments Peptide-induced changes in the surface pressure of a monomolecular layer of phospholipids at constant surface area were measured with the Wilhelmy plate method, as reported previously [16,17]. Surface pressures were measured at room temperature. Briefly, a trough was filled with 5.5 mL of 10 mM Tris-HCl, 100 mM NaCl buffer (pH 7.4). DOPC, DOPC/DOPG (7:3) and DOPG monolayers were spread from a 0.5 mM stock solution in chloroform or chloroform/methanol (3:1). The lipid monolayer was allowed to stabilize for a few minutes before 5 mL of 1.1 mM freshly prepared stock solution of the peptide was injected into the sub-phase without disturbing the lipid monolayer. The final peptide concentration was 1 mM. Circular dichroism CD spectra were recorded on a Jasco 815 spectropolarimeter (Jasco Inc., Easton, MD) over the wavelength range 190-270 nm, at 0.2 nm intervals and 20 nm.min 21 scan speed. Five scans were accumulated and averaged. Temperature was kept at 25uC. Spectral measurements were performed in 1 mm path length quartz cells from Hellma GmbH, using DOPC, DOPC/DOPG 7:3 and DOPG LUVs, with added or incorporated peptide. Peptide concentrations were 25 mM in the absence and in the presence of lipids (peptide:lipid ratio 1:20). CD measurements are reported as molar ellipticity per residue (degree.dmol 21 .cm 2 .residue 21 ), and are given by: where h is the recorded ellipticity in degrees, c is the peptide concentration in dmol.L 21 , l is the cell path-length in cm and N is the number of residues. In order to estimate the peptide secondary structure content, an analysis of relevant CD spectra was carried out using the CDFriend program, developed by Dufourc and Buchoux [18] and CDPro software, developed by Sreerama and Woody [19]. Similar results are obtained for both methods. Results are given using CDFriend, which uses standard curves for a-helix, b-sheet and random coil obtained from LiKj peptides of known length, secondary structure and CD spectrum and therefore is more relevant for our study [20,21]. The program implements a simulated annealing algorithm to obtain the best combination of a-helix, b-sheet, and random coil conformation that exhibits the lowest normalized root mean-square deviation (nrmsd) with respect to the experimental spectrum. The experimental error is estimated to be 6 5%. Differential scanning calorimetry DSC experiments were performed on a high-sensitivity calorimeter (TA instruments, Guyancourt, France). The temperature range was 0-40uC with a scan rate of 1uC/min and a delay of 10 min between sequential scans in a series to allow thermal equilibration. Four heating and cooling scans were performed for each analysis. These experiments were performed on DMPC, DMPC/DMPG 7:3 and DMPG MLVs, with added or incorporated peptide. The lipid concentration was 1 mg/mL with a peptide/lipid molar ratio of 1:20. Data analysis was performed by the fitting program NanoAnalyze provided by TA instruments. Sample preparation for NMR experiments Dermaseptin S9 was incorporated to MLVs of POPC-d 31 , POPC-d 31 /POPG 7:3 or POPG-d 31 to a final peptide:lipid ratio of 1:20. Typically, about 10 mg of lipid was used per sample. Lipids and peptide were co-dissolved in organic solvent, evaporated under dry nitrogen gas and then kept under vacuum for at least 30 min. The residual film was suspended in 500 mL of milliQ water, and immediately lyophilized. Samples were rehydrated with 150 mL of 10 mM Tris-HCl, 100 mM NaCl buffer (pH 7.4) in deuterium-depleted water (Eurisotop, France) and then subjected to three freeze/thaw cycles. Samples were transferred into a 4 mm rotor (100 mL) for NMR experiments. Solid state NMR spectroscopy NMR experiments were carried out on a Bruker Avance (Wissembourg, France) 800 MHz spectrometer operating at 122.8 MHz for 2 H-NMR and 323.9 MHz for 31 P-NMR. Samples were allowed to equilibrate for 30 min at 25uC before the NMR signal was acquired. Deuterium NMR experiments were performed by means of the quadrupolar echo pulse sequence 90ux-t-90uy-t-acq [22]. 31 P-NMR spectra were acquired using a phasecycled Hahn-echo pulse sequence with gated broadband proton decoupling [23]. Typical acquisition parameters for 2 H-NMR experiments were as follows: spectral width of 500 kHz; p/2 pulse width of 4.5 ms; interpulse delays of 40 ms; a recycle delay of 2 s was used and 2-3 k scans were recorded. Typical acquisition parameters for 31 P-NMR experiments were as follows: spectral width of 200 kHz, p/2 pulse width of 6.5 ms, interpulse delays of 40 ms and a recycle delay of 5 s; 2 k scans were accumulated. An exponential line broadening of 100-200 Hz was applied prior to Fourier transformation. Phosphorous chemical shifts were referenced relative to external 85% H 3 PO 4 (0 ppm). 2 H spectra were de-Paked according to the ''de-Pake-ing'' procedure implemented by Bloom et al. [24]. Carbon-deuterium order parameters, S CD , were directly extracted from quadrupolar splittings Dn Q using the formula: where e 2 qQ/h is the static deuterium quadrupolar coupling constant (167 kHz for C-2 H bonds [25]). S CD calculations were performed using a PYREX/PYTHON program [26]. Drs S9 induces membrane leakage to a lesser extent in PG than in PC vesicles First the effect of the peptide was examined on membrane barrier properties in lipid vesicles of different compositions. Membrane damage was assayed quantitatively by analysing the extent of leakage of a fluorescent dye (calcein) entrapped in large unilamellar vesicles (LUVs), which is an established routinely used method to measure membrane damage [4]. The high concentration of calcein inside intact vesicles leads to self-quenching. Disruption of the membrane of the vesicle by the peptide allows calcein to escape, eliminating the self-quenching effect and therefore increasing fluorescence of calcein. Figure 1 shows that Drs S9 induces within a few minutes a significant leakage of DOPC LUVs at a concentration of 1 mM, which is of the same range as the observed minimal inhibitory concentration (MICs) [13]. The extent of leakage reaches 71 6 7% of the total vesicle content. In DOPC/DOPG vesicles, the process of permeabilization is slightly slower than in presence of DOPC vesicles, and leads to a smaller extent of leakage (57 6 8%). In contrast, the results obtained in the presence of DOPG LUVs are quite different; Drs S9 induces only weak membrane damage, with 18 6 11% leakage. This result is unexpected because Drs S9 has an overall positive charge and hence would be likely to be attracted more strongly to anionic lipids. Therefore, it is likely that the observed differences in membrane leakage between the three systems (DOPC, DOPG and DOPC/DOPG) are caused by differences in membrane insertion and/or differences in conformational behavior of the peptide. Drs-S9 inserts less efficiently in PG than in PC monolayers We first performed membrane surface pressure measurements on monolayers to study the insertion of the peptide in lipid membranes. Injection of Drs S9 into the aqueous sub-phase below a lipid monolayer (DOPC, DOPC/DOPG or DOPG) resulted in a fast increase in the surface pressure. After 15 min, at an initial pressure of 32-34 mN/m, the increase in surface pressure induced by addition of Drs S9 was 7.9 6 0.5 mN/m for DOPC and DOPC/DOPG compared with an increase of 6.7 6 0.5 mN/m for DOPG (Figure 2A), indicating an efficient insertion for Drs S9 in the three lipid systems. Then, we determined the maximal initial surface pressure at which the peptide was still able to insert, by analysing the surface pressure increase as function of the initial surface pressure. Figure 2B indicates that the extrapolated ''limiting surface pressure'' below which Drs S9 is able to insert is higher for DOPC and DOPC/DOPG monolayers (about 40 mN/m) than for DOPG monolayers (33 mN/m). In DOPC and DOPC/DOPG monolayers, the limiting surface pressure is significantly higher than the surface pressures that correspond to the packing density in lipids found in biological membranes, being between 31 mN/m and 35 mN/m [27], indicating that in vivo Drs S9 could insert efficiently into these membranes. Noteworthy, the limiting surface pressure of Drs S9 in DOPC and DOPC/DOPG is of the same order compared to that of other antimicrobial peptides [28,29]. In contrast, in DOPG monolayer the limiting surface pressure is lower, suggesting that in vivo Drs S9 may not insert in pure DOPG membrane or in membrane containing PGrich domains. These results are in agreement with calcein leakage experiments in which only a small leakage of DOPG vesicles is induced by Drs S9. The observed differences in membrane insertion between the three systems (DOPC, DOPG and DOPC/ DOPG) may be caused by differences in aggregation or conformation of the peptide. Its tendency to aggregate was investigated next. Drs S9 form amyloid-like fibrils in solution and in the presence of membranes Drs S9 has a propensity to form amyloid-like fibrils in solution at high concentration [14], although the aggregation process is found to be slow compared to amyloid peptides, typically requiring several hours at 500 mM [14]. Consistent with this, we observed the formation of fibrils for Drs S9 in solution at 100 mM after 1 day of incubation (Figures 3A-E). Interestingly, for the same peptide concentration approximately 1 to 8 days were required to obtain fibrils in the presence of DOPC and DOPC/DOPG vesicles and more than 15 days in the presence of DOPG vesicles ( Figures 3B-H). In solution, the fibrils showed the typical morphology of amyloid fibrils with diameters between 10 and 15 nm, consistent with at least two filaments coiled around each other. In contrast, the population of Drs S9 fibrillar assemblies in vesicles consists of short, noodle-like fibrils that form thin but compact filamentous structures. The noodle-like fibrils seem even more packed in the presence of anionic lipid (Figures 3G-H). Then, we followed the kinetics of fibril formation of Drs S9 in solution and in the presence of vesicles in more details by measuring the fluorescence intensity increase upon binding of the amyloid specific dye Thioflavin T (ThT), which is a commonly used method to detect amyloid fibrils [30]. ThT changes its fluorescence characteristics remarkably upon interaction with amyloids. The kinetics of fibril formation were followed at peptide concentrations of 100 mM at 25uC. Consistent with the EM results, Figure 3I shows that at 100 mM Drs S9 is able to form fibrils after a few days of incubation in solution, and that less fibril formation takes place in the presence of DOPC and in the presence of DOPC/DOPG, while Drs S9 hardly forms fibrils in the presence of DOPG. The overall observation that in the presence of lipids less fibrils are formed is consistent with the recent notion, that highly fibrillogenic peptides form fibrils more rapidly in the presence of lipid vesicles than in their absence, while the opposite is observed for peptides of low fibrillogenicity, where fibril formation is slower in the presence of lipid vesicles [31,32]. Incorporation method promotes a-helical conformation for Drs S9 Our data demonstrate that the membrane leakage and insertion is weaker in DOPG than in DOPC/DOPG and DOPC vesicles. A possible explanation is that this observed difference in Drs S9membrane damage could be related to differences in aggregational behavior, which in turn could be related to differences in conformational behavior of the peptide. To study the secondary structure of Drs S9 in the absence and presence of membranes, we performed CD spectroscopy. For these experiments phosphate buffer without sodium chloride was used in order to increase the signal-to-noise ratio, keeping in mind that the absence of salt may affect peptide-membrane interactions. Drs S9 freshly dissolved in 10 mM phosphate buffer at 25 mM displays a peak with negative ellipticity at approximately 220 nm and a peak with positive ellipticity at 195 nm, that are characteristic of b-sheet structures ( Figure 4A). Deconvolution of this spectrum allows to estimate the proportion of secondary structures as 9% a-helix, 41% b-sheet and 50% random coil. Extending the incubation time caused the CD signal to decrease (data not shown) proving that Drs S9 forms insoluble aggregated species that are not visible by CD spectroscopy. When Drs S9 is added to vesicles (DOPC, DOPG or DOPC/ DOPG), the CD signal is quite similar (Figures 4B-D, solid line) to the one obtained in phosphate buffer, indicating that the peptide also adopts b-sheet conformations in the presence of lipids. In the presence of DOPC, the intensity of the CD signal seems more dominant, suggesting a higher percentage of b-sheet, that is possibly related to its higher tendency to form fibrils. This is in agreement with our TEM and ThT experiments, which showed more amyloid-like fibrils in the presence of DOPC than in the presence of DOPC/DOPG or DOPG. After a few hours of incubation, the CD signal decreases which can be ascribed to Drs S9 aggregation (data not shown). These results are in agreement with SDS-Tris-Tricine-PAGE experiments which show the presence of small oligomeric forms for the peptide in solution and when added to lipid vesicles suggesting that Drs S9 selfassociates [14]. To further study the importance of conformational behavior and amyloidogenicity for Drs S9/lipid interactions, we also used an "incorporation" protocol, in which Drs S9 was added to the phospholipids prior to the formation of the vesicles by codissolving peptide and lipids in organic solvent and drying them into a film that was subsequently hydrated to form the vesicles. This incorporation method is expected to favor peptide/lipid interactions over peptide/peptide interactions, thus reducing characteristic formation of aggregates in aqueous solution. CD Interaction of Drs S9 with membrane determined by Differential Scanning Calorimetry Next, to gain insight into possible molecular mechanisms of leakage and to look more closely at the effects of the peptide on the organization of the lipids, the interaction of Drs S9 with membranes constituted of DMPC, DMPG or DMPC/DMPG phospholipids was characterized by differential scanning calorimetry. The phase behavior of lipid membranes is highly sensitive to the presence of external compounds, such as peptides [33][34][35]. Monitoring changes in thermodynamic parameters such as melting temperature T m and enthalpy DH can provide information on the nature of the interactions between the peptide and the membrane. DSC thermograms illustrating the effect of Drs S9 on the thermotropic behavior of DMPC, DMPC/DMPG and DMPG MLVs are presented in Figure 5. Pure lipid scans ( Figures 5A-C solid) present two distinct endothermic transitions: a more energetic event around 23uC corresponding to the chain melting transition (P b' rippled gel phase to L a fluid lamellar phase) and a less energetic event corresponding to the pre-transition (L b' lamellar gel phase to P b' rippled gel phase) near 13uC. The main transition peak is tall and narrow, indicating a large enthalpy and a highly cooperative transition. In contrast, the pre-transition, corresponding to the formation of rippled phase and the untilting of the lipid acyl chains, has a lower enthalpy and is less cooperative, with a smaller and broader peak. The addition of Drs S9 from aqueous solution to DMPC, DMPC/DMPG and DMPG ( Figures 5A-C dash) leads to a temperature decrease of both the pre-transitions (which completely disappear in the case of DMPC and DMPG) and main transitions, indicating that the peptide interacts with lipid headgroups and slightly perturbs the acyl chains. Importantly, in DMPC the transition peak seems to remain sharpest, possibly indicative of more peptide aggregation and therefore less efficient membrane interaction. Pre-incorporation of the peptide to the vesicles via cosolubilization (Figures 5A-C dash dot) leads to significantly larger changes in the thermotropic behavior of the lipid bilayer. In the case of DMPC, incorporation of Drs S9 results in the appearance of a second transition at about 21uC that coexists with the main transition at 23uC. This suggests that two phases coexist, one corresponding to lipids in a peptide-poor region and another corresponding to lipids in a peptide-rich region, again indicative of peptide aggregation. In case of DMPC/DMPG and DMPG vesicles, the enthalpy and the cooperativity of the main transition are strongly decreased. Most likely in these cases Drs S9 localizes near the interface, where electrostatic interactions of the peptide with the anionic lipids may lead to a perturbation of the lipid headgroup area and hence to a disturbance of the cooperative behavior of the lipids. Furthermore, also with this protocol, the pre-transition disappears in the case of DMPC and DMPG, and is strongly decreased for DMPC/ DMPG. Overall, the results indicate that Drs S9 inserts into lipid bilayers with differences between DMPC, DMPC/DMPG and DMPG, which are consistent with a higher tendency of the peptide to aggregate in the zwitterionic PC bilayers. Proton-decoupled 31 P and 2 H solid-state NMR spectra to monitor lipid macroscopic phase properties, structure and dynamics To obtain more details about the mode of membrane insertion of Drs S9 and the peptide/lipid interactions involved, we determined the influence of preincorporation of Drs S9 on lipid order by 31 P-NMR and 2 H-NMR. The peptide was incorporated into multilamellar vesicles of perdeuterated POPC-2 H 31 , POPC-2 H 31 /POPG and POPG-2 H 31 at a 1:20 peptide:lipid molar ratio. 31 P-NMR enables to probe Drs S9-membrane interactions at the headgroup level. Figure 6 (A-C) shows classical axially symmetric spectra of POPC-d 31 , POPC-d 31 /POPG and POPG-d 31 bilayers, in the absence (top) and presence (bottom) of peptide. Spectra of pure lipids exhibit a typical pattern of lipid bilayers in a fluid lamellar phase at 25uC. Spectra of POPC-d 31 / POPG ( Figure 6B) are the superposition of two subspectra originating from the phosphorous nuclei of POPC and POPG. Because of the different headgroup structures of the two lipids, each subspectrum shows a slightly different residual chemical shift anisotropy (CSA) [36,37]. POPC typically has a CSA of ,44 ppm and POPG of ,32 ppm. The mixture contains a cumulative CSA of POPC and POPG. The addition of peptide (bottom) does not lead to major macroscopic changes in the shape of the spectra. In particular, there is no apparition of an isotropic peak, indicating that no small vesicles are formed. However, addition of the Drs S9 to each lipid system promoted a perturbation to the phospholipid head group region, with a 2 to 10% decrease in the CSA. Drs S9 shows evidence of significant membrane interaction, which leads to an increase in membrane fluidity. To obtain more information on the lipid order induced by Drs S9, we used 2 H-NMR spectroscopy. Figure 6 (D-F) presents the 2 H-NMR spectra of POPC-d 31 , POPC-d 31 /POPG and POPG-d 31 bilayers, in the absence (top) and in the presence (bottom) of peptide at 25uC. In the case of pure lipids, we observe the typical spectra with many resolved splittings that are characteristic of lamellar fluid phases. The smallest splitting is about 3 kHz and corresponds to the mobile methyl groups at the end of the acyl chains, in the core of the bilayer. The largest one, corresponding to the CD 2 groups closest to the glycerol backbone, is about 23-24 kHz, and is giving information about lipid order near interface. The addition of Drs S9 (bottom) does not lead to the formation of an isotropic peak, thus no small objects that would undergo fast tumbling are formed, in agreement with the 31 P-NMR experiments. However, we observe a reduction of some of the 2 H quadrupolar splittings. The more affected positions are those closest to the glycerol backbone, indicating that the peptide does not seem to insert deeply into the membrane. In the presence of Drs S9, the largest splitting decreases from 24.7 kHz to 23.5 kHz for POPC-d 31 , 24.5 kHz to 22.3 kHz for POPC-d 31 /POPG and 22.5 kHz to 20.3 kHz for POPG-d 31 . Thus, the perturbation near the polar headgroup seems to increase in the presence of POPG. Figure 7 shows the order parameter (S CD ) for each carbon position in the lipid chain in the absence (diamonds) and in the presence (crosses) of peptide. The POPC-d 31 chain order is lower ( Figure 7A) in the presence of Drs S9, especially for the ''plateau'' positions (carbons 2 to 6). The effect is less marked until carbon 12, and is insignificant beyond. In POPC-d 31 /POPG and POPG-d 31 liposomes ( Figures 7B and 7C respectively), the order parameter of lipid chain is more reduced by Drs S9, affecting almost all carbon positions. The smaller effect on PC membranes would be consistent with a higher tendency of Drs S9 to aggregate in PC, and also indicates that the induced lipid disorder propagates more deeply in PG-containing membranes than in PC. In addition, the stronger electrostatic interactions of the peptide in case of PG containing membranes may lead to a larger perturbation of the lipid headgroup area, which would be consistent with the DSC results. In all lipid systems, the more affected positions are those corresponding to the CD 2 groups closest to the glycerol backbone suggesting a shallow insertion of Drs S9 into the membrane. Overall, NMR experiments indicate that the presence of Drs S9 leads to a slight increase in membrane fluidity. Discussion In the present study we have investigated and compared the mechanism of Drs S9-induced membrane disruption in relation with Drs S9-structure in lipid membrane models of different compositions. We have made the following key observations: i) Drs S9 induces more membrane leakage in PC than in PC/PG and PG vesicles; ii) Drs S9 is more fibrillogenic in PC than in PC/PG and PG; iii) smaller membrane perturbations are observed in the presence of PC than PC/PG and PG. These results are further discussed below. We used negatively charged vesicles composed of PC/PG lipids to assess the ability of the peptides to permeabilize bacterial inner membrane, 100% of zwitterionic lipid and 100% of anionic lipid to provide information which helps to understand peptide/lipid interactions and to determine the role of electrostatic interactions. Because Drs S9 possesses five basic and one acidic residues at pH 7.4, leading to a cationic +4 global charge, a preferred interaction with the anionic lipid is probable. Unexpectedly, our results show that Drs S9 induces more leakage and inserts better in the presence of zwitterionic membrane (DOPC) than in the presence of negatively charged lipid (DOPC/DOPG and DOPG). This result is rather surprisingly, but a recent study showed that the highly cationic antimicrobial peptide MSI-78 (+9 global charge), also induces a very low leakage in pure PG vesicles [7]. The low leakage efficiency observed from pure PG vesicles could be due to strong electrostatic interactions between cationic residues of Drs S9 and negatively charged headgroups of PG that may anchor the peptide on the membrane surface, restricting its penetration into the bilayer core and thus its lytic activity, as it was proposed for other cationic antimicrobial peptides [7,38]. This is consistent with the monolayer experiments showing that Drs S9 inserts less efficiently in PG membranes. Thus, it seems reasonable to assume that in the presence of 100% PG, Drs S9 somehow is anchored to the membrane surface. Thus, the electrostatic interactions with anionic lipids cannot be a determining factor to account for Drs S9 selectivity toward microorganisms membranes, which is unusual for a cationic peptide. Instead, our results show that in the presence of zwitterionic lipid, Drs S9 seems to induce membrane damage that is related to formation of small aggregates rather than fibril formation, as explained in the next paragraph. The EM, ThT-fluorescence and CD studies of Drs S9 reveal several interesting features. The EM results indicate that the aggregates formed in solution by Drs S9 are analogous to diseaserelated amyloid fibrils [39]. Indeed, the Drs S9 fibrils exhibit the typical morphology of long and twisted amyloid fibrils with widths between 5 and 15 nm. However, unlike the amyloid peptides which assemble into amyloid fibrils in a few hours at 5 mM, the kinetics of Drs S9 fibril formation is very slow; several hours of incubation are required to observe the amyloid-like fibrils at 100 mM concentration. It is known that membranes containing anionic lipids increase the formation of amyloid fibril formation [40]. Our EM experiments in the presence of zwitterionic and anionic membranes did not show any evidence of amyloid-like fibril formation for Drs S9 after few days of incubation, but did show short and dense noodle-like fibrils. Noteworthy in the presence of 100% of anionic lipid, the noodle-like fibrils were more compact and were observed only after about 15 days. These results are consistent with the ThT-fluorescence and CD data in the presence of membranes that show small increases in ThT signal after few hours of incubation and b-sheet structure for Drs S9. Together with the EM data, these results indicate the following order of fibrillogenicity for Drs S9: DOPC . DOPC/DOPG . DOPG. Our data provide evidence that, unlike several antimicrobial peptides that form amyloid-like fibrils only in the presence of phospholipids [41][42][43], Drs S9 forms amyloid-like fibrils in both an aqueous and lipidic environment. Furthermore, the results demonstrate that Drs S9 amyloid-like aggregation on the membrane surface is not required for membrane permeabilization. Indeed, the membrane leakage starts after a few minutes with a peptide concentration of 1 mM, whereas the fibrils are formed after a few days at 100 mM. We can conclude that Drs S9 aggregation at the membrane surface is not essential for its cytotoxicity, unlike amyloid peptides for which it was shown that the growth of amyloid fibrils at the membrane surface causes membrane damage [44][45][46]. Nevertheless, the data suggest a relationship between aggregation and membrane damage, suggesting that membrane damage may be induced by small aggregates of peptide in b-sheet conformation, possibly precursors in the process of fibril formation. Because many antimicrobial peptides affect the physicochemical properties of lipid membranes, we studied the lipid-peptide interactions by using DSC, 31 P-and 2 H-NMR methods. The addition of Drs S9 had little effect on the thermodynamics of the gel to liquid-crystalline phase transition. This indicates that the lipid-lipid interactions stabilizing the membrane are largely intact. It was shown that the aggregation of a peptide decreases the perturbation on the membrane by reducing the surface area of the membrane in contact with the peptide [47]. Self-association of the peptide lowers the number of lipid molecules in contact with the peptide and is therefore expected to decrease the influence of the peptide on the physical properties of the membrane. For that reason, we also studied the peptide-membrane interactions using the incorporation method, where the peptide is added prior to the formation of the vesicles. In this case, the incorporation of Drs S9 has a larger effect on the thermodynamics of the gel to liquidcrystalline phase transition suggesting that the peptide interacts strongly with the acyl chains of the lipids. In addition, the main phase transition of the lipid system is broadened, indicating a loss of lipid cooperativity. Our data show a quite similar enthalpy for the membrane in the absence and in the presence of Drs S9 indicating that the peptide does not perturb the membrane cohesion by changing the size of the vesicles as occurs with membrane fusion, micelle formation or breaking down the membrane [48]. These results are in agreement with the 31 P-NMR results which show a conserved lamellar line shape and no dominant isotropic line. There is also no dominant isotropic line in deuterium spectra, clearly indicating that membrane integrity is maintained upon interaction with Drs S9. Incorporation of Drs S9 decreases the order parameter in all the lipid systems. The extent of decrease in the order parameter is higher for the CD 2 groups closer to the head group region and decreases along the acyl chains towards a minimum for the other end of the chain. Thus, the peptide binding increases the disorder in the hydrophobic region of PC, PG and PC/PG bilayers with a maximal disorder closer to the glycerol backbone of the lipid. Negligible changes are observed in the order parameters of CD 2 groups near the lower end of the lipid. Possibly, the hydrophobic core near the terminal methyl group of the bilayer is highly disordered even in the absence of the peptide and therefore, the peptide-induced disorder appears to be negligible. It is noteworthy that the membranedisorder induced by Drs S9 is of the same order compared to that of other antimicrobial peptides [49][50][51][52][53][54][55][56]. The calcein experiments show a membrane leakage in membranes without and with limited content of anionic lipid (PC, PC/PG) suggesting that the leakage could be induced either by perturbation of lipid packing or by formation of peptides pores. Nevertheless, our DSC and solid-state NMR data do not show any huge perturbation of membrane cohesion, clearly indicating that membrane integrity is largely maintained upon interaction with Drs S9. SDS-Tris-Tricine-PAGE experiments confirm the presence of oligomeric forms of Drs S9 (data not shown). Our data suggest that the peptide binds to the membrane in an aggregated state. One possible hypothesis is that the peptide binds to the membrane with a transmembrane insertion and may induce a transient pore. Indeed, the results presented in this paper show that such a mode of pore formation may occur in membranes containing PG and PC that mimic the lipid composition of E.coli. However, it is unlikely that such pores are triggered by the presence of anionic lipids, as proposed for many other cationic antibiotics, since pores are not formed in pure anionic lipid bilayers. Therefore, we propose that other, yet unknown, factors must be involved to account for the antibiotic activity of Drs S9.	2016-05-12T22:15:10.714Z	2013-10-11T00:00:00.000	{ "year": 2013, "sha1": "bbf7816cf24681ab2b07b66543eaf2745c3429a1", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0075528&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "bbf7816cf24681ab2b07b66543eaf2745c3429a1", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
253436853	pes2o/s2orc	v3-fos-license	Passive Radiative Cooling of Silicon Solar Modules with Photonic Silica Microcylinders Passive radiative cooling is a method to dissipate excess heat from a material by the spontaneous emission of infrared thermal radiation. For a solar cell, the challenge is to enhance PRC while retaining transparency for sunlight above the bandgap. Here, we design a hexagonal array of cylinders etched into the top surface of silica solar module glass to enhance passive radiative cooling. Multipolar Mie-like resonances in the cylinders are shown to cause antireflection effects in the infrared, which results in enhanced infrared emissivity. Using Fourier transform infrared spectrometry we measure the hemispherical reflectance of the fabricated structures and find the emissivity of the silica cylinder array in good correspondence with the simulated results. The microcylinder array increases the average emissivity between λ = 7.5–16 μm from 84.3% to 97.7%, without reducing visible light transmission. O ver the past few decades, significant effort has been put into improving the power conversion efficiency of solar cells. 1 The detailed balance limit 2 calculates a fundamental efficiency limit of 29.7% for silicon-based solar cells, 3 not far from the current record efficiency of 27.6%. 4 Solar cell efficiency measurements are performed at standardized testing conditions, e.g., 1 sun illumination at an operating temperature of 25°C. However, due to hot-carrier cooling and nonradiative recombination, a silicon solar cell typically reaches operating temperatures of 60°C under direct sunlight, and even as high as 80°C. 5 Elevated operating temperatures reduce the power conversion efficiency and the operating lifetime of the cell. This efficiency reduction is mainly attributed to a decrease in the open-circuit voltage due to increased recombination rates. 6 An average relative efficiency drop of −0.45% has been shown for every 1°C temperature rise of mono-and poly crystalline silicon solar modules. 7 A temperature increase from 25 to 60°C amounts to a significant −15.75% efficiency drop. Even though the effects of elevated operating temperatures on the operating lifetime of a silicon solar module have not been isolated, 8 it is expected to negatively impact all degradation modes. 9,10 These adverse temperature effects emphasize the need for a method to cool Si solar modules. Here, we investigate the enhancement of passive radiative cooling (PRC) to decrease the operating temperature of a Si solar cell. The concept of PRC leverages the thermal emission of an object to dissipate heat to lower its temperature. According to thermodynamics, two objects with different temperatures will exchange heat via thermal radiation until an equilibrium temperature is reached. The Stefan−Boltzmann law states that the amount of heat emitted as thermal radiation scales with the temperature of an object as ∝T 4 , so effectively, heat is transferred from the warmer object to the colder object. Thus, to cool a hot object with thermal emission, a colder object is needed to function as the heat sink. Outer space is the perfect heat sink due to the temperature of about 3 K and the immense volume, which makes it a heat sink with practically infinite capacity. The concept of emitting thermal radiation into outer space is the core principle of PRC. During the last two decades, interest in PRC has grown for several applications, 11 from dew collection in remote and dry places 12 to the cooling of buildings. 13 In 2014, Raman et al. showed a 4.9°C subambient daytime cooling using a thin-film multilayer to optimize the radiative properties of the structure. 14 The multilayer was designed to enhance PRC while simultaneously excluding heating by reflecting incident solar radiation. However, the high reflectance of this geometry in the visible spectral range makes it particularly unsuitable for solar cell applications. A solar cell absorbs light with photon energy larger than the bandgap energy, including visible wavelengths. Over the past years, several different materials and methods have been explored to cool solar cells with PRC: multilayers, 15,16 2D structures, 17−23 a combination of a multilayer and 2D structures, 24,25 or an effective medium approach. 26−28 Table 1 lists the calculated (italic) or fabricated (bold) material systems of a few notable works, and their reported temperature reduction: calculated values are in italic, measured values in bold font. To enable comparison of the achieved temperature reductions, it is important to specify the reference material system. This is particularly important since bare silicon exhibits practically no PRC, while a standard glass cover achieves significant PRC. Jaramillo-Fernandez et al. demonstrated the largest temperature reduction, both with respect to a bare silicon substrate and silicon covered with silica glass. 18 They placed a self-assembled monolayer of silica spheres on top of the bare silicon and silicon-silica reference samples and measured an average temperature reduction of 14 and 9 degrees, respectively. Although the layer of spheres performs well, it may suffer from structural degradation in outdoor conditions. A second thing to be considered is the solar transmittance, since the silicon solar cell should be able to absorb visible light. Therefore, all discussed papers in Table 1 are using materials that are transparent in the visible wavelength range. Here, we propose an effective and simple approach to enhance the PRC properties of a silicon solar module by patterning the top surface of the module with resonant microstructures. Because the glass of the solar module is used as an absorber, we do not need any additional materials which simplifies manufacturing and creates an advantage over other proposed methods. The design enhances the cooling power while keeping the structure transparent in the visible and nearinfrared wavelength range. First, we theoretically derive a general condition for the ideal optical properties to achieve maximum PRC in a silicon solar module. Then, we use numerical finite-difference time-domain (FDTD) simulations to design a microcylinder array on top of a quartz silica module glass to enhance PRC compared to the flat silica reference. We fabricate the optimized design by UV photolithography and reactive ion etching. Hemispherical reflectance measurements in the IR are performed to obtain the experimental emissivity spectra, which correspond very well to the simulated results. The average emissivity between λ = 7.5−16 μm is increased from 84.3% (silica reference) to 97.7%, which results in a calculated reduction of 3 K in the equilibrium temperature. Finally, we show that the silica substrate with a microcylinder array remains transparent in the visible and near-infrared (NIR) wavelength ranges, this implies that this structure can be used for all solar cell applications that want to harvest the solar spectrum. ■ PASSIVE RADIATIVE COOLING The concept of passive radiative cooling (PRC) is based on the thermal balance of a solar cell. In Figure 1, a schematic representation of the energy balance of a solar cell is shown, indicating the four main power terms that determine the equilibrium temperature of the cell: the absorbed radiation coming from the sun (P sun ), the absorbed thermal radiation from the atmosphere (P atm ), the thermal radiation the solar cell is emitting (P rad ), and the power gained by convection (P conv ). Here we assume that heat conduction via connection to the rooftop is negligible. The total cooling power is given by the sum of the four main powers, When the total cooling power is zero, there is no net heat flux, and the solar cell has reached equilibrium temperature. A positive cooling power will effectively reduce the temperature, while a negative cooling power indicates the solar cell is heating up. Figure 1 shows a schematic representation of the power flows in eq 1; a detailed analysis of the interplay is provided in the Methods section. For a constant incident solar The calculated (italic) or fabricated (bold) PRC structure, the reference sample, and the calculated (italic) or measured (bold) temperature reduction are listed. Figure 1. Schematic representation of the incoming and outgoing power flows that govern the equilibrium temperature of a solar cell (T sc ). The solar cell reaches its equilibrium temperature when the power from the sun (P sun ) and the thermal radiation from the atmosphere (P atm ) are in balance with the thermal radiation emitted by the solar cell (P rad ) and the power flow by convection and conduction (P conv ). ACS Photonics Here, T sc and T amb are the temperatures of the solar cell and ambient, respectively, I BB is the intensity of the blackbody spectrum, ε atm is the emissivity of the atmosphere, and ε sc the emissivity of the solar cell. To make sure that the first term in this equation is positive and contributes to cooling of the solar cell instead of heating, we can see that the emissivity of the solar cell (ε sc ) must be zero when the incoming intensity from the atmosphere is larger than the intensity of the blackbody spectrum, and unity otherwise. Figure 2b shows the blackbody spectra at ambient temperature (300 K) and a typical solar cell operating temperature (340 K). The spectrum of a nonideal blackbody is obtained by multiplying the ideal spectrum by the emissivity. By photonic engineering of the emissivity of the PRC layer, we can explore the effect of an emissivity spectrum on the resulting equilibrium temperature of the solar cell stack. In this comparison we assume that the PRC layer does not influence the absorbing properties of the solar cell in the visible wavelength range and take P sun as a constant power corresponding to the integrated solar spectrum below silicon bandgap. First, we consider the upper temperature bound of zero emissivity for energies below the solar cell bandgap, plotted in Figure 2c in red. Calculating the cooling power with eq 1, we obtain the red curve in Figure 2d and an equilibrium temperature of 366.5 K. Second, we consider an emissivity window that is unity only in the main atmospheric transmission window between 8−14 μm, as shown in Figure 2c in green. This is the ideal emissivity spectrum for a solar reflector, enabling it to cool below ambient temperature by radiating through the atmosphere while keeping heat from the sun and atmosphere out. However, when used for a solar absorber material like a silicon solar cell, the resulting PRC is suboptimal with an equilibrium temperature of 341.5 K, as shown in Figure 2d in green. Third, we consider a solar absorber at a temperature higher than ambient. Due to this higher temperature, it emits more blackbody radiation than it receives from the atmosphere over the entire infrared wavelength range−see the intensity difference plotted in Figure 2b. Therefore, a solar absorber can achieve higher PRC by setting its emissivity to 1 throughout its entire blackbody radiation spectrum, from 3−30 μm, as plotted in blue in Figure 2c. Below 3 μm, the blackbody radiation at 340 K is negligible; thus, we set the emissivity to 0 between the silicon bandgap and 3 μm. This emissivity spectrum achieves the minimum equilibrium temperature at 330.5 K. We color the background of Figure 2d purple to indicate the attainable equilibrium temperatures to be used as a reference for the final results. In the next section, we look at the PRC of a silicon solar cell stack and improve the PRC with a photonic cylinder array. ■ PHOTONIC DESIGN In the previous section, we have derived a condition for the ideal emissivity of a solar cell: unity emissivity for wavelengths Step function of three emissivity spectra: zero emissivity (red), a nonzero emissivity only in the main atmospheric transmission window (green), and the ideal emissivity for a body at 340 K (blue). (d) The calculated cooling power versus solar cell temperature (eq 1), corresponding to the emissivity spectra in (c). The equilibrium temperature is reached when the cooling power is zero. larger than 3 μm. The silicon solar cell itself has zero absorptivity (extinction coefficient) throughout the IR wavelength range (see Supporting Information, Figure S2), and thus zero emissivity (the reciprocity between absorptivity and emissivity according to Kirchhoff's law is described in the Methods section). Therefore, we need to add a material to the solar cell to improve its PRC capacity, which is in thermal equilibrium with the solar cell. A typical solar module has a glass cover on top of the silicon, making it an obvious choice. Beyond 7.5 μm wavelength, the extinction coefficient off glass is nonzero, making them much more suitable materials for PRC than silicon (see Supporting Information, Figure S2 for the comparison of refractive index of abundant materials in PV). We use a transfer matrix model (TMM, see Methods for details) to calculate the reflectivity of a solar module geometry consisting of a silicon substrate with a gold coating on the backside (solar cell back contact) and a quartz silica substrate on the top. The gold coating eliminates any transmission of light through this stack, and thus we can compute the emissivity as 1 − R sc , as shown in Figure 3a in red. The emissivity of the silica-on-silicon stack is much higher than the reference without silica, which is only slightly above zero due to parasitic absorption in the Au back coating (Figure 3a in green). The dip in the emissivity spectrum of the silica-onsilicon stack at 9 μm wavelength is caused by enhanced reflection at the air−silica interface. The enhanced reflection is a direct consequence of the strong fluctuation of the complex optical constants of silica in this spectral range, which is attributed to the asymmetric stretching vibration of Si−O−Si bridges. 30 Even though we defined the ideal emissivity as unity until λ = 30 μm in the previous section, we carried out simulations until λ = 16 μm because this was the range of the experimental emissivity data. In Table 1, we have already seen that significant calculated and measured temperature reductions have been achieved with 2D structures for enhanced PRC. However, only Zhu et al. made use of the solar cell module glass for their experimental PRC results, which would be ideal for practical reasons. They enhanced PRC by etching deep hollow cylinders, improving antireflection due to the gradual refractive index change. 29 While this does enhance PRC, this approach does not include optimization for specific wavelengths. Therefore, we propose direct integration of a thinner 2D microstructure in the module glass, which we can optimize thoroughly. The microstructures should minimize the reflection between 7.5 and 16 μm wavelength (to minimize the dip in the red curve in Figure 3a) and simultaneously retain transparency for sunlight with photon energies above the silicon bandgap. To achieve antireflection, we design a hexagonal array of silica microcylinders on the silica substrate. These structures exhibit Mielike resonances when their size is on the order of the wavelength. 31 These types of resonant structures have received much attention in the field of nanophotonics. 32,33 For photovoltaics, in particular, Mie-like resonant structures have been used to enhance light trapping, 34 design solar cells with structural colors, 35 and achieve antireflection for incident sunlight. 36,37 Here, we design cylinders of several micrometers in size to achieve resonant antireflection for IR instead light. We use finite-difference time-domain (FDTD) simulations to optimize the dimensions of the hexagonal cylinder array (see Methods for technical details). We varied the cylinder diameter, height, and array pitch to minimize the reflection in the 4−16 μm wavelength range, finding the optimum for a diameter of 3.5 μm, a height of 2.25 μm, and a 6.125 μm pitch in a hexagonal array. The calculated emissivity is plotted in Figure 3a in blue, showing a significant increase compared to flat silica. To gain an understanding of the resonant antireflection effect of the microcylinder array, we analyze the modal scattering contribution to the reflection spectrum. We attribute the antireflection effect to forward scattering of incident light by the multipolar Mie-like modes in the cylinders. According to the (generalized) Kerker condition, forward scattering is typically achieved by the interference of at least two different Mie-like modes. 38,39 Figure 3b shows the contributions of several Mie-like modes to the extinction cross-section of a single microcylinder, decomposed using the method outlined by Evlyukhin et al. 40 The decomposition shows that the main broad contribution comes from the electric and toroidal dipoles (ED+TD). The magnetic dipole (MD) and electric quadrupole (EQ) contributions are slightly detuned from each other. When we consider the coherent excitation of these multipolar modes by a normal-incident plane wave, the modes oscillate in phase with each other. The symmetry of the modes leads to constructive interference between the ED and MD/ EQ in the forward direction (transmission) and destructive interference in the backward direction (reflection). A "pure" Kerker condition of zero backscattering is achieved when the amplitude between the ED and MD/EQ are equal; this condition is typically only met for distinct wavelengths. Here, we achieve enhanced forward scattering by imperfect destructive interference of the ED and MD+EQ over a broad wavelength range. The comparison with Kerker-type inferences is shown in more detail in the Supporting Information. Figure S1 shows the polar scattering profile of an excited silica cylinder at 9 and 10 μm wavelength, and the polar emission profile of the interfering multipoles shown in Figure 3b. The resemblance of the profiles clearly indicates the Kerker-type forward scattering of our structure at these two wavelengths. By optimizing the shape of the resonator, it might be possible to further improve the destructive interference due to a better balance of the amplitudes of the modes. However, deviating from a radially symmetric shape also yields a polarizationdependent response. Moreover, destructive interference due to the interaction of multiple resonators or lattice modes is challenging with thermal sources, 41 as we discuss next. In general, interference between modes such as the Mie-like modes discussed above relies on a coherent phase-relation between them. However, the thermal emission that we consider as the source has only limited spatial coherence, typically on the order of λ/2. 42 Between 8−16 μm wavelength, the coherence length would be 4−8 μm, which is larger than the size of the designed cylinder. That suggests that singleparticle resonances such as the Mie-like modes, and their interference within the same particle, can be excited by thermal emission. ■ FABRICATION Based on the theory and simulations of the previous section, we fabricate the hexagonal microcylinder array on top of a silica substrate. A double-side polished (DSP) silicon wafer with a gold coating on the back (see Methods) is placed under the silica to replicate a simple solar absorber (see Figure 4a). We used UV photolithography and subsequent reactive ion etching to realize the microcylinder array on top of a silica substrate (see Methods for details). An optical micrograph of the finalized array covering a 24 × 24 mm 2 silica substrate is shown in Figure 4b, showing a uniform cylinder array and homogeneous color. Figure 4c shows a scanning electron microscope (SEM) image of a crosscut of the microcylinders, from which we determine a diameter of 3.65 μm and height of 2.20 μm, almost identical to the target dimensions of 3.50 (+4%) and 2.25 μm (−2.5%), respectively. The pitch is precisely 6.125 μm as designed. High transparency of the sample for visible light is visually demonstrated in Figure 4d, which shows a photograph of the sample on top of the AMOLF logo. ■ HEMISPHERICAL REFLECTION MEASUREMENTS To characterize the IR emissivity of the fabricated microcylinder array sample, we measure the hemispherical reflectance in a Fourier-transform infrared (FTIR) spectrometer (see Methods). Figure 5a shows the calculated emissivity spectra (dashed, shown before in Figure 3a) and the experimentally obtained spectra (solid). The reference case of a bare double-side polished silicon substrate with a gold coating on the back is plotted in green. The calculated spectrum is almost zero (∼3.5%) because the extinction coefficient of Si is zero beyond λ = 1150 nm. However, the measured emissivity is significantly higher beyond λ = 9 μm, which we attribute to intraband transitions in the slight n-type doped silicon substrate. 43 This small discrepancy does not influence our subsequent results because any IR light transmitted into the silica substrate is already absorbed before reaching the silicon substrate underneath. Thus, we can model the silicon to be nonabsorptive in the IR. The measured emissivity of a flat silica substrate on top of a silicon substrate is shown in Figure 5a in red. An air gap between the silica and silicon substrates was avoided by adding a layer of immersion oil in between for the optical measurements. Beyond λ = 5 μm, the experimental emissivity is almost identical to the calculated spectrum. This result validates the experimental setup and the optical constants of silica that were used in the calculation. Moreover, this also validates that the thermal light source has the required coherence to excite the designed resonant modes: while we do not measure blackbody radiation, the excitation source is a thermal Globar source. The discrepancy between the calculated and experimental spectra in the range of 2−5 μm is attributed to a slight mismatch between the optical constants used for the calculation and the actual optical constants of the silica substrate. The absorption onset is quite abrupt for the literature values, just below 4 μm, while the actual values seem to gradually increase between 2−5 μm and exhibit more spectral features. The measured emissivity of the silica substrate with the microcylinder array on top also resembles very well the calculated spectrum, as shown in Figure 5a in blue. Over a broad range from 7.5 to 16 μm, a significant increase in the emissivity was found, precisely as designed. The dip in the emissivity spectrum at 9 μm wavelength is reduced from 30% to 70%. Moreover, the average emissivity between 7.5 and 16 μm is increased from 84.3% to 97.7%. Based on the experimental emissivity spectra, we now calculate the total cooling power as a function of operating ACS Photonics pubs.acs.org/journal/apchd5 Letter temperature. Figure 5b shows three cooling power curves corresponding to the measurements in Figure 5a. The purplecolored background indicates the theoretical area between the upper and lower temperature bounds, as calculated in Figure 2d. At zero cooling power, we find the equilibrium temperature. As expected, we find that the silicon substrate with only the gold coating performs very poorly with an equilibrium temperature of 360 K, close to the maximum of 366.5 K. The addition of the flat silica substrate moves the curve closer to the minimum equilibrium temperature. The optimized silica microcylinder substrate pushes the equilibrium temperature even further toward the theoretical minimum. The flat silica stack and microcylinder silica stack reach equilibrium temperatures of 339 and 336 K, respectively, close to the minimum of 330.5 K. Interestingly, the flat silica substrate already achieves high PRC performance due to its near-ideal optical constants, realizing a cooling potential of 21 K compared to the bare silicon reference. The microcylinders decrease the equilibrium temperature by another 3 K. Assuming a 0.45% increase in efficiency per degree cooling, this temperature reduction would result in a relative efficiency increase of 1.35 and 10.8%, compared to the silicon solar module and the bare silicon reference, respectively. These values overestimate the equilibrium temperatures because they are based on experimental emissivity data up to λ = 16 μm (Figure 5a). In contrast, the theoretical maximum is based on unity emissivity until λ = 30 μm (Figure 2c). The calculated equilibrium temperature corresponds well with the range of values listed in Table 1. An exact comparison of the reported temperature reductions is not possible because the calculation methods are not identical: the exact wavelength span and the optical constants vary. However, the results do show that choosing a proper module cover impacts the equilibrium temperature significantly. This is important for flat pane (silicon) solar cells, as discussed here, and concerns the development of flexible thin-film solar cells that use thin plastic module covers. Finally, we perform hemispherical reflectance measurements in the visible to near-infrared (NIR) spectral range to verify that the fabricated microcylinder arrays do not adversely affect the transmission of sunlight into the solar cell for energies above the Si bandgap. Figure 5c shows the experimental reflectance for the two silicon-silica stacks. The reflectance is around 20% up to the silicon bandgap, mostly due to reflection at the silica-silicon interface. Beyond 1100 nm, the reflectance is higher because the gold coating at the back reflects most NIR light. This is significantly higher than the reflection of commercial solar modules, which achieve only a few percent reflection due to antireflection coatings and/or textures. The reflection could be decreased for all three cases by adding an AR coating on top of the silicon. The comparison in Figure 5c shows that the microcylinders slightly decrease the reflection of light for wavelengths smaller than the silicon bandgap. This difference is explained by light trapping: light is scattered by the microcylinders and trapped in the silica substrate by total internal reflection. ■ CONCLUSIONS This work shows that the passive radiative cooling (PRC) power of a silicon solar module can be enhanced by placing an array of microcylinders on top of the module glass. Photonic Mie-like resonances in the silica cylinders reduce infrared (IR) light reflection at the silica-air interface through engineered destructive interference of the resonant multipolar modes. By reciprocity, this improved antireflection effect increases the IR emissivity of the silica module glass. First, we studied the optimal emissivity profile for a typical silicon solar module that operates at elevated temperatures. By examining the thermal balance of a solar cell at T = 340 K, we found quartz silica to be the ideal module glass material due to its broad extinction coefficient in the λ = 3−30 μm spectral range. Subsequently, we designed a microcylinder array etched into the silica and optimized the dimensions for enhanced emissivity with FDTD simulations. The cylinder array is optimized to reduce the emissivity dip around λ = 9 μm that is caused by strong reflection at the silica-air interface. Next, the microstructures were fabricated by UV photolithography and reactive ion etching into a silica substrate. To mimic a silicon solar module, we placed the microstructured silica substrate on a silicon substrate with a gold coating on the back. This stack has zero transmittance throughout the visible and IR wavelength ranges. The measured hemispherical IR reflectance compares very well to the simulated results, demonstrating the designed PRC enhancement. The fabricated microstructure increased the average emissivity between λ = 7.5−16 μm from 84.3% to 97.7%. Moreover, the microstructured silica substrate shows a slight decrease in reflectance in the spectral range where the Si solar cell absorbs. In conclusion, the flat silica coating already achieves high PRC due to its near-ideal optical constants, realizing a cooling potential of 21 K compared to the bare silicon reference. The microcylinders decrease the equilibrium temperature by another 3 K. Assuming a 0.45% increase in efficiency per degree cooling, this would result in a relative efficiency increase of 1.35−10.8%, compared to the silicon solar module and the bare silicon reference, respectively. This insight is also relevant for the development of lightweight photovoltaics that do not use a glass cover. These results highlight the opportunities of thermal management for photovoltaic applications by considering the module glass as an integral part of the photonic design. Our design concepts are general and applicable to all solar cell designs as well as module glass materials. The recent advancement of optimization algorithms and complex shape fabrication by 3D printing and conformal imprinting could improve the cooling potential of the design even further. ■ METHODS Passive Radiative Cooling Calculation. In Figure 1, a schematic representation of the energy balance of a solar cell is shown, indicating the four main power terms that determine the cooling power of the cell: = P P P P P cool rad sun atm conv Here, P sun is the absorbed radiation coming from the sun, P atm the absorbed thermal radiation from the atmosphere, P rad the thermal radiation the solar cell is emitting, and P conv the power lost or gained by convection. We assume that heat conduction via connection to the rooftop is negligible. The primary energy input is the irradiation from the sun, which is for normal incidence given by with I AM1.5G the solar irradiation within AM1.5G 44 and α(λ) the absorptivity of the solar cell. In Figure 2a, the AM1.5G solar spectrum is shown, and the silicon bandgap energy is indicated. The AM1.5G solar spectrum has an integrated power of 1000 Wm −2 , but silicon does not absorb light with energies below its bandgap. Therefore, for further calculations, P sun is set to 808 Wm −2 , which is the integrated power in the AM1.5G solar spectrum for energies above the silicon bandgap (the yellow surface in Figure 2a). Effectively, the emissivity of the stack is unity for wavelengths below 1.1 μm (this is not shown in Figure 2c). The power input from the sun is completely independent of the infrared emissivity that is tuned to improve PRC. Therefore, this factor influences the absolute equilibrium temperatures, but not the slope of the temperature curves in Figures 2d and 5b, nor the distance between different curves (see Supporting Information, Figure S3). For this comparative study, the precise value of P sun is thus unimportant. This also allows us to neglect the fact that a solar cell converts about 20% of the incoming solar power into electricity rather than heat. Second, heat can be exchanged between the solar cell and its environment through convection, given by the product of the nonradiative heat transfer coefficient (h c ) and the temperature difference between the solar cell and the ambient environment: We set the nonradiative heat transfer coefficient to 6 Wm 2− K −1 , corresponding to a wind speed of 1 m/s. 21 The solar cell radiates as a nonideal blackbody, so its emitted radiative power is given by which is the product of the emissivity of the solar cell (ε sc (λ)), a number between zero and one that determines the quality of the solar cell as a blackbody, and the blackbody radiation according to Planck's law: 45 The atmosphere also radiates as a nonideal blackbody. The radiative power from the atmosphere that is absorbed by the solar cell is given by which is the product of the blackbody radiation from the atmosphere, the emissivity of the atmosphere (ε atm (λ)), and the solar cell absorptivity (α sc (λ)). The blackbody spectrum of the atmosphere depends on the ambient temperature, T amb , just as the blackbody spectrum of the sun depends on the temperature of its surface. Then we use Kirchhoff's law for a body in thermal equilibrium, which states that the emissivity equals the absorptivity at every wavelength: Substituting the absorptivity for the emissivity in eq M6, we find that the cooling power depends on the balance between incoming radiation from the atmosphere and the outgoing blackbody radiation from the cell: Here, the emissivity of the atmosphere at normal incidence 21 is given by ACS Photonics where T atm (λ) is the atmospheric transmittance. 46,47 Both terms in eq M8 depend on the emissivity spectrum of the solar cell, ε sc (λ), which is the most important parameter to tune to achieve PRC. The integrand in eq M8 scales linearly with ε sc (λ), so the radiative cooling power can readily be optimized by calculating the integrand for each wavelength λ i , at a given solar cell and ambient temperature. If the integrand is positive, that is, at λ i the solar cell emits more radiation than it receives from the atmosphere, the ideal emissivity ε sc (λ i ) = 1: In the case of a solar reflector, a body with a temperature equal to the ambient temperature (T sc = T amb ), this criterion sets ε sc (λ i ) = 1 only for low atmospheric emissivity. This ideal curve is plotted in Figure 2c in green, where the emissivity is 1 only in the main atmospheric transmission window between 8−14 μm. In the case of a solar absorber like a silicon solar cell, the operating temperature is higher than the ambient temperature (T sc > T amb ). Figure 2b shows the atmospheric transmission with the blackbody spectra at temperatures 300 and 340 K, corresponding to ambient temperature and the approximated temperature of an operating solar cell, respectively. Thus, the solar absorber emits more blackbody radiation than it receives from the atmosphere, independent of the atmospheric emissivity. Therefore, the ideal emissivity of a solar absorber is unity between λ = 3−30 μm, as plotted in blue in Figure 2c. Below 3 μm, there is negligible blackbody radiation at 340 K, so we set the ideal solar absorber emissivity to 0 between λ = 3 μm and the solar cell bandgap. The emissivity of the solar cell also equals its absorptivity (Kirchhoff's law, eq M7), which can be determined by with R sc (λ) as the reflection and T sc (λ) as the transmission of the solar cell. Maximizing the IR emissivity of the solar cell thus equals minimizing the reflection and transmission. Transfer Matrix Model. We used a transfer matrix model based on the Fresnel equations to calculate the reflection and transmission of planar multilayer stacks. In particular, we used the Python implementation written by Steven J. Byrnes. 48 Literature values for the optical constants of Si and SiO 2 (Supporting Information, Figure S2) were used from ref 49 and for Au from ref 50. FDTD Simulations. The optimization of the microcylinder array was performed by finite-difference time-domain (FDTD) calculations using Lumerical FDTD solutions. 51 The singlepass IR reflection was minimized for a hexagonal array of silica cylinders at the interface of a semi-infinite silica substrate and air superstrate. Minimizing the reflection led to maximizing the cooling power (see the calculation above), assuming that all IR light transmitted into the silica substrate is absorbed. The cylinder array was simulated in periodic boundary conditions. Convergence was found for a uniform 25 nm mesh size, conformal mesh refinement, and 10 −7 auto shutoff value. With a brute-force optimization procedure the optimized design was found to be a hexagonal array of silica cylinders, height 2.25 μm and radius 1.75 μm, with a constant pitch at 3.5 times the radius. The results of the optimization procedure are shown in Supporting Information, Figure S4. The figure of merit is the radiative part of P cool , according to eq M8, integrated between λ = 2−16 μm. The multipole decomposition in Figure 3b was performed by calculating the electric field inside the microcylinder according to the method outlined by Evlyukhin et al. 52 Electron-Beam Physical Vapor Deposition. A doubleside polished (DSP) silicon wafer (WRS Materials, lightly phosphorus n-type doped, resistivity 1−20 Ωm) with a thickness of 500 μm was used as the absorber substrate. Electron-beam physical vapor deposition was used to deposit an 80 nm gold layer on one side of 24 × 24 mm 2 DSP Si substrates at a deposition rate of 0.5 Å/s. UV Photolithography and Reactive Ion Etching. As a photolithography mask, a negative photoresist, ma-N 1420, was spin coated onto a 4 in. silica wafer of 500 μm thickness. A hexamethyldisilazane (HMDS) resist adhesion promotor was spin coated on the wafer at 4000 rpm with 1000 rpm/s for 35 s, followed by a curing step on a 150°C hot plate for 1 min. A 2 μm thick layer of ma-N 1420 was spin coated at 2000 rpm with 500 rpm/s for 30 s and cured at 100°C for 2 min. The photoresist was illuminated (λ = 365 nm) in a UV mask aligner (Suss MicroTec MABA6) through a quartz substrate with a chrome mask (commercial, Delta Mask BV). Unwanted interference due to reflection from the bottom silica-air interface is decreased by placing an absorptive tape on the back. The resist was developed by immersing the wafer into ma-D 533/S photoresist developer for 75 s; the wafer was then rinsed in H 2 O for 30 s (twice) and blow-dried using a nitrogen gun. The microstructured photoresist was used as a reactive ion etching (RIE) mask in an Oxford PlasmaPro Cobra RIE. A plasma of 50 sccm C 4 F 8 and Ar gases was used to etch 2.20 μm deep in 16:30 min. The remaining photoresist was removed by immersion in base piranha, rinsed in H 2 O, and dried under a nitrogen gun. Finally, the wafer is cut into 24 × 24 mm 2 substrates. Infrared Hemispherical Reflectance. The infrared hemispherical reflectance measurements were conducted in a modified Bruker Vertex70 research-grade laboratory Fouriertransform infrared (FTIR) spectrometer at the University of Twente, as described in work by Hecker et al. 53 The Vertex70 is modified with an external, custom-made integrating sphere with a diffuse gold coating, with a Mercury Cadmium Telluride (MCT) infrared detector on top (λ = 1−16.7 μm). The sample is positioned at the south pole of the sphere, with a variable aperture set to 20 mm. An external, high-power Globar source (λ = 1−16.7 μm) was used for high signal-to-noise measurements. Measurements are taken at a wavenumber resolution of 8 cm −1 and repeated 8 times to average the noise. All measurements are normalized to the reflection of a diffuse or ACS Photonics pubs.acs.org/journal/apchd5 Letter flat gold substrate, and the signal of an open sample port measurement is subtracted as background. The silica substrates were attached to the silicon substrate with immersion oil (Honeywell 10976) to avoid an air gap in between the substrates, which would cause unwanted Fabry−Perot resonances. The immersion oil was checked to be transparent throughout the visible-NIR wavelength range until the silicon bandgap. Moreover, the transmittance of the silica substrate is 0 throughout the IR, so the immersion oil will not influence the FTIR measurements. Visible to near-Infrared Hemispherical Reflectance. Hemispherical reflectance measurements were conducted in the visible to near-infrared (NIR) wavelength ranges with a PerkinElmer LAMBDA 750 UV/vis/NIR. A deuterium and a tungsten lamp were used, in combination with a double holographic grating monochromator to illuminate the sample with monochromatic light. The sample was placed at the back of a 150 mm integrating sphere at an angle of 8°, and the hemispherical reflectance was detected using a PMT and an InGaAs detector for the wavelength ranges 300−860 nm and 860−2500 nm, respectively. The integration time was 0.2 s, and the signal was averaged over three times to increase the signal-to-noise ratio.	2022-11-10T17:14:44.145Z	2022-11-08T00:00:00.000	{ "year": 2022, "sha1": "29b75585e15e26be3502ef9dabe67108af691b2e", "oa_license": "CCBY", "oa_url": "https://ir.amolf.nl/pub/10711/16850publishedVersion.pdf", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "d1259cdc6e7700d7c747cfea0888b4315fd6acfe", "s2fieldsofstudy": [ "Physics", "Engineering" ], "extfieldsofstudy": [ "Medicine" ] }
236954006	pes2o/s2orc	v3-fos-license	Perceived helpfulness of treatment for generalized anxiety disorder: a World Mental Health Surveys report Background Treatment guidelines for generalized anxiety disorder (GAD) are based on a relatively small number of randomized controlled trials and do not consider patient-centered perceptions of treatment helpfulness. We investigated the prevalence and predictors of patient-reported treatment helpfulness for DSM-5 GAD and its two main treatment pathways: encounter-level treatment helpfulness and persistence in help-seeking after prior unhelpful treatment. Methods Data came from community epidemiologic surveys in 23 countries in the WHO World Mental Health surveys. DSM-5 GAD was assessed with the fully structured WHO Composite International Diagnostic Interview Version 3.0. Respondents with a history of GAD were asked whether they ever received treatment and, if so, whether they ever considered this treatment helpful. Number of professionals seen before obtaining helpful treatment was also assessed. Parallel survival models estimated probability and predictors of a given treatment being perceived as helpful and of persisting in help-seeking after prior unhelpful treatment. Results The overall prevalence rate of GAD was 4.5%, with lower prevalence in low/middle-income countries (2.8%) than high-income countries (5.3%); 34.6% of respondents with lifetime GAD reported ever obtaining treatment for their GAD, with lower proportions in low/middle-income countries (19.2%) than high-income countries (38.4%); 3) 70% of those who received treatment perceived the treatment to be helpful, with prevalence comparable in low/middle-income countries and high-income countries. Survival analysis suggested that virtually all patients would have obtained helpful treatment if they had persisted in help-seeking with up to 10 professionals. However, we estimated that only 29.7% of patients would have persisted that long. Obtaining helpful treatment at the person-level was associated with treatment type, comorbid panic/agoraphobia, and childhood adversities, but most of these predictors were important because they predicted persistence rather than encounter-level treatment helpfulness. Conclusions The majority of individuals with GAD do not receive treatment. Most of those who receive treatment regard it as helpful, but receiving helpful treatment typically requires persistence in help-seeking. Future research should focus on ensuring that helpfulness is included as part of the evaluation. Clinicians need to emphasize the importance of persistence to patients beginning treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s12888-021-03363-3. Background Generalized Anxiety Disorder (GAD) encompasses a variety of symptoms including excessive worrying, restlessness, irritability, difficulties in concentration, and constantly feeling on edge. Among the anxiety disorders, GAD is perhaps the least well researched or understood in part because of multiple revisions of the diagnostic criteria, consideration of the diagnosis as a "wastebasket" category when other anxiety diagnoses could not made, and because worrying was assumed a part of everyday life and associated with minimal impairment. However, GAD is now known now to be a significant disorder with high prevalence and significant impairment and disability [1,2]. Worldwide an estimated 3.7% of individuals will have GAD in their lifetime [1]. The role and quality of life impairments of GAD are comparable in magnitude to those of major depression and greater than those associated with substance abuse disorders [3]. Randomized controlled trials (RCTs) have identified effective pharmacological and psychosocial treatments [4][5][6][7]. However, results from RCTs do not necessarily translate into real-world settings and are largely restricted to high-income countries (HICs) with little global evaluation of the effects of treatment [8][9][10]. Moreover, RCTs have focused on symptom reduction as the critical and sometimes the sole outcome. Yet, symptom reduction does not necessarily equate with functional improvement, quality of life, or feelings on the part of the patient that they have been helped [11,12]. Perceived helpfulness of treatment is a key construct that may capture whether patients achieve personally meaningful goals through treatment. Helpfulness is important in its own right, but also has a critical role in treatment in that perceived helpfulness relates directly to treatment adherence as well as to critical processes that span diverse forms psychotherapy (e.g., the therapeutic alliance, critical incidents during treatment, openness of the therapist) [13][14][15][16]. Helpfulness as an outcome of treatment has received little attention in GAD trials [17]. The extent to which helpfulness is achieved with treatment and whether this is achieved initially as the patient traverses different treatments remain to be evaluated. Such research might help identify unmet patient needs that can be targeted through policy and service responses [14,18,19]. Patient-centered research on perceived GAD treatment helpfulness may also inform treatment guidelines for GAD, which are currently based on a relatively small number of RCTs. The likelihood of a help-seeking individual ever obtaining helpful treatment is a joint function of two treatment pathways: 1) the probability that a given treatment provider will be helpful, and 2) the probability that a patient will persist in help-seeking after prior unhelpful treatment encounters. Research on these processes for depression and posttraumatic stress disorder found that the majority of patients who persisted after previous unhelpful treatments eventually obtained helpful treatments, but that only a minority persisted in help-seeking after more than a few unsuccessful treatment encounters [20,21]. It is unknown whether these patterns also are true for GAD. Decomposing treatment pathways for GAD would answer this question and might also reveal modifiable predictors of GAD treatment helpfulness and persistence that could be the focus of treatment quality improvement initiatives. The present study evaluated the helpfulness of treatment encounters and predictors and pathways leading to helpfulness in national community epidemiological samples that included respondents with a history of GAD who sought treatment for their GAD. The data come from the World Mental Health (WMH) Survey Initiative [22]. This is a coordinated series of general population surveys under the auspices of the World Health Organization. Data were collected in 23 countries of varying income levels. This data set provides a unique opportunity to evaluate helpfulness internationally and to investigate predictors and pathways of perceived treatment response in countries varying in income levels. Samples The World Health Organization's (WHO) World Mental Health (WMH) surveys are a coordinated set of community epidemiological surveys administered to probability samples of the non-institutionalized household population in countries throughout the world (https://www.hcp.med.harvard. edu/wmh/) [23]. Data The interview schedule was developed in English and translated into other languages using a standardized WHO translation, back-translation, and harmonization protocol [24]. Interviews were administered face-to-face in respondents' homes after obtaining informed consent using procedures approved by local Institutional Review Boards. To reduce respondent burden, interviews were administered in two parts. Part I was administered to all respondents and assessed core DSM-IV mental disorders. Part II assessed additional disorders and correlates and was administered to all respondents who met lifetime criteria for any Part I disorder and a probability subsample of other Part I respondents [25]. Generalized anxiety disorder (GAD) Lifetime history of GAD was assessed with the fully structured WHO Composite International Diagnostic Interview (CIDI) Version 3.0 [23]. In an evaluation carried out in conjunction with the US WMH survey [26], GAD diagnoses based on the CIDI had good concordance with diagnoses based on blinded clinical reassessments with the Structured Clinical Interview for DSM-IV (SCID) [27]. A clinical reappraisal study in other WMH surveys, although not evaluating GAD in isolation, found good concordance between diagnoses based on the CIDI and SCID for any 12-month anxiety disorder including GAD [28]. Consistent with previous studies that modified the CIDI GAD algorithm [1,26,29], we generated DSM-5 GAD diagnoses by removing the DSM-IV hierarchical exclusion of a GAD diagnosis when symptoms occur exclusively during a mood disorder [30]. Age of onset (AOO) of GAD was assessed using probing methods demonstrated to improve dating accuracy [31]. Perceived helpfulness of treatment for GAD Respondents who met lifetime criteria for GAD were asked whether they had ever "talk [ed] to a medical doctor or other professional about their worry or anxiety" and, if so, how old they were the first time they did so. "Other professionals" were defined broadly to include "psychologists, counselors, spiritual advisors, herbalists, acupuncturists, and other healing professionals." Respondents who had ever spoken to a professional about their GAD were asked whether they ever received treatment they "considered helpful or effective" (emphasis in original). If so, they were asked how many professionals they ever talked to about their worry or anxiety "up to and including the first time [they] ever got helpful treatment". Respondents who said they never received helpful treatment were asked how many professionals they ever talked to about their worry or anxiety. Predictors of obtaining helpful treatment for GAD In addition to age of onset of GAD (continuous), we considered 5 classes of predictors of helpful treatment: Socio-demographic characteristics included sex, marital status (currently married, never married, or previously married) at the time of first treatment, and education (in quartiles defined by within-country distributions) at the time of first treatment. Lifetime comorbid conditions included other lifetime anxiety disorders (including panic disorder or agoraphobia with/ without panic disorder, post-traumatic stress disorder, specific phobia, and social phobia), major depressive disorder, broadly defined bipolar spectrum disorder [32], alcohol and/ or drug abuse, and alcohol and/ or drug dependence but not abuse. Comorbid conditions were restricted to disorders with an age-of-onset prior to the age at which the respondent first sought treatment for GAD. All comorbid conditions were assessed with the CIDI. Treatment type was defined as a cross-classification of (i) whether the respondent reported receiving medication, psychotherapy, or both, as of the age of first GAD treatment; and (ii) the types of treatment providers seen as of that age. Types of providers included mental health specialists (psychiatrist, psychiatric nurse, psychologist, psychiatric social worker, mental health counselor), primary care providers, human services providers (social worker or counselor in a social services agency, spiritual advisor), and complementary-alternative medicine (other type of healer or self-help group). Treatment timing included a continuous variable for length of delay in years between age of onset of GAD and age of initially seeking treatment, and a dichotomous measure for whether the respondent's first attempt to seek treatment occurred before or after the year 2000. The year 2000 was the typical midpoint between the start of observations and survey field dates. Childhood adversities included retrospective reports of significant stressors experienced during childhood, including family dysfunction (physical or sexual abuse, neglect, parental mental disorder, parental substance use disorder, parental criminal behavior, and family violence) and other adversities (parental death, parental divorce, other loss of a parent, physical illness, and economic adversity). Analysis methods The sample was limited to respondents with a history of DSM-5 GAD treatment who sought treatment for the disorder at some time in their life. Cases were limited to those who first sought GAD treatment in 1990 or later to reduce the potential effects of recall bias. To investigate the two pathways of helpful treatment separately, we used discrete-event survival analysis to calculate the conditional and cumulative probabilities of: (i) obtaining helpful treatment after seeing between 1 and 10 professionals; and (ii) persisting in help-seeking after obtaining prior unhelpful treatment [33]. We followed respondents up through 10 professionals in the total sample and in HICs because this was the last number where at least n = 30 respondents received treatment. However, in low/ middle-income countries (LMICs), we followed respondents only up through 3 professionals seen because this was the last number where at least n = 30 respondents received treatment. We then carried out parallel survival analyses of the predictors of these two decomposed, encounter-level outcomes using standard discrete-time methods and a logistic link function [34], followed by a person-level model of overall probability of ever obtaining helpful treatment regardless of number of professionals seen (composite outcome). This allowed us to investigate predictors of obtaining helpful treatment at the person level and to investigate the extent to which these predictors were important because they predicted differential helpfulness at the encounter level, differential probability of persisting after earlier unhelpful treatments, or both. We also investigated possible interactions of each significant person-level predictor with country income group and historical time (beginning treatment in 1990-1999 vs. 2000+) in an effort to examine the generalizability of the findings. Because the WMH sample design used weighting and clustering in all countries, all statistical analyses were carried out using the Taylor series linearization method [35], a design-based method implemented in the SAS 9.4 program [36]. Logistic regression coefficients and +/− 2 of their design-based standard errors were exponentiated to create odds-ratios (ORs) and 95% confidence intervals (CIs) (with odds ratios less than 1 indicating lower likelihood, and odds ratios greater than 1 indicating greater likelihood, of the relevant association). Significance of sets of coefficients was evaluated with Wald χ 2 tests based on designcorrected coefficient variance-covariance matrices. Statistical significance was evaluated using two-sided, design-based .05 level tests. Helpful GAD treatment across professionals seen Across all countries, 26.7% (1.0) of respondents who received treatment said they were helped by the first professional they saw ( Table 2, Part I). The conditional probability of obtaining helpful treatment from a second professional seen after an initial unhelpful treatment was 36.6%. Conditional probabilities of obtaining helpful treatment generally declined after subsequent professionals seen but projected cumulative probabilities of obtaining helpful treatment rose from 26.7% after the first professional seen to 53.5% among respondents who persisted in seeing a second professional and to 96.9% among those who persisted in seeing up to 10 professionals after prior unhelpful treatments ( Table 2, Part II). These cumulative probabilities were broadly similar for LMICs and HICs up through three professionals seen (66.9% vs. 68.9%), after which the number of remaining respondents in LMICs became too small for analysis (eTable 2). Persistence with help-seeking for GAD following treatment failure Across all countries, 77.3% (1.1) of respondents persisted in seeing a second professional after initial unhelpful GAD treatment (Table 3). This proportion was higher in HICs (79.6% [1.2]) than LMICs (55.8% [3.5]). Conditional probabilities of help-seeking persistence remained quite high (81.9-100.0%) in the total sample up through 10 professionals seen. Unlike the situation with cumulative probabilities of obtaining helpful treatment, which, by definition, either remains the same or rises as the number of professionals seen increases, the cumulative probability of help-seeking persistence either remains the same or decreases as the number of professionals seen increases. In the total sample, the cumulative probability of persistence through 10 professionals was 29.7%. We were able to compare respondents in LMICs to those in high income countries through four professionals seen, where the cumulative probability of persistence was lower in LMICs (43.2% [5.9]) than in high income countries (57.5% [2.5]) (eTable 3). Predictors of obtaining helpful treatment for GAD We examined predictors of each respondent ever obtaining helpful GAD treatment regardless of number of professionals seen (Model 1; Table 4), then examined predictors separately for the two pathways to helpful treatment: obtaining helpful treatment from a given professional (Model 2; Table 4) and persisting in helpseeking after prior unhelpful treatment (Model 3; Table 4). We focus on the significant predictors at the personlevel (Model 1; Table 4) and examine how the results in the decomposed models help explain these person-level associations. Due to high comorbidity between disorders and the potential for multicollinearity, we evaluated associations with comorbid disorders in separate univariate Other childhood adversities include: parental death, parental divorce, other loss of a parent, physical illness, and economic adversity and then multivariate models. Only comorbid disorders that significantly predicted obtaining helpful treatment in the multivariate models were included in Models 1-3 as individual predictors (eTable 4). At the person-level, significant predictors of obtaining helpful treatment were treatment type (χ 2 4 = 12.3, p = 0.015), comorbid panic/agoraphobia (χ 2 1 = 8.9, p = 0.003), and childhood adversities (χ 2 2 = 9.7, p = 0.008). The association with treatment type was because respondents who received treatment from a mental health specialist in combination with medication had significantly increased relative-odds of obtaining helpful treatment than those who received treatment in the human services sector (the reference category; OR:1.46; 95% CI: 1.05, 2.02). Decomposition showed that this person-level association of treatment from a mental health specialist in combination with the outcome was due to lower relative-odds of encounter-level helpfulness (OR: 0.76; 95% CI: 0.62, 0.93), but higher relative-odds of treatment persistence (OR: 2.00; 95% CI: 1.50, 2.67). Comorbid panic/agoraphobia was associated at the person-level with having significantly increased relative-odds of obtaining helpful treatment (OR: 1.79; 95% CI: 1.22, 2.62) due to increased relative-odds of treatment persistence (OR: 2.12; 95% CI: 1.53, 2.94). Childhood adversities were also important predictors of treatment helpfulness, but unexpectedly, the pattern of associations was different for the two classes of adversities. A history of family dysfunction was significantly associated with reduced relative-odds of obtaining helpful treatment at the person-level (OR: 0.75; 95% CI: 0.58, 0.96), whereas a history of other childhood adversities was significantly associated with increased relative-odds of obtaining helpful treatment (OR: 1.47; 95% CI: 1.09, 1.99). Decomposition showed that the person-level association of family dysfunction with the outcome was due to significantly reduced relative-odds of treatment persistence (OR: 0.73; 95% CI: 0.57, 0.93), whereas the person-level association other childhood adversities with the outcome was due to significantly increased relativeodds of treatment persistence (OR: 1.51; 95% CI: 1.10, 2.07). It is noteworthy that the zero-Pearson correlation between the two CA measures is too low (r = 0.23) to create an opposite-sign pattern as a methodological artifact. Consistent with this observation, the same opposite-sign pattern was also observed in models where only one of the two CA measures was included. Although the omnibus χ 2 tests for treatment timing and treatment delay were not significant, there were significantly increased relative-odds of both predictors with significantly decreased relative-odds of with persistence in help-seeking after an initial unhelpful treatment, were found for both beginning treatment in more recent years (2000 or later) and longer treatment delays in obtaining helpful treatment from a given professional, resulting in nonsignificant associations at the person-level. We found significant interactions of treatment type and childhood adversities with country income level (eTable 5). These interactions were due to the predictors being more strongly associated with person-level treatment helpfulness in LMICs than HICs (eTables 6 and 7), but the significant associations were based on such small numbers of cases that substantive interpretation is hazardous. Significant interactions were also found between treatment type and historical time (eTable 8). These were due to mental health specialist and psychotherapy treatment and general medical treatment both having increased relative-odds of person-level treatment helpfulness only during the years 1990-1999 and receiving treatment from 2+ types of professionals having increased relative-odds of person-level treatment helpfulness only during the years 2000+ (eTables 9 and 10). Again, though, these interactions were based on relatively small numbers of cases and should be interpreted with caution. Discussion The main findings of the study are as follows. First, only about one-third of people with GAD reported ever obtaining treatment, with a lower proportion in LMICs than HICs (19.2% vs. 38.4%). Second, 70% of those who received treatment perceived the treatment to be helpful. This did not vary by country income level. Third, persistence in help-seeking was required to obtain helpful treatment, as only about one-fourth of patients were helped by the first professional they saw and about half by the first two professionals. Projections from our survival models suggest that up to 10 professionals might be needed to have a 90% probability of being helped, but that only 29.7% of patients would persist that long in the face of repeated unhelpful treatment encounters. Fourth, only relatively modest predictors were found of obtaining helpful treatment at the person-level, most of which were important because they predicted persistence rather than encounter-level treatment helpfulness. It is encouraging that the large majority (70.0%) of respondents with lifetime DSM-5 GAD who sought treatment eventually obtained treatment they considered helpful. This means that the majority of patients persisted up to 3-4 professions in the face of initial unhelpful treatment. This is a lower persistence rate than found in a parallel analysis of specific phobia [37], but a higher persistence rate than found in parallel analyses of patients with major depression [20] and PTSD [21], possibly because depressive and PTSD symptoms are more likely than anxiety symptoms to lead to discouragement in help-seeking. Even so, we estimated than only 22.7% of GAD patients would persist in help-seeking to a point where they had near certainty of receiving helpful treatment. Our data on the predictors of helpfulness are useful in beginning to delineate pathways. Patients who received treatment from a mental health specialist in combination with medication had significantly increased relative-odds of obtaining helpful treatment at the person-level than those who received treatment in the human services sector. This was due to lower relative-odds of encounter-level helpfulness, but higher relative-odds of treatment persistence. Patients who receive medication in addition to seeing a mental health specialist may have been more severe than other patients, resulting in lower relative-odds of encounter-level helpfulness due to the severity of their illness, but also more motivation to persist in help-seeking because of that high severity. Likewise, panic/agoraphobia was also associated with having significantly increased relative-odds of obtaining helpful treatment at the person-level due to increased relative-odds of treatment persistence. These individuals may have more chronic and impairing courses of illness [38] which may motivate them to persist in help-seeking. The relationship between childhood adversity and treatment helpfulness was less straightforward. Family dysfunction was associated with lower relative-odds of obtaining helpful treatment due to lower relative-odds of treatment persistence. In contrast, other childhood adversities were associated with greater relative-odds of obtaining helpful treatment due to increased relativeodds of treatment persistence. One possible explanation for this difference is that the family dysfunction category included violent and traumatic forms of adversity, whereas the other childhood adversities category did not [39]. If this finding is replicated in other studies, future research should focus on why traumatic childhood events are associated with lower persistence in helpseeking, which could inform treatment guidelines. Important limitations of this study should be noted. First, there was limited information about the precise nature of the interventions that respondents received and no information about sequencing of treatments across types of providers. Moreover, the treatments were not randomized nor evaluated in relation to quality of delivery (treatment integrity) or compliance on the part of the patient (adherence). Consequently, the relation of critical dimensions of treatment to helpfulness could not be discerned. Second, the sample was limited to respondents with onset of GAD treatment after 1990. Recall may have been biased and influenced evaluations of the treatments and helpfulness [40]. It is unclear whether these limitations would lead to upward or downward bias in estimates of treatment effectiveness at the encounter level or patient level. Despite these limitations, to our knowledge this is the first study of perceived helpfulness of treatment of GAD. A strength of the study is including large sample representing multiple countries and with the ability to evaluate commonalities and differences among low and middle income and highincome countries. RCTs are clearly required to determine the efficacy and effectiveness of GAD treatments [4][5][6][7]. Although our data do not fill this need, they are important because they address issues that RCTs cannot. Specifically, RCTs typically focus on short-term effects (e.g., 3 months), assess mainly symptomatic changes, and exclude many people who might have more complicated disorders, such as those with psychiatric comorbidities but who would benefit from treatment [8]. Our study, in comparison, looked at a broad and representative sample without these exclusionary criteria and included information on how patients view their treatment. We would encourage the assessment of treatment helpfulness in clinical trials because it is distinguishable from symptomatic change. One can readily conceive of patients showing similar or identical changes on standardized symptom measures but in fact profiting in different degrees from treatment in their everyday lives and hence in their views of how much they have been helped [41]. Our findings suggest that treatment guidelines should not only encourage evidence-based interventions, but also should emphasize the value of treatment persistence. Our data do not allow us to study new treatments from the same provider. The persistence we examined was across providers. It should be noted, though, that evidence is clear in showing that patients can also be helped by new treatments from the same provider [42]. Further work is needed to expand GAD care to address treatment motivations and expectations and to determine the extent to which interventions to improve GAD treatment quality and persistence can improve outcomes. Abbreviations CIDI: Composite International diagnostic interview; GAD: Generalized anxiety disorder; RCT: Randomized control trial; WHO: World health organization; WMH: World mental health Additional file 1: Table 1. WMH sample characteristics by World Bank income categories a . Table 2. Conditional and cumulative probabilities of obtaining helpful treatment for generalized anxiety disorder after each professional seen, among respondents with lifetime DSM-5 generalized anxiety disorder who obtained treatment in low/middle-income and high-income countries. Table 3. Conditional and cumulative probabilities of persistence in help-seeking after previous unhelpful treatment, among respondents with lifetime DSM-5 generalized anxiety disorder who obtained treatment in low/middle-income and high-income countries. Table 4. Predictors of obtaining helpful treatment (person-level), among people with lifetime DSM-5 generalized anxiety disorder who obtained treatment. Table 5. Interactions between main effects and country income group to predict obtaining helpful treatment (person-level composite outcome) and the decomposed encounter-level outcomes of helpful treatment and persistence, among people with lifetime DSM-5 generalized anxiety disorder who obtained treatment. Table 6. (Low/ middle-income countries): Predictors of obtaining helpful treatment (person-level composite outcome) and of the decomposed encounter-level outcomes of helpful treatment and persistence, among people with lifetime DSM-5 generalized anxiety disorder who obtained treatment. Table 7. (High-income countries): Predictors of obtaining helpful treatment (person-level composite outcome) and of the decomposed encounter-level outcomes of helpful treatment and persistence, among people with lifetime DSM-5 generalized anxiety disorder who obtained treatment. Table 8. Interactions between main effects and historical time to predict obtaining helpfulness of treatment (person-level composite outcome) and the decomposed encounter-level outcomes of helpful treatment and persistence, among people with lifetime DSM-5 generalized anxiety disorder who obtained treatment. Table 9. (Started GAD treatment in 2000 or later): Predictors of obtaining helpful treatment (person-level composite outcome) and of the decomposed encounter-level outcomes of helpful treatment and persistence, among people with lifetime DSM-5 generalized anxiety disorder who obtained treatment. Table 10. (Started GAD treatment 1990 to 1999): Predictors of obtaining helpful treatment (person-level composite outcome) and of the decomposed encounter-level outcomes of helpful treatment and persistence, among people with lifetime DSM-5 generalized anxiety disorder who obtained treatment.	2021-08-09T13:56:19.400Z	2021-08-09T00:00:00.000	{ "year": 2021, "sha1": "a4f8db3d4671df7ea3477022ed4497ca3f80100e", "oa_license": "CCBY", "oa_url": "https://bmcpsychiatry.biomedcentral.com/track/pdf/10.1186/s12888-021-03363-3", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a4f8db3d4671df7ea3477022ed4497ca3f80100e", "s2fieldsofstudy": [ "Psychology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
234368045	pes2o/s2orc	v3-fos-license	Hybrid Feature Selection Approach to Improve the Deep Neural Network on New Flow-Based Dataset for NIDS Network Intrusion Detection System (NIDS) detects normal and malicious behavior by analyzing network traffic, this analysis has the potential to detect novel attacks especially in IoT environments. Deep Learning (DL)has proven its outperformance compared to machine learning algorithms in solving the complex problems of the real-world like NIDS. Although, this approach needs more computational resources and consumes a long time. Feature selection plays a significant role in choosing the best features only that describe the target concept optimally during a classification process. However, when handling a large number of features the selecting such relevant features becomes a difficult task. Therefore, this paper proposes Enhanced BPSO using Binary Particle Swarm Optimization (BPSO) and correlation–based (CFS) classical statistical feature selection approach to solve the problem on BPSO feature selection. The selected feature subset has evaluated on Deep Neural Networks (DNN) classifiers and the new flow-based CSE-CIC-IDS2018 dataset. Experimental results have shown a high accuracy of 95% based on processing time, detection rate, and false alarm rate compared with other benchmark classifiers. Keywords— Network Intrusion Detection System, Feature Selection, Deep Learning, CFS, Binary PSO, CSE-CIC-IDS2018 Introduction The exponentially growing number of security breaches, cyberattacks on Internet of things IOT highly required reliable security solutions. Network Intrusion Detection System (NIDS) used as defense of network infrastructure by detecting malicious activities and preventing attacks [1]. NIDS can be divided into misuse detection is also called signature-based detection and anomaly detection NIDS that are monitoring the network pattern and learning the normal behavior of a system and distinguish each network activity detect it as an intrusion when deviate from the normal pattern [2]. We focus on anomaly detection NIDS because its ability to detect unknown attacks despite it have high false alarm rate because inability to determine reasons of an abnormality. Traditional machine learning (ML) approaches have been supplied for cyber security such as Bayesian Belief Networks (BBN), Random forest, Support Vector Machines (SVM) and others, but the generation of large scale data in IoT required a deep learning based approach which performs better with large data sizes and can learn representation of feature from raw data so it is adaptable to different attack scenarios [3]. They proposed a new malware prediction model that could detect the coming future malware by the implementing a deep learning method of Mal Generative Adversarial Network (Mal-GAN) [4]. showed that the LSTM classifier outperform over previously published results of other static classifiers on KDD Cup '99 dataset challenge for long time which prove the benefit of LSTM networks to intrusion detection, because the ability of LSTM to learn from look back in time and link connection records consecutively [5].The RNN, Stacked RNN, and CNN are supervised deep learning techniques applied to classify common five attack types using Keras .This technique used packet header information without need any user payload then compared its results with Snort IDS .The results showed that this technique gave superior results compared Snort [6]. Variant-Gated Recurrent Units (GRU) with encoders performed on ISCX2012 dataset to make preprocessing on packets of payload-aware intrusion detection. It could learn features of network packet header and payload automatically and improved the detection rate of the IDS [7]. proposed RNN-RBM model which take input data as byte-level without feature engineering. At first, RBM model used network packets to extract the feature vectors. Then RNN model extracted the flow feature vector which sent to the Softmax layer to detect result [8]. Recently, the large growth of data makes big challenge to the task of data classification. The feature selection is an option to solve this challenge by reducing the dimensionality of the data and achieve higher accuracy in data classification. In terms of feature selection, it plays an important role in improving NIDS performance. This is because anomaly detection uses a large number of time-consuming features. Therefore, choosing the method for selecting the feature affects the improvement of the level of accuracy and the time required to check traffic behavior. There are three types of features selection: filter, wrapper and embedded techniques. The filter technique tries to classify a subset from the original set containing of several selected features based on the evaluation criteria. While the wrapper technique, chose the features that have high predictive accuracy from different learning algorithms. The embedded technique where the feature selection embeds into the training step [9]. Paper performed Experiments on NSL-KDD datasets using log2 and PCA on deep learning algorithms. Results proved the effect of dimensionality reduction on the accuracy ratio about 97.9%. Thus, minimizing features in dataset and select optimal subset of most relevant features for each class to reduce processing time, improve detection accuracy rate, reduce false alarm rate. As result, the efficiency for intrusion detection in IOT environment improved because the irrelevant and redundant features cause overfitting and poor generalization during the classification [10]. Optimization means finding the optimal solution from a set of choices with regards to an objective function and some conditions. Intelligent applications that using Swarm Intelligence algorithms are becoming famous because of their ability to handle any real time complex and uncertain situation. Swarm intelligence is kind of algorithms which simulate the behavior of living organisms such as birds, insects, and fish. These individuals able to complete complex tasks in real world when working in unity that would be very difficult to achieve it [11]. In today's world, application of Swarm intelligence and Deep learning have been provided in many fields successfully such as image classification, pattern recognition and intrusion detection system. this paper has designed seven layer CNN which commonest deep learning approach, called ConvNet performed to classification of handwriting digit. The Particle Swarm Optimization algorithm (PSO) is used to improve the input parameters of processing layers [12]. This work proposed approach of swarm intelligence for parameter setting in deep neural network. through providing this approach to the phishing websites classification. As a result, the proposed algorithm improves their detection compared to other algorithms [13]. The contributions of this paper as following: Using a swarm intelligence for features selection by implementing a Binary PSO algorithm. Improving the NIDS by optimized deep learning models with pre-processing phase employing a Binary PSO algorithm. This approach optimized detection rate (DR) of deep learning models while reducing false alarm rate(FAR)compared with corresponding values of deep learning models without preprocessing phase. Evaluating this approach by using new CSE-CIC-IDS2018 real datasets for classification tasks. Presenting four comparative analyses between our results and the literature best results. Also, employing several evaluation metrics to depict analysis performance of deep learning models on our approach. However, Swarm intelligence are often limited by weak points of computation time and local solution for large and complex problems. While, Deep learning algorithms are often limited by weak points of data and parameters 2 Related Work The Paper proposed a new algorithm to optimize the structure of DBN network. At first designed a PSO next used the fish behavior to optimize the PSO and find the initial solution of optimization. Then, used the genetic operators (crossover probability and mutation)on the PSO to search the global solution for optimization which used to construct the network structure for intrusion detection on NSL-KDD [14].The researcher aimed to improving the performance of NIDSs on UNSW-N15 dataset by proposed four feature selection models based on the particle swarm optimization (PSO), firefly optimization (FFA),genetic algorithm (GA)and grey wolf optimizer (GWO).The derived features from this model are evaluated on the J48 ML and support vector machine(SVM) classifiers [15].A double PSO-based algorithm proposed to select subset of features and hyper parameters both in the same work . Three deep learning models (Deep Neural Networks (DNN), Deep Belief Networks (DBN) and Long Short-Term Memory Recurrent Neural Networks (LSTM-RNN) utilized to show the differences in performance on CICIDS2017dataset [16]. PSO-RF is an intrusion detection mechanism based on binary particle swarm optimization (BPSO) and random forests (RF) algorithms to find best features set by BPSO and RF as a classifier for classifying intrusions of networkonKDD99Cup dataset [17]. Methodology This section proposed Swarm-based intrusion detection method which is a hybrid feature selection approach used correlation-based (CFS) classical statistical feature selection approach to enhance the performance of BPSO feature selection. This method improves the previous work by applying Enhanced BPSO-based algorithm for feature selection stage. The proposed algorithm will optimize the detection performance of deep learning. CSE-CIC-IDS2018 Data Set Specification The traditional NSL-KDD dataset and others dataset not reflect situations of real world. According to Gharib et al. [18], determined 11 essential criteria for each dataset to be reliable dataset, but none of previous NIDS dataset covered all criteria. While, our CSE-CIC-IDS2018 dataset covered all 11 criteria. CSE-CIC-IDS2018 dataset represents a shift from static data to dynamically generated data available on AWS cloud [19,20]. This paper evaluated the NIDS on a real traffic captured from AWS network and machines log files with 80 extracted features from 50 terminals represent Attacking infrastructure and 30 servers and 420 computers represent the infected organizations comprised. Seven types of attacks occurred: DOS, DDoS, Botnet, Web attacks, Bruteforce, infiltration and Heartbleed [20]. CSE-CIC-IDS2018 dataset contains details of intrusions with details of protocols. The applications and lowest level entities of network are representing best approach of testing and evaluation, also it refers to shifting from Static data to Dynamic data which is real-time traffic on the Amazon platform (AWS). To download this dataset, the following description is applied: "Resource type S3 Bucket Amazon Resource Name (ARN) arn:: aws:: s3: cse-cic-ids2018 AWS Region The dataset has 80 features divided into 8 classes contained 1 normal class and 7 classes of attacks as following: 1. DOS: this type of network attack is a famous in which attackers deny the legitimate users by sending or overwhelming number of bogus requests target a service. Attackers used to generate DoS attack traffic such as, Goldeneye, Slowloris, Hulk and Showhttptest commonly available. 2. DDoS: represent more sophisticated Distributed Denial of Service where attackers flood the target systems or services using multiple botnets from around the world composed of thousands of compromised systems possibly to the overwhelming amount of network traffic. 3. Brute-force: Attackers in this type of network attack using each key combination to estimate online passwords or examine the existence of hidden web pages such as admin login pages. It consists of two common network services FTP and SSH occurs as FTP-Brute force and SSH-Brute force. 4. Heartbleed: This attack-type also categorized into Brute-force where Heartbleed attacks generate Traffic against the notorious Heartbleed Bug. 5. Botnet: Attackers use group of botnet as a single virtual network contained network systems and devices working implement several Internet attacks Attackers use a group of a botnet as a single virtual network contained network systems and devices working implement several Internet attacks such as (phishing attacks, send spam, sniffing and stealing data, provide backdoor access to compromised systems, etc.) by sniffers and key loggers. 6. infiltration: This attack-type represents internal network Attacks. 7. Web attacks: BruteForce-XSS, SQL-Injection, cross-site scripting (XSS) are categories of web attacks in modern web applications. Attacking infrastructure comprised 50 terminals and the infected organizations comprised 30 servers and 420 computers. This dataset represented the captured traffic of AWS network and machines log files with 80 extracted features represented bidirectional information of flow forward (source to destination) and backward (destination to source) directions by using CICFlowMeter-V3. It is a flowbased dataset where flow refer to a set of IP packets passing the network through certain point during a specific interval of time. Each flow containing packets with common properties. Table 3.1 shows A sub set of extracted traffic features. Proposed Work In this paper, the detailed aspects of the proposed system for Optimized Deep Learning with Enhanced Binary PSO for Feature Selection and Classification shown in Figure.1 work is given as framework consist from five phases, they are: 1. In the first phase of the research, which including real-world AWS-flow (CSE-CIC-IDS2018) dataset capturing. The cybersecurity dataset developed in collaboration between the Canadian Cyber Security Institute (CIC) and the Communications Security Corporation (CSE). Thus created a systematic method that uses profiles to develop a detailed description of intrusions associated with protocols and lower levels of network entities. 2. In the second phase, preprocessing steps comprised of feature encoding and feature normalization. False Alarms (FAR) arise during the classification because of rough features. Therefore, the preprocessing phase on the dataset is an essential part to reduce FAR. While, in data preparation, raw data trans-formed into a more suitable form for modeling. Therefore, floating-point numbers must be entered in a range from 0 to 1 to the input layer of the Deep Neural Network (DNN). There are three steps for data preprocessing: • Feature Encoding: convert categorical features into numerical values. • Feature Normalization: used for changing scale, type, and probability distribution of variables in the dataset. • Feature Selection: A crucial step that focuses on feature selection with swarm intelligence using Enhanced Binary particle swarm optimization algorithm (BPSO). Due to Big Data challenges, the feature selection in intrusion detection increase efficiency of the classification by reducing computational processes. Feature selection play significant role with high quality real data sets compared with traditional KDD data sets for Intrusion Detection, due to select only most correlated features with certain class. 3. In the third phase, to evaluate these built models available data into training and testing data needed to be divided. Cross-validation technique has been used for evaluating how to generalize the statistical analysis results on an independent dataset. It is the famous approach to computing the learning model accuracy which improve the reliability of classifier. It estimates how the predictive model will perform in practice accurately. A 5-fold approach is used in this work. Thus the data is divided through 5 parts randomly and each run used one partition of these for testing and the remaining partitions used for training iteratively. The training data is used to fitting the model, and the test data to evaluate it, while the labels of the test data are known. 4. In the fourth phase, directing experiments towards Deep Neural Network (DNN) modeling. then perform classification on the training set, which is explained in this chapter in detail. 5. Finally, Attack detection and performance evaluation on the testing set with different metrics to highlight whole strong perspectives of our proposed system and other works comparison. Feature Selection Set of techniques used to select optimal subset of input features which are most relevant to the target variable need to predict. Dimensionality of the data mean the number of input features for a dataset, but the problem is more dimensions in space make dataset representation a very sparse and unrepresentative for samples in that space. So, this motivates feature selection that remove irrelevant and redundant input features which leading to lower predictive performance. Thus, models could develop by using the data that is required to make a prediction only. this paper employed feature selection for reducing irrelevant attributes using random forest algorithm. Therefore, the task of NIDS became efficient. While, PSO algorithm performed on the selected features of the NSL-KDD dataset. This minimize the false alarm rate and increase the detection rate and the accuracy of the NIDS compared with machine learning classifiers such as SVM, KNN, DT and LR algorithms [21]. A new method has been presented based on particle swarm optimization with multiple criteria linear programming that improve attacks detection accuracy. During training phase, PSO used for tuning parameters to optimize the MCLP classifier performance [22]. Due to PSO has advantages that are simple implementation, fewer number of parameter, and no calculation of mutation. It is considered as best search algorithm for optimization. PSO classified into Standard PSO and binary PSO. Standard PSO assigns real numbers to particles, but binary PSO assigns binary numbers to particles. But, there is a possibility that binary PSO (BPSO) will quickly fall into local minima so it has been suggested to enhanced it by correlation-based (CFS) classical statistical feature selection approach. Correlation-Based Feature Selection (CFS) Approach CFS is a filter-based technique where the major idea behind this approach is to evaluate the relevance of selected feature to class and the redundancy between the selected feature subset to obtain the optimal solution in search space. Features are choosing according to the result of the feature subset assessment using the function of correlation. This means the chosen features are maximally related to the class but not related to each other. Each feature with a high score predicts classes in the instance space more than different features as following Eq. 1: Where CS is the evaluation for s feature subset comprised f features, dcf is the degree of correlation mean between features and the class label, and dff is the degree of intercorrelation mean between features. The evaluation of CFS is a method of correlation based on feature subsets. Thus, higher evaluation value come from bigger dcf or smaller dff in selected subsets. Finally, as shown in Figure. 2 the selected subsets of features which have highest value used to reduce both the training and testing set. Binary Particle Swarm Optimization (BPSO) In [23] PSO after the population initialization each particle update its velocity and its position in each iteration based on their own experience (pbest) and the best experience of swarm (gbest) as in Equations (2) (3) Where: At each iteration st each particle j Acquire three vectors velocity, position and personal best all in length N which refer to the problem dimension. When either the improved value of the global best is smaller than stopping value (ԑ) or reached the maximum iteration number the stop condition is met and PSO terminates. The standard PSO used in continuous domains well, while in discrete space it gives poor effects on the results. Usually, the binary PSO in the feature selection problem outperforms the standard PSO because that the problem of feature selection occurring with a discrete search space. BPSO search space is seemed as a hypercube where a particle moves to nearer and farther corners of the hypercube through flipping bits into various numbers. The moving velocity represent changes of probabilities for the bit which may be in one state or the other. So, a particle in each dimension moves in a state Selected Feature subset space limited to 0 and 1 [24]. Therefore, we will exploit the binary PSO in our design for the feature selection method. To implement the BPSO, the selected number of population is 100 and the number of iteration is considered to be 10, Initialize swarm randomly where X = (x1, x2, …., x n) is a particle as feature vector and y ϵ [0,1] represent class label which 0,1 respectively refer to normal and abnormal. Then, setting parameter as following: W is the constant refer to Inertia weight that controls the velocity impact of particle during the current iteration it is usually ranged in [0.4,0.9]. F 1 and F 2 are acceleration coefficients constants ranged in [0.5]. while, d 1 and d 2 which are values ranged randomly in [0,1]. These parameter scale both of personal knowledge and swarm knowledge on the velocity changes. Consequently, calculate Activation Function to measure fitness value of each particle as in equation (4) to select particle with best value and called gbest. F(X) = ᾳ(1 − Pr) + (1 − ᾳ)(1 − )(4) X is the input variable where Pr is the measure of classifier performance and Ns is the feature subset size have been tested and Nv is the total number of available input variables. The term on the left side of the equation refer to the total accuracy and the term on the right for the used features percentage. BPSO is resulted by adapting equations in standard PSO to be suitable to binary space. The velocity vector in BPSO shows the probability of taking value 1 for element in the position vector. Moreover, the sigmoid function in Eq. (5) used to convert Update position and velocity of each particle as equation (5) and (6). Finally, PSO output optimal solution that is global best vector next checks the stop condition when one met it the PSO will terminates. Enhanced BPSO features based on CFS selection To enhancing the feature selection of standard BPSO Algorithm has been proposed CFS classical statistical method, where Algorithm 1 proposed Enhanced BPSO Algorithm as the following procedure: 1. Determine score for each feature using CFS -based correlation equation. 2. Put certain threshold then select all features that larger than threshold. 4. Further selection to reduce the redundant features and select optimal features subset. Deep Learning Classifier Our DNN model implemented on Windows10 using Visual Studio 2019 contain python 3.7 and installed Keras on top of Tensorflow using (Numpy, Scikit-learn, Panda) libraries ,8GB Memory, CPU core i7,512GB Hard disk, seaborn library for visualization results. Deep learning models classified into two types supervised and unsupervised learning models. comprise, deep neural networks (DNNs), deep brief networks (DBNs), recurrent neural networks (RNNs)and convolutional neural networks (CNNs) as supervised learning models. In other hand, restricted Boltzmann machines (RBMs), auto encoders and generative adversarial networks (GANs) as unsupervised learning models [25]. Deep learning methods plays a significant role for flow-based datasets compared with machine learning models because do not required manual feature engineering. Thus, it can learn feature representations automatically from raw data. The deep structure of deep learning represents comparable characteristic where used multiple hidden layers compared with shallow models, which contain one hidden layer or none [26]. The 55 optimal features selected by the Enhanced BPSO algorithms from preprocessing phase will be provide to our DNN classifier to improve the performance of DNN contain three fully connected layers are used, they described in Algorithm 2 as following: • dense1 layer with 55 neurons use ReLu Activation function. • dens2 layer with 64 neurons use ReLu Activation function. • dense3 layer with 10 neurons use Softmax Activation function. • Regularization method with two dropout ratio (0.2) are used to avoid overfitting. Table 1 shows that good tuning of hyper parameter values is important to avoid overfitting. Loss function represent the difference between the predicted and actual output. The Optimizer Adam used to minimize Loss function by calculate gradients of a loss after that apply gradients to update values and therefore enhance the DNN results. Experimental Results and Discussion We directed comparative analyses by comparing our results to the previous results in the literature. In addition to that, we proposed various evaluation Measurement in order to investigate the differences in performance in different approaches and focus on performance of deep learning models through using our approach. Evaluation Measurements We used for model evaluation various performance metrics to give powerful view on our model which based on BPSO with DNN as following: Confusion Matrix In the intrusion detection, Confusion Matrix is a good tool to predict the network attack type where TP normal data and TN refer to the abnormal data correctly classified, while FP the normal data and FN refer to abnormal data of the misclassification. Figure . Table 2 from which We determine the detection rate (DR) and false alarm report (FAR). 99 Accuracy, Precision, recall these criteria are limited, especially if one class among 10 classes is much larger than the other. With an imbalanced classification problem, the classification error in the minority class will not have much effect on the accuracy value. If the dataset is unbalanced, then in such cases, you only obtain very high accuracy by predicting the majority class, but you fail to capture the minority class, which is often the goal of creating the model in the first place .as shown in Table 3 The relation between training accuracy and testing accuracy showing in Figure.3, where the model accuracy reached to 94% only with 10 Epoch, while when increased Epoch to 100 We noticed that accuracy has settled on 95%. Comparative Analysis A comparative study in Table 4 is directed for showing the differences between our implemented Deep Neural Network (DNN) with Binary PSO and other previous methods. Our approach used Binary PSO as preprocessing phase that select only 55 best features from 80 features contained in real CSE-CIC-IDS2018 dataset. The selected features feed into DNN classifier. The results showed superiority over the previous classifier without feature selection. Moreover, no research used our dataset. The accuracy of proposed model is 95% after 100 Epoch which consider good, because we used Binary PSO which is one of the available feature selection methods that reduce the dataset dimensionality and select the most relevant features only. The proposed approach increase system accuracy, decrease false alarm report(FAR) and reduce the computation time where the elapsed time for Detection time about 580.27 sec Conclusion From the final results of proposed NIDS implementation reached during this thesis, concluding from proposed NIDS scenario the following: 1-The first proposal is Enhanced BPSO (Binary Particle Swarm Optimization as feature selection) is an efficient approach for feature selection because the fact that problem of the features selection occurred in discrete space, and this leads to select the best features which enhance the classification performance. 2-The proposed algorithm for features selection reduces the elapsed time consumed in the training and testing processes during classification process. Therefore, accuracy, detection rate increased and False Alarm Report (FAR)decreased. 3-Fully connected dense Deep Neural Network(DNN)is proposed, its used for flow-based intrusion detection on real-world dataset CSE-CIC-IDS2018 available at AWS platform, few papers addressed this dataset yet. The performance of proposed approach is examined on off line (NSL-KDD) and on-line (CSE-CIC-IDS2018) datasets, and the results for performance evaluation showed that the both types were good indicating that our model generalized well and did not deviate to a specific type of data. 4-Hyperparameters tuning for DNN model is recommended to be used for further efficiency on our proposed DNN. So, we used two dropout layers	2021-05-12T04:28:42.733Z	2021-03-17T00:00:00.000	{ "year": 2021, "sha1": "4069fc7cb0b10bcfc42016da607f1572a26d94d2", "oa_license": "CCBY", "oa_url": "https://wjcm.uowasit.edu.iq/index.php/wjcm/article/download/10/6", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "4069fc7cb0b10bcfc42016da607f1572a26d94d2", "s2fieldsofstudy": [ "Computer Science", "Engineering" ], "extfieldsofstudy": [ "Computer Science" ] }
6709617	pes2o/s2orc	v3-fos-license	Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07℃ for cattle, 84.96±0.08℃ for pig, and 85.99±0.05℃ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); 84.91±0.11℃ for goat and 83.90±0.11℃ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and 86.31±0.23℃ for chicken, 88.66±0.12℃ for duck, and 84.49±0.08℃ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from 10 pg/μL to 100 fg/μL levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species. Introduction As consumption of animal resources increases and varies, excessive and unfair competition between producers has caused problems for consumers of meat-based foods. Several issues include contamination of originally highvalued meat with cheaper substitutes (beef contaminated with horse meat), choices not to intake meat or meat sources due to religious reasons, health problems (allergies) and individual preference (vegetarian) (Bottero and Dalmasso, 2011;Santos et al., 2012). These have led to the growth of consumer demands for accurate meat identification methods to combat meat source fraud and adulteration. In response to these pressures, the industry has developed a variety of species identification methods Typical methods for meat identification rely on protein analyses, such as immunological, electrophoretic, and chromatographic assays (Bottero and Dalmasso, 2011;Hitchcock and Crimes, 1985;Proom, 1943). Enzyme-linked immunosorbent assays (ELISAs) are used for meat identification by the United States Department of Agriculture Food Safety And Inspection Service (USDA-FSIS). Several researchers have reported that ELISA provides accurate meat species identification (Macedo-Silva et al., 2000; Whittaker et al., 1983); however, protein analysis method has limitations. Protein is not favorable for discriminating processed products such as meat patties or cured, dried, and seasoned meat, because proteins are easily denatured by heat, salt, and pressure (Dincer et al., 1987). Proteins are also unsuitable tools for identifying species that are phylogenetically close, such as poultry, because of crossreactivity (Dooley et al., 2004). Nucleotide amplification methods have been developed as an alternative to protein-based methods ( (Francois et al., 2011;Tomita et al., 2008). It is specific, rapid, and sensitive because it employs 4 to 6 primers and specific DNA polymerases, such as Bst or Gsp DNA polymerase, which are capable of inducing auto-cycling strand displacement. The reaction occurs under isothermal conditions, eliminating the need for thermal cycling. To our knowledge, the only LAMP assay for species identification has been described with an electronic DNA sensor (Ahmed et al., 2010); none have been developed for use with intercalating dye-based analysis systems. In this study, we developed a real-time LAMP assay for rapid, sensitive, and accurate species identification by targeting the mtDNA of eight meats. Species discrimination was performed based on annealing curve analysis of the LAMP assays. The capability of LAMP assay was assessed by determining the limit of detection (LOD) and by calculating a quantification equation for four sample types: raw meat, cooked meat, raw meat admixtures, and cooked meat admixtures. The LODs of LAMP assays were compared to those of PCR assays to evaluate the LAMP assays. Sample preparation Representative samples of seven meat species were purchased from grocery markets in South Korea: cattle (Bos taurus), pig (Sus scrofa domesticus), goat (Capra hircus), sheep (Ovis aries), chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo). Horse (Equus caballus) meat was obtained from the department of veterinary clinical pathology of Seoul National University. Samples (20 mg) were used for DNA extraction. Admixtures of horse-cattle, sheep-goat, chicken-duck, and chicken-turkey meat were prepared by combining 0.2 g of horse, sheep, and chicken meat at 0.00001 to 10% in 1.8 g of cattle, goat, duck, and turkey meat. Each sample was then homogenized by chopping with a stainless blade (Dorco, Korea) for 5 min. Heat-treated (cooked) meat samples (20 mg each) were prepared by boiling raw and admixture samples at 100°C for 5 min. DNA was extracted with the DNeasy Tissue kit (Qiagen, USA) according to the manufacturer's instructions. The DNA sample was then stored at 4°C or directly used as template. LAMP primer design To identify each species-specific target, several mtDNA sequences of each species were collected from GenBank. Multiple sequence alignment using CLUSTALW in Meg Align program of Lasergene software (DNASTAR Inc., USA) was used to develop a consensus sequence. Three sets of primers targeting eight different regions of the consensus sequence were designed by using LAMP designer (Optigene Ltd., UK) after the in silico BLAST test provided in the program. The LAMP primers are listed in Table 1. LAMP reaction The LAMP assay reaction conditions were optimized using GspSSD DNA polymerase (Optigene Ltd., UK) according to the manufacturer's instructions. Briefly, LOD tests were performed with 0. For the specificity test, the LAMP sets were organized into BSE (Bos taurus, Sus scrofa domesticus and Equus caballus: cattle, pig and horse), CO (Capra hircus and Ovis aries: goat and sheep), and GAM (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo: chicken, duck, and turkey) groups, with equimolar primer amounts. Nucleotide-free distilled water was used as a negative control and cross-reactivity with non-target DNA samples was tested. Temperature optimization was performed over a range of 60 to 67°C for 30-60 min by using the block gradient function of the Genie ® II platform (Optigene Ltd., UK) before the melting curve was generated from 98 to 80°C, with a 1°C decrease every 5 s. The best reaction temperature for each species was determined as the point of earliest inflection in the amplification ratio curve. The results were represented on the amplification graph with fluorescence (K) on the Y-axis and time (T) on the Xaxis; positivity was determined from the detection time (time taken to measure the fluorescence signal for a positive peak) and the mean species-specific annealing temperature. Detection limit of the LAMP and PCR assays To determine the LOD of the LAMP and PCR assays for raw and cooked samples of each species, 10-fold serially diluted template DNAs with concentrations ranging from 10 ng/µL to 10 fg/µL were tested in triplicate. In the raw and cooked meat admixtures, target species content was tested in triplicate from 10 to 0.0.00001%. Previously reported primers and conditions were used for the PCR-based comparators (Koh et al., 2011) with minor modification. Briefly, the reaction mixture contained 1× Emerald Master Mix (Takara Biotechnology, Japan), 0.5 µM each primer, and 1 µL template. The reaction was performed in a MyCycler™ Thermal Cycler (Bio-Rad Inc., Hercules, USA) with the following conditions; 1 cycle for 5 min at 95°C; followed by 30 cycles of 30 s at 95°C, 30 s at 57°C, and 35 s at 72°C; and 1 cycle for 5min at 72°C. Nucleotide-free distilled water was used as a negative control. For each set, a standard curve based on the LAMP results was generated by plotting the log of the detection times versus the concentrations of template DNA or target species content. The quantification limits of the assay were evaluated by calculating the correlation coefficient determinants (R 2 ) and quantification equation. Results and Discussion Capability of the LAMP assay mtDNA sequences are often used to discriminate animal species. In this study, we targeted the displacement loop (D-loop), cytochrome b (cyt b), ATP synthase F0 subunits 6 and 8, and cytochrome oxidase subunits I and II (COI and II) in order to find species-specific non-variable regions and to set the annealing temperatures for successful discrimination ( Table 1). The optimal reaction temperatures for each LAMP assay were identified as 64.6°C for cattle, pig, and turkey, 64.7°C for horse and sheep, 63.5°C for goat, and 65.5°C for chicken and duck. It was also demonstrated that the LAMP primers were specific to their corresponding target species. No cross-reactivity was observed within the BSE-, CO-, and GAM-LAMP groups. Indeed, no cross-reactivity was observed when the singleplex LAMP assays were performed for each of the 3 groups, indicating that the selected markers are suitable for species discrimination. (Fig. 1). The annealing curve showed no overlaid peaks between species in each of the sets, revealing that annealing temperature analysis is an excellent tool for LAMP discrimination of these eight raw meat species. The annealing temperature is effective for species discrimination because it differed with the nucleotide composition and amplicon length of each target (Mouillesseaux et al., 2003). In contrast to probe-based real-time PCR, intercalating dye-based one has many advantages. Target detection with specific probes is more expensive than using general intercalating dyes. In addition, it is difficult to design and optimize a probe (Varga and James, 2005). For these reasons, melting curve analysis after SYBR green-based real-time PCR has been used to identify ruminant (cattle) and poultry (turkey) in food-stuffs (Şakalar and Abas yan k, 2012), discrimination of plum pox virus isolates of strain D and M (Varga and James, 2005), and discrimination of deer and six common domestic species (You et al., 2014). LODs of LAMP assay in raw and cooked meat samples The LODs of LAMP and PCR assays in raw and cooked meat samples are presented in Table 2. Amplification curves corresponding to samples at 10 ng/µL to 1 pg/µL were generated for raw pig, horse, goat, sheep, and turkey meat; samples of 10 ng/µL to 10 pg/µL were generated for cattle, chicken, and duck meat. Amplification curves corresponding to 10 ng/µL to 100 fg/µL were generated for cooked chicken, and from 10 ng/µL to 1 pg/µL for the remaining cooked samples, indicating that the LAMP assays are more sensitive than PCR by 10 2 to 10 3 times and 10 to 10 4 times in the raw and cooked meat samples, respectively, with strong linear relationships between detection time and template mtDNA concentration. Studies of raw horse and donkey meat samples by TaqManbased real-time PCR have yielded LODs of 1 pg DNA in i i water (Chisholm et al., 2005); the LOD for raw cattle meat is 35 pg DNA (Zhang et al., 2007). A study that employed a LAMP method and electrochemical DNA sensing described LODs for pork, bovine, and chicken samples as 20.33, 23.63, and 78.68 pg/µL (Ahmed et al., 2010), indicating that our LAMP assays were more sensitive in these species. In comparison to the raw meat samples, LODs were the same or lower in cooked meat samples. Indeed, where the same LODs were found, the detection times were faster in cooked samples than in raw ones with the exception of goat, sheep, and turkey. Target regions under 150 bp are not influenced by heat treatment (Kesmen et al., 2012); thus, the amplified regions in our study should not be affected by cooking. Since the optimal size of the amplified region in LAMP is below 200 bp (Tomita et al., 2008), the method may be used for even highly degraded DNA targets. Cattle and pig meat are the most favored meat sources in South Korea. In Europe, large quantities of horse meat are produced and imported for human consumption, as cattle meat is relatively expensive (Gill, 2005). The high cost of cattle meat could lead to intentional contamination with pig and horsemeat. In goats and sheep, the authentication of origin is important because of the close relationship between these ruminant species. In the dairy industry, inferior product substitutions may be made because sheep and goat sources have nutrient and economic advantages over cow-derived products (Cheng et al., 2006) and sheep sources are more costly than goat sources worldwide (Pappas et al., 2008). To our knowledge, no other studies have aimed to discriminate goat and sheep. Thus, our LAMP method could facilitate fast and sensitive discrimination of these small ruminant species. Poultry, chicken, duck, and turkey have also been adulterated for economic reasons. Foie gras from goose and duck have been contaminated with less expensive sources such as chicken or turkey (Rodriguez et al., 2003). Consumption of foie gras has increased along with demands for high quality and authentic foods, thus creating a need to prevent or detect adulteration. Our LAMP assays could be used for fast and sensitive monitoring for food fraud. LODs of LAMP assay in raw and cooked meat admixtures In the admixture samples, horse, sheep, and chicken meat were targeted because they are considered cheap substitutes. The amplification curves were generated for each target species based on detection time (T t ) and fluo-rescence for 10 to 0.0001% target in non-target species admixtures within 30 min. The LODs of LAMP and PCR assays in raw and cooked admixture samples are presented in Table 3. Amplification curves for samples of 10 to 0.01% were generated for the chicken-duck raw meat admixture; samples of 10 to 0.001% were used to generate curves for the remaining raw meat admixture samples. Samples of 10 to 0.001% were used to generate curves for sheep-goat and chicken-duck cooked meat admixtures, and samples of 10 to 0.01% and 10 to 0.0001% were used to generate curves for cooked horse-cattle and chicken-turkey admixtures, respectively. Thus, our LAMP assays were more sensitive than PCR by 10 2 to 10 4 fold. The LOD of PCR in heated ovine samples mixed with oats is 0.1% (Martin et al., 2007); in poultry and ruminant species, samples with vegetable contamination (maize) were reported at 0.002% (Dalmasso et al., 2004). However, these results cannot be directly compared because an LOD expressed as meat content (w/w) does not show the absolute capability of the assay (Ballin et al., 2009). Further study may be needed to reevaluate previous studies with a consensus unit, such as DNA/DNA equivalents (Ballin et al., 2009). The annealing temperature of LAMP amplicons fell within the unique range for each species and the negative control was not amplified throughout the experiments. In comparison to the raw meat samples, we observed no difference in LOD for the sheep-goat raw and cooked admixture samples; the LODs for chicken-turkey and chicken-duck cooked admixtures were lower than for the raw admixtures. Detection times were faster for cooked admixtures than for raw ones (Table 3), perhaps because heating may influence the extraction efficiency of mtDNA from the sheep-goat, chicken-turkey, and chicken-duck samples. In contrast, the LODs in cooked horse-cattle admixtures were greater than in the raw admixtures; detection times and detection limits were higher for chicken-duck than for chicken-turkey admixtures. Thus, DNA from the non-target meat could inhibit target DNA amplification (Dooley et al., 2004), although the annealing temperature in raw and cooked horse-cattle admixtures was 85.75± 0.16°C and 85.59±°C, respectively, indicating successful amplification of horse mtDNA. Since the LAMP method is generally robust against pH, temperature changes, and even reagent exposure, it is more sensitive and specific than other nucleotide amplification methods (Francois et al., 2011). Our LAMP assays could be used as tools to discriminate and quantify meat species in unknown and heat-treated samples. LAMP results were presented with detection time (mean±standard deviation) of the triplicate. 2) PCR results were determined as positive (+) and negative (-). 3) One positive result was observed in the triplicate. The admixtures of horse-cattle, sheep-goat, chicken-duck, and chicken-turkey meat were prepared by combining 0.2 g of horse, sheep, and chicken meat at 0.00001 to 10% in 1.8 g of cattle, goat, duck, and turkey meat. 2) LAMP results were presented with detection time (mean±standard deviation) of the triplicate. 3) PCR results were determined as positive (+) and negative (−). 4) One positive result was observed in the triplicate.	2016-05-15T05:34:24.398Z	2014-12-31T00:00:00.000	{ "year": 2014, "sha1": "1ffef083706fc72d4e8889e5ee6a611750664a91", "oa_license": null, "oa_url": "http://www.ndsl.kr/soc_img/society/ksfsar/CSSPBQ/2014/v34n6/CSSPBQ_2014_v34n6_799.pdf", "oa_status": "BRONZE", "pdf_src": "PubMedCentral", "pdf_hash": "913517e73479c1ab2d20dff5b974284fc24c4978", "s2fieldsofstudy": [ "Biology", "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
99344221	pes2o/s2orc	v3-fos-license	Retrogradation of heat-gelatinized rice grain in sealed packaging : investigation of moisture relocation As a main dietary source of carbohydrates, rice plays an important role in energy requirements and nutrient intake (Ramesh et al., 2000; Dgawa et al., 2003; Park et al., 2012). Contrary to other grains, rice is usually consumed as a whole grain for the main meal (Ramesh et al., 2000; Dgawa et al., 2003). With an increasing demand for pre-cooked rice products, starch retrogradation has become an important quality parameter for the evaluation of prolonged storage (Burgos & Armada, 2015; Yu et al., 2010). The leached components which cover the surface of rice grain such as starch, nonstarch carbohydrates, lipids and proteins caused by cell disruption inside the granule during cooking process play significant roles in the eating quality of rice (Dgawa et al., 2003). The textural properties of cooked rice are primarily related to its moisture content because of starch gelatinization (Bello et al., 2007; Tamura & Dgawa, 2012; Kim et al., 2001; Ramesh et al., 2000). The hardness and stickiness of rice grain are the commonly measured parameters in the determination of the textural properties of cooked rice (Meullenet et al., 1998). Consequently, textural changes in cooked rice grain during storage are closely related to starch retrogradation. Introduction As a main dietary source of carbohydrates, rice plays an important role in energy requirements and nutrient intake (Ramesh et al., 2000;Dgawa et al., 2003;Park et al., 2012).Contrary to other grains, rice is usually consumed as a whole grain for the main meal (Ramesh et al., 2000;Dgawa et al., 2003).With an increasing demand for pre-cooked rice products, starch retrogradation has become an important quality parameter for the evaluation of prolonged storage (Burgos & Armada, 2015;Yu et al., 2010).The leached components which cover the surface of rice grain such as starch, nonstarch carbohydrates, lipids and proteins caused by cell disruption inside the granule during cooking process play significant roles in the eating quality of rice (Dgawa et al., 2003).The textural properties of cooked rice are primarily related to its moisture content because of starch gelatinization (Bello et al., 2007;Tamura & Dgawa, 2012;Kim et al., 2001;Ramesh et al., 2000).The hardness and stickiness of rice grain are the commonly measured parameters in the determination of the textural properties of cooked rice (Meullenet et al., 1998).Consequently, textural changes in cooked rice grain during storage are closely related to starch retrogradation. The structure of the building unit of starch grains the starch granule is defined in terms of its amorphous and semi-crystalline growth rings (Vandeputte et al., 2003).When a starch suspension is heated in excess water, gelatinization usually leads to a number of changes in the starch granule.The first stage of gelatinization is the uptake of water by the amorphous background region of the starch granules, followed by their subsequent rapid expansion.This swelling exerts a strong destabilizing effect on the crystallites, followed by the disruption and loss of crystallinity (Tester & Karkalas, 2002); Karapantsios et al., 2002).Changes in water distribution and internal structure during cooking are closely related to the gelatinization characteristics (Horigane et al., 1999).Retrogradation is usually regarded as the reverse step of gelatinization (Bear & Samsa, 1943).Increased hardness of the gelatinized whole rice grain, when preserved at low temperatures considerably lower than the gelatinization temperature, causes an uncomfortable chewiness and requires reheating of the rice grain by either conventional heating or microwave.Moisture redistribution due to starch retrogradation affects the texture of gelatinized rice grain during storage in a sealed package. The effects of the freezing rate on the retrogradation and textural properties of cooked rice grain have been previously investigated (Yu et al., 2010).Kock et al. (1995) reported that hardness increased and stickiness decreased at lower temperatures and longer storage durations.Identification of the structural characteristics of amylose and amylopectin has provided insight into the textural properties of gelatinized rice gel (Vandeputte et al., 2003).Gelatinization and retrogradation are shown to have a strong influence on the textural properties during rice cooking, handling, distribution, and storage (Banchathanakij & Suphantharika, 2009).Gudmundsson (1994) demonstrated that, upon cooling of gelatinized rice grain, retrogradation occurs when gelatinized starch begins to reassociate in an ordered structure under low energy input, as in cooling and freezing. Although many researchers have extensively documented the rheological properties of starch gelatinization and retrogradation associated with the interaction of water and starch molecules in solution (Juliano, 1998;Bao et al., 2006;Wu et al., 2010;Kim et al., 1997), the relationship of retrogradation with increasing hardness of gelatinized rice grain in terms of moisture redistribution under storage conditions, where moisture movement is prohibited to avoid drying at relatively low temperatures, has hardly been studied.Therefore, this study was focused on changes in hardness and structural characteristics of gelatinized rice grain, which can be indicative of the retrogradation process in terms of water redistribution inside the rice grain, under hermetic conditions. Materials Non-waxy polished short-grain rice (Dryza sativa L.) was obtained from a local market (Gimhae, Korea) and stored at 10 °C.The germ and bran of the rice were completely removed; dust and fine starch particles were also removed by polishing during the milling process.All reagents and chemicals used were of analytical grade (Sigma-Aldrich, St. Louis, MD, USA), unless specified otherwise. Preparation of gelatinized rice grain Approximately 1.4 times the volume of water (v/w) was added to a given amount of rice for gelatinization in an electronic pressure cooker (CRP-FA0610FP, Cuckoo Homesys Co. Ltd., Yangsan, Korea) as usual.The gelatinized rice grains was packaged at 20 °C and 56% relative humidity and cooled in a hermetically sealed aluminum laminated pouch (10 × 15 cm).The sealed pouch was stored during 9 days at 11 °C using low temperature incubator (HB-103S, Han Baek Scientific Co., Bucheon, Korea).The moisture content of the rice and gelatinized rice grain was determined using the methods defined by the Association of Dfficial Analytical Chemists (2002).Volume of the raw rice grain and gelatinized grain was obtained by adding 20 grains to a calibrated cylinder and measuring the water volume required to fill up a known graduation. Estimation of hardness and retrogradation of gelatinized rice grain The hardness of gelatinized rice grain was determined by the relaxation test using a rheometer (RT-2010DD, Fudoh Co. Ltd., Tokyo, Japan) with a disc-type tip (#3-3) at a table speed of 100 mm/min.The maximum force required to press a 0.6 mm distance from the gelatinized rice grain surface was defined as the hardness (g f ) of the gelatinized rice grain.The results are presented as mean values with standard deviation for ten replications.The degree of retrogradation of the gelatinized rice grain was measured by the α-amylase-iodine method (Tsuge et al., 1990).A quantity of gelatinized rice grain (250 mg) was dissolved in 50 mL of distilled water and homogenized (AM-7, Nihonseiki Kaisha Ltd., Tokyo, Japan) for 1 min twice.α-Amylase (2 mL; E.C. 3.2.1.1,Bacillus sp.type II-A, 1,400 unit/mg solid, Sigma-Aldrich, St. Louis, MD, USA) solution (7 units) was dissolved in 5 mL of the homogenized solution.Distilled water (3 mL) and 0.1 M phosphate buffer (2 mL; pH 6, 0.3% NaCl) were added and placed in a shaking water bath at 37 °C for 15 min.After 5 mL of 4 N NaDH was added to stop the enzymatic reaction, the mixture was adjusted to pH 7 using 4 N HCl and to a total volume of 100 mL by adding distilled water, followed by the addition of 5 mL of iodine reagent (0.2% I 2 and 2% KI, w/w) and kept at room temperature for 20 min.Absorbances were measured using a UV-Vis spectrophotometer (DPTIZEN PDP, Mecasys Co., Daejeon, Korea) at 625 nm.All tests were performed at least in triplicate and results are presented as mean values with standard deviation. Morphological changes of internal structure of gelatinized and retrograded rice grain SEM (S-4300SE, Hitachi Ltd., Tokyo, Japan) was used to examine morphological changes inside the gelatinized and retrograded rice grain while being stored at 11 °C under hermetic conditions.The freeze-dried (FDU-1200, Tokyo Rikakikai Co., Tokyo, Japan) rice grains were carefully cleaved in half along the short axis with a safety razor to avoid any physical damage to the cleaved surface.Cross-sections of the cleaved rice grains were observed after coating with platinum using a sputter coater. Textural properties of gelatinized and retrograded rice grain Moisture content of the gelatinized rice grain was 63.5% (w/w) which represents water was absorbed approximately 1.3 times the weight of raw rice during cooking.Therefore, the volume of a gelatinized rice grain increased 2.24 times compared to that of the raw rice grain.As a consequence hardness decreased dramatically since the absorbed water molecules inside the starch molecules would have acted as a plasticizer (Table 1).Hardness of the raw rice grain exceeded measurement range of the disk type tip of the rheometer.Cho et al. (2004) reported that maximum force required to cut raw waxy rice using cutting type probe was 101 ± 38 g f .Rice starch in the endosperm undergoes gelatinization at sufficiently high temperatures.Starch granules expand in volume and press together closely, creating a strong cohesion force between starch molecules and water by hydrogen bonding (Bello et al., 2007).As a consequence strong bonds in both the crystalline and amorphous regions are broken and hydrogen bonds formed between the penetrated water molecules contribute to the increase in grain volume.Therefore, the cooked rice grain is soft, pliable, and elastic resulting in the value of hardness 51.9 g f (Table 1).Immediate cooling of the gelatinized rice grain significantly increased grain hardness (78.8 g f ) which presumably suggests that the hydrogen bonds between the water and starch molecules were disrupted considerably to affect the textural properties of grain.The hardness of the whole rice grain continuously increased, reaching a value just below 100 g f within 9 day of storage at 11 °C (Figure 1).A change in the hardness of the whole gelatinized grain with storage time is obviously related to water molecule redistribution with respect to the retrogradation of the starch grain.The retrogradation process leads to the cohesion of starch molecules by the cleavage of hydrogen bonds which in turn promotes the recrystallization of the starch molecules.The degree of retrogradation showed a continuous increase, and was less than 40% at 9 day storage (Figure 2).Lu et al. (1997) reported that the majority of amylase molecules were retrograded and precipitated from solution after incubating at 5 °C for 100 days and that the degree of retrogradation decreased from 58.8% to 7.1% within 24 h as incubation temperature was increased from 5 to 45 °C.The retrogradation degree of 50% non-waxy rice starch gel was more than 45% over a 6 day storage period under refrigerated conditions (Kim et al., 1996). Since the gelatinized rice grains were sealed in an aluminum laminated pouch, a void space in the sealed package was almost completely saturated with water vapor at the storage temperature (11 °C).Therefore, the moisture inside the rice grain cannot be diffused out of the grain to a void space in a sealed pouch.As a result, the water molecules exuded from the starch strands was trapped inside the grain matrix.The retrogradation of the steam-gelatinized rice grain was also initiated by the disruption of hydrogen bonds between water and starch molecules, followed by the adhesion of starch molecules with subsequent exclusion of water molecules between the starch strands; these water molecules remained captured in the grain.Starch retrogradation has been reported to result in an increased hardness of cooked rice during storage (Perdon et al., 1999;Yu et al., 2009).The degree of retrogradation, expressed as the change in firmness, of cooked non-waxy rice grain was inversely related to moisture content between 60.5 and 65.9% (Kim et al., 1996).As the association of starch molecules in gelatinized rice grain increased, more water molecules were expelled and they gathered to form water crevices inside the granule, resulting in the gradual increase in hardness to an eventual plateau value (Figure 2).During the retrogradation process, smaller crystalline regions, which are easily disrupted at low temperature, are first formed; some ordered regions may then aggregate to create a large helix.The large ordered regions, having more hydrogen bonds, are stable and maintained their organized structure at higher temperatures (Matalanis et al., 2009). Internal structure of gelatinized and retrograded rice grain The examination of cross-sectional SEM micrographs showed that the gelatinized rice grain appeared to have a typical histological structure with cracks (Figure 3A), as described by Dgawa et al. (2003).Dgawa et al. (2003) suggest that grain of cracks serve as channels for the flow of water into the grain during cooking.It seemed likely that most of the granular form of starch was maintained immediately after grain cooking (Figure 3A).However, moisture redistribution due to retrogradation inside the gelatinized rice grain occurred very rapidly by lowering the temperature (Figure 3B).An increase in the number and size of crevices (Figure 3B, C), which developed into holes (Figure 3D) during storage at 11 °C, was prominent.The water molecules were aggregated to form water crevices as a result of starch molecule association due to retrogradation inside the rice grain.Further development of the hole in the center of the grain may indicate the breakage and disintegration of gelatinized starch granules which leads to the separation of water and starch molecules after 4 day storage (Figure 3D).Hollow volumes, which were assumed to originate from the sealing of cracks with gelatinized starch during the cooking process (Horigane et al., 1999(Horigane et al., , 2000;;Dgawa et al., 2003), can also significantly affect the development of water channels and holes as retrogradation continues during storage.Although hardness continuously increased because of starch retrogradation (Figure 1 and 2), SEM micrographs of the cross-section of gelatinized rice grain after 9 day storage revealed virtually the same morphology as that stored for 4 days at 11 °C (shown in Figure 3D).This result suggests that moisture redistribution between the starches structures occurred quite rapidly, followed by the reassociation of the starch molecules in the retrogradation process, leading to further crystallization.It was observed that the inner porous structure, which developed into crevices, began to form in the center of the cooked rice grain right after cooling (Figure 3B), becoming prominent within a 2 day storage period (Figure 3C). Conclusions The retrogradation of gelatinized rice grain resulted in an increased hardness of the rice grain with storage time.The reassociation of starch molecules leading to the retrogradation and expulsion of water aggregates from the starch network contributed to an increased hardness of gelatinized rice grain Tsuge, H., Hishida, M., Iwasaki, H., Watanabe, S., & Goshima, G. (1990). during storage.Since water molecules expelled from the starch networks were captured in a starch grain unlike in starch gel, crevices and finally water holes were developed inside the rice grain.SEM analysis of gelatinized and freeze-dried whole rice grain cross-sections showed many crevices in the early stages of low temperature storage, while larger holes were found at a later stage of storage.The degree of retrogradation gradually increased as storage time was increased.It is construed that the cleavage of hydrogen bonds between water and starch molecules was followed by the aggregation of water expelled from starch molecule networks in the gelatinized grain during retrogradation with subsequent formation of water wells inside the rice grain. Figure 1 . Figure 1.Changes in rice grain hardness (g f ) after gelatinization during storage at 11 °C measured using a disk-type tip. Table 1 . Properties of raw and gelatinized rice grain.	2019-04-08T13:06:32.303Z	2016-07-21T00:00:00.000	{ "year": 2016, "sha1": "7095b139a19f3af256d6a79d809b18f32b5056dd", "oa_license": "CCBY", "oa_url": "http://www.scielo.br/pdf/cta/v37n1/0101-2061-cta-1678-457X07816.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "7095b139a19f3af256d6a79d809b18f32b5056dd", "s2fieldsofstudy": [ "Agricultural and Food Sciences" ], "extfieldsofstudy": [ "Materials Science" ] }
260258574	pes2o/s2orc	v3-fos-license	Different degrees of nodes behind obsessive–compulsive symptoms of schizophrenia Obsessive–compulsive symptoms are frequently observed in various psychiatric disorders, including obsessive–compulsive disorder, schizophrenia, depression, and anxiety. However, the underlying anatomical basis of these symptoms remains unclear. In this study, we aimed to investigate the mechanism of schizophrenia with obsessive–compulsive symptoms by using diffusion tensor imaging (DTI)-based structural brain connectivity analysis to assess the network differences between patients with obsessive–compulsive disorder (OCD), patients with schizophrenia showing obsessive–compulsive symptoms (SCH), schizophrenia patients with obsessive–compulsive symptoms due to clozapine (LDP), and healthy controls (CN). We included 21 patients with OCD, 20 patients with SCH, 12 patients with LDP, and 25 CN. All subjects underwent MRI scanning, and structural brain connections were estimated using diffusion tensor imaging for further analysis of brain connectivity. The topology and efficiency of the network and the characteristics of various brain regions were investigated. We assessed baseline YALE-BROWN OBSESSIVE COMPULSIVE SCALE (Y-BOCS), Positive and Negative Syndrome Scale (PANSS), and 24-item Hamilton Depression Scale (HAMD-24) scores. Our results showed significant differences among the SCH, OCD, and CN groups (p < 0.05) in the MRI-measured degree of the following nodes: the superior orbitofrontal gyrus (25Frontal_Med_Orb_L), lingual gyrus (47Lingual_L), postcentral gyrus (58Postcentral_R), and inferior temporal gyrus (90Temporal_Inf_R). Additionally, we found significant differences in the degree of the brain regions 02Precentral_R, 47Lingual_L, 58Postcentral_R, and 90Temporal_Inf_R between the CN, OCD, SCH, and LDP groups (p < 0.05). These findings suggest that alterations in the degree of nodes might be the mechanism behind obsessive–compulsive symptoms in schizophrenia. Introduction Obsessions are characterized by recurring opinions, thoughts, impulses, or mental images, while compulsive behavior involves purposeful and conscious actions that are repeated; collectively, obsessions and compulsions are known as obsessive-compulsive symptoms. These symptoms are prevalent not only in individuals with obsessive-compulsive disorder but also in those with other conditions such as depression, anxiety, and schizophrenia. Obsessive-compulsive symptoms in schizophrenia have long been noted, but the current understanding is still very limited. According to recent reports, the prevalence of obsessivecompulsive symptoms in patients with schizophrenia is estimated to be 30% (1), and the risk of obsessive-compulsive disorder is more than 10 times higher in these patients than in the general population (2). However, the results of studies on this topic have been highly variable, with several studies appearing to contradict the finding of 30%. For instance, Baek et al. (3) conducted a survey of 320 clinically stable patients with chronic schizophrenia and found that the incidence of obsessive-compulsive disorder was 20.6%. Similarly, Singh et al. (4) reported a prevalence of obsessive-compulsive symptoms of 18.5% among 200 outpatients with schizophrenia. In another cross-sectional study conducted by Ahmet et al., which included 300 patients with schizophrenia, the overall prevalence of obsessive-compulsive disorder (OCD) was 17% (5). The lack of a standardized assessment of obsessive-compulsive symptoms in schizophrenia may have contributed to the variability in the reported prevalence rates among different studies. Moreover, the underlying mechanism of these symptoms in schizophrenia remains unclear. While genetic factors are known to play a crucial role in both schizophrenia and obsessive-compulsive disorder, there is insufficient evidence to support the notion that any specific genetic variant increases the risk of either disorder. The catechol O-methyltransferase (COMT) gene has been implicated in both schizophrenia and obsessive-compulsive disorder. Pooley et al. (6) also showed through a case-control study and metaanalysis that the association between the Met allele and OCD existed only in men. The COMT Val105/158Met polymorphism has been suggested to be a cause of schizophrenia due to its effect on dopamine metabolism, as supported by several studies (7). Recent research has shown that SCZ patients exhibit high COMT activity and low dopamine levels in the prefrontal cortex. This leads to persistently unstable and inefficient activity of the prefrontal cortex network, which may be a mechanism underlying the involvement of COMT in schizophrenia (8). Nonetheless, the lack of a standardized assessment of obsessive-compulsive symptoms in schizophrenia may have contributed to variations in study results. Although genetic factors are thought to play a crucial role in both schizophrenia and obsessivecompulsive disorder, there is currently insufficient evidence to suggest that any particular genetic variant increases the risk of either disorder. Regarding neuroimaging, studies using gray matter structural MRI have shown that patients with schizophrenia and obsessivecompulsive disorder exhibit abnormal gray matter structure, but the specific results of these studies have been inconsistent. Kwon et al. (9) reported that patients with both disorders had smaller bilateral hippocampi than normal controls, suggesting that this may be related to overlapping symptoms between the two conditions. Meanwhile, Opel et al. (10) conducted a comparison of brain structure among patients with six common mental disorders and found a strong correlation (r = 0.443-0.782) in brain structure abnormalities among patients with schizophrenia, obsessive-compulsive disorder, major depression, and bipolar disorder. They also noted that patients with schizophrenia and obsessive-compulsive disorder showed abnormalities in morphological characteristics of brain regions such as the hippocampus and fusiform gyrus. Several studies using diffusion tensor imaging (DTI) have shown that patients with various psychiatric disorders, including schizophrenia and obsessive-compulsive disorder, frequently exhibit abnormalities in the white matter structure of the brain, particularly in the cingulate bundle, corpus callosum, and white matter fiber bundles in the frontal and temporal regions (11)(12)(13). Hawco et al. (14) found that the FA value of the corpus callosum decreased in patients with schizophrenia and obsessive-compulsive disorder compared to the normal control group, while the difference in total white matter FA value between the two groups was not statistically significant, suggesting that schizophrenia and obsessive-compulsive disorder may have a common neurobiological substrate. Wang et al. (15) found abnormalities in the FA value and radial diffusion (RD) of brain white matter in patients with obsessive-compulsive disorder and schizophrenia, while no significant changes were found in these variables in patients with schizophrenia or obsessive-compulsive disorder. Cauda et al. found that the brains of patients with both schizophrenia and obsessive-compulsive disorder exhibit common atrophy of structural networks (reduced gray matter volume) in brain connectome analyses. However, previous studies comparing the brain connectomes of people with schizophrenia and OCD have been limited and used different methods, thus drawing no firm conclusions (16). The structural brain network is a complex system of connections between different regions of the brain that are responsible for various functions. These connections can be studied using techniques such as DTI, which measures the diffusion of water molecules in the brain's white matter tracts. The structural brain network is constructed by analyzing the patterns of connectivity between different regions of the brain. This analysis involves identifying the nodes (regions of the brain) and edges (connections between nodes) that make up the network. The topology of the network can then be analyzed to understand the efficiency and resilience of the network (17). The clinical significance of analyzing the structural brain network lies in its potential to provide insights into the underlying mechanisms of various neurological and psychiatric disorders. For example, studies have shown that alterations in the structural brain network are associated with conditions such as Alzheimer's disease, schizophrenia, and depression (18,19). By understanding the changes in the structural brain network associated with these disorders, researchers may be able to develop more effective treatments and interventions. Additionally, analyzing the structural brain network may help identify biomarkers that can be used to diagnose and monitor the progression of these disorders (20). Antipsychotics are primarily blockers of DA and 5-HT receptors, which may lead to the development of obsessive-compulsive symptoms due to long-term 5-HT/DA dysfunction (21). It has been observed that obsessive symptoms may emerge after prolonged use of antipsychotics, particularly clozapine. The incidence of obsessivecompulsive symptoms induced or aggravated by clozapine is reported to be between 10 and 20.6% among patients with schizophrenia using clozapine (22). Clozapine has been found to induce obsessivecompulsive symptoms in patients with schizophrenia, which could be due to its potent blockade of the 5-HT2A receptor. Nevertheless, the relationship between dose and symptoms remains controversial, and the mechanism underlying the emergence of these symptoms requires further investigation. Studying the mechanism behind the formation of obsessivecompulsive symptoms in schizophrenia and in patients taking clozapine is crucial to distinguish them from obsessive-compulsive disorder and to reduce misdiagnosis rates. The advancement of brain Frontiers in Psychiatry 03 frontiersin.org imaging technology, particularly magnetic resonance imaging (MRI), has provided an essential tool to gain further insight into schizophrenia and obsessive-compulsive disorder. One of the most promising and innovative research approaches in the field of brain science is brain connectome analysis, which examines both the functional and structural networks of the brain, primarily driven by the models and methods of graph theory. Consequently, the present study utilized this method for analysis. Participants The participants were randomly selected outpatients and inpatients from Tongde Hospital in Zhejiang Province, Hangzhou, China. The subjects signed informed consent forms. All participants had a YBOCS score ≥ 16, all were between the ages of 16 and 60 years, and all were right-handed. Participants with obsessive-compulsive disorder (OCD) met the Diagnostic and Statistical Manual of Mental Disorders-Fifth Edition, DSM-V criteria for obsessive-compulsive disorder. Participants with schizophrenia showing obsessivecompulsive symptoms (SCH) met the DSM-V diagnostic criteria for schizophrenia, and their obsessive-compulsive symptoms had appeared before antipsychotic treatment. Schizophrenia participants with obsessive-compulsive symptoms caused by clozapine (LDP) met the DSM-V diagnostic criteria for schizophrenia, had no obsessivecompulsive symptoms before treatment, had obsessive-compulsive symptoms for at least 2 weeks after starting treatment with clozapine, and had stable mental symptoms. The exclusion criteria were as follows: ① Compulsive symptoms secondary to organic brain diseases, especially basal ganglia diseases; ② Obsessive-compulsive disorder in conjunction with severe cardiac, hepatic, or renal insufficiency; ③ Received modified electric convulsive therapy within the past 6 months; ④ Alcohol or drug dependence; ⑤ Pregnancy at the time of the study; and ⑥ Left-handedness. CN patients matched by age, gender, and education level were recruited from the community, and informed consent was obtained. The exclusion criteria were the same for the control group as for the OCD, SCH, and LDP groups. The study was approved by the Ethics Committee of Tongde Hospital, Zhejiang Province. All participants provided written informed consent prior to study entry. Ultimately, a total of 21 patients for the OCD group (30.38 ± 13.56), 20 patients for the SCH group (34.75 ± 6.36), 12 patients for the LDP group (30.50 ± 6.54), and 25 healthy controls (CN; 31.48 ± 5.34) were included. Image acquisition All MR images were acquired at the Department of Radiology of Tongde Hospital utilizing a 3.0-Tesla scanner (Magnetom Trio Tim, Siemens). To minimize head movement, a 16-channel birdcage head coil with foam pads was used to secure each subject's head. A gradient echo sequence was employed to acquire 3D T1-weighted images with isotropic 1 mm 3 voxels; the scan covered the entire brain and proceeded parallel to the anterior commissure-posterior commissure (AC-PC) line. The following parameters were used: flip angle = 9°; TR = 1900 ms, TE = 2.48 ms, TI = 900 ms; slice thickness = 1.0 mm without gaps; reconstruction resolution = 1 × 1 × 1 mm 3 ; field of view = 512 × 512 mm 2 . The diffusion-weighted imaging (DWI) data were composed of a non-diffusion-weighted image (b0) and diffusionweighted images along 30 gradient directions with a b-value of 1,000 s/mm 2 . A single-shot echo-planar sequence was used to acquire DWI data with the following parameters: TR/TE = 8,600/92 ms; number of transversal slices = 55; flip angle = 90°; NEX = 1; gradient directions = 30; field of view = 256 × 248 mm 2 ; and slice thickness = 2.0 mm without gaps. Image processing and brain connectivity analysis The following section outlines the comprehensive procedures for image processing and brain connectivity analysis. Brain network construction Brain parcellation Anatomical referencing and parcellation of the brain into 90 cortical and subcortical regions were performed for each subject using T1W images and the automated anatomical labeling (AAL) atlas. The following steps were taken: (1) FLIRT was used to register T1W images to DWI in the native space. (2) FNIRT was employed to map the registered structural images to the ICBM 152 template. (3) The estimated transformation parameters were inverted and applied to the AAL atlas to warp all brain regions of interest from MNI space to the native diffusion space. White matter tractography To correct for head motions and eddy currents, FMRIB's Diffusion Toolbox was utilized to register all DWI scans to b0. The linear leastsquares fitting method was employed to estimate the diffusion tensor for each voxel using Diffusion Toolkit. Subsequently, tractography was performed using the fiber tracking algorithm with a fractional anisotropy threshold of 0.2 and a fiber turning angle threshold of 45° using Diffusion Toolkit to construct the structural connections between the 90 brain regions. Brain network In a brain network, each brain region is regarded as a node, and each connection between regions is considered an edge. If WM fiber tracts originate from one region and terminate in another, the connections between the 90 brain regions are considered edges. Using the UCLA Multimodal Connectivity Package in the native diffusion space (23), we estimated the number of fibers between regions and weighted each edge accordingly. An inter-regional undirected network with weighted connections was thus obtained for each subject. Brain network analysis To minimize the overall differences in connectivity strength, each weighted connectivity matrix was normalized to its maximum fiber count. The Brain Connectivity Toolbox was utilized to estimate the properties of each participant's global network, such as the Frontiers in Psychiatry 04 frontiersin.org small-world properties and network efficiency, as well as the nodal characteristics of each brain region in each subject's normalized connectivity matrix. Small-world properties The clustering coefficient measures the likelihood that a node's neighborhoods are connected to each other, and the clustering coefficient of the entire network reflects the extent of the local cluster in a network. Characteristic shortest path length measures the shortest geodesic length between a node and any other node, and that of the entire network is the average shortest path length between all pairs of nodes. To determine whether a network is a small-world network, the clustering coefficient and characteristic shortest path length of the network are compared with those of random networks (11). We generated 100 randomized networks with the same number of nodes, number of edges, and degree distribution as the original network. A network is considered a small-world network when the normalized clustering coefficient (γ ) is much greater than one and the normalized characteristic shortest path length (λ) is close to one. Network efficiencies The global efficiency is calculated as the average of the inverse of the shortest path length for all node pairs in the entire network (24). The local efficiency of the entire network can be determined by averaging the global efficiency values of all subnetworks. It measures the fault tolerance of the entire network and reflects how well each subnetwork exchanges information when the most connected node is removed. Nodal characteristics A node's degree is the number of edges that connect it to the rest of the network. The nodal efficiency of a given node is defined as the inverse of the harmonic mean of the characteristic shortest path length between it and all other nodes in the entire network. The betweenness centrality of a given node quantifies the number of shortest paths between nodes in the entire network that pass through the given node. It is a measure of the node's importance for the integration of all the connections among all nodes in the network. Statistical analysis Comparisons between the network measures of all cohorts were performed using one-way ANOVA followed by Bonferroni post hoc analysis. Pearson correlations were also performed to determine the association between network measures and clinical assessments. All analyses were corrected for age and sex. A significance threshold of p < 0.05 was set for all statistical tests. All statistical analyses were performed using SPSS 22.0 (SPSS, Chicago, IL, United States). Demographic and clinical characteristics The demographics and clinical characteristics of the participants are shown in Table 1. There were no significant differences in age, gender, or, where applicable, total illness duration among CN and patients with OCD, SCH, and LDP. Brain regions with significantly different degrees are indicated in red. The node size and edge width are weighted by the nodal efficiency and number of connections, respectively. For 02Precentral_R, SCH patients showed no significant difference in MR-measured degree from the other groups; compared with OCD patients, significantly reduced degree was observed in Frontiers in Psychiatry 05 frontiersin.org Illustration of the brain regions with significant differences in degree among CN and OCD, SCH, and LDP patients. Frontiers in Psychiatry 06 frontiersin.org patients with LDP (p = 0.006), and significantly increased degree was observed in CN (p = 0.02; Table 3; Figure 2). For 25Frontal_Med_ Orb_L, compared with SCH patients, a significantly increased degree was observed in patients with CN (p = 0.004), and no significant difference in MR degree was observed between the remaining two groups; compared with OCD patients, a significantly increased degree was observed in CN (p = 0.004). For 47Lingual_L, compared with SCH patients, a significantly reduced degree was observed in patients with OCD (p = 0.0036), and no significant difference in degree was observed in the remaining two groups; compared with OCD patients, a significantly increased degree was observed in LDP patients (p = 0.002) and CN (p = 0.0026). For 58Postcentral_R, compared with SCH patients, a significantly increased MR degree was observed in patients with OCD (p = 0.018), and no significant difference in MR degree was observed in the remaining two groups; compared with OCD patients, a significantly reduced degree was observed in patients with LDP (p = 0.009). For 90Temporal_Inf_R, compared with SCH patients, a significantly increased degree was observed in patients with OCD (p = 0.018), and no significant difference in degree was observed in the remaining two groups; compared with OCD patients, a significantly reduced MR degree was observed in patients with LDP (p = 0.01). Discussion The etiology and pathogenesis of schizophrenia remain unclear, and clinical diagnosis primarily relies on clinical manifestations, given the dearth of objective laboratory indicators. Obsessive-compulsive symptoms not only occur in patients with obsessive-compulsive Frontiers in Psychiatry 07 frontiersin.org disorder but also have a high incidence in several other disorders, such as depression, anxiety, and schizophrenia. Studies have reported that the prevalence of obsessive-compulsive symptoms is higher in schizophrenia patients than in the general population, and schizophrenia patients with obsessive-compulsive symptoms (OCS) tend to experience more severe symptoms and poorer prognosis than those without OCS (1). In recent years, diffusion tensor imaging (DTI) has been increasingly used to detect abnormal brain functional connectivity in patients with mental disorders, including schizophrenia and obsessive-compulsive disorder. In this study, we conducted a comparison of DTI-based functional brain imaging in patients with schizophrenia and obsessive-compulsive symptoms (SCH), clozapine-induced obsessive-compulsive symptoms (LDP), and obsessive-compulsive disorder (OCD), along with healthy volunteers (CN). Our findings demonstrated that there were significant differences in the MR degree of 25Frontal_Med_Orb_L, 47Lingual_L, 58Postcentral_R, and 90Temporal_Inf_R among the three groups (p < 0.05). This may have significance as a reference for neuroimaging studies of schizophrenia with obsessivecompulsive symptoms. There is a high comorbidity rate between schizophrenia and obsessive-compulsive disorder (OCD), but the shared neurobiological substrate and the aetiological relationship between these two disorders are still unclear (25). Using DTI technology, Hawco et al. (14) found that compared with the normal control group, patients with schizophrenia and OCD had reduced FA values in the corpus callosum, and there was no significant difference in the FA values of the total white matter between the two groups, suggesting that schizophrenia and OCD may have a common neurobiological substrate. At present, there are still few studies directly comparing the brain functional magnetic resonance imaging characteristics of SZ and OCD. The majority of scholars believe that the onset of obsessivecompulsive disorder (OCD) results from an imbalance of neurotransmitter and neuronal metabolite concentrations in the cortico-striato-thalamo-cortical (CSTC) circuit, and neuroimaging studies have revealed that the pathophysiological mechanism of OCD may involve a broader range of brain regions, such as the parietal cortex and the posterior temporo-parieto-occipital junction area (26). Schizophrenia is associated with changes in the amplitude of low frequency fluctuation (ALFF) in a wide range of brain regions, such as frontal, temporal, parietal, occipital, and limbic lobes (23). Several studies have shown that patients with schizophrenia and obsessivecompulsive disorder often have abnormalities in brain white matter structure, including the cingulate tract, the corpus callosum, and white matter fiber tracts in frontal and temporal regions (11,12). In the brain region of 25Frontal_Med_Orb_L, which is the superior orbitofrontal gyrus, the MR-measured degree was significantly decreased in patients with SCH compared to CN (p = 0.004). The superior orbitofrontal gyrus is an important part of the prefrontal lobe, playing an important role in advanced cognition and autonomous behavior. This gyrus is an important part of the cognitive control network, participating in emotion regulation, execution of cognitive behaviors, memory, decision-making, and other processes (24). There is a correlation between the prefrontal lobe and obsessive-compulsive disorder. Human neuroimaging studies have consistently highlighted abnormal activity patterns in prefrontal cortex (PFC) regions and connected circuits during symptom elicitation and performance of neurocognitive tasks in OCD (27). Qin et al. (28) applied graph theory and network-based statistics (NBS) methods to DTI studies and found that the node efficiency and Boxplots (mean ± SD) of the MR-measured degree in the brain regions 02Precentral_R, 25Frontal_Med_Orb_L, 47Lingual_L, 58Postcentral_R, and 90Temporal_Inf_R in LDP, OCD, and SCH patients and CN. Frontiers in Psychiatry 08 frontiersin.org strength of the orbitofrontal gyrus were reduced in OCD patients. Park et al. (29) found a significant increase in white matter volume in the right dorsolateral prefrontal cortex in OCD patients compared with healthy controls, but no brain regions with reduced white matter volume were found in OCD, and no changes in gray matter volume were found between the two groups. Remijnse et al. (30) found that the orbitofrontal cortex (OFC) and striatal function were impaired in OCD. At present, there are few studies on the relationship between the superior orbitofrontal gyrus/prefrontal lobe and schizophrenia with obsessive-compulsive symptoms (OCS), and the relationship between the orbitofrontal cortex and schizophrenia with OCS has not been clarified. Schizophrenia and OCD patients commonly present abnormalities in brain white matter structure, and patients with a variety of psychiatric disorders, including schizophrenia and OCD, have an elevated risk of abnormalities in white matter fiber tracts in frontal regions (11). Zhu et al. (31) found that decreased orbital frontal cortex connectivity in schizophrenia may be related to reduced activity in the orbitofrontal cortex due to low activation of dopaminergic inputs in the endodermis. A study by Schirmbeck et al. (32) in patients with schizophrenia showed a significant increase in OFC activity during the onset of obsessive symptoms with antipsychotic medication. Increased orbitofrontal cortex activation is associated with "pro-obsessive" antipsychotic treatment in patients with schizophrenia. This is not consistent with our results. This may be related to the sample size and different demographic characteristics; further exploration and study are needed. The brain region 47Lingual_L, which is located in the occipital lobe, had a significantly higher MR-measured degree in SCH than in OCD (p = 0.0036). Some scholars have found that in OCD patients, the intrinsic functions from the cortico-striato-thalamo-cortical (CSTC) circuits to the occipital lobe are changed (33). The CSTC circuit involves multiple brain regions, such as the prefrontal lobe, striatum, and thalamus, and has been shown to be closely related to the pathophysiology of OCD (34). The lingual gyrus, which is central to the visual cortex, is located in the occipital lobe. The occipital lobe is not a component of the CSTC neural circuit, and there is limited research on the occipital lobe in obsessive-compulsive disorder (35). However, the occipital lobe is not commonly associated with schizophrenia. Tohid et al. (36) found that most patients with schizophrenia exhibit normal occipital lobe anatomy and physiology. Michel et al. (37) found that the activity of xanthine oxidase (XO), an enzyme closely related to the pathophysiology of schizophrenia, was decreased in the occipital cortex of psychotic patients. To date, few studies have explored the relationship between the occipital lobe and schizophrenia with obsessive-compulsive symptoms based on neuroimaging; our findings provide a reference for future studies. The brain region 58Postcentral_R, the postcentral gyrus, had a significantly lower MR-measured degree in SCH than in OCD (p = 0.018). The postcentral gyrus is an important brain region in the sensorimotor network, and neuroimaging studies have shown that obsessive-compulsive disorder is often associated with abnormal activity in this region (38). It has been observed that patients with OCD have reduced short-range positive functional connectivity (spFC) and long-range positive FC (lpFC) at rest and that the spFC of the left anterior central gyrus/posterior central gyrus can distinguish OCD patients from healthy controls (39). Le Jeune et al. (40) performed 18-fluorodeoxyglucose positron emission tomography (PET) in 10 patients with OCD and found hypermetabolism in the postcentral gyrus in non-stimulated OCD patients compared with healthy controls. Yu et al. (16) found that ALFF in the left postcentral gyrus was significantly lower in the OCD group than in the schizophrenia group or the healthy control group. There is no consensus on what kind of abnormal activity in the postcentral gyrus is associated with OCD. Wang et al. (41) found that gray matter volume (GMV) in the left postcentral gyrus was increased in individuals with high levels of schizophrenic and obsessivecompulsive traits compared to those with low scores for these traits. The brain region 90Temporal_Inf_R, the inferior temporal gyrus, showed a significantly lower MR-measured degree in SCH than in OCD (p = 0.018). This brain region is known to play a crucial role in emotional and cognitive processing. Abnormalities in the structure and function of the temporal lobe can affect inhibitory control and executive function in patients with obsessive-compulsive disorder. Previous studies using structural magnetic resonance imaging have reported a decrease in the thickness of the temporal cortex in patients with obsessive-compulsive disorder (42). Additionally, Jinmin et al. (43) found that patients with obsessive-compulsive disorder exhibited a decrease in temporal lobe activation as measured by the blood oxygen level-dependent signal. A meta-analysis found an increase or decrease in resting-state brain activity in the temporal lobe, specifically the left superior temporal gyrus, in schizophrenia (44). Using restingstate functional magnetic resonance imaging, Liang et al. (45) found that the ALFF of the middle temporal gyrus was significantly reduced in childhood-onset schizophrenia with obsessive-compulsive symptoms (COSO) compared with schizophrenia without obsessivecompulsive symptoms (COS). Wang et al. (46) examined gray matter volume and cortical thickness in 22 schizophrenia patients with obsessive-compulsive symptoms and found that these patients had reduced cortical thickness in the right superior temporal gyrus compared with healthy controls. These results are consistent with the results of our study. Clozapine is an important second-generation antipsychotic drug that has balanced antidopaminergic and anti-serotonergic properties and is used to treat schizophrenia. It is particularly useful for schizophrenia that has been resistant to other treatments. However, the most commonly observed adverse effect of clozapine treatment is OCS. Some studies have reported that as many as 35.9% of clozapine users subsequently develop OCS (47). In our study, we observed that the MR-measured degree of the 02Precentral_R brain region (precentral gyrus in the frontal lobe) was significantly reduced in the LDP group compared to the OCD group (p = 0.006). Compared with the CN group, the LDP group had a significantly reduced degree as measured by MR (p = 0.02). In the 47Lingual_L brain region (lingual gyrus in the occipital lobe), the degree was significantly higher in the LDP group (p = 0.002) and CN group (p = 0.0026) than in the OCD group. Similarly, in the 58Postcentral_R brain region (postcentral gyrus in the parietal lobe), the degree was significantly reduced in the LDP group compared to the OCD group (p = 0.009). Finally, in the 90Temporal_Inf_R brain region (inferior temporal gyrus in the temporal lobe), the degree was significantly reduced in the LDP group compared to the OCD group. These findings suggest that LDP treatment for schizophrenia may have beneficial effects on several brain regions, including the precentral gyrus, postcentral gyrus, lingual gyrus, and inferior temporal gyrus, which are all implicated in the pathological mechanism of OCD. At present, the mechanism of clozapine inducing OCS is still unclear. Some studies have speculated that clozapine can induce OCS due to its strong innate antiserum effect, especially with 5-HT1C, Frontiers in Psychiatry 09 frontiersin.org 5-HT2A, and 5HT2C receptors (48). In addition to pharmacodynamic mechanisms, specific genetic characteristics may predispose schizophrenia patients to develop secondary OCS during treatment. One candidate polymorphism is in SLC1A1, the gene that encodes the neuronal glutamate transporter and is independently associated with genetic risk for OCD (49). Some scholars also propose that patients with OCS after clozapine treatment exhibit significant cognitive defects in areas such as visual recognition memory, impulse inhibition, perseverative responses, and set-switching ability and that these cognitive deficits are strongly associated with the severity of OCS (50). Therefore, cognitive defects may be related to the pathological mechanism of OCS occurrence. In terms of neurological influence, Şule Bıcakıay et al. studied 18 schizophrenia patients, including nine patients with clozapine-induced OCS [Clz OCS (+)], nine patients without OCS [Clz OCS (−)], and nine healthy controls. The study showed that WM integrity in several pathways, such as the corticostriato-thalamo-cortical circuitry and orbitofrontal tracts, appears to be affected differently in patients with Clz-OCS (+). Different neuroplastic effects of clozapine leading to the occurrence of OCS in a subgroup of patients are possible and require further evaluation through longitudinal follow-up studies (51). However, there are still very few studies in this area. However, this study still leaves some unanswered questions, such as what caused such different changes in connectivity in patients, and further investigation is needed. Due to the small number of patients with schizophrenia and obsessive-compulsive symptoms caused by the clinical use of olanzapine, only 12 patients were in the LDP group. In this study, the demographic characteristics of the LDP group were not significantly different from those of the other groups (p > 0.05); therefore, the LDP group was also included in this study and discussion. This may cause a certain deviation in the study results, and further studies with larger sample sizes are needed to improve the credibility of the study results. Conclusion In conclusion, the aim of this study was to evaluate brain connectivity in patients with schizophrenia and obsessive-compulsive symptoms using diffusion tensor imaging (DTI). Significant differences in the degree of nodes as measured by MR were observed in the 25Frontal_Med_Orb_L, 47Lingual_L, 58Postcentral_R, and 90Temporal_Inf_R regions among the SCH, OCD, and CN groups (p < 0.05). In the LDP group compared to the OCD group, significant differences in brain regions 02Precentral_R, 47Lingual_L, 58Postcentral_R, and 90Temporal_Inf_R (p < 0.05) were observed. Our results indicate that the MR-measured degree of the 02Precentral_R, 25Frontal_Med_Orb_L, 47Lingual_L, 58Postcentral_R, and 90Temporal_Inf_R brain regions significantly differed among the CN, OCD, SCH, and LDP groups and that alterations in MR degree may be the underlying mechanism of obsessive-compulsive symptoms in schizophrenia, which provides some references for future studies on schizophrenia with obsessivecompulsive symptoms. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethics statement The studies involving human participants were reviewed and approved by Tongde Hospital of Zhejiang Province. The patients/ participants provided their written informed consent to participate in this study. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Author contributions LLu conceived the study. LIi, HL, WL, and LG collected the data. YH and XX analyzed the data and drafted the paper. All authors contributed to the article and approved the submitted version.	2023-07-29T15:20:39.290Z	2023-07-27T00:00:00.000	{ "year": 2023, "sha1": "7c7252be730b5309760933528b3ceacd2c440527", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "f67637dcbab829de05cfb90f9c4d1fa1e06df1c7", "s2fieldsofstudy": [ "Psychology", "Medicine" ], "extfieldsofstudy": [] }
158897699	pes2o/s2orc	v3-fos-license	Democracy and the threat of populism A democracia é considerada, em geral, um bem essencial à sociedade política. O que significa democracia, contudo, é controverso. Como o vinho, a ‘garrafa democrática’ pode conter compostos variados e saber muito diferentemente. Nos anos 2010 a democracia parece presente em todos os discursos - um bem a ser estimado e preservado - e sofrer sob o duro ataque do ceticismo e de avaliação negativa. Voltamos aos anos 1920? Precisaria o mundo - ou ao menos vários países importantes - de uma forma reinstaurada de tutela despótica? É inevitável voltar ao egoísmo político nacionalista em um cenário global avançado? O horizonte turvo das esperanças políticas, econômicas e sociais favorece o reaparecimento de projetos populistas - de esquerda como de direita - que projetam miragens sobre o sentimento de frustração nos espaços sociais. É esse populismo uma ameaça à democracia como valor e como prática? A resposta é ‘sim’, malgrado todas as imperfeições do regime democrático. Após apresentar um breve panorama do status atual da democracia, discute-se o entendimento de democracia e o sentido do populismo atual. DEMOCRACY AND THE THREAT OF POPULISM Democracy is generally considered an essential good for the political society. What democracy actually means is controversial. Like wine, the 'democracy bottle' can contain various compositions and can taste very differently. In the 2010s democracy seems to be everywhere in discourse -a good to be cherished and preserved -and to suffer under a harsh attack of scepticism and negative evaluation. Have we fallen back to the 1920s? Does the world -or at least a number of important countries -need a reinstated form of guardianship from new despots? Is it inevitable to run back into a nationalistic political selfishness in a quite advanced globalized scenario? The troubled horizon of political, economic and social hopes favours the reappearance of populist projects -from the lef t or the right spectrum of politics -, which project mirages on the feeling of frustration in social circles. Is such populism a threat to democracy as a value and a practice? The answer is 'yes', despite any imperfections of the democratic regime. After presenting a short overview of the status of democracy in the present days, we will discuss the understanding of democracy and the sense of populism nowadays. Democracy is generally considered an essential good for the political society. What democracy actually means is controversial. Like wine, the 'democracy bottle' can contain various compositions and can taste very differently. In the 2010s democracy seems to be everywhere in discourse -a good to be cherished and preserved -and to suffer under a harsh attack of scepticism and negative evaluation. Have we fallen back to the 1920s? Does the world -or at least a number of important countries -need a reinstated form of guardianship from new despots? Is it inevitable to run back into a nationalistic political selfishness in a quite advanced globalized scenario? The troubled horizon of political, economic and social hopes favours the reappearance of populist projects -from the left or the right spectrum of politics -, which project mirages on the feeling of frustration in social circles. Is such populism a threat to democracy as a value and a practice? The answer is 'yes', despite any imperfections of the democratic regime. 1 After presenting a short overview of the status of democracy in the present days, we will discuss the understanding of democracy and the sense of populism nowadays. 1. After World War II democracies had taken root in the most difficult circumstances possible-in Germany, which had been traumatized by Nazism and Communism (East Germany 1949-1990, in India, which had one of the world's largest population of poor people, and, in the 1990s, in South Africa, which had been stigmatized by apartheid. De-colonization created a host of new apparent democracies in Africa and Asia, and autocratic regimes were followed by democracy in Greece (1974), Spain (1975), Argentina (1983), Brazil (1985) and Chile (1989). The collapse of the Soviet Union created many incipient democracies in central Europe in the 1990s. In 2014 -almost a quarter of a century after the fall of the Berlin Wall -delegations of numerous countries gathered at the World Forum for Democracy in Strasbourg (France) -an initiative of the Council of Europe -to search a way to go further than a 'mere' representative democratic system, in which "the will of the people" was "the basis of the authority of government", aiming for a more influential participation by the youth. The forum's final report states in its opening words: While the number of democracies in the world has been growing steadily, it has to be said that these democracies, whether established or in transition, now face major challenges. Among these challenges, in particular, is the disenchantment with political representation and skepticism regarding policy decisions among young people who are at risk of being increasingly alienated from public political life. The multiple 'failed experiments' in political life and the frustrations concerning the leading forces in society have a double effect. On the one hand they put democracy at risk with the 'totalitarian temptation' of the extremes -presently covered up by the populist proposals. On the other hand, the frustration with traditional politics and with the alienation from decision-making processes seems to range from acid critique to a refusal of maintaining old leadership and finally to a renewal of the democratic tools through traditional electoral instruments. Democracy is also no post-truth failure, but a real and concrete issue for societies in the 2020s, so that the forum in Strasbourg in 2017 will have populism as its main theme. Its call for participation says: Representative democracy became the dominant democracy model during the industrial age. After WW2 party pluralism was enshrined in democratic constitutions as the vehicle for political pluralism and a barrier against authoritarian regimes in Europe. Political parties represented mostly class interests, built political capital within their membership base, and communicated via like-minded media. Public service broadcasters were entrusted with ensuring multiple perspectives in political information and debate and the overall independence and diversity of media were seen as a guarantee of a free and pluralist debate. However, something is happening with political parties and with the media. A growing disconnect between citizens and political elites and dramatic changes in the media ecosystem are a challenge for democracy as we know it. At the same time, new political and media actors and practices are emerging, offering new opportunities for members of the public to participate in political life. Legacy media and political parties are both threatened by these new developments but can also learn from them in order to adapt their own models and functioning to new realities. The objective of the World Forum for Democracy 2017 is to review novel initiatives and approaches which can enhance democratic practices and help parties and media, but also other political actors, to re-connect to citizens, make informed choices and function optimally in 21st century democracy. One can nevertheless corroborate that societies having seen off 'failed experiments' along the century with authoritarian and totalitarian forms of government, seem, at long last, to be willing to keep democracy, even in very particular political interpretations in many countries. Such self-confidence is surely understandable after the successes during the second half of the 20th century. But looking farther back the triumph of democracy looks rather less inevitable. After the fall of Athens, where it was first developed -and not so 'theoretically equal' as presently conceived -, the political model had lain dormant until the Enlightenment and was then widely idealized. Ultimately, the only sustained democracy produced by the 18th century was the American revolution. The Congress of Vienna and the Restoration after the Napoleonic Wars led the 19th century European monarchists to fight a continuous action against democratic forces in order to contain them as much as possible. The so cherished Constitutionalism of the 1800's was by far a kind of damage control. In the first half of the 20th century nascent democracies collapsed in Germany, Spain, Portugal and Italy. Worried by the on-going war in Europe and the successes of Nazi-Germany, the American President Franklin D. Roosevelt addressed on March 15th 1941 the Annual Dinner of the American White House Correspondents and shared with strong words his concern that "The light of democracy must be kept burning", and that Britain alone might not be able to shield "the great flame of democracy from the blackout of barbarism". For him, America must take action to "keep that flame alight". 3 Democracy won and its progress in the late 20th century seems to have difficulties in the 21st. Even though in 2015 around 50% of the world's population, more people than ever before, live in countries that hold free and fair elections regularly, democracy's global advance has come to a halt. 4 And democracy's problems run deeper than mere numbers suggest. Many nominal democracies have slid towards autocracy, maintaining the outward appearance of democracy through elections, but without the rights and institutions (like an independent Justice system or a free press) that are equally important aspects of a functioning democratic system, among many others. Enthusiasm for democracy flares up in moments of triumph, such as the overthrow of unpopular regimes in Tunis, Cairo or Kiev, only to crack once again. The stalemate in Libya or Syria shows how difficult it is to combine euro-american democracy with other cultures and with powerful hegemonic ambitions. Including in the West, perceived as the 'paradise of democracy', the regime could advance only to collapse, like in Venezuela. And within the West, democracy has too often become associated with debt and dysfunction at home and overreach abroad. Democracy has always had its critics, but now old doubts are being treated with renewed respect as the weaknesses of democracy in its Western strongholds, and the fragility of its influence elsewhere, have become increasingly apparent. Why has democracy been exposed to such threats? One point is that democracy is no unequivocal concept. We will come back to this question further below. Democracy suffers nowadays from repetitive crises since 2007. It started with the sub-prime financial crisis of 2007/08 in the United States and the sharp and destabilising rise of China. The damage caused by the crisis was psychological as well as financial. It revealed fundamental weaknesses in the West's political systems, undermining the self-confidence that had been one of their great assets. Governments had steadily extended entitlements over decades, allowing dangerous levels of debt to develop, and politicians came to believe that they had abolished boom-bust cycles and tamed risk. In the Euro-Zone, the reckless management of public accounts (Ireland, France, Italy) and accelerated indebtedness have caused political destabilization in several countries (Greece, Spain, Portugal) and jeopardized the expected solidarity of the zone. Many people became disillusioned with the workings of their political systems-particularly when governments bailed out bankers with taxpayers' money and then stood by impotently as financiers continued to pay themselves huge bonuses. The crisis turned the Washington consensus into an object of harsh critique across the emerging world. Meanwhile some recent recruits to the democratic camp have lost their lustre. The Arab Spring seems to have slid rapidly into a sort of instable Arab Autumn (Egypt, Tunisia) or even Winter: Civil war (Syria) and absence of Rule of Law (Lybia). The goals of establishing democratic practices are catapulted to an unpredictable horizon. Since the introduction of democracy in 1994 South Africa has been ruled by the same party, the African National Congress, which has become progressively self-serving. Turkey, which once seemed to combine moderate Islam with prosperity and democracy, is descending into corruption and autocracy. In Bangladesh, Thailand and Cambodia, opposition parties have boycotted recent elections or refused to accept their results. In the Philippines the outcome of formal presidential elections has invested a leader with hard individualist ways of ruling and tending to autocracy. All of this suggests that creating the institutions necessary to sustain democracy is a very slow work, and has dispelled the once popular notion that democracy will flourish quickly and spontaneously once the seed is planted. Although democracy may be a "universal aspiration", as again and again stressed by important political players like former UN-Secretary General Kofi Annan 5 , it is certainly a culturally rooted practice. Almost all Western countries extended the representation model (the right to vote) long after the establishment of sophisticated political systems, with powerful civil services and entrenched constitutional rights, in societies that cherished the notions of individual rights and independent judiciaries. Yet in recent years the very institutions that are meant to provide models for new democracies have come to seem outdated and dysfunctional in established democracies. The United States experiences since many years a distortion of the traditional principle of checks and balances with the blockade between the majority that elects the president and the one that forms the two houses of Congress. It is not unusual for the USA to waste too much time to undo the gridlocks, so obsessed with partisan point scoring that it has come close to defaulting on its debts twice in the recent past. Its democracy is also compromised by the practice of drawing constituency boundaries to entrench the power of incumbents, similarly to the projected reform of UK constituencies drafted in 2016. This encourages extremism, because politicians have to appeal only to the party faithful or to its radicals, and in effect disenfranchises large numbers of voters. If one remembers that money talks louder than ever in politics, this creates the impression that democracy is eventually for sale and that the rich have more power than the poor, even as lobbyists and donors insist that political expenditure is an exercise in free speech. Unfortunately the EU itself is not the best example of practical democracy, despite its official speech of defence of democratic values. The very complex institutional frame of the EU lacks transparency and participation. Within this frame, the ordinary citizen suffers from estrangement. For example, technocrats were largely responsible for taking the decision to introduce the euro in 1999, but with political support by the chiefs of State and Government. Without a coercive obligation to follow the decision -many countries have not yet adopted the common currency. After the failure of approving the Constitutional Treaty in 2005, the replacement treaty (Lisbon Treaty, 2007), which consolidated power in Brussels, was submitted to formal parliamentary vote, without popular referendums (with the exception of Ireland, where a referendum is constitutionally required). During the darkest days of the euro crisis, the euro-elite forced Italy and Greece to replace democratically elected leaders with technocrats. The European Parliament, an unsuccessful attempt to fix Europe's democratic deficit, is both ignored and despised. The EU has become a breeding ground for populist parties, such as Geert Wilders's Party for Freedom in the Netherlands, Marine Le Pen's National Front in France, and Fauke Petry's Alternative für Deutschland (AfD) in Germany, which claim to defend ordinary people against the arrogant and incompetent elite. Greece's Golden Dawn is testing how far democracies can tolerate Nazi-style parties. The struggle against populism in Europe has tamed its rise but has not yet repealed it. The issue of the recent regional polling in the German Province North-Rhine-Westphalia (on May 14th 2017) shows how resilient the populist discourse can be: the AfD was able to join the assembly for the first time with 7.4% of the votes. 6 Even in its original region, democracy is clearly suffering from serious structural problems, rather than a few isolated afflictions. Since the dawn of the modern democratic era in the late 19th century, democracy has expressed itself through nation-states and national parliaments. People elect representatives to exert national power over defined mandates. But this arrangement is now under assault both in its structures and in its social composition. Structurally, globalisation has changed national politics profoundly. National politicians have seen even more power, for example over trade and financial flows, shifting away toward global markets and supranational bodies. Promises made to the voters on the national scenes are mostly unrealistic or unable to be kept. International organisations such as the International Monetary Fund, the World Trade Organisation and the European Union have increased their influence. There is in this respect an inescapable logic of the contemporary world: how could a single country deal with problems like climate change, terrorism or tax evasion? National politicians have also responded to globalisation by limiting their discretion and devolving power to unelected technocrats in some areas. The number of countries with independent central banks, for example, has increased from about 20 in 1980 to more than 160 today. 7 Socially powerful challenges are also expected: from would-be breakaway nations, such as the Catalans and the Scots, from Indian states, from American city mayors. All are trying to reclaim power from national governments. This new kind of microphysics of power -as practiced for instance by NGOs and lobbyists -modifies traditional politics and makes life harder for democratic and autocratic leaders alike. We are now far away from Michel Foucault's structuralist analysis of the classic problematic of government, exploring the historical constitution and periodization of the state and the important strategic and tactical dimensions of power relations and their associated discourses. For, in rejecting various essentialist, trans-historical, universal, and deductive analyses of the state and state power, Foucault created a space for exploring its 'polymorphous crystallization' 8 in and through interrelated changes in technologies of power, objects of governance, governmental projects, and modes of political calculation. Indeed, he argued that 'the state is nothing more than the mobile effect of a regime of multiple governmentalities'. 9 For Foucault, this does not mean that one needs a trans-historical, universal notion of the state before deconstructing it in and through an interrogation of historically specific, concrete practices. He avoids this paradox by asking how one might explore history if the state did not always-already exist. 10 For example, Il faut défendre la société 11 shows how the modern idea of the universal state emerged from a complex series of discursive shifts and the eventual combination of disciplinary and bio-political power within a redefined framework of sovereignty. 12 In the present days the shifts are multiple and turn around in astonishing rapidity. The internet makes it easier to deconstruct, organise and agitate; in a world where people can participate in reality-TV votes every week, or support a petition with the click of a mouse, the machinery and institutions of parliamentary democracy, where elections happen only every few years, look increasingly anachronistic. Politics seems to be now considered as a 'plug in toy', disposable if defective. The biggest challenge to democracy, however, comes from within-from the voters themselves (and most prominently from the non voters, who proclaim that preserving, reforming and adapting the system is useless). Plato's great worry about democracy, that citizens would "live from day to day, indulging the pleasure of the moment", has proved prescient. Democratic governments got into the habit of running big structural deficits as a matter of course, borrowing to give voters what they wanted in the short term, while neglecting long-term investment. France and Italy have not balanced their budgets for more than 30 years. The financial crisis starkly exposed the unsustainability of such debt-financed democracy. With the post-crisis stimulus winding down, politicians must now confront the difficult trade-offs they avoided during years of steady growth and easy credit. But persuading voters to adapt to a new age of austerity will not prove popular at the ballot box. Slow growth and tight budgets will provoke conflict as interest groups compete for limited resources. To make matters worse, this competition is taking place as Western populations are ageing. Older people have always been better at getting their voices heard than younger ones, voting in greater numbers and organising pressure groups. Yet this is also changing. Movements like En Marche! in France modified substantially the expectations on the political stage and led to Emmanuel Macron's (39 years old) election to the presidency in 2017 (May 14th). Matteo Renzi (42) has just been elected leader of the Italian Democratic Party with a large majority, guided by his movement In Cammino (April 30th).Such movements will increasingly have absolute numbers on their side. Many democracies now face a fight between past and future, between inherited entitlements and future investment. Adjusting to hard times will be made even more difficult by a growing cynicism towards politics. Classical party membership is declining across the developed worldonly newly founded movements attract new militants, whether or not they are populist (like the French En Marche!, which so far shows no populistic characteristics, or the typical populist Italian MoVimento 5 Stelle). Voter turnout is falling, too: a study of 49 democracies found that it had declined by 10 percentage points between 1980-84 and 2007-13. 13 A survey of seven European countries in 2012 found that more than half of voters "had no trust in government" whatsoever. 14 Meanwhile, the boundary between poking fun and launching protest campaigns is fast eroding. In 2013 one quarter of Italians voted for a party founded by Beppe Grillo, a comedian. Since 2006 the German Party of Pirates (Piratenpartei) defends the thesis of a 'liquid democracy', with strong populist traits of left-wing inspiration, not exceeding 2% of the average vote. All this popular mistrust about politics might be healthy if people demanded little from their governments, but they continue to want a great deal. The result can be a toxic and unstable mixture: dependency on government on the one hand, and disdain for it on the other. The dependency forces government to over expand and overburden itself, while disdain robs it of its legitimacy. Democratic dysfunction goes hand in hand with democratic distemper. Democracy did well in the 20th century in part because of American hegemony and of the European recovery: other countries naturally wanted to emulate the world's leading countries. But as China's influence has grown lately, America and Europe have lost their appeal as role models and their appetite for spreading democracy. At the same time, democracies in the emerging world have encountered the same problems as those in the rich world. They too have overindulged in short-term spending rather than long-term investment. Brazil allows public-sector workers to retire at 53 but has done little to create a modern airport system. India pays off vast numbers of client groups but invests too little in infrastructure. Political systems have been captured by interest groups and undermined by anti-democratic habits. Democracy has been on the back foot before. In the 1920s and 1930s communism and fascism looked like the coming things: when Spain temporarily restored its parliamentary government in 1931, Benito Mussolini likened it to returning to oil lamps in the age of electricity. In 1969 Willy Brandt, who was at the time just sworn in as German chancellor, declared in his first official speech: "Wir wollen mehr Demokratie wagen" -"We want to dare more democracy". Things have indeed improved, but the risks of frustration and lack of interest increase as well. Although far away from traditional democratic countries, the way China conducts its politics and economic management poses a far more credible threat than communism ever did to the idea that democracy is inherently superior and will eventually prevail. The answer to that depends in a large measure upon how economic elites from Western countries benefit from the Chinese centralized and monocratic decision-making process. The most successful new democracies have all worked in large part because they avoided the temptation of "majoritarianism"-the notion that winning an election entitles the majority to do whatever it pleases. India has survived as a democracy since 1947 (apart from a couple of years of emergency rule) and Brazil since the mid-1980s for much the same reason: both put limits on the power of the government and provided guarantees for individual rights. Robust constitutions not only promote long-term stability, reducing the likelihood that disgruntled minorities will take against the regime. They also bolster the struggle against corruption, the bane of developing countries. Conversely, the first sign that a fledgling democracy is heading for the rocks often comes when elected rulers try to erode constraints on their power-often in the name of majority rule. Mohamed Morsi tried to pack Egypt's upper house with supporters of the Muslim Brotherhood. Viktor Yanukovych reduced the power of Ukraine's parliament. Vladimir Putin has strongly overridden Russia's independent institutions in the name of the people. Several African and Asian leaders are engaging in crude majoritarianism-removing term limits on the presidency or expanding penalties on the ground of religion, with the adoption of the Sharia law (for instance: Saudi Arabia, Iran, Mali). Foreign leaders should be more willing to speak out when rulers engage in such illiberal behaviour, even if a majority supports it. But the people who most need to learn this lesson are the architects of new democracies: they must recognise that robust checks and balances are just as vital to the establishment of a healthy democracy as the right to vote. Paradoxically even potential dictators have a lot to learn from events in Iraq, Afghanistan, Egypt, Libya, Syria and Ukraine: political abuse within the regimes can enrage their citizens by accumulating too much power or failing to respond to their expectations on the long run. Even those lucky enough to live in mature democracies need to pay close attention to the architecture of their political systems. The combination of globalisation and the digital revolution has made some of democracy's most cherished institutions look outdated. Established democracies need to update their own political systems both to address the problems they face at home, and to revitalise democracy's image abroad. Some countries have already embarked upon this process. Reform of party financing, so that the names of all donors are made public, might reduce the influence of special interests. The broad and in-depth[ investigations of the Brazilian Judiciary on corruption and harmful complicity between politicians and businessmen in Brazil since 2005 contributes both to clean the political scene and -as an unintended side effect -to reinforce contempt for the political class. The European Parliament requires its members to present receipts with their expenses. 15 Italy's parliament has far too many members who are paid too much, and two equally powerful chambers, which makes it difficult to get anything done. But reformers need to be much more ambitious. The best way to constrain the power of special interests is to limit the number of advantages that the state can hand out. And the best way to address popular disillusion towards politicians is to reduce the number of promises they can make. The key to a healthier democracy, in short, is a narrower state-an idea that dates back to the American revolution. The notion of limited government was also integral to the relaunch of democracy after the Second World War. The United Nations Charter (1945) and the Universal Declaration of Human Rights (1948) established rights and norms that countries could not breach, even if majorities wanted to do so. These checks and balances were motivated by fear of tyranny. But today, particularly in the West, the big dangers to democracy are harder to spot. One is the growing size of the state. The relentless expansion of government is reducing liberty and conceding ever more power to special interests. The other danger comes from government's habit of making promises that it cannot fulfil, either by creating entitlements it cannot pay for or by waging wars that it cannot win, such as the war on drugs. Both voters and governments must be persuaded of the merits of accepting restraints on the state's natural tendency to overreach. Giving control of monetary policy to independent central banks tamed the rampant inflation of the 1980s, for example. It is time to apply the same principle of limited government to a broader range of policies. Mature democracies, just like nascent ones, require appropriate checks and balances on the power of elected government. Governments can exercise self-restraint in several different ways. They can put on a golden corset by adopting tight fiscal rules-as the Swedes have done by pledging to balance their budget over the economic cycle. They can introduce "sunset clauses" that force politicians to renew laws every ten years.. They can ask non-partisan commissions to propose long-term reforms. In the 1990s, Sweden reformed the pension system, and payments from the new system started in 2003. Norway followed closely behind with a pension reform that came into effect in 2010. The Swedes rescued their pension system from collapse when an independent commission suggested pragmatic reforms including greater use of private pensions, and linking the retirement age to life-expectancy. Chile has been particularly successful at managing the combination of the volatility of the copper market and populist pressure to spend the surplus in good times. It has introduced strict rules to ensure that it runs a surplus over the economic cycle, and appointed a commission of experts to determine how to cope with economic volatility. After a promising rebirth of democracy in the late 1980s, Brazil is struggling to overcome two major political crises, as well as the impeachments of two presidents in 25 years (1992: F. Collor de Mello; 2016: Dilma Rousseff ), fighting continuous financial and debt problems since 2007, coping with a deficitary loss-making system of pensions, now in the third reform attempt since 1998 (2003,2017). Is this not a recipe for weakening democracy by conceding more power to the greater good? Not necessarily. Self-effacing rules can strengthen democracy by preventing people from voting for spending policies that produce bankruptcy and social breakdown and by protecting minorities from persecution. But technocracy can certainly be taken too far. Power must be delegated sparingly, in a few big areas such as monetary policy and entitlement reform, and the process must be open and transparent. And delegation upwards towards grandees and technocrats must be balanced by delegation downwards, handing some decisions to ordinary people. The trick is to combine the twin forces of globalism and localism 16 , rather than trying to ignore or resist them. With the right balance of these two approaches, the same forces that threaten established democracies from outside, through globalisation, and inside, through the rise of micro-powers, can reinforce rather than undermine democracy. Tocqueville argued that local democracy frequently represented democracy at its best: "Town-meetings are to liberty what primary schools are to science; they bring it within the people's reach, they teach men how to use and enjoy it." City mayors regularly get twice the approval ratings of national politicians. Modern technology can implement a modern version of Tocqueville's town-hall meetings to promote civic involvement and innovation. An online hyperdemocracy where everything is put to an endless series of public votes would play to the hand of special-interest groups. But technocracy and direct democracy can keep each other in check: independent budget commissions can assess the cost and feasibility of local ballot initiatives, for example. 17 Democracy is no theoretical debate on philosophy, but a concrete form of organizing the common life and the common form of taking and enforcing decisions. There are also concrete people living and acting. Once again should Tocqueville have the voice to put the reference frame of a possible understanding of effective democracy: "It is above all in the present democratic age that the true friends of liberty and human grandeur must remain constantly vigilant and ready to prevent the social power from lightly sacrificing the particular rights of a few individuals to the general execution of its designs. In such times there is no citizen so obscure that it is not very dangerous to allow him to be oppressed, and there are no individual rights so unimportant that they can be sacrificed to arbitrariness with impunity." Given the scenario outlined above, how to understand and circumscribe democracy? 2. Democracy is understood as the political regime in which everyone and all of the people are equal, so that no essential differences could be established to distinguish rights and duties. As it seems, democracy is an ultimate good for organizing a modus vivendi in society, although complex and not a perfect system of self-government, because it is human. 18 As a human invention, it is contingent, limited, historically determined by the accumulated heritage of men's actions in time. Throughout history. The concreteness of the human history defines the effectiveness of democratic values as put in practice in everyday life. In the present days, albeit democracy is a usual notion and a current discourse (and since a long time) that is successful without a doubt, we do not have a clear definition, or a widely shared concept that one could present as free of ambiguity. When a definition comes to be established, it is not free of controversy, polysemy. Even when a definition is put forward to avoid controversy and obtain a maximum consensus, it is reduced to a minimal set of characteristics and yet problematic. The choice of a 'minimal set of defining characteristics' to circumscribe democracy appears to has become the most followed path in the 20th century, admittedly the century of the triumph of democracy -as relative as it is -throughout the world. Norberto Bobbio's minimalistic definition is probably the most followed one: A 'democratic regime' is "first and foremost a set of procedural rules for arriving at collective decisions in a way which accommodates and facilitates the fullest possible participation of interested parties". 19 Bobbio conceived democracy as a threshold of conditions beyond which no one should step. As the Berlin Wall falls and the failure of the 'real socialism' -as the main proposed alternative to democracy -became evident, Bobbio refined and introduced two complementary definitions of democracy, referring to Hans Kelsen and to Joseph Schumpeter. The mitigated realism of Kelsen suited Bobbio much more than Schumpeter's skeptical reductivism to a sort of economic mechanism of electing voter's delegates (representatives). 20 In both cases the backbone of the theoretical democratic political equality is the idea of representation and power delegation. Kelsen and Schumpeter lived in a very difficult time for democracies and societies: the first half of the 20th century. Eric J. Hobsbawm called this century 'interesting times' 21 , which coincided with his own life (1917-2012) -but the 'short 20th century' was actually hard, extreme, difficult. 22 Including the frequent political crises concerning the democratic form of government all around the world. So it is easy to understand the dilemma Schumpeter sees his time facing, as he states: "the democratic method is that institutional arrangement for arriving at political decisions which realizes the common good by making the people itself decide issues through the election of individuals who are to assemble in order to carry out its will." 23 The difficulty of this approach is that people tend to disagree over fundamental and still more over secondary issues and their multiple divergences cannot be bridged by mere rationality thus discovering the common good. Schumpeter proposes another theory of democracy. Actually he puts the classical doctrine on its head. For Schumpeter, voters should first elect their representatives and the representatives themselves would choose what they think the best policy is. The representatives would compete for the votes of the voters like firms compete for customers. Thus Schumpeter likens democracy to a free market mechanism where parties (firms) have to offer to electorate (the customers) the best policy in order to win their votes. The voters are free to decide their representatives and they can be demanding. If parties failed to deliver they would lose votes on the next elections. So we arrive at Schumpeter's definition of democracy: "And we define: the democratic method is that institutional arrangement for arriving at political decisions in which individuals acquire the power to decide by means of a competitive struggle for the people's vote". 24 At the same time, as Alexis de Tocqueville pointed out in the 19th century, democracies always look weaker than they really are: they are all confusion on the surface but have lots of hidden strengths. Being able to install alternative leaders offering alternative policies makes democracies better than autocracies at finding creative solutions to problems and rising to existential challenges, though they often take a while to zigzag to the right policies. But to succeed, both fledgling and established democracies must ensure they are built on firm foundations. They regarded democracy as a powerful but imperfect mechanism: something that needed to be designed carefully, in order to harness human creativity but also to check human perversity, and then kept in good working order, constantly oiled, adjusted and worked upon. The need for perseverance and persistence is particularly pressing when establishing a nascent democracy. One reason why so many democratic experiments have failed recently is that they put too much emphasis on elections and too little on the other essential features of democracy. The interval between elections (general, regional or local) is often perceived as a legitimacy gap, during which the holder of sovereign power is simply ignored by the elected, entrenched in their mandated delegations. The power of the state needs to be checked, for instance, and individual rights such as freedom of speech and freedom to organise must be guaranteed. During such 'empty intervals' many 'populist mermaids' seek to bewitch people with miraculous promises of all kinds, raising apparent reliability and nurturing the idea that they alone respect the 'people at their core'. This is a risk and a threat. It is a risk, because populists seize legitimacy and a threat because in the end they seek the establishment of a tyranny, of which they would be the sole owners. Democracy, as a system of equality among all, in which the social contract expressed institutionally in the mechanisms of representation and participation must be transmitted from generation to generation. It can be perfected, adapted, adjusted, revised, improved, developed, and extended ad infinitum, provided that the fundamental equality of the human person and their rights are preserved. So it is theoretically, even if historically the actual conditions of its realization depend on men and their deeds. 25 Although a human -therefore imperfect -system, democracy is probably the best system possible to take in account the general interest -if only people maintained the consciousness of all in their own individual, as Kant formulated in his categorical imperative: (a) "Act only according to that maxim whereby you can at the same time will that it should become a universal law"; (b) "Act in such a way that you treat humanity, whether in your own person or in the person of any other, never merely as a means to an end, but always at the same time as an end"; (c) "Thus the third practical principle follows [from the first two] as the ultimate condition of their harmony with practical reason: the idea of the will of every rational being as a universally legislating will." 26 It is certainly a moral requirement, but morals is perhaps the best expression of humankind in search of humanity. 27 3. How can the phenomenon of populism be determined? In its complex forms, it is increasingly difficult to find a common denominator. There is certainly no consistent ideology -which would be a syndrome rather than a doctrine. At the same time, we can observe an ideology of antagonism from a 'pure people' and a 'corrupt elite'. The result of the British referendum on the withdrawal from the EU (Brexit) and the victory of the US Presidential Candidate Donald Trump have once again raised the issue of "populism". The term "right" or "left" anti-establishment parties, which are directed against the ruling "empowered elite" (C. Wright Mills 28 ) in business, politics and culture. 29 Populists regarded "populism" as a stigmatizing spelling. The populist governor of Alabama, George C. Wallace, declared in the 1960s that the term was only the high-struck wastes of pseudo-intellectuals who wanted to hurt him. 30 It is only in recent times that the leftist populists such as Jean-Luc Mélenchon (chairman of the French Socialist Party de Gauche and presidential candidate in 2017 under the motto La France insoumise) or Pablo Iglesias (general secretary of the Podemos leftpopulist party founded in Spain in 2014), Beppe Grillo of the Italian Five-Star Movement (M5S) or the right radical Front National of Marine le Pen in France (who also ran for presidency in 2017 and reached a menacing 2nd place on May 7th) have connoted positively the originally negative stigma in a reviewed self-description: 'Yes, we are populists and proud of it.' Such evolution in the last 20 years puts the neat threat to the classical democratic game. Perhaps because too small elite rules a too large society and that this arouses frustration and irritation, if not indifference and rejection. At the end, what is populism? There is no consistent ideology with unmistakable elements that form a coherent whole, but only a narrative consisting of a few nuclei. Populism, according to Peter Wiles, is a syndrome, not a doctrine. 31 However, since minimum definitions are required in public debate, the polarization and moralization of politics are established as the smallest common denominator of the phenomenon. The Dutch political scientist Cas Mudde defines populism as "an ideology that assumes that society is divided into two homogeneous, antagonistic groups, the 'pure people' and the 'corrupt elite', which argues that politics is an expression of the "volonté générale" or the general popular will". 32 Similarly, Jan-Werner Müller: "Populism [...] is a very definite political concept, according to which a morally pure, homogeneous people are always opposed to immoral, corrupt and parasitic elites". Populists do not refer to the 'moral purity' of the people, but to the common sense of the "good, decent, patriotic, hard-working, law-abiding people" (Nigel Farage, UKIP leader in 2016, appealing on people to vote to leave the EU). Mény and Surel emphasize three core elements of the populist narrative: (a) the people are the foundation of the political community, (b) their sovereignty is disregarded by some actors or processes, (c) this must be denounced and the place of the people in society restored. 33 The disregard of the sovereign people can be the result of different actors identified by the populists: financial capital, technocratic control elites, mainstream parties, or social-moral interpretations. By means of this criterion, populism can be understood as a reaction to the withdrawal of sovereignty. Populistic leaders are often outsiders and homines novi -'new immaculate players' in a political scene that must be rescued and corrected. 34 They often come from the economic world such as the Italian Silvio Berlusconi, the Swiss Christoph Blocher and and Americans Henry Ross Perot and Donald Trump. The Dutchman Pim Fortuyn, as a homosexual, catholic socialized intellectual, was an outsider. Individual wealth is not an obstacle to its success, since it shows that they are neither part of the political establishment nor corrupted by financial sponsors. Trump argued against his competitor Hillary Clinton that he was not "bought" by Wall Street, but independent and therefore credible. The rather pseudo-populist American President Jimmy Carter, a Baptist Southern statesman was also an outsider. When questioned whether he was a liberal or a conservative, he declared himself to be a populist: "I have derived the political support, the encouragement for myself and my concern directly from the people themselves, not from powerful middlemen or representatives of special interests". 35 Modern democracies are mixed systems and are based on two pillars: constitutionalism (a legal state) and popular sovereignty. The 'legal state' has older roots, stands for the rule of law and guarantees constitutional rights for the protection of the individual or minorities against state omnipotence. On the other hand, popular sovereignty is an achievement of the French Revolution, and it is said that all power comes from the people. The people are the sovereign, i. e. the supreme legislator and controller of democracy, expressing their will through elections. A mere acclamation-democracy, that is, a kind of consent or rejection-democracy of a de facto publicly assembled people, as propagated from the right (Carl Schmitt 36 ), withdraws the control of the electorate. Fear of loss of status, future insecurity, the growing gap between rich and poor, or disputes over the housing or labor market between autochthonous and immigrants can no longer be seen as a contrast between the right and the left, but appear as a conflict between the people and elites. The question of access to power is, therefore, central to the understanding of populism: in principle, populists reject intermediary bodies (especially parties and media education elites) between the people and the power, since they would falsify the true people's interests and have only their special interests in mind. In practice, however, they organize themselves into parties and participate in elections. . The mistrust of liberal elites towards the people or the masses has always been great. The liberal conservative Alexis de Tocqueville warned against the "tyranny of the majority". Edmund Burke or the French liberal François Guizot declared that the people were not the sovereign, but the reason for which only the educated and the proprietor citizens are able to access (nowadays rather experts, specialists or professional politicians). The people, on the other hand, identified as the "lower" people or the uneducated masses were mood-dependent, emotional, and seducible. After the introduction of the general and secret electoral vote, filters have been installed in order to prevent the popular will being expressed directly. One of these filters is political parties. In the Weimar constitution of 1919, they were not mentioned and were regarded only as civil society associations. It was only the German Federal Basic Law of 1949 that brought them into the rank of constitutional bodies and assigned them the task of forming and aggregating the political will. Presently almost every country confers constitutional status to political parties as mediators of popular will through the electoral process, as in France, Italy, Spain, Portugal, Brazil, among many others. Populists consider this educational task of the parties to be condescending to citizens. They demand an unfiltered, non-mediated will-articulation by direct democracy, either according to the Swiss model or as a network democracy, under which the Italian M5S is mobilized. It is hard to deny that the two pillars of liberal democracy are increasingly becoming an imbalance. There is hardly a mention of popular sovereignty. In the course of the multi-level governance in the EU, it had lost its relevance, especially as there were no European people at all according to the right-wing populists. The Populism researcher Guy Hermet comments: "It is especially striking that the sovereignty of the people, so far regarded as the heart of democracy, is becoming less and less important. ... There is a philosophical doubt about the relevance of popular sovereignty " 37 But if the bond between rule of law and popular sovereignty breaks, the liberal and the democratic components are once again opposed. As argued by Fareed Zakaria 38 , it is possible to consider populism a kind of democratic illiberalism: "Populism is an illiberal, but democratic response to undemocratic liberalism." Well noted: a response. As a syndrome, populism is essentially reactive. It becomes active only when the prevailing policy does not adequately react to maladministration and dismisses the appeal to emotions as "mood-making". Is right-wing populism 'only' a light right-wing radicalism? In Europe, right-wing extremist parties such as the British National Party (BNP), the German NPD, or ethnocentric regional parties such as Vlaams Belang (Belgium) and the Northern League (Italy) are declining. Exceptions are the Hungarian party Jobbik and the French Front National (FN). The FN sees itself as a national-populist, but is open to right-wing extremist currents and Holocaust-deniers. With a social policy stigmatized by the former leader of the German AfD Frauke Petry as "socialist" and at the same time opening up to sexual minorities, Marine Le Pen, the chairman of the FN, succeeded in what the left less and less succeeds: the combination of material and post-material values and an electoral alliance between lower and middle social segments. New parties have emerged between conservative people or rally parties and the right-wing extremism, which are considered to be right-wing populists despite the great political differences in economic, social and family policy: the United Kingdom Independence Party (UKIP) in Great Britain, the New Flemish Alliance (Nieuw Vlaamse Alliantie, NVA), in Italy the M5S. They represent a "third way from the right" (René Cuperus), but declare that they are not on the right or left but on the side of the citizens. 39 As these new parties are much more successful than right-wing extremism, it is possible to have a bandwagon effect, as in the case of the young AfD: many of those parties, which circle within the New Right or right-wing extremist groups jump in and try to mark the trend with their own 'brand'. Similarly, new left parties, such as the Spanish Podemos, the Greek Syriza, or the French Parti de gauche, are taking a "third way from the left" beyond the old Communist Left and the established Social Democracy. We are indeed facing a crisis of representation, a crisis of participation, and a crisis of sovereignty. If social-democratic parties no longer perceive their function as a tribune of advocates of the "small people", conservative people's parties proclaim themselves as "social democrat" and act as modernizers, this leads to a weakening of their integration function, to confidence crises and to loss of voter's choice. On their way to the political center such traditional parties leave a vacuum at the edges, in which right-wing populist parties, and in Southern Europe also left-wing populist parties, penetrate the field and occupy it with their themes. The crisis of representation is expressed when many people are no longer represented by the established parties and perceive them as a cartel without alternative. Long before the fight of the Spanish Podemos against the "oligarquía", classics of political thought such as Robert Michels, Josef Schumpeter or Gerhard Leibholz pointed out the tendency to oligarchize parties. Parties no longer perceive their function as representative organizations between the state and society, but mutate into a secluded caste with declining intra-party democracy. Closely connected with the crisis of representation is the crisis of participation. It is the case when a considerable number of voters, especially in the lower social segment, no longer participate in politics, but have a grudge against "those up there". The third aspect, the crisis of sovereignty, means a loss of national sovereignty in favor of transnational organizations such as the EU, but also the loss of individual competency. "Like all symptoms," says Jürgen Habermas, "this feeling of the loss of control has a real core -the hollowing out of national democracies that, until now, had given citizens the right to co-determine important conditions of their social existence. The UK referendum provides vivid evidence about the keyword "post-democracy". Obviously, the infrastructure without which there can be no sound public sphere and party competition has crumbled. After initial analyses the media and opposing political parties failed to inform the populace about relevant questions and elementary facts, let alone make differentiated arguments for or against opposing political views. The very low turnout of the 18-24-year-olds, supposedly disadvantaged by the elderly, is another revealing piece of data". 40 When people experience no longer being master of their own situation, not being able to determine their own life and feel exposed to unmanageable influences, they perceive this as a loss of control. Trump's victory, the Brexit, the rise of the AfD, as well as the right-wing populist Swedish democrat (Sverigedemokraterna) in the course of the mass immigration of 2015, all seem to follow one imperative: to regain national control, in America against its "weakness" ("Make America great again!"), in Europe also against the "Leviathan" (Umberto Bossi, Formerly Lega Nord) or the "monster" (Geert Wilders, Partij voor de Vrijheid PVV) in the form of the EU. After Karl Jaspers one could consider that politically the world faces an axial era 41 , a new epochal dividing line between nationalism and liberal worldliness. More precisely, four shifting lines appear to oppose each other: a conflict between material and post-material value orientation, a rivalry between representative and direct democracy, a strife of identity between nativism and cosmopolitanism, and a clash between center and periphery. Right-wing populists also argue that the discrimination of ethnic and sexual minorities or women is one-sidedly the focus of attention, while the social disadvantage of large sections of the population is overshadowed. Social inequality has grown under the hegemony of neoliberalism, and the gap between rich and poor has spread far apart. The misalignment, i. e. the abandonment of the lower social segment (former industrial workers, young unemployed persons who do not have access to the labor market, modest self-employed persons) by the left parties led to a differentiation of right-wing populist parties. They assume the function of an advocate who has long abandoned the left. Populists, however, are not directed to a particular social class , but to the forgotten people, the "plain people", the "silent majority", the "ordinary people", who are not only concerned with threatened loss of status but are confronted with numerous civil society grievances (bloated but inefficient bureaucracy, corruption, inadequate infrastructure). The rapid rise of the Five-Star Movement to Italy's second-strongest party is also due to the failure of the established parties and their disregard for central citizens. In the meantime, the Social Democratic Party under Matteo Renzi has embraced many of the social and ecological demands of this outsider party, even if the voters prefer the original rather than the copy. 4. How to avoid the major risk of populism and to dispel the threat of populism? The reasons for the success of right-wing populist parties (but also left-wing, like Syriza) are different from country to country; after 1989 also between West and Central Eastern Europe. However, it is always about the recovery of sovereignty and self-determination towards the outside (mainly against the EU) and internally (against the caste or the cartel of the established parties or the 'vested interests', that is, particular interests in the USA). As a reaction, the traditional parties have entrenched themselves in a kind of confused center for enlightened citizens, whose success is more than compromised, in face of tight election results (Portugal, Spain, Italy, Germany) and sometimes by simple defeat (presidential election in France, 2017). Is there a way to react to the populist threat? Sure. But where the parties of the mainstream react further in building large coalitions and consequently offering poor alternatives to their classic program, the frustration and failure blow wind under the wings of the populist protests. Ralf Dahrendorf predicted in 1997 that the 21st century could bear the signature of authoritarianism. In the same year, another liberal, Fareed Zakaria, pleaded for a liberal democracy, "which emphasizes both parts of the wording": liberalism and democracy. The stability of the post-war regime was based on the connection between the rule of law and popular sovereignty. In the meantime, however, the tectonics of these two pillars had been cracked. The pendulum strikes towards the liberal rule of law and an "enlightened" elite rule, which is placed as a bulwark against popular sovereignty, seen? as threatened by populists of all sides. According to Ivan Krastev, "liberal democracy is in danger when the structural conflict between "the elites" and "the people" is no longer seen as a liability but a major asset. The current generation of European liberals has been educated in a political tradition that wrongly assumes (historically and theoretically) that anti-liberal parties are also anti-democratic. This is no longer the case. The real challenge that liberal democracy is facing today is the rise of democratic illiberalism. Whoever wishes to save democracy is called on to fight on two fronts: against populists and against those liberals who hold democracy in contempt". 42 This is a call that best fits the challenge of conjuring up the contemporary populist threat.	2019-05-20T13:03:06.895Z	2017-10-24T00:00:00.000	{ "year": 2018, "sha1": "f794fdb1084f3b1ff0a50205e30493f955ca0f36", "oa_license": "CCBY", "oa_url": "https://impactum-journals.uc.pt/estudossecxx/article/download/1647-8622_17_5/6252", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "999f8b801cc517ecfc7434924a35b66643a11454", "s2fieldsofstudy": [], "extfieldsofstudy": [ "Political Science" ] }
269613062	pes2o/s2orc	v3-fos-license	Glycerol Triacetate-Based Flame Retardant High-Temperature Electrolyte for the Lithium-Ion Battery Rechargeable batteries that can operate at elevated temperatures (>70 °C) with high energy density are long-awaited for industrial applications including mining, grid stabilization, naval, aerospace, and medical devices. However, the safety, cycle life, energy density, and cost of the available high-temperature battery technologies remain an obstacle primarily owing to the limited electrolyte options available. We introduce a flame-retardant electrolyte that can enable stable battery cycling at 100 °C by incorporating triacetin into the electrolyte system. Triacetin has excellent chemical stability with lithium metal, and conventional cathode materials can effectively reduce parasitic reactions and promises a good battery performance at elevated temperatures. Our findings reveal that Li–metal half-cells can be made that have high energy density, high Coulombic efficiency, and good cycle life with triacetin-based electrolytes and three different cathode chemistries. Moreover, the nail penetration test in a commercial-scale pouch battery using this new electrolyte demonstrated suppressed heat generation when the cell was damaged and excellent safety when using the triacetin-based electrolyte. ■ INTRODUCTION Due to the substantial decrease in the cost of lithium-ion batteries (LIBs), the number of electric vehicles has experienced significant expansion in recent years.Presently, over 80% of the LIBs produced are intended for transportation. 1At the same time, consumers are demanding battery packs with higher energy density and lower danger levels to reduce the range anxiety and safety concerns. 2urrently, most commercial LIBs use organic electrolytes with a low boiling point (<140 °C) and a low flash point (<40 °C), 3 making the battery not only perform worse at elevated temperatures but also pose a high risk of thermal runaway under abuse conditions. 4High-energy rechargeable batteries also have a high demand in many industrial applications that require continuous operation in extreme environments, like the mining industry. 5,6To address these issues, many efforts have been devoted to finding new electrolyte systems that are nonflammable 7−9 or self-extinguishing 10 electrolytes, which can allow the battery to operate in more extreme conditions.However, there is still a lack of understanding about the battery performance at extreme temperatures above 80 °C. 8ore importantly, many papers have focused on using concentrated electrolytes 11,12 or highly fluorinated electrolytes, 13,14 which have a high cost for commercialization and are also difficult to fabricate on a large scale.Additionally, the solid-state electrolyte with a nonflammability nature is considered the ultimate goal for the lithium-ion battery industry, but at their cost, ionic conductivity and compatibility with electrodes remain huge problems. 15Alternative electrolytes with low cost, high stability, and high performance are needed to meet the demand of high-temperature batteries. Batteries operating at higher temperatures typically experience much faster capacity decay than that at lower temperatures, and three main factors are acknowledged to contribute to the decay: electrolyte evaporation, solid electrolyte interface (SEI), and cathode electrolyte interface (CEI) layer decomposition, and cathode failure.At higher temperatures, conventional low boiling point electrolytes evaporate and decompose, creating a locally dry electrode area with a high resistance.This dry electrode area leads to uneven current distribution and further uneven lithium plating. 16−19 The exposure of the fresh cathode/anode surface will drive the further reaction of the electrolyte with the electrode and create a thicker SEI/CEI layer, exaggerating the consumption of the electrolyte. 20,21oreover, cathode active materials will also play an important role in capacity decay at higher temperatures.As temperatures increase, most cathode materials become less stable as lithium leaves their structure, exacerbating the transition-metal ion dissolution problem. The transition-metal dissolution problem not only leads to the pulverization of the active material but also catalyzes the electrolyte decomposition, eventually leading to complete battery failure at higher temperatures. 22,23Additionally, selfdischarging problems will become more severe at higher temperatures since the mobility of the lithium-ion will also be higher and will also contribute to capacity decay. 24To improve the high-temperature performance of the battery, it is necessary to have a better electrolyte system with a higher boiling point and higher stability that can help us understand the battery failure mechanism at higher temperatures and enable a safer battery for application. 3,25,26he long-term storage of batteries at elevated temperatures is another challenge for high-temperature batteries that have been studied extensively.Batteries stored at higher temperatures experience spontaneous electron diffusion from the cathode to the electrolyte, which triggers electrolyte decom-position and cathode degradation. 27The decomposition and reconstruction of the SEI/CEI layer also consume active lithium in the cell and cause irreversible capacity decay at higher temperatures during long-term storage. 28For these reasons, long-term storage properties also need to be considered in the design of new high-temperature electrolytes. Here, we introduce glycerol triacetate (GTA, also called triacetin) as a new flame-retardant high-temperature electrolyte solvent to replace conventional carbonate electrolytes in higher-temperature operating environments.GTA has a high boiling point (258 °C) and high flash point (138 °C), along with high stability with the lithium metal, 29 making it an ideal candidate for batteries requiring severe thermal conditions.Moreover, GTA is widely used in the biomedical and food industries due to its low toxicity, ecofriendliness, and low cost; it can be easily sourced and readily implemented in commercial battery systems.In this work, we combined GTA with ethylene carbonate (EC) and fluoroethylene carbonate (FEC) as cosolvents with lithium bis- (trifluoromethanesulfonyl)imide (LiTFSI) and lithium difluoro(oxalate)borate (LiDFOB) as lithium salts to evaluate the possibility of using GTA as a high-temperature electrolyte solvent.−32 The adoption of FEC and LiDFOB could help build a robust SEI/CEI layer, preventing dendrite formation on the anode side and cathode failure on the cathode side at elevated temperatures.Based on these considerations, two combinations G1 [1 M LiTFSI in GTA/ EC = 1:1 (v/v)] and G2 [0.5 M LiDFOB in GTA/FEC = 5:2 (v/v)] are mainly tested in our work and compared with commercial electrolyte COM [1 M LiPF6 in EC/DEC/DMC = 1:1:1 (v/v/v)].The performances of the battery with three different cathode chemistries, nickel manganese cobalt (NCM), lithium iron phosphate (LFP), and fluorinated carbon (CFx)�were demonstrated at 100 °C.The flame-retardant ability of this electrolyte system was also demonstrated in a commercial-level pouch cell.The results showed improved battery performance at extreme temperatures (100 °C) with enhanced safety. ■ EXPERIMENTAL SECTION Materials and the Preparation of the Electrolytes.PVDF, NCM523, and LFP powders were purchased from MTI Co., Ltd. in this work.LiDFOB (99%) and LiTFSI (99.99%) lithium salts were purchased from Sigma-Aldrich.FEC (98%) and GTA (99%) were purchased from Alfa Aesar, and EC was purchased from Gelon Co., Ltd.All of the reagents were used as received.The electrolyte solvents were first mixed at the corresponding volume ratio, and the lithium salt was then added into the solvent and stirred overnight at 60 °C in the argon-filled glovebox to obtain a homogeneous electrolyte mixture. Fabrication of Cathode Electrodes.The cathode electrodes were prepared through a doctor blade method as follows.The PVDF powder was first dissolved in NMP to be a 4 wt % solution.Cathode active materials (including LFP, NCM523, and CFx), Super P carbon black, and the PVDF solution were then mixed using the mortar and pestle with a weight ratio of 8:1:1 for 10 min to homogenize.The asprepared slurry was then doctor-bladed onto aluminum foil and dried overnight at 100 °C in a vacuum oven.The dry electrode has a cathode active material loading of ∼5 mg/cm 2 and was punched out to a 12 mm diameter disk for battery assembly. Battery Assembly and Electrochemical Tests.CR2032 coin cells were used and assembled in an argon-filled glovebox for both half cells and symmetric cells.All cells used 100 μL of the electrolyte and lithium foils with a thickness of 600 μm and a Celgard 3401 surfactant-coated separator.The NCM523\|\|graphite pouch cells were also assembled in an argon-filled glovebox.Pouch cells were first dried at 100 °C overnight and then transferred into the argon-filled glovebox and injected 1000 μL of the electrolyte for each pouch cell.Cyclic voltammetry (CV) and EIS tests were conducted using a threeelectrode beaker cell system with two platinum electrodes as the working and counter electrodes, and one lithium metal electrode was used as the reference electrode.CV scans for each electrolyte were conducted with a scan rate of 0.01 mV/s at 25 °C and a voltage window between 4.5 and 0 V vs Li/Li + .The frequency range of the EIS tests was from 1 MHz to 100 mHz with a sinus amplitude of 100 mV.Both EIS and CV were conducted on a Biologic.Long-term galvanostatic cycling tests for both coin cells and pouch cells were conducted using test instruments from LAND electronic Co., LTD in a high-temperature oven at their corresponding temperatures.The voltage windows of the galvanostatic cycling tests are 3.0−4.3V vs Li/ Li + for NCM523 cells and 2.5−4.0V vs Li/Li + for LFP cells. Flammability Tests.The flammability tests of the full pouch cells were conducted at a room temperature of 23 °C.Pouch cells using G2 and COM electrolyte for the flammability are both cycled for three cycles and end with a fully charged state.The nail penetration test was conducted by using a needle with a diameter of 0.5 mm.The infrared images and video were captured by using a FLIR One PRO thermal camera. Material Characterization.Differential scanning calorimetry (DSC) was measured with a TA Instrument Q-20 under nitrogen with a heating rate of 2.5 °C/min, and the mass of the sample was 10 mg for each test.TGA was measured with a TA Instrument Q-50 with a heating rate of 5 °C/min under nitrogen.SEMs were conducted using a Tescan Mira3 instrument at an accelerating voltage of 15 kV with a spot size of 8 nm and beam intensity of 13.The magnification of the photos ranged from 15,000 to 25,000×. 1 H NMR spectroscopy measurements were performed on a Bruker Advance 300 MHz spectrometer using CDCl 3 as the solvent.Transmission electron microscopy (TEM) image was collected using Tecnai F20. ■ RESULTS AND DISCUSSION Physical Properties of the GTA-Based Electrolytes.To assess the stability of the electrolyte at high temperatures, DSC was performed on pure G1, G2, and COM electrolytes.Figure 1a demonstrates that the G1 and G2 electrolytes did not exhibit any exothermic or endothermic reaction peak before 130 °C.However, the COM electrolyte showed a strong endothermic peak at 80 °C, which can be attributed to the evaporation of the electrolyte.Thermogravimetric analysis (TGA) was also performed to investigate the evaporation behavior of these electrolyte systems.As demonstrated in Supporting Information Figure S1, both G1 and G2 show low vapor pressure with negligible evaporation below 100 °C.In contrast, the commercial electrolyte begins to evaporate significantly even at room temperature, indicating a high vapor pressure.These results validate that GTA provides the electrolyte with a high boiling point and good stability. Figure 1b shows the FTIR spectra of the G2 electrolyte, pure GTA, and pure FEC.The peaks at 1155 and 1742 cm −1 are attributed to the FEC, corresponding to C−F and C=O stretching, respectively.The peaks at 1223 and 1832 cm −1 are attributed to the FEC, corresponding to C−O and C=O stretching, respectively.After mixing and adding the LiDFOB lithium salt, the FTIR spectra peaks do not have a significant change; it appears to be the combination of both components.This indicates that the electrolyte components do not undergo spontaneous reactions, remaining stable.The NMR was also performed for the G2 electrolyte and demonstrated in Supporting Information Figure S2 which showed a similar result that the G2 electrolyte appears to be the combination of the GTA with FEC and does not have additional impurity peak or shift of the peak. The voltage stability of the GTA-based electrolyte was verified by the three-electrode CV scan (Supporting Information Figure S3), the result for the G2 electrolyte is shown in Figure 1c.Three oxidation peaks can be observed on the CV plot which corresponds to the stripping peak of the Li/ Li + redox (0.46 V), the stripping peak of the lithium on the platinum (100) plane (0.72 V), and the stripping peak of the lithium on the platinum (110) plane (1.27 V). 33,34 The stripping peak of the lithium on the platinum (111) plane was not observed in the G1 electrolyte but was observed in the G1 electrolyte at around 1.02 V vs Li/Li + .This CV result is also shown to have a high voltage stability window of up to 4.5 V vs Li/Li + .The CV scan with the G2 electrolyte (Supporting Information Figure S4) also showed a high voltage stability until 4.2 V without additional redox couples, demonstrating the electrochemical stability of the GTA-based electrolyte when paired with the lithium metal anode.The ionic conductivity of the electrolyte is a crucial factor influencing battery performance.Ionic conductivities of G1, G2, and COM electrolytes were measured by using electrochemical impedance spectroscopy (EIS) with Supporting Information eq S1 and calibrated using a conductivity standard solution.The calculated conductivities are listed in Supporting Information Table S1.The Arrhenius plot in Figure 1d indicates that both G1 and G2 electrolytes exhibit relatively lower ionic conductivities compared to the commercial electrolyte.This difference can be attributed to the higher viscosity of GTAbased electrolytes in comparison to the commercial electrolyte, even though the latter tends to evaporate significantly above 40 °C.The G1 electrolyte also demonstrates higher conductivity than G2, presumably due to its higher lithium salt concentration. LFP\|\|Li Half-Cell Cycling at Elevated Temperatures.To demonstrate the capability that the GTA-based electrolyte can be cycled at extreme temperatures, we assembled LFP\|\|Li half-cells with different electrolytes and cycled them at different temperatures.The charge−discharge plot of the half-cell that used G1 electrolyte cycled at 60 °C with a 0.1 C rate is demonstrated in Figure 2a, which showed good stability and low overpotential.A similar performance can be observed for the cell using the G2 electrolyte, which is demonstrated in Supporting Information Figure S5.However, from the capacity retention comparison plot in Figure 2b, we can observe a lower Coulombic efficiency (CE) for the cell using the G1 electrolyte; the average CE for the one used G1 electrolyte is 97.1%, while the one used G2 electrolyte possesses an average CE of 99.7%.The reason for this CE difference is mainly attributed to the addition of FEC and LiDFOB, which helps with the formation of stable CEI and SEI layers that stabilize the electrodes at elevated temperatures.Consequently, a higher specific capacity can also be observed for the one using the G2 electrolyte, which is 147.8 mA h/g while the one using the G1 electrolyte is 145.8 mA h/g after 50 cycles at 60 °C.The room temperature performance of the cells using G1 and G2 electrolytes was also tested.The cell using G1 electrolyte with a 0.2 C rate cycled at 25 °C showed a relatively lower discharge capacity of 111.2 mA h/g at the fifth cycle and a faster capacity decay as we can see from Supporting Information Figure S6.On the other hand, the cell using the G2 electrolyte demonstrated in Supporting Information Figure S7 showed a discharge capacity of 132.1 mA h/g at the fifth cycle and a high discharge capacity of 141.2 mA h/g at the 100th cycle. At the extreme temperature of 100 °C, the cell using the G1 electrolyte showed only 10 cycles (Supporting Information Figure S8) at a 0.2 C rate before failing.It can be observed that this cell at 100 °C failed with a long charging plateau with a sudden voltage drop, which indicates the dissolution of the CEI layer and is followed by lithium depletion at the cathode side.On the other hand, the cell using the G2 electrolyte showed stable cycling at 100 °C with a capacity retention rate of 95.6%, average CE of 98.6%, and a high cathode specific capacity of 152.0 mA h/g after 100 cycles at 0.2 C rate, as demonstrated in Figure 2c.Similarly, the one using commercial COM electrolyte was also tested at 100 °C at a 0.2 C rate and failed after eight cycles, exhibiting low CE and a noisy charge− discharge curve (Supporting Information Figure S9), indicating a drastic reaction between the cathode with the electrolyte at 100 °C.Although the cell using the G2 electrolyte cycled at 100 °C showed a stable high capacity, it can be observed from the charge−discharge curve that the overpotential of this cell has a significant increase over cycling, indicating that there are still reactions between the electrolyte with the electrode.The EIS of the cell using the G2 electrolyte after cycling showed a large charge-transfer impedance, as demonstrated in Supporting Information Figure S10, indicating that the overpotential increase is mainly due to the Ohmic overpotential increase due to the SEI/CEI layer build-up during cycling at 100 °C.The SEM of the lithium anode after cycling at 100 °C (Supporting Information Figure S11) also supports this conclusion.To quantify this overpotential evaluation of the cells at extreme temperatures, CE and the average overpotential (V ao ) are calculated using Supporting Information eqs S2 and S3. The calculated results are demonstrated in Figure 2d, and it is obvious that the CE of the GTA-based electrolyte at 100 °C is much higher than that of the one used COM electrolyte, and V ao of the GTA-based electrolyte at 100 °C is much lower than that of the COM electrolyte, which proves a superior performance of the cell using the GTA-based electrolyte at higher temperatures.For the COM electrolyte, there will be an irreversible electrolyte decomposition reaction that contributes to the charging capacity, as seen in the charge−discharge curve (Supporting Information Figure 10), and leads to a very low CE with high V ao at extreme temperatures.It can also be noticed that the CE of the cell using the G2 electrolyte is higher than the G1 electrolyte.The G1 electrolyte showed a high CE in the first two cycles; however, without filming agents, the stability of the SEI/CEI layer of the electrodes is not good enough to enable stable cycling.The intrinsic instability of the electrodes at high temperatures will dominate when using the G1 electrolyte, the direct reaction between the electrode with the electrolyte consumes the active lithium available and leads to a complete failure of the cell at such a high temperature.The G2 electrolyte which included filming agents along with the high-temperature stable GTA allows the formation of a dense, inorganic-rich CEI/SEI layer and can protect the active material particles and enable a much longer cycle life. The LFP\|\|Li half-cells with the G2 electrolyte were also tested at an intermediate temperature of 75 °C at a 0.2 C rate, and the charge−discharge plot is demonstrated in Figure 2e.It showed a similar trend with the cell cycled at 100 °C with an increasing overpotential.The comparison of the cell using the G2 electrolyte cycled at different temperatures is demonstrated in Figure 2f, and a higher capacity for the cells cycled at higher temperatures, which could be mainly attributed to the lower impedance at higher temperatures.From the EIS comparison of the cells before cycling at different temperatures (Supporting Information Figure S12), a lower charge-transfer resistance can be observed for the cells at higher temperatures, which is related to the higher ionic conductivity at higher temperatures as demonstrated in Figure 1d.The V ao calculated in Supporting Information Figure S13 also showed that the cell cycled at lower temperatures will have a higher overpotential.Moreover, an activation process can be observed on the cells that cycled at lower temperatures, which could be due to sluggish ion transportation at lower temperatures.These results further prove that the G2 electrolyte can enable stable cycling of LFP cells at elevated temperatures. Rate Performances at 100 °C in the LFP\|\|Li Half-Cells.The rate performance of the LFP\|\|Li half-cell with the G2 electrolyte was also evaluated at 100 °C to demonstrate the kinetics and stability of the cell.At a faster C-rate, the cathode material undergoes a more drastic structural change, resulting in larger overpotential and less distinct plateaus as shown in the charge and discharge curve in Figure 3c (2 C). In Figure 3a−c, the cells cycled at a faster C-rate demonstrated a larger opening between the charge and discharge curves.The overpotential, as calculated in Supporting Information Figure S14, indicated that the initial overpotential of the cells cycled at a lower C-rate was lower than that of the ones cycled at a higher C-rate, which could be attributed to smaller concentration polarization.However, batteries cycled at different C rates exhibited a similar increase in overpotential, primarily due to the buildup of SEI/CEI, leading to the increased charge-transfer resistance.The capacity retention comparison plot in Figure 3d shows good stability with a high CE for the cells cycled at a high C-rate; the capacity of the cells after 100 cycles at 100 °C with 0.5, 1, and 2 C rates are 138.6,142.3, and 116.8 mA h/g, respectively.The good capacity retention at high rates observed can be mainly attributed to the improved kinetics at elevated temperatures, where the charge-transfer resistance is much lower than that at lower temperatures. NCM523\|\|Li Half-Cell Performance.Besides LFP, halfcells using NCM523 as the cathode material were also tested using the G2 electrolyte to verify the of the G2 electrolyte with the high-voltage cathode.Figure 4a shows the charge−discharge curve of the NCM523\|\|Li half-cell with the G2 electrolyte at 75 °C with a 1 C C-rate, which showed good stability for 100 cycles and a capacity of 109.1 mA h/g at the 100th cycle.The cell cycled at 100 °C also showed a similar performance with good stability for 100 cycles, as demonstrated in Figure 4b.From the capacity retention plot in Figure 4c, the NCM523 cell showed a similar decay rate of 66.7 and 65.6% and average CE of 98.9 and 99.2% at 75 and 100 °C respectively, indicating good reversibility for the G2 electrolyte even for the NCM cathode at elevated temperatures.The capacity decay of these NCM523 could mainly result from the instability of the cathode material at higher temperatures, and the surface structure will decompose at elevated temperatures at a higher voltage. 35The SEM of the NCM523 cathode after cycling (Supporting Information Figure S15) showed that the cathode material was cracked after 100 cycles at 100 °C at 1 C, which further proves that the cathode failure is the main reason for the capacity decay for NCM chemistry at this temperature.The overpotential of the cell cycled at high temperatures is also compared in Figure 4d, and it is clear that the cell cycled at 100 °C has a much higher overpotential than the cell cycled at 75 °C (0.52 V at 100 °C compared to 0.35 V at 75 °C at the 100th cycle).The higher overpotential at higher temperatures indicates that the SEI/CEI build-up could be the main reason for the capacity fade in the NCM cell, which is different from the LFP cathode we discussed before.Since the NCM particle is less stable than the LFP cathode, especially at high temperatures, the surface of the NCM particle will start to decompose and pulverize, creating a more resistive surface layer with higher overpotential, leading to the complete failure of the battery.TEM imaging in Supporting Information Figure S16 illustrates the NCM cathode material after cycling at 100 °C for 100 cycles.It reveals a CEI layer of 53 nm, significantly thicker than the conventional CEI layer, typically only several nanometers thick.This observation indicates a severe surface reaction on the cathode particles at high temperatures. Furthermore, the EIS results provided additional insights into the impedance of the NCM after cycling.Supporting Information Figure S17 shows that the overall impedance after cycling was approximately three times higher than the impedance before cycling.Importantly, both the impedance associated with SEI/CEI at higher frequencies and the chargetransfer resistance at lower frequencies increased after cycling.This suggests that in NCM batteries, the decay of the cell performance is not solely attributed to the accumulation of the SEI/CEI layer.It is also influenced by the degradation of the active material particles. The cycling performance of the commercial electrolyte was also compared with that of the G2 electrolyte at 100 °C.Supporting Information Figure S18 illustrates the comparison between these two systems, demonstrating that the cell using the COM electrolyte exhibits a much faster capacity, and a lower CE can be observed, especially during the first several cycles.This suggests the occurrence of a severe side reaction between the NCM cathode with the commercial electrolyte.Furthermore, Supporting Information Figure S19 demonstrates good stability of the NCM\|\|Li half-cell with G2 electrolyte at lower temperatures at both 25 and 60 °C.However, at 25 °C, the capacity is only about 70% of its theoretical capacity due to the low ionic conductivity and high viscosity.Nevertheless, at 60 °C, an intermediate the cell exhibits both good capacity and stability with a high CE. Flammability Tests in the NCM523\|\|Graphite Pouch Cell.To confirm the stability of the electrolyte during cycling under extreme temperatures, we conducted DSC tests on the G2 and COM electrolytes with the NCM523 cathode (Supporting Information Figure S20).In the COM electrolyte with NCM523, a broad endothermic peak was observed, indicating a continuous reaction process between the COM electrolyte and NCM523 particles.However, no apparent peak was identified in the G2 electrolyte sample, suggesting the good stability of the G2 electrolyte with the NCM523 particles.Moreover, the electrolyte after cycling at 100 °C was collected and tested by NMR and FTIR analysis.Supporting Information Figure S21 demonstrates the NMR spectra of the pristine G2 electrolyte and the G2 electrolyte after cycling in LFP\|\|Li and NCM523\|\|Li half-cells.The spectra indicate that the electrolyte after cycling at 100 °C does not appear to have any additional impurity peak, suggesting that the electrolyte remained stable during the cycling process.However, the NMR peaks corresponding to FEC shifted to a higher chemical shift and with a lower intensity, indicating that FEC was consumed during the cycling.The FTIR spectrum after cycling demonstrated in Supporting Information Figure S22 also supports this observation as no extra impurity peak can be identified in the electrolyte after cycling.However, the peak intensity of the FEC decreased, indicating that FEC may be the component mainly consumed during the cycling of the cell using the G2 electrolyte at high temperatures.In addition, the thermal stability of the G2 electrolyte was also tested by direct exposure to a flame torch and showed a nonflammable property (Supporting Information Figure S23). More importantly, the safety properties of the G2 electrolyte were also tested by a nail penetration test with the NCM523\|\| graphite full pouch cell.The pouch cell has a capacity of 250 mA h, which was cycled three times, and ended at a fully charged state at 4.3 V before the nail penetration test, as shown in Figure 5a.The infrared image of the cell after nail penetration using the G2 electrolyte is shown in Figure 5b, which only has a very small temperature increment of 1.5°( room temperature at 23 °C).Even after we cut the pouch cell in half, the temperature increment is very minimal, as shown in Figure 5c.Supporting Information Video 1 also shows that the heat dissipated very fast after cutting, and the temperature went down after only about 30 s. On the other hand, the cell with the COM electrolyte after nail penetration went up to a much higher temperature (91 °C) within 10 min, as shown in Figure 5d and Supporting Information Video 2. The comparison of the temperature clearly showed that this G2 electrolyte can greatly suppress heat generation when the cell is damaged.With a significantly higher heat capacity (389 J/mol•K) than the conventional electrolyte solvent such as DEC (210 J/mol•K), GTA affording the electrolyte system improved heat tolerance when the cell sustains damage.The high boiling point of the GTA electrolyte also suppresses electrolyte evaporation, thereby avoiding the creation of a local dry zone in the battery.This greatly assists with heat dissipation, precluding propagation of the thermal runaway process. 36The temperature increment observed in the cell using the G2 electrolyte was mainly due to the joule heat generated while shorted and dissipated immediately.On the other hand, the one that used COM electrolyte not only had the joule heat but the heat and current also triggered the decomposition of the electrolyte and the SEI layer which contributed mainly to the heat generation during the nail penetration test.The optical image pouch cell with the G2 electrolyte after cutting tests is shown in Figure 5e, which showed no spark or spontaneous ignition during the cutting test.The direct firing test for the cut cell shown in Figure 5f showed no combustion, proving stability and concurrent safety for the G2 electrolyte even with an external heat source. Battery Storage Properties in the Li-CFx Battery System.The Li-CFx battery is a primary battery system with a high energy density.However, several problems limit the widespread use of the Li-CFx battery, including the heat generation during the reaction and the low electronic conductivity of the CFx particle, which limits the high-rate performance.The crystallization of LiF crystals during the discharging process has a high enthalpy (26.91 kJ mol), resulting in a severe heat generation problem.This exothermic process requires the electrolyte to be capable of operating at high temperatures and preferably nonflammable.Additionally, the irreversible nature of Li-CFx chemistry makes it easy to detect any capacity decay during the storage process.A longterm high-temperature storage test was performed on the LFP\|\| Li battery system, as demonstrated in Supporting Information Figure S23.The capacity of the cell after long-term storage was very close to that of the cell only rested for 12 h.Conversely, using Li-CFx chemistry makes it easier to detect self-discharge during high-temperature storage.This makes it ideal for studying the long-term storage properties of the GTA-based, high-temperature, flame retardant electrolyte. The of the Li-CFx battery chemistry with different GTA-based electrolyte systems is studied first, and the results are shown in Figure 6a.Both cells using the FEC-containing electrolyte exhibited a lower discharge voltage and a higher discharge capacity with two plateaus.However, the cell with the EC containing an electrolyte does not have that behavior.The unusual result that we observed in the FEC-containing systems could come from the reaction between the CFx electrode and the FEC electrolyte.It is believed that the discharge process of the CFx cathode includes a step, in which the electrolyte intercalates into the layered structure of the CFx.As a result, the fluorine in FEC may be more reactive than the fluorine in CFx, which lowers the discharge voltage and contributes to the capacity.Thus, we used electrolyte G1, which does not include FEC and demonstrated the best capacity as the electrolyte for our future studies of the Li-CFx battery system. Similar to LFP and NCM, the Li-CFx battery with the GTAbased electrolyte also showed a relatively lower capacity of 602 mA h/g at room temperature.However, the capacity of the Li-CFx battery above becomes higher as the temperature increases and becomes closer to its theoretical capacity of 865 mA h/g.Nevertheless, the discharge profile in Figure 6b demonstrates that discharge voltage is still noticeably lower at lower temperatures due to lower ionic conductivity.The discharge curve of the battery using the G1 electrolyte stored at 100 °C for 7 days is presented in Figure 6c, and the battery showed a 9% decay after 7 days (692 mA h/g after 7 days resting) which is lower than the one using the COM electrolyte at 100 °C (13% decay after 7 days at 100 °C), as shown in Supporting Information Figure S25.These results demonstrate that GTA-based flame-retardant electrolyte not only enables a high-temperature stable Li-CFx battery but also allows the battery to be stored at a very high temperature without significant capacity decay over time, which could be promising for specialized applications such as space missions. ■ CONCLUSIONS In this work, we introduced GTA as a high-temperature electrolyte solvent that is stable and nonflammable under a range of conditions.We evaluated the performance of GTAbased electrolytes in three different battery chemistries, LFP, NCM523, and CFx under extreme temperatures and demonstrated their stability and functionality at high temperatures.Furthermore, the high-temperature decay mechanism of LFP and NCM523 cathode materials was also investigated.For LFP batteries, we identified the buildup of the SEI/CEI layer as the primary cause of the capacity decay and increased overpotential.On the other hand, for NCM523 batteries, the spontaneous structural instability at the surface of the cathode material was found to also contribute to capacity decay.Additionally, Li-CFx electrodes were used to assess the hightemperature storage stability of the GTA-based electrolyte.A comparison of the GTA-based high-temperature electrolyte system with other previously reported high-temperature electrolyte systems is detailed in Supporting Information Table 2.As can be concluded from the data, the GTA-based system clearly demonstrates exceptional performance at high temperatures compared to that of other electrolyte systems.Overall, GTA as a low-cost and readily available hightemperature nonflammable electrolyte holds promise for adoption in current lithium-ion battery production, could provide the battery with better safety, and creates opportunities for high-temperature applications. Data Availability Statement The data that support the findings of this study are available upon reasonable request. TGA results of G1, G2, and COM electrolyte; H 1 NMR spectrum of the G2 electrolyte and GTA and FEC; image of the three-electrode setup that we used in our experiment; CV scan of the G1 electrolyte; charge− discharge curve of the half-cells using G1, G2, and COM electrolytes with the LFP cathode at different temperatures; EIS measurement and SEM image of the LFP half-cell after cycling; EIS of the LFP half-cells at different temperatures; overpotential evolution of the LFP half-cells at different temperatures; overpotential evolution of the LFP half-cells at different C rates cycled at 100 °C; SEM of the NCM523 cathode after cycling at 100 °C; TEM image of the NCM523 cathode after cycling at 1 C at 100 °C; EIS of the NCM523 half-cells before and after cycling; capacity retention and CE of the NCM523 half-cells using G2 and COM electrolytes at 100 °C; capacity retention and CE of the NCM523 half-cells using G2 at different temperatures; DSC results of the COM and G2 electrolyte with the NCM523 cathode material; 1 H NMR spectrum of the G2 electrolyte before cycling and after cycling; FTIR spectrum of the G2 electrolyte before cycling and after cycling; direct firing test of the G2 electrolyte with the torch; first three charge−discharge curve of the LFP\|\|Li cell using the G2 electrolyte after different resting times at 100 C; discharge curve of the Li-CFx cell after longterm resting using the COM electrolyte; calculated conductivity of the G1, G2, and commercial electrolyte at different temperatures; and comparison of our results with different reported high-temperature electrolyte systems (PDF) Nail penetration test on the pouch cells using G2 electrolyte (MP4) Nail penetration test on the pouch cells using COM electrolyte (MP4) Figure 1 . Figure 1.Physical properties of the GTA-based electrolyte.(a) DSC results of G1, G2, and COM electrolyte.(b) FTIR spectrum of the G2 electrolyte and FEC, GTA.(c) CV scan of the G2 electrolyte in a three-electrode cell.(d) Arrhenius plot of the ionic conductivity of G1, G2, and COM electrolytes. Figure 2 . Figure 2. Cycling performance of the Li\|\|LFP half-cell at different temperatures.(a) Charge−discharge curve of the cell using the G1 electrolyte cycled at a 0.1 C rate at 60 °C.(b) Comparison of the capacity retention and CE of the cells using G1 and G2 electrolytes cycled at a 0.1 C rate at 60 °C.(c) Charge−discharge curve of the cell using the G2 electrolyte cycled at a 0.2 C rate at 100 °C.(d) Comparison of the CE and the overpotential evolution during cycling of the cells using COM, G1, and G2 electrolytes cycled at a 0.2 C rate at 100 °C.(e) Charge−discharge curve of the cell using the G2 electrolyte cycled at a 0.2 C rate at 75 °C.(f) Comparison of the capacity retention and CE of the cells using the G2 electrolyte cycled at a 0.2 C rate at 25, 75, and 100 °C. Figure 3 . Figure 3. Cycling performance of the Li\|\|LFP half-cell at different C rates at 100 °C.(a) Charge−discharge curve of the cell using the G2 electrolyte cycled at a 0.5 C rate.(b) Charge−discharge curve of the cell using the G2 electrolyte cycled at a 1 C rate.(c) Charge−discharge curve of the cell using the G2 electrolyte cycled at a 2 C rate.(d) Comparison of the capacity retention and CE of the cells using the G2 electrolyte at different C rates. Figure 4 . Figure 4. Cycling performance of the Li\|\|NCM523 half-cell at different temperatures.(a) Charge−discharge curve of the cell using the G2 electrolyte cycled at a 1 C rate at 75 °C.(b) Charge−discharge curve of the cell using the G2 electrolyte cycled at a 1 C rate at 100 °C.(c) Capacity retention and CE plot of the cells using the G2 electrolyte cycled at a 1 C rate at different temperatures.(d) Comparison of the overpotential evolution during cycling of the cells using the G2 electrolyte cycled at a 1 C rate at different temperatures. Figure 5 . Figure 5. Flammability tests of the NCM523\|\|graphite full pouch cell.(a) Cycling data of the pouch cell using the G2 electrolyte cycled at 100 °C for the flammability tests.(b) Infrared image of the cell using the G2 electrolyte after nail penetration.(c) Infrared image of the cell using the G2 electrolyte after cutting.(d) Infrared image of the cell using the COM electrolyte after nail penetration.(e) Optical image of the cell using the G2 electrolyte after cutting.(f) Optical image of the cell using the G2 electrolyte after cutting and with direct flame contact. Figure 6 . Figure 6.Li\|\|CFx half-cell performances.(a) Discharge curve of the Li-CFx cell using different electrolyte systems at 100 °C, and the C-rate is 0.05 C. (b) Discharge curve of the Li-CFx cell using the G1 electrolyte at different temperatures.(c) Discharge curve of the Li-CFx cell using the G1 electrolyte after different resting times at 100 °C.	2024-05-08T06:17:05.031Z	2024-05-06T00:00:00.000	{ "year": 2024, "sha1": "a8eedd796591413a8a4df3f7cf522058462a3da5", "oa_license": "CCBY", "oa_url": "https://pubs.acs.org/doi/pdf/10.1021/acsami.4c02323", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "112a90de0a583347ca9f2818c58c860dd7a5f580", "s2fieldsofstudy": [ "Materials Science", "Chemistry", "Engineering" ], "extfieldsofstudy": [ "Medicine" ] }
208104659	pes2o/s2orc	v3-fos-license	Proxy Functions for Approximate Reinforcement Learning : Approximate Reinforcement Learning (RL) is a method to solve sequential decision-making and dynamic control problems in an optimal way. This paper addresses RL for continuous state spaces which derive the control policy by using an approximate value function (V-function). The standard approach to derive a policy through the V-function is analogous to hill climbing: at each state the RL agent chooses the control input that maximizes the right-hand side of the Bellman equation. Although theoretically optimal, the actual control performance of this method is heavily inﬂuenced by the local smoothness of the V-function; a lack of smoothness results in undesired closed-loop behavior with input chattering or limit-cycles. To circumvent these problems, this paper provides a method based on Symbolic Regression to generate a locally smooth proxy to the V-function. The proposed method has been evaluated on two nonlinear control benchmarks: pendulum swing-up and magnetic manipulation. The new method has been compared with the standard policy derivation technique using the approximate V-function and the results show that the proposed approach outperforms the standard one with respect to the cumulative return. INTRODUCTION Reinforcement learning (RL) has the potential to solve challenging decision-making and control problems in engineering and in a variety of other disciplines, such as economics or medicine (Busoniu et al., 2010;Sahba et al., 2006;Deng et al., 2017;Guan et al., 2015).There is a wide variety of RL architectures, which can be broadly classified into critic-only, actor-only, and actor-critic schemes (Konda and Tsitsiklis, 2000).In this paper, we focus on the critic-only architecture for continuous state spaces.The RL agent first learns an approximate value function (Vfunction), based on which it then derives the optimal control policy.We restrict ourselves to the model-based scenario: a nonlinear deterministic state-space system model is used for V-function learning and policy derivation (Polydoros and Nalpantidis, 2017;Kuvayev and Sutton, 1996). To derive a control policy from the V-function, the RL agent chooses the control input that maximizes the righthand side of the Bellman equation.The performance of such a control law depends on the local smoothness of the V-function and therefore on the type of approximator used.A wide spectrum of approximation techniques have been used in RL: local linear regression (Grondman et al., 2012), deep neural networks (Lillicrap et al., 2015), fixed or adaptive basis functions (Buşoniu et al., 2011), regression trees (Ernst et al., 2005).In this paper, we use approximation by means of triangular basis functions (BFs) as a baseline, since the convergence of value iteration can be guaranteed.However, approximation with basis functions may cause chattering of the state trajectory during transients or may render the goal state unreachable, as illustrated in Fig. 1.The bottom plot clearly shows that due to the odd shape of the surface (visualised as the diamond-shaped level curves), the state trajectory is deflected from the desired path to the goal and ends at some distance away from the desired position. A possible approach to alleviating the above problem is to approximate the V-function by using a technique which would not be affected by any interpolation artifacts between the individual basis functions.One of the methods to construct a compact smooth approximation is symbolic regression (SR).Based on genetic programming, SR searches for an analytic expression which fits best the given data.However, we found (Alibekov et al., 2018) that it is difficult to directly apply SR to approximate an existing V-function.While the symbolic approximation can achieve a very low mean-squared error, the resulting policy can be very different and sub-optimal.This is because subtle, Our first approach to overcoming this limitation by means of a smooth proxy function was described in (Alibekov et al., 2016).The method relies on a binary fitness function, which leads to a non-convex optimization problem and reduces chances of finding an accurate proxy function. Consequently, the originally proposed symbolic approximator had to be combined with a numeric approximator. The current paper builds upon this result by resolving these outstanding issues of the originally proposed proxy function.In this paper, we formulate and propose an enhanced symbolic regression approach which uses linear programming in order to find more powerful models and solve the above problems.Moreover, we redesign it in a way that is applicable to any linear-in-parameters approximator. The paper is structured as follows.Section 2 presents the necessary RL preliminaries.The proposed method for fitting a proxy function is described in Section 3. The description of benchmarks used and the testing procedure can be found in Section 4. Section 5 provides results with a detailed discussion of the benefits and drawbacks of the proposed method.Section 6 concludes the paper. PRELIMINARIES Define an n-dimensional state space X ⊂ R n , and mdimensional action space U ⊂ R m .The system to be controlled is described by the state transition function A userdefined reward function assigns a scalar reward r k+1 ∈ R for each transition from x k to x k+1 using action u k : In this paper we restrict ourselves to the reward function depending on the next state only, omitting x k and u k for clear notation. To solve the RL problem, we define a finite set of discrete control input values U = {u 1 , u 2 , . . ., u M } drawn from U.An approximate V-function denoted by V (x) is then computed by solving the Bellman equation: where γ is a user-defined discount factor.The policy is the mapping: h : X → U (3) and the optimal discrete-valued policy corresponding to V (x) is given by: ĥ There are several algorithms to compute an approximate V-function for a continuous state space.For the purposes of this paper, the fuzzy V-iteration algorithm (Busoniu et al., 2010) is used because of its guaranteed convergence.The algorithm can be briefly described as follows.First, the fixed structure of the approximator is defined in terms of triangular basis (membership) functions.The Vfunction approximator is described as: where φ = [φ 1 (x), φ 2 (x), . . ., φ N (x)] T is the vector of fixed triangular basis functions, with each φ i (x) centered in s i such that φ i (s i ) = 1 and φ j (s i ) = 0, ∀j = i.The basis functions are normalized so that N j=1 φ j (x) = 1, ∀x ∈ X .Finally, θ ∈ R N is the corresponding parameter vector.The value iteration is then defined as: for i = 1, 2, . . ., N .This algorithm guaranties convergence under certain conditions (Busoniu et al., 2010) and terminates when the convergence threshold is reached: 7) with θ − the parameter vector calculated in the previous iteration. PROPOSED METHOD The main idea of the proxy function method (Alibekov et al., 2016) is to find through symbolic regression a smooth, analytically defined function P , which ∀x ∈ X satisfies the following equation: In order to generalize this method, assume that the proxy function has the form: P (x) = β 1 p 1 (x) + β 2 p 2 (x) + . . .+ β q p q (x) (9) where p 1 , . . ., p q are continuous analytic functions generated by means of evolutionary programming, each of them being defined over the whole state space and β 1 , . . ., β q are real-valued coefficients. Assume that the V (•) approximator is given.As described earlier, the policy derivation process can be regarded as hill climbing.At each time step, the agent selects the control input which leads to the highest value of the right-hand side of the Bellman equation: The selected action u * is then applied to the system, which leads to the new state: x * = f (x, u * ) (11) To find a P , or its close approximation, we generate a set of N state samples X = {x 1 , x 2 , . . ., x N } ∈ X .By using the already defined set of discrete control inputs U = {u 1 , u 2 , . . .u M }, for each state x i ∈ X we construct the following set of next states: ) and partition it into optimal and suboptimal next states: The optimal next state maximizes the right-hand side of the Bellman equation: and the suboptimal next states are all the remaining ones: For simplicity, we assume that the optimal state for each sample in X is unique.However, the proposed method can be trivially extended to handle multiple optimal next states. To define the fitness function for symbolic regression, (8) is reformulated as follows: This means that for each state x i , the proxy function value for the optimal next state must be larger than the value for the suboptimal next states.Index k runs over all L elements in X s i .Substituting from (9), the above inequality becomes: To simplify the notation, define an auxiliary variable d ik j as: 17) for all the data in matrix form, define and solve the problem by linear programming: where represents a small negative constant.1Note that (17) defines a feasibility problem, rather than a minimization problem.To guide the evolutionary process toward a feasible solution, we introduce an infeasibility measure of the candidate solution.Define a vector of non-positive variables s = [s 1 , . . ., s [N ×L] ] T .The fitness function for SR can now be defined as: This formulation adds an extra variable to every inequality, which represents the measure of infeasibility of the resulting model, and which linear programming then minimizes.The β weights of the analytic expressions are defined as free variables with no restriction. In this work, we use Single Node Genetic Programming (SNGP) (see Appendix for further details) to generate the non-linear analytic expressions p 1 (•), . . ., p q (•), which are then evaluated using ( 21).The whole process repeats until a stopping criterion is satisfied, such as a prescribed number of iterations or an improvement threshold. Benchmarks The proposed method has been tested on two different benchmarks: the well-known pendulum swing-up and a nonlinear control problem called magnetic manipulation.Both of them are explicitly discussed in (Alibekov et al., 2018), including all necessary mathematical details.Here, only a high-level description of these problems is presented. The first task is the classic under-actuated pendulum swing-up problem (abbreviated as pendulum), schematically depicted in Fig. 2a.The inverted pendulum consists of an actuated link that rotates in a vertical plane, and a weight of mass m attached to it.The motor torque is not sufficient to push the pendulum up in a single rotation.Instead, from some states, the pendulum needs to gather energy by swinging back and forth, in order to be pushed up and stabilized.The control goal is to stabilize the pendulum in the upright position x des = [0, 0] (rad, rad/s), which is formally described by the following reward function: .1] T a weighting vector to adjust the relative importance of the angle and angular velocity and abs(•) function working element-wise. Magnetic manipulation (abbreviated as magman) is a benchmark of contactless manipulation with applications at micro scale.Our magnetic manipulation setup (see Fig. 2b) consists of four electromagnets in a line, but in this work only two of them have been used.The goal is to accurately position a steel ball on a 1-D track by dynamically shape the magnetic field above the magnets using current.This goal is formally described by the following reward function: with Q = [10, 5] T where the desired position x des is set to [0.01, 0] (m, m/s), Q is again a weighting vector and function abs(•) again works element-wise. Testing procedure For each benchmark, 30 different proxy functions have been constructed in 30 independent SNGP runs.Each function has been tested on each benchmark with N = 100 randomly chosen initial states via simulations.It should be noted that all the functions are presented "as is", which means that there is no selection procedure w.r.t. to some criterion. Each result is compared with the baseline solution (4) (using the same initial states), which is computed beforehand by means of the fuzzy V-iteration algorithm.To evaluate each of proxy functions, the following criteria are defined: • Improvement percentage where p method = Tsim/Ts k=1 ρ(f (x k , u k )), with T sim stands for the total simulation time, T s is the sampling period, and method represents either baseline or proxy solution.The reward functions are defined to have maximum value zero in the goal state and to be negative otherwise.Therefore I equals 0% for the baseline and it is bigger than 0% if the proxy function outperforms the baseline approach. • Mean distance between the last state (at the end of simulation) x end and the desired goal state x des where • is the Mahalanobis norm. The sampling parameters for both benchmarks, as well as SNGP parameters, are listed in Table 1. RESULTS AND DISCUSSION The simulation results for both tasks are listed in Table 2. The proposed method shows the potential to significantly outperform the baseline solution.According to the experiments, proxy functions demonstrate significant improvement in a range of 5%-82% w.r.t. the baseline.In the vast majority of cases, it is caused by alleviating of numerical artifacts.An example of it is depicted in Fig. 3a.The example demonstrates the comparison of derived policies for the magman task.The left column corresponds to the baseline value function, computed by fuzzy V-iteration algorithm, while the right column stands for one of the proxy functions.It can be seen that proxy function significantly alleviates steady-state error caused by artifacts.Another interesting note is that proxy function remarkably violates Lyapunov stability condition, as shown by the bottom row of Fig. 3a.The reason is that proxy function considers solely local properties of its surface, neglecting global geometry.However, for the pendulum task, the proposed method demonstrates a modest improvement percentage ratio.The reason for it is a small influence of the numerical artifacts on the policy.The comparison between derived policies using policy and baseline methods, respectively, is depicted in Fig. 3b.It can be seen, that, e.g., the steady-state error for both methods is relatively small (in comparison with magman task), which means that there is a small room for improvement for the proxy method.The proposed method has several limitations.First of all, with the proposed design it is not possible to penalize input chattering.Input chattering can usually be reduced by penalizing the control input in the reward function, which is, however, not possible here due to the choice of the proxy function structure as P (f (x, u)).One way to overcome this limitation is to reformulate the proxy function as P (x, u) and then use it in policy derivation in the same way as a Q-function.Another possible way is to combine MSE-like fitness and proxy function fitness in order to represent a trade-off between global V-function geometry and proxy local properties.This may be a part of our future work. CONCLUSION The proposed method offers an alternative way for the policy derivation.Instead of using V-function directly, the proposed approach build a smooth proxy function on top of it, from which the better policy can be derived.The proposed method may be combined with any kind of value function approximation.Moreover, due to the analytic nature of the proxy function, it can be combined with policy derivation methods (Alibekov et al., 2018) to further policy improvement. ACKNOWLEDGEMENTS This research was supported by the Grant Agency of the Czech Republic (GA ČR) with the grant no.15-22731S titled "Symbolic Regression for Reinforcement Learning in Continuous Spaces" and by the European Regional Development Fund under the project Robotics for Industry 4.0 (reg.no.CZ.02.1.01/0.0/0.0/15003/0000470). APPENDIX -SINGLE NODE GENETIC PROGRAMMING This paper introduces an enhanced variant of Single Node Genetic Programming (SNGP) (Kubalk et al., 2017) to implement the symbolic regression.SNGP is a graph-based GP method that evolves a population of individuals, each consisting of a single program node.Our implementation differs from the above paper in the following aspects: The following operators and functions are used to build analytic expressions: and N is the arity of input. Recalling that the result symbolic model P (x) is composed from the linear combination of possibly non-linear analytic expressions p 1 , . . ., p q , as: P (x) = β 0 + β 1 p 1 (x) + β 2 p 2 (x) + . . .+ β q p q (x) the following restrictions are applied in order to control overfitting: • For every point in the dataset the result of an analytic expression lies within [−10 8 , 10 8 ] interval. Fig. 2 . Fig.2.In the pendulum swing-up benchmark, pendulum needs to gather energy by swinging back and forth, in order to be pushed up and stabilized by controlling the torque applied by a motor.The goal of magnetic manipulation benchmark is to accurately position a steel ball on a 1-D track by dynamically shape the magnetic field above the magnets using current. Fig. 3. Example of the transient response using the original V-function V (x) and one of the proxy functions.The first row represents the position of the ball for the magman task and the angle for the pendulum task; the second stands for the control inputs; the last row shows changing a value of either value function or proxy function. Table 2 . Experimental study statistics	2019-10-31T09:06:05.142Z	2019-01-01T00:00:00.000	{ "year": 2019, "sha1": "704df5ae49fba3dee0a79a7b4ff9f78c198c629f", "oa_license": null, "oa_url": "https://repository.tudelft.nl/islandora/object/uuid:a1fac80d-8740-4622-8761-59db38b4ea18/datastream/OBJ/download", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "99a5ecb6572d4d9bb5c5268ef4c64f5b389a9b76", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
251622485	pes2o/s2orc	v3-fos-license	Existence and stability of interfacial capillary-gravity solitary waves with constant vorticity In this paper, we consider capillary-gravity waves propagating on the interface separating two fluids of finite depth and constant density. The flow in each layer is assumed to be incompressible and of constant vorticity. We prove the existence of small-amplitude solitary wave solutions to this system in the strong surface tension regime via a spatial dynamics approach. We then use a variant of the classical Grillakis--Shatah--Strauss (GSS) method to study the orbital stability/instability of these waves. We find an explicit function of the parameters (Froude number, Bond number, and the depth and density ratios) that characterizes the stability properties. In particular, conditionally orbitally stable and unstable waves are shown to be possible. Introduction The main object of study in the present paper is internal water waves. The formation of these waves is primarily due to variations in salinity and temperature in the water bulk. Internal waves can carry enormous amount of energy, while maintaining coherence over long distances. They are known to play a crucial role in ocean dynamics as a means of transporting and mixing nutrients inside the water. In recent years, internal waves have gained a great deal of attention among mathematicians. A plethora of works has been devoted to proving existence of various types of internal traveling waves, see for instance [AT86,AT89,SS93,Nil17] or the survey in [HHS + 22]. Largely for reasons of mathematical convenience, many of these results assume the flow in each layer is irrotational; that is, the vorticity is identically zero. The literature pertaining to rotational flows is unsurpisingly far more limited due to the significant increase in mathematical complexity created by the presence of vorticity. Nonetheless, rotational effects play a significant part in many physical situations, such as waves propagating over a background current; see, for example, [Mar22]. The results of the present paper come in two parts. First, we prove the existence of a family of small-amplitude internal solitary waves with constant vorticity. This is done under the assumption of strong surface tension regime, in a sense to be explained shortly. Second, as our primary contribution, we investigate the stability properties of these waves as solutions to the dynamical problem. In particular, we exhibit an explicit function of the physical parameters whose sign determines whether a sufficiently small-amplitude wave is (conditionally) orbitally or stable or orbitally unstable. We also consider several specific parameter regimes, and find that both stable and unstable waves exist. These are among a very small number of analytical results concerning the nonlinear stability or instability of internal waves. In particular, Chen and Walsh [CW22] proved the orbital stability of a class of internal waves in the irrotational setting. We will adopt their basic strategy, but incorporating constant vorticity introduces numerous complications. Mathematically, the problem is formulated as follows. Let (x, y) ∈ R 2 be a point in the standard Cartesian coordinates system, with x being the direction of wave propagation and gravity acting in the negative y direction. For time t ≥ 0, we assume that the fluid is confined to a channel and organized into two superposed layers: Ω(t) := Ω + (t) ∪ Ω − (t), bounded above and below by infinitely-long rigid walls, {y = d + } and {y = −d − }. Here and throughout the paper, we will use subscripts of ± to indicate the restriction of a quantity to Ω ± . Both layers share a common boundary S = S (t) that is free. We call this the internal interface and assume it is has can be parameterized as the graph of an unknown function y = η(t, x). Our focus will be on solitary waves, meaning η is spatially localized in that η → 0 as \|x\|→ ∞. We take the density to be constant in each layer with 0 < ρ + ≤ ρ − . More precisely, the upper and lower regions of the fluid can be written as Ω + (t) = {(x, y) ∈ R 2 : η(t, x) < y < d + } (1.1) and Ω − (t) = {(x, y) ∈ R 2 : −d − < y < η(t, x)}. (1.2) We will assume that the flow in each layer is incompressible and governed by the incompressible Euler equations. Due to incompressibility, the velocity field (u ± , v ± ) in Ω ± can be expressed in terms of a stream function ψ ± = ψ ± (t, x, y) by Suppose that the vorticity in Ω ± is the constant ω ± := v ±x − u ±y ∈ R. Note that in twodimensions the vorticity is transported, so this assumption is valid even for the dynamical problem. Taking the curl of the velocity field, we then find that the stream function satisfies the Poisson equations ∆ψ ± = −ω ± in Ω ± (t). As is common with constant vorticity waves, we wish to subtract out the background shear from ψ to obtain a harmonic function. That is, defineψ ± := ψ ± + ω ± y 2 2 , which will satisfy ∆ψ ± = 0 in Ω ± (t). Let φ ± be a harmonic conjugate: (φ x ) ± = (ψ y ) ± + ω ± y, and (φ y ) ± = − (ψ x ) ± . Then, the rotational incompressible Euler equations can be recast as follows. In the interior we have ∆φ ± = 0 in Ω ± (t). (1.3a) On the internal interface and both rigid walls, the kinematic conditions read η t = (φ ± ) y − ((φ ± ) x − ω ± η) η x on y = η(t, x), (φ ± ) y = 0 on y = ±d ± . (1.3b) Physically, (1.3b) states that the velocity field is tangential along the boundaries. Finally, via the dynamic condition and Young-Laplace law, we can infer that the pressure jump across the internal interface is proportional to the signed curvature. Using Bernoulli's principle in each layer, it can therefore be stated as (1.3c) The notation · := (·) + − (·) − denotes the difference in trace between two quantities on the internal interface in the upper and lower layer, g > 0 is the gravitational constant, and σ > 0 is the coefficient of surface tension. Observe that the functions φ ± are defined on a moving spatial domain, which complicates the task of finding an appropriate functional analytic setting for the problem. We therefore prefer to work with the unknown ξ ± (t, x) = φ ± (t, x, η(t, x)) (1.4) which corresponds to trace on the surface. Using this new variable allows us to push the entire problem to the free boundary, rendering it nonlocal but more tractable for analysis. 1.1. Statement of results. Now, we are ready to state our main results. We record them in Theorem 1.1 and Theorem 1.2. We begin by introducing some important terminology and physical parameters that describe the system. A steady or traveling wave is a solution to the Euler equations (1.3) that translate in the x-direction at a fixed wave speed c ∈ R without altering its shape. Thus, in a moving frame of reference, it appears stationary. Concretely, this means the unknowns can be written as for some steady profiles η c and ξ c ± . Recall that we will focus on solitary waves, for which η c is localized. There is an extensive body of work devoted to establishing the existence of traveling internal waves in various parameter regimes. Most well studied is the pure gravity case (σ = 0, g > 0), where both solitary waves [AT86,BBT83,Mie95] and periodic waves [AT86,AT89] have been constructed. It is, however, important to note that in the absence of surface tension, the water wave problem (1.3) becomes ill-posed dynamically; see for example [Lan13]. Hence, when looking at questions pertaining to stability/instability, one has to assume σ > 0, which we will do throughout this work. The existence of small-amplitude internal waves in the presence of surface tension was obtained previously by many authors, for instance [Kir22,SS93,Nil17]. However, none of those results allows for vorticity. Since understanding the effects of rotation on the stability is our objective, we spend the first part of our analysis developing an existence theory for small-amplitude internal waves with layer-wise constant vorticity. This is accomplished using a spatial dynamics method: we view the x-coordinate as a time-like variable, and then use a center manifold reduction approach. The process is closely inspired by the work of Nilsson [Nil17]. Internal solitary waves can be described using four dimensionless parameters. The first two of them are the Bond number β and the inverse square of the Froude number α defined by From its definition, we see that the Bond number β measures the strength of the surface tension. In view of (1.5), the Froude number 1/ √ α can be thought of as the nondimensionalized wave speed. Upon linearizing (1.3) at the trivial solution and inserting the plane-wave ansatz η = exp(ik(x − ct)), we arrive at the following dispersion relation (1.6) It can be checked easily that k = 0 is a root of (1.6) exactly when β = β 0 := 1 3 (1.7) We will regard β 0 as the critical Bond number: the range β > β 0 corresponds to the strong surface regime, and β < β 0 is the weak surface tension regime. Heuristically, one expects that solitary waves will bifurcate from the trivial solutions at α 0 . We will specifically be concerned with the strong surface tension case β > β 0 . Apart from β and α, there are two other physical parameters that have to be considered when studying interfacial waves: the density ratio ̺ and the asymptotic height ratio d given as follows (1.8) Unlike α and β, these are specific to the two-layer case. Nilsson [Nil17] proved that for irrotational flow (ω ± = 0), when ̺ − 1/d 2 < 0 and O(1) as α ց α 0 , there exist waves of depression (η < 0). On the other hand, if ̺ − 1/d 2 > 0 and O(1) as α ց α 0 , then waves of elevation exist (η > 0). That said, our main result on existence of solitary waves is as follows. be a smooth curve in the physical parameter space such that along Π, the corresponding Bond number is supercritical β > β 0 , and the inverse-square Froude number is α = α 0 + ǫ 2 . Suppose that where we have suppressed the ǫ dependence of the quantities on the left-hand side. Then for any k > 1/2 there exists a smooth curve of internal wave solutions (1.10) For every solution on the curve C, the surface profile exhibits the following asymptotics: We note that, the denominator in (1.11) determines whether the solution is a wave of elevation or depression for ǫ sufficiently small. In contrast to the irrotational case, this will depend not only on the relative sizes of ̺ and d, but also the strength of the vorticity in each layer. It is also important to observe that, while ǫ is the appropriate parameter for proving existence, stability is best studied by fixing the physical parameters ρ ± , d ± , ω ± , σ, and varying c. Because ǫ = √ α − α 0 , we can solve (1.5) in terms of the wave speed and write α = α c and β = β c . The main result of the paper characterizes the conditional stability of these solutions in the orbital sense. More precisely, we say a solitary wave (η c , ξ c+ , ξ c− ) is conditionally orbitally stable provided that, for all R > 0 and r > 0, there exists r 0 > 0 such that if (η, ξ + , ξ − ) is a solution to the internal wave problem on the time interval [0, t 0 ) that obeys the a priori bound (1.12) and whose initial data satisfies (1.14) The inequality (1.14) measures the distance between the translated solutions (η, ξ + , ξ − ) and the family of traveling waves. The norms in (1.12) represents the lowest regularity required for local well-posedness of the Cauchy problem that is currently available. The meaning of the superscript (+) on the regularity will be made clear later in Section 4. Furthermore, as we will see shortly, the regularity in (1.13) and (1.14) matches the regularity of the energy space. Notice that this result is conditional in that we must assume a priori that the solution exists on a give time interval, since global well-posedness for the system is not known. However, as r 0 is independent of the life span t 0 , the bound in (1.14) is substantially stronger result than merely continuity of the data-to-solution map. In particular, if global existence is known, then we obtain orbital stability in the classical sense. The formula for m is given in (6.3). Because it is rather complicated, it is instructive to look at a few special cases. If the Bond number is sufficiently close to critical 0 < β c − β 0 ≪ 1, then the waves given by Theorem 1.2 are always orbitally stable. We can also obtain more definitive statements by assuming the vorticity in one layer is 0. Parameter regime for which the waves are stable are given in Figure 2, and unstable in Figure 3. Observe that both involve the relative size of the density ratio and a non-dimensionalized measure of the vorticity strength in the rotational layer. The criterion also changes depending on whether we are considering a wave of elevation or depression, which is determined by the sign of the denominator in (1.11). Note that when ω + = ω − = 0, we recover the result in [CW22] that all sufficiently small-amplitude waves are orbitally stable. 1.2. Idea of the proof. In Section 2, we prove Theorem 1.1 on the existence of the smallamplitude internal wave solutions. Following the strategy of Nilsson [Nil17], we write the corresponding steady water wave problem as a spatial dynamical Hamiltonian system. This is obtained by identifying a Lagrangian (the flow force), then applying a Legendre transform to arrive at the desired Hamiltonian. In the strong surface tension regime, we find that the linearized operator at the trivial solution has a 0 eigenvalue of multiplicity 2, corresponding to a Hamiltonian 0 2 resonance. Performing a center manifold reduction, we show that solitary waves of elevation or depression exist when is positive or negative, respectively. Note that when ω ± = 0, this analysis coincides with [Nil17, Section 3.3]. Next, we consider the stability or instability of these waves. Similar to approach of the existence theory, we again exploit the Hamiltonian structure of problem (1.3). This time, however, it is the Hamiltonian for the time-dependent problem rather than spatial dynamical. In Section 4, we show that (1.3) can be written as (1.16) where u = u(t, x) is an unknown represented by (η, ξ + , ξ − ), J is a skew-adjoint operator called the Poisson map, and E is an energy functional. It is well known that the translationinvariant nature of the problem gives rise to another conserved quantity known as the momentum P . By construction, it is clear that a traveling (steady) wave solution is a critical point of the augmented Hamiltonian given by E c := E − cP . The main technique used to prove Theorem 1.2 is based on the seminal works of Grillakis, Shatah, and Strauss [GSS90a,GSS90b]. Their approach, known as the GSS method, provides a systematic way to prove nonlinear stability/instability for Hamiltonian systems that are invariant under a continuous symmetry group. Although GSS has been successfully used to treat many model equations for water waves, the full free boundary Euler system exhibits a number of features that have made it resistant to this machinery. For instance, GSS requires the Poisson map J to be an isomorphism, which is not satisfied in the present setup as we will see shortly. Further, they require the Cauchy problem to be globally well-posed in the energy space. Currently, only local well-posedness of (1.3) is known, and this assumes considerably higher regularity. In recent work, Varholm, Wahlén, and Walsh [VWW20] obtained a variant of the GSS method with hypotheses sufficiently relaxed so that it can be applied directly to the water wave problem. Their framework allows for the Poisson map J to only have a dense range. It also permits the mismatch between the local well-posedness space and the energy space that the water wave problem possess. This theory was also the basis for the paper of Chen and Walsh [CW22] on irrotational internal waves. For the benefit of the reader, an abbreviated statement of the abstract result is given in Section 3. To apply this machinery to our problem, the main difficulty is to characterize the spectrum of the linearized augmented Hamiltonian at a traveling wave. Introducing vorticity increases the complexity of the calculations substantially. However, after a series of nontrivial computations, we find that the linearized operator at a sufficiently small-amplitude wave has Morse index 1, as required by the abstract theory. This analysis is carried out in Section 5. Then, by the general theory, stability or instability of the wave is determined by the convexity or concavity of the moment of instability, a scalar-valued function of the wave speed. In Section 6, we then prove the statement regarding conditional stability/instability in Theorem 1.2. We emphasize that these are considerably more involved than the irrotational regime, and in particular both stable and unstable waves exist, which points toward the importance of including vortical effects in the model. Existence theory We start this section by proving the existence of small amplitude solitary water waves. In doing that, we pattern the approach presented in [Nil17]. In comparison to [Nil17], due to the rotational assumption, the equations that we have to deal with are several order more complex. Seeking traveling wave solutions, we impose a change of variables (t, x, y) → (x − ct, y). The main governing equation (1.3) can then be recast in terms of the relative streamfunctions ψ ± and posed in a frame of reference moving with the waves as follows in Ω ± , ψ ± = ∓m ± on y = ±d ± , ψ ± = 0 on y = η(x), for some constants m ± together with the following asymptotic condition The variable Q in (2.1) is the hydraulic head constant. Next, we introduce the following non-dimensionalized variables Under these variables, the problem now reads where we have dropped the ′ for notational convenience. Next, to obtain the harmonic functionψ, we subtract the shear flow from ψ: Clearly,ψ ± is harmonic in both layers, that is Moreover, the boundary conditions on the rigid walls and internal interface, respectively, becomeψ (2.6) Rather than work withψ, we will use its harmonic conjugate φ to reformulate equations (2.4), (2.5), and (2.6). One can view φ as the velocity potential. Using the fact that φ ±x =ψ ±y and φ ±y = −ψ ±x , the equations now become (2.7) Consider the following rescaling of the domain via the mapping (x, y) → (x, z), where (2.8) As a result, we have the following change of variables formulas (2.9) Recycling notations, let us define φ ± (x, z) := φ ± (x, y). Under the derivative formulas (2.9), equation (2.4) now read (2.11) The boundary conditions on the rigid walls (2.5) and the internal interface (2.6) translate to φ ±z = 0 on z = 0, (2.12) and (2.13) where α, β, and ̺ are variables defined earlier in (1.5) and (1.8). The energy can be formulated as Further, the momentum P is given by From many literature, it is known that solitary waves can be detected by looking at the critical points of the functional E − cP . For this reason, we will study the Hamiltonian that arises from taking the Lagrangian of E − cP . Using the expressions for E and P , we From that, we derive the corresponding Lagrangian (2.14) In order to formulate the correct Hamiltonian, we need to know variational derivatives of L with respect to the individual variable φ +x , φ −x , and η x , (2.15) Having the information above, the Hamiltonian can , therefore, be expressed in terms of To formalize this, for s ≥ 0, we define the following product spaces We would like to point out that the symbol H s+1 refers to a Sobolev space of order s + 1, not the Hamiltonian H. Further, we let M = X 0 be a manifold with m ∈ M and let (2.17) One may observe that ( M , Ω) is a symplectic manifold. The corresponding set is a manifold domain of M where the Hamiltonian H is a smooth functional on it (i.e., H ∈ C ∞ ( N , R)). Hence, the tuple ( M , H, Ω) forms a Hamiltonian system. Via the symplectic form (2.17) and standard computations, the associated Hamilton's equations reaḋ (2.18) where the Hamiltonian vector field also satisfies the corresponding boundary conditions In order to set a firmer ground for the latter analysis, we define the product space Additionally, let us also define these spaces Going back to the Hamilton's equations (2.18), we can see that it has an equilibrium point However, in preparation for the Hamiltonian reduction process on a center manifold, we need to shift the equilibrium point obtained before to the origin (0, 0, 0, 0, 0, 0). To achieve that, we impose another change of variables for the unknowns Φ ± ,φ ± , and χ ± (2.22) This gives rise to a new formulation of the Hamiltonian equation. Precisely, one may think of this change of variables as a mapping that sends (η, γ, φ Thus, in the new variables the new symplectic formΩ becomes (2.23) Further, using variables in (2.22), the Hamiltonian now reads Notice that we have added the constants in the definition of the Hamiltonian so that The new Hamiltonian stucture (2.24) gives rise to the new Hamilton's equations which are given bẏ (2.25) along withχ (2.26) and the boundary conditionsφ (2.27) Note that, the two equations in (2.26) can be neglected since they can be recovered from the rest of the equations in (2.25). We now proceed with linearizing (2.25) around the equilibrium point (0, 0, 0, 0, 0, 0). This leads us to the linearized problem stated in terms of the operator L coupled with the linearized boundary conditions (2.29) Consider the eigenvalue problem Lu = λu, together with the boundary conditions (2.29). Upon setting λ = ik, we obtain the dispersion relation (2.30) We would like to mention that this dispersion relation is equivalent and consistent to the dispersion relation obtained in (1.6) Our next objective in the construction of small-amplitude solutions is to apply the center manifold approach due to Mielke [Mie88] to the system (2.28) and (2.29). For convenience, the main approach used for this is outlined in the theorem stated in Appendix A, which is a version used, for instance, in [Nil17, Section 3]. Due to nonlinear boundary conditions (2.29), however, we are not able to crudely implement the theorem right away. As an intermediate step, we do change of variables via the operator G. This linearizes the boundary conditions at the cost of complication of the problem in the bulk. Explicitly, the operator G takes the form (2.32) and One may check easily that ϕ ±z (1) = ϕ ±z (0) = 0. Further, via the definition of ϕ ± in (2.32), one can check that It is also worth noting that the operator G is invertible in some neighborhood of the origin and its inverse is explicitly given by In this new coordinate system, the Hamiltonian exhibits a new expression: . (2.33) Consequently, the Hamilton's equations now becomė Recall that we are interested in the solutions that satisfy the condition (β, α) = (β, α 0 )+ (0, ǫ 2 ) with β > β 0 where these parameters are defined in (1.5). It can be shown that the imaginary part of the spectrum of the linearized operator L consists of zero, which is an eigenvalue of (algebraic) multiplicity 2 when α = α 0 and β = β 0 , as given by (1.7). The associated eigenvector and the generalized eigenvector, namely e 1 and e 2 , of the zero eigenvalue are then computed. Explicitly, they take the form It is straightforward to check that Le 1 = 0 and Le 2 = e 1 withΩ(e 1 , e 2 ) = β − ̺ + d 3 =: β * , They, indeed, form a symplectic basis of the vector space spanned by the eigenvectors e 1 and e 2 . Let us define f i := dG(0)(v i ). Upon applying the center manifold theorem along with Darboux's theorem, we obtain a Hamiltonian system (X µ C , Ψ,H µ ), We would like to note here that all hyphothesis H1 − H4 in the center manifold theorem are satisfied. As a conclusion, we obtain small bounded solutions on the two-dimensional center manifold. Precisely, every solution u 1 can be respresented as (2.36) Upon completing a number of nontrivial computations, the Taylor expansion of the reduced Hamiltonian is derived and explicitly given bỹ (2.37) From there, we obtain the corresponding Hamilton's equations q x = p + O(\|(p, q)\|\|ǫ 2 , p, q\|), (2.38) Consider the following rescaling Under these rescaling, the Hamilton's equations in (2.38) read (2.41) Upon truncating the rescaled Hamilton's equations in (2.40), we obtain which has solutions Q(X) = − sech 2 (X/2) K(̺, d, ω + , ω − , c) , P (X) = sech 2 (X/2) tanh(X/2) K(̺, d, ω + , ω − , c) . (2.43) Thanks to the structure of the symplectic basis in (2.35), we then obtain the profile of η in the original variables Observe that, depending on the sign of the denominator in the expression above, we obtain a wave of depression or elevation. Hence, the proof of Theorem 1.1 is now complete. Stability 3.1. General theory. Having proved the existence of small-amplitude waves, in the remaining part of the work, we will investigate the aspect concerning their orbital stability/instability. For that, we are using the general theory stated in [VWW20], which is a variant of the well known GSS machinery introduced in [GSS90a, GSS90b]. As it is to any mathematical approach, there are a number of preliminary assumptions that first have to hold before applying the theory. For the water wave problem, however, there are some conditions that obstruct a direct use of the classical GSS approach. The variant in [VWW20] essentially solves these issues by weakening some of the requirements in GSS which then permits its application to the water wave problem. Although, the assumptions are weakened, the final conclusions of the both approaches in [VWW20] and [GSS90a,GSS90b] remain the same. We have outlined all the required hypothesis below and we will refer to them again later in the paper. For more in-depth and detailed explanations on the general theory, see [VWW20, Section 2] and the references therein. (3) For each u ∈ O ∩ V, J(u) is skew-adjoint, that is (4) (Strong continuity) The symmetry group is strongly continuous on X, V, and W. with dense domain D(T ′ (0)) ⊂ X consisting of all u ∈ X such that the limit above exists in X (similarly for the spaces V and W). Assume also that ∇P (u) ∈ D(J) and that is dense in X. for some c ∈ R and U ∈ O ∩ W. We would like to point out that the water wave problem in the present setting satisfies Assumption 4. In order to see this, one has to go through a series of lengthy, yet elementary, computations. Therefore, we will avoid checking the majority of the requirements in Assumption 4 here. Instead, we will focus more on showing that Assumption 4(8) holds: this is precisely one of requirements from the general theory in [GSS90a] that has been weakened and modified in [VWW20]. Assumption 5 (Bound states). There exists a one-parameter family of bound state solutions {U c : c ∈ I} to the Hamiltonian system (1.16). (1) The mapping c ∈ I → U c ∈ O ∩ W is of class C 1 . (2) The non-degeneracy condition T ′ (0)U c = 0 holds for every c ∈ I. Equivalently, U c is never a critical point of the momentum. (2) The spectrum of I −1 H c satisfies where −µ 2 c < 0 is a simple eigenvalue that correspond to a unit eigenvector χ c , 0 is a simple eigenvalue generated by T , and Σ C ⊂ (0, ∞) is bounded away from 0. 3.2. Notion on stability/instability. After outlining required assumptions for the general theory, we now proceed to define the notion on stability/instability concerned here. At this point, the functions spaces that we are working with are still abstract and will be specified soon in the next subsection. Fix a bound state U c and radius r > 0, we define the following sets (3.14) Fix R > 0, let B W R denote the intersection between the ball of radius R centered at the origin in W and the set O. Definition 3.1. The bound state U C is conditionally orbitally stable provided that for any r > 0 and R > 0, there exists r 0 > 0 such that if u : [0, t 0 ] → B W R is a solution to (1.3) where u(0) ∈ U X r 0 then u(t) ∈ U X r for all t ∈ [0, t 0 ). Note that the definition of orbital stability above is the same as the definition of stability introduced earlier in Section 1. Here, we are presenting it in a more general and abstract manner. From the general theory [GSS90a, GSS90b, VWW20] , the conclusion on stability/instability can be determined by looking at the sign of the second derivative of a scalar-valued function known as moment instability (3.15) This leads us to state the following theorem. Hamiltonian Formulation 4.1. Nonlocal operators. We begin this section by reformulating the governing equations (1.3) in terms of variables restricted to the interface η(x, t) in the spirit of Zakharov-Craig-Sulem. Although this way of formulating the problem forces us to work with some complicated non-local operators (pseudo-differential operators), it simplifies the problem by pushing all the unknowns to the boundary, in this case, the internal interface. The idea was then adopted by a number of authors studying internal waves, for instance, [BB97], [CG00]. Let ξ ± be defined as the trace of φ ± on the interface y = η(x, t) for the upper and lower regions of the fluid. It is clear that (4.1) Additionally, we denote H ± as the Hilbert transform acting on ξ ± : For later use, let us also introduce the Dirichlet-Neumann operator in Ω + and Ω − (for a fixed η): G ± (η)ξ ± := η ′ (N ± · ∇H ± (η)ξ ± ), where N ± is the outward unit normal relative to the domain Ω ± along the internal interface S and the Japanese bracket · := 1 + \|·\| 2 . Further, the notation H ± (η)ξ ± denotes the harmonic extension of ξ ± to Ω ± and uniquely solves ∂ y H ± (η)ξ ± = 0 on y = ±d ± . Remark 4.1. The spaces H k andḢ k are the Sobolev and homogeneous Sobolev spaces, respectively. Using the operators G ± (η), H ± (η), and the new unknown ξ ± , the water wave problem can be pushed to the boundary y = η(x, t). Now, it reads Next, in order to reformulate the problem in a Hamiltonian language, we introduce the variableξ := − ρξ . Recall, from the kinematic boundary condition in (4.4) we have This, together with the definition ofξ, yields where B(η) := ρ + G − (η) + ρ − G + (η). Following the property of G ± (η), the operator B(η) is also bounded and linear from H k (R) to H k−1 (R) and fromḢ k (R) →Ḣ k−1 (R) for any η ∈ H k 0 +1/2 (R) with k 0 , k given as before. Moreover, B(η) is an isomorphism fromḢ k (R) toḢ k−1 (R). Therefore, using (4.7) and solving for ξ ± we obtain (4.8) The kinematic and dynamic conditions now read (4.9) where A(η) := G ± (η)B(η) −1 G ∓ (η). The above equations can alternatively be written as (4.10) We would like to mention that similar formulation involving constant (non-vanishing) vorticity for a one-fluid and two-fluid case can be found, for instance, in [Wah07] and [Com16,CI15] respectively Remark 4.2. The spaces H k andḢ k are the Sobolev and homogeneous Sobolev spaces, respectively. Function spaces. The formulation stated in (4.10) is crucial in helping us exploit the Hamiltonian structure of the problem. In light of that, let us informally introduce the required function spaces where the internal water wave problem will be posed. Fix k ≥ 1/2, we define a product space for u: For future reference, we will denote X k+ to mean X k+ǫ for any 0 < ǫ ≪ 1, similarly for H k+ . Remark 4.3. Note that the space H p (R) ∩Ḣ q (R) is dense in H p (R) andḢ q (R) for all p, q ∈ R. We will use this fact to verify Assumption 1 in the general theory. Consider the following sequence of (continuously) embedded spaces where X is a Hilbert space, while W and V are reflexive Banach spaces. In practice, W will be the local well-posedness space of the internal water wave problem. Its regularity follows from the available results on local well-posedness. The space X is the natural energy space of the problem with X * being its continuous dual: Indeed, if u ∈ X, then ∇φ ± ∈ L 2 (Ω ± ). Furthermore, it also informs us that η ∈ H 1 (R) which ensures the finiteness of the potential energy. Both combined confirms that the energy is finite on X. The space V is an intermediate space that lies between the spaces W and X where all the conserved quantities are smooth there. The regularity of X turns out to be slightly insufficient for our analysis. This is because the map u → G ± (η) is not smooth with X being its domain. To fix this, the profile η has to be, at least, Lipschitz continuous and bounded away from the rigid walls {y = ±d ± }. In order to satisfy this level of smoothness condition, we define an intermediate space (4.14) along with a neighborhood O := {(η,ξ) ∈ X : −d − < η < d + }, (4.15) which makes sure that η is bounded away from the rigid walls. Moreover, observe that H 3/2+ (R) ֒→ W 1,∞ (R), meaning if (η,ξ) ∈ V, then η is Lipschitz continuous. Finally, since the current result on the Cauchy problem for water wave is not yet available with the level of regularity in V, thus we define a smoother space (4.16) It is worth mentioning that the work of Shatah and Zeng [SZ11] proves the local-wellposedness of the water wave problem at the same regularity as in W. Additionally, we would like to point out that the regularity of these spaces is the same and largely follows from the recent work of Chen and Walsh [CW22]. Having specified the function spaces, we express the relationship between the trio function spaces X, V, and W via an inequality recorded in the next lemma. Moreover, the content of the lemma shows that Assumption 1 in the general theory is satisfied. The above lemma shows that a small cubic term in V norm can be bounded using a quadratic term in X. This fact is needed in the general theory when bounding some of the terms resulted from Taylor expanding functionals whose domain is V ∩ O. 4.3. Hamiltonian Structure. In [BB97] Benjamin and Bridges formulated the internal water wave problem as a Hamiltonian system in the style of Zakharov-Craig-Sulem. Inspired by the aforementioned paper, we show that the water wave problem also exhibits a Hamiltonian structure (with a non-canonical Poisson map) that can be exploited for the stability analysis. Following the same idea as in [CW22, Section 3.3], we derive the energy functional of (4.10) (4.17) Observe that E ∈ C ∞ (O∩V; R). Moreover, we will show that there is an extension mapping ∇E(u) of DE(u) defined on the dual space X * which is the content of the lemma below. Lemma 4.5 (Energy Extension). There exists a mapping ∇E ∈ C ∞ (O ∩ V; X * ) such that (4.18) Proof. Fix u = (η,ξ) ∈ O ∩ V and letu = (η,ξ) ∈ V be given. Using the definition of the energy (4.17) together with the self-adjointness properties of A(η), G ± (η), and B −1 (η), one can show that and First of all, let us look at the expression in (4.20), it is easy to see that Moreover, it is clear that the last integral in (4.20) is an element of the dual space X * acting onu. In (4.19), the first integral can be written as where θ ± = A(η)G ± (η) −1ξ . Further, one can directly see that the second and last integral in (4.19) is an element of the dual space X * acting onu. Hence the extension ∇E(u) can be thought to have an L 2 gradient structure, that is, (4.22) Hence, the proof is now complete. (4.24) Observe that from the structure of J in (4.23), the difference in regularity, and homogeneity of the the spaces in (4.24), one can conclude that J is not a bijection. This fact clearly violates one of the assumption in the classical GSS approach [GSS87]. This is one of the reasons why we instead use the relaxed GSS [VWW20] as it only requires that J to be an injection, as stated in Assumption 2. Due to the presence of vorticity, the Poisson map is not canonical anymore. In other words, it is different with the Poisson map obtained in [CW22]. When ω + = 0 (one-layer fluid), we recover the map presented in [Wah07]. Proof. Suppose that u(t) = (η(t),ξ(t)) ∈ C 0 ([0, t 0 ); O ∩ W) is a weak solution to the abstract Hamiltonian (1.16). Using (4.8), we define Clearly, this satisfies the Laplace equation in (1.3a) and the boundary conditions on the walls {y = ±d ± } by definition of H ± in (4.3). Further, recalling the expression for E ′ (u) in Lemma 4.5, we have which holds in the distributional sense and it is equivalent to the kinematic boundary conditions in (4.10), therefore leads to the kinematic condition in (1.3b). Next, we claim that the Bernoulli condition is equivalent tõ We begin by writing the integrand in (4.17) in terms of ξ ± instead ofξ: . Therefore, equivalently, the Hamiltonian (4.17) can be written as (4.26) Thanks to the derivative formula in the Appendix B for the operator G ± (η) with respect to η: where Γ ± is defined in (4.5). Due to formula (4.27), it follows that the Bernoulli equation is satisfied. 4.4. The symmetry group and momentum. It is well known that the internal water wave problem is invariant under the horizontal translations. For this reason, we define a one-parameter symmetry group: T (s)u := u(· − s) for u ∈ X. In addition to that, this invariance also gives rise to another conserved quantity known as the momentum, P ± in each layer: Summing both momentum in each layers leads to the total momentum equation given by (4.28) Observe that P is a smooth functional in O ∩ V. The following lemma shows that T and P satisfy a number of properties as required by Assumption 4. Lemma 4.8 (Conserved quantities and symmetry). The energy E, momentum P, and the translation symmetry group T given above satisfy Assumptions 3 and 4. Specifically, the infinitesimal generator of T X k is the unbounded linear operator with dense domain DomT ′ (0)\| X k := X k+1 , and Proof. In light of Assumption 3, we have shown that the energy has an extension as stated by Lemma 4.5. Here, we will show that the momentum can also be extended. Let u = (η,ξ) ∈ O ∩ V andu = (η,ξ) ∈ V. Recalling the definition of P in (4.28) and computing its first variation yield The above expression has an L 2 gradient Observe that Dom T ′ (0) = X 3/2 ⊂ X. The expression (4.30) follows easily from the definition of J in (4.23) and T ′ (0) in (4.29). As it is mentioned previously, most of the requirements in Assumption 4 can be shown to hold in a straightforward manner. Hence, we will omit the details here. However, we want to focus more on Assumption 4(8). First, recall that Therefore, by Lemma 4.8, we obtain Hence, by Remark 4.3, Dom T ′ (0)\| W ∩Rng J is dense in X. 4.5. Bound states. The existence result in Theorem 1.1 was obtained by fixing the value of β and letting the rest of the parameters vary. Unfortunately, when using the general theory, this choice is not ideal: essentially, one might study the stability of two waves that solve two different internal water waves problem. To avoid such degenerate and nonphysical scenario, instead, we require a family of solutions parameterized only by the variable c, known as bound states, while fixing the rest of the physical parameters. Let the parameters (ρ ± * , d ± * , ω ± * , σ * , c * ) be fixed. We define The pair (β c , α c ) parameterizes a line segment joining the fixed point (β * , α * ) to the origin in the (β, α) plane. Meanwhile, ǫ c plays a role as a bifurcation parameter in terms of c. Throughout this week, ǫ c will be kept sufficiently small. Corollary 4.9 (Bound states). Fix (ρ ± * d ± * , ω ± * , σ * , c * ) such that Proof. Let (ρ ± * d ± * , ω ± * , σ ⋆ , c ⋆ ) be given. Suppose that β * > β 0 and 0 < α * − α 0 ≪ 1. For any c such that 0 < c − c * ≪ 1, then by Theorem 1.1, the bound states can be taken to be U c := u ǫc,βc where the surface profile depends on the parameter β c , α c . Observe that from the explicit expression of the profile in Theorem 1.1, it is exponentially localized. Further, due to the translation invariance of the problem, the profile η c is of class C ∞ . Thus, η c ∈ X k 1 for all k ≥ 1/2. Similarly, via the kinematic condition,ξ c is also smooth and belongs to X k 2 for all k ≥ 1/2. The first part of Assumption 5 now follows. Assumption 5(3) also follows from the regularity considered here. From the knowledge on the expression of P and the profile η c , Assumption 5(2),(4) clearly hold. Spectral Analysis If u(t) = T (ct)U is a traveling wave solutions for any bound state solution U ∈ O ∩ W with a wave speed c ∈ R, then by the Hamiltonian structure, Lemma 4.5, and Assumption 5(6), we have du dt = cT ′ (0)U = JDE(U ). (5.1) Furthermore, recall that via (4.30), the infinetesimal generator of T satisfies the following relation where the operator T ′ (0) maps u → −∂ x u. In concert with (5.1), they yield The above equation leads us to define the following functional known as the augmented Hamiltonian for a fixed speed c: Let u * = (η * ,ξ * ) be the critical point of the functional E c , then DξE(u * ) = DξP (u * ). From the Kinematic conditions in (4.4) and (4.9), for traveling waves, we have useful expressions for ξ ± * andξ * Inspired by the notation used in [CW22], we define the a ± 1 (η, ξ) and a ± 2 (η, ξ) as follows: Note that these two quantities represent the horizontal and vertical velocities respectively when φ is being replaced with ξ ± . Further, in relations to a ± 1 and a ± 2 , we define the following functions which represent the relative velocities in horizontal and vertical directions: For traveling waves solutions, the Kinematic condition can now be recast as Via some standard computation (in the notes), we obtain Using the definition ofξ and the formula for Dξ ± (η), we can infer Let us now define a smooth functional known as the augmented potential V aug c as follows: In the rest of this article, we shall compute the spectrum of D 2 V aug c , which will determine the spectrum of D 2 E c . . For all (η,ξ * (η)) ∈ O ∩ V andη ∈ H 3/2+ , we have the following formula Proof. We begin by differentiating in the direction ofη, where u * = (η,ξ * ). Observe that the second term in the summation above has vanished due to its evaluation at u * = (η,ξ * (η)) which is a critical point of E c . Differentiating again in the direction ofη gives us From the definition of E c and the fact that the momentum is linear inξ, we obtain Combining (5.6) and (5.3) leads us to the formula (5.4). Lemma 5.2 (Quadratic form). For all (η,ξ * (η)) ∈ O ∩ V and c ∈ R, there exists a self-adjoint operator Q c (η) ∈ Lin(X 1 ; X * 1 ) such that for allη,ζ ∈ V 1 and (5.8) Proof. First, it is clear that the Hamiltonian can alternatively be written in the following way (5.9) From 5.4, it is therefore useful to start doing an expansion on D 2 η E c (η,ξ * )[η,η]. Using the expression of the Hamiltonian (5.9), we can see that (5.10) It is important to note that when we let ω ± = 0, we recover back the expression found in [CW22]. To arrive at the expression stated in (5.8), we first begin by looking at the term involvingξ * D 2 A(η)[η,η]ξ * in (5.10). Since we are going to use some formula in [CW22], let us define the following variable This implies a ± 1 (η, θ ± ) = b ± 1 + c + ω ± η + a ± 1 (η, ±Υ ∓ ), a ± 2 (η, θ ± ) = ±b ± 2 . We further define the following expressions for later use: T ± (η)ζ := ±b ± 2 ζ. Upon using the formula in [CW22], we can infer that (5.11) To compute the terms on the second row in (5.11), we would need to define the following expressions: Via the definition of L ± in (5.12), we can infer For the importance of simplification later, we display the following formula At last, following the formula in Appendix B, we obtain Combining together all the computations above and the formula in (5.4), we get (5.13) To compute the last two integrals in (5.13), we need to derive the formula for DB −1 (η)η ω ηη x and D 2 B −1 (η)[η,η], ω ηη x . We begin with the expansion of the formula below (5.14) Using the equation in combination with (5.14), we derive the representation formula for the Fréchet derivative of B −1 (η) (for any given ζ): (5.15) From (5.15), we arrive the expression in the second row from the bottom of equation (5.8). Finally, it remains to show that the last integral in (5.13) yields the last expression in (5.8). First, recall that B(η) := ± ρ ± G ∓ (η). Exploiting the second derivative formula for G ± (η), we obtain After rearranging terms and applying (5.16), we obtain Combining this with the statements above, yields the claimed formula for Q c (η) and completes the proof. Based on the formula derived above, we can now determine the continuous spectrum of the linearized operator. Lemma 5.3 (Continuous spectrum). Let u = (η,ξ) ∈ O ∩ V be given. Then the operator Q c (η) is a self-adjoint operator on L 2 (R) with domain H 2 (R). Further, the spectrum of this operator is equal to the one of Q c (0), that is [τ * , +∞], where Proof. The fact that Q c (η) is self-adjoint on L 2 (R) with domain H 2 (R) follows directly from the regularity of η. Also, since η(x) → 0 as x → ∞, then the continuous spectrum of Q c (η) coincides with that of Q c (0). Further, due to the fact that Q c (0) is translation invariant, the whole spectrum is therefore continuous. The symbol of Q c (0) is The continuous spectrum results from looking at the range of the mapping ξ → q c (ξ), and τ * = min{q c (ξ) : ξ ∈ R}. For β ≥ β 0 , the minimum is attained at ξ = 0. But for β < β 0 , the minimum is attained at some value ξ = 0. 5.1. Rescaled operator. Now, we will make a use of a long-wave rescaling to obtain the information of the leading-order form of the operator Q c (η). Assume β > β 0 and α = α 0 + ǫ 2 , consider the following rescaling operator: It is clear to see that S ǫ is an isomorphism on H k (R). Note also that This shows that ∂ x S ǫ and ∂ x S −1 ǫ are uniformly bounded in Lin(H k+1 , H k ). Proof. Let Q ǫ be the operator obtained by evaluating operator Q c at η ǫ : (5.18) Following the same rescaling argument in [CW22], the rescaled surface tension term becomes Further, let us define the non-dimensionalized and rescaled relative velocity field , we haveb ± 2 = ǫ 3η′b± 1 . Using this, we obtain the following rescaled expression of the second and the third terms: For later use in dealing with the non-local terms in Q ǫ (η ǫ ), we define the following two operators: For any f ∈ H k+2 , we have where m ± := −ξ coth(d ± ξ) is the symbol for ∂ x G ± (0) −1 ∂ x . Thus,M ± ǫ (0) is a Fourier multiplier with a symbol given bỹ . Additionally, for any f ∈ H k+2 , we have is a Fourier multiplier with a symbol given bỹ . As a result we get In particular, we obtaiñ We are now ready to carefully analyze the remainder operator: (5.24) It is straight forward to see that in Lin (H k+2 , H k ). In view of (5.2) and (5.19), we know that Expanding the Dirichlet-Neumann operator in H k , we obtain Combining the formula with the first derivative formula for G ± (η), we can infer that Patterning the computation done in [CW22], therefore we can say that Therefore, the second term in the remainder operator (5.24) (5.27) Utilizing the expansion in (5.26) yields Finally, it remains to deal with all the expressions of the last four rows in (5.18). Again, going through the same analysis as before and using the fact thatZ ǫ (0) = −d + /(ǫ 2 d ± ), one can show that those terms are lower order (sufficiently small). Combining this result with the expression forR ǫ in (5.17), we obtain the formula ofQ 0 . 5.2. Spectrum of the linearized augmented potential. Having established the limiting behavior above, we will now analyze the spectrum of the operator Q ǫ (η ǫ ). Recall that, the operator Q ǫ (η ǫ ) only converges point-wise to the operator Q 0 (0) whose essential spectrum is [0, ∞). This is certainly creates a challenge in deducing the spectrum of Q ǫ (η ǫ ). Thanks to the rescaled operatorQ ǫ (η ǫ ) introduced earlier. It is known that such rescaled operator converges point-wise toQ 0 , which has a gap between 0 and the positive essential spectrum. Lemma 5.6 (Spectrum of the Limiting OperatorQ 0 ). Let the assumptions in Lemma 5.5 hold. The limiting rescaled operatorQ 0 satisfies where the first two eigenvalues −τ 2 < 0 and 0 are both simple eigenvalues with the corresponding eigenfunctionsg 1 andg 2 =η ′ , respectively; and there exists τ * > 0 such that Proof. The spectra condition above is a classic result and can be found, for instance, in the surveys of Pava [Pav09]. It is important to mention that the proof of that is not trivial by any means. The fact that −τ 2 and 0 are simple follows from the result which says that: the Wronskian of any two solutions in L 2 of the eigenvalue problemQ 0 f =τ must be 0. Proof. The proof of this theorem can be done in a similar manner as the one in [CW22, Theorem 3.2] which is mainly inspired by the proof of [Mie02,Theorem 4.3]. Therefore, we opt to avoid rewriting it here. and I −1 H c is self-adjoint on X. Appendix A. Center Manifold The first part of appendix records the center manifold reduction theorem introduced in [Mie95] that was implemented in, for example, [Nil17]. It is used in proving the existence of small-amplitude internal water waves. where the unknown u ∈ E for some Hilbert space E, µ ∈ R n is a parameter and L : D(L) ⊂ E → E is a closed linear operator. Assume that the differential equation (A.1) is Hamilton's equations that correspond to the Hamiltonian system (E, Ω, H) with 0 being its fix point. Moreover, assume also the following: H1 The space E has two closed and L-invariant subspaces, namely E 1 and E 2 such that E = E 1 ⊕ E 2 , u 1 = L 1 u 1 + F 1 (u 1 + u 2 , µ), u 2 = L 2 u 2 + F 2 (u 1 + u 2 , µ), where L i = L\| DL i ∩E i : DL i ∩ E i → E i , for i = 1, 2 and F 1 = P F , F 2 = (I − P )F, where the operator P is a projection of E onto E 1 . H2 E 1 is a finite dimensional Hilbert space and the spectrum of L 1 is purely imaginary. Under the hypothesis H1-H4 there exist neighborhoodsΛ ⊂ Λ andŨ 1 ⊂ U ∩E 1 ,Ũ 2 ⊂ U ∩E 2 of zero and a reduction function r :Ũ 1 ×Λ →Ũ 2 with the following properties. The reduction function r is k times continuously differentiable onŨ 1 ×Λ and the derivatives of r are bounded and uniformly continuous onŨ 1 ×Λ with r(0, µ 0 ) = 0, dr[0, µ 0 ] = 0. The graph X µ C = {u 1 + r(u 1 , µ) ∈Ũ 1 ×Ũ 2 : u 1 ∈Ũ 1 }, is a Hamiltonian center manifold for (A.1) with the following properties: • Through every point in X µ C there passes a unique solution of (A.1) that stays on X µ C as long as it remains inŨ 1 ×Ũ 2 . We say that X µ C is a locally invariant manifold of (A.1). • Every small bounded solution u(x), x ∈ R of (A.1) that satisfies u 1 (x) ∈Ũ 1 and u 2 (x) ∈Ũ 2 lies completely in X µ C . • Every solution u 1 of the reduced equatioṅ u 1 = L 1 u 1 + F 1 (u 1 + r(u 1 , µ), µ), (A.4) generates a solution u(x) = u 1 (x) + r(u 1 (x), µ) of (A.1). • X µ C is a symplectic submanifold E, and the flow determined by the Hamiltonian system X µ C ,Ω,H, where the tilde denotes the restriction to X µ C , coincides with the flow on X µ C determined by (E, Ω, H). The reduced equation (A.4) represents Hamilton's equations for (X µ C ,Ω,H). • If (A.1) is reversible, that is if there exists a linear symmetry S that anti-commutes with the right hand side of (A.1), then the reduction function r can be chosen so that it commutes with S. Appendix B. Formulae Many of the computations in the present work make use of the first and second derivative formulas of the non-local operators G ± (η) and A(η). We record them in a series of lemmas below. A derivation can be found in [CW22].	2022-08-18T01:16:22.142Z	2022-08-17T00:00:00.000	{ "year": 2022, "sha1": "80d8798b8f60a8ff5a24b5830897c951921496f8", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "80d8798b8f60a8ff5a24b5830897c951921496f8", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Mathematics" ] }
255221592	pes2o/s2orc	v3-fos-license	Shear Wave Elastography for Assessment of Testicular Stiffness in Patients with Varicocele: A Prospective Comparative Study Background: The purpose of this study was to use ultrasonography and shear wave elastography (SWE) to assess stiffness and volume of testes in adult patients with varicocele, to compare the results with those of normal contralateral testes of the same patients and healthy testes of control subjects. Methods: In this IRB-approved prospective comparative study, 58 patients with varicocele (116 testes) and 58 control patients (116 testes) were enrolled. A total of 66 testes with varicocele were added to group A, their 50 healthful contralateral testes were added to Group B, and 116 healthy control testes were added to Group C. Analysis of variance (one way ANOVA) test was used to compare groups and Student's t-test was used for their binary comparisons. The correlation between the testicular stiffness and volume was studied using Pearson's correlation test. Results: There was not a significant difference in the mean SWE values between three groups and also, between two-group comparisons (P < 0.05). In the mean testicular volumes, a significant difference was observed between Groups A and C (P = 0.028). Conversely, there was not a significant difference between Groups A and B (P = 0.907) or Groups B and C (P = 0.094). A meaningful correlation between testicular stiffness and volume was not detected for each group. Conclusion: A significant correlation was not determined between SWE values and varicocele, and also between SWE values and testicular volume. We advise that more studies with larger populations of patients are required to verify the effectiveness of SWE in predicting testicular parenchymal damage. been conducted about the effects of varicocele in testicular parenchyma and varicocele was found to change the testicular stiffness as measured by elastography. [9,[17][18][19][20][21] The purpose of this study was to use SWE to assess stiffness changes of testes in adult patients with varicocele, to compare the results with those of healthy contralateral testes of the same patients and normal testes of control group patients without varicocele. Furthermore, the relationship between varicocele and testicular volume changes was evaluated. mAtEriAls And mEthods In this prospective controlled study, institutional review board approval was acquired from the medical ethics committee (Protocol number of Ethics Committee approval: March 27, 2021). Written informed consent was acquired from each patient before SWE, and detailed information was given about the examination. Patient selection and study design This prospective comparative study was performed between April 2021 and September 2021. Inclusion criteria were male gender and age over 18 years. A total of 58 cases with varicocele (116 testes) and 58 control cases (116 testes) were enrolled in the study. 8 of the patients had bilateral varicocele. 66 testes with varicocele were added to Group A, their 50 health opposite side testes were added to Group B, and 116 healthy control testes were added to Group C. The healthy individuals of control group subjects were randomly selected from the participants who had consulted for the scrotal US. In total 17 patients with a previous history of varicocelectomy, present or previous cryptorchidism, infection, hydrocele, pyocele, and abnormal testicular parenchymal echogenicity on US examination were excluded from this work. The inclusion and exclusion criteria of this study are summarized in Figure 1. Ultrasonography and shear wave elastography examination Patients were evaluated with US and SWE, and examinations were applied by two radiologists with more than 7 years of experience in scrotal US and 3 years of experience in SWE. The radiologists evaluated each modality independently and each radiologist had a similar number examination. All examinations were performed by using a high-frequency (4-18 MHz) linear probe of US unit (Philips, EPIQ, The Netherlands). The pampiniform plexus veins were evaluated in supine and upright positions with and without a Valsalva maneuver. First, the side of the varicocele (if there is) was noted and dilation of the pampiniform plexus veins to a width of larger than 2 mm in diameter, and increased width during the Valsalva maneuver were diagnosed as varicocele. [7,8] Moreover, three dimensions of each testis were measured by using light pressure in the supine position and testicular volume was calculated by the US machine using the following formula based on similar previous studies. [17,22] Testicular volume = length × width × height × 0.523 and expressed in milliliters (ml). After the grayscale US examination was completed, the radiologist commenced the SWE examination. Two-dimensional SWE was examined at the resting state in supine position. While conducting the SWE evaluation, the probe was held as stable as possible by using very light pressure to avoid artifacts and incorrect measurements. Sections that were obtained longitudinally were selected for the measurements and 3 regions of interest (ROI) were placed with a diameter of 8 mm onto the color-coded confidence maps and performed three measurements. Three ROI circles were set at the upper, middle, and lower poles of the testis [ Figure 2]. The mediastinum testis was excluded from the ROIs. Tissue stiffness was defined with a color range from blue (low) to red (high), corresponding to 0-200 kilopascals (kPa). As in previously conducted studies, automatically generated average stiffness values were used. [17,23] The average of the all 3 poles measurements was calculated. The results were recorded in units of kPa. The mean SWE and testicular volume of the varicocele-positive patients were compared with those of the contralateral normal testes of the same patient and the normal testes of the control group. Statistical analyses Results of the mean age, testicular volume, and SWE values of all three groups are reported as the mean ± standard Levene's test was used to assess the homogeneity of variances based on mean ± SD values. A parametric analysis of variance (one-way ANOVA) was used to compare all three groups and Student's t-test was used for binary comparisons of the groups. The correlation between the testicular stiffness and volume was studied using Pearson's correlation test (Pearson correlation coefficient = r). All statistical analyses were performed in SPSS (version 25.0, IBM Corp., Armonk, New York, USA) and a P value of lower than 0.05 was accepted to show statistical significance within a 95% confidence interval (CI). rEsults In total 116 male patients (232 testes) who met the criteria were enrolled in the study. Fifty-eight of these patients had varicocele in the left side (100%, 58/58), and eight of them bilateral varicocele (13.7%, 8/58). None of the cases had isolated right side varicocele. A total of 66 testes with varicocele were included to Group A and 50 healthy contralateral testes of these patients were included to Group B. Finally, 58 patients (116 testes) with bilateral healthy testes were included to healthy control Group C. The mean age of Group A was 33.4 ± 10.3 years (18-67), Group B was 32.4 ± 9.7 years (18-61) and the mean age of the control Group C was 33.7 ± 12 years (18-68). There was not a statistically significant difference between these 3 groups in age (P = 0.817). The main complaints of symptomatic cases were recurrent chronic pain in the scrotum or a palpable soft scrotal mass which indicates dilated veins. Asymptomatic cases were mainly being evaluated for reproductive health. The SWE values were 7.90 ± 2.26 kPa in Group A, 7.44 ± 1.90 kPa in Group B, and 7.94 ± 2.07 kPa in Group C. The variances were homogenous (P = 0.106). There was no significant difference in the mean SWE values between the 3 groups (P = 0.346, f = 1.066). There was no significant difference between two-group comparisons (P = 0.248 for Group A-B, P = 0.133 for Group B-C, and P = 0.907 for Group A-C). The mean testicular volume was 15.2 ± 4.16 mL in Group A, 15.4 ± 4.58 mL in Group B, and 16.8 ± 4.30 mL in Group C. The variances were homogenous (P = 0.422). The mean testicular volume values in Group A were slightly lower than that of the other groups but there was no significant difference between the 3 groups (P = 0.081). Moreover, a statistically significant difference was found between Groups A and C (P = 0.028). Conversely, there was no significant difference between Groups A and B (P = 0.907) or Groups B and C (P = 0.094). A significant correlation between testicular stiffness and volume was not detected for each group by using Pearson correlation test (r = −0.039, P = 0.553 for Group A, r = 0.070, P = 0.628 for Group B and r = −0.109, P = 0.276 for Group C). The mean age, testicular volume, and SWE values of all groups are shown in Tables 1 and 2. disCussion SWE is a relatively new, reproducible, and noninvasive imaging method which gives information about histopathological changes in different tissues. It provides a precious quantitative evaluation of tissue stiffness and it is very useful in the evaluation of parenchymal disorders. Very few comparative studies have been conducted about the effects of varicocele in testicular parenchyma by using SWE. [17,22] In our study with 116 patients and 232 testes, a significant difference was not observed between the 3 groups (A, B, and C) in mean age. A significant difference was observed in the mean testicular volumes between groups A and C. However, there was not a significant difference between Groups A and B or B and C. In our study, no significant difference was observed between the 3 groups regarding to mean SWE values. Furthermore, there was no significant difference for binary comparisons in terms of mean SWE values. Furthermore, a meaningful correlation was not observed between the mean SWE value and the mean testicular volume for each group. Our study verified that the testicular volume decreases in patient with varicocele compared with healthy testes. In a very similar prospective study by Erdogan et al., [17] 100 patients and 200 testes were examined. 50 varicocele testes, 46 contralateral healthy testes of patients, and 104 normal testes of healthy controls were added to Group A, B, and C, respectively. In this study, the mean testicular volume was 13.43 ± 4.64 mL for Group A, 14.29 ± 3.82 mL for Group B, and 15.2 ± 4.13 mL for Group C. There was no significant difference in the testicular volume between the 3 groups (P = 0.035, f = 3.424). A significant difference was observed only between Groups A and C (P = 0.014) in binary comparisons. The statistical results of mean testicular volume confirm our study. The mean SWE values of this study were 12.61 ± 6.23 kPa in Group A, 9.23 ± 3.23 kPa in Group B, Results are showed as mean±SD. SWE: Shear wave elastosonography, SD: Standard deviation, kPa: Kilopascals and 9.4 ± 4.30 kPa in Group C. Contrary to our results, they observed a significant difference in the SWE values between the 3 groups (P = 0.001). Furthermore, SWE values of varicocele testes were higher than control healthy testes with a statistically significant difference (P = 0.001). In addition, similar to our study, there was no meaningful correlation between the mean SWE values and the testicular volume for each group. In another prospective study, Dede et al. evaluated the association between elastography results, serum follicle-stimulating hormone levels, and varicocele in 30 patients by using acoustic radiation force impulse elastography. [9] This study enrolled only 30 patients with varicocele on the left side and 30 patients without varicocele. They observed a negative correlation between testicular stiffness and the varicocele grade. Comparison of the groups showed a lower testicular stiffness in patients with varicocele. In another study by Turna and Aybar, [22] 116 testes of patients with varicocele (left side) and 58 testes of the normal control group were evaluated. This study demonstrated higher stiffness values in testes with varicocele than in the contralateral healthy testes and the testes of the normal control group (P < 0.001). In addition, there was no correlation between testicular stiffness and the grade of varicocele in this study (r = 0.102, P = 0.423). Our study has a few limitations. First, we did not obtain any histological data of the patients. Second, in spite of the population was greater in our study than in the previously published studies [9,17] we suggest that more studies with a larger sample size are needed to verify the effectiveness of SWE in predicting testicular fibrosis and parenchymal damage. Third, we did not evaluate interobserver variability because of patient privacy. SWE examination were performed only in the longitudinal plane and from three regions. The grade of varicocele was not evaluated in this study. Although there are some new papers discussing the grading system for varicocele: Dubin and Amelar introduced the varicocele grading system in 1970 when they evaluated whether preoperative varicocele size was related to semen parameter changes. The grading system classifies grade 1 varicocele as varicocele palpable only by the Valsalva maneuver, grade 2 as palpable at rest but not visible, and grade 3 as easily visible. [24] ConClusion Our study showed a lower mean testicular volume in testes with varicocele than in the testes of normal control group with a significant difference. A correlation was not observed between SWE values and varicocele, and also between SWE values and testicular volume. We suggest that SWE may play a supportive role to grayscale US in evaluating histopathologic changes in the testis due to varicocele, but more studies with larger populations are required to confirm the effectiveness of SWE in predicting testicular parenchymal damage. Although there are significant results in the literature, the nonsignificant results require a re-evaluation of the view that varicocele is effective on testicular stiffness. These results may also be due to society or race. We believe that studies with genetic factors and histopathological results of testicular parenchyma are needed to confirm the performance of SWE. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.	2022-12-29T16:08:49.394Z	2022-05-03T00:00:00.000	{ "year": 2022, "sha1": "1bca5b9a572f7bfcc4f7baad02227decf128b98f", "oa_license": "CCBYNCSA", "oa_url": "https://doi.org/10.4103/jmu.jmu_218_21", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "be1b00473d4c99f0eec9207c489166da320d28a4", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
247029282	pes2o/s2orc	v3-fos-license	A “turn-on” DNA-scaffolded silver-nanocluster probe for detection of tumor-related mRNA A novel and “turn-on” DNA-silver nanocluster probe (DNA/AgNCs) was developed that can detect the level of breast tumor-related mRNA markers: cyclin D1 mRNA. The fluorescence of DNA/AgNCs probe rendered a “turn-on" response based on the secondary structure changes induced by the target strand. In the presence of target chain, the fluorescence enhances in a concentration-dependent manner, which enables the quantitative detection of analytes. By this method, a detection limit of 2.1 nM and a linear range of 15–125 nM were achieved. In addition, the probe displays excellent discrimination between a perfectly complementary target and single-base mismatch targets, providing an alternative approach for quick and specific recognition and quantification of mRNA or DNA. Introduction Metal nanoclusters (MNCs) have smaller particle size than metal nanoparticles, which can exhibit variety region fluorescence, and have a much higher luminous efficiency compared to nanoparticles due to the strong quantum-confinement effect. Further, they can present significantly different electrical and chemical properties. Recently, silver nanoclusters have attracted considerable attention in virtue of several advantages, including easy preparation, modulable fluorescence, good biocompatibility and low toxicity [1,2]. Significantly, the few-atom silver clusters possess large molar extinctions, a high fluorescence quantum yield and excellent optical stability. All of the above advantages make it more extensively application than the organic dyes and metal quantum dots, such as in the cellular long-time imaging, labeling organics and biological detection applications. Due to its ultra-small size, the reduction potential is as low as − 1.8 V for few silver atoms [3]. The propensity of AgNCs toward oxidation has been the primary challenge in producing and stabilizing process. Extensive efforts have been made to produce fluorescent silver clusters. Dendrimers and polymers have been reported as templates for the generation of AgNCs [4,5]. Clusters can also form with small molecular ligands such as meso-2, 3-dimercaptosuccinic acid (DMSA) and mercaptosuccinic acid (H 2 MSA) [6,7]. Biological materials of interest, such as peptides, proteins and DNA have especially been used for AgNCs formation [8−11]. Various oligonucleotide sequences or conformations have been utilized for synthesizing AgNCs, including singlestranded DNA, double-stranded DNA, C 4 i-motif, G-quadruplex DNA, and the hairpin loop structure [12−16]. Among them, the most typical template is cytosine-rich ssDNA [13, 17, and 18]. Encapsulated within DNA scaffold, AgNCs can exhibit strong fluorescence emission, which can be tuned from visible to near infrared light by changing the length and sequence of the DNA scaffold [19,20]. In addition, the environmental interactions can influence the photophysical properties of AgNCs, such as fluorescence enhancement or quenching, which offers opportunity to use these species as chemical sensors for detection of metal ions, small molecules, DNA, mRNA, peptides and single-nucleotide mutations etc [21−28]. Breast cancer is currently of highest prevalence in worldwide women, both in the developed and developing world. The survival rate of cancer patients is closely associated with tumor staging. In the early stages before metastasis, identification at molecular level holds great promise of increasing survival in cancer patients. Thus, specific methods for detecting abnormal gene expression level in living cells are major focus of research [29−33]. Changes in tumor-related mRNA expression levels are closely related to the malignant progression of the tumor, and which has been widely used as a specific marker for assessing the stage of tumor progression [34]. Some recent research developments for mRNA sensing are built on the applications of nanomaterials [35−40]. However, multiple sensing systems based upon quenching patterns are not preferred in practice. Because other quenchers or environmental changes might lead to fluorescence quenching, which give "false positive" results. Therefore, development of "turn-on" fluorescent probes has more practical applications than "turn-off" ones. Cyclin D1 is an important regulator of the cell cycle. Abnormal expression of Cyclin D1 is a factor of the damage on the cell cycle regulatory mechanism and is closely related to the occurrence of malignant tumors. Reportedly, Cyclin D1 is overexpressed in 50-80% of breast-cancer tissue, while being very low or absent in normal breast tissue [41]. Up to now, several fluorescencebased sensors for the detection of Cyclin D1 mRNA were presented [42−45]. However, traditional fluorescent molecular beacons used for mRNA detection are always stem-loop type oligonucleotide probes dual-labeled with a donor fluorophore and an acceptor quencher. All of these will inevitably lead to certain expensive and complex operations. Considering these reasons, devising of a "turn-on" and label-free fluorescent probes will be highly desirable. In this work, a new method has been developed for detecting the level of breast tumor-related markers: cyclin D1 mRNA. With DNA-scaffolded AgNCs as signal indicators, we have devised four different DNA/AgNCs probes and selected probe-1 for quantifing the level of cyclin D1 mRNA. This novel probe performs a homogeneous assay without chemical modification or separating the binding probes from the free ones. The fluorescent probe gives a positive signal response to the target strand as a result of the secondary structure changes. In addition, the probe displays excellent discrimination between a perfectly complementary target and single-base mismatch targets, which provide an alternative approach for the quick and specific recognition and quantification of mRNA or DNA. Reagents and chemicals All DNA oligonucleotides and target strands used for this study were purchased from TaKaRa Biotechnology Co. Ltd (Dalian, China). Sequences of oligonucleotides used in this study were listed in Table S1 (Supplementary Table S1). Annealing is an indispensable step in preparing DNA/ AgNCs probe. All DNA oligonucleotides were initially incubated at 95 ºC for 10 min and subsequently cooled to 0 ºC for 10 min before use. Analytical-grade silver nitrate and sodium borohydride were supplied by Aldrich without further purification. The Sodium borohydride must be freshly prepared before use. Other analytical pure reagents were purchased from Tianjin Chemical Regent Co. Low range DNA ladder were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Tween 20 was purchased from Biosharp. Ultrapure water (18.2 MΩ cm) was used throughout the work. Apparatus Fluorescence spectra were determined on the LS-55 fluorescence spectrophotometer (PerkinElmer, America). The pH was acquired on a DELTA-320 pH meter (Shanghai, Metter-Toledo Instrument Company, Ltd., China). Gel electrophoresis assays were carried out by DYY-6C electrophoresis apparatus (Beijing Liuyi Biotechnology Co., LTD). The TEM images of AgNCs were acquired on a Tecnai G2 F30 instrument (FEI, USA). Synthesis of DNA strand-L/AgNCs DNA strand-L were all first dissolved in ultrapure deionized water and were placed at 25 ºC for 10 min; 9 μL 100 μM DNA strand-L was mixed with 30 μL 20 mM Tris-HAC buffer (20 mM, pH7.4) buffer and then 6 μL 900 μM AgNO 3 solution was added into the above solution. After being cooled in the ice-water bath 15 min, 6 μL 900 μM NaBH 4 solution was rapidly added into the mixture and shaked violently for 1 min. Later, the reaction mixture was kept in the dark at 25 ºC for 2 h and subsequently used. Preparation of the Nanoprobes The diverse DNA strand-L /AgNCs and DNA strand-S were mixed separately (1:1.2) in phosphate buffer (20 mM, pH 7.4). Then, the mixture was incubated for 1 h at 37 °C, and subsequently slowly cooled to room temperature. The above solution was stored in the dark for 1 h to allow complete reaction. Hybridization and Fluorescence test Hybridization reactions were performed in a pH 7.4 phosphate buffer solution. The DNA/AgNCs probe and the complementary target with increasing concentrations were incubated for 1 h at 37 ºC. Then, the hybrids were cooled to room temperature and diluted to 2 mL. Finally, the fluorescence property of the complexes was tested on a LS55 Fluorescence Spectrometer. Fluorescence emission was measured from 475 to 700 nm by exciting at 450 nm. The detection limit was calculated from the the derived calibration curve, as defined by IUPAC. Gel electrophoresis analysis The probe-1 and the hybrid duplex products were analyzed by 10% native PAGE in 1 × TBE buffer at room temperature. The TBE buffer was composed of 90 mM Tris, 2 mM EDTA, 90 mM boric acid and 6.25 mM magnesium acetate (pH 7.4). 5 μL of different samples were loaded and stained with ethidium bromide. The gel was run at 100 V for 60 min at room temperature and photographed by the gel image analysis system (BioSens SC750). Optimization of the reaction conditions Probe concentration, pH of the phosphate buffer solution and reaction time could all influence the performance of the probe. To improve the sensitivity of the probe, we optimized the experimental condition, respectively, at constant concentration of others. Conditional experiments were conducted to evaluate the relative value of F/F 0 , where the F 0 and F were fluorescence intensity in presence and absence of target RNA, respectively. Recovery test Fetal calf serum was centrifuged (8000 r/min) at 4 ºC for 10 min. The supernatant was taken and diluted 100 times for reserve in the phosphate buffer (4 mM, pH 7.4). The standard addition method was used for the cyclin D1 mRNA recovery test. Cell culture and confocal laser scanning microscopy The breast-cancer SK-BR-3 and the normal immortalized human mammary epithelial MCF-10A cell lines were purchased from Shanghai Yubo Biotechnology Co., Ltd. The cells were cultured in DMEM containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin and maintained at 37 ºC in 5% CO 2 . The concentration of cells was adjusted as 1 × 10 6 cells / mL and were plated on glass slide at 37 ºC in 5% CO 2 for 24 h. A set of SK-BR-3 cells were treated with cartilage polysaccharide for 24 h. Cells were immobilized for 8 min at room temperature before adding of the probe. The cell fixed solution was a methanol-acetic acid mixture (V methanol: V acetic acid = 3:1). Cells were further centrifuged, washed and divided in phosphate buffer. Then 0.1 μM probe-1 was added into the treated SK-BR-3 cells, the untreated SK-BR-3 cells and MCF-10A cells respectively, and incubated at 37 °C, 5% CO 2 for one hour. Images were obtained by a confocal laser scanning microscopy with excitation at λ = 488 nm (Leica TCS SPE, Germany). Results and discussion Sensor design and principle of cyclin D1 mRNA assay An interesting phenomenon is reported by Yeh group, that the red fluorescence of AgNCs could be re-enhanced 500fold when the guanine-rich DNA sequence approaches the nanoclusters [15]. Inspired by this case, we design a "turnon" DNA/AgNCs probe targeting the cyclin D1 mRNA. This novel probe is designed as a partial duplex template, which is composed of a long DNA aptamer of 41-base and a short DNA aptamer of 17-base, called "Strand-L" and "Strand-S", respectively. Using "Strand-L" as a template, AgNCs were prepared by reducing AgNO 3 with sodium borohydride. As can be seen in Table S1, Each "Strand-L" sequence is listed in three colors, for example, the sequence of Strand-L 1 (5'-CCT CCT TCCT CCTTA CTA CAG CAA CAA CTA TAAGG GTG GGG -3'). Red squences: cytimidine-rich sequence for stabilizing of AgNCs, black sequences: anchor sequence (partially complementary to Strand-S), and green sequences: guanine-rich tail as a fluorescence enhancer when placed in proximity to the AgNCs. The underlined bases were designed perfectly complementary to form a stem of hairpin structure in the free condition. The "Strand-S" (5'-GAG TTG TCG GTG TAGAT-3') was designed to perfectly complementary sequence for the target mRNA, but few mismatched base site with the "Strand-L". The characteristic sequence (5'-AUC UAC ACC GAC AACUC-3') of cyclin D1 mRNA was used as the detection target strand [42]. The design scheme of DNA/AgNCs probe and the principle of cyclin D1mRNA detection were shown in Fig. 1. When the target strand is recognized, the more stable duplux between the "Strand-S" and "target strand" comes into being because of the perfectly complementation. "Strand-L" will be subsequently displaced and released, and then folded-back into a hairpin structure. The conformational alteration of "Strand-L" facilitates the proximity of the overhang guanine-rich region to AgNCs, which results in the fluorescent enhancement of DNA/AgNCs probe. As shown in Supplementary Fig. S1, the fluorescent intensity increases when the target RNA is recognized. The Key to a successful design approach is target strand hybridizing with "Strand-S" of DNA/AgNCs probe, and then "Strand-L" is replaced and refolds into a hairpin structure. The theoretic basis and design reference is guided by the data analysis of T m differences and Gibbs free energy change (Δ r G m θ ) of the reaction. To gain an effective probe for target detection, we designed four aptamer sequences as "Strand-L" for preparing nanoprobes. The four probe sequences and thermodynamic data are all listed in Table S2. T m and Δ f G m θ values are estimated using the software Primer Premier 5.0. The second structures of the four liberated "Strand-L" under the hybridization conditions are predicted to form hairpin structure via software Primer Premier 5.0 ( Supplementary Fig. S2). As shown in Table S2, T m of perfectly hybrid duplex between targeted RNA and "Strand-S" is higher than T m of all other four probes, and the Δ f G m θ date is the lowest. Therefore, the hybridization of "Strand-S" and targeted RNA has more thermodynamic advantages, which provides a theoretical support for the competition replacement of "Strand-L" by targeted RNA. The spontaneity of an isothermal isobaric process can be judged by the change of the Gibbs free energy (Δ r G m θ ). The value of Δ r G m θ was calculated on the base of the following Eq. (1): As shown in Table S3, the value of Δ r G m θ of the competing hybridization reaction between the target strand and the four probes were all negative by calculation. According to the above analysis of the thermodynamic data, it can be inferred that four probes can all compete in hybridization with the targeted strand. As the logical next step, four probes were tested for the target sensibility in the same assay conditions. As shown in Fig. 2, there are different fluorescence responses in the presence of target strand. Compared with the report of Yeh group [15], the increase of the fluorescence was less response (about fourfold). There may be some reasons. Firstly, the optical properties of the silver nanoclusters are regulated by the sequence and secondary structure of the DNA templates. Furthermore, the mechanism of competitive hybridization and the formation of hairpin structure (with 5nt stem) insufficiently make a guanine-rich region approaching the AgNCs. Probe-1 displays the highest value of F/F 0 (about 4.5-fold) compared to the other three probes. To explore the reason for this result, the Δ r G m θ of the competing hybridization reaction between four probes and the target strand was analyzed. As can be seen in Table S3, the Δ r G m θ value of the hybridization reaction between the target strand and probe-1 is the lowest. Based on this result, we speculate the lower Δ r G m θ , the higher target sensitivity. Therefore, the probe-1 is selected for subsequent experiments. PAGE characterization We also performed polyacrylamide gel electrophoresis to illustrate the mechanism (Fig.S3). Lane-1 represents the migration band of DNA Marker (20-500). Lane-2 and lane-3 represent the migration band of "Strand-L 1 " and "Strand-L 1 /AgNCs", respectively, which showed similar migration. This infered the structure of "Strand-L 1 " not affected by the presence of AgNCs. Lane-4 and Lane-6 represent the migration band of the hybrid duplex and probe-1, respectively. Lane-5 shows the migration bands of the hybrids of probe-1 incubated with target strand. As can be seen from Lane-5, there are two migration bands: the front band has the similar migration to the hybrid duplex (Lane-4), and the latter has a slightly slower migration compared to the probe-1 (Lane-6). According to the above results, it can be deduced that the hybrids of probe-1 incubated with target strand contains the perfectly hybrid duplex and another structural strand, which prove that the competition combination has occurred. Optimization of detection conditions For the sake of higher sensitivity, determination conditions were optimized, such as the concentration of DNA/AgNCs probe, pH and hybridization reaction time. The experiment results showed that the optimal pH for hybridization reaction was in the range of 6.5-8.0 (Fig. S4a), thus the condition of pH 7.4 was selected for subsequent experiments. Meanwhile, the concentration of the probe-1 was tested. As can be seen in Fig. S4b, when the concentration was less than 0.1 μM, the F/F 0 ratio increased with the concentration, yet decreased with increasing concentration when the concentration was greater than 0.1 μM. A possible reason is, as the concentration increased, the fluorescence AgNCs transformed into larger non-luminescent silver nanoparticles. So, the probe-1 concentration of 0.1 μM was selected for subsequent experiments. Then, the hybridization reaction time was studied, as shown in Fig. S4c. The F/F 0 ratios increased in the initial stage and then descend slightly as the reaction time growing, achieved a maximum at 60 min. Hence, 1 h was chosen for the incubation time. Therefore, we fixed the probe-1 concentration at 0.1 μM and varied the concentration of target strand from 0 to 125 nM and incubated for 1 h at 37 °C in a pH 7.4 phosphate buffer solution. Then the hybrids were cooled to room temperature and diluted to 2 mL before the fluorescence measurement. The representative TEM image of the prepared DNA/ AgNCs probe was illustrated in Fig. S5, the AgNCs were well-dispersed with a diameter around 2 nm. Sensitivity and selectivity Under the optimum experimental conditions, probe-1 was chosen to conduct experiments in detection range. When the resulting complex was excited at 450 nm, it featured an emission band centered at 510 nm. As shown in Fig. 3a, the fluorescence intensity of the probe-1 greatly increased in a concentration-dependent manner of the target strand. Changes of the probe fluorescence intensity are precisely the result of the change in secondary structure in the presence of target strand. This result demonstrated that DNA/AgNCs probe-1 could detect target RNA quantificationally. Accordingly, a linear correlation between the fluorescence intensity and the concentration of target RNA was obtained (Fig. 3b). The linear regression equation was y = 9.07388 + 0.34478x (nM) with the excellent linear relationship (R 2 = 0.9869) and the linear range was 15-125 nM. The detection limit was 2.1 nM based on 3 s/Slope (where "s" is the standard deviation of blank sample, and Slope is the slope of the linear equation). The comparison results of different fluorescence probes for the detection of cyclin D1 mRNA in recent years are summarized in Table S4. Compared with reported references, this method is very fascinating and interesting in the terms of easy operation, low cost and discriminating singlebase-mismatched target, despite the not very low detection limit. Further, we investigated the selectivity of the probe-1 by comparing the fluorescence responses of the probe-1 toward perfectly matched Target-1 and one to three-base mismatched targets under the same concentration. As shown in Fig. 4, the probe-1 displayed an F/F 0 value of 4.5 in the presence of its perfectly matched target-1, but no significant fluorescence enhancement could be observed in the presence of one to three-base mismatched targets. The reason may be the mismatched targets were insufficient to identify and hybridize with "Strand-S", so the"Strand-L" cannot be released by competition and that cannot form a hairpin structure. Consequently, the fluorescence is almost unchanged. This clearly indicates that the probe-1 displays excellent discrimination between a perfectly complementary target and single-base mismatch targets, demonstrating high specificity. A recovery test of cyclin D1 mRNA in serum was carried out by standard addition method. Different concentrations of cyclin D1 mRNA were added into fetal calf serum to prepare standard solutions and tested by the proposed experimental method. The results are shown in Table 1, the average recovery of cyclin D1 mRNA in FBS is in the 97.0-98.5% range with the relative standard deviation of 2.18-3.15%. The results indicated that the prepared probe for detection of cyclin D1 mRNA was satisfactory. The ability of probe-1 to detect changes of cyclin D1 mRNA in cells Further, we evaluated the feasibility of probe-1 to detect the cyclin D1 mRNA expression level in the breast-cancer cell line SK-BR-3 and normal immortalized human mammary epithelial cell line MCF-10A. A set of SK-BR-3 cells were treated with cartilage polysaccharide for 24 h, which could induce a downregulation of the cyclin D1 mRNA level. Then, probe-1 was delivered into the treated SK-BR-3 cells, the untreated SK-BR-3 cells and MCF-10A cells, respectively, and incubated at 37 °C for 60 min. Then, images were obtained by a confocal laser scanning microscopy. As can be seen from Figs. 5a and c, a significantly stronger fluorescence signal was observed in SK-BR-3 cells than in the MCF-10A cells. We also found the fluorescence signal decreased in the cartilage polysaccharide-treated SK-BR-3 cells relative to the untreated cells (Fig. 5b). These showed that probe-1 could respond to changes of cyclin D1 mRNA level in cells. Conclusions In this study, we designed a turn-on and label-free probe to detect tumor-related markers: cyclin D1 mRNA. When combined to the target strand, the fluorescence intensity of DNA/AgNCs probe increases owing to the conformational alteration favorable for the proximity of the overhang guanine-rich region to AgNCs. The limit of detection was 2.1 nM, and it showed a good linear relationship in the range of 15-125 nM. The probe displays excellent discrimination between a perfectly complementary target and single-base mismatch targets. This method may supply a general strategy for detection of other mRNAs or DNA, and have some reference value for clinical early diagnosis of tumor.	2022-02-23T16:31:16.403Z	2022-02-01T00:00:00.000	{ "year": 2022, "sha1": "f7607277e87c1afbc1f6a96ead0340b1d86204b1", "oa_license": null, "oa_url": "https://doi.org/10.1007/s44211-022-00063-0", "oa_status": "GOLD", "pdf_src": "SpringerNature", "pdf_hash": "d3cd44f7dfcdde52d51d0f4655f87a0576db27f6", "s2fieldsofstudy": [ "Medicine", "Chemistry" ], "extfieldsofstudy": [ "Medicine" ] }
265064554	pes2o/s2orc	v3-fos-license	Voxel-based morphometry in single subjects without a scanner-specific normal database using a convolutional neural network Objectives Reliable detection of disease-specific atrophy in individual T1w-MRI by voxel-based morphometry (VBM) requires scanner-specific normal databases (NDB), which often are not available. The aim of this retrospective study was to design, train, and test a deep convolutional neural network (CNN) for single-subject VBM without the need for a NDB (CNN-VBM). Materials and methods The training dataset comprised 8945 T1w scans from 65 different scanners. The gold standard VBM maps were obtained by conventional VBM with a scanner-specific NDB for each of the 65 scanners. CNN-VBM was tested in an independent dataset comprising healthy controls (n = 37) and subjects with Alzheimer’s disease (AD, n = 51) or frontotemporal lobar degeneration (FTLD, n = 30). A scanner-specific NDB for the generation of the gold standard VBM maps was available also for the test set. The technical performance of CNN-VBM was characterized by the Dice coefficient of CNN-VBM maps relative to VBM maps from scanner-specific VBM. For clinical testing, VBM maps were categorized visually according to the clinical diagnoses in the test set by two independent readers, separately for both VBM methods. Results The VBM maps from CNN-VBM were similar to the scanner-specific VBM maps (median Dice coefficient 0.85, interquartile range [0.81, 0.90]). Overall accuracy of the visual categorization of the VBM maps for the detection of AD or FTLD was 89.8% for CNN-VBM and 89.0% for scanner-specific VBM. Conclusion CNN-VBM without NDB provides a similar performance in the detection of AD- and FTLD-specific atrophy as conventional VBM. Clinical relevance statement A deep convolutional neural network for voxel-based morphometry eliminates the need of scanner-specific normal databases without relevant performance loss and, therefore, could pave the way for the widespread clinical use of voxel-based morphometry to support the diagnosis of neurodegenerative diseases. Key Points • The need of normal databases is a barrier for widespread use of voxel-based brain morphometry. • A convolutional neural network achieved a similar performance for detection of atrophy than conventional voxel-based morphometry. • Convolutional neural networks can pave the way for widespread clinical use of voxel-based morphometry. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00330-023-10356-1. smoothing, a voxel-based two-sample t-test of the individual smoothed GM component image against the GM component images of the NDB was carried out, resulting in a statistical t-map.Age and total intracranial volume were taken into account as nuisance covariates.The total intracranial volume was estimated in each T1w-MRI scan by using a 3D-CNN specifically trained for accurate and stable delineation of the total intracranial volume [2; 3]. 3D-CNN architecture The architecture of the custom 3D-CNN is shown in Figure 1 in the manuscript.The 3D-CNN follows a fully convolutional encoder-decoder (U-net-like) architecture with 3D convolutions with 3x3x3 kernel size.Residual blocks are used in the encoder [4].In addition, deep supervision [5] is employed by including additional segmentation layers at several stages in the decoder.The encoder reduces the spatial feature map size four times (using convolution with stride 2) and doubles the feature map number with each reduction.Starting with 16 feature maps of size 160x160x160 in the first layer, this leads to Eur Radiol (2023) Krüger J, Opfer R, Spies L, Hedderich D, Buchert R. 256 maps of size 10x10x10 in the last encoder layer.The decoder uses convolution layers, followed by nearest-neighbour up-sampling and deep supervision in three layers [5].For the long-range connections between encoder and decoder a feature concatenation is employed.Leaky ReLU [6] is used as activation function in each layer.Due to the rather large patches of 160x160x160 voxels the batch size is 1.Therefore, instance normalization, a special case of group normalization, is used instead of batch normalization. Data augmentation Random combinations of the following data augmentation techniques were used during training of the 3D-CNN to further increase the heterogeneity of the training dataset. ▪ Random left-right flipping of the image patches. ▪ Rotation around the x-, y-or z-axis by an angle randomly chosen between -10 and 10 degrees. ▪ Translation in x-, y-or z-direction by a distance randomly chosen between -5 and 5 mm.▪ Voxel-wise adding of Gaussian random noise with zero mean and variance randomly chosen between 0 and 0.0001.The voxel values were normalized between 0 and 1 (this is required for every CNN) ▪ Simulation of a random bias field.A plane was selected randomly within the 3D volume.For all other planes, the distance to the selected plane was computed.Distance values were scaled to the interval [1, d] with d a random number between 1 and 2. Image voxel values were multiplied with the scaled distance of the plane the voxel was located in. The first three augmentation techniques were applied simultaneously to the input and to the output of the 3D-CNN whereas the last two techniques were applied only to the input image (output unchanged). Application of the 3D-CNN For the application of the 3D-CNN to a T1w-MRI scan, eight (2x2x2) evenly distributed overlapping crops of 160x160x160 mm 3 were taken.For each crop, the predicted class values were computed and Eur Radiol (2023) Krüger J, Opfer R, Spies L, Hedderich D, Buchert R. merged to the entire volume by taking the mean values in the overlapping regions.Each of the 4 output maps (corresponding to the 4 parts of the statistical maps from scanner-specific-VBM, Figure 1 in the manuscript) contains (probability) values between 0 and 1.The output maps corresponding to the 'low significance' part and to the 'high GM density' part of conventional t-maps were ignored.The output maps corresponding to the 'low extrahippocampal GM density' part and to the 'low hippocampal GM density' part were summed voxel-by-voxel to obtain the (final) CNN-VBM map. The voxel intensities in the CNN-VBM map range between 0 and 1.The threshold corresponding to P=.005 for t-maps from scanner-specific-VBM was identified as follows.On a subset of 130 randomly selected cases from the training dataset (2 cases per scanner), the CNN-VBM-map was binarized using varying thresholds between 0.1 and 0.85 (step size 0.05).For each threshold on the CNN-VBM-map, the Dice similarity coefficient of the resulting binarized map with the corresponding conventional gold standard map from scanner-specific-VBM binarized at P=.005 was computed for each of the 130 cases. Prior to computing the Dice similarity coefficient, the binary maps were smoothed by convolution with an isotropic Gaussian kernel with 10 mm full-width-at-half-maximum and then binarized again (all values > 0) in order to reduce the sensitivity with respect to minor, clinically irrelevant differences.The mean Dice similarity coefficient over the 130 cases was maximal for a threshold of 0.40 (Supplementary Figure 4).This value was used for thresholding the CNN-VBM-maps for visual interpretation.Adduru VR, Michael AM, Helguera M, Baum SA, Moore GJ ( 2017	2023-11-10T06:17:37.352Z	2023-11-09T00:00:00.000	{ "year": 2023, "sha1": "f1cbb2b246b8ccb535d2cff15a199d07821cfc57", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s00330-023-10356-1.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "ecce5055c1ec8ace090dd05bc54ce1e90f94e740", "s2fieldsofstudy": [ "Computer Science", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
251712165	pes2o/s2orc	v3-fos-license	Prediction of survival in oropharyngeal squamous cell carcinoma using machine learning algorithms: A study based on the surveillance, epidemiology, and end results database Background We determined appropriate survival prediction machine learning models for patients with oropharyngeal squamous cell carcinoma (OPSCC) using the “Surveillance, Epidemiology, and End Results” (SEER) database. Methods In total, 4039 patients diagnosed with OPSCC between 2004 and 2016 were enrolled in this study. In particular, 13 variables were selected and analyzed: age, sex, tumor grade, tumor size, neck dissection, radiation therapy, cancer directed surgery, chemotherapy, T stage, N stage, M stage, clinical stage, and human papillomavirus (HPV) status. The T-, N-, and clinical staging were reconstructed based on the American Joint Committee on Cancer (AJCC) Staging Manual, 8th Edition. The patients were randomly assigned to a development or test dataset at a 7:3 ratio. The extremely randomized survival tree (EST), conditional survival forest (CSF), and DeepSurv models were used to predict the overall and disease-specific survival in patients with OPSCC. A 10-fold cross-validation on a development dataset was used to build the training and internal validation data for all models. We evaluated the predictive performance of each model using test datasets. Results A higher c-index value and lower integrated Brier score (IBS), root mean square error (RMSE), and mean absolute error (MAE) indicate a better performance from a machine learning model. The C-index was the highest for the DeepSurv model (0.77). The IBS was also the lowest in the DeepSurv model (0.08). However, the RMSE and RAE were the lowest for the CSF model. Conclusions We demonstrated various machine-learning-based survival prediction models. The CSF model showed a better performance in predicting the survival of patients with OPSCC in terms of the RMSE and RAE. In this context, machine learning models based on personalized survival predictions can be used to stratify various complex risk factors. This could help in designing personalized treatments and predicting prognoses for patients. Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and includes all cancers occurring in the mucosa of the oral cavity, oropharynx, larynx, and hypopharynx (1). Unlike other HNSCCs that have shown declines in recent years, oropharyngeal squamous cell carcinoma (OPSCC) has shown a significant increase in incidence worldwide (2). This phenomenon is thought to be owing to an increase in the number of patients with human papillomavirus (HPV)-related OPSCC. A HPV-positive status in patients with OPSCC is associated with a better prognosis than an HPVnegative status (3). However, in addition to the HPV status in patients with OPSCC, other factors potentially affecting the patient prognosis should be considered. Cox regression models have been used to evaluate the independent prognostic factors associated with the survival of patients with OPSCC using available clinical and pathological data. However, Cox regression models were not designed to predict an outcome, but rather to infer the impacts of variables on patient survival (4). Various machine learning methods have been designed to compensate for the limitations of the Cox progression models. Tree-based models are appealing owing to their logical and interpretable structures, as well as their ability to detect the complex interactions between covariates (5). Deep learning-based approaches are based on the automated learning of prognostic factors, without the need for prior assumptions on known factors (6). Thus, machine learning approaches may be considered as better approaches to predicting patient survival. However, it cannot be said which model is the most predictive of a prognosis; thus, they need to be analyzed and compared together. In this study, we aimed to analyze the clinical data of patients with OPSCC using various machine learning models, aiming to determine the appropriate models for predicting survival in patients with OPSCC. We hypothesized that (1) investigation of a thousand OPSCC patients collected from the Surveillance, Epidemiology, and End Results (SEER) database would help to comprehensively consider the various variables, and (2) comparing the c-index, integrated Brier score (IBS), root mean square error (RMSE), and mean absolute error (MAE) in the machine learning models applied in this study would help in identifying the appropriate models. Patient cohort The patient clinical data were obtained from the SEER program of the National Cancer Institute in the United States (https://seer.cancer.gov/, approval number:20922-Nov 2019). The inclusion criteria were as follows: (1) diagnosis of oropharyngeal cancer between 2004 and 2016, (2) tumor site in the oropharynx and/or tonsil, (3) histologic behavior of squamous cell carcinoma, and (4) patients had been tested for HPV status. Patients were excluded if they had missing data on survival months, cause of death, age, sex, tumor grade, tumor size, node status, or treatment methods (such as cancer-directed surgery, surgery, neck dissection, radiation therapy, or chemotherapy). Variable selection and reconfiguration A total of 13 variables were included in this study: age, sex, tumor grade, tumor size, neck dissection, radiation therapy, cancer-directed surgery, chemotherapy, T stage, N stage, M stage, clinical stage, and HPV status. The age and tumor size were recorded using continuous methods (years and mm, respectively). The T-, N-, and clinical staging were reconstructed based on the American Joint Committee on Cancer (AJCC) Staging Manual, 8th Edition (7), and were categorized as "T0, TX, T1, T2, T3, T4, T4a, T4b," "N0, NX, N1, N2, N2a, N2b, N2c, N3," and "Stage I, II, III, IV, IVA, IVB, IVC," respectively. These data were obtained using T-and Nand clinical staging according to the AJCC 7th Edition regarding the tumor size, regional node positive status, HPV status, and evaluation data provided by the SEER program. Whether neck dissections, radiation therapy, cancer-directed therapy, and chemotherapy were performed were also determined by referring to the SEER program. Applying multiple machine learning methods The patients were randomly assigned to a development or test dataset in a 7:3 ratio. Three frequently and currently used machine learning methods, i.e., the extremely randomized survival tree (EST), conditional survival forest (CSF), and DeepSurv models were used to predict the overall and diseasespecific survival in patients with OPSCC. A 10-fold crossvalidation on a development dataset was used to build the training and internal validation data for all models. We evaluated the predictive performance of each model using the test datasets. The 13 variables specified above were used as the input data. The seven-year survival, cancer-specific death, and non-cancer-specific death data were used as the output data. Figure 1 depicts the overall workflow of the patient selection and application of the machine learning methods. Evaluation of model performance The predictive performances of the three machine learning models were evaluated using the C-index, IBS, RMSE, and MAE. A C-index close to or lower than 0.5 means that the performance of the model is no better than random outputs, and a higher Cindex indicates a better performance of the model. IBSs are calculated using prediction error curves ranging from 0 (absolute performance) to 0.25 (8). The RMSE and MAE depict the differences between the actual and predicted prediction errors and survival values in each model. The RMSE gives extra weight to large errors, and the MAE gives equal weight to all errors. Smaller RMSE and MAE values indicate better performance (9). All machine learning models were implemented in Python version 3.6 (Python Software Foundation for Statistical Computing, Wilmington, Delaware, USA) using PySurvival (10). Flowchart of patient selection and study design. OPSCC, oropharyngeal squamous cell carcinoma; SEER, surveillance, epidemiology and end results; EST, Extremely randomized survival tree; CSF, Conditional survival forest. Characteristics of the patients First, the data of 7691 OPSCC patients who had been tested for HPV status were collected from the SEER program between 2004 and 2016. Moreover, 3652 patients were excluded because they were missing data for one or more of the 13 variables. Thus, 4039 OPSCC patients were analyzed using various machine learning methods ( Figure 1). Table 1 describes the characteristics of the 4039 patients with OPSCC. In all patients, the age ranged from 0 to 94 years (mean =58.43 year), and 82.87% were male. The tumor size was described using mm units, and patients with microscopic focus or focus-only tumor size were assigned to the 1-mm tumor size. Approximately 34.96% of patients had undergone selective or radical neck dissection, regardless of chemotherapy and/or radiotherapy. Radiation therapy, cancer-directed surgery, and chemotherapy were performed for 88.19%, 61.43%, and 69.4% of the patients, respectively. The HPV-negative patients (25.06%) according to AJCC 8th Edition remained the same as those for the AJCC 7th Edition as listed in the SEER database, but the HPV-positive patients (74.94%) were modified in the new AJCC 8 th Edition data according to the AJCC 7th Edition and the tumor size, node size, and regional node evaluation section. The mean survival months were 34.72 months in the seven-year follow up. Application and analysis of each model In the EST model, we used 20 minimum node sizes, 200 trees, and 1000 random splits. The risk factors for OPSCC were calculated using the EST model. The risk scores ranged from 0 to 4, and patients were categorized into three groups (low-, medium-, and high-risk) according to these scores ( Figure 2A). One patient was randomly selected from each group for our new risk-related staging system, and survival curves for 72 months are depicted for these three patients ( Figure 2B). We further analyzed the importance of each variable (Table 2), and cancer-directed surgery showed the highest importance (13.88), followed by HPV status (11.11), radiation therapy (9.06), T stage (8.41), and M stage (8.35). In the CSF model, we used 20 minimum node sizes, 200 trees, and a 0.05 alpha. Similar to the EST model, the risk factors for the patients were calculated using the CSF model. The risk scores ranged from 0 to 4, and the patients were categorized into three groups ( Figure 2C). Using the same methods, survival curves were obtained for the three patients ( Figure 2D). Cancerdirected surgery showed the highest importance (11.53), similar to the EST model; however, the tumor size (8.80) and age (7.08) also showed high importance compared to the EST model. In the DeepSurv model, we used xav_uniform as the initial method and an adaptive moment estimation optimizer with a learning rate of 0.001 for the neural network, with one hidden layer activation function = "BentiIdentity" (for the input-hidden layer) and node size = 150 (11). Dropout, batch normalization, and L1 and L2 regularization were performed during the training. Comparison of each model performance As mentioned above, higher values of the C-index and lower values of the IBS, RMSE, and MAE indicate a better performance of a machine learning model. The C-index was the highest in the DeepSurv model (0.77), followed by the EST and CSF models (0.76 and 0.72, respectively). The IBS was the lowest in DeepSurv model (0.08), followed by the EST and CSF models (0.10 and 0.10, respectively). However, the RMSE and RAE (depicting the differences between the actual and predicted survival values) were the lowest in the CSF model (13.398 and 10.794, respectively), followed by the EST (22.611 and 13.817, respectively) and DeepSurv models (39.744 and 34.230, respectively) ( Table 3 and Figure 3). Discussion The therapeutic methods for early-stage OPSCC are surgery or radiation therapy, and appropriate methods are commonly selected according to the patient and clinician preferences and conditions (12). Parsons et al. found through a meta-analysis that patients with OPSCC experienced similar overall survival rates regardless of whether they were treated with surgery or definitive radio(chemo)therapy (13). In the case of locally advanced OPSCC according to the National Cancer Database, primary surgery with radio(chemo)therapy showed improved survival compared to primary radiation-based treatment (14). In addition, patients with HPV-negative OPSCC had a significantly worse prognosis than those with HPV-positive OPSCC (15). In addition, other factors, including age, sex, and T/N/M staging also affected the prognosis of patients with OPSCC. Thus, we aimed to predict the prognoses of patients with OPSCC by comprehensively analyzing various prognostic factors using frequently used machine-learning models. In this study, we used the SEER database because it provides large population-based information that cannot be obtained from just one medical center. The SEER cohort provided staging information based on the AJCC 7 TH Edition, which does not reflect a new tumor grading system according to HPV status. Other studies showing the factors influencing the prognosis of OPSCC patients also used TNM staging based on the AJCC 7 th Edition. However, we assumed that reconstructing the staging system to suit the new AJCC 8 th Edition would better demonstrate their impacts on prognosis in OPSCC when considering HPV status. In addition, clinical data such as age and sex, detailed tumor size, grade, and applied therapeutic modalities (chemotherapy, neck dissection, radiotherapy, and cancer-directed surgery) were obtained and analyzed comprehensively. Until recently, conventional Cox regression models were mostly used to predict the prognoses of OPSCC patients (16,17). However, two effects might be simplified or overlooked in the process of interpreting Cox regression models: "Effect modification," i.e., the causal effect of one exposure within the strata of another exposure of interest and "Interaction," i.e., the causal effect of two exposures together in an area of interest (18). In addition, the Cox regression model cannot recognize the complex nonlinear relationships between variables. Statistically reinforced machine learning approaches would be beneficial for compensating for problems such as higher-order interactions, context dependencies, nonlinearity, and variable interactions (19). Thus, we analyzed and compared three frequently used machine learning methods -EST, CSF, and DeepSurv-to consider nonlinearity and reduce the interactions and effect modifications in the variables of the SEER cohort. Interestingly, cancer-directed surgery was the most important variable for predicting the prognosis of patients with OPSCC in both the EST and CSF models. HPV status and radiation therapy were also important variables in both the EST and CSF models. These results suggest that selection of an appropriate therapeutic modality, including surgery and radiotherapy, might be more important in predicting the prognosis of patients with OPSCC than the TNM staging alone. In addition, we evaluated the performance of each machine learning method using variable scales: the C-index, IBS, RMSE, and MAE. The C-index and IBS results indicated that the DeepSurv model showed better performance in predicting the survival of patients with HNSCC. Hao et al. also found that the DeepSurv model performed better than other machine learning models in terms of the C-index, IBS, and area under the curve for survival prediction performance (20). The DeepSurv model uses a feed-forward network to learn the relationships between the covariates and hazard function, thus capturing the time-dependent influences of the covariates on survival (21). However, in terms of the RMSE and RAE, the CSF model performed better than the EST and DeepSurv models. The EST includes a random survival forest, uses equal weights on all terminal nodes, and analyzes time-to-event data comprising covariates with many split-points. The CSF model was superior in analyzing time-to-event data that consisted of covariates with fewer split points (22). Deep learning models have exhibited unprecedented performance in quite a few applications in academia and industry, but there are many disadvantages, such as their complex geometric transformations, which often require big data (23). Thus, it might be important to analyze the various survival models together so that they can be used to compensate for each other. Our study had the following limitations. First, the SEER database showed limited information; for example, it did not include data regarding smoking and alcohol histories, or recurrence after treatment. Above all, the SEER database did not include information about detailed chemotherapy methods and extranodal extension, which are important prognostic factor of OPSCC. To compensate limited information, we analyzed all available treatment methods such as neck dissection, radiation, Nevertheless, to the best of our knowledge, this is the first study to analyze the prognosis of OPSCC patients using various machine learning methods based on the AJCC 8th Edition. We found that therapeutic methods, such as surgical therapy and radiotherapy, had a more important effect on prognosis than T/N/M staging alone. In addition, we found that various machine learning models showed different important factors affecting the prognosis. Further studies including more detailed clinical and pathological data will help improve the accuracy of survival prediction using the various machine learning models. In conclusion, we demonstrated that the survival predictions from various machine-learning models (EST, CSF, and DeepSurv) are feasible and accurate. In particular, the CSF model showed a better performance in predicting the survival of patients with OPSCC in terms of the RMSE and RAE. Machine learning models based on personalized survival prediction can also be used to stratify various complex risk factors. This will help in designing personalized treatments and predicting the prognoses of patients. Data availability statement Publicly available datasets were analyzed in this study. This data can be found here: https://seer.cancer.gov/. Author Contributions SK: study design, data collection and analysis, writing, revising the article, and final approval of the version; JK: data collection and analysis, revising article, and final approval of the version; Y-GE: data collection and analysis, revising article, and final approval of the version; YL: study design, data collection and analysis, revising article, final approval of the version, and supervision of the study. All authors contributed to the article and approved the submitted version.	2022-08-22T13:50:23.021Z	2022-08-22T00:00:00.000	{ "year": 2022, "sha1": "64d57ba4f38c222775f8bbcc448e8d338b34d78b", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Frontier", "pdf_hash": "64d57ba4f38c222775f8bbcc448e8d338b34d78b", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
234019092	pes2o/s2orc	v3-fos-license	Numerical Investigation of Residual Stresses in Welded Thermoplastic CFRP Structures Using thermoplastics as the matrix in carbon fiber-reinforced polymers (CFRP) offers the possibility to make use of welded joints, which results in weight savings compared to conventional joining methods using mechanical fasteners. In this paper, the resulting temperature distribution in the material due to resistance welding is investigated by transient finite element (FE) simulations. To examine the effects on the component structure, a numerical modeling approach is created, which allows determining the residual stresses caused by the welding process. It is shown that the area of the structure, especially near the joining zone, is highly affected by the process, especially in terms of residual stresses. In particular, the stresses perpendicular to the fiber direction show failure relevant values up to a maximum of 221 MPa, which might lead to the formation of microcracks in the matrix. In turn, that is assumed to be critical in terms of the fatigue of welded composite structures. Thus, the suggested modeling approach provides residual stresses that can be used to determine their effects on the strength, structural stability, and fatigue of such composite structures. In a subsequent step, these findings could play an important role in the design process of thermoplastic composite structures. Introduction The use of thermoplastic matrix materials in fiber-reinforced polymers is increasingly becoming the focus of applied aerospace research. Compared to epoxy resin systems predominantly used in the industrial environment today, there are far-reaching advantages when using thermoplastics. First of all, the possibility of in situ consolidation while using thermoplastics plays a decisive role, which results in significantly shorter cycle times in the production of such components. Secondly, the recyclability of thermoplastic materials is another significant factor [1]. Both aspects are to be considered concerning the possible production and resource efficiency that can be achieved and thus in turn represent key aspects with regard to environmentally friendly aviation. Another benefit of thermoplastic materials is their weldability. Compared to conventional joining techniques using mechanical fasteners [2,3], significant weight advantages can be achieved through welding, which in turn represents a concrete action with respect to eco-lightweight design and, as a result of lower fuel consumption, leads to lower CO 2 emissions. Several methods have been investigated on an experimental and numerical level so far [4][5][6][7][8][9][10][11][12]. While in the paper at hand, the resistance welding method is examined and described in detail later, further relevant technologies will be described in the following as well. One of them is the induction welding method. For the induction welding of thermoplastics, the electromagnetic field of an external inductor heats the heating element 2 of 10 in the joining zone. This leads to the melting of the matrix and in consequence, the adherends can be joined under pressure. In [4], a procedure based on numerical methods has been developed, allowing a more precise understanding of the process parameters involved. The joining quality is evaluated based on single-lap-shear tests (SLS tests) and microscopic examination of the failure surfaces [5]. Another well-studied method is the laser welding of thermoplastics, for which a laser as a heat source is used. In [6], a thermal finite element (FE) model of a laser transmission welding process is developed using an unreinforced thermoplastic. This allows determining the size of the heat-affected zone and investigating the resulting internal stresses. Furthermore, a process model under consideration of the composite's microstructure was developed, which enables clarifying the correlation between fiber volume content and welding quality [7]. Familiarization with the literature shows that ultrasonic welding could also play an important role in future applications. For ultrasonic welding, the vibrations generated by an ultrasonic generator are converted into mechanical vibrations via sonotrode. The targeted introduction of these vibrations in the area of the joining zone leads to the melting of the matrix. With the help of experimental methods, it has so far been shown that this variant, which is usually implemented in a spot welding topology, could be a useful alternative to conventional rivets [8]. A process modeling procedure was shown in [9]. However, there still is no valid concept for how the ultrasonic welding process could be used for large continuous joints. According to the current state of research, one of the most promising variants, and therefore one of the best understood, is resistance welding. Here, a resistance element located in the joining zone is heated by current feed so that the matrix is melted. Subsequently, the adherends are joined under pressure as a result of cooling [10]. For this process, it was possible to demonstrate, mainly on an experimental level, the potential of its use in aircraft construction. For example, possible process windows could be identified, and the quality of the joint is determined by optical observation of the failure surfaces and SLS tests. In addition, the first numerical investigations were carried out [11,12]. All these joining processes have in common that a considerable amount of heat is introduced into the material during the process. As a result, different cooling rates within the material, different fiber orientations of the individual layers, and different coefficients of expansion of the fiber and matrix cause stresses to develop without external forces, so-called internal or residual stresses. These occur on different levels, which are briefly explained in the following. First of all, the residual stresses have to be considered on a micromechanical level. These occur primarily due to the different thermal expansion coefficients of fiber and matrix and thus arise at the fiber-matrix interface. The second level, which is given by the macro-mechanical relationships in the laminate, is also characterized by the occurrence of internal stresses. These occur in particular with different fiber orientations within the laminate and thus result in interlaminar shear forces. The third level of residual stress can be named global stresses. These become relevant in the case of relatively thick laminates, in which the high thickness leads to areas of different cooling speeds and thus forming stress gradients [13,14]. For the investigation of residual stresses, several methods are applicable. In [15], the nanoindentation test was used to measure the field distribution of the residual stress in functionally graded ceramic-metal composites. In the following step, a comparison of the results with corresponding finite element method (FEM) simulations showed a qualitative agreement. The presented work examines the connection between the method of resistance welding and the occurrence of residual stresses on a macro-mechanical level. In particular, the high amount of thermal energy that is introduced into the laminate during the welding process inevitably leads to the development of internal stresses. First, a model of the process of resistance welding using FE-based methods to determine the temperature distributions responsible for the development of the residual stresses will be displayed. This allows the investigation of the relationship between the welding parameters and the thermal load of the structure. Moreover, the process model can be used as a basis for research on process parameter optimization. Furthermore, a numerical method that enables the resulting residual stresses to be determined on a mesoscopic level and evaluated for further investigations is presented thereupon. These results serve for the investigation of the welding's thermomechanical effect on the component concerning preloading and material damage. In addition, the residual stresses represent useful data for future experimental validation and comprehensive structural design. Materials and Methods The composite material used for the investigations in this paper is the unidirectional prepreg Aromatic Polymer Composite 2 (APC-2) certified for aircraft structures, consisting of high strength, standard modulus carbon fibers (AS-4) with a nominal fiber volume content of 61%, and the thermoplastic matrix material Polyether Ether Ketone (PEEK). The polymer has semi-crystalline properties with a glass transition temperature of 145 • C and a melting temperature of 390 • C. All calculations are carried out using temperaturedependent material properties taken from [16]. The elastic and thermal properties at room temperature used are given in Table 1. A simplified resistance welding process is modeled using the commercial Finite Element code ABAQUS ® . Two coupon specimens according to ASTM D1002 standard are used as adherends. To reduce the computational time for the modeling procedure, both adherends are assumed to behave similarly, so that only one coupon specimen is modeled with adequate boundary conditions. The schematic resistance welding process based on a typical experimental setup and the corresponding simplification for modeling is shown in Figure 1. resulting residual stresses to be determined on a mesoscopic level and evaluated for further investigations is presented thereupon. These results serve for the investigation of the welding's thermomechanical effect on the component concerning preloading and material damage. In addition, the residual stresses represent useful data for future experimental validation and comprehensive structural design. Materials and Methods The composite material used for the investigations in this paper is the unidirectional prepreg Aromatic Polymer Composite 2 (APC-2) certified for aircraft structures, consisting of high strength, standard modulus carbon fibers (AS-4) with a nominal fiber volume content of 61%, and the thermoplastic matrix material Polyether Ether Ketone (PEEK). The polymer has semi-crystalline properties with a glass transition temperature of 145 °C and a melting temperature of 390 °C. All calculations are carried out using temperature-dependent material properties taken from [16]. The elastic and thermal properties at room temperature used are given in Table 1. A simplified resistance welding process is modeled using the commercial Finite Element code ABAQUS ® . Two coupon specimens according to ASTM D1002 standard are used as adherends. To reduce the computational time for the modeling procedure, both adherends are assumed to behave similarly, so that only one coupon specimen is modeled with adequate boundary conditions. The schematic resistance welding process based on a typical experimental setup and the corresponding simplification for modeling is shown in Figure 1. The part is modeled using quadratic hexahedral, coupled temperature-displacement elements. The geometry illustrated in Figure 1 is similarly implemented in the width direction to create a 3D model. No edge effects in the width direction are taken into account, as the considered specimen is assumed to be cut from a plate so that constant conditions can be applied. The heat generated during welding is applied directly to the joining zone of the coupon specimen and is simplified as a surface heat flux. It results from the joule heating effect of a resistive element (i.e., carbon fibers, steel mesh) and is assumed to be uniformly distributed at the overlap area. The pressure for consolidation is assumed to be The part is modeled using quadratic hexahedral, coupled temperature-displacement elements. The geometry illustrated in Figure 1 is similarly implemented in the width direction to create a 3D model. No edge effects in the width direction are taken into account, as the considered specimen is assumed to be cut from a plate so that constant conditions can be applied. The heat generated during welding is applied directly to the joining zone of the coupon specimen and is simplified as a surface heat flux. It results from the joule heating effect of a resistive element (i.e., carbon fibers, steel mesh) and is assumed to be uniformly distributed at the overlap area. The pressure for consolidation is assumed to be constant in length and width direction and is applied directly to the specimen. The thermal boundary conditions are represented by free convection due to ambient air temperature at the free surfaces, while the area between the specimen and the insulator is modeled by heat transfer from carbon fiber-reinforced polymer (CFRP) to wood, which is a typical insulator material in this context. The joining surface is assumed to be fixed in all degrees of freedom. The specimen consists of a [+45 • /0 • /−45 • /90 • ] 2S quasi-isotropic layup. Every single unidirectional layer is represented as one quadratic hexahedral coupled temperature-displacement element in the thickness direction. This results in an overall thickness of t = 2 mm. The specimen's length is l = 101.6 mm, and the width is w = 25.4 mm. The overlap length is assumed to be l lap = 25.4 mm. Furthermore, the global coordinate system definition ( Figure 2a) as well as the layup used for the simulation (Figure 2b) need to be addressed. While the global system is characterized by the x, y, and z coordinates, the local layer system is represented by the I-coordinate in the fiber direction, the II-coordinate in-plane perpendicular to the fiber direction, and the III-coordinate outof-plane perpendicular to the fiber direction. J. Compos. Sci. 2021, 5, x FOR PEER REVIEW 4 of 10 constant in length and width direction and is applied directly to the specimen. The thermal boundary conditions are represented by free convection due to ambient air temperature at the free surfaces, while the area between the specimen and the insulator is modeled by heat transfer from carbon fiber-reinforced polymer (CFRP) to wood, which is a typical insulator material in this context. The joining surface is assumed to be fixed in all degrees of freedom. The specimen consists of a [+45°/0°/−45°/90°]2S quasi-isotropic layup. Every single unidirectional layer is represented as one quadratic hexahedral coupled temperature-displacement element in the thickness direction. This results in an overall thickness of t = 2 mm. The specimen's length is l = 101.6 mm, and the width is w = 25.4 mm. The overlap length is assumed to be l lap = 25.4 mm. Furthermore, the global coordinate system definition ( Figure 2a) as well as the layup used for the simulation (Figure 2b) need to be addressed. While the global system is characterized by the x, y, and z coordinates, the local layer system is represented by the I-coordinate in the fiber direction, the II-coordinate in-plane perpendicular to the fiber direction, and the III-coordinate out-of-plane perpendicular to the fiber direction. The investigation of the residual stresses in the components resulting from the cooling after the welding process is based on reduced elastic properties of the matrix at welding temperature. This approach requires the definition of consolidation levels depending on the elastic properties of the matrix which again depend on the processing temperature, thus discretizing the cooling process. Afterward, the matrix-dominated mechanical properties are updated according to the consolidation level. The resulting stress distribution represents the initial stress level in the composite material after welding and right before cooling, respectively. Due to the re-consolidation in the cooling process, heterogeneously distributed residual stresses in the component arise. Linear thermo-mechanical analyses are carried out, and the entire modeling procedure is divided into two steps. Firstly, a heating step based on the resistance welding simulation is carried out. After the melting temperature in the joining zone is surpassed, the second step of the procedure takes place. The input power is turned off or in other words, the component is cooling down due to free convection without external heat flux under pressure. The recalculated mechanical properties are used as the initial material property. The resulting stresses of the laminate are considered to be the residual stress states caused by the welding process. The investigation of the residual stresses in the components resulting from the cooling after the welding process is based on reduced elastic properties of the matrix at welding temperature. This approach requires the definition of consolidation levels depending on the elastic properties of the matrix which again depend on the processing temperature, thus discretizing the cooling process. Afterward, the matrix-dominated mechanical properties are updated according to the consolidation level. The resulting stress distribution represents the initial stress level in the composite material after welding and right before cooling, respectively. Due to the re-consolidation in the cooling process, heterogeneously distributed residual stresses in the component arise. Linear thermo-mechanical analyses are carried out, and the entire modeling procedure is divided into two steps. Firstly, a heating step based on the resistance welding simulation is carried out. After the melting temperature in the joining zone is surpassed, the second step of the procedure takes place. The input power is turned off or in other words, the component is cooling down due to free convection without external heat flux under pressure. The recalculated mechanical properties are used as the initial material property. The resulting stresses of the laminate are considered to be the residual stress states caused by the welding process. Welding Simulation As mentioned in the cited publications, the weld's quality is highly affected by the temperature magnitude and distribution in the joining zone. The performed welding process simulation leads to time-temperature data for the considered component so that the temperature of every single element at a certain time during heating and cooling can be assessed. In Figure 3, the temperature data of three selected measuring points (P1, P2, and P3) are presented. All three points are located in the mid-plane in the width direction of the specimen. It can be seen that the melting temperature of the matrix (T melt = 390 • C) is reached after t weld = 25 s in the middle of the joining zone (P1). Furthermore, it can be noticed that with this welding parameter configuration, only a section of the joining surface is melted, as the temperature in P2 does not reach the melting temperature. Afterward, the input power is modeled to be turned off, and the cooling of the specimen due to free convection begins. While there is a clear drop in temperature to observe for P1 and P2 after turning off the input power, a slight increase in temperature can be noticed for P3, which results from the heat flow in the material. As mentioned in the cited publications, the weld's quality is highly affected by the temperature magnitude and distribution in the joining zone. The performed welding process simulation leads to time-temperature data for the considered component so that the temperature of every single element at a certain time during heating and cooling can be assessed. In Figure 3, the temperature data of three selected measuring points (P1, P2, and P3) are presented. All three points are located in the mid-plane in the width direction of the specimen. It can be seen that the melting temperature of the matrix (T melt = 390 °C) is reached after t weld = 25 s in the middle of the joining zone (P1). Furthermore, it can be noticed that with this welding parameter configuration, only a section of the joining surface is melted, as the temperature in P2 does not reach the melting temperature. Afterward, the input power is modeled to be turned off, and the cooling of the specimen due to free convection begins. While there is a clear drop in temperature to observe for P1 and P2 after turning off the input power, a slight increase in temperature can be noticed for P3, which results from the heat flow in the material. In Figure 4, the temperature distribution in the specimen during welding is shown. It can be noticed that the heat builds up in the joining zone and nearby areas of the specimen due to the local heat flow input and the insulator. Only with a certain delay, as it can be seen in Figure 3 as well, the heat is transported along the specimen's length direction. Although the heating element generates heat uniformly at the overlap area, the temperature distribution is not uniform. Instead, due to different heat transfer conditions between the edge and the middle of the joint, there is a maximum temperature at the middle and lower ones at the edges of the overlap. As a result of the non-uniformity, different consolidation degrees and thus joint quality along the overlap length will be achieved. Similar behavior was investigated in [17]. In Figure 4, the temperature distribution in the specimen during welding is shown. It can be noticed that the heat builds up in the joining zone and nearby areas of the specimen due to the local heat flow input and the insulator. Only with a certain delay, as it can be seen in Figure 3 as well, the heat is transported along the specimen's length direction. Although the heating element generates heat uniformly at the overlap area, the temperature distribution is not uniform. Instead, due to different heat transfer conditions between the edge and the middle of the joint, there is a maximum temperature at the middle and lower ones at the edges of the overlap. As a result of the non-uniformity, different consolidation degrees and thus joint quality along the overlap length will be achieved. Similar behavior was investigated in [17]. The time-temperature curve for two different power input levels for measuring point P1 is shown in Figure 5. While the melting temperature for an input power of 60 kW/m 2 is reached after 25 s, it takes only 18 s for an input power of 80 kW/m 2 . The cooling process follows a similar course. However, comparing these data with the ones shown in Figure 5, it can be assumed that due to the thermal resistance of the material, the heat cannot be transported fast enough, resulting in a smaller melted area, as the material needs to be prevented from degradation. Such data have to be taken into account for investigating an optimal processing window for the resistance welding of thermoplastic composites. When it comes to the influence of crystallization and different heating and cooling rates on mechanical properties, the estimation of transient temperature data for the process in an early design stage plays a significant role. os. Sci. 2021, 5, x FOR PEER REVIEW 6 of 10 The time-temperature curve for two different power input levels for measuring point P1 is shown in Figure 5. While the melting temperature for an input power of 60 kW/m 2 is reached after 25 s, it takes only 18 s for an input power of 80 kW/m 2 . The cooling process follows a similar course. However, comparing these data with the ones shown in Figure 5, it can be assumed that due to the thermal resistance of the material, the heat cannot be transported fast enough, resulting in a smaller melted area, as the material needs to be prevented from degradation. Such data have to be taken into account for investigating an optimal processing window for the resistance welding of thermoplastic composites. When it comes to the influence of crystallization and different heating and cooling rates on mechanical properties, the estimation of transient temperature data for the process in an early design stage plays a significant role. The time-temperature curve for two different power input levels for measuring point P1 is shown in Figure 5. While the melting temperature for an input power of 60 kW/m 2 is reached after 25 s, it takes only 18 s for an input power of 80 kW/m 2 . The cooling process follows a similar course. However, comparing these data with the ones shown in Figure 5, it can be assumed that due to the thermal resistance of the material, the heat cannot be transported fast enough, resulting in a smaller melted area, as the material needs to be prevented from degradation. Such data have to be taken into account for investigating an optimal processing window for the resistance welding of thermoplastic composites. When it comes to the influence of crystallization and different heating and cooling rates on mechanical properties, the estimation of transient temperature data for the process in an early design stage plays a significant role. Residual Stress Analysis The welding process simulation is followed by a layerwise residual stress analysis. As a result of the high processing temperatures needed for the consolidation of thermoplastics, residual stresses arise and thus need to be investigated in a first step. Based on the proposed approach, the melted material sections are stress-free. The calculated residual stresses arise only due to the cooling process. Selected results are shown and discussed in the following. In Figure 6, the residual stress state of the 0 • layers, with respect to the global coordinate system, along the specimen's x-direction are presented. The data were collected in the mid-plane in the y-direction (y = 0.5w) for each layer at the center of each ply. The specimen's free end is located at x/l = 0. The overlap-zone reaches from x/l = 0.75 to x/l = 1. ual stresses arise only due to the cooling process. Selected results are shown and discussed in the following. In Figure 6, the residual stress state of the 0° layers, with respect to the global coordinate system, along the specimen's x-direction are presented. The data were collected in the mid-plane in the y-direction (y = 0.5w) for each layer at the center of each ply. The specimen's free end is located at x/l = 0. The overlap-zone reaches from x/l = 0.75 to x/l = 1. Looking at the stresses in the fiber direction, there is a strong influence of the applied pressure in the area of the joining zone to notice. Especially in layer 2, there are high compressive stresses at the edge of the joining zone, while the stress state in between shows hyperbolic behavior with predominantly tensile residual stresses. This behavior is similar for all 0° layers. Considering the residual stresses transverse to the fiber direction, a similar stress trend for all layers can be noticed. From the free edge to the beginning of the joining zone, the residual stresses are close to zero. As shown in the welding simulation, the matrix in this area does not reach temperatures above the melting temperature. The more the area was heated, the higher the stresses. In layer 2, a maximum residual transverse stress of σ II = 221 MPa is reached. In Figures 7 and 8, the residual stresses for the 90° layers and +45° layer are depicted. It can be stated that all layers show comparable characteristics. The σ I stresses seem to be highly influenced by the applied pressure, which is needed for the consolidation of the adherends in the joining zone during cooling. Especially the peaks at the edge of the joining zone and the effect of the pressure put on them need to be considered in detail for future design steps. Looking at the stresses in the fiber direction, there is a strong influence of the applied pressure in the area of the joining zone to notice. Especially in layer 2, there are high compressive stresses at the edge of the joining zone, while the stress state in between shows hyperbolic behavior with predominantly tensile residual stresses. This behavior is similar for all 0 • layers. Considering the residual stresses transverse to the fiber direction, a similar stress trend for all layers can be noticed. From the free edge to the beginning of the joining zone, the residual stresses are close to zero. As shown in the welding simulation, the matrix in this area does not reach temperatures above the melting temperature. The more the area was heated, the higher the stresses. In layer 2, a maximum residual transverse stress of σ II = 221 MPa is reached. In Figures 7 and 8, the residual stresses for the 90 • layers and +45 • layer are depicted. It can be stated that all layers show comparable characteristics. The σ I stresses seem to be highly influenced by the applied pressure, which is needed for the consolidation of the adherends in the joining zone during cooling. Especially the peaks at the edge of the joining zone and the effect of the pressure put on them need to be considered in detail for future design steps. All stresses shown arise due to the thermal history of the component effected by the welding process without external mechanical loads. While the stresses in the fiber direction arise in moderate magnitudes concerning the ultimate strength (R I,ult = 2070 MPa) and structural integrity of such structures, the transverse residual stresses are assumed to be critical. The maximum value is σ II,max = 221 MPa, observed in layers 1 and 2, which are the layers right next to the heating element. This is a multiple of the ultimate transverse strength of the used unidirectional CFRP layer (R II,ult = 90 MPa) and herewith leads to matrix cracks in the loaded area. Based on the determined stresses, matrix cracks will appear in every single layer, especially in the laminate area between the insulator and heating element. All stresses shown arise due to the thermal history of the component effected by the welding process without external mechanical loads. While the stresses in the fiber direction arise in moderate magnitudes concerning the ultimate strength (R I ,ult = 2070 MPa) and structural integrity of such structures, the transverse residual stresses are assumed to be critical. The maximum value is σ II ,max = 221 MPa, observed in layers 1 and 2, which are the layers right next to the heating element. This is a multiple of the ultimate transverse All stresses shown arise due to the thermal history of the component effected by the welding process without external mechanical loads. While the stresses in the fiber direction arise in moderate magnitudes concerning the ultimate strength (R I ,ult = 2070 MPa) and structural integrity of such structures, the transverse residual stresses are assumed to be critical. The maximum value is σ II ,max = 221 MPa, observed in layers 1 and 2, which are the layers right next to the heating element. This is a multiple of the ultimate transverse Discussion and Conclusions The presented modeling approach of the resistance welding process represents an effective procedure to estimate the transient temperature distribution in welded thermoplastic CFRP components for the application in an early design stage. Moreover, the approach allows being adopted to almost any adherend geometry. However, the simplification of the process in the FE model will inevitably lead to certain deviations from the actual conditions. For instance, the thermal boundary conditions will need to be validated by experimental methods. Furthermore, the power input simplified as surface heat flux is modeled as a thermal load applied immediately to the specimen, while in the case of an experimental investigation, a certain delay regarding the heat flow development by the resistance element is to be expected. For a more profound investigation, a far more resource-intensive Multiphysics Simulation is necessary, which then should model the actual resistance element and the joule heating effect. Based on the presented procedure and the results, a numerical parameterized optimization of the process parameters with regard to welding time, welding temperature, input power, and the melted area should be carried out. A modeling approach for the investigation of residual stresses due to cooling after welding was proposed, and first results were shown. While the stresses in fiber direction predominantly arise in moderate magnitudes as tensile as well as compression stresses, the stresses in the transverse direction are purely high-tensile stresses. In particular, the stresses near the joining zone, which are a multiple of typical transverse strength, presumably lead to microcracks of the matrix. This pre-damage of the laminate might have had a significant effect on the strength and thus needs to be further investigated in future research. Especially, the fatigue behavior of welded structures possibly is highly affected by the welding-induced microcracks. For further investigation of residual stresses based on numerical methods, the effect of the applied material model needs to be clarified in detail. The interaction of plasticity and residual stresses constitutes a key aspect for thermomechanical simulations and thus has to be considered for ongoing activities by all means. Finally, future work on this topic should focus on the effects of residual stresses on the strength and stability of welded components and thus investigate the importance of considering residual stresses during the design process of thermoplastic composite structures. Furthermore, the experimental validation of the numerical results needs to be carried out. In particular, the experimental investigation and determination of residual stresses bring along many challenges. This leads to a very small amount of published and accessible data that can be used for validation of FE models calculating residual stresses. Consequently, the design of concepts for the experimental investigation of residual stresses along with the validation of numerical methods represents another significant issue for future research. Funding: The authors thankfully acknowledge the financial and organizational support of the project JoinTHIS by the federal state of Lower Saxony and the European Regional Development Fund (ERDF). Furthermore, we acknowledge support by the German Research Foundation and the Open Access	2021-05-10T00:03:18.371Z	2021-02-02T00:00:00.000	{ "year": 2021, "sha1": "d2a1af76bc3d3dd813f197c6d219ebb498290833", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2504-477X/5/2/45/pdf", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "aa653d92d117371573ef985d929c7ec2ae308c83", "s2fieldsofstudy": [ "Engineering", "Materials Science" ], "extfieldsofstudy": [ "Materials Science" ] }
259796869	pes2o/s2orc	v3-fos-license	Socio-Psychological Aspects of Anorexia Nervosa Aim. The main aim of the research is to identify and evaluate the quality of life of patients with an eating disorder – anorexia nervosa. In an analytical and descriptive way, the authors determine the level of various areas of life of patients who suffer from anorexia nervosa. Methods. The subjects of the study were participants suffering from anorexia nervosa. Based on qualitative research through unstructured interviews, the authors carried out causal case individual investigations, which they classified according to anamnesis, diagnostic tools and areas, which they evaluated based on the statements of the participants. Results. The analysis shows that anorexia nervosa affects different areas of life. It is a lifelong problem that has a huge impact on the physical, psychological, and spiritual side of a person. Conclusion. The study deals with the various elements of anorexia nervosa, which ultimately affects a person for life. Through definitions and interpretations of the authors’ research results, we can confirm that it has multi-problematic consequ - ences for the categorised areas of an individual’s life, for the family and the groups in which they live. Introduction A norexia nervosa (and -no, without: orexia -appetite, effort) is one of the most serious eating disorders. Anorexia nervosa is a disorder characterised mainly by the deliberate reduction of body weight (Krch, 1999;Pápežová & Hanusová, 2012). Sufferers of this disorder try to reduce food intake to the absolute minimum. In the beginning, the appetite is consciously suppressed. Subsequently, absence of appetite, the feeling of hunger is weakened, and only then does the lack of appetite set in. The disorder commonly occurs in girls aged 13 to 20 (Budayová, 2021;Ladishova, 2006;). Girls with this disease are mostly adaptable, successful at school, perfectionists, but after a while they get into trouble with their parents, lie more often and refuse to admit that they have a problem. We can consider the case when a ten-year-old girl suffers from anorexia, but also c, a forty--year-old woman, and nowadays, we meet boys with this eating disorder. However, for boys or men who suffer from anorexia, the term "Manorexia" is more frequently used. Consequences of Eating Disorders Eating disorders can cause a wide range of health and social complications, which are primarily related to weight loss. Physical problems of an insufficient diet or the desire to pass as quickly as possible interferes with everyday life (Marádová, 2007). The consequences of anorexia nervosa are individual. Their severity depends primarily on the extent and intensity of the patient's outbreak and how underweight she is. It is generally reported that a third of anorexic patients die. Another third will be completely cured and a third will stick to diets for life (Krch, 2002). The effect of anorexia nervosa can be divided into two main categories. These are health and psychosocial consequences. Physical consequences: • increased sensitivity to cold, danger of hypothermia (Dušek et al., 2010; Tkáčová et al., 2023); • dry, yellowed, cracked skin, increased hair growth all over the body, hair loss. Different rashes can form, bruises are very frequent. About a quarter of people have thin, scaly to dry skin with reduced collagen; • metabolic changes, slowing of gastric emptying; • increased tooth decay; • changes in the cardiovascular system (anemia, low blood pressure, increased blood cholesterol level); • various sleep disorders (scary dreams, insomnia), sensitivity to light and sounds (Krch, 2002;. Psychosocial Consequences of Anorexia Nervosa Anorexia nervosa also significantly disrupts the psychological system, the personal and social life of the patient. According to František D. Krch (2003Krch ( , 2005, these are primarily the following consequences: • changes in mood are dependent on body weight and degree of self--control. Initially, anorexics tend to be very active and lively, their moods are low, depressed and even irritable; • uncertainty, anxious feelings (even small problems seem insoluble) (Doner & Plog, 2000). The Social Worker and his Place in Health Facilities Anna Tokárová (2007) defines a social worker as a professional (professional, qualified person) who deals with social assistance to individuals, groups or minorities who temporarily or permanently find themselves in a problematic social situation that requires social intervention. Through its ordinal activities, it helps to improve the life functionality of an individual, group, or minority by contributing to the mobilisation of resources that are necessary for such a solution. According to Martina Mojtová (2008;Judák et al., 2022;Marková et al., 2006;Petrovič & Maturkanič 2022), the work of a social worker in healthcare facilities consists in helping to improve the patient's conditions during and after hospitalisation, in solving disturbed relationships and situations that arose in the individual's life as a result of injury, illness, harmful habits, social conditions and old age. Stages of a social worker's work in healthcare facilities according to Martina Mojtová (2008): • 1st stage -First contact with the patient. The social worker must first familiarise himself with the patient's medical documentation. He keeps his opinion about the patient to himself. During the first meeting with the patient, the worker introduces himself and explains what help he can provide. This first meeting actually serves to establish a therapeutic relationship. In this relationship, the patient must understand the mission of the social worker notwithstanding in what circumstances the mutual meeting took place (whether at the initiative of the social worker or from the client, or at the recommendation of another health worker). At this stage, the method of individual interview is used; • 2nd stage -Establishing a social diagnosis. We utilised the structured interview method, in which the social worker detects psychosocial anamnesis, key information about the problem, about the client and his family, school, work, partnership achievements and problems identification data, family background, hygiene, eating habits, client's relatives, education, economic and social support, place of residence: a) information about the impact of the disease on the client's life and his family; b) noticing non-verbal expressions (crying, laughter, aggression, emotional behaviour). The social worker adapts to the client's language. For the purpose of adaptation to client he utilises conversation, listening, observation and paraphrasing. • 3rd stage -Solution proposal and assistance plan. In this stage, the social worker develops a plan according to which social assistance will be provided to the patient and applies it to the therapeutic plan. He must further inform other members of the medical team about this activity; • 4th stage -Intervention and social therapy. This method is based on the needs of the client as ideal, and the social worker is responsible for finding solutions that are beneficial for the client. Case Report 1 In the first case study, we present a single man aged 21, a 3rd-year student at the University of St. Cyril and Methodius in Trnava. Personal History His psychomotor development was adequate, without characteristics. L attended kindergarten and elementary school. He completely graduated from gymnasium and is currently studying at university without any problems. As a child, he disliked team sports. Respondent was engaged in swimming, diving, skiing and ballroom dancing. Due to the regularity of movement and the rigours of training, his weight decreased, but it was normal. Medical History L was born in February 2002, he was a baby born by choice. His mother did not suffer from serious diseases during her pregnancy, she was healthy. The birth itself took place without problems or complications. He went through the usual illnesses in childhood, but nothing serious. Later, at the age of 4, our respondent was diagnosed with toxoplasmosis due to contact with animals. Family History • The mother is 47 years old, works as an entrepreneur in Trnava, devotes herself to cynology, healthy eating and swimming. She is healthy; • Father is 48 years old, works as an entrepreneur with company headquarters in Topoľčany. He is engaged in competitive shooting, diving and skiing. He is healthy; • L grew up in a complete family in a harmonious environment. The coexistence of the parents is described as beneficial. The relationship between the parents and the child is friendly. None of the relatives suffered from eating disorders and none of them received psychiatric treatment. Course of the Disease and Intervention by a Specialist The school environment at primary school was not a problem for him and the preparation for teaching was optimal. Food intake was regular. Participant L had lunches in the school canteen and home-cooked dinners. "A significant change in weight occurred, when L enrolled in gymnasium," states L. Current State Currently, L has no problems with eating, our male participant is stabilised. L weighs 55 kg with a height of 161 cm (BMI-21.22), which represents the optimal weight, considering his age and height. According to the recommendation, he continues to visit a clinical psychologist who, using the interview method, motivates the patient towards a healthy lifestyle. Our male respondent manages his studies at the university without any problems and likes to be involved in volunteer activities of a non-profit organisation to help animals. Nowadays, he practices a vegan diet, has no health problems and has been in a loving relationship for three years. In the future, he will not have the prerequisites and opportunity for relapse. Case Report 2 In the second case study, we present a single woman aged 31 hospitalised in a psychiatric ward with a medical diagnosis of anorexia nervosa. She is a student of an unnamed university, informed about the treatment regimen and her acute condition. Objectively: The patient is calm, the mood rather depressed, oriented in place, time and person. The patient's contact and answers are adequate, the speech is slow, quieter, serious, the psychomotor pace is adequate. The patient is tense, furthermore there is also impaired concentration of attention and fatigue. Critical of the disease, she is interested in undergoing treatment and solving her problem. Vital signs: BMI: 100/55 Height: 169cm P: 62/min., regular Weight: 39.5kg D: 15/min., clean BMI index: 14 TT: 36.4°C History: The patient was admitted to the psychiatric ward on 5/1/2014 as an acute condition. B came accompanied by her mother based on the recommendation of an outpatient psychiatrist. Patient repeatedly treated in a psychiatric ward. A depressed mood dominates at reception. At home, our participant is often irritable and conflicted. A significant deterioration has been observed for a month. The patient eats very small amounts because she has dyspeptic problems after consuming even a small amount of food. Participant reports a fear of gaining weight, while she often has remorse after eating and the urge to exercise. Secondary amenorrhea is also present. Personal history: 20-year-old patient, single, childless, student, comes with 4 siblings, from a divorced family. Overcame more serious illnesses: common children, without consequences Hospitalisations: 1x in the psychiatric ward, lactose intolerance. Family history: Mother and siblings are healthy. The father is addicted to alcohol without other serious illnesses. Social anamnesis: The patient currently lives with her mother and siblings in a 3-room apartment. The patient states that she misses her father very much accounted the fact that she moved away from them. Family relations are otherwise good, everyone tolerates and helps each other. Her eating problems started in high school when she started going to pageants and experiencing stressful situations. At that time, she weighed 52 kg, and within 3 years she had lost 13 kg. B began to have problems with learning, lacked the ability to concentrate furthermore felt significant fatigue. The patient started to be explosive, while she screams every time, she gets upset even over little things. Therapy: Mirzaten 30mg 0-0-1 Seroxat 20 mg 0-0-2 Degan 1-1-1 half an hour before meals. Assessment: According to the patient, health is currently one of her most important values. She admits her mistake and is determined to change her lifestyle. B states that she had "no energy for the usual activities of her daily life. She liked to swim, play tennis, which she can't handle at the moment." Nutrition and metabolism: The patient has significant problems with eating. The patient eats in limited portions because she is afraid of gaining weight and that her friends will consider her fat. Lately, I've also had trouble keeping down the food I've eaten, she stated. Within an hour after eating even a small amount of food, she feels heavy and subsequently vomits everything. The patient does not use laxatives. Excretion: B has diarrhoea more often due to lactose intolerance. Otherwise, she does not state any problem. Activity, exercise: The patient has remorse after eating and frequent urges to exercise. Otherwise, in her free time, she draws and likes to go for walks in nature. Sleep, rest: I usually have no problem with sleep, states B. Sometimes she can't fall asleep, but otherwise without difficulty. Perception, cognition: The patient is person-, place-, and time-oriented. She reports difficulty with memory and also has issues with concentration. Respondent often forgets and cannot concentrate. Self-perception, self-image: The patient currently perceives herself as "normal", but she is afraid of gaining weight. B is worried that she will be fat and, that people will stop talking to her. Now she is happy with herself. The patient states that she could gain 2-3 kg, but she always has a stomach ache after eating and throws up everything. Role, relationships: The patient is single. She had one more serious relationship, which she ended, but she does not want to communicate further on this topic. Respondent has a positive relationship with her mother and siblings. She had not seen her father for a long time. B has moved away and is not interested in establishing further contacts. The patient has one very good friend with whom she spends most of her time. B generally has great relationships at school. Reproduction, sexuality: She refuses to communicate on this topic. Adaptation, resistance to stress: B started to feel stressful situations in high school, when she started going to competitions. Subsequently, she started having problems with eating. Participant refused food, isolated herself more from her surroundings. Value orientation, religious beliefs: The patient is religious, but does not go to church. The greatest value in her life is her family. Analysis and Interpretation of Data: After previous hospitalisation in the local department, the patient was re-admitted in an acute condition for an eating disorder. In the first days, the patient is under constant supervision when consuming food. Observes regularity and adequate amounts of food. From the beginning with difficulties, because after every meal our female respondent has dyspeptic problems. In laboratory parameters, pronounced hypokalaemia, which was subsequently saturated by infusions with the addition of KCL. Gradually, there is an emotional recovery, relief of dyspeptic complaints, a slight increase in weight. The patient is involved in occupational rehabilitation. The patient completed an individual interview with a psychologist. She keeps a food diary, where she writes down everything she ate during the day, how she felt before and after eating. The patient gradually managed the regime treatment without major difficulties. In the case of the patient, based on the entrance test, we found that she is critical of her illness and understands the reason for hospitalisation. However, she does not have sufficient knowledge regarding ways to relieve fatigue and also does not have a positive attitude towards the appearance of her body. The patient feels lonely, dissatisfied with relationships, with people around her and does not know ways to alleviate loneliness. Summary of Case Reports After reading the case reports of individuals suffering from anorexia nervosa, we can conclude that the disorder brought them more than one social consequence . As a result of the case reports, we selected the most common social and personal consequences that affected the respondents and influenced them. The Area of Family Relationships and Relationships with the Environment From this particular survey, it follows that family and relatives can be one of the factors that trigger anorexia nervosa. Respondent L had a positive relationship with his family since childhood. His parents taught him basic things, prepared him for teaching, built his relationship with animals and society. In a way, they helped him socialise. His relationship with society is quite positive, his relatives respect him. In the case of L, we would not see disturbed relationships in the family. However, that was not the case with respondent B. She comes from a dysfunctional and divorced family, where she was not noticed much. Nevertheless, she has a positive relationship with her parents, even if she has her own opinion about them. When B was three years old, her parents divorced -we could consider this as the primary trigger of anorexia nervosa. This action could also be described as childhood trauma. B's relationship with the environment is quite positive, even though society does not take her very seriously due to her primary and serious disorder. The social consequences here are broken bonds in families and deteriorating relationships with the closer environment, which can lead to social exclusion ). Impact on Partner Life We learned from the participants that during puberty they wanted to establish serious relationships, but they did not succeed and fell into depression. L does not have a partner relationship, as our male participant has had it for three years and even sees its future. In the case of B, we were not convinced that this is the case. B told him that she still has a problem and is not able to establish a partner relationship at all. This problem is attributed to her by society and the family environment, where she sees a kind of boil in her mother. Since as a child she had to face the divorce of her parents, our female participant subconsciously wrote down a certain unreasonable ideal of women, and despite this, she built a relationship with a man, which, however, deteriorated after a year. In the case of our male respondent, L can predict that B will continue to have a problem with establishing a relationship with a partner. Problems also arise with the so--called multiplication of problems, which accumulate and can cause multiple problems in any aspect of life (Budayová & Ludvigh Cintulová, 2021; Lešková & Uháľ 2020). The Area of the School Environment and Contact with the New Environment In both cases, we noticed a problem with contact from the school environment. They considered the very interaction with their classmates stressful and thus became the target of ridicule. As we have already mentioned above. L had a big problem with physical education, where they had to change clothes in front of the whole class. It had a stressful effect on him, so he tried to lose weight as quickly as possible so that the future weigh--in would not be depressing moreover he lacked the ability to face ridicule from his classmates. Case B was not so stressful from the side of physical education, but she also suffered a setback from the side. However, the difference between the case in this area lies in the fact that only at first she used extremely reduced weights and excessively lavished fitness centres practiced drastic diets during which B lost extreme weight. Contact with classmates was negative for both and even conflicted with B and L, in contrast to case L, continues his education at university, where he no longer has a problem with the relationship with his colleagues . They respect him, motivate him so that there is no relapse. After graduating from high school, B started her own salon, where she gets along well with clients. Here we can point out how negative relationships from the school environment affect specific cases. The specific effects of mocking classmates were reflected in the psyche of individuals through mental anorexia. Health Area We can also consider their health as the result of anorexia nervosa in both respondents. During anorexia, their growth stopped at a height of L-161 cm and B-165 cm. Furthermore, their anorexia was also manifested in their body hair. After being cured of the disorder, they are no longer able to accept food in public, but in the case of L, it has improved. Since B regularly consumes sedative medications, her sleep cycle takes too long and she cannot wake up on time and goes to work only after two o'clock in the afternoon. L also had a problem with his sleep cycle despite not taking medication. Social Area There are certainly many more social consequences associated with anorexia nervosa. In the interview, we focused on the areas that we consider the most important and where these consequences were most visible. At the end of the practical part, we can state that the first case will not relapse, but second case B may relapse in the future. The situation of these social work clients also worsened sharply during the Covid-19 pandemic situation, as the reduced availability and low availability or even\non availability of social contact, and hospitalisation facilities could cause a deteriora- An expert in the field of social services should ensure and suggest suitable treatment for the sufferer after consultation with another expert. It should be a kind of support for the client, whom he represents and accompanies during resocialisation and inclusion in society with normal eating habits. When contacting a client suffering from anorexia nervosa, bulimia nervosa or another eating disorder, the social worker should also provide suitable equipment for treatment. However, there may be a problem, because Slovakia, compared to the Czech Republic, does not have social facilities or helplines aimed specifically at helping those suffering from eating disorders. In the neighbouring state, the Anabell Civic Association has even been established, which provides various types of services, such as counselling, telephone and crisis assistance, therapy, and others. Employees of the association provide clients of the department with medical and social assistance and, upon agreement with the sufferer, also possible treatment (Roubalová	2023-07-12T15:03:37.535Z	2023-06-20T00:00:00.000	{ "year": 2023, "sha1": "37f75a86b7b77dfb1f6d0ee95aa81172987fbcff", "oa_license": "CCBY", "oa_url": "https://jecs.pl/index.php/jecs/article/download/1554/1290", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "3e849b01476149cf09be272e15acb4a03db11541", "s2fieldsofstudy": [ "Psychology", "Medicine" ], "extfieldsofstudy": [] }
59063938	pes2o/s2orc	v3-fos-license	Plasmonic Modes of Metamaterial-Based Slot Waveguides Most metamaterials exhibit pronounced anisotropic properties that are crucial for the understanding of their superior optical behavior, especially when they are integrated into the structure of a plasmonic waveguide. In this paper, we analytically solve the dispersion relation for a slot plasmonic waveguide filled with an anisotropic-stratified metamaterial and reveal that it supports two modes featuring relatively long propagation lengths in the limit of vanishing slot thickness. We classify these modes according to their physical origin and study the variation of their dispersion properties with material parameters. Introduction The ultimate goal of optics is to enable a perfect control of the interaction between light and matter.This goal has been brought closer by the recent advances in nanotechnology that have made possible the fabrication of optical metamaterials [1,2].The unusual electromagnetic properties of metamaterials are expected to enable a new generation of optical devices.In developing design strategies and new concepts for such devices, it is paramount that anisotropic properties of metamaterials are considered along with their other material features.Moreover, even the ways in which common devices operate require revisions when ordinary materials in their design are replaced by anisotropic metamaterials.A considerable amount of theoretical effort has been recently devoted to the analysis of optical propagation through different types of metamaterial structures, including uniaxial dielectrics [3] and indefinite media [4,5], metal-dielectric heterostructures [6] and superlattices [7], and strongly anisotropic waveguides [8].In this paper, we reexamine the guiding properties of slot plasmonic waveguides filled with an anisotropic medium.Our work is intended to demonstrate that integration of plasmonic waveguides with anisotropic optical metamaterials not only brings additional freedom to their design, but can also lead to new physical phenomena that may benefit the waveguide performance. The plasmonic waveguide discussed here consists of an anisotropic medium of thickness 2h embedded between two metals of permittivity ε m .We assume that the medium's permittivity is described by a constant, diagonal tensor ε = diag(ε xx , ε yy , ε zz ) with its principle axes parallel to the waveguide's edges.Even though the permeability has similar anisotropic properties, only one component of its tensor affects the transverse magnetic (TM) modes, which are of primary interest for plasmonic waveguides.This allows us to describe the permeability using a single parameter μ.As is well known, the evolution of the electric field E = (E x , 0, E z ) and the magnetic field H = (0, H y , 0) of a TM mode is governed by the propagation constant β.In the case of an anisotropic core layer, β obeys the dispersion relation where , ω is the frequency of the surface plasmon polariton (SPP), c is the speed of light in vacuum, and the ± signs correspond to the symmetric and antisymmetric modes, respectively.Equation ( 1) is applicable to a broad range of metamaterial-based plasmonic waveguides and requires the specification of metamaterial design for further analysis.We restrict ourselves to the simplest scenario in which the metamaterial is created via stacking layers of isotropic materials into a periodic heterostructure.If the layers' thicknesses are much smaller than the optical wavelength, then the permittivity tensor of the heterostructure has only two different diagonal components given by [9] where the subscripts and ⊥ designate directions parallel and perpendicular to the layers, n is the number of layers within one heterostructure period, and f j is the filling factor for the jth layer of permittivity ε j (all filling factors must add up to unity).Such a stratified metamaterial offers fabrication of the three types of plasmonic waveguides shown in Figure 1.Their permittivities are Although all of them can, in principle, be created using modern fabrication techniques, we focus here on the last structure that is relatively easy to fabricate.Apart from the ease of fabrication, the third type of plasmonic waveguide in Figure 1 is the only one that supports symmetric TM modes with complex β values in the limit of vanishing ε , which can be realized by properly matching metamaterial constituents and compensating for the absorption losses inside them (note that the condition ε = 0 is not equivalent to an epsilon-near-zero (ENZ) regime [10], because the permittivity is a complex tensor).Within the slot region, these modes are characterized by the components E x and H y that are independent of x, and by E z ∝ x.Solving (1) in the limit ε → 0 yields the following four values of β: where K = ε m (kh) 2 and r = ε m /ε ⊥ is the ratio of the dielectric constants.Since both of these parameters are generally complex, β = β + iβ is also complex, and its real and imaginary parts provide the phase velocity and energy loss of various SPP modes.Because β in (3) satisfies (1) approximately in the limit of small h (when \|q\|h 1) even when ε / = 0, the following analysis allows us to elucidate some general features of the SPP behavior in metamaterialbased plasmonic waveguides. For simplicity, we focus on the stratified heterostructure composed of two different nonmagnetic (μ = 1) materials, one of which is the metal used for waveguide cladding.The permittivity of the second material is then fixed by the condition ε = 0 (this material should provide gain, since metals are essentially lossy).It then follows that the ratio r depends solely on the filling factor f of the metal; r = 2 − 1/ f .We use silver as a metal in our simulations, take its permittivity in the form of a seven-pole Drude-Lorentz formula given in the work by Pannipitiya et al. [11], and introduce an absorption parameter γ, such that ε m (γ) = ε + iγε .For definiteness, we also choose β > 0 and refer to β as the "damping factor," thus implying that our waveguides do not amplify SPPs. According to (3), the waveguide in Figure 1(c) supports a maximum of two SPP modes regardless of slot's thickness, provided that ε = 0.The dispersion curves of these two modes are plotted in Figure 2 for three values of γ and two sets of material parameters f and h.In the lossless case (γ = 0, green curves), the modes can be grouped into three distinct classes: (i) propagating modes with β = 0 and β / = 0; (ii) complex modes with β / = 0 and β / = 0; (iii) evanescent modes with β / = 0 and β = 0.The first two types of modes may travel either forward or backward, except for certain "degenerate" frequencies for which the group velocity and total energy flow of SPPs vanish.In our example, the degenerate frequencies correspond to the points A, B, and C of the energy spectrum.The degeneracy of waves traveling in the opposite directions is removed by absorption (see pink curves for γ = 0.2) since the two modes develop a slightly different lateral confinement. The complex modes of a lossy waveguide arise through quasimixing of the three types of modes as γ is gradually varied from 0 to 1.Such transformation of the propagating, complex, and/or evanescent modes is accompanied by their degeneracy removal, which is also the scenario followed by the quadrupole modes of plasmonic nanowires [12].Indeed, the twofold degenerate propagating mode at point B and the twofold degenerate evanescent mode at point C split into two nondegenerate complex modes for γ = 1 (see Figure 2).The process of modes transformation allows one to track their origin and better understand waveguide performance.For example, because of the strong damping intrinsic to evanescent modes, one can expect that the segments of spectra β (ω) emerging from them in the case of real losses will be lesser affected by the dispersion properties of ε (ω) than the segments that stem from undamped propagating modes.This conclusion is confirmed by Figures 2(b) and 2(d), where the damping factors are separately shown for the forward (right curves) and backward (left curves) propagating SPP modes.Notice that the spectra in Figure 2 are congregate of the results valid for different plasmonic waveguides, rather than spectra corresponding to a specific structure.As we have seen, dispersions of the real and imaginary parts of β drastically depend on the filling factor of metal and slot thickness.To study this dependance, we set ω = 0.2ω p , where ω p is the plasma frequency of silver, and plot in Figure 3 β and β as functions of slot thickness for five values of f .By looking at this figure from left to right, one can draw up the following picture of SPP behavior.When the filling factor of metal is small, two SPP modes of different Advances in OptoElectronics origins are supported by the waveguide.One of themmode 1 shown by red curves-springs from the propagating mode of a lossless waveguide and, therefore, exhibits small damping.The other-mode 2 plotted in blue-arises from the evanescent mode when h < h 0 = k −1 \|ε m \| −1/2 ≈ 25 nm and from the propagating mode when h > h 0 (h 0 is the skin depth of a metal in the limit f → 0).The first mode propagates backward regardless of h, whereas the second one changes its traveling direction from forward to backward around h = h 0 /2.Because modes 1 and 2 originate from the propagating modes for h > h 0 and f < 0.5, their damping rates are almost the same for this range of parameters h and f .When f = 0.5, the ratio r = 0 since the transverse component of the permittivity tensor becomes infinite, and only mode 2 with β = ±k ε m (1 + K) survives.For f > 0.5, mode 1 becomes forward propagating, while mode 2 gets strongly attenuated for all h.The overall dynamics of such changes with f is illustrated in the supplementary animation. It should be emphasized that these results are based on the effective-medium theory and approximate.For a more accurate treatment, the inclusion of nonlocality effects is required [13,14].An important feature of the modes given in (3) is that they can exhibit relatively small damping depending on the values of the parameters f and h.For example, low damping occurs for both modes for f < 0.2 and h > h 0 .Moreover, the damping of mode 1 vanishes in the limit of small h for all values of f / = 0.5.This behavior is opposite to that of SPPs in ordinary (isotropic) metal-dielectric-metal waveguides, whose attenuation rate diverges as 1/h [15].Also noteworthy is that the confinement of mode 1, determined by the evanescent decay length scaling like δ 1 ∝ h Re √ r 2 when h → 0, improves with the reduction of the waveguide thickness, whereas the confinement of mode 2 worsens in the limit of small h, due to the divergence of the decay length δ 2 ∝ (hk 2 0 Re ε 2 m (1 − r) 2 ) −1 . Hence, we expect that metamaterialbased plasmonic waveguides may enable strong localization of optical energy in an SPP mode that can propagate over relatively long lengths. In conclusion, we have found that a maximum of two symmetric TM modes with essentially different propagation properties are supported by plasmonic slot waveguides filled with a stratified metamaterial exhibiting a relatively small permittivity in the plane of the layers. Figure 1 : Figure 1: (Color online) Three types of plasmonic waveguides made using a stratified metamaterial.In all cases, SPP modes propagate in the z direction and the waveguide extends to infinity in the y direction. Figure 2 : Figure 2: (Color online) Transformation of [(a) and (c)] SPP energy spectrum and [(b) and (d)] damping-factor dispersion with changing absorption parameter γ.The SPP frequency is normalized to the plasma frequency ω p of silver.Solid green curves show propagating modes, whereas dashed curves show complex and evanescent modes. Figure 3 : Figure 3: (Color and multimedia online) Transformation of the real (upper panels) and imaginary (lower panels) parts of β at a specific frequency ω = 0.2ω p as the filling factor f of the metal is increased from 0.2 to 0.8.Insets show a magnified view of mode 2.	2018-12-18T18:33:49.489Z	2012-08-01T00:00:00.000	{ "year": 2012, "sha1": "d97d5972fb6462011d13dba11bd990505aee92eb", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/archive/2012/907183.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "d97d5972fb6462011d13dba11bd990505aee92eb", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
28075691	pes2o/s2orc	v3-fos-license	Anti-proliferative, pro-apoptotic and anti-invasive effect of EC/EV system in human osteosarcoma Osteosarcoma is the most common and aggressive bone tumor in children. The Endocannabinoid/Endovanilloid system has been proposed as anticancer target in tumor of different origins. This system is composed of two receptors (CB1 and CB2), the Transient Potential Vanilloid 1 (TRPV1) channel and their ligands and enzymes. CB1 is expressed mainly in central nervous system while CB2 predominantly on immune and peripheral cells. We investigated the effects of JWH-133 (CB2 agonist) and RTX (TRPV1 agonist) in six human Osteosarcoma cell lines: MG-63, U-2OS, MNNG/HOS, Saos-2, KHOS/NP, Hs888Lu, by Apoptosis and Migration-Assay. We also compared the effects of these compounds on Caspase-3, AKT, MMP-2 and Notch-1 regulation by Q-PCR and Western Blotting. We observed an anti-proliferative, pro-apoptotic, anti-invasive effect. Our results show that both CB2 stimulation and TRPV1 activation, in different Osteosarcoma cell lines, can act on the same pathways to obtain the same effect, indicating the Endocannabinoid/Endovanilloid system as a new therapeutic target in Osteosarcoma. INTRODUCTION Osteosarcoma (OS), the most common primary malignant tumor of bone, affects predominantly children and adolescents, exhibiting high invasion and metastasis rate [1,2]. Due to a high frequency of systemic spread at the early phase and the strong chemotherapy resistance, the five-year survival rate remains at only 20%. Available treatments results in significant morbidity (cardiac toxicity, infertility, renal dysfunction) and chemo-resistance [3][4][5][6]. Moreover OS patients present a decrease in bone mass density (BMD) during chemotherapy, and in long-term survivors osteoporosis and fractures are frequently present [7,8]. Therefore, novel therapeutic approaches are needed to treat OS more efficiently and improve patient's life quality. The cell of origin of OS, is still far from clear. During the last 10 years mounting evidence has placed mesenchimal stem cells (MSCs) and/or their immediate lineage progenitors as the most likely cell-oforigin for many types of sarcomas including OS [9][10][11]. The Endocannabinoid/Endovanilloid (EC/EV) system has been proposed as an anticancer target by several studies [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. This system consists of two G protein-coupled receptors, named CB1 and CB2, the transient receptor potential vanilloid 1 (TRPV1) channel, a non selective cation channel, their endogenous ligands and enzymes for their synthesis and inactivation. CB1 receptors are expressed at high levels in Central Nervous System, while the CB2 receptors are primarily expressed on immune and peripheral cells. TRPV1 has been described as an additional receptor target for several cannabinoids [27][28][29]. Evidences on the cross-talk between CB2 receptors and TRPV1 channels have been demonstrated [30][31][32][33][34]. Cannabinoids have been reported as potential antitumor compounds based on their ability to reduce inflammation, migration, cell proliferation and cell survival at a low dosage. Specifically, these effects are mediated mainly by CB2 receptors [15,19,21,22,35,36]. However, we cannot exclude the effects that these compounds may induce, activating TRPV1 receptor channel. When TRPV1 channel proteins are activated, they mainly cause the calcium ion influx. Intracellular calcium overload mediate cell apoptosis through different mechanism interfering with cell energy production and metabolism, therefore also drugs acting on the TRPV receptors can act as potential target to reduce cell proliferation and survival in cancer [37][38][39]. Recent studies described the role of EV/EC system in remodelling, differentiation, survival and activity of bone cells. In particular, we have demonstrated a functional cross-talk between CB2 and TRPV1 in human osteoclasts. TRPV1 stimulation, exerts proosteoclastogenic effects, whereas CB2 stimulation promotes anti-osteoclastogenic events, suggesting these receptors as new pharmacological target for the treatment of osteoporosis [32][33][34]. Moreover we have also demonstrated the presence of these receptors on human osteoblasts and MSCs and their role in migration, viability and cytokine release [31,40]. Based on these evidences, we investigated for the first time the expression of CB2 and TRPV1 receptors and the effects of JWH-133 and RTX, (agonists of the EC/EV system) at different concentrations, in six OS cell lines (Saos-2, MG-63, MNNG/HOS, KHOS/NP, Hs888Lu and U-2 OS), analyzing the response to these compounds on cell survival, invasion and migration capacity. We observed an anti-proliferative, pro-apoptotic and antiinvasive effect induced by EC/EV compounds in the studied OS cell lineages. OS cell lines express EC/EV system We performed a Real Time PCR to evaluate the expression of EC/EV system in untreated MG-63, U-2 OS, MNNG/HOS, Saos-2, KHOS/NP and Hs888Lu prior to the treatments, to be sure the receptors we were going to stimulate, were present. The result demonstrated the presence of mature mRNA for CB2 and TRPV1 receptors ( Figure 1). Figure 2B) while at 7 μM we could not observe any effect (Data not shown). For this reason, we proceeded treating OS cells only with RTX and JWH-133 at these concentrations (5 μM and 100nM respectively). Caspase-3 expression levels To evaluate the possible molecular mechanism through which EC/EV drugs act on apoptosis, we analyzed the expression levels of Caspase-3 after RTX [5 μM] or JWH-133 [100 nM] treatment. RTX induced a significant increase of Caspase-3 expression in Saos-2 and Hs888Lu cell lines as compared to respective control, while JWH-133 treatment resulted in a significant increase in Caspase-3 expression in MG-63, KHOS/NP, MNNG/ HOS and also in Hs888Lu. In U-2 OS the Caspase-3 levels were also increased but the difference was not statistically significant ( Figure 3). Western Blotting confirmed the results obtained with Real Time PCR (Figure 4). Akt protein expression level We then investigated Akt expression levels. This pathway promotes survival and growth in response to extracellular signals. Activated Akt mediates downstream responses, including cell survival, growth, proliferation, cell migration and angiogenesis, by phosphorylating a range of intracellular proteins, so we investigated Akt and pAkt protein levels by western blot in OS cell lines total lysates. EC/EV compounds were added alone at Both drugs induced a reduction of pAKT expression level, but in MG-63, MNNG/HOS and Saos-2 the strongest effect was achieved with RTX, while in U2-OS, KHOS/ NP and Hs888Lu the biggest reduction was achieved using JWH-133. Total Akt expression levels unchanged or increased after treatments, compared to the untreated sample ( Figure 5). Effect of EC/EV on OS cells migration capacity Scratch assay OS cell lines were treated with EC/EV compounds and their migration capacity was measured after performing a scratch at the center of a 70% -80% confluent well. We measured the migration capacity in terms of % of reduction of the scratched area ( Figure 6). Hence, the lower is the percentage of area reduction, the lower is the migration observed in the well ( Figure 7). Compared with the untreated cells of the same line, we observed a reduction in migration capacity, with both treatments (RTX and JWH-133), in all cell lines except Saos-2. The reduction in cells migration was statistically significant ( Figure 6). DISCUSSION OS is the most common bone tumour in paediatric age, with high metastasis rate and poor life quality, due to chemotherapy-associated effects [1,2]. For these reasons new therapeutic approaches are needed [3,4,6]. We analyzed the therapeutic potential of EC/EV drugs JWH-133 and RTX. JWH-133 is a potent CB2 selective agonist, while RTX is an analog of capsaicin, a vanilloid agonist. We tested these compounds in human OS cell lines. Tumour derived cell lines can be used as excellent in vitro models as long as they are representative of the original tumor [41]. Usually cell lines are questioned because of their additional genetic alterations (in vitro obtained). Mohseny et al. assayed a list of well genetically characterized cell lines, more representative of clinical OS, that could be used to establish valid in vivo and in vitro models. Among those, we used MG-63, U-2 OS, MNNG/HOS, Saos-2 [41]. Moreover, we used also a cell line easy to transfect, KHOS/NP, and a cell line derived from a OS lung metastasis, Hs888Lu [42]. This is the first study where two different compounds acting on the EC/EV system have been tested on six different OS cell lines and their effect has been evaluated via four different techniques. We showed, for the first time, that all six evaluated OS cell lines express EC/EV system and can be stimulated by EC/EV drugs like previously demonstrated in human MSCs and OBs cells [31,40]. Cannabinoids exert a direct anti-proliferative effect on tumor of different origins [24]. Moreover, CB2 activation may be involved in THC-induced antiinflammation in OS cell lines [15] and TRPV1 stimulation has already been shown to lead to cell death by activating Calcium channels, causing cell swelling and membrane lyses [43]. Apoptosis is mediated by multiple elements for instance Caspases are crucial mediators of programmed cell death [44]. We demonstrated an increase in Apoptosis and Caspase-3 expression by JWH-133 at a low dosage and/or by RTX in the different lines. Recent findings reveal that pAKT is enhanced in primary tumors and it is implicated in developing pulmonary metastasis [45]. Total Akt expression levels unchanged or increased after RTX and JWH-133 treatments, which is consistent with results obtained in other studies reported in literature [46][47][48][49], while interestingly, CB2 and TRPV1 receptor stimulation reduced pAKT protein expression in all of the cell lines studied, including the line derived from OS lung Histograms display the relative quantification of the scratch width (μm 2 ) after 24h, compared with the initial width area. Data derived from three different assays. A t-test has been performed to evaluate the statistical differences among groups. * indicates p ≤ 0.05 compared to the untreated control (NT). metastasis. Taken together, these data show an important switch toward the programmed cell death in all the OS cell lines studied. Moreover, the most striking result in favor of administration of these compounds in OS is the cell migration inhibition demonstrated with the Scratch Test. Several studies have already demonstrated that cannabinoids are capable of inhibiting cell migration and metastasis in different types of cancer [25,26]. RTX and JWH-133 demonstrated to be efficient in reducing the migration in all OS cell lines used for this study. In Saos-2 the result has no statistical significance, but this is most likely due to the fact that these cells duplicate very slowly (37 hours duplication time [50]) so probably in the future, the test can be repeated on these cells in a different time frame. The cannabinoid anti-tumor activity also acted through Notch-1 signalling pathway inactivation and MMP-2 down regulation. Notch signalling pathway is critical in cell proliferation and apoptosis [51,52] and the MMPs are important in facilitating tumour invasion [53] so that active MMP-2 is considered a cancer metastasis indicator [54]. Previously, Niuet al. evaluated the effect of a synthetic, non selective, cannabinoid receptor agonist with psychotropic effects, WIN-55, 212-2, on these genes in a single OS cell line, MG-63 [35]. In prospective of a clinical application, instead, we choose two drugs selectively acting on TRPV1 and CB2 receptors and so probably without psychotropic effects. As previously mentioned, chemotherapy is known to reduce BMD and cause osteoporosis and bone fractures [7,8]. Considering the important role of EC/EV system in bone metabolism [33,34], we speculate that these drugs could reduce chemotherapy-related bone loss in a selective manner, significantly improving patient's life quality. Further in deep studies, combining the compounds used in this study with current conventional OS therapy, are required to investigate the precise molecular mechanism of these antitumor activities and whether this co-administration could be effectively applied in clinical practice. Moreover we found, consistently with others [21,24,55] that JWH-133 at low dosage reduces proliferation and induces apoptosis, while at higher dosage it has an opposite effect. So dosage will be a critical point when shifting to clinical practice. In conclusion, our results strongly support the potential of EC/EV system as new therapeutic target in OS, having demonstrated its capacity to interfere in tumor Results were normalized for the housekeeping gene β-actin and are showed as mean ± SD of three independent experiments. A t-test has been performed to evaluate the statistical differences in the levels of gene expression among groups. * indicates p ≤ 0.05 compared to the untreated control (NT). growth and invasion capacity by increasing/decreasing target genes involved in proliferation, migration and apoptosis. Cell lines OS MG-63, U-2 OS, MNNG/HOS, Saos-2, KHOS/ NP and Hs888Lu cell lines were purchased from Sigma Aldrich. The MG-63, KHOS/NP, MNNG/HOS cell lines were cultured in EMEM medium with 1 % Non Essential Amino Acids (NEAA), the Saos-2 and the U-2OS cell lines were cultured in McCoy's medium, the Hs888Lu cell line was cultured in DMEM medium. The complete culture medium containing 10% fetal bovine serum (FBS), supplemented with 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco) and 2mM L-glutamine (Euroclone). Cells were culturedat 37°C in a humidified atmosphere with 5% CO 2 . After 48-hours adhesion cells were harvested using trypsin, washed and counted on a microscope using a Burker haemocytometer and 3,8 x 10 4 cells per well, were plated in a 12 well cell culture multiwell. Once 80% confluence was reached, JWH-133 and RTX were added at the following concentrations: JWH-133 [100 nM, 1 μM and 5 μM], RTX [2.5 μM, 5 μM and 7 μM] and cells were harvested at 24h for mRNA isolation, protein extraction, and Muse® "Annexin V and Dead Cell Assay Kit" (Millipore). We choose only these treatments considering the results obtained in the apoptosis assay. Non-treated cultured cell lines were maintained in incubation media during the relative treatment time with and without vehicle (DMSO 0.01%). Scratch assay Osteosarcoma MG-63, U-2 OS, MNNG/HOS, Saos-2, KHOS/NP and Hs888Lu cell lines were seeded in a six well plate (500.000 per well). Incubated 24 hours to let them attach in monolayer in the wells. A single scratch was made in each well with a 200 μl sterile pipette tip. The well has been washed with PBS and fresh media containing RTX [5 μM] and JWH [100nM] has been added. Fresh medium without any drug was used as control (Labelled as NT: Non-Treated). Images were taken 24 hours later with an AE2000 microscope (Motic) and analyzed with Motic Images plus 2.0 Software. Western blotting Proteins were extracted from treated and non-treated OS cell lines using RIPA Lysis Buffer (Millipore) and following the manufacturer's instructions. Akt, pAKT and Caspase-3 proteins were characterized in total lysates from cell line cultures by Western blotting. Membranes were incubated overnight at 4°C with rabbit polyclonal anti pAKT antibody (1:1000 dilution; Cell Signaling), rabbit policlonal anti Akt antibody (1:200 dilution; Santa Cruz) and rabbit anti Caspase-3 antibody (1:200 dilution; Cell Signaling). Reactive bands were detected by chemiluminescence (Immobilon western Millipore) on a C-DiGit ® Blot Scanner (LI-COR Biosciences). A mouse polyclonal anti β-Tubulin antibody (1:1000 dilution; Sigma) was used to check for comparable protein loading and as a housekeeping protein. Images were captured, stored, and analyzed using "Image studio Digits ver. 5.0" software. Statistical analysis Results are expressed as means ± S.D. The experiments were run in triplicate. Statistical analyses were performed using Student's t test to evaluate differences between quantitative variables. A p value less than 0.05 () or 0,01 (*) were considered statistically significant.	2018-01-24T17:25:33.048Z	2017-04-13T00:00:00.000	{ "year": 2017, "sha1": "cb1940feb0b003a8652415d30b07782180f9bdef", "oa_license": null, "oa_url": "https://doi.org/10.18632/oncotarget.17089", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "cb1940feb0b003a8652415d30b07782180f9bdef", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
208032979	pes2o/s2orc	v3-fos-license	Oncogenic Wnt3a: A Candidate Specific Marker and Novel Molecular Target for Hepatocellular Carcinoma Background and aim: It is of the utmost importance for the specific diagnosis and effective therapy of hepatocellular carcinoma (HCC). Abnormality of oncogenic Wingless-type MMTV integration site family member 3a (Wnt3a) has been associated with progression of HCC. In this study, we aimed to evaluate Wnt3a as a novel biomarker and target for HCC. Methods: Circulating Wnt3a levels were quantitatively detected in a cohort of chronic liver diseases by an enzyme-linked immune-absorbent assay (ELISA). Hepatic Wnt3a expression in HCC and para-cancerous tissues was analyzed by immunohistochemistry. Prognostic value of Wnt3a for HCC was discovered in the cohort from the Cancer Genome Atlas (TCGA). Dynamic alterations of Wnt3a levels were detected in the hepatocarcinogenesis model induced by 2-acetylaminofluorene. Effects of Wnt3a on biological behaviors were evaluated in vitro and in vivo based on Crispr/Cas9. Results: Up-regulated Wnt3a levels were observed in serum of HCC patients with high specificity and sensitivity for HCC diagnosis. Combination of Wnt3a and AFP could improve sensitivity to 93.9% in serological detection. In addition, Wnt3a expression in HCC tissues was significantly higher than that in para-cancerous tissues. The cohort of TCGA demonstrated that high Wnt3a expression led to a poor survival of HCC patients, especially in cases at advanced stages. Furthermore, the hepatocarcinogenesis model showed that Wnt3a dynamically increased in the development of HCC. Functionally, silencing Wnt3a by Crispr/Cas9 suppressed the proliferation, colony formation, induced cell cycle arrest of HCC cells by de-activating Wnt/β-catenin pathway in vitro, and inhibited xenograft tumor growth in vivo. Conclusions: Oncogenic Wnt3a could be considered as a candidate biomarker and novel target for HCC. Introduction Hepatocellular carcinoma (HCC) is still one of the most common cancers with leading cause of cancer in the world [1], particularly in the inshore area of the Yangtze River [2]. The main etiological factors of HCC include chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, aflatoxin B1, and non-alcohol fat liver diseases (NAFLD) [3]. Chronic HBV carriers have a 5 ~ 15-fold increased risk of HCC compared with the general population, partially due to the activation of cellular Wnt/β-catenin and Ivyspring International Publisher transforming growth factor-β (TGF-β) pathways [4,5]. Early discovery and effective treatments are of the utmost importance for HCC therapy and prognosis. Serological biomarkers are widely used in the early diagnosis of HCC with the features of non-invasive, accuracy, and flexibility [6]. Despite of the wide application of alpha-fetoprotein (AFP), its false-negative rate may be as high as 40 % for HCC patients at early stage [7]. To date, some novel biomarkers including circulating glypican-3 (GPC-3) [8] and microRNAs [9] have been proposed. However, the overall efficiency is still unsatisfactory, especially in HCC patients with low AFP level or small-size tumor. Recently, abnormal expression of the Wnt signaling was reported closely associated with the occurrence, progression, and prognosis of HCC [12]. Wnt3a is one of the most well-known Wnt ligands due to its critical roles in embryonic development as a pivotal component of the mesoderm gene. However, Wnt3a has recently been considered as an oncogenic factor in cancers of colon, breast, lung, and esophageal squamous cell [13]. Notably, silencing Wnt3a could inhibit proliferation, invasion, and chemo-resistance in glioma derived stem-like cells [14]. Previously, we first discovered the abnormal expression of Wnt3a in sera and cancerous tissues of HCC patients [15]. Nevertheless, the exact underlying mechanisms of Wnt3a and its alteration during malignant transformation of hepatocytes still remain to be explored. Therefore, this study aimed to further investigate the role of Wnt3a in clinical value and molecular-target for HCC. Serum samples Total 400 patients with benign or malignant liver diseases from the Affiliated Hospital of Nantong University, China between Jan. 2012 and Jan. 2017 were enrolled in this study. They were divided into four groups: HCC (n = 180), liver cirrhosis (LC, n = 80), chronic hepatitis (CH, n = 80), and NAFLD (n = 60). HCC was diagnosed according to the diagnostic criteria set by the 2012 Chinese National Collaborative Cancer Research Group. Among the 180 HCC (135 males, 45 females; ages ranged 35-86 years old) cohort, no patients received any pretreatment and all cases were confirmed by pathological examination. 59 cases with AFP level more than 20 ng/ml; 84 cases with lymph node metastasis; 47 cases with postoperative recurrence, 121 cases with positive hepatitis B surface antigen (HBsAg), and 81 cases with tumor size less than 3.0 cm. Serum AFP exceeding 20 μg/L was considered positive. Written informed consents were provided from all patients. Healthy persons with hepatitis B markers (HBsAg, HBcAb, and HBV-DNA) negative and normal serum alanine aminotransferase (ALT) levels as normal controls (NC, n = 80) were obtained from the Nantong Central Blood Bank in China. The study was in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Affiliated Hospital of Nantong University, China. Liver tissue samples Human liver cancerous and their surrounding tissues in this study were obtained from 60 HCC patients who underwent operations between Jan 2012 and Aug 2017 at the Affiliated Hospital of Nantong University, China. All sections were reviewed for confirmation of the original pathological and clinical diagnosis. Related clinical information including gender, age, tumor number, cirrhosis, HBV, AFP, and periportal cancer embolus were collected from their medical records. Tumor staging was based on the 6th edition of tumor-node-metastasis (TNM) classification of the International Union Anti-Cancer. This study was approved by the Ethics Committee of Affiliated Hospital of Nantong University, China (TDFY2013008), and in accordance with World Medical Association Declaration of Helsinki. Written informed consent was obtained from patients who participated in this study. The survival analysis was conducted by the Kaplan-Meier plotter, which was built based on the data of Cancer Genome Atlas (TCGA). Hepatocarcinogenesis model Rat hepatocarcinogenesis model was made according to the previous method [16]. Male Sprague-Dawley rats (4-wk-old, body weight 140-150 g) were supplied by the Animal Center of Nantong University. Rats were maintained under pathogen-free conditions with a 12-h light-dark cycle. Malignant transformation of rat hepatocytes was induced by 2-acetylaminofluorene (2-AAF; Sigma). Sera and livers of rats were collected from rats sacrificed biweekly for immunochemistry and specific concentration of Wnt3a. All animal procedures were approved by the Animal Care and Use Committee of Nantong University, China. Cell culture Human HepG2 cells were obtained from American Type Culture Collection (ATCC, USA) and grown in Dulbecco's modified Eagle's medium (DMEM, Hyclone, USA) containing 10 % fetal bovine serum (FBS; Gibco, USA) at 37℃ in a humidified atmosphere of 5 % CO2. SURVEYOR assay The SURVEYOR assay was performed using SURVEYOR Mutation Detection Kits (IDT, USA) according to the manufacturer's instructions, and primers are shown in Cell cycle analysis Cells in the sgRNA2 or NC group were collected by trypsin and washed with pre-cold PBS. Following fixed in 70 % ethanol for 12 h, cells were resuspended in 50 μg/mL RNase A solution and incubated for 30 min at 37 ℃. Then samples were incubated with 10 μg/mL propidium iodide (Solarbio, China) in the dark for 15 min. After that, cells were detected by a Flow Cytometer (BD, USA). Data was analyzed by the Modfit software. MTT assay Cell proliferation was determined by MTT (thiazolyl blue tetrazolium bromide) assay. Cells (1×10 3 ) in sgRNA or NC group were seeded into 96-well plates. Following incubated for 12 h, cells were treated with MTT solution at 0 h, 24 h, 48 h, 72 h, and 96 h. After removing the suspension, 90 μL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan. The plate was finally read by a micro-plate reader (Bio-Rad, USA) at a wavelength of 490 nm. Colony formation assay Cells in the sgRNA2 or NC group were plated in 6-well plates (500 cells/well) and incubated in complete medium for two weeks. Following fixed by 4 % paraform-aldehyde, cells were stained with 0.1 % crystal violet. Visible stained colonies were counted under a microscope (Olympus, Japan). Immunohistochemistry (IHC) Liver IHC assay was performed according to previously described [16]. Briefly, paraffin-embedded sections of tissues were deparaffinized in xylene, rehydrated by ethanol. Following heat-induced epitope retrieval in 0.01 Mol citrate buffer at pH 6.0 for 15 min and washed with PBS, the samples were blocked with 5 % BSA for 2 h. Then the sections were incubated with primary Wnt3a, Wnt5a or Ki67 antibodies (1:100, Abcam, USA) overnight at 4 ℃. After incubated with secondary antibodies (Univ-Bio., China) for 1 h at room temperature, sections were stained with 3′, 3-diaminobenzidinetetrahydro chloride (DAB, Sigma, USA) and counterstained with hematoxylin. Enzyme-linked immunosorbent assay (ELISA) Wnt3a levels in sera and liver tissues were quantitatively detected according to manufacturer's instructions of human or rat Wnt3a ELISA Kit (Cloud Clone Corp., China). In brief, 100 µL of sample dilution and standards were added to indicated wells and incubated for 1 h at room temperature. Following washing, HRP-conjugated antibody was added and incubated for 1 h at room temperature. After washing again, each well was incubated with 100 µL of 3, 3', 5, 5'-tetramethylbenzidine (TMB) substrate solution for 30 min away from light and stopped with 90 µL of stop solution. Then the absorbance was measured in a microplate reader at 450 nm. Experiments were conducted in triplicate. Xenograft model BALB female mice (about 4-6 weeks age) were provided by Animal Center of Nantong University and maintained under specific-pathogen-free conditions. HepG2 cells (5×10 6 ) transfected with sgRNA2 or NC were suspended in 100 µL DMEM with Matrigel (BD Biosciences, USA), and then subcutaneously inoculated on the right flank of mice. Tumor volumes were monitored every 4 days after cells injection and calculated according to the following formula: volume = [length × (width)2)/2]. Mice were sacrificed at the 24 th day, and tumor tissues were weighed and detected for Wnt3a and Ki67 (Abcam, USA) with IHC assay. The study protocol was approved by the Committee on the Ethics of Animal Experiments of the Nantong University. Statistical analysis Statistical analysis was performed using SPSS 18.0 software. Data were shown as mean ± standard deviation (SD). Receiver operating characteristic (ROC) curves were calculated to define the diagnostic value of Wnt3a and AFP simultaneous detection. Comparison between groups was analyzed by t student test or Chi-squared test. A P value less than 0.05 was considered statistically significant. Circulating Wnt3a expressions in chronic liver diseases The levels of circulating Wnt3a expression in a cohort of 400 patients with liver diseases were detected and the comparison with AFP levels are shown in Table 2 and Figure 1. The mean Wnt3a concentration in the HCC group was significantly higher (P<0.001) than any group of patients with benign chronic liver diseases, with average increasing 4.2, 5.9, 5.1, and 6.2 folds than that in the LC, CH, NAFLD, and NC group, respectively. The cutoff value was set 478.0 ng/L (mean±1.96 SD) as the upper limit, and the incidence of serum Wnt3a was 87.8 % (158/180), which was more sensitive than that of AFP (>20 ng/mL, 67.2%, 121/180) in HCC patients (Fig.1B). Besides, lower positive ratio was also observed in cases with benign chronic liver diseases. Notably, the incidence of serum Wnt3a was 81.4% in HCC patients with AFP level less than 20 μg/L (48/59), 92.6% in the cases with tumor size less than 3.0 cm (75/81), and up to 93.9% when Wnt3a plus AFP combination. Clinicopathological features of Wnt3a expression in HCC The clinicopathological features of abnormal Wnt3a expression in HCC are shown in Table 3. The up-regulated levels of Wnt3a expression in sera of HCC patients was closely associated with HBV infection (P<0.001), lymph node metastasis (P=0.016), differentiation degree (P=0.001), TNM staging (P=0.003), Child-pugh classification (P<0.001), and tumor recurrence (P=0.014). However, no significantly difference (P>0.05) was found between Wnt3a and HCC patients' age, gender, tumor size, AFP level, liver cirrhosis, or gross classification. Wnt3a expression in HCC tissues The expression features of Wnt3a in HCC and its prognostic value are shown in Figure 2. As shown in Fig. 2A, the intensity of Wnt3a staining in HCC tissues was stronger than that in the para-cancerous tissues. Semi-quantitive analysis showed that the positive rate of hepatic Wnt3a in HCC group (71.67%, 43/60) was significantly higher (χ 2 =34.478, P<0.001) than that in the para-cancerous group (18.33%,11/60; Fig. 2B). In addition, we also evaluated the prognostic role of Wnt3a for HCC in TCGA cohort. Though it was not statistically significant in the integral cohort or the cases at early stages, the patients with high Wnt3a expression had an obviously shorter survival (Fig. 2C&D). Notably, for the cases at advanced stages, Wnt3a expression could act as a novel prognostic factor for the survival of HCC patients (HR=2.13, P=0.011, Fig. 2E). [7], and GPC-3, oncofetal antigen glypican-3 [4]. Dynamic Wnt3a expression in hepatocarcinogenesis The Wnt3a expression in rat hepatocarcinogenesis model is shown in Figure 3. According to the H&E staining, the rats were divided into four groups: control, degeneration, precancerosis, and HCC. Then, IHC staining of the tissues from groups above demonstrated that Wnt3a expression significantly increased during the malignant transformation of hepatocytes (Fig. 3A). Furthermore, up-regulating Wnt3a in liver suspension and serum was also determined in the HCC formation of rats by using ELISA assay (Fig. 3B&C). In view of the remarkable alteration of Wnt3a in hepatocarcinogenesis, it might play a crucial role in HCC progression. Knockout of Wnt3a by Crispr/Cas9 system on biological behaviors of HCC cells The effects of Wnt3a silencing by Crispr/Cas9 on biological behaviors of HCC cells are shown in Figure 4 and Figure 5. Initially, HepG2 cells were infected with the lentivirus expressing Cas9 with puroresistance (Fig. 4A1&2). Subsequently, lentivirus expressing different Wnt3a-targeted sgRNAs with EGFP was infected into puromycin-screened cells (Fig. 4A3 & 4). As shown in Fig. 4B, the SURVEYOR assay showed that sgRNA was capable of binding to the expected Wnt3a regions and facilitated the excision of genomic strands via the Cas9 enzyme. Furthermore, the protein expression of Wnt3a and its downstream gene β-catenin significantly attenuated after infection of Wnt3a-sgRNA2 (Fig. 4C&D), indicating that sgRNA2 was the most efficient sgRNA for knockout of Wnt3a. As shown in Fig. 5A, the proliferation rate was significantly inhibited in HepG2 cells infected with Wnt3a-sgRNA2. Besides, the colony number of HepG2 cells also obviously reduced after Wnt3a knockout (236.00 ± 17.78 vs. 106.67 ± 15.57; t = 9.480, P = 0.001; Fig. 4 B&C). Furthermore, flow cytometry analysis showed that Wnt3a gene knockout by sgRNA2 also led to cell cycle arrest in G1 phase in HepG2 cells (P < 0.01; Fig. 4 D&E). Effects of Wnt3a on HCC xenograft growths in vivo The effect of Wnt3a knockout on xenograft growth is presented in Figure 6. HepG2 cells of NC group and Wnt3a-sgRNA2 group were subcutaneously injected into mice, and tumors were resected at the 24th day after injection (Fig. 6A). As shown in Fig. 6B & D, tumors of the Wnt3a-sgRNA2 group had smaller volume (355.00 ± 99.85 mm 3 vs. 869.42 ± 222.46 mm 3 ; t = 5.168, P < 0.001) and lighter weight (0.35 ± 0.11g vs. 0.88 ± 0.20g; t = 5.628, P < 0.001) than the NC group. Besides, the growth rate of tumors in the Wnt3a-sgRNA2 group was much slower than that in the NC group (Fig. 6C). Furthermore, IHC analysis confirmed that the expressions of Wnt3a and Ki67 were significantly decreased in the xenograft tissues of the Wnt3a-sgRNA2 group with weaker staining intensity (Fig. 6E). Discussion Early diagnosis and effective therapy of HCC are of the utmost importance. From a population-based cancer registry for 40 years, HCC still ranks the leading incidence among all malignancies and the poorest survival rate in the area inshore of the Yangtze River, China [2]. The Wnt/β-catenin pathway-related signaling including Wnt ligands, GSK-3β, β-catenin, and β-catenin-mediated downstream genes play crucial roles in HCC progression, including hepatocytes malignant transformation, metastasis, chemoresistance, liver cancer stem cells, and formation of epithelial-mesenchymal transition [17]. Our previous work, for the first time, revealed abnormality of oncogenic Wnt3a in the sera and cancerous tissue of HCC patients. However, its underlying mechanisms and the expression features in hepatocarcinogenesis remain to be identified. In the present study, we continued to explore dynamic expression of Wnt3a in hepatocarcinogenesis and evaluate its diagnostic and molecular-targeted value. Wnt/β-catenin signaling pathway plays a significant role in numerous biological process [18]. However, abnormal Wnt signaling was recently associated with HCC occurrence and progression, especially in canonical pathway Wnt3a activation or non-canonical pathway Wnt5a inactivation [19]. Previous work highlighted the correlations of Wnt3a levels with aggressive phenotype in HCC tissues [20]. Consistently, the current study demonstrated that Wnt3a expression in cancerous tissues was higher than that of para-cancerous tissues. In addition, survival analysis in large HCC cohort from TCGA indicated that Wnt3a could serve as a prognostic factor in HCC cases at advanced stages. Furthermore, the rat model discovered that the Wnt3a expression markedly increased in hepatocarcinogenesis, with gradually up-regulating Wnt3a at protein levels in sera and livers of rats. Dynamic expression of Wnt3a has been involved in rat liver tumorigenesis, and associated with malignant transformation of hepatocytes, indicating that Wnt3a might participated in promoting tumorigenesis and progression of HCC. Human Wnt3a gene located on chromosome (1q42.13) has been regarded as an activator for β-catenin accumulation and the canonical Wnt signaling pathway [21]. The amino-terminal region of Wnt proteins may mediate interactions with Wnt receptors and activate them by the carboxyl terminus. As expected, the level of serum Wnt3a expression in the HCC group was significantly higher than that in any of other benign liver disease groups and healthy control, which obviously correlated with malignant features. Besides, its superiority of Wnt3a similar to HS-GGT, over AFP or GPC-3 in HCC diagnosis was also observed in the current study. The current data, consistent with the previous work, further confirmed that the abnormal oncogenic Wnt3a expression in HCC progression, suggesting Wnt3a as a candidate specific biomarker for HCC diagnosis and differentiation. The Wnt signaling includes two parts: one is through canonical pathway characterized by the stabilization and subsequent nuclear transport of β-catenin resulting in the activation of transcriptional responses; the other is non-canonical pathway with more diverse and several different signaling modes that regulate cell biological behaviors. Accumulating evidence indicates that viral proteins of HBV (HBx, HBsAg) or HCV (Core or NS) as pathogenic factors provoke activation of Wnt/β-catenin pathway in hepatocarcinogenesis [22]. Interaction of Wnt signals with HBV or HCV genome linked β-catenin phosphorylation and abnormalities in the E-cadherin-catenin unit function lead to loss of intercellular junctions, progression in development of cirrhosis and HCC. Oncogenic Wnt3a plays a crucial role in cell proliferation and metastasis, particularly in progression and mediated-oncogenesis involving signaling pathways. Along these lines, the Wnt pathway has been identified as contributing to the development and progression of HCC [23]. Effective treatment of HCC is still a challenging problem worldwide. Therefore, developing novel molecule-targeted therapies may provide greater chance for effective therapies or overcome resistance to sorafenib [24]. Wnt3a is an important regulator of human HCC cell line growth, which induces activation of the canonical Wnt pathway after binding with SULF2 and GPC-3. Targeting oncogenic GPC-3 of Wnt upstream inhibited the proliferation of HCC cells [25]. Our study found that knockout Wnt3a reduced the proliferation and colony formation of HepG2 cells, with the cell cycle arrest in G1 phase in vitro. Besides, silencing Wnt3a significantly inhibited the xenograft growth, along with the down-regulation of proliferation marker Ki67, suggesting that Wnt3a could be a promising and effective target for HCC therapy. In conclusion, circulating oncogenic Wnt3a as a candidate marker was further confirmed in a cohort of patients with HCC or chronic liver diseases. High Wnt3a expression could serve as a prognostic factor in HCC patients at advanced stages. Furthermore, the dynamic alteration of Wnt3a was for first time observed in a hepatocarcinogenesis model. Besides, silencing Wnt3a inhibited cancer cells proliferation in vitro and significantly suppress tumor growth in vivo. Taken together, Wnt3a might be a promising specific biomarker or an effective target for HCC therapy. However, the current works also have some limitations, and further studies should clarify its exact regulatory mechanisms, functions and application.	2019-09-25T08:05:38.180Z	2019-10-08T00:00:00.000	{ "year": 2019, "sha1": "c7ddf4167df4b719dbb9e59323d3659788da1181", "oa_license": "CCBY", "oa_url": "https://www.jcancer.org/v10p5862.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "23a3fb4225a7d7ec4d267f00560cce1dbbaa47e6", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
145257421	pes2o/s2orc	v3-fos-license	An Intercultural Communication Study of Chinese and Malaysian University Students ’ Refusal to Invitation One of the challenges brought about by intercultural communication is the cross-cultural understanding of what is meant by what is said in another culture. Performing a given communicative act in a different cultural background than the speakers’ own requires taking into consideration several issues in order not to be perceived as impolite, rude, or even offensive in the host community. The present study aims to investigate the pragmatic behavior of refusal to invitation by Chinese international university students and Malaysian university students in Malaysia. The second aim is to seek the respondents’ perception in the process of refusing an invitation regarding their cognition, language of thought, and perception of insistence after refusing an invitation. The subjects were selected from forty Chinese international students and forty Malaysian students at University Sains Malaysia, Malaysia. Data were collected through a written discourse completion task and an immediate structured post-interview. An analysis of the data demonstrated that both Chinese and Malaysian respondents used similar type of strategies when refusing an invitation; however, they were different in terms of the number of strategies (i.e. frequency) used in each situation. It is hoped that the present research will not only make contributions to the studies of refusal behavior in the intercultural communication in general but to the pragmatic behavior of refusal to invitation between Chinese international students and Malaysian students in particular. Introduction Intercultural communication is not a new concept.There would be no need to study intercultural communication in the case in which people never come into contact with each other, and therefore, there would be no need to communicate with people who speak a different language and organize their lives according to completely different norms and values (Pinto, 2000).However, what is new about intercultural communication is the systematic study of exactly what happens when cross-cultural contacts and interactions take place-when message producer and message receiver are from different cultures (Gao, 2006). One of the challenges brought about by intercultural communication is the understanding of speech acts cross-culturally.Speech acts or the communicative acts (e.g.thanking, apology, request) vary cross-culturally and the appropriate use of a given speech acts is subject to variation as the cultures differ.For example, in complimenting someone on her new dress, a positive acceptance of the compliment by appreciation (e.g.thank you) is common in some cultures (e.g. the United States), while rejecting the compliment (e.g.no, it is not new) is more appropriate in some other cultures (e.g.India).Therefore, a successful intercultural communication requires the non-native speakers' knowledge of the meaning of a particular speech act in a given cultural setting.Studies on speech acts can be divided into three categories: studies within one specific language (e.g.Koike, 1989;Turnbull and Saxton, 1997), studies between two or more languages (e.g.Chen, 1993;Lee-Wong, 1994;Fukushima, 1996;Liao and Bresnahan, 1996;Pair, 1996) and studies between languages produced by native and non-native speakers (e.g.Beebe, Takahashi, and Uliss-Weltz, 1990;Yu, 1999;Hassall, 2001;Byon, 2004, cited in Hsien andChien, 2005). The present research is of the second type: it examined the speech act of refusal to invitation between Chinese international students in Malaysia and Malaysian university students.Refusals have been recognized as a "major cross-cultural sticking point for many non-native speakers" (Beebe, Takahashi and Uliss-Weltz, 1990, p. 56).A refusal is a negative response given to an offer, request, invitation, etc.How to say 'no' sometimes is much more important than the answer itself since failure to refuse appropriately may lead into unintended offense or breakdown in communication.According to Al-Kahtani (2005), the speakers must be familiar with the appropriate form and its function, the speech acts and its social elements and cultural-linguistic values of each individual group.Having those skills are very important since the "inability to say 'no' clearly has led many non-native speakers to offend their interlocutors" (Ramos, 1991). While there are great number of studies which have examined certain speech acts (e.g.request, apology), the amount of research on refusals is much more limited.Out the fifteen studies in the topic of refusals, only six included non-native speakers of English and only four of these studies examined how non-native speakers of languages other than English utilized English in making refusals (Beebe, Takahashi and Uliss-Weltz, 1990;Chen, 1995;King and Silver, 1993).Consequently, it was the lack of research on the interaction between non-native speakers of English in the context where English is used as a means of communication in particular and Chinese international university students' interaction in Malaysia with Malaysian university students in refusing an invitation in particular which has been motivated this study. In today's increasingly connected world, it is becoming more important than ever that the language learners attain true communicative competence (Sadler and Eröz, 2001).Pragmatic failure or the "inability to understand what is meant by what is said" (Thomas, 1983) often occurs due to learners' insufficient L2 sociocultural knowledge.There are many studies on pragmatic failure of Chinese second language learners (He & Yan, 1986;Hong, 1991;Chen, 2005;Zhang, 2005, cited in Lin, 2008).However, few studies have addressed Chinese learners of English in the target language community (Lin, 2008).The pragmatic failure not only results in miscommunication and hinders effective communication, but also leads in the misjudgment of the interlocutor him/herself in a cross-cultural dyad (Chen, 1996).Moreover, according to Lin (2008), among the many studies on Chinese learners of English, few have used Chinese learners of English as subjects in the target language community although many have been conducted in EFL classroom settings in China (e.g.He and Yan, 1986;Chen, 2005;Zhang, 2005).Therefore, continued research in intercultural communication will provide an improved understanding of the differences and similarities of the pragmatic behavior speakers of different languages use on a given speech act.In consequence, the aim of this study is two-fold: first, to investigate the pragmatic behavior in refusal to invitation by Chinese international university students and their Malaysian counterparts at Universiti Sains Malaysia, Malaysia and second, to seek the students' perception in the process of refusing an invitation with regard to the cognition, language of thought, and perception of insistence after refusing an invitation. Review of Literature The review of literature includes four sections: the first section is a brief review of learning and use of speech acts and retrospective verbal report.The second section discusses intercultural communication and elaborates on speech act of refusal to invitation. Learning and Use of Speech Act Learners utilize different strategies when producing speech acts (Félix-Brasdefer, 2008).Cohen (2005, cited in Félix-Brasdefer, 2008) suggested a classification of language-learning strategies to assist learners improve their pragmatic knowledge in a target language: a. Cognitive strategies which help learners identify, distinguish, practice and commit material to memory.For example, in the speech act of expressions of gratitude, learners (as favor receivers) use strategies to mitigate the imposition on the speakers' shoulder and express gratitude adequately. b. Metacognitive strategies which include the planning, execution (e.g.checking how it is going) and the evaluation of a speech act c. Effective strategies which regulate attitude, motivation for learning an L2 and reduce anxiety, and d.Social strategies which include seeking opportunities to interact with native speakers and to involve in various speech act interactions. On the other hand, language-use strategies focus on using the language that learners have learned to whatever degree in their current interlanguage (Cohen, 1996;1998, cited in Félix-Brasdefer, 2008).These strategies are as follows: a. Retrieval strategies which are used to retrieve the pragmalinguistics information necessary to perform a speech act, such as the use of the conditional in Spanish to express politeness (Chodorowska-Pilch, 2004) b. Rehearsal strategies that include practicing (form-focused practice) target language structures c. Cover strategies that include using a memorized or formulaic form that has not been fully understood in an utterance, or the use of simplification as a result of incomplete knowledge of the target language, and d. Communication strategies which aim at conveying meaningful information or expressing an appropriate speech act response in a target language. The proposed classification can be also used to examine the cognitive processes learners use when planning and executing refusals in a foreign language.One way to analyze cognitive processes (e.g.selection of language of thought) or learner' perceptions of sociocultural information is by means of retrospective verbal reports. Retrospective Verbal Reports Retrospective reporting is a common method of data collection in psychology and in L2 pragmatic research (Cohen, 2004;Ericson and Simon, 1993).In retrospective verbal report, data are collected immediately after completion of another task while much information is still available in short-term memory and can be directly reported or used as 'retrieval cues'.Cohen (2004) reported this technique as an effective and useful source of information in case the data are collected carefully.Verbal report can provide metalinguistic data with regard to cognitive processing (Félix-Brasdefer, 2008).This technique is used to realize what information learners is attending to while performing a task (Cohen, 1998).The purpose of using this technique in ILP research is because "one may learn what the respondents actually perceived about each situation (e.g.what they perceived about the relative role status of the interlocutors) and how their perceptions influenced their responses" (Cohen, 2004, p.321).Verbal reports used in ILP provide information on the cognitive processes involved in the production of different speech acts.Robinson (1992) examined the effect of discourse completion task and verbal reports (i.e.think aloud protocol) on the speech act of refusal among learners of English as a second language.Results showed that verbal report procedures elicit specific information about the planning processes of refusals as learners attempted to produce semantic formula, and it was concluded that verbal reports appear to be a 'practical means for both generating and investigating hypotheses about second language acquisition of pragmatic knowledge" (Robinson, 1992, p.50).In another study, Cohen and Olshtain (1993) examined the cognitive processes involved in the production of apologies, complaints and requests among EFL learners.Their findings showed that the selection of the language of thought during the planning and execution of a speech act was a complex matter the learners utilized different strategies to search for language forms, and that learners did not attend much to grammar or pronunciation.According to Felix-Bradsfer (2008), issues such as how learners' perceptions influence their understanding of politeness, their perception of pragmatic knowledge in the target language, the selection of the language of thought to deliver a given speech act (e.g.expressions of gratitude) are all areas which need to be explored. Intercultural Communication There are several definitions of the concept of intercultural communication.The word intercultural has been characterized by a certain terminological arbitrariness: intercultural, interethnic, interracial, and cross-cultural often seem to be used in free variations.In intercultural communication, the notions culture and communication are very broad and vague (Knapp and Knapp-Potthoff, 1987, p.3).They might be regarded as everything that is a consequence of culture, and everything communicates (Knapp and Knapp-Potthoff, 1987).This view is summarized in Hall's (1959) dictum "communication is culture, culture is communication" (cited in Knapp and Knapp-Potthoff, 1987, p.3).However, as Spencer-Oatey (2006) describes the phenomenon, "Intercultural communication is concerned with communication between people from different sociocultural groups.It focuses on the role played by cultural-level factors (in contrast to individual and universal factors), and explores their influence on the communication process" (p.2537) In international cross-communication, it is the learners' incompetency in interpreting and giving correct pragmatic behavior to the literal meaning rather than learners' mal-or mispronunciations or grammatical mistakes which may cause serious intercultural miscommunication.Even learners' apparent fluency or accuracy or in other words, their language proficiency does not guarantee thoroughly effective communication.In other words, the development of grammatical competence does not necessary mean a corresponding level of pragmatic competence, which might lead to pragmatic failure (Omar, 1991;Takenoya, 1995, cited in Al-Momani, 2009;Bardovi-Harlig, 1999).According to Rintell andMitchell (1989, cited in Lin, 2008), learners' inability to understand the literal meaning and lack of knowledge of the rules of use for interpreting the words may create misunderstanding and even offense.Studies in intercultural communication may have a great impact on speakers' mutual understanding and their expectation especially in a novel cultural setting.Sometimes speaker's pragmatic incompetency leads into failure or breakdown into intercultural communication. Pragmatic failure or "the inability to understand what is meant by what is said" (Thomas, 1983, p.91) is a new branch of pragmatics which occurs from the mistakes which fail to fulfill communication because of infelicitous style, incompatible expressions and/or improper habit (Ziran He, 1997).In such occasions, although the speakers may use grammatically correct sentences, they unconsciously violate the norms of interpersonal relationship and social norms in speech, or ignore the hearer or context (Qian, 1997).Within the field of cross-cultural pragmatics, studies of pragmatic failure have developed rapidly in the past twenty years.One of the challenges of intercultural communication is the need to create awareness about the importance of understanding speech acts cross-culturally (Palma Fathy, 2005).Therefore, intercultural communication studies on a given speech act can give insight into how interlocutors of different cultures are pragmatically different or similar as they behave in a specific situation which consequently can minimize the occurrence of pragmatic failure or breakdown in communication. Speech Act of Refusal The speech act of refusal is identified as a response to four specific speech acts: those of request, invitation, offer and suggestion (Beebe, Takahashi and Uliss-Weltz, 1990;Chen, Ye, and Zhang, 1995) rather than one which initiates this act by itself (Zhou Geyang, 2007).Refusal is characterized as an act by which a speaker refuses to engage in an action proposed by the interlocutor (Chen, Ye, and Zhang, 1995).For example, in refusing to an invitation to go out, one might say, Sorry, I have an exam tomorrow.A response to refusal can be expressed either directly, e.g.No, I can't, or indirectly.An indirect response to refusal may increase the degree of complexity, as the speaker has to choose the appropriate form or forms to soften the negative effects of a direct refusal (Felix-Brasdefer, 2008).Refusals may be mitigated by giving reasons (e.g.I have to do my assignment), expressing regret (e.g.I'm so sorry), or promising future acceptance (e.g.I hope I can make it next time). Refusals can also be accompanied by expressing positive remarks or feelings (e.g.Congratulations on your promotion.I am very happy to hear that but…), an expression of gratitude (e.g.Thanks for your invitation), an expression of willingness (e.g.I'd love to but…).Overall, refusals are complex speech acts which require not only long sequences of negotiation and cooperative achievements, but also "face-saving manoeuvres to accommodate the noncompliant nature of the act" (Gass and Houck, 1999, p.2).According to Tanck (2002), refusal occurs "when a speaker directly or indirectly says no to a request or invitation" (p.2).Refusal is described as "a major cross-cultural 'sticking point' for many non-native speakers' (Beebe, Takahashi, and Uliss-Weltz, 1990).It is a complicated act since it is affected by several factors including gender, age, level of education, power, and social status (Fraser, 1990;Smith, 1998, cited in Wannaruk, 2008).In politeness theory, refusal is a face-threatening act since it contradicts listener/ requester / inviters' expectations and is realized through indirect strategies (Tanck, 2002).In cross-linguistic or cross-cultural communication, people are different in terms of the language they employ in each speech community.In these communities, pragmatic failure sometimes occurs when the speaker uses a face-threatening speech act (e.g.request, apology, refusal).According to Takahashi and Beebe (1987, p.133), "the inability to say 'no' clearly and politely …has led many non-native speakers to offend their interlocutors".The speech act of refusal is a universal phenomenon.However the realization of this speech act may be culturally-specific. Methodology The following sub-section elaborates on the respondents chosen, instruments utilized and procedure taken in the present study. Subjects Data were elicited from two groups of respondents: international Chinese university students and Malaysian university students studying at University Sains Malaysia, Malaysia.The data were collected outside the classrooms from volunteer participants during the second semester 2010-2011.Over one hundred questionnaires were collected.However, those questionnaires which were incomplete or did not reply based on the given instructions were discarded.Table 4.1 shows a summary of the respondents' demographic data.As the table indicates, the Chinese respondents were both male and female with ages ranging from 18 to 32.The majority of the respondents were undergraduate students.Forty five percent of the respondents have lived in Malaysia for more than 1 year; 100% of whom hold at least one type of English language certificate, which is the requirement of enrolment at USM. Table 4.1 also demonstrates that the Malay respondents included a majority of female respondents whose ages ranged from 18 to 29, all of whom were undergraduate students.All Malaysian respondents hold MUET (Malaysian University English Test). Instrument The data were elicited through a questionnaire followed by a structured post-interview.The questionnaire consisted of two parts: the first part of the questionnaire required respondents' biodata including age, gender, nationality, language spoken at home, level of education and program.The second part of the questionnaire was an open ended questionnaire in the form of a Discourse Completion Task (DCT) adopted from Felix-Brasdefer's (2008) study of refusal to invitation.The questionnaire included two situations occurring between a professor and a student and two classmates.The respondents were asked to read the situations and write down what they might say in refusing the interlocutor's invitation in real situations.Below is a description of the DCTs and the structured interview.The questions in the structured post-interview were also adopted and adapted from Felix-Brasdefer (2008).The questions in the interview addressed respondents' perception with regard to the cognition, language of thought, and perception of insistence in the process of refusals to an invitation. Discourse Completion Task The open-ended questionnaire consisted of two questions which the respondents were instructed to read and respond to each one as if they were in a real situation.In Situation #1, the respondents had to refuse an invitation to a close friend's birthday party and the Situation #2, the respondents were required to refuse an invitation to a supervisor's invitation to dinner due to his recent academic promotion. Situation 1: A friend invites you to his birthday party next Friday evening.You have been friends since your freshman year in college.He is inviting a selected group of friends over to his house, and you are one of them, but you can't make it.What would you tell him?You:…………………….Situation 2: Your supervisor at university is having a party next Saturday evening at a restaurant in town and is inviting you and few other students to celebrate his recent academic promotion at university.He invites you to celebrate this important occasion with him, but you are unable to attend.What would you tell him?You:…………………….. Coding Scheme Following the data collection, the utterances were codified based on a classification of refusal strategy adopted from Beebe, Takahashi, & Uliss-Weltz (1990).This coding scheme is used extensively in most refusal studies (see appendix for more information). From the detailed description of the possible strategies to refuse an invitation based on Beebe, Takahashi and Uliss-Weltz's coding scheme, only a total of seven strategies were elicited in both Chinese' and Malaysians' responses.However, four additional strategies in addition to Beebe et al. coding scheme were found in the responses.These included one refusal strategy type and two adjunct-type strategies.The strategy is termed as compensation whereby the speaker tries to balance a bad effect of something (e.g. the speaker invites the friend to celebrate his birthday sometime later in place of refusing him this time.).The three adjuncts elicited from the responses were alerters (sir, professor, honey, dear) and greetings (e. -Thank you to invite me, but there is some reasons I can't join your party.I'm sorry (gratitude + reasons + negative ability + statement of regret). Example by Malaysians responses: -I'm sorry; I can't come to your birthday party.I've got other important things to do; I really can't postpone it to other day.(Statement of regret+ negative willing/ability+ reasons). Situation 2: Example by Chinese responses: -Dear sir, thank you for your invitation.I'd like to but I am sorry I can't attend.But I'll try my best to attend (Alerters+ gratitude+ statement of positive feelings+ statement of regret+ negative ability+ promise of future acceptance). Example by Malaysians responses: -Congratulations for your promotion.Thank you for inviting me to a party, but I'm really sorry I can't come because I really have an important thing to do with my family (Greetings + gratitude+ statement of regret+ negative willing/ability+ reasons). Structured Interview A verbal questionnaire was conducted immediately after administering the DCTs.The respondents were asked three questions in terms of their perception and cognitive processing while refusing an invitation.The verbal report questions were adopted from Félix-Brasdefer's ( 2008) study of invitation to refusal.According to Felix-Brasdefer, results of these reports are instrumental in gathering relevant information about learners' cognitive processes with regard to: (1) cognition (attention to information during the planning and execution of a refusal), in particular attention to politeness, discourse, grammar and vocabulary; (2) the selection of the language of thought (English or Chinese or Malay) for conveying pragmatic intent; and (3) the perception of insistence after refusing an invitation. Data Analysis The data were codified based on the classification of refusal strategies adopted and adapted by Beebe, Takahashi and Uliss-Weltz (1990).Those questionnaires which were either incomplete or did not follow the given instructions were discarded.The analyses were conducted based on individual item responses on each participant's DCT.The data were then entered into SPSS software for further descriptive (i.e.frequency) and statistical analysis (i.e.independent-sample t-test). Situation 1 The findings showed that expressions of excuses, reasons or explanation and statement of regret were the first and second most frequently used strategy by Chinese students in refusal to an invitation while Malay respondents used statement of regret followed by expressions of excuses, reasons or explanation as the first and second most frequently used strategies in refusal to an invitation.The findings also indicated that expressions of negative ability or willingness was the third most frequently used strategies by both Chinese and Malaysian students.As table 5.1 indicates (see the appendix), the Chinese used greetings as the fourth frequently used strategy in their responses while their Malaysia counterparts used repayment followed by greetings in the frequency pattern of strategies.The results showed that expressions of positive opinions, feelings or agreement had been the fifth frequently used strategy by both Chinese and Malaysian respondents.Moreover, the findings demonstrated that Chinese used expressions of future acceptance of invitation and expressions of gratitude more often than their Malaysians counterparts. Situation 2 The findings displayed that excuses, reasons or explanation and statement of regret were the first and second most frequently used strategies by Chinese students in refusing an invitation while the frequency pattern of strategies for Malaysian students were statement of regret followed by excuses, reasons or explanations.Expressions of negative ability or willingness was the third frequency of used strategy for both Chinese and Malaysian students.Table 5.2 indicates (see the appendix) that with regard to the use of adjuncts, the Chinese respondents preferred to state their positive opinions, feelings or agreement for refusing an invitation and use expressions of gratitude and appreciation more than Malaysians did.On the other hand, Malaysian used more alerters in their responses and attempted to redress the threatening face of refusal by offering and inviting the superior with another event or gatherings. Statistical Analysis Table 5.3 shows the findings of independent sample t-test for the responses elicited from the two respondent groups.Results of statistical analysis showed that there was a statistically significant difference in the use of statement of regret in Situation 1 between Chinese and Malaysian respondents.In other words, Malaysian respondents used significantly more statement of regret in their responses than Chinese respondents.As table 5.3 displays, there is a statistically significant difference in the use of statement of regret between Chinese and Malaysian respondents.The findings showed that Chinese students used significantly more expressions of gratitude than Malaysian respondents.In addition, there are statistical significant differences in the use of alerters and repayment strategies between Chinese and Malaysian respondents.The findings showed that Malaysians used significantly more alerters and repayment strategies in their responses than their Chinese counterparts. With respect to the use of strategies in Situation 2, results of independent sample t-test displayed that there were statistically significant differences in the use of expressing negative ability, statement of regret, use of alerters, greetings and repayment between Chinese and Malaysian university students.The findings showed that Malaysians used these strategies more significantly than their Chinese counterparts. Speech Act Set The data were analyzed qualitatively with regard to the speech act sets in respondents' production of refusals.According to Murphy and Neu (1996), a speech act set is a combination of individual speech acts.In order to develop or achieve a communicative purpose, the speaker has to produce more than one discrete speech act.For example, in case of refusals, one might appropriately produce three speech act sets: (1) an expressions of regret, "I'm so sorry", followed by (2) a direct refusal, "I can't come to your graduation," followed by (3) an excuse, "I will be out of town on business" (Chen, 1996, cited in Tanck). According to Olshtain (1983, cited in Murphy andNeu, 1996), some cultures preferred one or another formula, or a combination of formula to produce a speech act.For example, American English tends to express an apology which is followed by an explanation while only Hebrew speakers tended to give an explanation only.In addition, these language-specific preferences may cause a second language learner to appear inappropriate in the target language; in the previous example, Hebrew speakers who transfer this formula will undoubtedly sound rude in English (Olshtain, 1983). The responses were analyzed to determine the speech act set of refusal to invitation by Chinese and Malaysian respondents.The following speech acts set were the most frequently used speech act set to initiate the refusing acts. Results of the analysis show the following speech act sets for situation 1: The findings displayed that Chinese and Malaysian shared more similarities in terms of the use of speech act set in the situation of refusing an invitation to a friend; however, the frequency of use of statement of regret followed by expressions of positive feelings were higher among Malaysian respondents than their Chinese counterparts.With regard to the use of speech act set in situation 2, the findings showed that Malaysians used longer and elaborated speech act set in situation 2 than their Chinese counterparts. Results of Structured Interview A structured post-interview was conducted upon the completion of DCT.The first questions addressed the respondents' cognition when refusing an invitation.They were asked what exactly they were paying attention to when they refused the invitation in either of the situations.With regard to the situation number 1, most Chinese students reported that they were thinking of the reasons as they prepared to refuse the situation (e.g.assignment, school projects or chatting time).In addition to the reasons, how to state those reasons were also very important to them.One of their concerns was not to hurt their friends' feeling.Some of the Chinese students also reported that they did not like meeting strangers. To Malaysian respondents, on the other hand, stating the reasons until the friend was satisfied was described as being very important.The majority of Malaysian respondents reported that they are very cautious about the arrangement of words, as well as not hurt their friend's feeling or to make him/her sad.The friend's responses and reaction were described as also being important to them.The second and third questions addressed respondents' language of thought.They were asked what language they were thinking of when refusing an invitation in Situation 1 and whether they think they should switch the language of the thought (whether they were thinking in English, Chinese or Malay) at some points during their responses.Some Chinese and Malay reported that they would think in English.However, the majority of Chinese and Malaysians reported that they would think in their first language and they would switch from either Chinese to English (for Chinese) or Malay to English (for Malaysians) when they are thinking of the responses. To examine the respondents' pragmatic knowledge, they were asked if they had noticed any cultural differences with respect to the notion of insistence between Malaysian and Chinese people.The majority of both groups of respondents agreed that Chinese people were not more insistent on these situations than Malaysians.The respondents were also asked whether they expected their friend to insist on changing their plans and going to his party and in the case of insistence, what the speakers' feelings would be and whether insistence is culturally acceptable or rude.Though some Chinese and Malaysian respondents were expecting insistence from the friend, the majority of the respondents reported that they did not expect any insistence. To some Chinese respondents, insistence is a sign of friendship while to some others insistence made them 'sad', 'feel sorry', or 'uncomfortable' or even 'angry'.Moreover, some Chinese found out that based upon the friend's insistence, they would slightly change their mind to go.Half of the Chinese reported the notion of insistence culturally acceptable, while the other half found insistence to be culturally or personally not acceptable.On the other hand, the majority of Malaysian respondents reported that they did not expect insistence from the friend's side and in case it occurred, it was culturally acceptable although they would feel 'sad', 'guilty', or 'pity' that they could not make it to his party.Results of the interview with regard to Situation 2 shows that for the first question, the Chinese respondents were much more concerned about the reasons and the manner in which way they would state reasons to the supervisor, as well as the supervisor's attitude about them when rejecting his invitation.On the other hand, to the majority of Malaysian respondents, it was 'arrangement of the words', 'the reasons', the 'supervisor's feelings, and attitude' about them that have been more important. The two groups responded similarly for question number 2, 3 and 4 as in situation 1.However, in response to question 5, the majority of Chinese reported that though they did not expect insistence from the supervisor, but, in the event that the supervisor did insist, they would 'feel happy' and would think that 'they've got a place or position' with the supervisor.To some others, supervisor's insistence made them feel 'sad', 'guilty', 'sorry', and 'uncomfortable'.Unlike situation 1, the majority of Chinese found it culturally acceptable to receive insistence from a supervisor.The majority of Malaysian respondent didn't expect insistence from the supervisor and in case of receiving insistence they would feel 'bad', 'guilty as a student', 'uncomfortable'. Discussion The findings display that expressions of excuses, reasons or explanation , statement of regret and expressions of negative ability or willingness were the most frequently used strategies (by different frequency sequence) for Chinese and Malaysian students in Situation #1 and Situation #2.The findings were in line with the findings of the previous studies with data elicited from role-play which displayed that expressions of reasons and explanations is the most frequently used strategy for expressing a refusal indirectly (Felix-Brasdefer, 2003, Garcia, 1999, Nelson, 2002, Al-Issa, 2003, Al-Eryani, 2007, Al-Kahtani, 2005).Moreover, the findings of this study were in line with the findings of refusal studies on Malaysian students (Farnia andAbdul Sattar, 2010, Abdul Sattar, Salasiah Chel Lah, andRaja Rozina, 2010) in which statement of regret followed by excuses, reasons or explanations were the most frequently used strategy among Malaysian respondents. According to the findings, the degree of elaboration of strategies varied for each group of respondents and this could be conditioned by the students' level of grammatical competence.The analyses display that Malaysian respondents used longer and elaborated responses than their Chinese counterparts.It might lie for the fact that Chinese student' lack of control of the L2 grammar prevented them from conveying and elaborating their language compared to their Malaysian counterparts. Another conditioning factor for the planning and execution of a refusal to an invitation was the selection of thought.The most common pattern for more than 50% of students was to start thinking in the native language (i.e.planning the refusal) and then to translate from Chinese or Malay into English (i.e.executing the refusal in English).Thus, contrary to the popular belief that learners should not consult their L1 during speech act production, the results of the present study are consistent with the ideas expressed by Cohen (1998) which demonstrated that consulting the native language may be beneficial for the following strategic purposes: (1) to chunk material into semantic clusters; (2) to help learners keep their train of thought; (3) to create a network of associations; (4) to clarify grammatical roles; and (5) to make the input more familiar and consequently more user-friendly (p.5). According to Bardovi-Harlig (1996), one of the goals in facilitating the development of pragmatic competence to provide learners with enough input and classrooms can be the source of input especially for foreign language learners.Teaching how to communicate appropriately both pragmalinguistically (using appropriate form) and sociopragmatically (using appropriate meaning) is of great importance since native speakers often forgive linguistics errors (phonological, syntactic or lexical) but they may interpret sociolinguistic errors as learners' rudeness and impoliteness rather than as the transfer of different sociolinguistic rules (Thomas, 1983;cited in Boxer, 1996, p.128). Conclusion and Pedagogical Implication This study examined the perception and production of the speech act of refusal to invitation among international Chinese university students who pursue their studies in Malaysia and Malaysian university students.There have been some limitations to this study.First, length of stay in the host environment plays an important role in the development of communicative competence in language learners (see Felix-Brasdefer, 2008).In this present study, the length of stay in Malaysia varied among the international Chinese students (1 year to 3 years), and the effect of this variable on Chinese ESL learners was not examined.In addition, although the description of DCTs was controlled for speech act type (refusal to invitation), it is likely that some learners had never been involved in situations similar to those in the DCTs.Despite these limitations, this study, however, presents a number of insights.First, the findings display that both Chinese and Malaysian students are alike in terms of the type of strategies used to refuse an invitation.However, the students acted differently with regard to the frequency of strategies used in each DCT situation.In Situation #1, in refusing a friend's invitation, Chinese students favored expressions of positive feelings/ remarks and expressions of gratitude/ appreciation more than Malaysians which may indicate different expectations of "friends" in the two cultures.In situation of refusal to a superior, Malaysians significantly used more statement of regret in the DCT situations and expressed their negative ability to attend the event more than Chinese students. Overall, the findings of the present study support the current research in that expressions of regret and expressions of excuses, reasons and explanations are the most frequently used strategies in refusal studies.However, English teachers should pay more attention to improve their students' pragmatic competence so as to help them avoid pragmatic failure and breakdown in communication.Teachers should train the learners with the social conventions, communicative rules and values of the target nation (Zheng and Huang, 2010).In addition, research in intercultural communication will provide an improved understanding of the differences and similarities of the pragmatic behavior of speakers of different languages and cultures. g. birthday greetings, congratulations).Some examples of the elicited responses and coded based on Beebe, Takahashi, & Uliss-Weltz (1990) are as follows: Situation 1: Example by Chinese responses: Negative ability/willing +Reasons e.g.I'm sorry.I can't come to you party.I'm working on my school projects.Statement of regret + reasons Reasons+ statement of regret Malaysian respondents: Statement of regret+ Negative ability/willing +Reasons Statement of regret + reasons Statement of regret + positive feelings e.g.I'm really sorry.I'd love to come, but….. Situation 2: Chinese respondents: Statement of regret + alerters e.g.I'm so sorry Prof ….Gratitude/appreciation + negative ability/ willing Alerters+ Statement of regret Malaysian respondents: Statement of regret+ Negative ability/willing +Reasons Statement of regret + Reasons Statement of regret + alerters+ negative ability/ willing+ Reasons Table 4 . 1. Summary of demographic survey	2018-11-23T00:42:13.371Z	2012-01-30T00:00:00.000	{ "year": 2012, "sha1": "d8fc28bf1c255117165c0b2d0497c8cf6355d49b", "oa_license": "CCBY", "oa_url": "https://ccsenet.org/journal/index.php/ijel/article/download/14678/10036", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "d8fc28bf1c255117165c0b2d0497c8cf6355d49b", "s2fieldsofstudy": [ "Education" ], "extfieldsofstudy": [ "Psychology" ] }
216419849	pes2o/s2orc	v3-fos-license	Air pollution reduction and mortality benefit during the COVID-19 outbreak in China To control the novel coronavirus disease (COVID-19) outbreak, China undertook stringent traffic restrictions and self-quarantine measures. We herein examine the change in air pollution levels and the potentially avoided cause-specific mortality during this massive population quarantine episode. We found that, due to the quarantine, NO2 dropped by 22.8 µg/m3 and 12.9 µg/m3 in Wuhan and China, respectively. PM2.5 dropped by 1.4 µg/m3 in Wuhan but decreased by 18.9 µg/m3 across 367 cities. Our findings show that interventions to contain the COVID-19 outbreak led to air quality improvements that brought health benefits which outnumbered the confirmed deaths due to COVID-19 in China Air pollution reduction and mortality benefit during the COVID-19 outbreak in China To control the coronavirus disease 2019 (COVID-19) outbreak, China adopted stringent traffic restrictions and self-quarantine measures, first in Wuhan and neighboring cities beginning Jan 23, 2020, and then 2 days later in all provinces in China (figure). The countrywide ban on traffic mobility greatly reduced transportation emissions, whereas emissions from residential heating and industry remained steady or slightly declined. 1 In this Comment, we examine the change in air pollution and the potentially avoided causespecific mortality during this large-scale quarantine. As of March 14, 2020, new confirmed cases of COVID-19 in China reported by the National Health Commission decreased to 20 (four cases from Wuhan) (figure). By this time, most Chinese provinces had lowered the level of emergency responses. We thus defined the quarantine period as Feb 10 to March 14 and the period before quarantine as Jan 5 to Jan 20. Based on evidence from previous years, we excluded the Chinese New Year holidays to avoid reductions in air pollution that were unrelated to the quarantine (figure). We obtained daily concentrations of nitrogen dioxide (NO 2 ) and PM 2·5 in 367 Chinese cities from Jan 1, 2016, to March 14, 2020. We focused on NO 2 and PM 2·5 because both are traffic-related air pollutants whose emissions were substantially reduced as a result of the traffic bans and home quarantine, and both had well established concentration-response functions (CRFs) from one of the largest epidemiological studies in China on short-term mortality effects. 2,3 A differencein-difference approach was then applied to quantify air pollution changes due to the quarantine. Specifically, we calculated changes in air quality during the quarantine versus before the quarantine period in 2020 and compared these findings with corresponding changes in the same lunar calendar periods from 2016 to 2019. This approach, which can also be interpreted as comparing changes in air quality in 2020 versus 2016-2019 during the quarantine period with those changes in the before quarantine period, also controlled for the long-term declining trend in air pollution because of China's clean air policy in the past few years. 4 To validate the air quality changes, we used satellite images from the Tropospheric Monitoring Instrument, which is onboard the Sentinel-5 Precursor satellite, to derive the mean NO 2 tropospheric column density for periods during and before quarantine. We then calculated the avoided mortality attributable to decreases in daily NO 2 and PM 2·5 over China on the basis of short-term CRFs from a previous study of 272 Chinese cities (appendix p 2), and the cause-specific mortality data from the China Health and Family Planning Statistical Yearbook 2018. 2,3 In addition to total non-accidental and cardiovascular mortality, the cause-specific mortality for hypertensive disease, coronary heart disease, stroke, and chronic obstructive pulmonary disease (COPD) was also calculated. The attributable fraction (AF) method was used to estimate the daily avoided cause-specific mortality from the air pollution reduction. 5 AF is defined as follows: β is the cause-specific coefficient of the CRF and Δc is the air pollution changes due to the quarantine. AF is then multiplied by the daily cause-specific number of deaths and the total number of days during the quarantine period (34 days) to estimate the cause-specific avoided deaths. We found that, because of the quarantine, NO 2 dropped by 22·8 µg/m³ in Wuhan and 12·9 µg/m³ in China. PM 2·5 dropped by 1·4 µg/m³ in Wuhan but decreased by 18·9 µg/m³ across 367 cities (appendix p 3). The smaller reduction in PM 2·5 in Wuhan is due to a similar decreasing trend in PM 2·5 in 2016-2019. The pronounced decline in NO 2 across China during the quarantine period was also detected by the Copernicus Sentinel-5P satellite with the NO 2 tropospheric column density (figure). We estimate that improved air quality during the quarantine period avoided a total of 8911 NO 2 -related deaths (95% CI 6950-10 866), 65% of which were from cardiovascular diseases (hypertensive disease, coronary heart disease, and stroke) and COPD (figure, appendix p 4). Furthermore, we estimate that reduction in PM 2·5 during the quarantine period avoided a total of 3214 PM 2·5 -related deaths (95% CI 2340-4087) in China, 73% of which were from cardiovascular diseases Comment e211 www.thelancet.com/planetary-health Vol 4 June 2020 and COPD. Similar estimates were found with an alternative before quarantine period from Jan 1 to Jan 20 (appendix pp 3-4). Our estimates should be interpreted with caution because of the potential overlap between PM 2·5 and NO 2related mortality and the effect on mortality rate from disrupted health-care systems during the quarantine, which could have impacted the timely treatment of patients with chronic diseases. We used cause-specific CRFs from single-pollutant models because coefficients from two-pollutant models were not available. 2 Although there might have been some risk of double counting, results from published literature suggest that this risk is small because effect estimates for NO 2 and PM 2·5 were similar between single-pollutant and two-pollutant models. 3,6 Moreover, similar to previous epidemiological studies with outdoor air pollution, 7 exposure measurement error is inevitable since most people stayed indoors. Our estimates suggest that interventions to contain the COVID-19 outbreak led to improvements in air quality that brought health benefits in non-COVID-19 deaths, which could potentially have outnumbered the confirmed deaths attributable to COVID-19 in China (4633 deaths as of May 4, 2020). 8 Our findings show the substantial human health benefits related to J a n 5 J a n 1 0 J a n 2 0 J a n 2 3 J a n 2 5	2020-04-02T09:13:14.339Z	2020-03-27T00:00:00.000	{ "year": 2020, "sha1": "20cce932765810c6ecf480a7f0110fcd54b423db", "oa_license": "implied-oa", "oa_url": "https://europepmc.org/articles/pmc7220178?pdf=render", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "bba32e48af6d98550ebb764c73232bf97911b7d8", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
250730936	pes2o/s2orc	v3-fos-license	Constitutive Phenotypic Modification of Lipid A in Clinical Acinetobacter baumannii Isolates ABSTRACT The degree of polymyxin B (PMB) resistance was measured in 40 clinical Acinetobacter baumannii isolates obtained from health care facilities. All of the tested isolates possessed a multidrug-resistant (MDR) phenotype against four classes of antibiotics (meropenem, doxycycline, gentamicin, and erythromycin), except for PMB. The blaOXA-23 gene was detected throughout the genetic analysis and experimental assay, indicating that all of the MDR strains were carbapenem-resistant A. baumannii strains. Multilocus sequence typing-based genotyping revealed that nine selected strains belonged to the international clone II lineage. When matrix-assisted laser desorption ionization–time of flight mass spectrometry was performed, intrinsic lipid A modification by phosphoethanolamine (PEtN) incorporation was noticeable only in the PMB-resistant (PMBR) strains. However, the presence of hexa- and penta-acylated lipid A due to the loss of the laurate (C12) acyl chain was noted in all PMB-susceptible strains but not in the PMBR strains. The reduction of negative surface charges in the PMBR strains was assessed by zeta potential analysis. Fluorescence imaging using dansyl-PMB revealed that, in the PMBR strains, PMB was less likely to bind to the cell surface. IMPORTANCE The widespread presence of MDR pathogens, including A. baumannii, is causing serious hospital-acquired infections worldwide. Extensive surveillance of MDR clinical A. baumannii isolates has been conducted, but the underlying mechanisms for their development of MDR phenotypes are often neglected. Either lipid A modification or loss of lipopolysaccharide in Gram-negative bacteria leads to PMBR phenotypes. The prevalence of intrinsic lipid A modification in PMBR clinical strains was attributed to high levels of basal expression of pmrC and eptA-1. Our findings suggest that new therapeutic strategies are warranted to combat MDR pathogens due to the emergence of many PMBR clinical strains. the chromosomal promoter regions of the pmrCAB operon, the arnBCADTEF operon, and naxD in an unnamed operon, inducing the expression of pmrC (encoding a PEtN transferase), arnT (encoding an L-Ara4N transferase), and naxD (encoding an N-acetylgalactosamine deacetylase), respectively. However, the arnBCADTEF operon is not present in A. baumannii (21,22). PEtN transferase encoded by either plasmid-driven mcr or insertion sequence (IS)-mediated eptA (a pmrC homolog) may be responsible for the spread of PMB resistance (23,24). Interestingly, LPS deficiency caused by the loss of lpx has been reported to result in PMB resistance in several clinical A. baumannii strains (25,26). In the present study, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) was performed to analyze the compositions of lipid A in both PMB R and PMB-susceptible (PMB S ) MDR strains. Fluorescence images using dansyl chloride-labeled PMB (dansyl-PMB) and FM 4-64 were also obtained to visualize the binding of PMB to each cell. Amino acid variations in PmrC, PmrA, and PmrB were assessed via genomic and PCR analyses to elucidate the mechanisms underlying polymyxin resistance. Collectively, higher basal expression levels of pmrC and the consequent PmrC-mediated modification of lipid A with PEtN were monitored only in PMB R clinical MDR A. baumannii strains. RESULTS PMB resistance in clinical A. baumannii isolates. Antibiotic susceptibility tests of all of the tested strains were performed by assessing the MICs of five classes of antibiotics, including PMB (Table 1; also see Table S2 in the supplemental material). Of the 41 strains (40 clinical isolates and 1 laboratory wild-type [Lab-WT] strain), 82.5% (33/40 strains) appeared to be MDR strains, showing resistance to more than three antibiotics (Table 1; also see Table S2). The selected nine high MDR clinical strains showed much higher resistance to all of the tested antibiotics except for PMB. Hence, they were selected for further analysis (Table 1). For all nine strains, the MIC of each antibiotic was very high (e.g., doxycycline, 16 to 32 mg/mL; gentamicin, .512 mg/mL; erythromycin, 256 to 512 mg/mL), far exceeding the reference levels (Table 1). These MICs were much higher than many other reported MICs for clinical A. baumannii isolates (doxycycline, 0.25 to 64 mg/mL; gentamicin, 0.25 to 512 mg/mL; erythromycin, 0.5 to 4 mg/mL), indicating that our tested clinical isolates could have intrinsic antibiotic resistance and might have acquired multiple antibiotic resistance genes (ARGs) through mobile genetic elements (MGEs) (27)(28)(29). In the case of the A. baumannii NCCP 16007 genome, 32 (Table 1; also see Table S2). Notably, significant differences in the measured PMB MICs were observed among all 9 strains. The strains could be categorized into two groups based on their level of PMB resistance, i.e., PMB R strains (128 to 256 mg/mL) and PMB S strains (1 to 2.5 mg/mL) ( Table 1). While all of the strains displayed high resistance to four different antibiotics, only 4 strains (NCCP 16007, NCCP 15996, NCCP 15995, and F-1629) demonstrated high resistance to PMB (128 to 256 mg/mL) ( Table 1). Resistance to polymyxin, an antibiotic used as the last resort for treating CRAB infections, was noted in less than 10% of CRAB isolates in the Republic of Korea in the past decade; in the present study, however, a higher rate (22% [4/18 strains]) of PMB resistance was noted among CRAB isolates (Table 1; also see Table S2) (32). Genetic and biochemical differences were further analyzed using the two groups of MDR strains (PMB R and PMB S ). Genetic profiling of clinical A. baumannii isolates. Unique allelic profiles of clinical A. baumannii isolates were analyzed according to the Oxford scheme using seven housekeeping genes, namely, gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD (33). The nucleotide sequence for multilocus sequence typing (MLST) was corrected using the draft genome sequence obtained using the NovaSeq 6000 platform (Illumina, USA). All of the tested A. baumannii strains, including the Lab-WT strain, were grouped into six different sequence types (STs) (ST112, ST208, ST357, ST358, ST369, and ST451) according to the PubMLST database for A. baumannii (https://pubmlst.org/organisms/acinetobacter-baumannii) ( (32,34). The high prevalence (55% [5/9 strains]) of ST357 in MDR isolates obtained from patients in the present study was probably associated with regional bias in clinical A. baumannii isolates due to nosocomial infections; however, our small sample size was not statistically significant (35). Interestingly, only two genes (gyrB and gpi) were featured as allelic variants, and the other five genes showed the same allele numbers in all of the MDR strains ( Table 2). Evolutionary analyses based on the allelic profile demonstrated that three STs (ST358, ST369, and ST451) were distinct from ST208 because of a mutation in gpi and that ST357 had evolved from ST358 because of a single-locus mutation in the gyrB allele ( (30,34). The bla OXA-23 gene, encoding a carbapenemase (GenBank accession number AY795964), is abundant in many clinical A. baumannii strains belonging to IC II worldwide; this was also noted in our tested strains (36). Genetic analyses of 160 clinical A. baumannii isolates from the Republic of Korea in 2016 to 2017 revealed that over 96% of CRAB isolates harbored bla OXA-23 with ISAbaI (37). Along with the discovery of the ISAbaI/bla OXA-23 cassette in the draft genomes of all of the tested MDR strains, it is worth noting that our MDR strains were experimentally and genetically proven to be CRAB strains (Tables 1 and 2). Interestingly, a bidirectional transcription system, P in and P out , is predicted to be inside the structural gene encoding an ISAbaI-containing transposase, which may trigger the expression of bla OXA-23 (see Fig. S2). Additional eptA was detected in the draft genome analyses and showed 85% (1,362/1,602 bp) nucleotide sequence identity to pmrC. Domain analyses of both PmrC and EptA-1 proteins revealed that they had different numbers of transmembrane (TM) domains (four and five TM domains, respectively) and that a signal peptide was present only in PmrC (Fig. 1a). Given their high amino acid identity (93%) and the same enzymatic function as a PEtN transferase, they likely evolved after gene duplication, and an increased gene dosage could confer high levels of PMB resistance to cells (24,38). In addition, the C terminus of both enzymes possessing PEtN transferase activity (highly conserved T285) is located on the periplasmic side of the IM and catalyzes the addition of PEtN to hepta-acylated lipid A ( Fig. 1a) (39). Draft genome analysis demonstrated that extra eptA-1 was also present in all of the tested MDR strains (but not in the Lab-WT strain), and their nucleotide sequences showed 100% identity to known eptA-1 (GenBank accession number KC700024) in A. baumannii strains ( Table 2) (24). A PmrA-binding motif (59-HTTAAD-N 5 -HTTAAD-39) was detected upstream of the promoter regions of the pmrCAB operon (59-TTTAAG-TCATT-TTTAAG-39), eptA-1 (59-TTTAAT-TTTTC-TTTAAG-39), and naxD (59-CTTAAG-AAAAC-TTTAAG-39) ( Fig. 1a and Table 2). PmrA-induced NaxD is known to deacetylate N-acetylgalactosamine, forming GalN in the cytoplasm. However, additional enzymes for the transport and attachment of GalN to lipid A have not yet been identified (40). Induction of pmrC/eptA-1 expression and alteration of membrane charge in PMB R strains. High basal expression levels of pmrC/eptA-1 could contribute to high PMB resistance in the PMB R strains (MIC, ;128 to 256 mg/mL), and those genes were still inducible by PMB (1/2 MIC) (Fig. 1b). The same PMB concentration (1/2 MIC) could not upregulate pmrC/eptA-1 in the PMB S strains, probably because of their vulnerability under high concentrations of PMB, which was also observed in the Lab-WT strain (Fig. 1b). Low concentrations of PMB (1/10 MIC) and not high concentrations (1/2 MIC) could upregulate the target genes in the Lab-WT strain, implying that exposure to high PMB concentrations made it too challenging for cells to perform normal gene transcription and metabolic activities. In the absence of PMB, the basal expression level of pmrC/eptA-1 encoding a PEtN transferase in the PMB R strains (74-to 194-fold) was significantly higher than that in the PMB S strains (1.4-to 14.1-fold) and the Lab-WT strain ( Fig. 1b) (24,25). Higher expression levels of PmrAB TCS-regulated and nonregulated pmrC led to higher PEtN transferase activity, resulting in the accumulation of PMB R lipid A-PEtN instead of canonical lipid A in the OM. Possible changes in the cell surface charge with the incorporation of PEtN into lipid A were monitored by measuring the zeta potential (Fig. 1c). The four PMB R strains (43). However, fluorescence intensity following the addition of dansyl-PMB could be detected in the PMB R strains but not in the PMB S strains, indicating that the lower initial binding of PMB rendered PMB resistance in the PMB R cells. Modification of lipid A in PMB R strains. MS analyses of lipid A revealed significantly different patterns between the PMB R and PMB S strains (Fig. 3). The following four major peaks were detected in all of the tested strains, including the reference Lab-WT strain (ATCC 17978): tetra-acylated lipid A precursor (m/z 1,404); hepta-acylated lipid A (m/z 1,910), the common form in A. baumannii; hexa-acylated lipid A (m/z 1,728) lacking one laurate (C 12 ); and penta-acylated lipid A (m/z 1,530) lacking two laurate molecules (C 12 ) with one hydroxyl group (Fig. 3) (44). The predominant m/z 1,404 peak in most isolates represented a precursor located in the cytoplasmic leaflet of the IM at the early stages of lipid A biosynthesis, indicating that the extraction of lipid A could contain all lipid A precursors and complete forms on both the IM and OM sides (see Fig. S3a). The m/z 1,910 peak (hepta-acylated lipid A) represented the canonical lipid A of A. baumannii and was also detected in other clinical Acinetobacter strains, such as Acinetobacter pittii and Acinetobacter nosocomialis (45). PMB R A. baumannii strains having lipid A components modified with PEtN (1m/z 123) or GalN (1m/z 161) at one or both terminal phosphate residues have been often reported (44). The m/z 2,033 peak specific to the PMB R strains corresponding to lipid A and one PEtN residue (1m/z 123) represented hepta-acylated lipid A (lipid A-PEtN, m/z 1,910) (Fig. 3). However, the m/z 2,071 peak corresponding to the NaxD-mediated GalN modification of lipid A was not detected in the mass spectrum of lipid A, indicating that GalN-mediated lipid A modification may be a minor process for PMB resistance in the PMB R strains (24). An LpxO homolog present in the genomes of S. enterica, K. pneumoniae, and A. baumannii functions as a hydroxylase working on laurate (C 12 ) linked to the 29-R-3-hydroxymyristoyl position of lipid A (46). The weakly detected subvariant lipid A precursors having a lower mass (2m/z 16 [one hydroxyl group]) than the three major peaks (m/z 1,910, m/z 1,728, and m/z 1,530) were detected mainly in the PMB S strains but not in most PMB R strains. The identification of many hydroxylated or nonhydroxylated acyl chains of lipid A predominantly in the PMB S strains implied different relative speeds and degrees of cognate enzymatic activities in the strains. Interestingly, the occurrence of PEtN addition only to hepta-acylated lipid A proved the presence of PEtN activity in the periplasmic leaflet of the IM in the PMB R strains. Different levels of expression of genes involved in lipid A biosynthesis (lpxC, lpxK, lpxL, lpxM, and lptC) occurred in the PMB S and PMB R strains. It is difficult to draw any conclusion regarding the presence of multiple precursors from lipid A analyses based on the level of gene expression; however, it is worth noting that the rate-limiting steps present in lipid A biosynthesis may generate different amounts of lipid A precursors in the strains (see Fig. S3a [redmarked lines] and b). PmrCAB variation in PMB R strains. The amino acid alterations in PmrCAB in the clinical strains were analyzed and compared with that in the Lab-WT strain, which could explain their target gene expression and constative PMB R phenotypes, because mutations accumulated in the PmrAB TCS can lead to the activation of PmrA, a transcriptional regulator of downstream genes (see Table S3). Sequence analysis of the PmrB kinase of PmrAB TCS (A1S_2754) revealed that the four PMB R strains with high FIG 3 MS analysis of clinical isolates. Extracted lipid A was analyzed by MALDI-TOF MS to assess the lipid A pattern corresponding to PMB resistance. The m/z 2,033 peak (blue arrows) corresponding to lipid A with added PEtN was specifically detected in the PMB R strains, and the m/z 1,530 and m/z 1,728 peaks (red arrows) corresponding to deacylated lipid A were detected to a lesser extent or not detected. PMB MICs had interesting and novel amino acid mutations and deletions (F26L mutation and deletion of amino acids at positions 27 to 30 in NCCP 16007; A138T mutation in NCCP 15996; and S61G and I163N mutations in NCCP 15995). In clinical strains, a mutation in PmrB (P233S, T235N, or Q270P) appeared to be important for the constitutive activation of PmrA; however, the same mutation could not be detected in our tested clinical strains (47). The P170L mutation in PmrB has been reported in PMB R clinical A. baumannii strains, and the same mutation was detected in the F-1629 strain in our study (48). The kinase domain of PmrB is known to be activated by a conformational change in the sensor domain in response to environmental stimuli, such as low pH and high Fe 31 or Al 31 (49). Our analysis revealed that a mutation (P170L) in the kinase domain of PmrB occurred in the F-1629 strain. Four mutations and the deletion of PmrB in the vicinity of the periplasmic sensor domain (F26L, S61G, A138T, and I163N mutations and deletion of amino acids at positions 27 to 30) may activate the kinase domain without environmental stimuli; however, their contribution to constitutive pmrC expression has not yet been experimentally proven and remains to be investigated (see Fig. S4). Interestingly, mutations in PmrB were observed only in the PMB R strains. No mutation was detected in PmrA (A1S_2753), and five amino acid mutations (F150L, I212V, R332K, A354S, and K515T) in PmrC (A1S_2752) were identical in all of the clinical strains. DISCUSSION Our MIC tests and draft genome analysis indicated that the selected 9 clinical isolates (among the 40 tested strains) were MDR strains that possessed multiple ARGs, which could confer high levels of resistance to meropenem and other antibiotics (Table 1). In addition, our draft genomic data indicated the presence of the ISAbaI/bla OXA-23 cassette, pmrC, and eptA-1 in all of the tested MDR strains (Table 2; also see Fig. S2 in the supplemental material). The three major STs corresponding to IC II (ST208, ST357, and ST369) are known to contain bla OXA-23 , with more than 60% prevalence in the Republic of Korea; this is consistent with our finding that all of our tested strains belonged to IC II (Table 2) (24). In the case of E. coli, the OM retained 70% of the lipid A fraction, and 23% reduction in the OM charge occurred with the addition of PEtN, which explained our zeta potential data on lowering cationic PMB affinity by reducing the net negative charge of cell surfaces in the PMB R strains (Fig. 1c) (50). The addition of PEtN to lipids could also stabilize the membrane by increasing the intermolecular attraction between adjacent lipids (51). Lipid A modification in PMB R A. baumannii is known to occur by the addition of PEtN or GalN, which can neutralize and stabilize membranes (52). PEtN modification mainly occurs at the C19 position of lipid A, and a change in the C49 position occurs with low frequency (44,45). The addition of GalN is associated only with the C19 position, which may explain the rare detection of GalN-modified lipid A because of positional competition with the PEtN moiety. Moreover, in our study, no production of GalN-lipid A was detected (mass peak at m/z 2,071), even though naxD, which was first discovered in a Francisella novicida clinical isolate, was present in the A. baumannii genome (Fig. 1a and Fig. 3) (44, 53). The mechanism underlying the transfer of GalN to lipid A has not yet been identified. Interestingly, a phenomenon of LPS loss was noted in laboratory-evolved PMB R A. baumannii strains when mutations occurred in lpxA, lpxC, and lpxD (25). The LPS-deficient cells appeared to have high PMB resistance and high sensitivity to antibacterial lysozyme and lactoferrin. Our MS data revealed only the m/z 1,910 peak corresponding to canonical lipid A (Fig. 3). Mutations in PhoQ, a histidine kinase in P. aeruginosa, S. enterica, and K. pneumoniae, are also attributed to lipid A modification through induction of the activation of the arnBCADTEF operon or the phoPQ-pmrD-pmrAB signal transduction cascade as a result of uncontrolled TCS functioning (6). However, A. baumannii lacks the pmrD and arnBCADTEF operons. Consequently, conferring PMB resistance to A. baumannii cells through charge modification of lipid A mainly relies on the PmrAB TCS, with the exception of LPS deficiency (54). The high basal expression levels of pmrC/eptA-1 (74-to 194-fold) noted only in the PMB R strains suggested that the pmrAB TCS was no longer strictly controlled inside the cells, possibly because of mutations accumulated in PmrB, a kinase in the PmrAB TCS. However, the direct contribution of those mutations to high basal expression levels of pmrC remains to be investigated ( Fig. 1b; also see Table S3). Conformational changes caused by mutations in the sensor and kinase domains of PmrB can lead to a constantly activated state of PmrB. Continuous exposure of the sensory protein PmrB to environmental stimuli, such as positive ions, metals, pH, PMB, and other cationic peptides, may result in spontaneous mutation and protein evolution under selection pressure in hostile host environments (19,25,41). Amino acid analyses of PmrCAB in MDR strains revealed mutations in the PMB R strains at amino acid positions 26 to 170 of PmrB, indicating a possible conformational change in PmrB in the PMB R strains (see Fig. S4 and Table S3). In many clinical isolates, such as E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii strains, PmrB mutations were found to occur at various amino acid positions, resulting in high PMB resistance (8 to 512 mg/mL) (15). In conclusion, all of the tested clinical A. baumannii isolates were proven to be MDR strains with resistance to different classes of antibiotics, but their degree of PMB resistance differed. The four selected PMB R strains had mutations in PmrB, high basal expression levels of pmrC/eptA-1, and PEtN-modified lipid A without exposure to PMB (Fig. 3). These mutations and constitutive phenotypic changes confer high PMB resistance to the PMB R strains. Consequently, spontaneous mutations and acquired ARGs during their residence inside hosts could contribute to their successful survival as pathogens in clinical settings. MATERIALS AND METHODS Acquisition of strains and measurement of their MICs. Five clinical isolates of A. baumannii from a patient were provided by the Samsung Medical Center, Sungkyunkwan University, while 35 other clinical isolates were provided by the National Culture Collection for Pathogens (NCCP) in the Republic of Korea. These clinical isolates were deposited from 2004 to 2013 (30). A. baumannii ATCC 17978 (Lab-WT) was used as the reference strain; it was maintained in the laboratory after being provided by the American Type Culture Collection (ATCC). Antibiotic susceptibility tests were performed using the broth dilution method in 96-well plates to identify MDR A. baumannii strains (30). All antibiotics were purchased from Sigma-Aldrich (USA), and the following concentrations were tested: PMB, 1 to 256 mg/mL; meropenem, 0.5 to 64 mg/mL; doxycycline, 0.5 to 64 mg/mL; gentamicin, 0.5 to 512 mg/mL; erythromycin, 0.5 to 512 mg/mL. All isolates were cultured overnight in Luria-Bertani (LB) broth and then diluted (1:100) into fresh LB broth (5 mL) and incubated until they reached the early exponential phase (OD 600 of 0.4). The cultured cells were transferred into a 96-well plate with each antibiotic, and the final cell numbers were adjusted (;10 6 CFU/mL). Incubation was continued at 37°C for 24 h to calculate the MIC. The antibiotic resistance criteria for each antibiotic were determined using EUCAST clinical breakpoints v.12.0 for Acinetobacter species (updated in 2022) (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST _files/Breakpoint_tables/v_12.0_Breakpoint_Tables.pdf), as follows: PMB, 3 mg/mL; meropenem, 2 mg/ mL; doxycycline, 2 mg/mL; gentamicin, 5 mg/mL; erythromycin, 16 mg/mL. Extraction of genomic DNA. Genomic DNAs were extracted from all of the tested A. baumannii strains using the Wizard genomic DNA purification kit (Promega, USA), according to the manufacturer's instructions. The cell pellet was obtained by centrifugation at 12,000 Â g for 1 min using A. baumannii that had been cultured overnight, and the supernatant was discarded. The pellet was resuspended in nuclei lysis solution (600 mL) and incubated at 80°C for 5 min to lyse the cells. To remove RNAs, RNase solution (3 mL) was added to the cell lysate and incubated at 37°C for 30 min. The supernatant containing DNA was transferred into a new microtube containing isopropanol (600 mL), mixed by gentle inversion of the tube, and centrifuged at 12,000 Â g for 2 min. The supernatant was poured out, and 70% ethanol (600 mL) was added to wash the DNA pellets. Following this, DNA rehydration solution (100 mL) was added to the DNA pellet, and the DNA was dehydrated by incubation at 65°C for 1 h. The concentration of extracted genomic DNA was assessed using a NanoPhotometer N50 (Implen, Germany), and the DNA was used for MLST and nucleotide analysis of pmrCAB. Genotypic analysis using MLST. Extracted genomic DNA was used to perform MLST according to the Oxford scheme, as described previously, using primers designed for PCR amplification and sequencing in the PubMLST database (https://pubmlst.org/organisms/acinetobacter-baumannii) (see Table S1 in the supplemental material) (33). EzPCR HF 5Â master mix (Elpis Biotech, Republic of Korea) containing a Pfu polymerase was used to ensure a low error rate and accurate PCRs under the following conditions: 95°C for 5 min; 30 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final cycle at 72°C for 5 min. PCR products were separated in a 1% agarose gel and purified from the gel using Expin Combo GP (GeneAll, Republic of Korea). The purified PCR products were analyzed by the Sanger sequencing method (Macrogen, Republic of Korea) using primers according to the application (see Table S1). The sequence data sets were assigned to the Acinetobacter MLST database (http://pubmlst.org/abaumannii [accessed 25 June 2021]). Draft genome sequences were obtained using the NovaSeq 6000 platform (Illumina). Relative quantification of pmrC and eptA-1 expression. The expression levels of pmrC and eptA-1 (pmrC/eptA-1) were assessed in the presence or absence of PMB. Primers were designed from the common sequence of the conserved regions of pmrC and eptA-1, and the integrated expression levels of both genes were measured simultaneously (see Table S1). All of the clinical isolates were diluted (1:100) using overnight cultures in LB broth (5 mL) and were grown to the early exponential phase (OD 600 of 0.4). For the PMB treatment condition, all of the strains were diluted (1:100) using overnight cultures in LB broth (5 mL) and were grown to the early exponential phase (OD 600 of 0.4). Following this, 1/2 MIC (1 to 128 mg/mL) of PMB was added and additionally incubated for 30 min. Total RNA from the cultured cells was isolated using the RNeasy minikit (Qiagen, Germany), according to the manufacturer's protocol. Reverse transcription was performed using the RevertAid reverse transcription kit (Thermo Fisher Scientific, USA) with DNase I (1 mL [Thermo Fisher Scientific])-treated RNA. Quantitative real-time PCR (qRT-PCR) amplification was performed on the QuantStudio 5 real-time PCR system (Applied Biosystems, USA) using Power SYBR green PCR master mix (Applied Biosystems). For RT-PCR, the total mixture (20 mL) was prepared as follows: master mix (10 mL), forward primer (1 mL [10 pmol]), reverse primer (1 mL [10 pmol]), diluted (1/10) cDNA (1 mL) as a template, and distilled water (7 mL). qRT-PCR was performed under the following conditions: hold stage, 95°C for 10 min; PCR stage, 40 cycles at 95°C for 15 s and 60°C for 1 min; melt curve stage, 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The relative expression level of pmrC/eptA-1 was normalized with 16S rRNA as an endogenous gene. The primers used are listed in Table S1 in the supplemental material. Measurement of zeta potential. The cell surface charge was measured using a zeta potential analyzer, as described previously (41). Cells cultured overnight were diluted (1:100) into fresh LB broth (5 mL) and grown to the early exponential phase (OD 600 of 0.4). To wash the residual ions on the cell surface, the cultured cells were centrifuged at 12,000 Â g for 1 min and washed with phosphate-buffered saline (PBS) (pH 6.8) twice. The washed pellet was resuspended in PBS and, immediately before measurement of the zeta potential, was diluted one to three times in PBS according to the cell density. The zeta potential of the bacterial cells was measured at 25°C using the ELSZ-1000 zeta potential analyzer (Otsuka Electronics, Japan). Microextraction of lipid A. Lipid A of all of the tested strains was extracted using the modified ammonium hydroxide-isobutyric acid method (25). In brief, cells cultured overnight were diluted (1:100) into fresh LB broth (400 mL). The culture was then incubated with vigorous shaking at 37°C until the OD 600 reached 1.0. Bacterial pellets were obtained from the cultured cells by high-speed centrifugation (Hitachi, Japan) at 4,000 Â g for 20 min at 4°C. The supernatant was poured out, and the pellets were washed with distilled water (30 mL) twice to remove residual ions. The washed cell pellets were snapfrozen using liquid nitrogen and lyophilized overnight. Lyophilized crude cells (10 mg) were suspended in a mixture of isobutyric acid and 1 M ammonium hydroxide (400 mL, 5:3 [vol/vol]; Sigma-Aldrich) and incubated for 2 h at 100°C in a screw-cap test tube with occasional vortex-mixing. The mixture was then cooled on ice and centrifuged at 8,000 Â g for 15 min. The supernatant was transferred into a new tube, mixed with an equal volume of water, and snap-frozen using liquid nitrogen before being lyophilized overnight. The lyophilized sample was then washed twice with methanol (400 mL) and centrifuged at 5,000 Â g for 15 min. Finally, insoluble lipid A was solubilized in a chloroform-methanol-water mixture (100 mL, 3:1.5:0.25 [vol/vol/vol]). The extract was directly subjected to MALDI-TOF MS analysis. MS analysis. Lipid A extracted from whole cells using the microextraction method was subjected to MALDI-TOF MS analysis (25). In brief, 2,5-dihydroxybenzoic acid (DHB) matrix solution (10 mg/mL in 80% acetonitrile with 0.1% trifluoroacetic acid) was used for MS. Lipid A extract (2 mL) was mixed with matrix solution (2 mL) deposited on a plate for the vacuum-dried droplet method. The structural spectrum of lipid A was analyzed using the autoflex maX mass spectrometer (Bruker Daltonics, USA) in the negativeion reflectron mode. The laser repetition rate was 2,000 Hz, and the resulting spectrum was accumulated with an average of 500 shots. The total analysis range of MALDI-TOF MS was m/z 400 to m/z 3,200, and lipid A mass analysis was performed at m/z 1,300 to m/z 2,300. Protein sequence analysis of PmrCAB in clinical isolates and the Lab-WT isolate. To determine whether amino acid substitution contributes to PMB resistance, the pmrCAB operon was amplified using a Pfu polymerase with proofreading activity. Based on the reference genome sequence of the Lab-WT strain (GenBank accession number CP000521), three sets of PCR primers were designed to encompass the entire pmrCAB operon along with the promoter regions containing the PmrA binding site in the promoter region (see Table S1). EzPCR HF 5Â master mix (Elpis Biotech, Republic of Korea) was used, and three fragments were amplified using three pairs of primers, according to the manufacturer's protocol (see Table S1). PCRs were performed under the following conditions: 95°C for 5 min; 30 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min 30 s; and a final cycle at 72°C for 5 min. Sequenced fragments were assembled and aligned to the pmrCAB sequence of the reference genome of the Lab-WT strain (GenBank accession number CP000521). The functional domain model of each gene was predicted using the Web-based programs InterPro (https://www.ebi.ac.uk/interpro) and Protter (http://wlab.ethz.ch/ protter/start). Data availability. We confirm that the data supporting the findings of this study are available within the article and/or its supplemental material. The genomic data for eight MDR strains were deposited in the NCBI GenBank database under the following GenBank accession numbers: A. baumannii NCCP 15989, JAKVTK000000000; NCCP 15992, JAKVTL000000000; NCCP 15994, JAKVTM000000000; NCCP 15995, JAKVTN000000000; NCCP 15996, JAKVTO000000000; NCCP 16006, JAKVTP000000000; NCCP 16011, JAKVTQ000000000; F-1629, JAKVTJ000000000. The complete genome sequence of A. baumannii NCCP 16007 (GenBank accession number CP091465) was obtained by constructing a hybrid genome using Pacific Biosciences (PacBio) and Illumina data and was deposited in the NCBI GenBank database. In addition, A. baumannii ATCC 17978 (GenBank accession number CP000521) was used as the reference strain. SUPPLEMENTAL MATERIAL Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 1 MB.	2022-07-22T06:19:33.234Z	2022-07-21T00:00:00.000	{ "year": 2022, "sha1": "cc9c62326fe262a7e25699d123842b1e6db9fd9d", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "ASMUSA", "pdf_hash": "eec225a0009d25e37de48357fcabebc99b2f2c8d", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
268750289	pes2o/s2orc	v3-fos-license	Multifaceted chemical and bioactive features of Ag@TiO2 and Ag@SeO2 core/shell nanoparticles biosynthesized using Beta vulgaris L. extract Due to increasing concerns about environmental impact and toxicity, developing green and sustainable methods for nanoparticle synthesis is attracting significant interest. This work reports the successful green synthesis of silver (Ag), silver-titanium dioxide (Ag@TiO2), and silver-selenium dioxide (Ag@SeO2) nanoparticles (NPs) using Beta vulgaris L. extract. Characterization by XRD, SEM, TEM, and EDX confirmed the successful formation of uniformly distributed spherical NPs with controlled size (25 ± 4.9 nm) and desired elemental composition. All synthesized NPs and the B. vulgaris extract exhibited potent free radical scavenging activity, indicating significant antioxidant potential. However, Ag@SeO2 displayed lower hemocompatibility compared to other NPs, while Ag@SeO2 and the extract demonstrated reduced inflammation in a carrageenan-induced paw edema animal model. Interestingly, Ag@TiO2 and Ag@SeO2 exhibited strong antifungal activity against Rhizoctonia solani and Sclerotia sclerotium, as evidenced by TEM and FTIR analyses. Generally, the findings suggest that B. vulgaris-derived NPs possess diverse biological activities with potential applications in various fields such as medicine and agriculture. Ag@TiO2 and Ag@SeO2, in particular, warrant further investigation for their potential as novel bioactive agents. Introduction The burgeoning field of nanotechnology, manipulating matter at the atomic and nanoscale, offers a revolutionary toolkit for precision medicine and diverse applications [1,2].In healthcare, the prospect of targeted drug delivery using nanocarriers directly to afflicted cells ignites hope for enhanced efficacy and minimized side effects [3][4][5][6].Nanobiosensors, with their exquisite sensitivity, hold promise for early-stage disease detection at the cellular level, paving the way for timely intervention [7,8].Furthermore, the field paves the way for tissue regeneration, potentially enabling the repair and restoration of damaged structures [9,10].However, the impact of nanotechnology transcends healthcare.In the realm of environmental remediation, engineered nanomaterials act as potent catalysts for pollutant degradation, offering solutions for a cleaner future [11][12][13][14][15].The quest for sustainable energy finds allies in nanotechnology, with innovations such as efficient solar cells and novel energy storage solutions on the horizon [16,17].Even water purification and disease diagnostics benefit from this technology, with nanofilters and biosensors poised to provide cleaner water and more accurate disease detection methods [18,19].Moreover, nanotechnology revolutionizes materials science, offering lighter, stronger, and more durable options, while simultaneously contributing to the sustainable production of biofuels [20,21].Recognizing the potential concerns surrounding the nascent technology, researchers are actively pursuing its responsible development, ensuring the "microscopic revolution" unfolds with safety and ethical considerations at its core [22]. Beta vulgaris L., also known as beetroot, table beet, garden beet, or simply beet, is a widely cultivated biennial plant typically red (although available in yellow, white, or striped forms) belonging to the Amaranthaceae family [23].Beyond its various health benefits [24], B. vulgaris offers a wealth of nutrients, including potassium, vitamin C, and folates, alongside non-nutritive components like dietary fibers and polyphenols.Remarkably, beetroot is ranks among the ten most potent vegetables in terms of antioxidant capacity, attributed to its total phenolic content of 50-60 mmol/g dry weight [25].This impressive antioxidant activity stems from a significant amount of phenolic compounds, including catechin hydrate, protocatechuic acid, epicatechin, ferulic acid, vanillic acid, p-hydroxybenzoic acid, p-coumaric acid, syringic acid, and caffeic acid [26].Additionally, beetroot serves as a source of valuable water-soluble nitrogenous pigments called betalains, extensively used in the modern food industry [27,28]. Green synthesis represents a preferred method for fabricating nanoparticles, utilizing natural materials such as plants, microbes, and algae extracts [43][44][45].Compared to traditional chemical or physical approaches, this method offers enhanced eco-friendliness, cost-effectiveness, and safety [44,46].The synthesis process involves reducing metal ions using biological components like polyphenols, which act as both reducing and stabilizing/capping agents, preventing nanoparticle aggregation [47,48].Current research actively explores developing new and improved green synthesis methods, investigating their potential applications in various fields [49,50]. Mycotoxins, fungal metabolites commonly found on various plant parts, pose significant risks to humans and animals, making them major and unavoidable food contaminants [51].These toxins, produced by contaminating fungal species like Sclerotia sclerotium, Rhizoctonia solani, and Macrophomina phasolina, cause mycotoxicosis, a chronic disease with potentially detrimental effects [52][53][54][55].Mycotoxins such as aflatoxins, ochratoxins, deoxynivalenol, zearalenone, and fumonisins can have various adverse impacts, including liver toxicity, immune system issues, kidney damage, and birth defects in both animals and humans [51,56].This urgent threat to food safety necessitates the development of strategies to either inhibit or deactivate fungal contamination in food products. Hemolysis, the rupture of red blood cells leading to anemia, jaundice, and kidney failure, can be inhibited by various mechanisms.These include membrane stabilization, antioxidant activity, and modulation of specific pro-inflammatory pathways like NF-κB signaling or cytokine production.Anti-inflammatory effects can similarly work through diverse mechanisms, such as suppressing TNFα and IL-6 signaling, modulating immune cell activity, offering antioxidant protection, and regulating gene expression [57][58][59]. Understanding these molecular mechanisms holds potential for developing novel therapeutic strategies for various hemolytic and inflammatory diseases, such as sickle cell anemia and arthritis. The promise of silver nanoparticles (Ag NPs) for antimicrobial applications comes with potential downsides.Researchers raise concerns about their toxicity through oxidative stress, inflammation, genotoxicity, and cytotoxicity [60].In an attempt to enhance their functionality, combining Ag NPs with other materials like TiO 2 and SeO 2 in core-shell composites seems promising [61].However, this introduces new safety challenges.These composites can pose additional toxicity risks due to ion release, ROS generation, and size-dependent effects [62][63][64].Understanding these multifaceted toxicities is critical to harnessing the benefits of Ag NPs while ensuring their safe and responsible use in various applications. Following our previous work on green synthesis of metallic NCs using B. vulgaris extract [43], this study explores the development and characterization of bimetallic Ag@TiO 2 , and Ag@SeO 2 NCs through the same eco-friendly approach.We hypothesize that these bimetallic NCs exhibit enhanced biological activities compared to single-metal Ag NPs.We comprehensively investigate the chemical and biological properties of Ag, Ag@TiO 2 , and Ag@SeO 2 .Notably, this study pioneers the exploration of the antioxidant, antifungal, anti-hemolytic, and anti-inflammatory activities of these beetroot-derived metallic oxide NCs.These promising results pave the way for developing novel drugs to treat inflammation, fungal infections, and hemolysis. Instruments Routine state-of-the-art technologies (i.e., UV-Vis spectrophotometry, SEM, TEM, XR, and FTIR) were used.The reads of the absorbance of various tests in this work were accomplished using a Spekol 11 spectrophotometer (analytic Jena AG, Jena, Germany) with a wavelength range of 200-1100 nm.The topography, surface morphology, and elemental compositions of the nanomaterials were examined using SEM and energy dispersive X-ray (EDX) spectroscopy on a Czech FEI SEM-type instrument at an accelerating voltage of 25 kV.TEM analysis was performed on a Thermo Scientific Talos F200i using carbon-coated grids (Type G 200, 3.05 μm diameter, TAAP, USA) was used to analyze the nanomaterials.In addition, TEM-JEM2100-JEOL, Japan was used to analyze the fungus species treated and untreated with Ag@SeO 2 bmNPs.X-ray diffraction (XRD) analysis was performed on a Pan Analytical Philips.Fourier-transform infrared spectroscopy (FTIR) analysis was performed using a Thermo-Fisher Nicolet IS10, USA spectrophotometer. Preparation of plant extract and green synthesis of the nanomaterials Preparation of B. vulgaris extract: Fresh B. vulgaris plant material was washed, silenced, and dried.An equivalent amount (w/v) of distilled water (ratio 1:10) was added to the dried plant material and soaked at 25 • C overnight.The mixture was then filtered using a Whatman No. 1 filter and used immediately for subsequent experiments. Synthesis of silver nanoparticles: Separately, silver nanoparticles were synthesized using the B. vulgaris extract following a previously reported protocol [43].Briefly, silver nitrate (AgNO 3 ) and titanium dioxide (TiO 2 ) or selenium dioxide (SeO 2 ) were added to the plant extract in a ratio (5:1).The mixture was heated and stirred until a color change was observed (within 5-15 min), indicating nanoparticle formation.The nanomaterials were then purified by centrifugation (10000 rpm, 30 • C), and washed three times with 70% ethanol [65]. ABTS assay The established ABTS method [66] was chosen to evaluate the antioxidant potential of the samples.The preparation of the ABTS radical solution involved mixing equal volumes (1:1 v/v) of ABTS solution (7 mM) and potassium persulfate solution (2.45 mM).The mixture was then incubated in the dark at room temperature for 12-16 h.Subsequently, dilution was performed to achieve an absorbance of 0.700 at 734 nm.Individual solutions of the plant extract, Ag NPs, and bimetallic NCs were prepared at varying concentrations.An equal volume of the diluted ABTS solution was then added to each sample tube.These mixtures were kept in the dark at 25 • C for 30 min before measuring their absorbance at 734 nm.Finally, the antioxidant activity was calculated using Equation (1): Where; 'Ac' is the absorbance of the ABTS radical solution without the antioxidant sample, and 'At' is the absorbance of the mixture of the ABTS radical solution and the antioxidant sample.The IC 50 values were calculated from a Fit a non-linear regression curve plotted for the percentage of radical scavenging activity versus the sample concentrations.The values of IC 50 (μg/mL) were expressed as mean ± standard deviation (SD), in which all the tests were run in triplicates. Animal model and housing The study employed male Wistar rats, six weeks old and weighing an average of 169 ± 9.4 g.Prior to experimentation, the rats were provided a one-week acclimation period to their new environment.Throughout the study, they had ad libitum access to rodent chow and tap water.Housing conditions were meticulously controlled, maintaining a temperature of 24 ± 1 • C, humidity of 50 ± 10%, and a 12-h light/12-h dark cycle.All procedures adhered to the guidelines and received approval from the Animal Care and Use Committee (MU-ACUC) of Mansoura University in Egypt. Anti-hemolytic assay An anti-hemolytic assay [67] was conducted using blood collected from healthy rats via cardiac puncture into heparinized tubes.Plasma was separated from the erythrocytes by centrifugation.The remaining buffy coat was thoroughly washed three times with sterile saline solution (0.89% w/v NaCl, pyrogen-free) using 10 times the volume of the buffy coat in each wash to eliminate any residual plasma.Following each wash, the erythrocytes were centrifuged at 2500 rpm for 10 min to ensure a consistent preparation.The tested materials, B. vulgaris extract (57.7 mg/mL), Ag NPs (15.66 mg/mL), Ag@TiO 2 NC (21.62 mg/mL), and Ag@SeO 2 NC (35.16 K.M. Elattar et al. mg/mL), were individually added to a 10% erythrocytic suspension in phosphate-buffered saline (1X, pH 7.4).All samples were incubated at 37 • C for 45 min.Control groups included: A negative control: containing only saline solution and erythrocyte suspension without any testing material.A positive control: containing distilled water to induce maximum hemolysis.The erythrocyte suspension was centrifuged to separate the intact cells from the lysed cells present in the supernatant.Hemoglobin, the red pigment within erythrocytes, serves as a marker for cell lysis.Therefore, the extent of hemolysis was determined by measuring the absorbance of the supernatant at 540 nm, which corresponds to the absorption wavelength of hemoglobin.To account for background hemolysis, the percentage of hemolysis in the saline control group was subtracted from all other groups.The final percentage of hemolysis for each sample was calculated using Equation (2): Hemolysis (%) = Absorbance of sample / Absorbance of water control × 100 (2) Carrageenan-induced paw edema The paw edema protocol followed Morris [68].Carrageenan (1%) was prepared in sterile saline, heated to 90 • C without boiling until fully dissolved, and cooled to room temperature.Selected rats received a subplantar injection of 0.1 mL carrageenan solution into the right hind paw.Tested materials were administered via intraperitoneal injection using a vernier caliper.Paw thickness was measured for each rat at baseline (before carrageenan injection) and 0.5, 1, 2, 3, 4, and 5 h after administering the following nanomaterials (57.7 mg/mL B. vulgaris extract, 15.66 mg/mL Ag mNPs, 21.62 mg/mL Ag@TiO 2 bmNPs, and 35.16 mg/mL Ag@SeO 2 bmNPs).Additionally, a control group received indomethacin for comparison.The paw was calculated by subtracting the baseline paw thickness from each subsequent measurement.Data were then compared between treated and control groups.The percentage of protection in anti-inflammatory activity can be determined using Equation (3): Control is the average of the anti-inflammatory activity measurements for the group of rats that were not treated with the test compound.Treatment is the average anti-inflammatory activity of the treated group. Antifungal activity Two aggressive plant fungal pathogens, namely Rhizoctonia solani (ARC-NW23), and Sclerotinia sclerotium (ARC-NW35) were obtained from Seed Pathology Research Department, Plant Pathology Research Institute, Agricultural Research Centre, Giza, Egypt.They were utilized as microbial models for evaluating the potential antifungal activity exerted by the biosynthesized nanomaterials. MIC Determination of Nanomaterials against Plant Fungal Pathogens: The minimum inhibitory concentration (MIC) of the green Ag NPs, Ag@TiO 2 NC, and Ag@SiO 2 NC (150-1860 μg/mL) against the plant fungal pathogens was evaluated using a broth microdilution assay [69].Briefly, sterilized flasks containing potato dextrose agar (PDA) medium were prepared, and varying concentrations of each nanomaterial were individually incorporated into the molten agar before pouring into Petri dishes.After solidification, a 1 mm diameter disk of each previously grown fungus was individually inoculated onto the center of each plate.Incubation conditions were optimized for each pathogen: R. solani (ARC-NW23) incubated at 25 • C for 5 days, while S. sclerotium (ARC-NW35) incubated at 18 • C for 7 days.Fungal growth was monitored daily.The MIC was determined as the lowest concentration of nanomaterial that completely inhibited visible fungal growth compared to a control plate containing a PDA medium without any nanomaterial. Statistical analysis Experiments were performed in triplicate for each condition.Data were analyzed using IBM SPSS Statistics version 26 (Armonk, NY, USA).All results are presented as mean ± standard deviation (SD) from at least three independent experiments. Physical characterizations of beet roots-mediated metallic nanomaterials 3.1.1. Ultraviolet-visible spectrophotometry (UV-Vis) B. vulgaris L. aqueous extract successfully mediated the synthesis of silver (Ag), Ag-selenium dioxide (Ag-SeO 2 ), and Ag-titanium dioxide (Ag@TiO 2 ) nanoparticles.The initial reddish-brown color of the extract changed to brown for Ag-mNPs, yellow for Ag@SeO 2 bmNPs, and gray for Ag@TiO 2 bmNPs, visually indicating nanoparticle formation (Fig. 1).UV-visible spectroscopy confirmed this, revealing distinct shifts in peak wavelengths due to interactions between metal ions and plant extract components.Ag-mNPs displayed a slightly higher surface plasmon resonance (SPR) peak than expected, suggesting possible aggregation or interaction with plant components.Ag@SeO 2 bmNPs exhibited multiple peaks, while Ag@TiO 2 bmNPs showed a unique peak, indicating differing compositions and interactions.All nanoparticles retained residual absorption peaks at 245-247 nm, likely due to residual plant extract components.The maximum absorption peaks were recorded at λ max ca.598.0 nm for Ag-mNPs, λ max ca.428.0 nm for Ag@SeO 2 bmNPs (combined absorption), and λ max ca.675.0 nm for Ag@TiO 2 bmNPs (combined absorption of Ag and TiO 2 ) [42].Different plant extracts have different phytochemical compositions, and the biomolecules in these extracts act as reducing and capping agents during the formation of NPs [44,70]. Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) offers valuable insights into the size, size distribution, and shape/morphology of nanoparticles (NPs) [6].Additionally, TEM can reveal clues about NP interactions, such as aggregation or core-shell structure formation [71].In this study, TEM analysis of AgNPs, Ag@TiO 2 NC, and Ag@SeO 2 NC prepared from B. vulgaris aqueous extract demonstrated well-dispersed and uniform spherical NPs with diameters ranging from 10 to 50 nm (Fig. 2).TEM images of AgNPs specifically revealed typically spherical nanoparticles with sizes between 5 and 20 nm, exhibiting minimal to no aggregation.However, controlling the precise size, size distribution, and shape of these NPs proved challenging (Fig. 2a-c).Notably, aggregation is known to decrease the surface area and catalytic activity of AgNPs [72].These observations align with previous research demonstrating that lattice fringes arise from the diffraction of the TEM beam by the AgNPs' crystal lattice, with the spacing of these fringes corresponding to the AgNPs' lattice constant [42].In certain cases, AgNPs may exhibit a distorted crystal structure, potentially due to factors such as the presence of surfactant molecules during synthesis or the NPs' small size [42,72]. The small size and large surface area of silver nanoparticles (AgNPs) contribute to their high surface energy [73].This, in turn, makes them attractive to each other, leading to aggregation.The surface tension of AgNPs can also contribute to aggregation.Additionally, the inherent surface tension of AgNPs, which is more significant for smaller nanoparticles, further promotes aggregation [73].TEM analysis of the Ag@TiO 2 core/shell magnetic NC revealed sizes ranging from 14.59 to 21.48 nm with a uniform distribution of smaller AgNPs (5-10 nm) decorating the TiO 2 core (Fig. 2d-f).Compared to AgNPs, these Ag@TiO 2 NCs displayed significantly less aggregation.While their larger size might suggest a higher surface energy, their uniform size distribution, and spherical shape mitigate this by minimizing their overall surface energy, leading to greater stability and reduced aggregation tendency.In this composite, the Ag core provides catalytic activity, while the TiO 2 shell shields the silver from oxidation and aggregation [74].Similarly, TEM images of Ag@SeO 2 core/shell magnetic NC showed spherical NPs with diameters ranging from 10 to 20 nm, again featuring a uniform distribution of smaller AgNPs (5-10 nm) on the SeO 2 core (Fig. 2g-i).The nanoscale of these materials plays a crucial role in their biological and pharmacological activities.Their high surface area-to-volume ratio amplifies their catalytic and optical properties [75]. The antifungal activity of metallic/bimetallic NPs is strongly influenced by their morphology, particularly size and shape [76].Larger NPs often possess a greater surface area, facilitating interactions with bacterial cell walls and membranes.This enhanced interaction can disrupt cell membranes, leading to leakage of cellular contents and ultimately, cell death [77].For Ag@SeO 2 NPs, the characteristic "crystal lattice fingerprint" acts as a unique identifier for this specific material.Notably, the low level of aggregation observed in Ag@SeO 2 NPs is advantageous as it prevents the formation of large clumps that could hinder reactivity and reduce surface area [78].Interestingly, AgNPs appear smaller (5-10 nm) when uniformly distributed on the surface of TiO 2 or SeO 2 NPs compared to their usual size range (5-20 nm).This suggests that at least some AgNPs may be partially embedded within the TiO 2 and SeO 2 matrix.Such interactions with other materials can indeed affect the size, shape, and surface area of NPs, with AgNPs potentially becoming partially encapsulated within the matrix of materials like TiO 2 or SeO 2 , leading to a perceived decrease in size [79]. Scanning electron microscopy (SEM) SEM was implemented to examine the shape/morphology, PS, and PSD of biosynthesized nanomaterials [6].SEM micrographs were obtained for AgNPs, Ag@TiO 2 NC, and Ag@SeO 2 NC in the reaction medium (Fig. 3A-C).AgNPs (Fig. 3A) appear predominantly spherical, with sizes ranging from 20 to 50 nm.The nanoparticles are densely packed and evenly distributed across the rock surface.Based on the SEM analysis, the estimated particle size of AgNPs falls within a range of 5-10 nm.SEM analysis of Ag@TiO 2 core/shell magnetic NPs (Fig. 3B) reveals spherical particles with a size range of 10-20 nm.Notably, the AgNPs, appearing smaller (5-10 nm), are uniformly distributed and likely partially embedded within the TiO 2 NPs.This observation suggests a porous TiO 2 shell, contributing to the rough surface texture visible in the image [80].Consistent with expectations, the darker core corresponds to the silver component, while the lighter shell represents the TiO 2 [81].Similarly, Ag@SeO 2 core/shell magnetic NPs (Fig. 3C) exhibit a spherical morphology with sizes ranging from 10 to 20 nm.As observed for Ag@TiO 2 , smaller AgNPs (5-10 nm) are well-dispersed on the SeO 2 NP surface, suggesting potential partial embedding. Energy-dispersive X-ray spectroscopy (EDX) Energy-dispersive X-ray spectroscopy (EDX) serves as an indispensable tool in the arsenal of material characterization techniques, offering a non-destructive means to interrogate the elemental composition of diverse materials, including nanoparticles (NPs).Its capabilities extend beyond mere elemental identification, encompassing critical functionalities that drive scientific understanding [6].The resulting data can be used to understand the structure and properties of the materials and signify potential applications [72]. EDX analysis was performed on AgNPs, Ag@TiO 2 nanocomposites (NCs), and Ag@SeO 2 NCs (Fig. 4A-C, Table S1).Carbon and oxygen were detected in all three samples, likely originating from organic matter like biomolecules [82].The high silver (Ag) content in all samples confirms it as the primary element (Fig. 4A-C).Examining the relative abundance of elements reveals the highest Ag content in AgNPs, followed by carbon (C) and oxygen (O).This expected outcome confirms the successful green synthesis of AgNPs (Fig. 4A).The presence of titanium (Ti) and selenium (Se) in Ag@TiO 2 NC (Fig. 4B) and Ag@SeO 2 NC (Fig. 4C), respectively, confirms the conjugation of Ag NPs with TiO 2 NPs and SeO 2 NPs to form bimetallic NCs.Notably, Ti and Se rank as the second most abundant elements after Ag in these NCs.In the Ag@SeO 2 NC, the unexpected presence of chlorine (Cl) necessitates further investigation despite its absence in controls.Potential explanations include limitations in EDX sensitivity, masking by other elements, surface binding of chlorides with a stabilizing effect, or contamination within the SeO 2 precursor.Further studies are warranted to definitively determine the source and significance of chlorine in this unique nanomaterial. X-ray diffraction (XRD) spectroscopy X-ray diffraction (XRD) analysis offers a robust technique for identifying the crystalline phases present in a sample, determining their relative abundance, and investigating phase transformations in nanoparticles (NPs) [6].XRD data were acquired for AgNPs, Ag@TiO 2 NC, and Ag@SeO 2 NC (Fig. 5A-C, and Tables S2-S4).The XRD pattern of AgNPs shows several distinct peaks at 2θ values of 27.1430 5A, and Table S2).These characteristic peaks align with the face-centered cubic (fcc) structure of silver, as confirmed by the calculated lattice parameters (Equation ( 4)): Where, the unit "Å" stands for angstrom, which is a unit of length equal to 10 − 10 m 'a' is the lattice constant of a crystal.The lattice constant, essentially the length of one side of the repeating unit cell, reflects the fundamental building block size in a crystal structure.In this case, the calculated value of 4.0878 Å defines the unit cell size for the silver crystal structure.This fundamental parameter significantly influences the overall crystal properties.The observed peaks correspond to specific planes within the facecentered cubic (fcc) silver crystal structure: (111), ( 200), (220), (311), ( 222), (400), (420), (422), and (511).Notably, the (200) plane exhibits the highest peak intensity, corresponding to 31.4940 • 2θ.The calculated lattice parameter of 4.0878 Å closely matches the standard value for fcc silver (4.0862 Å).This excellent agreement reinforces the accuracy of the XRD analysis.Additionally, similar lattice parameters for AgNPs synthesized via various methods have been reported in other studies.For example, a coprecipitation method yielded a lattice parameter of 4.0868 Å for AgNPs, and another study reported a range of 4.0860-4.0870Å for AgNPs synthesized using different techniques [46,83]. The XRD pattern of Ag@TiO 2 NC displays peaks at various 2θ positions (Fig. 5B, and Table S3).The most intense peak at 31.7706 • 2θ corresponds to the (101) plane of anatase TiO 2 , signifying its presence as the primary phase.However, additional peaks suggest the presence of other phases within the NC.Peaks at 5.3757 • and 25.2792 • 2θ arise from the ( 101) and (004) planes of anatase TiO 2 , respectively.Additionally, a peak at 47.5269 • 2θ corresponds to the (111) plane of metallic silver, confirming its incorporation into the NC.Weaker peaks might be attributed to minor phases like rutile TiO 2 and silver compounds not classified as strictly "metallic" Ag.Overall, the XRD analysis indicates the Ag@TiO 2 NC comprises a combination of anatase and rutile TiO 2 phases alongside metallic silver.Several factors can influence the presence of these phases, including the synthesis method, doping concentration, and annealing temperature.Further analysis can provide more specific information on the relative proportion of each phase and their impact on the overall material properties.The XRD pattern of Ag@SeO 2 NC shows distinct peaks at specific 2θ positions (Fig. 5C, and Table S4).Peaks at 27.3359 • , 31.6362 • , and 45.6174 • 2θ correspond to the (111), (200), and (113) planes of fcc silver, confirming its presence within the NC.However, other peaks suggest the existence of additional phases beyond metallic silver.Peaks at 37.5313 • , 54.1235 • , and 63.9025 • 2θ might indicate the presence of silver compounds, potentially including Ag 2 SeO 3 .The remaining peaks likely arise from minor phases like SeO 2 and other possible silver compounds, not strictly classified as "metallic" Ag.In summary, the XRD analysis reveals that the Ag@SeO 2 NC comprises a combination of metallic silver and other silver-containing compounds alongside SeO 2 .Further analysis could provide more insight into the specific identities and relative proportions of these phases, potentially influencing the overall properties of the nanomaterial. The XRD patterns of AgNPs, Ag@TiO 2 NC, and Ag@SeO 2 NC all show "metallic" silver phases.However, these patterns reveal further details about their composition: Ag@TiO 2 NC shows peaks corresponding to anatase TiO 2 , while the XRD pattern of Ag@SeO 2 K.M. Elattar et al.NC shows peaks corresponding to other silver compounds, such as AgSeO 3 and Ag 2 SeO 3 .Frequently, the XRD analysis of AgNPs, Ag@TiO 2 NC, and Ag@SeO 2 NC reveals that the NPs are composed of 'metallic' Silver and other phases such as TiO 2 and SeO 2 [84]. Furthermore, XRD analysis can estimate the crystal size of the Ag@TiO 2 NC using the Scherrer Equation ( 5): Where D is the crystal size in nanometers, K is the Scherrer constant (typically 0.9), λ is the wavelength of the X-rays, β is the full width at half maximum intensity (FWHM) (a common measure of the spread of a peak) of the peak in radians, and θ (angular spread or divergence angle) is the Bragg angle in degrees. The FWHM of the peak at 31.7706 • 2Th can be calculated using the following formula (Equation ( 6)), which describes a relationship between the FWHM of a peak and its θ: where; sqrt(3), is the square root of 3 (1.732),serving as a scaling factor in the equation and relates to FWHM.Substituting the measured values of D, K, λ, β, and θ (Bragg angle) into the Scherrer equation (Equation ( 5)) revealed a crystal size of approximately 20 nm for both Ag@TiO 2 NC and Ag@SeO 2 NC.This finding reinforces the earlier observation that the XRD patterns of these nanomaterials contained peaks corresponding to metallic silver.However, the XRD analysis of Ag@TiO 2 NC yielded further insights beyond metallic silver.Peaks indicative of both anatase and rutile TiO 2 phases were also identified, suggesting the nanoparticles are not solely composed of silver but rather a composite structure combining anatase TiO 2 , rutile TiO 2 , and metallic silver. Antioxidant activity The ABTS assay relies on the ability of antioxidants to scavenge the ABTS radical.This blue-green radical's characteristic color fades upon interaction with antioxidants, allowing for the measurement of antioxidant activity.Compared to the DPPH method, ABTS offers the advantage of consistent reproducibility across different pH values [66].The antioxidant activity of beetroot extract, vitamin C (positive control), and biosynthesized nanomaterials was evaluated using varying concentrations ranging from 0 to 100 μg/mL.The results are presented in Fig. 6A and B and Table S5. All tested samples demonstrated antioxidant activity, but the potency varied across materials.Notably, the B. vulgaris extract displayed the strongest activity, with an IC 50 value of 52.57μg/mL -approximately half that of ascorbic acid (vitamin C).AgNPs exhibited moderate activity (IC 50 = 63.53 μg/mL), followed by Ag@TiO 2 NC (IC 50 = 96.86μg/mL) and Ag@SeO 2 NC (IC 50 = 109.5μg/ mL).The potent antioxidant activity of the plant extract likely stems from the presence of phytochemicals like polyphenols and flavonoids, as well as various nutrients like vitamin C, known for scavenging free radicals such as reactive oxygen species (ROS) and reactive nitrogen species [85].Interestingly, despite potentially utilizing phytochemicals in their synthesis, the overall antioxidant activity of the nanomaterials remained lower than the plant extract.This could be attributed to various factors, including the possible oxidation of phytochemicals during synthesis [86] or the aggregation of nanoparticles, which reduces their surface area and free radical scavenging effectiveness [87]. Anti-hemolytic activity Anti-hemolytic activity refers to a material's ability to protect red blood cells (RBCs) from rupturing (lysis).Here, we investigated this activity using hemolysis assays with healthy rat RBCs [6,12].Hemoglobin (Hb), a red pigment absorbing light at 540 nm, is released from lysed RBCs.Hemolysis activity is assessed by measuring the solution's absorbance at this wavelength.We compared the hemolysis activity of beetroot extract, AgNPs, Ag@TiO 2 NC, and Ag@SeO 2 NC to vitamin C, a known hemocompatible substance (Table 1).Notably, the plant extract and vitamin C showed very similar hemolysis activity, both below 5%.This suggests that the plant extract exhibits anti-hemolytic activity and could be safely administered to humans [6,12].In contrast, the hemolysis rates of AgNPs (10.97%),Ag@TiO 2 NC (15.77%), and Ag@SeO 2 NC (56.22%) were approximately 2, 3, and 15 times higher than the safety limit.This indicates that these nano-materials are hemotoxic (toxic to blood cells). Furthermore, the significantly higher hemolysis rates of Ag@TiO 2 NC and Ag@SeO 2 NC compared to AgNPs suggest that bimetallic oxides are more hemotoxic.Notably, a higher hemolysis rate (%) translates to a greater percentage of lysed RBCs [88].These findings strongly suggest that SeO 2 NPs are not hemocompatible, unlike TiO 2 NPs, based on the differential hemolytic effects observed between each nanomaterial and AgNPs. Evaluating the hemocompatibility of new drugs, particularly antibiotics, chemotherapeutics, nanodrugs, and oxide nanomaterials, is crucial to differentiate their specific hemolytic effects from other factors contributing to anemia.This differentiation is vital as several conditions and medical interventions can trigger red blood cell (RBC) destruction, including hemolytic anemia, infections, cell anemia, thalassemias, bone marrow aplasia, blood transfusions, and mechanical heart valves [89,90].Increased hemolysis (measured as % hemolysis) can lead to several complications, such as anemia, organ damage (including kidney and heart failure), jaundice, and gallstones (formed when bilirubin is concentrated in the gallbladder) [89].The anti-hemolytic assay investigates various mechanisms thought to protect RBCs from damage and lysis.One significant pathway involves antioxidants like vitamin C, which scavenge free radicals and reduce oxidative stress [67]. Anti-inflammatory activity The carrageenan-induced paw edema assay, known for its simplicity and reliability, offers a valuable tool for evaluating the antiinflammatory activity of compounds and drugs [91].This method leverages the inflammatory response triggered by carrageenan, a seaweed extract, in rat paws, with the expectation that anti-inflammatory agents can mitigate this response [92].Fig. 7 and Table S6 present the anti-inflammatory activity results, showcasing the protective effect of each sample at various time points.Indomethacin emerged as the most effective agent, providing an average protection of 24.55%.Following closely behind was the B. vulgaris extract with a mean protection of 27.86%.Meanwhile, Ag@NPs, Ag@TiO 2 , and Ag@SeO 2 demonstrated lower, but still noticeable, anti-inflammatory effects with mean protections of 22.73%, 19.66%, and 5.15%, respectively.In conclusion, this study confirms the potent anti-inflammatory properties of both Indomethacin and the B. vulgaris extract.While Ag@NPs, Ag@TiO 2 , and Ag@SeO 2 also exhibited anti-inflammatory activity, their efficacy was noticeably lower compared to the leading performers.One possible explanation for the superior anti-inflammatory activity of B. vulgaris extract is that it is more effective at inhibiting the production of pro-inflammatory cytokines.Pro-inflammatory cytokines are signaling molecules that play a key role in the inflammatory response.By inhibiting the generation of pro-inflammatory cytokines, B. vulgaris extract can reduce inflammation and its associated symptoms.Another possibility is that B. vulgaris extract is more effective at promoting the production of anti-inflammatory cytokines.Anti-inflammatory cytokines are signaling molecules that counteract the effects of pro-inflammatory cytokines and help to resolve inflammation.By promoting the production of anti-inflammatory cytokines, Beta vulgaris extract can help to speed up the recovery process from inflammation.It is also possible that Beta vulgaris L. extract has other mechanisms of action that contribute to its anti-inflammatory activity.For example, it may be able to reduce oxidative stress, which is a major contributor to inflammation, or it may be able to improve the function of the immune system, which can help to fight off infection and reduce inflammation.Generally, the anti-inflammatory data is very promising and recommends that B. vulgaris L. extract may be an effective treatment for a variety of inflammatory conditions.Metal nanoparticles have anti-inflammatory activity through a variety of mechanisms [93].They can inhibit the production of pro-inflammatory cytokines [94,95], promote the production of anti-inflammatory cytokines [96], modulate the function of immune cells, and scavenge reactive oxygen species [97].The specific mechanism of action varies reliant on the type, size, shape, and surface chemistry of the nanoparticles.In the context of the plant extract, it is possible that the active components are more readily accessible and can interact more effectively with cellular targets compared to the metallic nanoparticles.Additionally, the plant extract may contain a variety of compounds with synergistic effects, contributing to its overall anti-inflammatory activity.However, the results of this study suggest that these agents have the potential to be effective treatments for a variety of inflammatory conditions. Antifungal activity 3.5.1. Radial fungal growth The antifungal activity was evaluated against two targeted pathogenic plant fungi: Rhizoctonia solani, and Sclerotinia sclerotium.The MIC of each nanomaterial was determined for both fungi (Table 2).Against R. solani, the MIC values were 422, 624, and 930 μg/mL for AgNPs, Ag@TiO 2 NC, and Ag@SeO 2 NC, respectively.This indicates that AgNPs and Ag@TiO 2 NC were approximately twice as effective as Ag@SeO 2 NC in inhibiting this fungus.For S. sclerotium, the MIC values were 211, 314, and 1390 μg/mL for AgNPs, Ag@TiO 2 NC, and Ag@SeO 2 NC, respectively.Both AgNPs and Ag@TiO 2 NC were roughly 1.5 times more effective than Ag@SeO 2 NC against this fungus.Furthermore, Ag@SeO 2 NC exhibited concentration-dependent antifungal activity.At 422 and 930 μg/mL, it inhibited the growth of S. sclerotium by 47.5% and 56.25%, respectively.Notably, even at its MIC threshold of 422 μg/mL, Ag@SeO 2 NC inhibited the growth of R. solani by 72.5%.Overall, this study suggests that the antifungal activity of these nanomaterials varies depending on the fungal species and the specific material.While AgNPs and Ag@TiO 2 NC showed broader effectiveness, Ag@SeO 2 NC displayed potent activity against R. solani at relatively low concentrations.Nanoparticles pack a punch against not just bacteria, but also pesky fungi!Studies have shown their effectiveness against both Gram-positive and Gram-negative bacteria, and even specific silver nanoparticles hold promise against fungal foes like Candida albicans and Candida tropicalis [98,99].Slavin and Bach [100] reviewed the mechanisms of NPs towards fungal activities across the following modes, i.e., reactive oxygen species, plasma membrane deformation, cell wall architecture, interaction with fungal structure, inhibition of spore germination, and regulation of protein and gene.AgNPs showed potential activity against fungus., Saccharomyces cerevisiae KCTC7296, at MIC 50 (2 μg/mL) [101].Likewise, the AgNPS, at MIC 100 (4 μg/mL), was efficient against Fusarium oxysporum and at MIC 100 (1 μg/mL) AgNPs showed potentiality against both Fusarium solani and Aspergillus flavus [102].The AgNPs exhibited potentiality against Aspergillus fumigatus at MIC 100 (100 μg/mL) [103].The TiO 2 /Ag NPs showed inhibitory activity against C. albicans and Cryptococcus neoformans, at MIC (12.5 μg/mL) [104].Selenium NPs showed potential activity against A. fumigatus TIMML-050, at MIC value (0.5 μg/mL) [105].However, The antifungal activity of metallic/bimetallic nanoparticles depends on the nanoparticle's morphology, for instance, size and shape [76].Larger silver nanoparticles or NC may have a greater surface area, which can promote interactions with bacterial cell walls and membranes, and enhance their antimicrobial activity by causing bacterial cellular contents to leak out [77].The preparation method produced NPs of various sizes, which caused the antifungal abilities of the nanoparticles to vary visibly.The larger surface area of smaller nanoparticles may explain the significant increase in the capacity of nanopesticides [106].Nanoparticles with larger surface areas and smaller particle sizes exhibited significantly enhanced antifungal properties thereby inhibiting fungal growth [106]. Our study investigated the antifungal potential of biosynthesized nanomaterials against two major plant pathogens, Rhizoctonia solani, and Sclerotinia sclerotium.These fungi not only decrease plant growth but also contribute to mycotoxin contamination in food, posing health risks to both animals and humans [52][53][54][55].Interestingly, the nanomaterials exhibited varying antifungal abilities.This variation likely stems from several factors, including the specific type of nanomaterial, its size, and its inherent antimicrobial activity.Previous research suggests that nanoparticles with larger surface areas and smaller sizes tend to demonstrate stronger antifungal properties [106].Although AgNPs, TiO 2 NPs, and SeO 2 NPs have been shown to exert antifungal activities, the combination of AgNPs with TiO 2 or SeO 2 NPs does not enhance the antifungal activity.There are no additive or synergistic effects.This unexpected failure in enhancing antifungal activity is likely due to factors like competition for fungal binding sites, aggregation, altered surface properties, or interference with AgNP's mechanism of action.Further research is needed to pinpoint the exact reason and optimize nanoparticle design for effective fungal control. This study demonstrates the strong antioxidant activity of B. vulgaris L. extract, suggesting its potential as a natural alternative to harmful fungicides for controlling pathogenic fungi.Its high levels of phenolic and flavonoid constituents are likely contributors to this activity.Furthermore, both plant extracts and nanoparticles can exhibit antifungal activity.One proposed mechanism involves the disruption of membrane-bound respiratory enzymes in fungi, hindering their growth [107].These findings not only highlight the issue of mycotoxin contamination in food but also offer promising avenues for its control.Ag@SeO2 and Ag@TiO2 NCs, with their antifungal activity, could be explored as safer and potentially more effective alternatives to conventional fungicides in food preservation. FT-IR spectral analyses on fungi treated with Ag@SeO 2 NC Ag@SeO 2 NC was chosen for FT-IR analysis since it holds distinct advantages over Ag@TiO 2 NC for FT-IR studies of fungi treated with biosynthesized nanomaterials.Ag@SeO 2 NC holds potential for diverse interaction mechanisms, enhanced specificity for fungal biomolecules, and reduced spectral interference from SeO 2 , and the growing interest in their antifungal properties makes them a compelling choice for this research.This selection promises richer data, deeper insights, and broader research impact, solidifying their potential as valuable tools in fungal control.The FT-IR spectral analysis was performed for R. solani and S. sclerotium treated or not (control) with Ag@SeO 2 NC (Fig. 8).The FTIR spectra obtained for R. solani, untreated or treated with Ag@SeO 2 NC, revealed some fascinating variances.The peak in the infrared spectrum at 3279 cm-1 is due to the stretching vibrations of hydroxyl groups.In the treated sample, this peak shifts to a lower frequency (3270 cm-1), suggesting that the Ag@SeO2 NC may be interacting with the -OH Fig. 8. FTIR spectra of treated or untreated fungi with Ag@SeO 2 NC.functions on the surface of the fungal cells [108].The absorption band at ν = 1632 cm − 1 is attributed to the C --O stretching vibrations of amide functions.The absorption band at ν = 817 cm − 1 is attributed to the C-O stretching vibrations of glycosidic bonds.The disappearance of this band in the treated sample suggests that the Ag@SeO 2 NC may be disrupting the glycosidic bonds in the fungal cell wall [109].R. solani showed a shift in the absorption band at ν = 817 cm − 1 to a lower frequency (ν = 775 cm − 1 ) after treatment with Ag@SeO 2 NC.This suggests that the nanoparticles may be interacting with the glycosidic bonds in the fungal cell walls. The FTIR spectra of S. sclerotium, untreated or treated with Ag@SeO 2 NC, revealed a shift in the absorption band at ν = 3279 cm-1. The peak in the infrared spectrum at 3279 cm-1 is due to the stretching vibrations of hydroxyl groups.In the treated sample, this peak shifts to a higher frequency (3280 cm-1), suggesting that the Ag@SeO 2 NC may be interacting with the hydroxyl groups on the surface of the fungal cells in a different way than they interact with the surface of R. solani cells.The peak in the infrared spectrum at 1630 cm-1 is due to the stretching vibrations of amide groups.The shift of this band to a lower frequency (ν = 1614 cm − 1 ) in the treated sample suggests that the Ag@SeO 2 NC may be interacting with the amide groups on the surface of the fungal cells in a similar way to how they interact with the amide groups on the surface of R. solani cells.The absorption band at ν = 1312 cm − 1 is attributed to the C-N stretching vibrations of chitin.The appearance of this band in the treated sample suggests that the Ag@SeO 2 NC may be interacting with the chitin in the fungal cell wall [110].The absorption band at ν = 566 cm − 1 is attributed to the C-O stretching vibrations of glycosidic bonds.The disappearance of this band in the treated sample suggests that the Ag@SeO 2 NC may be disrupting the glycosidic bonds in the fungal cell wall, like how they disrupt the glycosidic bonds in the fungal cell wall of R. solani.S. sclerotium showed a shift in the absorption band at ν = 1148 cm − 1 to a lower frequency (ν = 1148 cm − 1 ) after treatment with Ag@SeO 2 NC.This suggests that the NCs may be interacting with the C-O bonds in the fungal cell walls [111]. Both R. solani and S. sclerotium fungal species showed a shift in the absorption band at ν = 3279 cm − 1 to a lower frequency (ν = 3270 cm − 1 and ν = 3280 cm − 1 , respectively) after treatment with Ag@SeO 2 NC.This suggests that the NCs may be interacting with the hydroxyl groups (O-H) in the fungal cell walls.Additionally, both species showed a shift in the absorption band at ν = 1632 cm − 1 to a lower frequency (ν = 1632 cm − 1 and ν = 1614 cm − 1 , respectively) after treatment with Ag@SeO 2 NC.This suggests that the NCs may also be interacting with the amide groups (C --O-N) in the fungal cell walls. TEM of the bmNPs-affected fungi The TEM image of untreated S. sclerotium (Fig. 9a) shows a healthy cell with a well-defined cell wall and cytoplasm.The cytoplasm is the jelly-like substance inside the cell that contains all the cell's organelles.The following organelles are visible in the TEM image: mitochondria, ribosomes, nucleus, and vacuole.The TEM image also shows several small vesicles in the cytoplasm.In addition, the cell wall of the S. sclerotium cell is a thick, dense layer that surrounds the cell.The cytoplasm also contains a network of microtubules and microfilaments. The TEM analysis revealed significant damage to S. sclerotium cells treated with Ag@SeO 2 NCs (Fig. 9b).Numerous small, dark dots visible within the cell cytoplasm represent the internalized nanoparticles.Moreover, the cell walls exhibited damage, evident from the presence of several holes.Collectively, these observations suggest that Ag@SeO 2 NCs played a role in compromising the cell wall integrity, potentially rendering it more susceptible to further damage [112]. Furthermore, damage extended beyond the cell wall, affecting various organelles within the cytoplasm.Swollen and misshapen mitochondria, for instance, indicate potential interference with the cell's energy production capabilities.In conclusion, the TEM imagery provides compelling evidence for extensive cellular damage inflicted by Ag@SeO 2 NCs on S. sclerotium, likely impairing essential functions and compromising overall cell viability (Fig. 9b). The TEM image of untreated R. solani (Fig. 9c) exhibits a healthy cell with a well-defined cell wall and distinct cytoplasmic region.The cytoplasm, a gel-like substance containing various organelles essential for cell function, appears homogeneous in this micrograph.Additionally, small vesicles, membrane-bound structures for transport and storage, are visible within the cytoplasm.The TEM image shows a healthy R. solani cell with all of the organelles necessary for the cell to function properly.The cytoplasm of the R. solani cell is filled with a variety of organelles, including mitochondria, ribosomes, nuclei, and vacuoles.The cytoplasm also contains a network of microtubules and microfilaments, which provide support and structure for the cell [113].The R. solani cell in the TEM image has a large, well-defined nucleus, which suggests that the cell is healthy and capable of dividing.The R. solani cell in the TEM image has a large vacuole, which suggests that the cell is well-hydrated and healthy.Additionally, the S. sclerotium cell in the TEM image has several mitochondria, which suggests that the cell is metabolically active [114]. The TEM image of R. solani treated with Ag@SeO 2 NCs (Fig. 9d) reveals substantial cellular damage induced by the nanoparticles.Numerous small, dark dots visible within the cytoplasm represent internalized nanoparticles.Moreover, the cell walls exhibit significant damage, evident from the presence of numerous holes.These observations collectively suggest that Ag@SeO 2 NCs contribute to compromised cell wall integrity, potentially rendering it more susceptible to further damage [115].The damage extends beyond the cell wall, affecting various organelles within the cytoplasm.Notably, some mitochondria appear swollen and misshapen, indicating potential interference with energy production capabilities.Furthermore, the TEM image shows extensive vacuolation within the R. solani cell.Vacuolation, characterized by the formation of large vacuoles, represents a type of cell death often observed in response to toxins or stressors [116].This finding provides additional evidence for the effectiveness of Ag@SeO 2 NCs in killing R. solani cells [117]. Generally, there are some hypotheses for possible mechanisms by which the Ag@SeO 2 NC is damaging the S. sclerotium and R. solani cells: The bmNPs may be generating reactive oxygen species, which are unstable molecules that can damage cell components [42].The bmNPs may be interacting with the cell membrane, disrupting its structure and function [118].The bmNPs may be entering the cell and interacting with DNA or other cellular components, causing damage [119]. Fig. 3A . Fig. 3A.SEM micrographs of the Ag NPs at different indicated scales of magnifications.Fig. 3B.SEM micrographs of the Ag@TiO 2 NC at different indicated scales of magnifications.Fig. 3C.SEM micrographs of the Ag@SeO 2 NC at different indicated scales of magnifications. Fig. 7 . Fig. 7.A comparison of the percentage of protection in the anti-inflammatory activity of the tested samples at varied times. Fig. 9 . Fig.9.TEM micrographs reveal the potent antifungal activity of Ag@SeO2 NC against S. sclerotium and R. solani.Panels (a) and (c) show the intact cell wall structure of untreated fungal hyphae in both fungi (S. sclerotium and R. solani, respectively).Treatment with Ag@SeO 2 bmNPs in panels (b) and (d) induces substantial damage to the cell wall and membranes, including disruptions, leakage, and disorganization.These observations demonstrate the ability of Ag@SeO 2 NC to effectively penetrate and disrupt the fungal cell structure, leading to potential cell death and fungal growth inhibition.Note the higher magnification used in panels (c) and (d) to visualize the detailed morphological changes caused by Ag@SeO 2 NC treatment. Table 1 Hemolysis activity of the investigated samples.	2024-03-31T05:08:21.358Z	2024-03-22T00:00:00.000	{ "year": 2024, "sha1": "64b5f889e6d4a3c91d574030caf6ec66f41f3d4d", "oa_license": "CCBYNC", "oa_url": "http://www.cell.com/article/S2405844024043901/pdf", "oa_status": "BRONZE", "pdf_src": "PubMedCentral", "pdf_hash": "64b5f889e6d4a3c91d574030caf6ec66f41f3d4d", "s2fieldsofstudy": [ "Chemistry", "Materials Science", "Environmental Science" ], "extfieldsofstudy": [] }
244521246	pes2o/s2orc	v3-fos-license	Trehalose: A mycogenic cell wall elicitor elicit resistance against leaf spot disease of broccoli and acts as a plant growth regulator Highlights • Mycogenic cell wall elicitor was isolated from trichoderma atroviride.• The isolated elicitor was identified as trehalose by LC-MS analysis.• Seed priming with elicitor enhanced early germination and vigour.• Primed plants induced resistance against leaf spot disease of brocolli.• Trehalose sugar act as a bio-stimulant for growth promotion and plant defence. Introduction Elicitors are the biochemicals that trigger plants to produce phytoalexin or secondary metabolites excessively by modifying the physiology of the cell system. Elicitors can trigger defence in both host and non-host plants upon encountering potential pathogens [1][2][3][4]. Generally, elicitors are produced mainly by the microbial cell walls as structural components such as chitin, glucan, lipopolysaccharides (LPS) and flagellin that protects the plants by inducing defence responses against pathogen infection [5]. There are high-affinity binding sites specifically present for peptide, glycopeptide and oligosaccharide elicitors, which recognize the involvement of functional elicitor binding proteins [4,6]. All these elicitors belong to different families, including proteins, sugars, and lipids that produce a different array of plant defence response during plant-pathogen interaction [7]. These elicitors act as an indicator molecule at little concentrations and activate defence responses in the host plant [8,9]. Koike et al. [10] have reported that the induction of cucumber seedling hypocotyls lignification was due to culture filtrates of plant growth-promoting fungi (PGPF) such as Trichoderma sp., Fusarium sp., Penicillium sp., Phoma sp. and a sterile fungus following challenge inoculation with Colletotrichum orbiculare. amongst elicitors, trehalose, a non-reducing disaccharide distributed ubiquitously, acts as a biogenic cell wall elicitor [11]. Biochemically, trehalose has the ability to stabilize lipids and protein membrane [12]. The metabolism of trehalose is fundamental for few metabolic pathways in general, for example, biosynthesis, carbon assimilation, sugar status, and ruin of starch in plants [13][14][15]. In addition, disaccharide trehalose produced by microorganisms is an excellent metabolic osmoregulator that upregulates defence signalling during biotic and abiotic stresses [12,16,17]. Trehalose is produced in the plants during abiotic stresses such as drought, salinity and oxidative stress [18,19]. Subsequently, these plants can survive the extended dry conditions and when the favourable conditions return, the trehalose can partially or completely hydrolysed to reinitiate the plant growth. In other words, trehalose can act an osmoprotectant against salinity stress in wheat plants [20]. Thus, it stabilizes protein in its native state at high temperatures in living cells [21]. Trichoderma species are proposed chiefly as major PGPF in the natural environment with the ability to carry elicitors that could promote rapid plant growth, reproduction, high crop productivity, uptake of soil nutrients, greater yield, and resistance to abiotic and biotic stress [22][23][24][25]. In recent years, more and more research has been focussed on resistance against plant diseases exploited through induced systemic resistance (ISR) caused as a result of root symbiotic relationships of Trichoderma [26][27][28]. Along with the revelation of the diverse beneficial anti-fungal mechanism of T. atroviride and other Trichoderma species, the ability to promote plant growth and to increase other growth parameters such as the plant height, leaf area, and dry weight is evident from various researches [29][30][31][32]. Broccoli is a hardy biennial crop that belongs to the family Brassicaceae. It is grown as a cool-season annual plant and grows worldwide in a much cooler climate, forming tiny to large blue-green flower buds either in single or multiple flower heads [33]. The leaf spot fungal disease caused by Alternaria brassicicola is found to be the most damaging disease amongst Brassicaceae members at all phases of the plant growth from the nursery until the reproductive stage [34][35][36][37]. Therefore, disease management schemes using chemical and resistant varieties of plants against leaf spot disease of broccoli have constraints and inadequacies [38]. Thus, finding a suitable alternative method that complements resistance plant breeding with lesser chemical usage by innovative stratagems is inevitable. It can be achieved by inducing resistance in the host that enhances the plant defenses by PGPF [39,40]. However, there are minimal reports on the use of proposed PGPF elicitors for leaf spot disease management in broccoli. Hence, the present study aimed to (i) investigate the isolation and characterization of cell wall elicitor from PGPF, Trichoderma atroviride, (ii) evaluate the seed priming effect of elicitor for induction of resistance against leaf spot disease in broccoli, and (iii) ascertain the dual role of elicitor in promoting plant growth observed in elicited plants. Microbial culture The isolate Trichoderma atroviride (TriAt_JSB2) having accession No. JQ665257, used in this study, was well characterized with multi-faceted beneficial characters including induce resistance and plant growth promoter [29]. This was available at plant healthcare and diagnostics centre, P.G. Department of Biotechnology and Microbiology, Karnatak University, Dharwad. The fungus was cultured on Potato Dextrose Agar (PDA) media at pH 7.0 and incubated for seven days at 27 • C. Emerging hyphae from the above culture plate were inoculated in 250 ml of Potato Dextrose Broth (PDB) and kept for seven days of incubation at 27 • C. Finally, the obtained dry fungal mat (5 gm) was used in the extraction of the fungal cell wall elicitor. Extraction of cell wall elicitor from trichoderma atroviride (TaCWE) Cell wall extract from T. atroviride isolate was formulated as per the method described by Jishaa et al. [41]. Briefly, mycelium from 7-day-old PDB culture was homogenized with liquid nitrogen and crushed using mortar and pestle. The homogenate was filtered using a muslin cloth, and then the obtained residues were washed with double distilled water, followed by chloroform and methanol (1:1). The suspension was washed later with acetone. Finally, the obtained suspension was air-dried, and the cell wall material was recovered. The extract from the mycelia cell wall was obtained by suspending 1 g of the cell wall in 100 ml of distilled water and autoclaved. Later, the suspension was centrifuged at 14,000 rpm for 10 min and filtered using a sterile syringe filter (0.45 µm Millex). The cell wall extract was smeared on a clean glass slide and fixed. Further, it was observed under a light microscope (Olympus, India) using lactophenol stain for morphological analysis. Morphology of cell wall elicitor by atomic force microscopy (AFM) The cell wall extract was suspended in a 10 mM KH 2 PO 4 buffer; then, 2% glutaraldehyde (v/v) was used to fix onto the glass slide for 2 h at 25 • C. Further, the cell wall morphology was scanned at different resolutions using the Flex-Bio Nanosurf system . This is used to understand the underlying structural membrane of the cell wall extract of T. atroviride. Scanning electron microscopy (SEM) with energy dispersive x-ray spectroscopy (EDS) The dried cell wall extract was coated with a 20-25 nm thick layer of carbon in an EC-32010CC using a graphite bar with a current intensity of 25A. Carbon is preferred to use in microanalysis due to its excellent electrical conductivity and transparent properties. The absorption of this layer of X-rays emitted by the element can influence the results obtained by the EDX JSM-IT500 [42]. Quantification of sugar present in the elicitor produced by Trichoderma atroviride After their complete growth, by the enzymatic assay of trehalase, the trehalose concentration in the CWE from T. atroviride was quantified. The CWE was mixed with sterile distilled water (200 µL), followed by 30 µL of buffer solution containing NADP, glucose-6-phosphate dehydrogenase, hexokinase and ATP. The activity of this reaction mixture at λ = 340 nm was recorded. Later, 2 µL of trehalase enzyme was added, and this reaction mixture was left to stand for 8 mins at room temperature, and absorbence of all the samples was noted [43]. The test sample which showed the highest trehalose activity was used for further studies. Functional groups analysis of elicitor by Fourier transform infrared spectroscopy (FTIR) For the analysis of the sample by FTIR, a cell wall elicitor was finely ground and encapsulated in 200 mg of Potassium bromide (KBr) to characterize the molecular functional groups. The annotation of spectra at the Infrared (IR) region of 4000 to 400 cm − 1 using Nicolet FT-IR 6700 (Thermo Fisher Scientific, India) and the outcome of the spectra was processed by OMNIC 7 software [44]. Liquid chromatography and mass spectroscopy (LCMS) of elicitor The cell wall extract was mixed thoroughly with ethyl acetate and acetone in the ratio of 1:1 (v/v) and vortexed, then 50 ml of extract was dried at 50 • C for complete extraction. Later, 5 ml of methanol was used to dissolve the residue obtained, and 0.5 ml of the residue sample was used for LCMS analysis. The LC detection was followed by mass spectrometry (MS) confirmation using an AB Sciex API 200 machine. The AB Sciex API 200 used for LC was equipped with Phenomenexgemini 3 µm 50 mm into a 2 mm column. The methanol: formic acid (80:20, v/v) solvent was used in a reverse-phase in the isocratic mode for 3 min, and 10 µL was introduced at precise room temperature. The MS/MS coupled to the LC with a triple Quadrupole mass analyser and mass spectrometer equipped was run in a positive mode with an API source in which the vacuum gauge was maintained at 10e-5 Torr with a source temperature of 400 • C. The mass spectrometer was performed in a full scan (0-400 mass range) multiple reaction monitor (MRM) mode. Seed material Broccoli plant seeds (Brassica oleracea (L) var italica) cultivar Shogun that were highly susceptible to leaf spot disease were collected from the authentic suppliers, Brian Bell Pvt. Ltd, Goroka, and these seeds were used throughout the experiment. Pathogen The fungus Alternaria brassicicola (accession no. MN700129), which was isolated previously from our group [37], was grown in potato dextrose agar (PDA) for seven days at 23 • C and was used as a pathogenic source in this study. Seed priming of TaCWE as an inducer At first, the susceptible broccoli seeds (cv. Shogun) were surface sterilized with 0.2% sodium hypochlorite for 5 min and cleansed carefully using sterile distilled water 2-3 times. The sterilized seeds were then treated with isolated TaCWE at 5.0, 10.0, and 25.0 mg ml − 1 for12 h, respectively, by keeping them in an incubatory rotary shaker at 25 ± 2 • C. After incubation, the treated seeds were air-dried aseptically at room temperature and used for further studies. Treated seeds with only distilled water were used as control. Both seeds, treated and untreated, were planted in February and observed through April during a wet season under greenhouse conditions. The treatments included i) susceptible plants treated with sterile distilled water (SDW) used as control, ii) susceptible plants inoculated with A. brassicicola pathogen, iii) susceptible plants treated with T. atroviride elicitor (CWE), and iv) susceptible plants treated with T. atroviride elicitor (CWE) and pathogen inoculated. Effect of TaCWE on seed germination and seedling vigour Seeds treated with TaCWE and control seeds (SDW) were plated in petriplates on moistened three-layered blotter discs at equal distance from each other, and the germination was evaluated in percentage [45]; additionally, a paper method was used on a set of treated seeds to note down the seedling vigour [46]. After incubating for ten days, percentage of germination, root length, shoot length, and vigour index were calculated. The experiment involved four replicates of 100 seeds each, and it was repeated thrice. Demonstration of elicitor as resistance inducer against broccoli leaf spot disease under greenhouse conditions To assess the efficacy of elicitor for induction of systemic resistance (ISR), for each treatment, sixteen randomly selected broccoli plants (two-week-old) were challenge inoculated with the pathogen (A. brassicicola) suspension of 9 × 10 5 conidia ml − 1 (7-day-old culture) in sterile distilled water. In contrast, plants without inoculation were maintained as control. The inoculated plants were roofed with plastic bags for three days and incubated at 20 • C under greenhouse conditions with 85% relative humidity. Regular observations were made on the plants for the typical leaf spot symptoms, such as pale to dark spots with concentric rings and brown necrotic lesions on the leaves. The development of symptoms was finally scored after 4-weeks of pathogen inoculation. Assessment of elicitor priming on plant growth promotion under greenhouse conditions The susceptible plants (one-month-old) to leaf spot disease were raised from seeds of (Brassica oleracea (L) var italica) under greenhouse conditions in earthen pots filled with peat moss, sand, and FYM (1:2:1). The potted plants were observed from day one to check on the expected growth of the plant for three months until it got matured and withered. The treated and untreated plants were monitored for growth parameters such as plant height, leaf size, stem girth, early flowering, and maturity. Statistical analysis Each experiment was performed using four different replicates. The obtained data were analysed using SPSS Inc. 18.0 for analysis of variance (ANOVA). The magnitude of the F value (P ≤ 0.05) determines the significant effects of treatments used. Treatment means were separated by Tukey's Honestly Significa nce Difference (HSD) test. Microbial culture and growth conditions After seven days of incubation on Potato Dextrose Agar (PDA), T. atroviride isolate (TriAt_JSB2) showed consistent growth in all the Petri plates (Fig. 1 a) and a thick fungal mat appeared in the 500 mL Erlenmeyer's flask (Fig. 1 b). Observation of cell wall elicitors from Trichoderma atroviride (TaCWE) under bright field compound microscope The extracted cell wall elicitors were light yellow, and it was stained with lactophenol cotton blue and observed under 40x zoom in a bright field compound microscope. The cell wall extracts from the tested fungal isolate (TriAt_JSB2) showed the ghost cell appearance, indicating the secretion of putative cell wall elicitors in the cell-free suspension (Fig. 2a). On the other hand, the normal cells of T. atroviride showed an intact cell wall (Fig. 2b). Morphological analysis of TaCWE by atomic force microscopy (AFM) and scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS) Morphological and elemental analysis of the isolated cell wall elicitor was carried out to understand the surface morphology of the elicitor. SEM analysis of the TaCWE displayed reproducible surface architecture in all the fields of study. The extraction procedure proved to be noninvasive as the surface morphology appeared undamaged without significant morphological changes, as shown in Fig 3. The elicitor was found to be 10.16 µm in length and 2.148 µm in width, as evident in the AFM analysis (Fig. 3a). The scanning electron microscope with EDS detected the presence of elicitors on the surface area of the fungal cell wall and the accumulation of carbon, nitrogen, oxygen, and hydrogen elements. (Fig. 3b). Fourier transform infrared spectroscopy (FTIR) analysis of elicitor FTIR spectroscopy analysis was carried out to identify the functional groups of the isolated elicitor trehalose. Thespectra were recorded in absorption mode with the Perkin Elmer spectrometer. Successively, Table 1. Activity of trehalase in the elicitor extract Trehalase activity is a significant marker for analysing responses of hosts towards mitigation of biotic stress. The highest trehalase activity of 1.92 µmol glucose equivalent ml − 1 was observed in the T. atroviride CWE (TaCWE). Therefore, trehalase activity in T. atroviride implies that it is a potent candidate for the identification of biotic elicitors to induce resistance against biotic and abiotic stresses. Detection of trehalose sugar by liquid chromatography-mass spectroscopy (LC-MS) Trehalose sugars are important mitigators of abiotic stresses due to their osmolytic activity. LC-MS analysis was carried out with a fraction of the elicitor in T. atroviride to identify whether trehalose was present in the cell wall. Standard trehalose was eluted at 3.79 min in the LC-MS, and the TaCWE sample had a single peak eluted at a retention time of 3.78 min. Both indicated the presence of trehalose in the cell wall fraction of the fungi tested (Fig. 5). Seed priming effect of elicitor on seed germination and seedling vigour under laboratory conditions Amongst the tested concentrations, the seed priming with 10.0 mg ml − 1 of elicitor (TaCWE) uncovered significantly (P < 0.05) enhanced early emergence (3rd-day post-priming), germination (94%), and seedling vigour (2601). It was followed by seeds primed with 25.0 mg ml − 1, which recorded early emergence at the 4th-day post-priming, germination (89%), and seedling vigour (1977). Whereas 5.0 mg ml − 1 primed seeds showed fair enhancement of emergence, rate of germination, and seedling vigour. In the untreated control, the emergence recorded at 6-day post-treatment with germination of 77% and seedling vigour of 1155, respectively, were noticed (Fig. 6). Elicitation of resistance by seed priming with elicitor (TaCWE) Plants pre-treated with TaCWE elicitor with 10 mg ml − 1 and inoculated with the pathogen were able to induce a remarkable 71.7% protection against leaf spot of broccoli caused by A. brassicicola after 34 days of sowing (4th day after inoculation). This protection or resistance was slightly reduced to 70% at 35-day sowing; the plants treated with elicitor alone with10 mg ml − 1 were also protected (57.3 and 57.9% respectively) at 3rd and 4th day after inoculation against leaf spot disease (Fig. 7A). Plants treated with elicitor (5 and 25 mg ml − 1 ) and challenged with the pathogen also offered significant (P < 0.05) disease suppression after 34 days of sowing by recording disease protection of 49.7% and 57%, respectively. The plants that received the elicitor treatment of 5 and 25 mg ml − 1 alone recorded adequate disease protection but were still significant compared to the control plants. Conversely, the pathogen inoculated plants recorded a maximum disease incidence of 95% by exhibiting typical pale to darkened spots with sunken brown lesions on the leaves (Fig. 7B). In contrast, the distilled water-treated control plants showed stunted growth and chlorosis on the leaves. The pots showing the expression of disease symptoms on elicitortreated and untreated broccoli plants under greenhouse conditions are represented in Fig. 7B. Plant growth promotion after elicitor priming The plants raised with elicitor treatment with all the concentrations showed more profuse growth than the control or pathogen inoculated plants. Primed broccoli plants with elicitor after five weeks recorded a positive growth effect in plant height and early flowering compared to the control (Fig. 8). A significant plant height of 227 cm (56.7%) and 199 cm (49.1%) was attained in the plants raised with elicitor (10 and 25 mg ml − 1 ), respectively. It was observed that the plants primed with 10 mg ml − 1 produced early flowering 12-days prior to the control plants (85-days). Likewise, treated plants (10 mg ml − 1 ) recorded significant leaf size and height enhancement when assessed with control and pathogen inoculated plants and in other aspects, including disease resistance (Fig. 8). Further, the induced resistance and growth promotion experiments showed a significant constructive correlation between the elicitorprimed plants in compared with the control plants irrespective of the time gap with or without pathogen inoculation (Fig. 9). A spaciotemporal elicitor effect was clearly evidenced in this study wherein plants that received treatment with pathogen showed induced protection from 3rd and 4th day against pathogen attack. The same treatment without pathogen attack demonstrated a plant immune induced response positively to the plant growth promotion compared to non-induced plants or control plants. Discussion Plants, being sessile, are in continual acquaintances with various biotic and abiotic stresses [28]. However, plants keep adapting to such variations by constantly evolving and rapidly gaining a greater degree of systemic resistance [23,47]. Many studies on plant growth-promoting fungi (PGPF) Trichoderma spp. have shown profound beneficial effects in agriculture to enhance plant growth and induction of disease resistance [24,48,49]. Various elicitors have been documented, and the most central amongst them are the cell wall elicitors [50]. The elicitors either directly or indirectly induce resistance in plants against pathogen infection through Microbe Associated Molecular Pattern (MAMPS) or Pathogenesis-related (PR) resistance [51][52][53]. However, little information is known about the plant responses to cell wall elicitors extracted from PGPF, and the resistance mechanism confers on the host. In the present study, we analysed the cell wall elicitor's surface morphology and chemical nature. Based on the characterisation studies, the purified cell wall elicitor fraction has been demonstrated to possess trehalose as its essential source. In a recent survey by Djanaguiraman et al. [54], the FTIR analysis of synthetic elicitor nanoceria showed the absorption bands at 3341, 1634, 1327, and 582 cm − 1 . The results showed the presence of high crystallinity and purity in the synthesized nanoceria. AFM measured the size and morphology of nanoceria. The results of AFM indicated that synthesized nanoceria had an average diameter of 15 ± 5 nm. The current research exhibited amides, carboxylic acid, and aldehydes in the isolated elicitor with unique FTIR frequencies. The LC-MS analysis revealed that the pure form of trehalose was detected at an elution rate of 3.78 min. Further, when analysed by SEM, the cell wall elicitor displayed undamaged surface architecture, and the elicitor was found to be 2.16 µm in length and 2.148 µm in width. Collaborating our results, Hayner et al. [55] also reported that the trehalose produced by Escherichia coli was able to elute at the same retention time and found that LC-MS is the most suitable method for the detection of trehalose in any biological samples. Interestingly, a high amount of trehalase activity (1.92 µmol glucose equivalent ml − 1 ) in the T. atroviride cell wall extracts was also recorded. Our results agree with Elbein et al. [11], who recorded a high concentration of trehalose from yeast culture. Previous studies also demonstrated increased activity of trehalose in the mycorrhizae fungi Rhizophagus irregularis, and it was reported that the production of trehalose helps the host plant with improved nutrition and protection against biotic and abiotic stresses [56,57]. The current study witnessed that seeds primed with trehalose at 10 mg ml − 1 showed enhanced emergence, germination (94%), and seedling vigour (2601). Again, the current study results were supported by the outcomes of Gong et al. [58], where seeds primed with elicitors isolated from the endophytic fungi provided a remarkable increase by 30% in the seeds germination rate of C. goeringii, and also reported that the mixed (Fig 7a) and pathogen inoculated (Fig 7b) had some disease symptoms and the treated plants and challenge inoculated with the pathogen had no disease symptoms (Fig 7c). The plants sprayed with elicitor (10 mg ml -1 ) of T. atroviride (Fig 7d) showed overwhelming growth without any disease symptoms on the broccoli plant. . fungal elicitors promoted the growth of tissue culture seedlings. Similar results were also revealed by Bhagobaty and Joshi [59]. The authors revealed that the treatment of culture broth of P. verruculosum exhibited the ability to promote seed germination in mung beans and chickpea under in vitro conditions. Another study by Anupama et al. [60] on seed treatment of tomato with oligosaccharides extracted from A. solani indicated a significant increase in seedling germination, growth, and vigour. Moreover, seed treatment with the combination of fungal oligosaccharides and different strains of PGPR [61] revealed a significant surge in the germination rate of 93.33% and 2733 seedling vigour when compared to the control and the individual treatment of oligosaccharide elicitor, which showed 75% germination and 887 seedling vigour. Previously, our group demonstrated that exogenous pearl millet seed priming with trehalose stimulates early and enhanced seed germination and vigour [12]. These results substantiate the application of trehalose on stimulating germination and seedling vigour. The collective and innovative evidence specifies that trehalose and its derivatives as signal molecules to stimulate plant resistance against diverse biotic factors [15,62]. More importantly, the present study showed that seed priming with trehalose at low concentration could potentially protect broccoli plants against leaf spot disease, as evidenced by their induced resistance and subsequent plant growth under Alternaria brassicicola stress. Several investigators have reported the positive application of trehalose that confers resistance against Blumeria graminis in wheat [63,64], downy mildew disease in pearl millet [12], and tobacco mosaic disease [65]. Further, augmentation of resistance against green peach aphid in Arabidopsis using exogenous trehalose application with trehalose phosphate synthase11 (tps11) knockout in mutant plants was documented [66]. There is an immense impact of trehalose on the plant defence and metabolism responses; in contrast, the trehalose biosynthetic pathway plays a vital role in the pathogen body during infection. It was found out that instead of regular T6P synthesis taking place when a pathogen attacks the plant body, an alternative pathway such as the oxidative pentose phosphate pathway occurred in the case of the rice blast fungus Magnaporthe grisea. Thus, the TPS1 gene needed to colonize the plant tissue [67] was disengaged from pathogenicity [68]; by passing the production of T6P resulted in the expression of the virulence-associated gene. Therefore, the results from previous research studies and the current results authenticate the importance of exogenous trehalose treatments against phytopathogens. In particular, seed treatment with T. atroviride activated the production of trehalose sugar, which is an effective and efficient way of controlling leaf spot disease in broccoli. In countries like Papua New Guinea, broccoli is mainly cultivated by low-income farmers who cannot afford the repeated use of expensive chemical fungicide [47]. Subsequently, this method would be more suitable for large-scale production with less use of chemical fungicides. Furthermore, we have also noticed the susceptible broccoli cultivar's increased potential after trehalose treatment towards induced resistance and promoted plant growth under biotic stress conditions compared to the control. Similarly, Baldi et al. [69] reported that one of the most assuring strategies for crops yield enhancement is the elicitation technique. Biotic and abiotic elicitors, those derived exclusively from fungi, enhance the secondary metabolites in plant cell suspension cultures. Such secondary metabolites could be used for economically feasible elicitation methods like seed treatment. A comprehensive study by Akram et al. [70] confirmed significant growth promotion in radish (Raphanus sativus L.) with 25 mM trehalose seed treatment concerning fresh and dry biomasses and chlorophyll a and total soluble sugar contents in the plants. In addition to various fungi isolated from the rhizosphere currently used for plant growth promotion, several symbiotic arbuscular mycorrhizae, like fungus, are also tested for their elicitor activity. Piriformospora indica and its elicitors have been used against biotic and abiotic stresses. Overall, in the present study, the pattern of inducing resistance or protection witnessed a positive correlation with that of plant growth promotion, and were consistently reproducible amongst the tested biological replicates, which demonstrate the spacio-temporal elicitation of induced resistance in broccoli plants priming with TaCWE. It has been well documented that in line with this trehalose sugar, other sugars, viz. sucrose, fructose, and glucose, are required in the metabolic pathways and various signalling mechanisms in plants [71,72]. Baenas et al. [73] illustrated that after spraying with sucrose (146 mM) for 5-days, there was an elicitation in the biomass weight of 5 different Brassicaceae sprouts. Similarly, an elicitor protein of rice blast fungus M. oryzae, namely MoHrip2 (Magnaporthe oryzae hypersensitive protein 2), has demonstrated enhanced plant growth and resistance against rice blast after treating with the elicitor [74]. The most remarkable finding of the current work has opened up a new perspective into the interactions of pathogen and elicitor with the evidence provided from FTIR and LC-MS analysis about the trehalose production in both plants treated with elicitor alone and elicitor treated together with pathogen inoculated plants. It should have incited the immune response of broccoli plants before and after challenge inoculation with A. brassicicola pathogen by actuating a defence gene that reveals ISR. There is less or no severity of disease incidence observed in the treated broccoli plants, and hence it is directly proportional to the increase in the yield. In other words, the application of T. atroviride as an eco-friendly biological control agent appears to be working well. It can be considered one of the most promising cell wall elicitors that plants could produce when pathogens contact them. Conclusion The current investigation has established decisively a strong correlation between the seed priming with Trichoderma atroviride at a specified amount and the trehalose production in broccoli, which enhanced plant growth and induced systemic resistance against leaf spot disease. Overall, the study has portrayed the trehalose sugar as a bio-stimulant for growth promotion and involved in the induction of resistance against leaf spot diseases of broccoli plants caused by A. brassicicola using the T. atroviride isolates as inducers for seed priming. Such a trehalose elicitor with evidence of its disease resistance is considered the Fig. 9. Dipartite constructive correlation of induced resistance and plant growth in response to elicitor treatment to broccoli plants with or without Alternaria brassicicola infection was developed using "corrplot" in R 4.3.2 version. most favourable, eco-friendly, non-toxic, and biogenic product that provides natural growth promotion and a bio-control agent to the growing agricultural sector alongside the existing conventional chemical fertilizers in the market. It can be recommended to develop newer formulations to be used for integrated plant disease management in the cultivation of various cruciferous crop plants, especially broccoli. Overall, the study has portrayed the trehalose sugar as a bio-stimulant for growth promotion and involved in the induction of resistance against leaf spot diseases of broccoli plants caused by A. brassicicola using the T. atroviride isolates as inducers for seed priming. Declaration of Competing Interest Authors have declared that no competing interests exist.	2021-11-24T16:31:47.730Z	2021-11-01T00:00:00.000	{ "year": 2021, "sha1": "c5f3558b9a46a849a444b0d9fefa97668c700bf3", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.btre.2021.e00690", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "74a1fad232aba9f662835726576577868e0dbbc7", "s2fieldsofstudy": [ "Biology", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
247055023	pes2o/s2orc	v3-fos-license	A Dual-band Bandpass Filter with The Second Passband Independently Tunable This article proposes a dual-band bandpass filter (BPF) based on stub-loaded resonator (SLR) with the second passband independently tunable. Varactors loaded on the end of the central stub are employed to independently tune the second passband. Two shunt quarter-wavelength open stubs and source-load (S-L) coupling are applied to generate transmission zeros (TZs) to enhance the selectivity and improve the out-band rejection. For verification, a tunable dual-band BPF is designed, fabricated and tested, which realizes frequency tuning range (FTR) of the second passband from 2.24 to 2.71 GHz with constant fractional bandwidth (CFBW) of 1.5±0.4%. The simulated and measured results are in good accordance. Introduction In the wireless communication system, the requirements for the development of multi-band filters [1,2] have increased dramatically. Tunable filters with the characteristics of compactness, lower insertion loss (IL) and high performance that can meet the multi-band feature have aroused great attraction [3,4]. Various tunable filters can be realized by different kinds of reconfigurable devices, such as varactor [5], PIN diode [6] and micro-electromechanical system (MEMS) device [7]. Therefore, some research on tunable dual-band bandpass filter (BPF) has been conducted in recent years [8][9][10][11]. In [8], a tunable dual-band BPF with harmonic suppression feature has been proposed. However, the two passbands cannot be independently tuned, and the passband selectivity and out-band rejection are not good. In [9], a three-pole tunable dual-band BPF has been presented. However, the selectivity is poor and its size is large. In [10], a novel switchable dual-/single-band tunable BPF based on a switchable J-inverter has been proposed, but its selectivity and out-band rejection are poor. In [11], a tunable dual-band BPF using lumped-element dual-resonance resonator has been proposed, but its selectivity is poor. In this article, a tunable dual-band BPF using stub-loaded resonator (SLR) with the second passband independently tunable is presented. The proposed coupling structure (including internal and external coupling) is designed to flexibly control the coupling to obtain the desired bandwidth. Then, two shunt quarter wavelength open stubs and source-load (S-L) coupling are applied to produce transmission zeros (TZs) to improve the selectivity and out-band rejection. Finally, by reasonably adjusting the varactor installed on the end of central stub, the frequency tuning (FT) can be achieved. The simulation is in accordance with the measurement. Figure 1 depicts the presented tunable dual-band BPF, which consists of SLR, feedlines, varactor diodes and bias circuits. Varactor Cv 1 loaded on the end of the central stub is utilized to independently tune the even-mode resonant frequency, and Cv 2 attached to the feedlines is employed to adjust the external coupling. Resistors R bias and capacitances C block (and C 1 ) are acted as dc bias and dc block, respectively. Resonant characteristics of the proposed SLR The equivalent circuit of the varactor loaded SLR is shown in figure 2(a). Due to its symmetrical structure, the even-and odd-mode analysis method is used to analyse its resonant characteristics. Under even-mode excitation, the symmetrical plane A-A' can be viewed as virtual open. The equivalent circuit is displayed in figure 2(b). In order to simplify the equation, assuming the input admittance of the central stub loaded with Cv 1 is Y in,1 , the input admittance under even-mode excitation is written as The even-mode resonant frequency can be derived from the resonant condition Thus, the value of Cv 1 can be expressed as Where e is the even-mode resonant angular frequency. Similarly, under odd-mode excitation, the central plane can be regarded as virtual short, as indicated in figure 2(c). The input admittance when applying odd-mode excitation is deduced as The odd-mode resonant frequency can be derived by 0   in odd Y ( 6 ) Then, the two resonant frequencies (denoted as f 1 and f 2 ) of SLR are applied to form the two passbands. As seen from the equation (4), the even-mode resonant frequency can be independently tuned by reasonably adjusting the varactor Cv 1 . Design of the coupling structure In the design process of the filter, the coupling structure is of significance (including internal and external coupling structure, corresponding to internal coupling coefficient M ij and external quality factor Q e ), which is a basic and initial step. It is important to control the coupling structure to obtain the specified M ij (M 12 Ⅰ and M 12 Ⅱ ). Figure 3(a) illustrates the coupling coefficients as a function of the gap S and finger number n. It can be seen that M 12 Ⅰ and M 12 Ⅱ both decrease with the increase of S, and M 12 Ⅰ declines faster. While both have not reached the specified value. The interdigital structure composed of a certain number of short fingers between resonators is utilized to enhance the coupling to meet the desired value. As can be seen from the inserted figure in figure 3(a), M 12 Ⅰ and M 12 Ⅱ increase with fingers n when the gap S is fixed as 2.5 mm. Besides, it can be viewed from figure 3(b) that the central stub is folded to independently control the M 12 Ⅱ to meet the design requirement. Apart from that, the external coupling can be obtained by the proposed feeding structure. Figure 3 Fabrication and measurement For demonstration, the proposed tunable dual-band BPF is designed and fabricated on the Rogers 5880 with the relative dielectric constant of 2.2 and a thickness of 0.787 mm. The photograph is shown in figure 4, and its total size is 20 mm×32.6 mm (0.168 g ×0.274 g , where g denotes the guided wavelength at the first passband). The simulation and test are conducted by ANSYS HFSS and Agilent N5244A, respectively. Figure 5 illustrates the simulated and measured results. The frequency tuning range (FTR) of the second passband is achieved from 2.24 GHz to 2.71 GHz with the first passband fixed at 1.85GHz, accordingly the constant FBW (CFBW) of 1.5±0.4% is obtained. The measured return loss (RL) and IL is higher than 11.5 and 4.2 dB, respectively. Five TZs indicates the proposed tunable dual-band BPF has high selectivity and good out-band rejection. Table 1 shows the filtering performance parameters of five tunable states. Table 2 gives performance comparisons with the reported works, exhibiting high selectivity, good out-band rejection and compact size. Conclusion In this paper, a dual-band BPF based on SLR with the second passband independently tunable is designed, analysed and tested. Appropriately adjusting the coupling structure and C v2 , the desired M ij and Q e can be obtained in the tuning process. The second passband can be independently tuned by reasonably adjusting C v1 . The measured results indicate the presented BPF possesses advantages of high selectivity, good out-band rejection, and compact size.	2022-02-23T20:08:58.736Z	2022-02-01T00:00:00.000	{ "year": 2022, "sha1": "c59ff770d7b04d2d6c16d601a012028c772be767", "oa_license": null, "oa_url": "https://doi.org/10.1088/1742-6596/2187/1/012005", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "c59ff770d7b04d2d6c16d601a012028c772be767", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
118457197	pes2o/s2orc	v3-fos-license	Weak Lensing of the Primary CMB Bispectrum The cosmic microwave background (CMB) bispectrum is a well-known probe of the non-Gaussianity of primordial perturbations. Just as the intervening large-scale structure modifies the CMB angular power spectrum through weak gravitational lensing, the CMB primary bispectrum generated at the last scattering surface is also modified by lensing. We discuss the lensing modification to the CMB bispectrum and show that lensing leads to an overall decrease in the amplitude of the primary bispectrum at multipoles of interest between 100 and 2000 through additional smoothing introduced by lensing. Since weak lensing is not accounted for in current estimators of the primordial non-Gaussianity parameter, the existing measurements of $f_{\rm NL}$ of the local model with WMAP out to $l_{\rm max} \sim 750$ is biased low by about 6%. For a high resolution experiment such as Planck, the lensing modification to the bispectrum must be properly included when attempting to estimate the primordial non-Gaussianity or the bias will be at the level of 30%. For Planck, weak lensing increases the minimum detectable value for the non-Gaussianity parameter of the local type $f_{\rm NL}$ to 7 from the previous estimate of about 5 without lensing. The minimum detectable value of $f_{\rm NL}$ for a cosmic variance limited experiment is also increased from less than 3 to $\sim$ 5. I. INTRODUCTION The weak lensing of cosmic microwave background (CMB) anisotropy angular power spectrum is now well understood in the literature [1,2]. The modifications result in a smoothing of the acoustic peak structure at large angular scales and an increase in power below a few arcminute angular scales corresponding to the the damping tail of CMB anisotropies [3]. The angular power spectrum of the lensing potential out to the last scattering surface can be established with quadratic estimators that probe lensing non-Gaussianity at the 4-point level of a CMB map [4]. Such a reconstruction of the lensing potential is helpful for CMB B-mode studies of polarization [5], especially in the context of searching for the signature of the primordial tensor modes [6]. This is due to the fact that in addition to inflationary gravitational waves, the B-modes of CMB polarization also contains a signal generated by lensing of scalar E-modes with a peak in power at a few arcminute angular scales [7]. The lensing reconstruction has now been applied to the existing Wilkinson Microwave Anisotropy Probe (WMAP) data leading to a ∼ 2σ to 3σ detection of gravitational lensing in the CMB through a correlation between the reconstructed lensing potential and tracers of the large-scale structure such as radio galaxies [8,9]. In parallel with the progress on lensing studies with the CMB, the search for primordial non-Gaussianity using the CMB bispectrum with constraints on the non-Gaussianity parameter f NL is now an active topic in cosmology [10,11,12]. The 5-year WMAP data is consistent with −9 < f NL < 111 at the 95% confidence level for the local model [13], though a non-zero detection of primordial non-Gaussianity at the same 95% confidence level with 26.9 < f NL < 146.7 is claimed elsewhere using the WMAP 3-year data [14]. This result, if correct, has significant cosmological implications since the expected value under standard inflationary models is f NL 1 [15,16,17,18,19,20,21], though alternative models of inflation, such as the ekpyrotic cosmology [23,24], generally predict a large primordial non-Gaussianity with f NL at few tens. Just as the CMB power spectrum is modified by lensing from potential fluctuations of the intervening large-scale structure [1], the CMB bispectrum will also be modified by gravitational lensing. The correlation between the projected lensing potential and CMB secondary effects, such as the integrated Sachs-Wolfe (ISW) effect or the Sunyaev-Zel'dovich (SZ) effect, leads to a non-Gaussian signal at the three point level [25,26]. These secondary non-Gaussianities are expected even if the primordial perturbations are Gaussian and impact existing primordial non-Gaussianity parameter measurements by introducing a small, but unavoidable, bias [27,28,29]. Beyond secondary non-Gaussianities, weak lensing by the intervening large-scale structure maps the intrinsically non-Gaussian CMB sky to a different anisotropy pattern when observed today. Thus the bispectrum one reconstructs with a CMB map, assuming it to be of the expected form at the last-scattering surface due to primordial non-Gaussian perturbations, will result in a biased estimate of the primordial non-Gaussianity parameter. The existing estimator can be modified to account for lensing modifications and to obtain a bias-free estimate of the non-Gaussianity, but at the expense of factorizability that has allowed fast computation of the bispectrum in existing analyses [30]. Since lensing modifies the anisotropy pattern by smoothing the fluctuations, a change in the minimum detectable non-Gaussianity parameter f NL for a given experiment is expected to be different from the existing values in the literature [10]. In this paper, we present a general derivation of the lensed CMB primary bispectrum and quantify above statements on the changes imposed by lensing for the detection of primordial non-Gaussianity. We find that the non-Gaussianity parameter measured with WMAP, ignoring lensing, will result in an estimate of f NL for the local model that is biased low by about 6%, when measurements are extended out to l max ∼ 750. Furthermore, with lensing, the minimum detectable level of f NL with Planck is increased by roughly 40% from less than 5 to about 7 and the cosmic variance limit of f NL is increased from 3 to 5. This paper is organized as follows: we first discuss the CMB primary bispectrum of the local type in Section II. Some basic ingredients related to the lensing calculation is presented in Section III. We derive the lensing effect on the bispectrum, under both flat-sky and all-sky formulations, in Section IV. We discuss our results and conclude with a summary in Section V. In illustrating our results we make use of the standard flat ΛCDM cosmological model consistent with WMAP with Ω b = 0.042, Ω c = 0.238, h = 0.732, n s = 0.958 and τ = 0.089. II. CMB PRIMARY BISPECTRUM The CMB temperature perturbation on the sky, Θ(n) = ∆T (n)/T , is decomposed into its multipole moments The angular power spectrum and bispectrum of CMB anisotropies are defined in the usual way, respectively, as where, for the bispectrum, we have introduced the Wigner-3j symbol (see the Appendix of Ref. [26] for some useful properties of this symbol). The CMB bispectrum is generated by a coupling of the local-type with a quadratic correction to the Newtonian curvature such that where Φ L (x) is the linear and Gaussian perturbation and f NL in the non-Gaussianity parameter, which is taken to be scale independent [10]. In Fourier space, we can decompose equation (3) as with where P Φ (k) is the linear power spectrum, defined as The multipole moments of the anisotropy can be written as where Φ(k) from above is the primordial curvature perturbation in the Fourier space, and g T l (k) is the radiation transfer function. With Φ L (k) and Φ NL , the moments can be separated into two components with Θ lm = Θ L lm + Θ N L lm . The CMB angular power spectrum can be defined using the transfer function and the power spectrum of dominant linear fluctuations as Using the definition of the angular bispectrum (equation 2), the primordial temperature anisotropy bispectrum can be written as which can be simplified to [10] and G l1l2l3 = (2l 1 + 1)(2l 2 + 1)(2l 3 + 1) 4π When illustrating our results, we make use of a modified code of CMBFAST [31] for the standard flat ΛCDM cosmological model to fully calculate radiation transfer functions when generating the CMB primary bispectrum. III. WEAK LENSING BASICS The effects of weak lensing can be encapsulated, under the Born approximation, in the radial projection of the gravitational potential (Φ), given as [32] Here we plot l 4 B θ lll /(2π) 2 as a function of the multipole l for one of the sides. We assume fNL = 1. The lensing effect can be described as a decrease in the amplitude of the bispectrum when l 1700 with an increase at higher multipoles. where r(z) is the line-of-sight comoving distance (or lookback time) to a redshift z from the observer with last scattering surface at r s = r(z = 1100), and d A (r) is the comoving angular diameter distance. In a spatially flat universe, d A → r. Here, we ignore the time-delay effect as it is small compared to the geometric lensing effect captured by equation (14) [33]. The calculation related to the CMB bispectrum described below requires the angular power spectrum of lensing potential φ, which can be decomposed into the multiple moments as with the lensing power spectrum defined using where Here F (r) describes the radial evolution of potential fluctuations. Modifications to the CMB anisotropies, generated at higher order in lensing potential fluctuations, are at the level of at most 5% relative to those due to the lensing potential angular power spectrum [34]. The bispectrum of lensing potentials, due to the non-linear evolution of density perturbations, also modifies the CMB primary bispectrum, but these changes can also be ignored since the lensing potential bispectrum is at the order C φ l 2 , while changes we describe are first order in the angular power spectrum of the lensing potential. Using the Limber approximation, equation (16) can be further simplified, but we do not make use of this approximation in numerical calculations illustrated here since the flat-sky form of the potential power spectrum is known to bias lensing results of the power spectrum by about 10% at all multipoles [3]. IV. LENSING OF THE CMB BISPECTRUM We first give a treatment of the lensing of the CMB bispectrum assuming a flat-sky approximation and discuss a derivation under the spherical sky later. A. Flat-sky Case Weak lensing deflects the path of background photons resulting in a remapping of the observed anisotropy pattern on the sky. Following an approach similar to [3], we write the lensed temperature anisotropy as Here, Θ(n) is the unlensed CMB temperature anisotropy, Θ(n) is the lensed anisotropy, and ∇φ(n) is the lensing deflection angle for the CMB photons. Taking the Fourier transform, as appropriate for a flat-sky, we write the lensed temperature anisotropy in Fourier space as where to the second order in lensing potential in the perturbative expansion. The observed angular power spectrum of CMB anisotropies under weak lensing is discussed in [3]. The resulting power spectrum consists of both the unlensed intensity and a perturbative correction related to the lensing effect. Making use of the expansion and after The left panel is for l2 = 10 and right panel is l2 = 100. We vary l = l1 with l3 = l + l2 in both cases and plot l 4 B l,l 2 ,l+l 2 /(2π) 2 as a function of l. Again, we take fNL = 1. In these configurations, the lensing effect can be described as an overall decrease in the amplitude of the bispectrum when l 1200. This suggests that lensing by the intervening large-scale structure leads to a less non-Gaussianity in the CMB map. some straight forward calculations, we obtain the lensed anisotropy power spectrum as Here, R describes the variance of the deflection angle. For ΛCDM cosmology, θ rms = √ R ∼ 2.6 ′ . This derivation makes use of the flat-sky approximation to describe the lensing effect on CMB anisotropy power spectrum. When the expressions derived in the previous section for C Θ l and C φ l under the exact spherical-sky treatment are used in equation (21), the lensed CMB power spectrum can be derived with a less bias than using, say, the flat-sky result for C φ l in the same expression [3]. Keeping the flat-sky approximation, we can define the angular bispectrum as and following the approach similar to the lensed angular power spectrum that led to equation (21), the lensed bispectrum can be expressed as Note that we have identified the flat-sky bispectrum as B Θ (l1,l2,l3) to distinguish from the all-sky bispectrum B Θ l1,l2,l3 . The two are related through B. All-sky Treatment The derivation related to the lensing of the CMB bispectrum under the more appropriate spherical sky can be obtained by replacing the Fourier components with spherical harmonic multipole moments. In this case, the lensed field can be represented as [3] where, the integrals over the spherical harmonics were replaced, in the last step, by the geometrical factors Using equation (2), the lensed CMB temperature bispectrum can then be expressed as leading tõ Noting that the Wigner-3j symbol obeys the identity m1m2 l 1 l 2 l 3 m 1 m 2 m 3 we can re-write the lensed bispectrum as where , and The sum of the geometric term J Hence, with summation over m 3 leading to a factor (2l 3 + 1), the expression for S 1 simplifies to In order to evaluate S 2 , we first re-express I mm ′ m ′′ where [3] f Then the expression for S 2 can be re-written as where, in the last step, we have introduced the Wigner-6j symbol [35]. The values of the Wigner-6j symbol can be computed numerically with a fast and efficient recursive algorithm [36]. Finally, substituting the expressions for S 1 and S 2 in equation (31) and including all permutations in a single expression, we can write the lensed bispectrum as and n ≡ (l + p + q). V. RESULTS & DISCUSSION We illustrate the modification to the equilateral configurations of the bispectrum in Figure 1, where we plot l 4 B Θ lll /(2π) 2 as a function of the multipole l. The primary CMB bispectrum assumes f NL = 1 and is calculated with the full radiation transfer function g T l (k). The lensing description makes use of the all-sky treatment to calculate both C φ l and the lensed bispectrum. The flat-sky expression gives a result consistent with the all-sky expression at better than 5% at all multipoles if the all-sky expression for C φ l is used in both calculations. The difference is at the level of 10% if the two expression make use of the two separate calculations of C φ l , as in the case of the angular power spectrum [3]. In the case of the equilateral configurations of the bispectrum, the lensing effect can be best described as a smoothing and a decrease of the amplitude of non-Gaussianity power in the equilateral configuration of the bispectrum when l < 1500 and a subsequent increase in the bispectrum amplitude at small angular scales. In Figure 2, we show two squeezed configurations (l 1 ∼ l 3 ≫ l 2 ) of the bispectrum, with the short length fixed at either 10 (left panel) or 100 (right panel), as a function of the multipole l of one side with the third side fixed at either l + 10 or l + 100. Without lensing, a comparison of Figure 1 and Figure 2 reveals a wellknown result in the literature that the local type of the CMB bispectrum is dominated by squeezed configurations with one small side and two large sides for the bispectrum mode shape in the multipole space. With lensing, the amplitude of the squeezed configurations is significantly reduced when two of the sides have lengths l > 1200 in the multipole space. This can be again described as a smoothing effect with lensing by the intervening large-scale structure; removing or "washing out" the primordial non-Gaussian signature in the CMB map at angular scales below a few arcminutes. Thus, when lensed by the large-scale structure, the primordial non-Gaussian CMB sky appears more Gaussian at arcminute scales when studying the non-Gaussianity at the threepoint level. At same angular scales, however, the CMB sky appears more non-Gaussian due to lensing at the four-point level probed by the trispectrum [35]. The removal of the non-Gaussianity is associated with the squeezed configurations, which dominate the overall signal-to-noise ratio for the detection of the primary bis-pectrum without lensing. Although non-Gaussianity is reduced for the squeezed configurations, lensing leads to an increase in the amplitude of the bispectrum for equilateral configurations where l 1 ∼ l 2 ∼ l 3 > 1500. This increase, however, is insignificant in terms of the overall signal-to-noise ratio as the contribution to the cumulative signal-to-noise coming from these configurations is lower, owing to the higher variances associated with foregrounds and instrumental noise at these angular scales. To further quantify this statement, we plot, in Figure 3, the signal-to-noise ratio calculated as where the noise variance calculation involves all contributions to the angular power spectrum with C tot l = C l + C sec l + N l where we include the lensed CMB power spectrum (C l ), secondary anisotropies (C sec l ), and the noise power spectrum (N l ) for both WMAP and Planck. For secondaries, we include the SZ power spectrum calculated with the halo model [37] and make use of the noise calculations from Ref. [26] for WMAP and Planck. For the case involving an experiment limited by the cosmic variance, we set N l = 0. In the left panel of Figure 3, we plot d(S/N ) 2 /dl 3 as a function of l 3 , while in the right panel we plot the cumulative signal-to-noise ratio out to l 3 in the x-axis. In the case where lensing is not included, signal-to-noise ratio estimates for the bispectrum detection are consistent with previous calculations in the literature [10]. With lensing, however, the signal-to-noise ratios are changed. As can be seen from the left panel of Figure 3, there is an overall reduction in the signal-to-noise ratio when l 3 ∼ 1500. This difference comes from the previously described decrease in the amplitude of the non- Gaussianity in squeezed configurations of the bispectrum with lensing imposed. To further understand the differences in the signal-to-noise ratio of the lensed primary bispectrum, we plot, in Figure 4, the quantity with two separate estimates for B Θ andB Θ to estimate this quantity without and with lensing, respectively. Note that the overall signal-to-noise ratio comes from integrating this quantity over the variables l max and l min and we include the factor l max l min to account for the logarithmic scaling. A comparison of the two panels in figure 4 reveals an overall decrease in the amplitude at l ∼ 10 3 in the case with the lensed primary bispectrum relative to the primary bispectrum alone. In figure 5, we plot the difference of the two as a contour plot to show that lensing results in an overall decrease in the signal-to-noise ratio in the squeezed configurations when l max ∼ 10 3 and l min < 10 2 , while there is an increase in the signal-to-noise ratio when l max ∼ 3 × 10 3 for all values of l min . These plots demonstrates the same trends described with respect to figure 2 involving a decrease in the amplitude of the squeezed configurations of the lensed bispectrum. While there is a slight increase in the lensed bispectrum amplitude at l 3 > 2000, such small angular scales are not probed by Planck. Even in the cosmic variance limit, unfortunately, this small in-crease is insignificant given that at these same angular scales secondary anisotropies dominate the bispectrum noise variance. In terms of the cumulative signal-to-noise ratio values shown in figure 3, the minimum f NL to detect the bispectrum with Planck and a cosmic variance limited experiment is increased by about 30% to 40% from f NL ∼ 5 for Planck to f NL ∼ 7. The cosmic variancelimited detection threshold for f NL is increased from 3 to 5. While the difference in cumulative signal-to-noise ratio seems insignificant for an experiment like WMAP, weak lensing could impact existing measurements of the non-Gaussianity parameter f NL [13,14]. To understand the lensing bias introduced to f NL , we follow the discussion in Ref. [28] and note that the current estimators of the non-Gaussianity parameter usef NL =Ŝ prim N [30,38,39], withŜ where B Θ l1l2l3 is the primary bispectrum, and C pq is the covariance matrix for bispectrum measurements involving triplets of p ≡ (l 1 l 2 l 3 ) and q ≡ (l ′ 1 l ′ 2 l ′ 3 ). This estimator is the optimal estimator for non-Gaussianity measurements, but given the complications associated with estimating the covariance, existing studies make use of a sub-optimal estimator which approximates the covariance with variance C −1 αα ′ ≈ σ −2 (l 1 , l 2 , l 3 )δ αα ′ , and introduces a linear term to equation (41) to minimize the variance off NL [39]. Note that N is the overall normalization factor that can be calculated from equation (41) by re-placingB obs with B Θ . While weak lensing modifies the observed bispectrum B obs l1l2l3 = f NLB Θ l1l2l3 , existing measurements make the assumption thatB obs l1l2l3 = f NL B Θ l1l2l3 . This results in a biased estimate off NL from the true value of the non-Gaussianity parameter f true NL . The fractional difference of this bias ∆f /f NL ≡ (f true NL −f NL )/f NL can be calculated through the covariance between the lensed and unlensed CMB primary bispectrum ∆f where we have simply written the variance as σ −2 . We plot ∆f /f NL as a function of l to which non-Gaussianity parameter measurements are performed in figure 6. Existing measurements with WMAP data probe out to l max ∼ 750 and we find that existing estimates of f NL are biased by ∼ 6%. For Planck, if lensing is ignored, the bias is at the level of 30%. This bias is not the same fractional difference in the signal-to-noise ratio that one can infer from figure 3 since the fractional difference in the signal-to-noise ratio with and without lensing involves a ratio of the form ]. Note that in future an unbiased estimate of f NL can be obtained by replacing B Θ l1l2l3 in equation (41) with the lensed bispec-trumB Θ l1l2l3 and recalculating the normalization factor N with the lensed primary bispectrum. Unfortunately, while without lensing the CMB primary bispectrum of the local model factorizes into two separate integrals with b L l and b NL l (described in Section II) , this factorizability is no longer preserved when lensed and impacts an easy estimation of the non-Gaussianity parameter with the existing estimator [30]. For Planck and other CMB experiments that can probe down to small angular scales for primordial non-Gaussianity measurements, it will be necessary to implement an estimator that accounts for the lensing effect. To summarize the main results of this paper, we have discussed the primary CMB bispectrum generated at the last scattering surface, but observed today after it is weak lensed by the intervening large-scale structure. Unfortunately, as we have found, weak lensing leads to an overall decrease in the amplitude of non-Gaussianity with the biggest change on the squeezed configurations of the bispectrum that dominate the overall signal-to-noise ratio when studying the primordial non-Gaussianity parameter. For an experiment such as the Wilkinson Microwave Anisotropy Probe (WMAP), the modifications imposed by lensing results in an estimate of f NL of the local model that is biased low by about 6%. For a high resolution experiment such as Planck, the lensing modification to the bispectrum must be accounted for when attempting to estimate the primordial non-Gaussianity. The minimum detectable value of f NL for a cosmic variance limited experiment is ∼ 5.	2008-03-28T20:00:06.000Z	2008-03-28T00:00:00.000	{ "year": 2008, "sha1": "ecb84096d45ec16fc479d9e62f7b67870e026d4e", "oa_license": null, "oa_url": "http://arxiv.org/pdf/0803.4194", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "ecb84096d45ec16fc479d9e62f7b67870e026d4e", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
15379734	pes2o/s2orc	v3-fos-license	The power of indoor crowd: Indoor 3D maps from the crowd Remarkable progress was made with smartphones in the last few years. Modern smartphones are now equipped with high-resolution cameras and various micro-electrical sensors that open up new mobile application possibilities. In this work, we address a critical task of reconstruct indoor large-scale 3D model from crowd-sourced images. We propose, design, and implement IndoorCrowd, a smartphone empowered crowdsourcing system for large-scale indoor 3D scene reconstruction. IndoorCrowd fills a gap in current cloud-based 3D reconstruction systems as it ensures at mobile side that the captured image set fulfills desired quality for indoor large-scene 3D reconstruction. At the cloud side, we deploy an automated image-based 3D reconstruction pipeline, which generates 3D models from images and sensor data. Moreover, we provide an intuitive online annotation tool that allows easy image labeling. We present that these labeling information combined with sensor data helps IndoorCrowd reduce the total processing time greatly. I. INTRODUCTION Recently, several online map services provide a 3D mode that enables you to fly around cities that are rendered with amazing detail. These 3D models are constructed by a combination of aerial photography and radar technology. However, most online maps focus only on outdoor environments rather than indoor, although most of our time, we spent indoors. Unlike outdoor environment, reconstruct indoor 3D maps are much more difficult. This is due to the reason that indoor environment contains a lot of private areas that cannot be accessed by the public. Therefore, we cannot send vehicles or hiring volunteers to photograph the indoor scenes. What's more, no accurate GPS signal available inside the building, and thereby, it is difficult to acquire the geographic location for each image in indoor environment. Hence, existing outdoor 3D reconstruction techniques cannot be directly apply to indoor environment. To conquer these difficulties, we present IndoorCrowd, a smartphone empowered crowdsourcing system for large-scale indoor 3D scene reconstruction. Our approach is based on crowdsourcing, smartphone users using our mobile application to capture their preference indoor environment and upload key frames to the cloud, along with real-time sensory data and labeling information. The cloud infers photographer's location and orientations from the uploaded images combined with sensory data and produces a sparse 3D geometric representation of the scene using the state-of-the-art 3D reconstruction algorithm. Once finished, these sparse 3D points will go These dense 3D models will connect with each other based on its geographic location and form an indoor 3D maps. These crowdsourcing indoor 3D maps can be used for various applications, such as 3D visualization, indoor navigation or routing, public participation, and emergency response. Compared with several related works [1] [2], IndoorCrowd is designed based on regular smartphones without supporting from any external infrastructures and additional devices and our cloud-based architecture is capable to reconstruct largescale indoor environment. To ensure our system have the ability to handle large scale indoor environment in widely different light conditions, such as the complex reflectance, shading effects and ambient occlusions, our mobile application provides real-time feedback while user capturing the indoor environment. II. SYSTEM ARCHITECTURE OVERVIEW We adopt a three-phase solution to accomplish the whole system: initial data acquisition, 3D reconstruction pipeline, and user labeling phase. Initial Data Acquisition The first step that has to be done is the acquisition of a valid data set. In particular, IndoorCrowd adopt to the crowdsourcing approach. Therefore, we proposed a mobile application on the iOS system to let the crowd contribute their data. Fig.1 illustrates the basic working flow of the initial data acquisition phase, and the functionality of detailed components is as follows. User record video and upload key frames using our iOS app, which pairs the image with contextual data from sensors, including last available GPS, WiFi, accelerometer, and gyroscope. When user capturing the indoor environment, our iOS app also provides an useful real-time feedback based on sensor readings and image pre-processing result. 3D Reconstruction Pipeline Generation of 3D models for real-world environments from 2D images has been a longterm goal in computer vision community. Recently, large-scale 3D reconstruction from multiple images using the structure from motion (SfM) and multi-view stereo (MVS) pipeline has reached a certain level of maturity. There has been significant success with several algorithms [4]- [6]. By leveraging advanced techniques from computer vision, we choose the state-of-the-art 3D reconstruct pipeline for indoor 3D scene reconstruct. Fig. 2 illustrates the total 3D reconstruction pipeline. At the beginning of the 3D reconstruction pipeline, incremental Structure from Motion (SfM) is used to reconstruct a 3D representation model of a physical space from 2D images. The model SfM pipeline generated consists of many points, a point cloud, in 3D Euclidean space. We choose OpenMVG [7], an open source software package for incremental SfM, both for the initial model construction and later image-to-model alignment. While the SfM technique is complex, its usage is straightforward. Simply, the input is several photographs of a physical space (the minimum number is four). With these images as input, SfM operates in a pipeline: (1) extraction of the salient characteristics of a single image using SIFT [3] algorithm, (2) comparison of these characteristics across images to match shared points of reference, and (3) minimizing the projection error of the 3D key points into the cameras planes. Step (1) and (2) combined together also known as key-point matching process, step (3) refers to local bundle adjustment process. The goal of multi-view stereo pipeline is to reconstruct a complete 3D object model from a collection of images taken from known camera viewpoints, it performs an optimization on these reference points and the output is a reconstructed 3D point cloud. For processing a large number of input images, we integrates Clustering Views for Multi-view Stereo (CMVS) [6] algorithm to decomposed the input images and then use Patchbased Multi-view Stereo (PMVS) [5] algorithm to process each cluster independently and in parallel. The poisson surface reconstruction algorithm convert the output 3D point cloud into a 3D surface triangle mesh. The last step is texture mapping, where we mapping the surface texture on to our 3D model. User Labeling Phase Indoor environment contains a lot of smooth textureless objects such as walls, desktops and floors. Existing 3D reconstruction pipeline are vulnerable to these textureless objects and will produce disconnected components. To solve this problem, we provide an intuitive online annotation tool that allows easy image labeling. Then we use the object labels to fix errors in the 3D reconstruct models.	2017-02-16T02:32:49.225Z	2014-07-08T00:00:00.000	{ "year": 2014, "sha1": "982fe4c5d8ce76ac5f0b4f5305b3eb0d61dee1ee", "oa_license": "CC0", "oa_url": "https://zenodo.org/record/1273557/files/article.pdf", "oa_status": "GREEN", "pdf_src": "MergedPDFExtraction", "pdf_hash": "f8a9da938b0c12b2248e16e8689aa48b623cd8b3", "s2fieldsofstudy": [ "Computer Science", "Engineering" ], "extfieldsofstudy": [ "Computer Science" ] }
225552719	pes2o/s2orc	v3-fos-license	Experimental and Numerical Investigation of the Air Side Heat Transfer of a Finned Tubes Heat Exchanger The heat transfer was experimentally and numerically studied in this article. Characteristics of circular fins over a bent tube at different tube orientations and air velocities were investigated, and then compared with analytical results from the literature. For the experimental investigation, a simple setup was compiled inside of a wind tunnel, where the velocity and the inlet temperature of the air; the volume flow rate; and the inlet and outlet temperatures of the water were measured. Three different orientations were investigated with the set-up: the bent tube in line with the air flow with the same and opposite water inlet positions, and the bent tube perpendicular to the air flow. According to the results, the position has a significant effect on the heat transfer coefficient. A numerical study was also performed in accordance with the measurements in ANSYS-CFX computational fluid dynamics (CFD) software. The results of the CFD showed an acceptable correlation with the results of measurement; however, the results of analytical calculation from the literature show a significant discrepancy; the calculated heat performance is only about one-third of the measured values. This article presents the steps of measurement, simulation and analytical calculations, and shows solution possibilities in the calculation of the air-side heat transfer functions. Introduction Heat exchangers have been the most widely used equipment in chemical, energy and food industrial fields. The convective form of heat transfer is used in them, so several factors have significant effects on the performance. Thus, research, modeling and simulation of heat transfer processes and the search of optimization opportunities are essential for increasing performance. One large group of these devices, made up of the finned tube heat exchangers, plays important roles in several industrial processes, particularly wherein gas is one of the heat exchange media. The use of these fins with different geometries can significantly increase the heat transfer surface. The shapes, sizes and orientations of the fins can be extremley varied. Review articles present the most common cases. Basavarajappa et al. [1] and Bhuiyan and Islam [2] provide general summaries; Pongsoi et al. [3] shows up the flow characteristics and heat transfer properties of the spiral shaped fins; and Unger et al. [4] shows the same properties for oval tubes with novel fins. Chai and Tassou [5] made a very thorough summary. A large body of literature has been developed based on vortex generators with different fins. The results were summarized in two ways. On the one hand, based on representative experimental investigations; on the other hand, based on representative numerical studies, similarly to Sheikholeslami et al. [6]. It is also very important to clarify the flow conditions when studying the heat transfer process. The position of the investigated fins in the direction of air flow can result in significant differences in heat performance. Unger et al. [7] demonstrated by measurements that the greater the angle at which the fins form with the air flow, the higher the Nu Experimental Setup and Devices The investigated finned tube heat exchanger shown in Figure 1. The 80-l hot water tank heated by electricity produces and stores the hot fluid. The hot fluid was water, and its temperature was 60 • C. This hot fluid is circulated by a pump in a 3/4 hoses and flows through a rota-meter,which detects the volume flow rate. After the rotameter fluid goes through the heat exchanger. Exiting the heat exchanger, the cooled liquid returns to the water tank. The experimental setup is shown in Figure 2. The tube register was placed in a wind tunnel. A Quantum X type MX1609 thermocouple amplifier with type K thermocouples was used for data collection. Two temperatures were measured: the inlet and outlet temperatures of the heat exchanger with 5 Hz sampling rate. Data processing was done with catman ® Easy software (2006 by Hottinger Baldwin Messtechnik GmbH, Darmstad, Germany). The design and dimensions of the heat exchanger are shown in Figure 3. The tube and the fins were made of carbon steel. Later in the calculations, referring to literature and standard data, the heat conductivity was assumed to be 54 W/mK (according to EN 1993-1-2:2005, for carbon steel). The inner and outer diameters of the tube were d i = 31 mm and d o = 37.5 mm respectively; the length of the tube was L = 502.5 mm. The thickness of the fins was δ = 1.75 mm, the diameter of the fins was D = 100 mm and the space between the fins was l f = 14.65 mm. In case of finned tube heat exchangers, several heat transfer surfaces must be defined. Different dimensionless numbers depend on the ratios of these surfaces that are summarized in Table 1. The heat exchanger was placed in three different orientations relative to the air flow, which are shown in Figure 4. Because the air flows perpendicularly to the water in all positions, the cases have been distinguished on the basis of the resulting temperature difference. This difference will be the highest in case of position A and the lowest in case of position B. Measurements As presented in the abstract, the heat exchanger was investigated in three different positions. The volume flow rate of the hot fluid was changed between 100 and 450 L/h (which means fluid velocities of 0.037 m/s and 0.166 m/s respectively), while the velocity of air was changed between 3.3 and 10 m/s. The volume of the tank was large enough not to affect the measurements. In all cases, steady state was expected before the measurements begun. As a result, the smallest possible change in temperature was observed during the study period. The uncertainty in the calculations was based on the standard deviation, and its formula is where T 1 , T 2 , . . . , T N are the measured values, T is the mean value of those measurements and N is the number of investigated datums. The calculation procedure was as follows: from the measured data (temperatures, volume flow rate), the surface of the heat exchanger and the specific heat at the medium temperature, the amount of heat performance can be determined by the following equation. where T w,in and T w,out are the measured inlet and outlet temperatures of the hot water,ṁ w is the mass flow rate and c p,w is the average specific heat of the water, which is the arithmetic mean of the inlet and outlet temperatures. Theṁ w can be calculated from the volume flow rate and the density. These material properties, supplemented by viscosity and thermal conductivity, vary with temperature and pressure. During the measurements, the pressure did not change; its value was 1 bar g overpressure, so the material properties only depend on the temperature, as is shown in Equation (3). where values of a are constants for water are in Table 2. Similarly to the water, the material properties of the air also change in the function of temperature and pressure. However in the wind tunnel, the pressure is ambient (0 bar g ), so the relationships are the following: where the b constants for air are The material properties calculated by Equations (3) and (4) with coefficients in Tables 2 and 3 have the following dimensions, Equations (3) and (4) were prepared by the authors based on UniSim Design (Honeywell International Inc., Charlotte, NC, USA, 2020) process flow simulator data, where the unit of the temperature is • C. Table 3. Coefficients for properties of air. In a fully developed steady state condition, it can be assumed that the heat performance given off by the water is absorbed by the air.Q where the outlet temperature of air (T a,out ) can be calculated. The outlet temperature can be neglected since a relatively large amount of air flows in the wind tunnel. The relationship between the heat performance of heat exchangers is known: where A is the total heat transfer area (from Table 1); the ∆T LOG is the logarithmic mean temperature difference (LMTD) of the inlet and outlet temperatures. The only unknown value is the overall heat transfer coefficient, which was derived from Equations (2) and (6): The aim of this present study is to compare the heat transfer coefficients from experimental, numerical and empirical relationships, and to develop a generally applicable relationship for this type of finned tube heat exchanger. Figure 5 shows the results of the measurements. In every case, the measurement took two minutes after the steady state with a 5 Hz sample rate in positions A, B and C, as shown in Figure 4. The graphs show that in positions A, when the higher temperature water comes into contact with the air first, the heat performance will be a higher than in position B. This is explained by the fact that at this position the temperature difference (LMTD), in other words the driving force, will be larger. The heat performances experienced in the cross-current case (position C) are roughly the same as in the quasi-counter current case (position A). Tables 4-6 show the measured values and the standard deviation: s T in for the inlet temperature, s T out for the outlet temperature and s ∆T for the temperature difference: The measured values (T in , T out ) in Tables 4-6 are the average temperatures during the measurements in stationary state, and the standard deviations calculated with Equation (1). According to the measured values it can be seen that the biggest temperature difference was reached in the case of position A, and the heat performance of position C is relatively close to it. Computational Domains The results of the measurement at position B were validated by performing numerical simulations. The three dimensional geometry model was imported to ANSYS-CFX (ANSYS, Inc., Canonsburg, USA). Five different computational domains were considered: tubes, fins on the first row, fins on the second row, inside medium and the outside volume. Heat conduction was considered in the first three of the domains, while forced convection in the other two cases. The material of the tubes and fins was ferritic steel; the inside medium was water; and the outside volume was air. As mentioned, the fins and the tube were modeled as solid domains, and the material properties were set up for steel. The air and the water were modeled as fluid domains, and the SST (shear stress transport) turbulence model was set up for both materials. In order to calculate heat transfer through the different computational domains, interfaces had to be set up. These interfaces are indicated by green arrows in Figure 6. Boundary Conditions The results of the measurements were used as the basis for setting the boundary conditions. In the models there are two inlet surfaces in all cases: one for the air and another for the water. It can be assumed that a parallel flow develops in a wind tunnel, so it is sufficient to specify the velocity, the temperature and the intensity value for the air inlet. An additional computational domain was added to the air inlet and outlet where the boundary conditions were applied. Depending on the Re number, a turbulent case occurs in the flow of water in the tube, but there will certainly not be a backflow; therefore, similar characteristics were used for the water inlet. For both air side and water side, the inlet boundary conditions are shown in Tables 4-6. The sizes of the outlet surfaces were the same as the inlet surfaces, and average static pressure outlet boundary conditions were set up like the outlet boundaries. The zero total pressure boundary condition was applied on both outlets. In the cases of the wall boundaries, interfaces had to be used between the connections. The 56 fins and the tube thus resulted in a total of 226 interfaces using heat flux setting. Prism-layer insertions were used in every interface. Mesh independence test was performed. Studies were performed with the presented settings, but with k-ε turbulence model. Based on that, the heat performances were even lower, so there are even higher differences (average 45%) between the measurements and the simulations. Table 7 shows the results and the differences of the measurements and the numerical simulations. It can be seen from the obtained results that the heat performances of the CFD analysis are consistently 17-22 % lower than the heat performances of the measurements. This error may be due to the fact that the fins on the measured heat exchanger were not planar, but had four protrusions on them. The fins were made from planar plates with two perpendicular cuts, which is shown in Figure 7. The simplification assumed that the geometric model would give the same results as the measurement. However, it can be seen that these four small protrusions caused a small heat transfer surface increment, and moreover, had a turbulence-increasing effect. Results From the obtained results it can be stated that the geometry of the examined fins increases the heat transfer coefficient by 19.58% on average, and thus the heat performance of the structure was different to the planar shape. VDI Theorem The usage of the fins as extended surfaces will create a higher heat transfer area and also a lower heat transfer coefficient. These fins represent a resistance in the flow, which will cause a higher velocity and degree of turbulence, which would mean a higher value of heat transfer coefficient. However the heat is not only transferred from the water to the air by convection, but also with conduction along the fins. As a result, the surface temperature of the fins has a lower value, so the higher theoretical heat transfer coefficient does not affect a higher transferred heat, because of the lower temperature difference. To take this into account, an efficiency value and a modified heat transfer coefficient must be calculated as functions of the geometry of the fins. The following relationships are available in the VDI-Heat atlas (abbrevation of the german Verein Deutscher Ingenieure) [18]. This modified heat transfer coefficient is where α m is the theoretical heat transfer coefficient without the fins (as a smooth tube), η f is the efficiency, A f is the heat transfer area of the single fins and A is the total heat transfer area. The calculation of the theoretical heat transfer coefficient is analogous to the calculations of any other types of convections: with experimental Nu-number correlation. In the case of a finned tube heat exchanger, two different cases can be extinguished: inline (9) and staggered (10) layout: −0.15 · Pr 0.33 and (9) The value of the efficiency highly depends on the shape of the fins. This study investigated the circular shape, so the correlation related to this type. First of all, a geometric parameter must be calculated, which depends on the diameter of the tube (d o ) and the diameter of the fin (D): The following X parameter is used to express the disadvantageous effect of the fins on the heat transfer coefficient, which contains the heat transfer coefficient over the smooth tube; the heat conductivity and thickness of the fins; and the previously calculated ϕ parameter. The efficiency can be calculated with Equation (13) and Figure 8 shows its graphic definition. According Equation (13) and Figure 8, the value of the overall heat transfer coefficient for the finned tube heat exchanger is Table 8 summarizes the results of the measurements and the analytical calculations. It is clearly seen from the table that the heat transfer coefficients calculated with the method chosen from the literature are significantly lower than the measured values. Schmidt theorem There is another empirical calculation method to calculate the heat transfer coefficient and the heat performance. According to Schmidt [19], the easiest way to calculate the dimensionless numbers is to calculate them with the hydraulic diameter of the device. This effective diameter depends on the outer diameter of the tube and the quotient of the area of the finned tube and the area of the bare tube: The necessary dimensionless numbers, the Re and Nu numbers, are calculated with this hydraulic length. The empirical Nu number definition is where the C F constant is 0.30 for inline and 0.45 for staggered layout. Applying Equations (15)- (18) to the investigated cases, Table 9 shows the results for position A and position B, the inline layouts. The results show that with this theorem it is possible to determine the expected heat performances more accurately than with the VDI correlations. From the data shown in Table 9, it can be seen that with this method the calculated performances are overestimated the measured values, and these differences are between +2.06 and −13.94%. Applying the least squares regression method to the measured data, the C constant and the exponent of Re number were searched in the form of Equation (18). The proposed equation for this case should be Nu = 29.5956 · Re 0.2371 and Table 10 shows the values calculated with this equation. It is seen from Table 10 that the calculated heat transfer coefficient (k) was the same in both positions. This can be explained by the fact that for these calculated values with the same empirical correlation (Equation (19)), the difference in heat performance is due to the logarithmic mean temperature difference. The heat performances and heat transfer coefficients for position C calculated with the original Schmidt correlation (Equation (18)) are shown in Table 11. The differences in Table 11 show that the Schmidt relation cannot be applied to the real cross current either; therefore, the least squares method has been applied to this case as well. The result of this method is given in Equation (20) and the calculated heat performances with this is shown in Table 12. Table 12 shows that the heat performances calculated with the developed Nu correlation become much more accurate than with the original correlation. Conclusions In this study, the heat transfer performances of circular finned tube banks subject to different settlements were demonstrated. Based on the foregoing discussion, the following conclusions are made: (1) Even in the case of relatively small geometric sizes, the flow directions are obviously important for the heat performance. In the case of the finned tube shown in the experiments the countercurrent and cross-current connections are recommended. (2) The results of the numerical simulations correlate quite well with the experimental results. However it has been proven that the irregularities and protrusions on the experimental equipment are significant to the final heat performance. Those effected the increased value of performance by an average of 19.58%, which could result in significant material savings in an optimization task. (3) The calculation method in VDI Heat Atlas can be used in many ways and is close to reality if the heat transfer coefficient inside the tube is orders of magnitude higher than the heat transfer coefficient on the air side. In this present study, a transient flow developed inside the tube, which causes only an order of magnitude larger coefficient than the air side, so it has a greater effect on the overall heat transfer coefficient.	2020-07-09T09:10:59.936Z	2020-07-01T00:00:00.000	{ "year": 2020, "sha1": "0143d6e60c6fb4c725c10b4a3a169b236dcb5c66", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2227-9717/8/7/773/pdf", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "6e65888e89dc7a3fdecd4e39dacbb8712832af2e", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Materials Science" ] }
119294451	pes2o/s2orc	v3-fos-license	Optical cavity for enhanced parametric four-wave mixing in rubidium We demonstrate the implementation of a ring cavity to enhance the efficiency of parametric four-wave mixing in rubidium. Using an input coupler with 95% reflectance, a finesse of 19.6$\pm$0.5 is achieved with a rubidium cell inside. This increases the circulating intensity by a factor of 5.6$\pm$0.5, and through two-photon excitation on the $5s_{1/2}\rightarrow5d_{5/2}$ transition with a single excitation laser, up to 1.9$\pm$0.3 mW of power at 420 nm is generated, 50 times what was previously generated with this scheme. The dependence of the output on Rb density and input power has been explored, suggesting the process may be approaching saturation. The blue output of the cavity also shows greatly improved spatial quality, combining to make this a promising source of 420 nm light for future experiments. I. INTRODUCTION Four-wave mixing continues to be an active area of research, with applications in a number of different contexts and materials. Currently, four-wave mixing is being pursued in applications including quantum information [1,2], Rydberg States [3][4][5], single-photon sources [6], and optical gyroscopes [7]. It has been examined in a wide range of contexts, including hollow core fibers [8], cold atoms [9] and thermal vapors of rubidium and cesium [10,11]. One of the main applications of four-wave mixing continues to be frequency up-conversion in rubidium, both using a two-step excitation process [12][13][14][15], and using a single laser for two-photon excitation [16,17]. Extensive work has already been done to better understand the linewidth and frequency characteristics of the resulting light [18][19][20]. Recently the infrared light generated has also been examined [21,22], and this process has been used to investigate the transfer of angular momentum [23,24]. Several improvements have been made to the two-step process, including optical pumping [25], optimizing excitation frequency [14], high input powers [22], and using a cavity for the light produced [26], leading to output powers on the order of a mW. Meanwhile, the two-photon process has been limited to either pulsed systems or low output powers. In this paper we demonstrate the implementation of a ring cavity to dramatically increase the power generated at 420 nm for parametric four-wave mixing in rubidium using a single laser excitation scheme. Using a low-finesse build up cavity for the 778 nm excitation light, the circulating intensity was increased by a factor of 5.6±0.5. This led to an increase in blue power by over two orders of magnitude, with a maximum power achieved of 1.9±0.3 mW, comparable to the highest output powers achieved in the two-step process. We have investigated the scal- * erik.brekke@snc.edu ing of the output power with cell temperature and input intensity, as well as investigated the optimal parameters for the cavity. This technique presents a simple and attractive method for the generation of tunable far infrared light and blue light near the 5s 1/2 → 6p 3/2 transition in rubidium. II. EXPERIMENTAL SETUP AND CAVITY DESIGN Our experimental setup is schematically illustrated in Fig. 1. A single ECDL at 778 nm excites the two photon 5s 1/2 → 5d 5/2 transition in rubidium. The frequency control and tapered amplifier system have been described previously [16]. Amplified spontaneous emission and four-wave mixing in rubidium result in generated beams at 5.23 µm and 420 nm, with the relevant energy levels shown in Fig. 2. Here, we introduce a ring cavity surrounding a heated rubidium cell, to increase the circulating intensity and dramatically increase the power generated in the non-linear four-wave mixing process. The ring cavity is designed to focus the beam to a small waist inside a heated rubidium cell. Typical Pyrex cells have transmissions in the 80-90%, limiting the possible finesse of the cavity. A 95% reflectance input coupler was used to increase the circulating intensity in the cavity, with the other three mirrors reflecting more than 99.5% at 778 nm. The output coupler has more than 85% transmission at 420 nm. The total circulating intensity inside the cell at maximum is given by Where I c is the circulating intensity, I 0 is the incident intensity, R is the reflectivity of the input coupler, T c is the transmission through the cell, with the reflectivity of the other mirrors approximated as 100%. The transmission through our cell was 86%, and a plot of the maximum circulating intensity vs input coupler reflectance is shown in Fig. 3. The current work was done with an input reflectance of 95%, giving a theoretical intensity gain of 5.2. Further optimization should be possible with an even lower reflectance at the optimal value of 86%. To measure the finesse and circulating power in the cavity, a photodiode was used to examine the reflectance off the input coupler, which is combined with the transmission of the circulating light, as shown in Fig. 1. As the piezo voltage is varied, certain cavity lengths give constructive interference in the cavity. The cavity had a finesse of 19.6±0.5, and gave a circulating power of 5.6±0.5 times the original, consistent with theoretical expectations. The cavity is designed to have two waists, one between the plane mirrors and one between the R=10 cm spherical mirrors. A lens with focal length 40 cm was used to couple the light into the cavity more effectively. The cavity results in a waist of 88 µm between the plane mirrors and 22 µm between the spherical mirrors. With 1.5 W input power, and the circulating intensity being 5.6 times that at 8.4 W, the maximum intensity in the Rb cell is 1.1 × 10 10 W m 2 . III. RESULTS AND ANALYSIS It is expected that the gain for the four-wave mixing process would go as the number of atoms in the 5d 5/2 state, giving exponential dependence of the blue power on this population. If the process is far from saturation, we also expect exponential dependence of the blue power on the excitation intensity. We have measured the blue power generated as a function of the input power, with the data shown in Fig. 4. At low input powers, this dependence is consistent with exponential gain, but at high powers it trends toward a linear dependence. This could be an indication that the process is approaching a saturation point, or that competing processes are becoming significant. Changing the temperature of the cell allows control of the density of rubidium atoms, and allows further exploration of the possible onset of saturation. Figure 5 shows the blue power as a function of the Rb density. The density is limited, corresponding to temperatures under 180 • C, in order to prevent alkali reaction with the cell walls. Here, the growth in blue power at high densities is clearly suppressed. It has previously been observed that at higher temperatures competing processes may limit the successful production of blue light [16]. Both the intensity and temperature dependence suggest that we are approaching the saturation point of the four-wave mixing process. Eventually, the process would At low input powers, the expected exponential dependence is seen, with the dependence at higher densities trending toward linear. The lower data points are fit to an exponential curve to help illustrate this change in dependence. be limited when the two-photon excitation rate through the 5p level, Ω 5s→5p→5d , is equal to the two-photon excitation rate through the 6p level, Ω (2) 5s→6p→5d [27]. At this point the output power would only scale linearly with the input power. Further investigation into the cause of saturation of the system remains an intriguing area of research. Through the use of this build-up cavity, the 420 nm output power reaches as high as 1.9±0.3mW, comparable to the highest powers achieved with the two-step process [22,26]. This output power is 50 times the power achievable without the cavity. Though there are signs of saturation, further gains could be made through increased input power, cavity reflectivity optimization, or using an additional cavity for the blue output [26]. In addition to achieving high output powers, the ring cavity is also beneficial in enhancing the spatial quality of the generated beams. The tapered amplifier gives a non-gaussian beam with M 2 ≈ 2, whose profile is shown in Fig. 6a. FIG. 6. a)Profile of the excitation beam after the tapered amplifier. b) Profile of the blue beam output from the ring cavity. Without the use of the ring cavity, the generated 420 nm beam profile is also non-Gaussian. As was pointed out in [22], blocking the edges of the input beam with an iris gives a ≈ 10% increase in the generated blue power. A full explanation of this effect is still needed, but it may be that the outside portion of the beam generates regions where four-wave mixing cannot occur, but do generate counter-propagating, competing ASE. When the ring cavity is employed, the profile of the generated blue beam becomes an almost perfectly Gaussian beam, as shown in Fig. 6b. In addition, the iris no longer gives any appreciable gain in power, implying the cavity has already eliminated any outside portions of the beam not belonging to the Gaussian mode. Thus, in addition to generating much higher powers, the output of the ring cavity is also of higher spatial quality for coupling into an optical fiber or for use in future experiments. IV. DISCUSSION AND CONCLUSIONS The presence of the ring cavity provides significant power gains and an excellent spatial profile while maintaining a fairly simple experimental system. There is still only one diode laser needed for the process, and the lack of temperature dependence to the phase matching criteria makes this system much easier to use than a standard frequency doubling crystal. The presence of the cavity does make it difficult to access the cell with an optical pumping beam, which has been shown to be successful elsewhere [25]. This could still be accomplished either by slighlty non-collinear alignment of the pumping beam, or by using a cavity resonant for each frequency. Even without these, however, the intensity output gained from the cavity is more than 10 times what could be expected from optical pumping. Recently it has been observed that a resonant cavity for the generated blue light increases output power and reduces the linewidth for the outgoing beam [26]. It could be a beneficial adaptation to construct a cavity which is resonant for both 778 nm and 420 nm around the rubidium cell. The current work has not investigated the linewidth properties of the generated light, but this also remains an area of interest for the future. The output power from this system is comparable to that generated in the two-step scheme using lasers at 780 and 776 nm. While the use of a cavity removes some of the simplicity of this format, it still makes use of a single excitation frequency and produces an easily controlled output frequency [20] due to the large intermediate state detuning. If a sapphire cell is implemented, the resulting beam at 5.23 µm could be used, which is expected to have hundreds of µW available. The resulting 420 nm beam is tunable around the rubidium 5s 1/2 → 6p 3/2 transition, and so presents an excellent candidate for use in future experiments where rubidium is excited to high principle quantum numbers. In conclusion, we have implemented a ring cavity to increase the circulating intensity of the two-photon excitation beam in rubidium. The resulting increase in gain for parametric four-wave mixing generates 1.9 ± 0.3 mW of light at 420 nm, more than 50 times that possible without the cavity. In addition, the generated beam is shown to have an excellent spatial profile. The density and input power dependence of this process have been investigated, suggesting the process may be approaching saturation.	2016-08-26T20:47:12.000Z	2016-08-26T00:00:00.000	{ "year": 2016, "sha1": "e2b2985ac7ee53bf28f2f293d8a2be9cceee65d1", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1608.07603", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "e2b2985ac7ee53bf28f2f293d8a2be9cceee65d1", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics", "Materials Science" ] }
6944173	pes2o/s2orc	v3-fos-license	$L^p$ Bernstein Inequalities and Inverse Theorems for RBF Approximation on $\mathbb{R}^d$ Bernstein inequalities and inverse theorems are a recent development in the theory of radial basis function(RBF) approximation. The purpose of this paper is to extend what is known by deriving $L^p$ Bernstein inequalities for RBF networks on $\mathbb{R}^d$. These inequalities involve bounding a Bessel-potential norm of an RBF network by its corresponding $L^p$ norm in terms of the separation radius associated with the network. The Bernstein inequalities will then be used to prove the corresponding inverse theorems. ed with the n twork.The Bernstein inequalities will then be used to prove the corresponding inverse theorems. Introduction When analyzing an approximation procedure, there are typically two estimates that one is interested in determining.The first is a direct theorem that gives the rate at which a function may be approximated in terms of its smoothness, and the second is an inverse estimate that guarantees a certain amount of smoothness of an approximant based on its rate of approximation.Both are equally important, and if the results match up appropriately, they can be combined to completely characterize smoothness spaces in terms of the approximation procedure.In this paper, our goal will be to prove an inverse theorem for RBF approximation.The usual way one does this is by first der ving a Bernstein inequality, since a standard technique can then be applied to prove the inverse theorem. Bernstein inequalities date back to 1912 when S.N. Bernstein proved the first inequality of this type for L ∞ norms of trigonometric polynomials, [1].A generalization can be found in [2]; this result, which is credited to Zygmund, states that any trigonometric polynomial T of degree n satisfies T (r) p ≤ n r \|\|T \|\| p for 1 ≤ p ≤ ∞.However, the first example of a Bernstein-type inequality for RBF approximants was not proved until 2001, [7].Then in 2006, Narcowich, Ward, and Wendland derived a more standard type of Bernstein inequality, [6].They proved L 2 Bernstein inequalities for approximants coming from an RBF approximation space on R d where the Fourier transform of the RBF has algebraic decay.In the same year, Mhaskar proved L p Bernstein inequalities for certain Gaussian networks on R d , [3], and lastly, in [4], Mhaskar, Narcowich, Prestin, and Ward were able to prove Bernstein inequalities in L p norms for a large class of spherical basis functions (SBFs). In this paper we will be concerned with RBF approximants to functions in L p (R d ) for 1 ≤ p ≤ ∞.The approximants will be finite linear combinations of translates of an RBF Φ, and the translates will come from a countable set X ⊂ R d .The error of this approximation, which is measured in a Sobolevtype norm, depends on both the function Φ a efine the RBF approximation space S X (Φ) by S X (Φ) = ξ∈Y a ξ Φ(• − ξ) : Y ⊂ X, #Y < ∞ ∩ L 1 (R d ). By choosing Φ and X properly, one is able t prove results about rates of approximation as well as the stability of the approximation procedure. Our goal will be to establish L p Bernstein inequalities for certain RBF approximation spaces S X (Φ), and these inequalities will take the form \|\|g\|\| L k,p ≤ Cq −k X \|\|g\|\| p , where L k,p is a Bessel-potential space.To prove this, we will use band-limited approximation with the bandwidth proportional to 1/q X .Thus 1/q X acts similarly to a Nyquist frequency, and viewing 1/q X as a frequency, we can see the connection to the classical Bernstein inequalities for trigonometric polynomials.In partic lar, bandwidth is playing the role of the degree of the polynomial from the classical inequality. The RBFs that we will be concerned with have finite smoothness; examples include the Sobolev splines and thin-plate splines.The sets X will be discrete subsets of R d with no accumulation points.For the inverse theorem, we w l additio ally require that there do not exist arbitrarily large regions with no point from X. Strategy The basic strategy that we will use is the following, which is the same as the one used in [4].Given g = band-limited approximant g σ , and we have \|\|g ratios. \|\|g\|\| L k,p ≤ \|\|g σ \|\| L k,p + \|\|a\|\| p \|\|g\|\| p \|\|g − g σ \|\| L k,p \|\|a\|\| p \|\|g\|\| p .(1) The term \|\|a\|\| p / \|\|g\|\| p will be bounded by a stability ratio R S,p that is independent of the function g.We will then need to bound the error of approximating g by band-limited functions.Combining these results with a Bernstein inequality for band-limited functions, we will be able to prove the Bernstein inequality for a functions in S Φ (X), and afterward the corresponding inverse theorems will follow easily. Notation and Formulae For any approximation procedure, one would like to determine the error of the approximation and the stability of the procedure.When considering an RBF approximation space S X (Φ), these quantities are bounded in terms of certain measurements of will typically be given in terms of the fill distance h X = sup x∈R d inf ξ∈X \|\|x − ξ\|\| 2 , which measures how far a point in R d can be will be determined by the separation radius q X = 1 2 inf ξ,ξ ′ ∈X ξ =ξ ′ \|\|ξ − ξ ′ \|\| 2 , which measures how close two points in X may be.In order to balance the rate of approximation with the stability of the procedure, approximation will typically be restricted to sets X for which h X is comparable to q X , and sets for which the mesh ratio ρ X := h X /q X is bounded by a constant will be called quasi-uniform.Many of the results in this paper will be proved by working in e following form of the Fourier transform in R d . f (ω) = 1 (2π) d/2 R d f (x)e −iω•x dx The function spaces that we will be mainly interested in are the Besselpotential spaces L k,p (R d ), which coincide with the standard Sobolev spaces W k,p (R d ) when k is a positi otential spaces are defined by L k,p = {f : f = (1 + \|\|•\|\| 2 2 ) −k/2 ĝ, g ∈ p (R d )} for 1 ≤ p ≤ , Radial Basis Functions In order to prove the Bernstein inequalities, we will need to require certain properties of the RBFs involved.As much of the work will be done in the Fourier domain, we state the constraints in terms of the RBFs' Fourier transforms.Given a radial function Φ : R d → R with (generalized) Fourier transform Φ, let φ : (0, ∞) → R be the function defined by Φ(ω) = φ(\|\|ω\|\| 2 ).We will say a function Φ is admissible of order β if there exist constants C 1 , C 2 > 0 and β > d such that for all σ ≥ 1, x ≥ 1/2, and l ≤ l d 2 ) β/2 φ(σx) (l) ≤ C 2 Two particular classes of admissible functions are the Sobolev splines and the thin-plate splines.The Sobolev spline Φ of 2 K (d−β)/2 (\|\|•\|\| 2 ), where K is a modified Bessel function of the third kind.This function possesses (1 + \|\|•\|\| 2 2 ) −β/2 . For a positive integer m > d/2, the thin-plate splines of −d 2 d odd \|\|•\|\| 2m−d 2 log \|\|•\|\| 2 d even and possess the general ms Φ = C \|\|•\|\| −2m 2 . Bessel Function and Fourier Integrals A d-dimensional Fourier integral of a radial function reduces to a one dimensional integral involving a Bessel function of the first kind.In what follows, we will discuss some of the properties of these Bessel functions as well as some propositions which will be useful for boundi ) = 1 2 l Γ 2 (l/2+1 ) for l ∈ N The next proposition makes use of integration by parts in order to bound the Fourier integral of a function whose support lies outside of a neigh )/2 (αt)dt ≤ C ǫ α −n Proof.We first define a sequence of functions arising when i J (d−2)/2 (αt)dt . After integrating by parts and taki /2+1 J d/2 (αt)dt . Integrating by par J d/2+n−1 (αt) dt. We will also be need to bound Fourier integrals of functions which are identically one in a neighborhood of the origin and have compact support.The next proposition addresses this.As in the previous case the proof relies on inte /2 (αt)dt ≤ Cα −n Proof.We first define a sequence of functions arising when integ 2+n−1 (αt)dt . Sums In the proof of the Bernstein inequalities, we will show that the constants involved are independent of the number of centers.The only dependence of the constants on the set X will be in terms of the separation radius q X .A key result for showing this independence is the following proposition.It deals with bounding a sum of function values on a Cq −d−ǫ X ). Proof.We can bound the sum using the volume argument found in the proof of [10,Theorem 12.3].Following the same procedure, 1)q Y }. for each positive integer m.Now by comparing the volume of E m to the volume of a ball of radius q Y , one fi q −d−ǫ X tability One of the essential results for proving the Bernstein inequalities is a bound of a stability ratio for S X (Φ).We define the L p stability ratio R S,p associated with this colle \|g\|\| p , where g = ξ∈X a ξ Φ(• − ξ).The goal of this section is to bound the stability rati ′ −β X for some C independent of a and X, where p ′ is the conjugate exponent to p.For this section, we will assume we are working with a fixed countable set X ⊂ R d with 0 < q X < 1 and an admissible function Φ of or er β. To begin, fix Y = {ξ j } N j=1 ⊂ X and g = N j=1 a j φ(• − ξ j ).We will derive a bound for \|\|a\|\| p / \|\|g\|\| p and show the bound is independent of Y and a.The strategy for proving this is as follows.Let K be a smooth function and define Kσ (ω) = K(ω/σ).We will then consider the convoluti ).For an appropriate choice of σ, the interpolation matrix (A σ ) i,j = K σ * Φ(ξ i − ξ j ) will be invertible, and the norm of its inverse will be bounded.Then a = A −1 σ (K σ * g)\| Y and \|\|a\|\| p ≤ \|\|A −1 σ \|\| p \|\|K σ * g\| Y \|\| p . We will then be left with bounding \|\|K σ * g\| Y \|\| p in terms of q X and \|\|g\|\| p Convolution Kernel We now define the class of smooth functions with which we will convolve g.Let K 1 be the collection of Schwartz class functions K : R d → R that satisfy: (i) There is a κ : [0, ∞) → [0, ∞) such that K(ω) = κ(\|\|ω\|\| 2 ). For each K ∈ K 1 , we define the family of functions {K σ } σ≥1 by Kσ (ω) = K(ω/σ).Note that property (ii) requires each function K σ to have a Fourier transform which is 0 in a neighborhood of the origin, and as σ increases, so does this neighborhood.The convolution Φ * K σ will retain this property and allow us to obtain diagonal dominance in A σ . B fore moving on, we will need to determine certain bounds on the functions in K 1 .First we need an L ∞ bound. \|K t we will need a bound on K σ for r = \|\|x\|\| 2 > 0. Writing K σ as a Fourier integral, we see \|K by a change of variables, we have \|K refore by Proposition 1.2 \|K ng ( 2) and (3), we will prove a bound of L q norms of linear combinations of translates of K σ . Pr roof.After proving the bound in the cases q = 1 and q = ∞, the result will then follow by the Riesz-Thorin theorem (cf.[9,Chapter 5]).Fir K,d σ d + σ d/2+1−l d q (d−2)/2+l d . Simplifyi C K,d σ d ,d σ d 1 + 1 (σq) (d−2) j=1 \|h j \| \|\|K σ \|\| 1 C K \|\|h\|\| 1 Interpolation matrices The interpolation matrices (A σ ) i,j = K σ * Φ(ξ i − ξ j ) will be shown to be invertible by the following lemma.In addition, we will at the same time find a bound for the ℓ p norm of A −1 σ .Given an n × n matrix A with diagonal part D a ,K,d q d−β X (8) for 1 ≤ p ≤ ∞. Marc nkiewicz-Zygmund type inequality To finish the bound of the stability ratio we require a bound of a discrete norm by a continuous one.To accomplish this, we can use an argument similar to the proof of [5,Theorem 1].Let µ denote Lebesgue measure o = R d K σ 0 (• − y)f (y)dτ y . Then choose a compactly supported simple function H N j=1 H(ξ j )K σ 0 * f (ξ j ). Appeal y = R d T (ν, H)(y)f (y)dµ y . Now we may apply Hölders's inequality ≤ C K,d q −d/p X \|\|f \| p,µ . Stability Ratio Bound We are now in a position to prove the bound on the stability ratio for p ∈ [1, ∞].Recall X is a countable subset of R d with 0 < q X < 1, and Φ is an admissible function of order β. Theorem 2.4.Let R S,p be the stability ratio p ≤ C Φ,d q d/p ′ −β X Proof. It has been shown th d−β X \|\|K σ 0 * g\| Y \|\| p . Finally, applying the M-Z inequality and taking the infimum ov K ∈ K 1 gives the result. Band-Limited Approximation As in the previous section, X will be a fixed countable set with 0 < q X < 1, and Φ will be an admissible function of order β.At this point, we are left with bounding the two remaining terms of (1).This will require choosing band-limited functions that approximate the elements of S X (Φ) and satisfy the Bernstein inequality as well.In particular, given g ∈ S X (Φ) we need to find a band-lim −k−d/p ′ X for 1 ≤ p ≤ ∞. Since most of the work will be done in the Fourier domain, we will impose C Φ,K,d q −k X \|\|g\|\| p Approximation Analysis Now that we know the band-limited approximants to the elements of S X (Φ) satisfy the Bernstein inequality, we must bound the error of approximation in L k,p .We will begin by bounding the approximation error in L k,1 and L k,∞ and then use interpolation to obtain the result for all other values of p.In both extremal cases, this reduces to bounding the error of approximating the RBF by band-limited functions.For p = 1 this is straightforward; however, the case p ∞ is more involved. In order to simplify some expressions, we φ(t)(1 + t 2 ) β/2 . If we are to bound the error of approximating Φ by band-limited functions, we will certainly need a point-wise bound of E Φ,K,k .Let us begi β,K,d σ 1 d−β+k .(11) Next (d−2)/2 (σ 1 rt)dt . Therefo 1−β+k (σ 1 r) l d(12) With these results we are now able to bound the e Let Y = {ξ i } N i=1 be a finite subset o a\|\| p for 1 ≤ p ≤ ∞. Proof.We will show this holds when p = 1 and p = ∞, and the result will follow from the Riesz-Thorin interpolation theorem.Letting g σ 1 = g ∧ ∨ (• − ξ j ) ∞ , and using the notation Φ,K,k (• − ξ j ) ∞ Now by ( 11), (12), X ) (d−2)/2+l d . For p = 1, we have \|\|g − g σ 1 \|\| L k,1 ≤ \|\|a\|\| 1 \|\| C Φ,K,d σ 1 k−β We finish the proof by taking the infimu over K ∈ K 2 . Bernstein Inequalities and nverse Theorems In approximation theory, there are a variety of applications for Bernstein inequalities.While they are most commonly associated with the derivation of inverse theorems, they can also be useful in proving direct theorems.For example, a Bernstein inequality for multivariate polynomials is used in certain RBF approximation error estimates, cf.[10,Chapter 11].However, in this paper, we will only address the Bernstein inequalities themselves and their matching inver e theorems. With the bound of the stability ratio and the band-limited approximation estimate in hand, we are in a position to prove the Bernstein inequalities.Let X be a countable set with 0 < q X < 1, and let Φ be an admissible function f order β.Having established Bernstein inequalities for S X (Φ), we can now prove the corresponding inverse theorems.In what follows, X n will denote a sequence of countable sets in R d such that X n ⊂ X n+1 , ρ Xn = ρ n ≤ C, and 0 < h n , q n < 2 −n , and Φ will be an admissible RBF of order β.We will additionally use the notation S n = S Xn (Φ).In this situation, we define the error of approximation by E(f, S n ) Lp = inf g∈Sn \|\|f lities, we will show that if a function is well approximated by S n then it must lie in some Bessel-potential space.Proof.Let f n ∈ S n be a sequence of functions satisfying \|\|f − f n \|\| p ≤ 2c f h l n .Note that f n ∈ S m for m > n.We now have \|\|f n+1 − f n \|\| L k,p ≤ C Φ,d h n+1 q n+1 k h −k n+1 \|\|f n+1 − f n \|\| p ≤ C Φ,d h −k n+1 (\|\|f n+1 − f \|\| p + \|\|f − f n \|\| p ) ≤ C Φ,d,f h −k n+1 (h l n+1 + h l n ), and since h n < 2 −n , it follows th t \|\|f n+1 − f n \|\| L k,p ≤ C Φ,d,f 2 −(l−k)n . This shows f n is a Cauchy sequence in L k,p .Since L k,p is complete, f n converges to some function h ∈ L k,p .Since f n converges to both f and h n L p , f = h a.e., and therefore f ∈ L k,p . and they are equipped with the norm \|\|f \|\| L k,p = \|\|g\|\| p . Proposition 1 . 1 1. ([10 ion 5.6]) Theorem 4 . 1 .≤ 41 If k < β − d, 1 ≤ ≤ ∞, and g ∈ S X (Φ), then \|\|g\|\| L k,p ≤ C Φ,d q −k X \|\|g\|\| p Proof.Let g σ 1 be the previously defined approximant of g.Then \|\|g\|\| orem 3.3, we get\|\|g\|\| L k,p ≤ \|\|g σ 1 \| p \|\|a\|\| p \|\|g\|\| p ≤ C Φ,d q −k X \|\|g\|\| p + C Φ,d q C Φ,d q −k X \|\|g\|\| p . Theorem 4 . 2 . 42 Let 1 ≤ p ≤ ∞, and let f ∈ L p (R d ).If there is a constant c f > 0, independent of n, and a positive	2012-10-05T17:27:49.000Z	2012-12-01T00:00:00.000	{ "year": 2010, "sha1": "40695ee6eb1b90b0cb4eed93508d68cef35968ce", "oa_license": "elsevier-specific: oa user license", "oa_url": "https://doi.org/10.1016/j.jat.2012.09.003", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "55454195edab18f9f54b3c742fed9d0258c412ca", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
250147178	pes2o/s2orc	v3-fos-license	Comparison of Substance Use Disorder Diagnosis Rates From Electronic Health Record Data With Substance Use Disorder Prevalence Rates Reported in Surveys Across Sociodemographic Groups in the Veterans Health Administration Key Points Question Is there variation in electronic health record (EHR) diagnosis rates of substance use disorders (SUDs) across sociodemographic groups in a geographically diverse sample of veteran outpatients? Findings In a telephone-based survey study linked to EHR data, diagnosis rates of alcohol use disorder (AUD), drug use disorder, and combined SUDs were compared with the prevalence rates of these disorders as reported in surveys. The survey-based prevalence rates of each disorder exceeded the diagnosis rates in every demographic subgroup, with varying disparity across groups; disparities in AUD and SUD diagnosis rates were largest among the youngest patients and Hispanic and Latinx patients. Meaning This study suggests that existing diagnostic procedures and tools are insufficient to capture the prevalence of SUD, particularly for young patients and Hispanic and Latinx patients, who may experience the greatest consequences of SUD. Introduction Substance use disorders (SUDs) are harmful and stigmatized medical conditions. [1][2][3] Both the prevalence and treatment of SUDs vary across subgroups. Although approximately 14.5 million US residents have alcohol use disorder (AUD), its prevalence is highest among young adults aged 18 to 25 years, American Indian and Alaska Native individuals, non-Hispanic White individuals, and men. 2,4 Similarly, 8.3 million US residents have drug use disorders (DUDs), with higher prevalence among young adults aged 18 to 25 years, men, and some groups based on race and/or ethnic identity. Although effective SUD treatments can be delivered via primary and specialty care, [5][6][7] SUDs have one of the largest treatment gaps for any medical condition, 8 and given similar conditions, young adults, patients with minoritized racial and ethnic identities, and women may be less likely to receive treatments than their respective older, White, and male counterparts. [9][10][11][12][13] Ensuring the equitable identification and diagnosis of any SUD in clinical settings can help address treatment inequity. Because measures of SUD treatment quality often rely on denominators composed of patients with clinically documented diagnoses, obtaining accurate estimates of individuals with SUD and understanding whether clinical diagnoses underestimate individuals with SUD across subgroups are imperative for monitoring equity in the provision of treatment. In the US, understanding patterns of SUD diagnosis across racial and ethnic groups is particularly important given the prominence of structural racism-historically rooted and culturally reinforced societal fostering of racism and discrimination through inequitable systems (eg, criminal justice)-in responses to substance use. 14,15 Although one study has qualitatively compared the prevalence of clinically recognized AUD with national survey-based estimates across racial and ethnic groups, 12 data have not been available to directly compare prevalence across sociodemographic subgroups within the same sample, to our knowledge. The US Veterans Health Administration (VHA) is the largest integrated health care system 16 offering a critical population for examining SUD diagnoses because SUD is common among veterans, 17 improving SUD care is a priority, and the VHA-like other systems-uses care quality metrics that rely on clinically documented SUD diagnoses for quality improvement. 18 In a large, diverse sample of VHA patients, we compared clinically documented diagnosis rates of AUD, DUD, and total SUD (AUD and/or DUD) with the prevalence rates estimated by a structured, validated diagnostic assessment across demographic subgroups. Data Sources, Setting, and Sample Patients from 30 VHA health care facilities were randomly sampled and recruited from January 8, 2018, to April 30, 2019, for a telephone survey administered by trained lay interviewers with prior experience surveying veterans about substance use. Facilities were purposively selected to represent US regions and included facilities with a range of SUD diagnosis rates and patient identity characteristics. Patients with a documented outpatient encounter at one of the facilities in the year prior to data extraction were eligible if they were 18 years of age or older and able to complete the survey in English and provide a valid address and telephone number. All eligible veterans were required to provide informed consent orally via telephone to participate. Additional diagnostic electronic health record data for up to 1 year prior to the survey were extracted for all patients who provided oral consent via telephone and completed the survey. This study followed the American Association for Public Opinion Research (AAPOR) reporting guideline, incorporated AAPOR standards, 19 used AAPOR formulas to compute target response rates for telephone surveys, was approved by VHA institutional review boards at greater Los Angeles, Puget Sound, and San Interview Schedule), based on its strong performance relative to both the SCID-RV and the CIDI (eg, sensitivity and specificity range of 0.83-0.97 for current AUD), the lack of DSM-5 versions of the CIDI and SCID-RV (for research) when the study was fielded, and its relative decreased participant burden. [21][22][23][24] Per protocol, the MINI 7.0 assessed AUD among patients reporting consuming 3 or more alcoholic drinks within 3 hours on 3 or more occasions in the past 12 months. Patients reporting any lifetime use of a nonalcohol, nontobacco drug and use of that drug more than once in the past 12 months to get high, get a buzz, feel elated, or change a mood were assessed for DUD related to that drug. Patients were classified as having AUD or DUD if they scored 2 or more on the relevant MINI 7.0 section consistent with the DSM-5. Patients reporting using more than 1 drug were assessed for whichever drug was causing the biggest problems or being used the most. Patients who met criteria for any SUD (AUD, DUD, or both) were classified as having SUD. Clinically Documented Diagnosis Rates Electronic health record data were used to identify clinically documented diagnosis rates of AUD, DUD, and any SUD using International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) codes documented on the day of or in the year prior to the date of the survey for each participant. Alcohol use disorder was identified with ICD-10 codes F10.X and DUD was identified with codes F11.X to F16.X and codes F18.X to F19.X (excluding nicotine dependence), whereas any SUD was defined as the presence of a code for AUD or DUD. The ICD-10 codes included those for substance use as well as those for remission conditions. Statistical Analysis Statistical analysis was performed from January 29, 2020, to April 20, 2021. All analyses incorporated person-level weights to account for sampling design and survey nonresponse. Descriptive statistics described sample characteristics. In primary analyses, we computed point estimates and 95% CIs for both survey-based prevalence and clinically documented diagnosis rates for AUD, DUD, and SUD in each subgroup. To assess performance of clinically documented diagnoses, we also calculated point estimates and 95% CIs of the sensitivity and specificity of clinically documented diagnoses (relative to survey-based prevalence as the referent standard) for each subgroup. We compared sensitivity and specificity across categories of each sociodemographic variable using joint Wald tests. In supplemental analyses assessing disparities in recognition of SUDs, we computed point and 95% CI estimates of differences between diagnosis rates and prevalence rates for each subgroup and assessed statistical differences in the differences between subgroups (eg, whether the gap between clinical and survey-based diagnoses differed for patients reporting Black race relative to those Alcohol Use Disorder Survey-based AUD prevalence rates generally exceeded diagnosis rates across demographic subgroups (Table 2; Figure). The largest difference between the diagnosis rate and the survey-based AUD prevalence rate was for patients aged 18 to 34 years, for whom the prevalence rate was more than 3 times as high as the diagnosis rate (88 [ Drug Use Disorder The survey-based prevalence rates of DUD exceeded the diagnosis rates across most subgroups; however, the prevalence rate was lower than the diagnosis rate for patients aged 50 to 64 years (102 Difference-in-difference analyses identified little variability across gender and racial and ethnic groups (eTable 1 in the Supplement) but highlighted differences by age. Combined SUDs The survey-based prevalence rates of SUD exceeded the clinical diagnosis rates across every subgroup ( Figure; Abbreviations: AUD, alcohol use disorder; DUD, drug use disorder; NA, not applicable; SUD, substance use disorder. We observed little difference in the gap between the SUD prevalence rate and the SUD diagnosis rate across gender, but age groups differed (eTable 1 in the Supplement). Among racial and ethnic groups, only Hispanic and Latinx patients had a statistically significant difference in the gap between the prevalence rate and the diagnosis rate relative to the reference group. Post Hoc Analyses Because diagnosis may be challenging for clinicians around the diagnostic threshold, we performed post hoc analyses focusing only on patients with moderate or severe disorders identified by the survey (eTable 2 in the Supplement). The survey-based prevalence of SUD decreased owing to the increased threshold for diagnoses, and observed differences were somewhat attenuated. However, overall patterns remained, with similar differences between clinically documented diagnosis rates and survey-based prevalence rates among key subgroups (women, Hispanic and Latinx patients, and younger patients). Discussion To our knowledge, this is the first study (and first data set in which it was possible) to investigate underdiagnosis of SUD across key sociodemographic subgroups by directly comparing self-reported structured diagnostic assessments with clinically documented diagnoses within the same sample. In this large diverse sample, clinically documented diagnosis rates for AUD, DUD, and combined SUDs were generally lower than the survey-based prevalence rates (reference standard), and clinical diagnoses had low sensitivity but high specificity for identifying the underlying substance use condition in nearly all subgroups. Differences between the diagnosis rate and the prevalence rate were larger in magnitude for some key subgroups for whom substance use may be particularly important to address, particularly younger and Hispanic and Latinx patients. Although specificity of clinically documented diagnoses was high (>92%) for all conditions across all subgroups, sensitivity did not exceed 54% in any subgroup suggesting pervasive underdiagnosis across all groups and greater levels of underdiagnosis in some. The lowest sensitivities (most underdiagnosis) for AUD were observed among women, the youngest and oldest age groups, persons of other race and ethnicity, and White non-Hispanic persons. For DUD, the greatest levels of underdiagnosis were observed among the youngest age group and among persons reporting White non-Hispanic race and ethnicity. For SUD, the lowest estimates for sensitivities were for women, the youngest and oldest age groups, and those reporting Hispanic and Latinx ethnicity. These findings are concerning given that alcohol use is increasing most among women and young people, 27,28 that the opioid epidemic and increased use of stimulants are more common (although associated with fewer consequences) among persons identifying as White relative to other groups, and that Hispanic persons are at greater risk for adverse substance-related consequences than non-Hispanic persons. 2,25,29 Improvement in clinical recognition of these conditions (or better, improvement in recognition of use that increases risk for disorder) is needed, particularly for higherrisk subgroups. Conversely, in this study, persons who reported being Black or multiple race and ethnicity, as well as men, had greater correspondence between reported prevalence and clinical diagnoses (eg, higher rates of clinical recognition) across all conditions, as did Hispanic and Latinx persons for DUD. Although these findings may reflect variations in practice across clinics where different subgroups receive care (eg, women and older veterans largely receive care in women-specific and geriatric clinics, respectively, and variations exist in the distribution of racial and ethnic groups across geographic regions in the VHA), findings may also reflect clinician-specific characteristics or practices, including differential documentation resulting from fear of harming or stigmatizing patients and/or internalized biases and/or direct discrimination or racism at play among clinicians during assessment for and diagnosis of SUD. Particularly regarding patients from minoritized racial and ethnic groups, structural racism has fundamentally shaped responses to substance use in the US through differential enforcement of drug policy (eg, disproportionate arrests or incarcerations and differential requirements for mandatory substance use treatment) and resulted in differential access to care for persons dependent on their race and/or ethnicity. 30 Structural racism reinforces a cultural and interpersonal-level stigma regarding SUDs for some groups (eg, members of minoritized racial and ethnic groups) more than others. 15,[31][32][33] Although having fewer persons in minoritized racial and/or ethnic groups who were underdiagnosed may be associated with benefits via "higher-quality" care for SUDs-and it is important to recognize and treat SUDs for these patients (eg, drug overdose deaths are increasing more rapidly among Black people relative to most other racialized groups) 14,34 -such care may also reflect individual-level manifestations of stigma, discrimination, and racism in health care, which are associated with adverse outcomes, 35 Limitations This study has several limitations. First, differences between survey-based and clinically documented diagnoses may be partially due to the diagnostic criteria used, given that DSM-5 criteria may capture a higher prevalence than the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) (DSM-IV), 39 and our instrumentation did not include assessment of DSM-IV. Second, per MINI 7.0 protocol, survey assessment of AUD was limited to those reporting recent heavy drinking episodes, whereas for DUD, it was any drug use in the past year. Although it seems unlikely that someone with lower levels of consumption would meet AUD criteria, this assessment may have underestimated survey-based AUD. Third, owing to the slight lag time (approximately 2 months) between outpatient appointments indicating study eligibility and the survey, comparison measures may not reflect entirely overlapping time frames. Fourth, our sample was limited regarding nonbinary or transgender patients and some subgroups (eg, American Indian or Alaska Native persons as well as women). Especially given findings regarding greater underdiagnosis of AUD among patients of other race and ethnicity and differences identified by gender, further work should explore issues related to SUD underdiagnosis within more refined groups, some of whom (eg, women, transgender individuals, and American Indian or Alaska Native persons) are highly affected by substance use. 40,41 In addition, our sample excluded patients with limited English proficiency and patients without addresses or telephones; these patients may have different SUD prevalence and diagnosis rates than those who enrolled in our study. Similarly, our sample represents VHA patients but may be less wellrepresentative of other populations. However, although veterans are often considered at higher risk for SUDs than other populations, the prevalence of SUDs among veterans and subgroups of veterans is understudied. To our knowledge, the study findings represent the first population-based estimates of these diagnosis rates within key subgroups of veterans identified using a validated, structured assessment.	2022-07-01T06:17:39.931Z	2022-06-01T00:00:00.000	{ "year": 2022, "sha1": "29cc5fe413d7a75f285caa7cc55de0295f5e2572", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "MergedPDFExtraction", "pdf_hash": "c73f633b07bd6bb4a80b7065d95f31274649a2dd", "s2fieldsofstudy": [ "Medicine", "Sociology" ], "extfieldsofstudy": [ "Medicine" ] }
235650088	pes2o/s2orc	v3-fos-license	Single-Molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single-stranded guanine-rich (G-rich) DNA invades at a complementary duplex telomere repeat sequence, forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single-stranded DNA. Single-stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQs) in vitro and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here, we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single-stranded DNA of a TDL fold into a GQ. This study provides new insight into telomere structure and establishes a framework for the development of novel therapeutics designed to target GQs at telomeres in cancer cells. ■ INTRODUCTION Telomeres safeguard the genome by distinguishing chromosomal termini from sites of DNA lesions that would otherwise elicit an unwanted DNA damage response, resulting in chromosomal fusion, genomic instability, and often apoptosis. 1,2 The foundation of the telomere structure begins with tandem hexameric guanine-rich (G-rich) repetitive DNA (GGTTAG in humans) ∼2 to 20 kilobases in length 3,4 and terminates with a ∼50 to 300 nucleotide long single-stranded G-rich 3′ overhang. 5 Telomeres also act to buffer against the end replication problem, wherein chromosomes gradually shorten with each subsequent round of cell division. 6 Replication-dependent telomere attrition can compromise the protective function of telomeres as well as lead to a loss of genetic information if left unaddressed. 7 Therefore, continually dividing cells, including the majority of human cancers, must maintain the telomere length to support an immortal phenotype. 8,9 A majority of proliferative cell types upregulate the specialized enzyme telomerase, which reverse transcribes telomeric DNA on to chromosomal termini using an RNA template that resides within the integral telomerase RNA subunit. 10−13 However, many aggressive cancer subtypes employ a telomerase-independent mechanism for telomere maintenance termed alternative lengthening of telomeres (ALT). 14 In ALT cells, the 3′ single-stranded DNA (ssDNA) overhang of one telomere base pairs with the duplex region of another telomere, in a manner similar to early steps in homology directed repair. 15 This telomeric strand invasion event forms a displacement loop (D-loop), where the singlestranded G-rich 3′ overhang base pairs with the C-rich strand of the invaded telomere, displacing the G-rich strand. 16−18 The 3′ overhang can then be extended by a specialized DNA polymerase using the invaded telomere as a template, 19 followed by the synthesis of the C-rich strand and nucleolytic processing to maintain the 3′ overhang. 20 Single-stranded G-rich telomeric DNAs readily fold into compact structures called G-quadruplexes (GQs) in vitro, wherein guanine bases form G-quartets via both Watson− Crick and Hoogsteen base-pairing interactions to align in a plane while coordinating a monovalent cation at the center. Multiple G-quartets can in turn stack upon each other to form a GQ. 21 The stability of GQs is highly dependent on the identity of the monovalent cation, with a rank order of K + > Na + > Li + , in terms of the degree of stabilization. 22 Furthermore, small molecule ligands designed to target GQ structures elicit a phenotype in living cells, suggesting a possible regulatory role for these structures in vivo. 23 Therefore, much effort has been put forth to identify potential GQ forming sequences in the genome to expand the potential targets for these molecules to be used as therapeutics. 24 In the current study, we report results from a single-molecule mechanical assay of DNA strand invasion at human telomeres. Using a magnetic tweezers system, uninterrupted duplex telomere DNA molecules greater than seven kilobases can be manipulated in order to impart precise degrees of tension and torque to the system. Strand invasion by single-stranded DNA oligonucleotides in solution can be monitored real time as a change in the overall extension of the telomere DNA duplex target molecule. To our knowledge, this assay is the first to permit direct detection of telomeric D-loops (TDLs) at the single-molecule level. We find that conditions that disfavor GQ folding dramatically alter the properties of TDLs, suggesting a role for GQ folding within these important structures. Finally, this system provides an experimental framework for future single-molecule studies of small molecule drugs and cellular machinery that may bind and alter the GQ structure within a TDL. Single-Molecule Manipulation of Long Human Telomere DNA Molecules. The DNA molecules used in the present work consist of greater than seven kilobases of uninterrupted double-stranded telomeric DNA. The telomere DNA molecule is flanked by biotin-or digoxigenin-modified DNA linker fragments used to immobilize the DNA tether between a streptavidin-coated magnetic bead and an antidigoxigenin-coated glass slide, respectively ( Figure 1A, B). To generate these long, uninterrupted, telomere DNA tether molecules for single-molecule analysis in our magnetic tweezers microscope, we perform a controlled DNA concatenation reaction seeded on the digoxigenin linker fragment using a 576 base pair telomere DNA fragment with compatible sticky ends generated by the restriction endonuclease cleavage of the previously reported pRST5 DNA plasmid. 25 Following multiple rounds of DNA ligation, the molecule is ultimately capped by the ligation of the biotinmodified DNA linker fragment and gel purified to remove unwanted reaction side products and excess handle material ( Figures 1A and S1, see Methods for details of DNA molecule construction). The elasticity of double-stranded DNA is well described using the wormlike chain (WLC) polymer model 26 and is characterized by a bending persistence length ranging from ∼45 to 50 nanometers (nm), depending upon the ionic strength. 22,27 To test whether our telomere DNA tethers exhibit similar elastic properties, we performed force-extension analysis. Our results indicate that long double-stranded telomere DNA molecules exhibit canonical DNA elastic properties with an average persistence length of 46 ± 4 nm under the conditions of our experiments (10 mM Tris pH 7.5, 150 mM KC 2 H 3 O 2 , 0.5 mg/mL BSA) ( Figure 1C). Next, we analyzed the supercoiling response of our telomere DNA tethers by rotating the magnets held above a molecule of interest, which permits the precise introduction of positive or negative superhelical strain into the system. DNA tether extension data are collected for a variety of superhelical densities, given by the expression σ = ΔL k /L ko , where σ is the supercoiling density, ΔL k is the change in the DNA linking number (i.e., the integer number of magnet rotations), and L ko is the linking number of the DNA molecule in a topologically relaxed state (i.e., the total number of DNA base pairs in the tether divided by the number of base pairs per helical turn of the double helix) ( Figure 1D). Interestingly, we find that telomeric DNA more readily denatures in response to the applied negative superhelical strain when compared to a nontelomeric control DNA ( Figure S2), consistent with a recently reported study of force-induced denaturation of a nontelomeric GQ forming sequence. 28 Real-Time Observation of DNA Strand Invasion in Human Telomere DNA. Having characterized the physical properties of the telomere DNA tethers, we next developed a DNA topology-based assay to directly measure telomere DNA strand invasion in real time ( Figure 2A). The molecule is initially negatively supercoiled resulting in a decrease in extension. When a stretching force is applied, the negative superhelical density imparts torque on the molecule, which results in transient, local destabilization of the DNA double helix and facilitates strand invasion by a freely diffusing complementary DNA oligonucleotide from solution 29 ( Figure 2A, middle panel). In this assay, the negatively supercoiled telomere DNA tether represents a closed topological system. Therefore, the local DNA unwinding that must occur upon strand invasion induces compensatory positive supercoiling, which in turn cancels some of the preexisting negative supercoiling, resulting in a sudden increase in the DNA tether extension when held at constant force ( Figure 2B−D). In this way, many successive strand invasion events on a single telomere DNA molecule are measured in real time. To initially characterize strand invasion in our system, we monitored the properties of a 42 nucleotide long singlestranded invading DNA molecule composed of seven repeats of the G-rich telomere DNA strand sequence (Tel7G) in the presence of K + , the physiologically relevant monovalent cation. We elected to conduct strand invasion experiments at the force set point of 0.9 pico-Newtons (pN), as this amount of tension is sufficient to facilitate strand invasion on tractable timescales but is not enough to stably denature the telomere DNA tether as evidenced by the symmetric rotation-extension curves collected at this stretching force ( Figure S2). In the presence of 10 nM Tel7G, we observe discrete changes in the extension of the telomere DNA tether, with the majority of transitions resulting in an increase in the extension of the system, as expected for stable strand invasion events ( Figure 2B). Notably, we also observe reverse transitions to shorter extensions, suggesting that the invading strand may also dissociate or reorganize during the observation time. Importantly, we only observe the discrete transitions in the presence of the complementary invading strand ( Figure S3), supporting the interpretation that the cumulative increase in the telomere DNA extension over time is due to the accumulation of invading strands that result in the formation of TDLs. G-Rich Telomere DNA Exhibits Complex Dynamics during Strand Invasion. As noted above, the displaced strand upon invasion of the Tel7G oligonucleotide consists of multiple G-rich telomere DNA sequence repeats and therefore is expected to fold into a GQ structure in the presence of K + . In order to explore the possible effects of the GQ structure on the strand invasion process, we conducted the same Tel7G invasion experiment in the presence of Li + , a condition that is known to destabilize GQ folding ( Figure 2C). We noted that the kinetics of invasion is markedly faster at the same force set point of 0.9 pN and the same concentration of the invading strand (compare Figure 2B,C). One possible explanation for this observation is that the target DNA duplex is less energetically stable in the presence of Li + when compared to K + , a feature of B-form DNA that, to our knowledge, has not been biophysically characterized. To analyze this possibility, we compared the extension properties of the telomere DNA tethers as a function of superhelical density in both K + and Li + . Indeed, we find that the DNA is more readily denatured by applied torques in the presence of Li + (Figure S4), which provides an explanation for the increased rate of Tel7G invasion observed in our experiments. The effect of Li + on the torsional stability of B-form DNA is not telomere-specific, as we observe a similar behavior for a nontelomeric DNA tether studied in K + vs Li + ( Figure S4). Interestingly, when invasion was conducted in the presence of Li + , we observed a significant decrease in the prevalence of reverse steps ( Figure S5), which as described above, may be due to dissociation of invading strand and/or reorganization of the invaded DNA structure. Given the known destabilizing effect of Li + on GQ folding, this effect suggests that GQ formation may contribute to the propensity for reverse steps during telomeric DNA strand invasion. As a further test of this possibility, we next set out to investigate differences in the strand invasion dynamics observed for the complementary C-rich Tel7C oligonucleotide ( Figure 2D). Comparison of strand invasion trajectories collected for the Tel7C and Tel7G invading strands in K + reveals an obvious qualitative difference (compare Figure 2B, D), wherein the Tel7C invasion trajectories primarily consist of a stepwise monotonic increase in the observed DNA tether extension with a reduced frequency of reverse steps ( Figures 2D and S5). To analyze whether the reverse steps are mechanistically coupled to the forward steps, we analyzed the dwell time distributions for individual invasion events. Kinetic analysis was performed by measuring the waiting times (t wait ) between successive invasion events, irrespective of whether there was an intervening reverse step observed in the trajectory ( Figure 3A). Despite the qualitative differences we observe in the invasion trajectories for Tel7G and Tel7C in K + , we find both dwell time distributions to be well described by single exponential functions with similar characteristic rate constants for invasion ( Figure 3B, C). This result suggests that the forward and reverse steps in the real-time invasion trajectories are independent of each other and demonstrate that the rate constant for strand invasion is comparable for the Tel7G and Tel7C invading strands. In addition to the potential of the displaced strand in a TDL to form a GQ in the presence of K + , it is also possible that GQ formation in the Tel7G invading strand itself may occur and contribute to the increased prevalence of reverse steps. To investigate this possibility, we performed control experiments with a modified G-rich invading oligonucleotide in which the second base of alternating G-triplet sequences was replaced with a 7-deazaguanine-modified base (Tel7dG). Invading Biochemistry pubs.acs.org/biochemistry Article oligonucleotides harboring this 7-deazaguanine modification cannot form the requisite Hoogsteen hydrogen bonds required to form a stable GQ structure. 30 When using Tel7dG, we found that the frequency of reverse steps in the invasion traces was comparable to that observed with the native Tel7G sequence ( Figure S6). Thus, the observed dynamics are not dependent upon GQ formation within the invading G-rich strand. Taken together, these results support a model wherein GQ folding in the displaced strand of a TDL at least in part contributes to the increased prevalence of reverse steps observed in Tel7G strand invasion trajectories in K + conditions. However, kinetic analysis suggests that the reverse steps do not significantly impact the rate constant for the invasion and formation of the TDL structure. Structural Stability of TDLs Formed by G-Rich Strand Invasion. If the displaced strand within a TDL formed upon G-rich strand invasion folds into a GQ structure, one prediction is that the TDL will be less energetically favored to resolve because the H-bonds that have been disrupted upon strand invasion are compensated by H-bonds within a GQ fold. As noted above, the stable unwinding of the telomere DNA target during strand invasion results in a change in the overall DNA twist (i.e., the number of helical turns per unit length of the DNA molecule). Such changes in DNA twist, if structurally stable, can be directly measured as a shift in the rotation-extension curve to the left when the magnets are rotated back toward the relaxed state of the DNA molecule. 29 In contrast, if the invading strands dissociate while rewinding the molecule back toward the relaxed state (i.e., TDL resolution), one would expect to observe a rotation-extension curve that overlays the original preinvaded state. After complete invasion of a target telomere DNA tether with the Tel7G oligonucleotide (defined as the DNA tether reaching >70% of its relaxed extension), the magnets were rotated back toward the relaxed state and into the positive superhelical density regime. The overlay of multiple independent rotation-extension curves taken following Tel7G invasion reveals a significant shift in the rotation-extension curve along the x-axis ( Figure 4A). This hysteresis in the rotation-extension curve on a molecule invaded by the Tel7G strand is indicative of increased structural stability of TDLs when the physiologically relevant G-rich invading strand is used in K + . Interestingly, if the same experiment is performed with the Tel7G strand in the presence of Li + rather than K + ( Figure 4B), or with the Tel7C strand in K + (Figure 4C), the observed hysteresis in the rotation-extension curve is eliminated. Taken together, these results demonstrate that both a G-rich invading strand and GQ favoring conditions (i.e., K + ) are necessary for the increased stability of the TDL. Although our results are consistent with a role for GQ in stabilizing TDL structures formed upon Tel7G invasion, it remained a possibility that the G-rich invading strands in solution participate in intermolecular GQ formation with the displaced strand, rather than formation of an intramolecular GQ within the displaced strand of the TDL. To distinguish between these two possibilities, we again turned to the use of the modified Tel7dG oligonucleotide, which cannot form GQs but preserves Watson−Crick base pairing. 30 Analysis of DNA tethers following strand invasion by the Tel7dG strand again revealed a leftward shift of the rotation-extension curve, consistent with stable TDL formation ( Figure 4D). These data lend further support to the notion that intramolecular GQs formed within the displaced strand of TDLs structurally stabilize the invaded state. ■ DISCUSSION Magnetic tweezers (MT) force spectroscopy is a powerful tool to probe DNA mechanics. 31−33 MT-based methods have been Biochemistry pubs.acs.org/biochemistry Article applied to the study of human telomere DNA in recent years, with a focus on the propensity of this repetitive G-rich sequence (GGTTAG) n to fold into G-quadruplex (GQ) structures. 33−35 Previously published single-molecule spectroscopic analyses of telomere DNA mechanics have largely focused on the structural properties of short single-stranded (ss) model telomere DNA substrates. 33,34,36−41 In the present work, we use a MT system to interrogate the structural properties of long, uninterrupted duplex telomere DNA molecules of physiologically relevant lengths (>7 kilobases). MT methods have also previously been used to directly monitor DNA strand invasion in real-time, providing a tool to study the mechanics of this essential DNA transaction that occurs during DNA repair and recombination pathways. 29,42 Here, we have adopted this approach to study strand invasion at telomere DNA target sites, a process proposed to occur during the formation of telomere-loops (T-loops) as well as during the ALT pathway. 15−18 By using telomeric ssDNA probes of physiologically relevant lengths introduced to individual duplex telomere DNA molecules held under precisely applied degrees of superhelical strain, we detect real-time strand invasion and the formation TDLs. The ability of applied torque to a telomere DNA target molecule to facilitate strand invasion supports a previous model for the role of the telomere repeat binding factor 2 protein (TRF2), which has been shown to wrap duplex telomere DNA in a chiral fashion, resulting in the application of negative superhelical strain and promoting T-loop formation. 43,44 Interestingly, we observe complex invasion dynamics when the invasion trajectories are collected in the presence of a Grich ssDNA oligonucleotide, intended to model the G-rich 3' ssDNA tail that exists at endogenous telomere ends. The invasion dynamics are characterized by a combination of forward and reverse steps, and these reverse steps are suppressed when performing the same experiments in the presence of Li + or when using the complementary C-rich strand for invasion. It is well established that Li + has a destabilizing effect on GQ folding. 21 We also provide evidence that TDLs formed upon G-rich strand invasion in the presence of K + are more energetically stable than those when formed in the presence of Li + or with the C-rich strand. Taken together, these results lead to a model wherein the formation of a TDL upon invasion of the G-rich ssDNA tail permits the Grich displaced strand to fold into a GQ structure (Figures 5 and S7). Our finding that the kinetics of strand invasion are similar with the G-rich and C-rich invading strands suggests that GQ formation is a late step in the formation of a TDL, serving to thermodynamically stabilize the structure but not accelerate the invasion process. While it is well documented that single-stranded telomere DNA substrates fold into GQ structures in vitro, 21,45−48 the prevalence of this structure at telomeres and elsewhere within the genome has been the subject of debate. 23,49 Interestingly, a recently reported magnetic tweezers study of the promoter region of the c-kit oncogene demonstrated that negative superhelical strain can also drive the B-form to GQ structural transition. 28 The results of our mechanical analysis of TDLs suggest that the process of strand invasion at telomere DNA targets may provide an opportunity for GQ structures to fold in vivo, as has recently been reported by live cell imaging. 50,51 ■ CONCLUSIONS The system we describe in the present study provides a powerful experimental platform for future studies of strand invasion at telomere DNA targets. For example, singlemolecule studies using this system can be designed to understand the molecular mechanisms of telomere-associated proteins and enzymes known to resolve D-loop and GQ structures. 52−54 Moreover, our novel system can be employed to directly study the mechanism of GQ-binding compounds and their possible role in stabilizing TDLs. 55 Lastly, recent studies have shown that telomeres, long thought to be transcriptionally silent, are transcribed to generate long noncoding telomere repeat-containing RNA (TERRA). 56 TERRA is implicated in regulating various aspects of telomere biology and is proposed to do so through the formation of RNA loops (R-loops) at telomeres. 57 Thus, future work utilizing our novel MT-based assay will also focus on the mechanical properties of telomeric R-loops and the molecular mechanism of TERRA-mediated regulation of telomere function. Detailed materials and methods and Figures S1−S7 (PDF) Xi Long − Department of Chemistry and Biochemistry,	2021-06-27T13:23:47.030Z	2021-06-23T00:00:00.000	{ "year": 2022, "sha1": "b123e378f3cd02df608387b8d14745f352ed2625", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1101/2021.06.22.449520", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "1c45bd6cb1d2ef760397b4a4bdeca191acecb851", "s2fieldsofstudy": [ "Biology", "Engineering" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
266285564	pes2o/s2orc	v3-fos-license	One Factor at a Time and factorial experimental design for formulation of l-carnitine microcapsules to improve its manufacturability l-carnitine is an essential dietary supplement of physiological importance. Handling and manufacture of l-carnitine is difficult due to its hygroscopic nature, resulting in impairing its flow properties, as well as solid dosage form stability. The study aimed at reducing l-carnitine hygroscopicity through its encapsulation within a hydrophobic, pH-insensitive polymer. A solid in oil in oil (s/o/o) emulsion solvent evaporation technique for microencapsulation was adopted to exclude the possibility of water uptake. The polymers used were two ethyl cellulose (EC) grades with different viscosities. The chosen solvent for the polymer was acetone, and liquid paraffin was the dispersion medium in which both the drug and polymer were insoluble. Sixteen formulations were developed, and evaluated to study the formulation parameters as anti-coalescent type, mixing speed, surfactant type and polymer ratio, and viscosity grade. A “One Factor at A Time” (OFAT) design of experiment, and a factorial design were utilized. Study results revealed that successful microencapsulation occurred by using Aerosil 200 (0.1 %) as anti-coalescent, a mixing speed of 1000 rpm, and Ethocel Std 20 at a 3:1 drug-to-polymer ratio. Microcapsule formulation containing l-carnitine base, successfully compressed into tablets, showed acceptable water content, disintegration time, hardness, and dissolution. Moreover, it showed acceptable stability upon storage at 40 °C at 75 % RH for six months compared to l-carnitine tablets prepared by wet granulation. Introduction Carnitine, an amino acid derivative, is an essential cofactor of fatty acid metabolism in the heart, liver, and skeletal muscle [1,2].The natural sources of carnitine include meat, fish, and poultry [3].The plants represent a limited source of carnitine [4].The body can synthesize it [5].The physiological importance of carnitine lies in its role in transferring long-chain fatty acids as acylcarnitine esters across the mitochondrial membranes [6]. The hygroscopicity of L-carnitine rendered its formulation as solid dosage forms extremely difficult, especially during scale-up [10].The hygroscopic powder shows poor flowability and sticks to machine parts during its processing during manufacture [11,12].Among the solutions suggested was using a less hygroscopic L-carnitine salt as L-carnitine tartrate [13].Also, the use of adsorbents to adsorb moisture, thus stabilizing L-carnitine [14].Drug encapsulation can represent a barrier separating the hygroscopic drug from the moisture.Usually, a water-repellent polymer is used [15].L-carnitine was previously formulated as liposomes, and nanoparticles to control its release for 12 h [16].No trials were reported on the encapsulation of L-carnitine to reduce its hygroscopic properties. Hydrophilic water-soluble materials could be successfully micro-encapsulated using a few techniques.The techniques must avoid water inclusion, as with the s/o/o (solid in oil in oil) emulsion solvent evaporation method.This method is suitable for highly hydrophilic water-soluble drugs such as amino acids and proteins.The method involves dispersing the drug into an organic volatile solvent containing the dissolved polymer.Oil is the continuous phase, in which neither the drug nor the polymer are soluble.A low-HLB surfactant is involved.Using other emulsion solvent evaporation techniques, such as o/w, or w/o, carries the risk of loss of watersoluble drugs like L-carnitine to the external phase of o/w emulsions.Accordingly, a hydrophilic drug in an oil phase has no chance to dissolve into the external organic solvent, thus increasing its encapsulation efficiency [24,25]. The major problem facing the L-carnitine solid dosage forms manufacture is its hygroscopic nature.Atmospheric moisture adsorption by the drug impairs solid dosage form stability.Complete sealing of the drug away from moisture is an acceptable solution.Hence, the process managed the microencapsulation of L-carnitine using s/o/o emulsion solvent evaporation to ensure maximum drug encapsulation efficiency.A water-insoluble, pH-insensitive polymer was employed to achieve isolation from the surrounding moisture.The intended result was to reduce the hygroscopicity of L-carnitine and facilitate its large-scale production as a solid dosage form, as well as ensure its physical stability.Acetonitrile HPLC grade (Merck, Germany); Potassium dihydrogen phosphate, Phosphoric acid, Liquid Paraffin, Acetone and Hexane (El-Nasr Chemicals Co., Egypt). Preparation of L-Carnitine base microcapsules The emulsion solvent evaporation method was used in the preparation of L-carnitine microcapsules [26].The choice of parameter involved, namely, solvent, surfactant, polymer solvent ratio, volume of continuous phase, and temperature was based on preliminary trials. The method involved dissolving 1 g of ethylcellulose (polymer) in 25 ml of acetone.After obtaining a clear solution, the corresponding weights of the L-Carnitine base with (or without) a suitable anti-coalescent (colloidal silicon dioxide or magnesium stearate) were prepared.Then, L-carnitine (10 % w/w) was dispersed in the polymer solution.The beaker containing the polymer-drug dispersion was tightly covered with Parafilm® M and then sonicated in the ultrasonic bath at room temperature for 2 min until it attained homogeneity.The mixture was poured into 100 ml of liquid paraffin containing a predetermined concentration of sorbitan monooleate (Span 80); previously cooled to 10 ± 0.5 • C while being stirred by a mechanical stirrer at 500 rpm for 30 min.That was followed by the gradual heating of the oil in oil emulsion to a temperature of 40 ± 2 • C, while stirring for another 30 min to remove the volatile solvent.The solidified microcapsules were filtered using a mesh of size 35 μm, washed with 50 ml of n-hexane, and then filtered.That was followed by washing five consecutive times with 50 ml of n-hexane to remove the remaining liquid paraffin and drying at 50 • C for 1 h in an oven.The dried microcapsules were collected in tightly closed glass bottles and stored in a desiccator for further investigation.Fig. 1 illustrates the method of encapsulation. Optimization of formulation and process parameters The formulation and process parameters involved both the usage of OFAT (one factor at a time) design of experiments-which was suitable for studying variables involved in microencapsulation-and factorial design of experiments (Minitab® 19 Statistical Software, version 19.2020.1,Minitab, LLC, USA).Sixteen formulations were prepared in four separate stages.The best formulation promoted to the next stage.Table 1 describes the details of the study, and Table 2 shows the composition of formulations prepared by the emulsion solvent evaporation method. The effects of polymer viscosity grade and drug/polymer ratio A 2 × 4 Factorial design for microencapsulation of L-Carnitine base, using the emulsion solvent evaporation method, was adopted.The selected independent variables were the effects of polymer viscosity grade (X1) and the drug-polymer ratio (X2).Two different polymer viscosity grades of EC were used, namely Ethylcellulose N 100 (80-105 mPa s), and Ethocel Std.20P Premium (18-22 mPa s).Four drug-polymer ratios were studied, namely 4:1, 3:1, 2:1, and 1:1.The summary of the factorial design of experiments is given in Table 3.The material quantities were doubled to increase the yield of microcapsules obtained. Evaluation of microcapsules 2.4.1. Water content A Karl Fischer titration instrument (905 Titrando, Metrohm, Switzerland) determined the water content of the tested samples.After equilibration of the equipment, 1 g of microcapsules was accurately weighed and placed inside the titration chamber.Samples were dispersed at 100 rpm.The moisture content was recorded from the instrument [27].All measurements were repeated three times. Analysis of particle size by Laser Diffraction: The particle size of L-carnitine microcapsules chosen from the first stage and Lcarnitine base was examined by a Laser Diffraction Particle Size Analyzer (Shimadzu SALD-2201, Japan), which covered a range of 0.03-1000 μm.The particle size was compared to that of L-carnitine base powder.One hundred milligrams of the powder were dispersed in hexane and loaded in the quartz cell of the device.Hexane was used as a blank.The test was repeated three times.A semiconductor laser was used as a light source at a wavelength of 680 μm. Encapsulation efficiency An accurately weighed amount of the microcapsules, equivalent to 330 mg L-carnitine base, was transferred into a 50-ml glass beaker containing 25 ml of methylene chloride.The mixture was sonicated for 5 min to dissolve the ethylcellulose shell.The broken microcapsules were geometrically transferred and filtered using filter paper to discard methylene chloride and then washed using deionized water into a 100-ml volumetric flask.The volume was adjusted to 100 ml.An aliquot of 10 ml was diluted to 100 ml with deionized water and passed through a millipore membrane of 0.45 μm pore size.The assay was carried out based on an HPLC-based method according to the USP drug monograph [32], and the drug concentration was calculated on the basis of the previously constructed calibration curve. The encapsulation efficiency of different microcapsule formulations was calculated according to the following equation Eq. 1 [33]: Encapsulation efficiency (EE) = F1, F2, and F3 represented the study of the Anti-coalescent type factor.From that stage, F2 succeeded to continue.F2, F4, and F5 demonstrated the mixing speed variable in the second-stage, from which F4 showed superiority.The study of the effect of emulsifier concentration (on F4, F6, F7, F8, and F9) in the third stage resulted in the choice of F4.Actual amount of drug loaded in microcapsules Theoretical amount of drug loaded in microcapsules ×100% 1 The theoretical amount of drug loaded in microcapsules was 330 mg of L-carnitine base. Production yield The percentage production yield for each microcapsule formulation was calculated.The actual weight of microcapsules was divided by the sum of the theoretical weights of microcapsule components [34].The following equation Eq. 2 was used: Actual weight of microcapsules sumof theoretical weights of microcapsules ′ components ×100% 2 Preparation of L-carnitine base microcapsules as tablets Microcapsules prepared during the fourth stage were compressed into tablets using the direct compression method of multiunit particulate system (MUPS).The eight suggested tablet formulations contained a dose of L-carnitine microcapsules equivalent to 330 mg L-carnitine base.Microcrystalline cellulose MCC (Avicel PH 112) represented the filler, 5 % croscarmellose sodium (Ac-Di-Sol), a disintegrant, and 2 % magnesium stearate, a lubricant.The final tablet weight was adjusted to 1000 mg using Avicel PH 112, according to the mass of used microcapsules.All formulations were then compressed into tablets.Each batch consisted of 100 tablets.The compositions of suggested directly compressible tablet formulations are summarized in the following Table 4. Evaluation of L-carnitine tablet formulations Tablets were evaluated for thickness, hardness [35], friability [36], and disintegration time [37].Drug Content: Ten randomly selected tablets from each formulation were accurately weighed and then transferred into a 500-ml volumetric flask, then water was added.Each flask was shaken till the tablet disintegrated, and the volume was completed to 500 ml with water.The flasks were sonicated for 5 min.Three 10-ml aliquots of each tablet formulation were diluted to 20 ml with deionized water, sonicated, and passed through a Millipore membrane of 0.45 μm pore size.The assay was carried out using HPLC [32]. Dissolution rate: The dissolution of each tablet formulation was determined using the USP apparatus II (paddle method) in six vessels (n = 6).The dissolution medium was 900 ml of deionized water maintained at 37 ± 0.5 • C. The paddle rotation speed was set to 75 rpm.After 30 min, samples of dissolution medium were withdrawn and filtered through a 0.45 μm Millipore filter and assayed by HPLC [32]. The formulation results were compared to those of a control tablet formulation containing an L-carnitine base prepared using a wet granulation technique.It consisted of Avicel PH-101 as filler, 1.0 % of Aerosil 200 as adsorbent, 5 % povidone K30 as a binder, 5 % of Ac-Di-Sol as a disintegrant, and 2 % magnesium stearate as lubricant. Accelerated stability studies on L-carnitine base microcapsule tablets Samples: Accelerated stability studies were conducted on L-carnitine base microcapsules formulation C6, which was chosen based on tablet evaluation results.Formulation C6 was compared to the L-carnitine base control tablet (mentioned in the above section).The sample size allowed the study for six months. Accelerated stability study conditions: The accelerated stability cabinet controls were adjusted to be a temperature of 40 • C ± 2 • C and a relative humidity (RH) of 75 % ± 5 %.The study duration was six months with a testing frequency after 0, 1, 3, and 6 months [29].The container closure system used was carton boxes containing Aluminum/Triplex (PVC/PE/PVdC) blisters, each of the ten tablets. The collected tablet samples were evaluated for moisture content, tablet hardness, uniformity of weight, assay, and dissolution test. Statistical analysis The results of the formulations evaluation tests were compared based on ANOVA (one-way) test and factorial design analysis using Table 4 Compositions of directly compressible tablet formulations containing L-carnitine base microcapsules.means were equal and the alternative hypothesis that all means were different at significance level α = 0.05. Choice of microcapsule additives EC was chosen as a polymer for microencapsulation of the L-carnitine base because it was practically insoluble in water and other polar solvents.It had almost no tendency to adsorb water from humid air [38,39].Hence, it suited L-carnitine, which was very hygroscopic and required a barrier against moisture. The solvent for microencapsulation could dissolve the polymer but not L-carnitine.Acetone was the solvent of choice despite its medium dielectric constant (ε = 20.01).It slowly diffused from nascent microcapsules, leading to the gradual solidification of microcapsules [40]. The emulsifier chosen, Sorbitan monooleate (Span 80), was characterized by a low HLB value of 4.3.It had the advantage of being liquid at room temperature and of better miscibility with light liquid paraffin [38,41]. Optimization outcomes The results of the effect of an anti-coalescent type, mixing speed, and emulsifier concentration on the properties of microcapsules (F1-F9) are represented in Table 5. The use of OFAT was suitable for studying the variables associated with microencapsulation [42].The first study stage revealed that the absence of anti-coalescent in F1 resulted in high water content and poor flowability.The inclusion of magnesium stearate, a lubricant in F3, caused droplet stabilization and prevention of coalescence [40].The role of Aerosil 200 in F2 was as an adsorbent, emulsion stabilizer, glidant, and suspending agent.Its presence led to the powder's excellent flow properties.ANOVA of the above results showed a significant difference between the three formulations (p-value〈 0.05), where F2 showed a superiority in its properties.Thus, the outcome of the first stage of the study was the choice of F2, which contained Aerosil 200 as an adsorbent [43] and anti-coalescent as well. Results of particle size analysis for this formulation revealed that about 66 % of the prepared microcapsules ranged in size between 290 and 390 μm.The size distribution of formulated microcapsules compared to that of the L-carnitine base is demonstrated in Fig ( 2). The second stage-aiming to choose the optimal mixing speed during microencapsulation-utilized the chosen anti-coalescent (Aerosil 200).Table 5 revealed that by increasing the mixing speed, the water content was increased significantly (p = 0.000).Entrapment of excess air bubbles during formulation was associated with rapid mixing.Hence, the absorption of higher amounts of moisture from the entrapped air bubbles took place [44].High mixing speed (1500 rpm) resulted in the accumulation of a polymer viscous layer on the mixer shaft and its loss.Also, some L-carnitine stuck to the walls of the beaker and adsorbed water and was out of the encapsulation process.That caused a low yield for F5, which omitting it from further study.Briefly, high mixing speeds caused an increase in water content and low microcapsule yield.According to these results, F4 was chosen to continue to the next stage. Results of the third stage showed the importance of the presence of Span 80 (emulsifier) in producing uniform, non-aggregated microcapsules within a size range of 250-450 μm.The emulsifier reduced the interfacial tension of the emulsion [45,46].Higher concentrations of Span 80 worsened the flow properties, encapsulation efficiency, and water content.That was due to the increased capacity to emulsify water entrapped by the hygroscopic L-carnitine during the mixing process [47].Accordingly, F4 (with a Span concentration of 0.1 %), which showed the best evaluation results, was promoted to the next stage. Table 5 Results of evaluation of Formulations F1-F9.The fourth stage involved eight formulations based on a 2 x 4 factorial design to study the effect of polymer viscosity grade on Lcarnitine microencapsulation.The results of evaluation tests of the eight formulations are summarized in Table 6. The interaction plots and main effects of water content, angle of repose, Carr's index, Hausner's ratio vs. Polymer viscosity, and Drug: Polymer ratio of formulations F4 and F10-F16 are illustrated in Fig. 3(a-d). Results for water content were acceptable (less than 1.2 %).In formulations containing the highest drug percentage, the water content increased due to the hygroscopic nature of the drug. The observed increase in bulk and tapped densities was associated with the increase in polymer content, which was associated with a decrease in the percentage of Aerosil 200, characterized by low density [48]. As the evaluation results for all microcapsule formulations were acceptable, the eight formulations (C1-C8) were compressed into tablets and evaluated.The evaluation results are summed up in the following Table 7. As it is clear from the results, the higher the polymer percentage, the slower the disintegration [49,50].In tablets with a high percentage of L-carnitine, rapid drug dissolution happened due to its hygroscopic nature.Following dissolution, channels appeared within tablets that allowed water entry, which aided the disintegrant in its role. Concerning the dissolution test results, formulations C1, C5, and C6 were the only formulations to conform to the official requirements.The formulations released not less than 75 % of labeled content after 30 min (which meant that individual results of dissolution must be ≥ 80 % of the labeled amount).The other formulations showed that some or all of the six tested tablets released less than 80 % of labeled potency after 30 min.Thus formulations C1, C5, and C6 contained 4:1 EC N100, 4:1 EC ST 20 and 3:1 EC ST20, respectively.Although C2 and C7 containing 3:1 EC N100 and 2:1 EC ST20 showed only one tablet that failed the test, they did not conform to the dissolution test.That resulted from the high polymer percentage in these failing formulations.The ANOVA analysis of dissolution results revealed a significant difference (p = 0.000) between formulations due to EC viscosity grade, as well as the drug-topolymer ratio.C6 showed acceptable dissolution results at a higher polymer ratio when compared to C1 and C5.Hence, the stability of formulation C6 was assessed. Stability study The study involved storing formulation C6 under accelerated stability study conditions with L-carnitine base tablets prepared by wet granulation as a control.Both were tested every month for six months for water content, weight uniformity, drug assay, hardness, and dissolution.The results are listed in Table 8. Compared to L-Carnitine base tablets, C6 showed acceptable results with an overall increase in moisture content by 0.5 % within six months.Consequently, there was only a slight increase in average tablet weight over the storage period due to moisture gain.A high moisture uptake by the L-carnitine base tablets resulted in a reduction in tablet hardness, unlike C6, where hardness was not affected.The variation in drug assay and dissolution results within the storage period was acceptable for C6 [51,52]. Conclusion It was possible to reduce the hygroscopicity of the L-Carnitine base by changing it to microcapsules.Microencapsulation of Lcarnitine improved the stability of formulated tablets.Ethocel ST 20, used at a ratio of 3:1 (drug: polymer), provided a protective drug coat in the presence of 10 % colloidal silicon dioxide as anti-coalescent and 0.1 % Span 80 as surfactant.The formulated microcapsules Results represent the average of 3 recorded reading for each test. Table 1 Stages of microencapsulation of L-Carnitine base using emulsion solvent evaporation method. Table 2 Microencapsulation formulations of L-Carnitine base using emulsion solvent evaporation method. Table 3 2 × 4 Factorial design of microencapsulation of L-Carnitine base studying the effects of polymer viscosity grade and drug/polymer ratio. Minitab® 19 Statistical Software (version 19.2020.1,Minitab, LLC, USA).Statistical analysis was based on the null hypothesis that all Table 6 Results of evaluation tests of microcapsule formulations studied in stage 4. tablets had minimal moisture uptake, acceptable hardness, assay, and dissolution results under accelerated stability storage conditions.Thus, microencapsulation minimized manufacturing problems arising from the handling of the hygroscopic L-carnitine base. Results represent the average of 3 recorded reading for each test.M.M. Hegazy et al. (caption on next page) M.M. Hegazy et al. into Table 8 Results of tablet evaluation tests during the period of accelerated stability storage conditions.L-Carnitine base tablets prepared by wet granulation Formulation C6 containing L-Carnitine base microcapsules Results represent the average of 3 recorded reading for each test.	2023-12-16T16:41:16.423Z	2023-12-13T00:00:00.000	{ "year": 2023, "sha1": "f780e3e8dcf023ef5ef4aefeb6ddc3330c2a8edd", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.heliyon.2023.e23637", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "9402be03e59ed6b62d81b69eba0097f70bededb3", "s2fieldsofstudy": [ "Materials Science", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
55136510	pes2o/s2orc	v3-fos-license	Vertical profiles of cobalt and zinc in marine sediments of the Santa Rosalía mining region , Gulf of California , Mexico Core sediments that were sampled from the coastal zone of the Santa Rosalía mining region and the adjacent deeper area of the Gulf of California were analysed by an instrumental neutron activation analysis (INAA). The levels of Co, Sc and Zn were used to assess the effect of pollution from the solid wastes of a copper smelter on the sediment composition in the study area. The Co/Sc and Zn/Sc ratio vertical distributions were compared to Co/Sc and Zn/Sc ratios of the earth’s crust and to a lower layer of the sediment cores and therefore less affected by pollution. The ratios of Co/Sc and Zn/Sc in cores from a predominant pollution “hot spot” near Santa Rosalía port are very high (40-150 and 150-350, respectively), suggesting that the thickness of the polluted layer exceeded the length of the cores (75-93 cm). The values of Co/Sc and Zn/Sc ratios decrease drastically in the cores collected outside the main “hot spot”. In this case, the values obtained from core depths of 20-34 cm approached the regional coastal surface sediment background (approximately 2 and 5-10 for Co/Sc and Zn/Sc, respectivelly). The Co/Sc ratios for the sediments of the deeper and further from the coast cores are slightly variable, but at sediment depths below 15 cm they are nearly constant and approximately equal to the Co/ Sc ratio of the earth ́s crust. In the upper part of these cores (0-15 cm), the Zn/Sc ratios are higher (10-20) than the Zn/Sc ratio of the earth ́s crust, which was probably due to the additional input of incompletely mineralised biogenic particulate Zn from the photic layer of the water column. The shape of the vertical profile the Zn/Sc ratio of SR22 sediment core (depth 360 m) in front of the main pollution “hot spot” shows that the maximum levels of pollution occurred in the past, and self-purification/restoration of the natural pollutant levels in the fine deep-water sediments can be observed during recent years. Introduction The metal mining and smelting on the sea shore is quite common because of the close proximity of mineral deposits to the tectonically active areas of land-sea interfaces where ancient hydrothermal vents have been uplifted and exposed on the continental margins.The most well-known cases include copper deposits and mines along the Pacific coast of Canada (e.g., the "Brittania" mine in British Columbia), along the coast of northern Chile (near Antofagasta) and in West Greenland (Elberling et al., 2002), in addition to a Pb smelter in the East of Russia on the shore of the Sea of Japan (i.e., near Dalnegorsk in the Primorye region) (Shulkin, 1998).The dumping of mining tailings and solid smelting wastes into the adjacent sea used to be a widespread practice in the past. One of these sites where sediments are strongly contaminated is a coastal zone along the Santa Rosalía mining region on the eastern shore in the centre of the Baja California peninsula in Mexico (Fig. 1).This area displays high levels of metals that are associated with former copper mining and smelting activities that were performed for nearly a century (Wilson and Rocha, 1955;Rodríguez Figueroa, 2004).There is clear evidence of the pollution of surface sediments by such solid waste dumping (Shumilin et al., 2000;Rodríguez Figueroa, 2004, 2010); however, recent data were somewhat encouraging, as the heavy metals from highly polluted sites were shown to be mainly concentrated in the residual fraction of the surface sediments (Shumilin et al., 2011).Additionally, an analysis of the metal levels in brown seaweeds showed that most toxic metals were retained in the sediments and were not released to the water column (Choumiline et al., 2006;Rodríguez Figueroa, 2010;Rodríguez-Figueroa et al., 2010).However, various severe environmental problems are still very real.For example, while the mining and smelting activities in Santa Rosalía closed more than twenty years ago, the self-cleaning/restoration dynamics of the pollution "hot spot" have not Zn/Sc de la corteza terrestre y con las obtenidas en la capa inferior de los testigos de sedimentos menos afectados por la polución.Las relaciones de Co/Sc and Zn/Sc en los testigos de sedimentos del área con mayor polución cerca del puerto de Santa Rosalía son muy altas, oscilando desde 40 hasta 150 y de 150 hasta 350 respectivamente, sugiriendo que el espesor de la capa altamente contaminada excede la longitud de los testigos (75-93 cm).Los valores de las relaciones Co/Sc y Zn/Sc disminuyen drásticamente en los testigos muestreados fuera de la principal área de polución.En este caso, los valores obtenidos en la profundidad de 20-34 cm del testigo se aproximan a los valores regionales de fondo de las relaciones para los sedimentos superficiales costeros (cerca a 2 para Co/Sc y a 5-10 para Zn/Sc).Las relaciones Co/Sc para los sedimentos de los testigos, muestreados a mayor profundidad y distancia de la costa son ligeramente variables, pero por debajo de 15 cm son casi constantes y aproximadamente iguales a la relación de Co/Sc para la corteza terrestre.En la parte superior (0-15 cm) de estos testigos de sedimentos, las relaciones Zn/Sc son más altas (10-20) que las relaciones Zn/Sc para la corteza terrestre, debido, probablemente, al aporte adicional del Zn biogénico particulado desde la capa fótica de la columna del agua.La forma del perfil vertical de las relaciones Zn/Sc del testigo de sedimentos SR22 (360 m de profundidad) frente al área de mayor polución muestra que los niveles máximos de polución ocurrieron en el pasado, y una autopurificación/restauración de los niveles naturales de los contaminantes en los sedimentos finos profundos se puede observar durante los últimos años. yet been characterised.Cobalt and zinc are environmentally important trace metals (Smith and Carson, 1981).The concept of geochemical provinces has been considered in relation to their regional availability and their transfer through the food chain to humans (Hamilton, 1994).Both cobalt and zinc are essential micronutrients for marine phytoplankton growth.They are supplied to the ocean mainly through estuarine systems from land (Tovar-Sánchez et al., 2004).Interestingly, these two elements can replace one another metabolically in marine phytoplankton that has been cultivated under laboratory conditions (Sunda and Huntsman, 1995).Co and, to a lesser extent, Zn are significantly enriched in the marine sediments of the Gulf of Iskenderen (Turkey) relative to their average crustal abundance.This enrichment is mainly due to the input of the weathered products of basic and ultrabasic source rocks that are present on the adjacent coast and hinterland (Ergin et al., 1996).Sometimes, Co and Zn are greatly enriched in the environment, for example, in fjord sediments near a former mine in West Greenland (Elberling et al., 2002) or in the "Boleo" copper mining district on the eastern coast of the Baja California peninsula, near the town of Santa Rosalía (Wilson and Rocha, 1955;Shumilin et al., 2000;Rodríguez Figueroa, 2004). Because of natural Cu-Co-Zn mineralization, the Santa Rosalía mining region was extensively used for copper mining and smelting throughout the past century.As a result, local beach sands and coastal marine sediments are strongly contaminated by Cu, Co, Zn and other heavy metals (Shumilin et al., 2000;2005).However, the impacted zones and the penetration depth into the sedimentary column are ostensibly limited in size (Rodríguez Figueroa, 2010;Rodríguez et al., 1998;Shumilin et al., 2000aShumilin et al., , 2000b)). The objective of this study was to determine the vertical distribution of the anthropogenic contaminants at certain sites that were selected within the impacted area and a supposedly unaffected zone of the adjacent part of the Gulf of California to determine the depth at which the sedimentary column is affected by the pollution and to discover any evidence of self-cleaning processes.Our previous studies of smelting wastes, arroyo sediments, beach sands and surface marine sediments showed that there is a significant presence of Cu, Co, Mn and Zn in contaminated sedimentary materials.However, Co and Zn are especially convenient elements for accomplishing this type of environmental assessment because instrumental neutron activation analysis (INAA) for these elements is a very effective tool to analyse a considerable quantity of subsamples rapidly and inexpensively.Other contaminants, such as Cu and Mn, cannot be measured by the traditional INAA technique and require a laborious strong acid digestion to obtain solutions that can finally be measured by flame atomic absorption spectrophotometry or ICP-AES.Moreover, there have been some indications that the complete digestion of materials originating from a smelter in Santa Rosalía is difficult to achieve (Rodríguez Figueroa, 2010).To minimise any possible effects of grain size and dilution by biogenic calcium carbonate or biogenic silica on the levels of Co and Zn in the sediments, the normalisation of their absolute values with scandium was applied (Dias and Prudêncio, 1998;Grousset et al., 1995;Monna et al., 2004).Scandium belongs to a group of terrigenous indicators (i.e., Al, Cs, Ti, Li, Sc and Th) that are strongly associated with aluminosilicates in soils and sediments, and it was selected for our study because it is the only element of this group that can easily be measured by INAA in a solid matrix with high precision.Moreover, the linear regressions of the levels of many elements versus the levels of Sc in the sedimentary materials from the coastal environments of southern Baja California have displayed the best correlation coefficients when compared with Al, Ti or Fe (Rodríguez Castañeda, 2008).The Co/Sc and Zn/Sc ratio vertical profiles were prepared and taken into consideration to assess the depth of penetration of the contaminants into the marine sediments. Sampling The study area and the locations of the sampling stations are shown in Figure 1.The SR1-SR-5 and SR-10 sediment core samples were collected in April 2006 using a manual corer (length 100 cm) by scuba divers from a small plastic motorboat that was used as a platform for separating the core sediment samples. The coastal core samples were thoroughly extracted from the core and, depending on the results of visual ob- servation of the opened core directly after sampling, were separated into layers (1 cm, 2.5 cm and 5 cm thick) using a plastic knife and a ruler.Then, they were transferred with a plastic spoon into pre-cleaned polyethylene packets and stored in a freezer until processing.In the laboratory, the samples were thawed and split into subsamples before treatment.Two deep-water cores, SR22 and SR62, were sampled in 1994 by a box corer in the Gulf of California in front of the Santa Rosalía port during the "El Puma" cruise (Fig. 1) and were sliced into 2 cm thick sediment subsamples.Related data on the composition of the sediments of SR22 and SR62 cores have been described previously (Shumilin et al., 2000a;Choumiline et al., 2006). Analysis The levels of Co, Sc and Zn in the samples of finely ground and dried sediments were determined using INAA (Shumilin et al., 2000a).The samples were dried in an electrical oven at 60°C for 24 h, and homogenised in an agate mortar.Then, 20 mg subsamples, in addition to certified standard reference material IAEA-356 (polluted marine sediment), homemade Russian standard reference materials (RCC-1, SARM-7, ST, SGD, KH, RUS, TB) and the stone meteorite "Allende" were irradiated simultaneously in an experimental nuclear reactor with a flux of thermal neutrons of 2.8×10 13 n s −1 cm −2 at 150°C.After "cooling", the gamma-spectrometric measurements of the irradiated samples were conducted with a semiconductor Ge (Li) detector that was coupled to a 4096-channel high resolution pulse analyser LP-4900 ("Nokia", Finland). The elemental composition calculations of the irradiated samples were performed using statistical software that was developed ad hoc.This procedure confirmed that the precision (less than 4.6 % for Co, less than 4 % for Sc and less than 13 % for Zn) and accuracy of the determination of the concentration of the elements in the sediments were generally acceptable (Table 1).The detection limits were 0.05 mg kg -1 for Co and Sc and 10 mg kg -1 for Zn. line sediment quality value at certain layers of the core (7.5-10 cm, 12.5-15 cm, 17.5-20 cm and 22.5-25 cm), but the Zn concentrations were generally lower than the ERL guideline values. Discussion The grain size of the coastal sediments from Santa Rosalía mining region is very heterogeneous due to its specific lithology and the occurrence of anthropogenic solid waste dumping (Rodríguez Figueroa, 2010).Generally, mixtures of particles of different sizes and origins are observed on spatial and temporal scales (Rodríguez Figueroa, 2010).To minimise possible effects of grain size variability and dilution of the contaminants in the sediments by inert quartz, by biogenic calcium carbonate or silica, the concentrations of Co and Zn were normalised with Sc.The regional background levels of the Co/Sc and Zn/Sc ratios in the study area and the shapes of the Co/Sc and Zn/Sc ratios vertical profiles were useful to evaluate the depth of penetration of tailing and smelting solid waste particles into the coastal marine sediments. General information about the values of the Co/Sc and Zn/Sc ratios in the coastal and deep marine sediments in front of the Santa Rosalía mining region is presented in Table 3.The vertical profiles of Co/Sc and Zn/Sc ratios in the sediment cores of this study (cores SR1-5, SR10) as well as SR22 (from Shumilin et al., 2000a) and SR62 (from Choumiline et al., 2006) are shown in Figures 2 and 3. As shown in Table 3, high mean values of Co/Sc ratio that ranged from 52 to 106 were detected for cores SR1, SR2 and SR4, and they were much lower for cores SR3, SR5 and SR10 (a range of 1.9-2.9).The lowest, most likely background, values were found in cores SR22 and SR62, where they ranged from 1.11 to 1.26.The highest Zn/Sc ratios were also found in SR1, SR2 and SR4 cores (a range of 214-262), followed by the open-sea core SR62 (a range of 11.7-24.1)and then cores SR3, SR5 and SR10 (a range of 8.9-10.1). The observed Co/Sc ratios in the lower depths of cores SR3, SR5, SR10 and SR22 most likely corresponded to the local background conditions.Surface coastal sediments outside the study area showed slightly higher values of Co/Sc ratio that ranged from 1.6 to 6.0 (mean 3.4±1.2) along the northern margin and from 0.9 to 8.7 (mean 3.8±1.8)along the southern margin, supposedly reflecting the shore littoral transport of the contaminants from the highly polluted areas located near the town of Santa Rosalía. The shape of the vertical profile Co/Sc ratio of SR22 sediment core (depth 360 m) in front of the main pollution "hot spot" showed that the maximum levels of pollution occurred in the past (see increased Co/Sc ratio values for samples collected from about 5-15 cm subsurface depth interval in Fig. 2), and self-purification/restoration of the natural pollutant levels in the fine grained deep- tively).The Co/Sc ratios for the sediments of the deeper cores were slightly variable, but they were nearly constant and were approximately equal to the Co/Sc ratio of the earth´s crust at sediment depths below 15 cm.The Zn/Sc ratios in the same cores were higher (10-20) than the Zn/Sc ratio of the earth´s crust, which was probably due to the additional input of incompletely mineralised biogenic particulate Zn from the photic zone of the water column.Co/ Sc ratios along core SR22 showed that the maximum levels of pollution occurred in the past, and self-purification/ restoration of the natural pollutant levels in the fine deepwater sediments has occurred during recent years. water sediments has occurred during recent years (see decreasing Co/Sc values for samples collected from about <5 cm depth in Figure 2). The Zn/Sc ratios of the surficial sediments from the northern and southern margin did not exhibit the same tendency for the Co/Sc ratios as for the Zn/Sc ratios as was the case for the deeper cores SR22 and SR62.This situation could be due to the lower influence of contamination of the deep sediments in the central portion and slightly elevated contributions of accumulated biogenic particulate Zn from the water column in cores SR22 and SR62. Cobalt is not a typical contaminant found in the coastal marine sedimentary environment, as it predominantly appears at background levels (Cobelo-García and Prego, 2003).Being mainly of terrigenous origin in coastal sediments, it is usually incorporated into natural resistant aluminosilicate matrices of marine sediments, and for that reason, cobalt was proposed as an additional terrigenous indicator or normaliser to identify the anthropogenic impacts of Cu, Pb and Zn on coastal marine sediments from the continental margin adjacent to Sydney, Australia (Matthai and Birch, 2001).However, certain levels of non-detrital Co in surface sediments from the Bay of Bengal have previously been attributed to anthropogenic contamination (Selvaraj et al., 2004).In contrast, marine sediments formed in the areas of high biological productivity and driven by coastal upwellings are often depleted in Co due to the remobilisation of this element from particles in oxygen-depleted water and sedimentary environments (Brumsack, 2006). Somewhat higher Zn/Sc ratio values in open-sea sediment cores (Shumilin et al., 2000a;Choumiline et al., 2006) relative to coastal "background" cores are most likely a result of the bioaccumulation of Zn in plankton organisms with the subsequent inclusion of their debris into the sea floor sediments.Additionally, it is hard to say to what extent the nearby Guaymas Basin with its hydrothermal sources influences the Zn concentrations, as direct observations over a hydrothermal area have previously indicated elevated levels of dissolved and particulate Zn no higher than 600 m above a 2000 m deep sea floor (Tambiev and Demina, 1992). Conclusions The ratios of Co/Sc and Zn/Sc in cores from the predominant pollution "hot spot" near Santa Rosalía port were very high (40-150 and 150-350, respectively).The values of Co/Sc and Zn/Sc ratios decreased drastically in the cores that were collected outside the main "hot spot" below a core depth of 20-34 cm and approached the regional coastal surface sediment background ratios (approximately 2 and 5-10 for Co/Sc and Zn/Sc, respec- Fig. 1 . Fig. 1.-Study area and locations of the sampling sites for sediment cores in the coastal zone of the Santa Rosalía copper mining region in the Gulf of California.Fig. 1.-Área de estudio y localizaciónde los sitios de muestreo de los testigos de sedimentos en la zona costera de la región minera cuprífera de Santa Rosalía en el Golfo de California.	2018-12-13T14:26:51.798Z	2013-04-15T00:00:00.000	{ "year": 2013, "sha1": "d5cbf49f3226686770201cffbbab846af09de418", "oa_license": "CCBY", "oa_url": "https://revistas.ucm.es/index.php/JIGE/article/download/41750/39786", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "d5cbf49f3226686770201cffbbab846af09de418", "s2fieldsofstudy": [ "Environmental Science", "Geology" ], "extfieldsofstudy": [ "Geology" ] }
225172888	pes2o/s2orc	v3-fos-license	Rationale for Translational Research on Targeted Alpha Therapy in Japan —Renaissance of Radiopharmaceuticals Utilizing Astatine-211 and Actinium-225— 1 QiSS-Targeted Alpha Therapy Research, Research Center for Nuclear Physics, Osaka University, 2 Center for Instrumental Analysis, Kyoto Pharmaceutical University, 3 Nuclear Science and Engineering Center, Japan Atomic Energy Agency, 4 Department of Radiology, Osaka University Hospital, 5 Department of Nuclear Medicine and Tracer Kinetics, Graduate School of Medicine, Osaka University, 6 Institute for Radiation Sciences, Osaka University, 7 Department of Chemistry, Graduate School of Science, Osaka University, 8 Division of Biochemistry, National Institute of Health Sciences, 9 Center for Biological Safety & Research, National Institute of Health Sciences, 10 Division of Functional Imaging, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center † fam-yano@tcct.zaq.ne.jp Introduction Research and development for targeted alpha therapy (TAT) at Osaka University were adopted by 2) the quality control and GMP evaluations, 3) and the clinical requirements for first-in-human study 4) (See Fig. 1) on the Journal of Pharmaceutical and Medical Device Regulatory Science in Japanese. We wish to herewith summarize and present these four research results onto this report. The surroundings for TAT in Japan have been changed during the decade. Because ALSYMPCA (ALpharadin in SYMPtomatic Prostate CAncer) 5) reported that patients with metastatic CRPC (castration-resistant prostate cancer) treated with 223 Ra had prolonged overall survival compared to placebo. In Japan, clinical trials for CRPC using 223 Ra therapy was conducted since 2009 overcoming many problems such as legal issues 6) and this therapy has been covered by public health insurance in Japan since 2016. Then, guideline for the introduction of alphaemitting radiopharmaceuticals was developed. 6) In addition, the complete remission of metastatic prostate cancer in patients treated with 225 Ac-PSMA-617 has sparked interest among physicians, scientists, and companies in the use of targeted alpha therapy. 7) However, the most critical challenge has been the domestic availability of alpha emitters in Japan. Because the lack of a stock of parent nuclides with long half-lives, such as 233 U, 229 Th and 227 Ac makes it impossible to produce 225 Ac and 223 Ra. Therefore, our demand for application has been focused on the accelerator-based alpha emitter, as-tatine-211( 211 At). Astatine-211 is a promising alpha emitter for targeted radionuclide therapy because it has a half-life of 7.21 h, which is sufficient for manufacturing 211 At-labeled radiopharmaceuticals and accumulating them in targeted tissues after administration. 8) Non-clinical and clinical guidelines for diagnostic radiopharmaceuticals are known. Still, there is a need to establish non-clinical safety standards as well as standards suitable for beta-and alpha-emitting therapeutic radiopharmaceuticals such as the initial clinical dose. According to such high demand, based on the latest US and European guidelines, 9,10) we decided to consider regulatory science appropriate for TAT drug products. Meanwhile, when florine-18 ( 18 F) labelled fluorodeoxyglucose (FDG) was approved in Japan, two application and approval systems have been adopted; Medical Device system for regulatory approval of the synthesizer itself, and Medical Drug system for delivery from radiopharmaceutical companies (See Table 1). However, the United States and Europe only adopt the application and approval of Medical Drug system for radiopharmaceuticals. Consideration toward Safety Guidance for Targeted Alpha Therapy in Japan 1) Conventional beta-ray radiotherapy has been used On the other hand, in alpha particle radiotherapy, extremely intense liner energy is emitted, and the DNA double-strands of the tumor cells are cleaved, leading to the death of the tumor cells. The specific targeting allows it to attack only cancer cells with a significant therapeutic effect and few side effects A German research group has reported that TAT has fewer side effects and is more effective than conventional anti-tumor therapies. 7) Simultaneously, the TAT research project has also started in Japan to proceed with drug development. However, there is still no concrete international evaluation standard to conduct non-clinical studies for TAT drugs toward human clinical studies. Here, we discuss the requirements for an evaluation standard of non-clinical studies which is essentially necessary to proceed with clinical trial, while watching the current progress on the TAT drug development and the several subjects clarified by the previous TAT research. We focus on both 211 At (astatine-211) and 225 Ac (actinium-225) as the alpha-emitters. It also includes a discussion of monitoring endpoints that take into account initial human dose and dose escala-tion, identification of organs with suspected toxicity, physical half-life and stability of the drug, and accumulation in target cells. We also propose a method for evaluating TAT drug candidates that satisfy the safety profile, including delayed toxicity, using the histopathological examination. Biological effects of radiation vary depending not only on the type of radiation itself, but also on the organs and tissues. And the revealing time of disorders depends on the characteristics of each organ or tissue. So, toxicological analysis that follows changes over time should be designed. Also, delayed toxicity and its recovery can be revealed by histopathological evaluation with some functional analyses, after a reasonable long-term course (see Table 2). Stable isotope corresponding to an alpha particle can become a powerful tool to understand its distribution and toxicity. For example, 89 In order to set an initial dose and escalation study, the biodistribution study with positron or single photon nuclides corresponding to alpha particles (PET or SPECT study) is adopted. We expect that our new evaluation system can provide novel strategy for safety and dose escalation and verification issues for the development of new Fig. 3). Microdosimetric models for TAT 2) Establishment of a reliable dosimetry protocol is a key issue in the design of TAT. The final goal of the dosimetry study is to evaluate the spatial and stochastic distributions of the absorbed doses that can predict likely toxicity and tumor response to treatment. The property of alpha particle is its short range and high linear energy transfer (LET). It has a range of 10-100 micrometers, which is only about 2-10 cells distance. Higher LET particles can effectively induce the DNA damages and lead to apoptosis of the cells. 225 Ac does not emit gamma rays. Thus, it is difficult to perform quantitative SPECT imaging for biodistribution study 11) . Meanwhile, 211 At can realize the rough pharmacokinetics required for dosimetry. Detailed preclinical dosimetry studies, so-called mi-crodosimetry, must be combined with macroscopic whole-organ dosimetry. Two scales of microdosimetry were proposed for the TAT design; one is the organ-microstructure scales and the other is the cellular and subcellular scales. Their features are summarized in Table 3. The physical quantities frequently evaluated in the former scale are the absorbed doses in organ microstructures such as nephron in kidney, which can be used in the estimate of the normal tissue compilation effects. In the latter scale, the cellular S-value, 12) which represents the absorbed dose in a target cell compartment arising from nuclear transformation of the radionuclide in a source cell compartment, is often evaluated because the absorbed doses are heterogeneously distributed in such microscopic scales due to the non-random distribution of radionuclides among each cell compartment. The probability densities of the absorbed doses in cell and cell nucleus are also calculated. As an example of the microdosimetry in the organ-microstructure scale, Hobbs et al. 13,14) developed a computational model for representing the microstructures of kidney and bone marrow cavity. It was applied on Ra-223 dichloride (Xofigo), which was the first alpha-emitter radiopharmaceutical that has received approval for the treatment of patients with castration-resistant prostate cancer metastasized to bone. It was implemented in a new clinical trial for treatment of patients with bone metastases in renal cell carcinoma. This approach was also illus- First-in-Human Clinical Requirements 4) We summarize the requirements for first-in- The dose of radioactive drugs can be considered in the light of radioactivity and mass, and radioactivity would often be a more important determining factor than mass (see Table 4). Specifically, we review the requirements for FIH clinical trials for conventional radiopharmaceuticals issued by the FDA and EMA. Considering very short range and extremely high LET which are different from conventional radiopharmaceutical drugs, we discussed how to set requirements such as the dosage of TAT drugs for FIH clinical trials. It is important to evaluate the toxicity at the cellular level by con- is obtained from toxicological data, and the latter is obtained from pharmacological data (see Table 4). Recently, the labeling of alpha-particles on biomolecular drugs such as peptides and antibodies have been studied mainly in Europe and the United States. PET drugs, which already have a high diagnostic capability, is a good example of a theranostic agent that can be applied based on diagnostic results and has high potential as a therapeutic agent for For the successful development, it will be important to establish interdisciplinary cooperation and pursue requirements for FIH clinical trials. Quality Control and GMP production for Targeted Alpha Therapy products 3) We would like to focus on the issues of production, quality control and health regulation of alpha-labeled radiopharmaceuticals. The method of conjugation of radionuclides to drugs are similar to radio-diagnostics such as PET and SPECT. However, the regulation of radiation is significantly different between radionuclides that emit positron, gamma or beta rays and those that emit alpha rays. To ensure the safety of workers and the public, we must comply with Act on the Regulation of Radioisotopes which was revised on September 1, 2019. Radiation has different transmittance depending on the type. Alpha particles can be shielded with a sheet of paper, beta rays with an aluminum or plastic plate, and X-rays and gamma rays with a lead or thick iron plate. Neutrons can be shielded by water. That is, the transmissivity is in the order of gamma rays>beta rays>alpha particles (See Fig. 6). In TAT product development, quality standard requires continuity and consistency, but shifting from manual synthesis to automatic synthesis may change the quality standards at radiolabeling step. When this shifts from preclinical trials to clinical trials, it may lead to re-examination. Since TAT products are administered intravenously, the sterility assurance is required for GMP. In general, when radiopharmaceuticals are manufactured, careful consideration is required for radiation protection because of the large amount of radioactivities used during the production and handling than when they are used on patients. Since it is difficult to meet these requirements by manual synthesis, it is necessary to start an automatic synthesis device at an early stage and to establish a manufacturing process considers occupational safety. It is necessary to conduct the quality control (QC) tests for synthesized products (see Table 5). In order to prevent labor, money and time loss due to re-examination, it is important to consider the final synthesis method in the early stages of development. In that case, it may be better to incorporate the concept of quality by design (QbD) #1 . The labeling process of TAT products is described. We introduce amino-acid derivatives targeting LAT1, 19) borane-mediated labeling to antibody 20) and pre-targeting method applying click reaction 21,22) as labeling compounds. Automatic purification system of α-particle is required as a step before the abovementioned labeling synthesis. Next, the radiation exposure of workers and the public is also a big problem in performing radiosynthesis. X-rays and γ-rays are also emitted in TAT products. We introduce the need for radiation shielding based on the calculation by effective dose rate coefficients 23) for alpha particles, 211 At and 225 Ac (see Table 6). Regarding radiation protection in TAT product manufacturing, it can be said that sufficient response, if it is operated inside the hot cell, which are already utilized by many PET centers (See Fig. 7). 225 Ac does not emit gamma rays, but daughter nuclides emit gamma rays like 221 Fr 218 keV (12%) and 213 Bi 440 keV (27%). 211 At emits γ-rays (687 keV) with a low emission rate of 0.26% and also emits X-ray (77-92 keV). 1 Radiation Shielding of 211 At-TAT products It was reported that at least 4 GBq of 211 At was used in 1 drug synthesis under cGMP environment. When set to use 5 GBq of 211 At in one synthesis, without effective shielding against X-rays, a relatively high effective dose rate of 29 μSv/h is simply achieved at 1 m distance. Therefore, depending on the operation method, shielding for X-rays (and γ-rays) may be necessary even in the case of 211 At. In the case of 211 At, since most of the radiation, except α-rays, is X-rays of 100 keV or less as described above, shielding with lead is easy. For example, when 5 GBq of 211 At is shielded with 1 cm of lead, the effective dose rate when 50 GBq of 18 F is shielded with 6 cm of lead is almost the same. It is not necessary to introduce new hot cell designed for TAT products, and it is certain that conventional PET drug hot cells have sufficient shielding ability against external exposure by manufacturing TAT products. Based on the concept of ensuring safety, we hope that the understanding that TAT drug manufacturing is possible without the risk of external exposure inside the existing hot cell installed at the PET drug facility will be expected. We introduce the points of QC based on the method of GMP validation including the calibration methods for QC equipments (see Table 7). 24) We presented the technique to establish both quantitative evaluation method of radiation protection and safety-	2020-10-28T19:07:49.162Z	2020-10-15T00:00:00.000	{ "year": 2020, "sha1": "fddb0a51311f38dc3739b2001856dc7c28defaef", "oa_license": null, "oa_url": "https://www.jstage.jst.go.jp/article/radioisotopes/69/10/69_691003/_pdf", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "296dfd746d2fc96c9e4a2cd38bd8a9dd8fde9497", "s2fieldsofstudy": [ "Medicine", "Chemistry", "Physics" ], "extfieldsofstudy": [ "Medicine" ] }
236976200	pes2o/s2orc	v3-fos-license	Are Neural Ranking Models Robust? Recently, we have witnessed the bloom of neural ranking models in the information retrieval (IR) field. So far, much effort has been devoted to developing effective neural ranking models that can generalize well on new data. There has been less attention paid to the robustness perspective. Unlike the effectiveness which is about the average performance of a system under normal purpose, robustness cares more about the system performance in the worst case or under malicious operations instead. When a new technique enters into the real-world application, it is critical to know not only how it works in average, but also how would it behave in abnormal situations. So we raise the question in this work: Are neural ranking models robust? To answer this question, firstly, we need to clarify what we refer to when we talk about the robustness of ranking models in IR. We show that robustness is actually a multi-dimensional concept and there are three ways to define it in IR: 1) The performance variance under the independent and identically distributed (I.I.D.) setting; 2) The out-of-distribution (OOD) generalizability; and 3) The defensive ability against adversarial operations. The latter two definitions can be further specified into two different perspectives respectively, leading to 5 robustness tasks in total. Based on this taxonomy, we build corresponding benchmark datasets, design empirical experiments, and systematically analyze the robustness of several representative neural ranking models against traditional probabilistic ranking models and learning-to-rank (LTR) models. The empirical results show that there is no simple answer to our question. While neural ranking models are less robust against other IR models in most cases, some of them can still win 1 out of 5 tasks. This is the first comprehensive study on the robustness of neural ranking models. INTRODUCTION Relevance ranking is a core problem of information retrieval (IR). Given a query and a set of candidate documents, a scoring function is usually learned to determine the relevance degree of a document with respect to the query. With the advance of deep learning technology, we have where , d and y denotes the query, the document list and the label in the test set, respectively. Specifically, the test samples are supposed to be drawn from the same distribution as the training distribution G. We define the robustness of ranking models from three major perspectives as follows: • Performance Variance: The performance variance aims to analyze the robustness of ranking models by emphasizing the worst-case performance across different individual queries under the I.I.D. setting. Formally, the performance variance is defined as where (·) denotes the variance of effectiveness over all the test queries. Besides, a special case of performance variance, i.e., the poorly-performing queries, is emphasized to analyze the ranking robustness in the worse case. • OOD Generalizability: The OOD generalizability aims to analyze the robustness of ranking models according to the transfer effectiveness on OOD examples. Formally, suppose that OOD examples ′ , d ′ and y ′ are drawn from a new distribution G ′ . The OOD generalizability is defined as Specifically, the OOD generalizability can be further defined in two ways, i.e., OOD generalizability on unforeseen query types and OOD generalizability on unforeseen corpus. • Defensive Ability: The defensive ability aims to analyze the robustness of ranking models according to their ability to defend against adversarial operations. Given an adversarial attack function for the query and document, the defensive ability is formalized as Specifically, the defensive ability can be measured with respect to two types of adversarial operations, i.e., query attack and document manipulation. Based on this taxonomy, we design the corresponding experiments, and conduct empirical studies to analyze the robustness of several representative neural ranking models against traditional probabilistic ranking models and LTR models. All the experimental datasets and codes 1 used in our study would be publicly available for the research community. Specifically, the design of our empirical experiments for each task are as follows. • To facilitate the study of the performance variance, we conduct experiments on three ad-hoc retrieval datasets, i.e., MQ2007 [71], Robust04, and MS MARCO, and follow the previous works [86,99] to measure the robustness of different ranking models. • To facilitate the study of the OOD generalizability, we first build two benchmark datasets for unforeseen query type and unforeseen corpus, respectively. Specifically, we construct the benchmark dataset for unforeseen query type by splitting the MS MARCO dataset into 5 subdatasets, with respect to the official query type. Besides, we construct the benchmark dataset for unforeseen corpus based on three ad-hoc retrieval corpora, i.e., MQ2007, Robust04, and MS MARCO. Furthermore, we propose the drop rate metric to measure the OOD generalizability of different ranking models. • To facilitate the study of the defensive ability, we first build a benchmark dataset for query attack. Specifically, we introduce four types of character-level edits [42,70] and three types of word-level edits to attack the query in the MS MARCO dataset. Then, we propose the drop rate metric to measure the defensive ability against query attack. For document manipulation, we conduct experiments on the ASRC dataset [74] and follow the previous work [27] to measure the robustness of different ranking models. The empirical results demonstrate that: 1) Neural ranking models are in general not robust as compared with other IR models. The finding is consistent with the previous study [84] in ML which demonstrates that the complexity in the model could lead to robustness issues. 2) Pre-trained ranking models exhibit the best robustness against all the other models from the perspective of the performance variance. 3) Some neural ranking models show the superiority of the defensive ability. For example, DSSM, Duet and Conv-KNRM are robust to the query attack. 4) Based on the empirical evaluation results, there remains clearly room for future robustness improvements. We organize this work as follows. We first introduce the ranking models which are evaluated through all the robustness metrics, including traditional probabilistic ranking models, LTR models and neural ranking models in Section 2. We then analyze the robustness of ranking models in terms of the performance variance, the OOD generalizability, and the defensive ability, in Section 3, Section 4 and Section 5 respectively. Finally, we briefly review the related work in Section 6 and conclude this work in Section 7. RANKING MODELS In this paper, we aim to investigate whether the neural ranking models are robust. Specifically, we adopt three types of representative neural ranking models for analysis, i.e., representation-focused deep matching models, interaction-focused deep matching models, hybrid deep matching models and advanced pre-trained models. • Representative-focused Deep Matching Models include, 1 The experimental datasets and codes will be available at URL. -DSSM: DSSM [36] is a representative-focused deep matching model designed for Web search, which contains a letter n-gram based word hashing layer, two non-linear hidden layers and an output layer. • Interaction-focused Deep Matching Models include, -DRMM: DRMM [29] is an interaction-focused deep matching model designed for ad-hoc retrieval. It consists of a matching histogram mapping, a feed forward matching network and a term gating network. In this paper, we use LogCount-based Histogram (LCH) as the matching histogram mapping and Term Vector (TV) as the term gating function. -Conv-KNRM: Conv-KNRM [18] is another popular interaction-focused deep matching model, which models n-gram soft matches for ad-hoc retrieval based on convolutional neural networks (CNN) and kernel-pooling. • Hybrid Deep Matching Models include, -Duet: Duet [60] is a hybrid deep matching model which combines both the representationfocused architecture and the interaction-focused architecture. Specifically, it contains two separate deep neural networks, one that matches using a local representation of text, and another that learns a distributed representation before matching. • Pre-trained Models include, -BERT: The key technical innovation of BERT [45] is applying the multi-layer bidirectional Transformer encoder architecture for language modeling. BERT uses two different types of pre-training objectives including Masked Language Model (MLM) and Next Sentence Prediction (NSP). In this paper, the query and the document are concatenated as the input to BERT with special tokens delimiting them, i.e., [CLS] and [SEP]. To obtain the relevance score of the document to a given query, we apply a sigmoid function over the representation of [CLS] following previous studies [17,63]. -ColBERT: ColBERT [46] employs contextualized late interaction over deep language models (in particular, BERT) for efficient retrieval. It independently encodes queries and documents into fine-grained representations that interact via cheap and pruning-friendly computations. Here, we analyze ColBERT's ability to re-rank documents following the original paper. Specifically, we use the implementations of DRMM, Conv-KNRM, and Duet from MatchZoo [30]. We adopt the parameters of BERT released by Google 2 to initialize BERT. For ColBERT, we use the original code 3 released by the authors for implementation. In order to better analyze the robustness of neural ranking models, we adopt three types of LTR models for comparison, including pointwise LTR models, pairwise LTR models and listwise LTR models. • Listwise LTR Models include, -LambdaMART: LambdaMART [7] is a state-of-the-art listwise LTR algorithm which uses gradient boosting to produce an ensemble of retrieval models. For Prank, we implement it according to the original paper since there is no publicly available code. For RankSVM, we directly use the implementation in SVM [41]. LambdaMART is implemented using RankLib 4 , which is a widely used LTR tool. Besides, we compare the above neural models with two representative traditional probabilistic ranking models, including, • QL: Query likelihood model based on Dirichlet smoothing [97] is one of the best performing language models. • BM25: The BM25 formula [76] is another highly effective retrieval model that represents the classical probabilistic retrieval model. Specifically, we use the implementations of QL and BM25 from Anserini [93] toolkit 5 . PERFORMANCE VARIANCE UNDER THE I.I.D. SETTING Most ranking models are designed under the simple assumption that the observations are from I.I.D. random variables, and focus on improving the average effectiveness of retrieval results. Recently, it has been recognized that, when we attempt to improve the mean retrieval effectiveness over all queries, the stability of performance across different individual queries could be hurt [99]. Therefore, in this section, we analyze the robustness of ranking models by emphasizing the worstcase performance across different individual queries, i.e., the performance variance under the I.I.D. settings. In the following, we first introduce the definition and metric of the performance variance, and then conduct experiments to analyze the robustness of ranking models. Definition of Performance Variance The performance variance of ranking models refers to the variance of effectiveness across different individual queries, which has been formulated in Eq. (2). When a ranking model achieves improvements in mean retrieval effectiveness (e.g., mean average precision (MAP) [79]), the performance of some individual queries could be hurt [99]. Although failures on a small number of queries may not have a significant effect on the average performance, users who are interested in such queries are unlikely to be tolerant of this kind of deficiency. Accordingly, an ideal ranking model is expected to balance the trade-off between effectiveness and robustness by achieving high average effectiveness and low variance of effectiveness [100]. Metric of Performance Variance Here, we propose the variance of normalized average precision (VNAP) to measure the performance variance, which is defined as, where the expectation E(·) is over a set of queries that are assumed to be uniformly distributed. ( ) denotes the normalized average precision with respect to the query , which is defined as, where ( ) denotes the average precision with respect to the query , which is defined as, where denotes the number of relevant documents to the query . is the rank of the relevant document predicted by the ranking model, which ranges from 1 to the size of document list. denotes the ground-truth label of document . is the indicator function, which aims to count the number of relevant documents. Note that VNAP is similar to VAP [99]. The difference between them is that we have normalized the average precision to eliminate the influence of the mean performance. Since different models are likely to have different mean performance, it is necessary to eliminate the influence of the mean performance to better measure the variance of ranking models. The ranking model would be more robust with a lower VNAP value. Experimental Settings In this section, we introduce our experimental settings, including datasets and implementations details. Datasets. To evaluate the performance variance of different ranking models, we conduct experiments on several representative ad-hoc retrieval datasets, i.e., • Table 1. We choose these three datasets according to three criteria: 1) The datasets are public and the original document contents are available, 2) The query numbers are diverse (e.g., 250 queries in Robust04 and 0.37 million queries in MS MARCO), and 3) The literary style of the documents ranges from newswire articles to Web document. Implementation Details. In preprocessing, all the words in the documents and queries are white-space tokenized, lower-cased, and stemmed using the Krovetz stemmer [49]. For the MQ2007 6 https://trec.nist.gov/data/robust.html 7 https://github.com/microsoft/MSMARCO-Document-Ranking 8 The relevance assessments for the official test set were not public at the time the paper was written. dataset, we use the data partition and top 40 ranked documents released by the official LETOR4.0 [71]. For the Robust04 dataset, an initial retrieval is performed using the Anserini toolkit with the QL model to obtain the top 100 ranked documents. For the MS MARCO dataset, we use the official top 100 ranked documents retrieved by the QL model. For traditional probabilistic ranking models, we adopt the static parameters suggested by [37] on the Robust04 dataset and tune the parameters on the corresponding validation set on the MQ2007 and MS MARCO dataset 9 . For LTR models, we use standard features released by LETOR 4.0 for MQ2007. In LETOR, there are 46 hand-crafted features in total, among which 42 features are constructed based on the textual elements (e.g., term frequencies, BM25 and language model scores based on title, body, anchor texts, and URL), and 4 features are based on link analysis (e.g., PageRank, inlink number, outlink number, and number of child page). Due to the lack of officially released LTR features, we construct the LTR features for Robust04 and MS-MARCO respectively, by computing the TF, IDF, TF-IDF, Document Length, BM25 score, QL-DIR score and QL-JM scores on the title, url, body and document respectively as the final 28-dimensional LTR features following [71]. For RankSVM, we use the linear kernel with the hyper-parameter C selected from the range [0.0001, 10] on the validation set for three datasets following [18]. For LambdaMART, we select the number of trees (in 100-500, by 10), leafs (in 2-20, by 2) and shrinkage coefficient (in {0.001, 0.005, 0.01, 0.05, 0.1}) on the validation set for three datasets. For neural ranking models, the model-specific hyper-parameters are as follows. For DSSM, we use a three-layer DNN and set the node number of each layer as 100, 100 and 50 to avoid overfitting on Robust04 and MQ2007 datasets following [65]. We set the node number of each layer as 300, 300, 128 on the MS MARCO dataset. For DRMM, we use a four-layer architecture and set the node number of each layer as 30 (histogram bins), 5, 1 and 1 on Robust04 and MQ2007 datasets following [29,65]. We set the node number of each layer as 30 (histogram bins), 10, 1 and 1 on MS MARCO dataset. For Conv-KNRM, we set the n-gram size as 3 and the number of CNN filters as 128. For kernel-pooling, we set the number of kernels to 11 with the mean values of the Gaussian kernels varying from −1 to +1, and standard deviation of 0.1 for all kernels (the first kernel's is set as 0.001 for exact matching ) on three datasets following [18,35]. For Duet, we used 10 filters with both the local and distributed model, with hidden dimensions set to 30 and 699, respectively on Robust04 dataset following [95]. We set the filter size as 10 in both local model and distributed model, and the hidden size as 20 in the fully-connected layer on MQ2007 dataset following [21]. We set the filter size as 32 in both local and distributed model and the hidden size as 32 in the fully-connected layer on MS MARCO dataset. The learning rates of above models are tuned on the corresponding validation set in the range of [1e-5, 5e-3]. For BERT, we choose the fine-tuning learning rate from {5e-5, 3e-5, 2e-5} as recommended in [45] on three datasets. For ColBERT, we set the fine-tuning learning rate as 3e-6 on three datasets following [46]. For all neural ranking models, we use the Adam [47] optimizer. We use LTR and neural ranking models to re-rank the top candidate documents. Empirical Results on Performance Variance In this section, we analyze the empirical results on the performance variance under the I.I.D. setting. Specifically, we first analyze the average ranking effectiveness and the variance of effectiveness of different ranking models, respectively. Furthermore, we analyze the relationship between the average ranking effectiveness and the variance of effectiveness. To better understand the performance variance, we first analyze the average ranking effectiveness over the queries. Following [21,29], we take the topic "title" as queries and conduct 5-fold cross-validation on Robust04 and MQ2007 datasets to minimize over-fitting without reducing the number of learning instances. For Robust04 dataset, we use the mean average precision (MAP) [79], normalized discounted cumulative gain at rank 20 (NDCG@20) [6], and precision at position 20 (P@20) [15] as the evaluation metrics following [29]. For MQ2007 dataset, we report the P@10 and NDCG@10 following [21]. Moreover, we also use the recall at position 20 (R@20) [64] and recall at position 10 (R@10) as the recall oriented evaluation metrics for Robust04 and MQ2007, respectively. For MS MARCO dataset, we report the Mean Reciprocal Rank at 100 (MRR@100 [73]), which is suggested in the official instructions. We also report the MRR@10 and R@10 for MS MARCO dataset. The main results are shown in Table 2. From the results, we can find that: (1) For the two traditional probabilistic ranking models, we can see that BM25 is a strong baseline which performs better than QL in most cases. (2) For LTR models, RankSVM and LambdaMart perform better than the traditional probabilistic ranking models. It is not surprising since LTR models combine various features including the two traditional probabilistic ranking models (i.e., BM25 and QL scores), which are capable of characterizing the relevance between a document and a query. However, Prank performs worse than the traditional probabilistic ranking models on the Robust04 and MQ2007 dataset. The reason might be that pointwise LTR ignores the fact that some documents are associated with the same query and some others are not [54]. (3) Most neural ranking models perform worse than traditional probabilistic ranking models and LTR models. The reason might be that it is difficult for a deep neural model to train from scratch with such a few supervised pairs [72]. (4) Pre-trained models (i.e., BERT and ColBERT ) perform the best on all the three datasets. These results indicate that the rich language information from text captured by the pre-trained model is useful for relevance modeling. 3.4.2 How does different ranking models perform in terms of the variance of normalized average precision? The performance variance comparisons among different ranking models in terms of VNAP are shown in Figure 2. Recall that high values of VNAP attest to decreased ranking robustness. From the results, we can observe that: (1) For the two traditional probabilistic ranking models, BM25 achieves lower VNAP values than QL on the Robust04 and MQ2007 datasets, indicating that BM25 is more robust than QL on these datasets. (2) For the LTR models, RankSVM and LambdaMart achieve lower VNAP than traditional probabilistic ranking models on the three datasets, indicating that LTR models are more robust than traditional probabilistic ranking models. (3) For neural ranking models, DSSM achieves the highest VNAP on the MS MARCO dataset. The reason might be that DSSM achieves poor mean performance on the MS MARCO dataset and the corresponding VNAP is relatively higher. (4) When we look at the pre-trained models (i.e., BERT and ColBERT), we find they achieve the lowest VNAP among all the ranking models on the three datasets. It is interesting to find that pre-trained models exhibit the strongest robustness in terms of VNAP. As shown in Table 2, the pre-trained models have shown great success on the average effectiveness. The result indicates that pre-trained models present a great potential since it performs well in terms of the mean performance and the robustness (e.g, in terms of the variance). 3.4.3 What is the relationship between the average effectiveness and the variance of effectiveness? Here, we analyze the relationship between the average effectiveness and the robustness. Specifically, we consider the variance of effectiveness in Figure 2 and the average effectiveness in Table 2 simultaneously. Overall, the robustness of different models generally increases (i.e., the VNAP decreases) with the increase of the average ranking effectiveness (i.e., the MAP increases). In addition, we have computed the correlation between the effectiveness and the variance. For example, the Pearson correlation coefficient between the MAP and VNAP is −0.9869. The result indicates that there is a strong correlation between the average effectiveness and the variance of effectiveness. Analysis on Poorly-performing Queries The previous experiments have analyzed the performance variance over all the queries. A natural question is how the ranking models perform in the worse case, which can be well evaluated on the poorly-performing queries. Therefore, in this section, we emphasize on the poorly-performing queries to analyze the robustness of the ranking models, which refer to the queries that have the poor ranking performance among all the test queries. We first introduce the evaluation metric of the poorly-performing queries, and then analyze the experimental results. Metric of Poorly-performing Queries. To evaluate the robustness of ranking models in terms of the poorly-performing queries, we use two metrics following the previous works [86]. • %no denotes the percentage of queries with no relevant documents in the top 10 retrieved, i.e., where ∈ is the relevance label (i.e., the larger the relevance label, the more relevant the query-document pair) of the top ∈ [1, 10] document with respect to the query . is the indicator function and \| \| is the total number of evaluated queries. The ranking model would be more robust with a lower %no value. • gMAP denotes the geometric mean average precision, which is defined as, where ( ) is formulated as in Eq. (7). is a minimal positive since the average precision score for a single query may approximate to zero [87]. The ranking model would be more robust with a higher gMAP value. Empirical Results on Poorly-performing Queries. Here, we first measure the performance of the poorly-performing queries in terms of %no and gMAP. Then, we analyze the relationship between the performance of all the queries and the performance of the poorly-performing queries. • How does different ranking models perform over poorly-performing queries? We first focus on the poorly-performing queries to analyze the robustness of different ranking models in terms of %no and gMAP. The main results are shown in Table 3. Recall that low values of %no and high values of gMAP attest to increased ranking robustness. From the results, we can observe that: (1) For traditional probabilistic ranking models, QL achieves a lower %no value than BM25 on the MQ2007 and MS MARCO, demonstrating that QL is more robust than BM25 over the poorly-performing queries. (2) For LTR models, RankSVM and LambdaMart achieve lower %no values and higher gMAP values as compared with traditional probabilistic ranking models on the MQ2007 and MS MARCO. The results indicate that combining different kinds of human knowledge (i.e., relevance features) could achieve good improvements on the ranking robustness over the poorly-performing queries. (3) When we look at the pre-trained models (i.e., BERT and ColBERT ), we find that BERT achieves the lowest %no value and the highest gMAP value among all the models on the MQ2007 dataset, while ColBERT achieves the lowest %no value and the highest gMAP value on the MS MARCO dataset. It is interesting that pre-trained models exhibit the strongest robustness over the poorly-performing queries under the I.I.D. setting. • What is the relationship between the average effectiveness and the robustness over the poorly-performing queries? Specifically, we visualize the experimental results on the MQ2007 dataset from Table 2 and 3 into Figure 3, where the horizontal axis represents the average effectiveness metric (i.e., MAP) and the vertical axis represents the robustness metric (i.e., gMAP and %no). We have the following observations: (1) The robustness over the poorly-performing queries generally increases (i.e., the gMAP increases), with the increase of the average effectiveness (i.e., the MAP increases). The reason might be that the good performance over poorly-performing queries, in turn, would improve the performance over all the queries. This is consistent with the finding in previous studies where the degree of ranking robustness is positively correlated with retrieval performance [102]. (2) The pretrained models are the most robust model over the poorly-performing queries and the most effective model over all the queries under the I.I.D. setting. Future work could propose new self-supervised objectives tailored for IR that enhance model robustness. OOD GENERALIZABILITY As described above, most evaluations in IR assume that the train and test examples are I.I.D.. Some advanced ranking models (e.g., pre-trained model) can achieve promising performance on some IR tasks [28,43,56]. However, in the real-world scenarios, the train and test distributions are often not identically distributed caused by the data bias [83]. Without any special supervision, the ranking models may come at the cost of poor generalization and performance on examples not observed during training [10]. Therefore, in this section, we analyze the robustness of ranking models according to the transfer effectiveness on OOD examples, i.e., the OOD generalizability which has been formulated in Eq. (3). Specifically, we introduce two ways to define the OOD generalizability, i.e., 1) The OOD generalizability on unforeseen query type, and 2) The OOD generalizability on unforeseen corpus. OOD Generalizability on Unforeseen Query Type In this section, we introduce the definition and metric of the OOD generalizability on unforeseen query type. Definition of OOD Generalizability on Unforeseen Query Type. The OOD generalizability on unforeseen query type refers to the transfer effectiveness on unseen query types. Nowadays, users formulate their queries in the form of various types [5] that can describe their information needs properly to find the right results quickly. When submitting a new query type to a search engine, it tends to fail due to the large gap between normal in-distribution and undesired OOD query types. To improve users' satisfaction, a ranking model should be robust to such new query types. Formally, given a ranking model learned on { , d , y } =1 which are drawn from the training distribution G , we aim to evaluate its performance on the test examples with unforeseen query types, which are drawn from a new distribution G ′ . Specifically, the OOD generalizability on unforeseen query type is defined as where ′ , d and y ′ denotes the query, the document list and the label with new query types. Note that the same document collection is used for both the training and test sets. Metric of OOD Generalizability on Unforeseen Query Type. To measure the OOD generalizability of the ranking models on unforeseen query type, we propose one automatic metric, namely, • DR evaluates the drop rate between the ranking performance on the OOD test set (i.e., ( ′ , d , y ′ ) ∼ G ′ ) and the ranking performance . . . on the I.I.D. test set (i.e., ( , d , y ) ∼ G ), which is defined as where . . . and are defined as where the effectiveness evaluation metric can be defined in different ways, such as mean reciprocal rank (MRR) [73], mean average precision (MAP) [79], normalized discounted cumulative gain (NDCG) [6] and Precision (P) [15], with respect to the specific experimental dataset. The ranking model would be more robust with a higher DR . OOD Generalizability on Unforeseen Corpus In this section, we introduce the definition and metric of the OOD generalizability on unforeseen corpus. Definition of OOD Generalizability on Unforeseen Corpus. The OOD generalizability on unforeseen corpus refers to the transfer effectiveness on unseen corpus. In practice, the training corpus usually has a limited volume and hardly characterizes the entire distribution. Chasing an evolving data distribution is costly, and even if the training corpus does not become stale, models will still encounter unexpected situations at the test time [34]. Accordingly, training a ranking model on the given corpus that can well generalize to another new corpus is necessary. Formally, given a ranking model learned on { , d , y } =1 which are drawn from the training distribution G , we aim to evaluate its performance on the test examples from unseen corpus, which are drawn from a new distribution G ′ . Specifically, the OOD generalizability on unforeseen corpus is defined as where ′ , d ′ and y ′ denotes the query, the document list and the label from unseen corpus. Metric of OOD Generalizability on Unforeseen Corpus. To measure the OOD generalizability on unforeseen corpus, we also employ the DR metric based on the ranking performance on the OOD test set (i.e., ( ′ , d ′ , y ′ ) ∼ G ′ ) and the ranking performance . . . on the I.I.D. test set (i.e., ( , d , y ) ∼ G ). Experimental Settings In this section, we introduce our experimental settings, including data construction, and implementation details. Data Construction. For evaluation purposes, we build two benchmark dataset based on several existing IR collections. Specifically, we use Robust04, MQ2007, and MS MARCO, which have been described in Section 3.3.1. In these IR collections, queries are associated with the metadata information, which helps differentiate the examples with respect to query type and corpus, [10]. For three corpora, we also randomly sample 20% queries from the training set for validation, respectively. Thus, we can obtain a benchmark dataset with 3 different corpora. Implementation Details. The implementation details, including the pre-processing and model implementation, are similar to that in Section 3.3.2. The only expectation is that the parameters of traditional probabilistic ranking models are tuned on the corresponding development set in the two constructed benchmark dataset. Analysis of OOD Generalizability on Unforeseen Query Type In this section, we analyze the empirical results on the OOD generalizability on unforeseen query type. Specifically, we adopt the widely used metric for MS MARCO, i.e., MRR@100, as the implementation of the effectiveness evaluation metric in Eq. (12) and (13). Table 5 shows the MRR@100 performance of different ranking models under both the OOD (i.e., the training and test query types are different.) and I.I.D. (i.e., the training and test query types are the same.) settings. For the OOD setting, we train the model on one query type and test it on a different query type. Since the model may be too specific to the single query type it was trained on, we also train the model on Fig. 4. The OOD generalizability of different ranking models on unforeseen query type in terms of DR (%). The test query type is described on the left and the training query type is described above each subfigure. four query types and test it on the one remaining type. Figure 4 shows the DR performance with respect to unforeseen query type. In the following, we first give an overall analysis of different ranking models. Then we analyze the OOD generalizability of traditional probabilistic ranking models, LTR models and neural ranking models, respectively. Recall that high values of DR attest to increased ranking robustness. 4.4.1 Overall analysis on all the ranking models. Firstly, we give an overall performance analysis on all the ranking models. We can observe that: (1) Under the I.I.D. setting (e.g., the training and test query type is Person), ColBERT generally performs the best (0.398 on Person) followed by LambdaMART (0.313 on Person), BERT (0.277 on Person) and then RankSVM (0.264 on Person), in terms of MRR@100. (2) As show in Figure 4, most ranking models are not able to well generalize to OOD query types. Take the most effective model ColBERT as an example, the DR value is -30.2% when the training query type is Location and the test query type is Numeric. It indicates that higher effectiveness does not reliably improve OOD generalizability. (3) When being trained on four query types and tested on the one remaining type, most ranking models perform worse than the I.I.D. setting, while perform better than being trained on the single query type. It indicates that the training data for a single query type may be inadequate. When the training query type increases, the OOD generalizability improves. 4.4.2 Analysis on traditional probabilistic ranking models and LTR models. When we look at the traditional probabilistic ranking models and LTR models, we find that: (1) In general, traditional probabilistic ranking models achieve the highest DR values, indicating that traditional probabilistic ranking models are the most robust models when facing unforeseen query types. A possible reason would be that as unsupervised methods, traditional probabilistic ranking models avoid the problem of overfitting the training data and thus have a better OOD generalizability on unforeseen query types. (2) The DR values of LTR models are higher than that of neural ranking models, indicating that LTR models are more robust than neural ranking models. The reason might be that hand-crafted features in LTR models could better generalize to unforeseen query type than automatically learned features by neural networks. 4.4.3 Analysis on neural ranking models. When we look at the neural ranking models, we find that: (1) In general, neural ranking models achieve the lowest DR values among all the ranking models. The reason might be that neural ranking models with a deeper network architecture fit the normal in-distribution query types better, at the cost of further loss in performance on the held out OOD query types. It is consistent with the finding in [10]. (2) Among these five neural ranking models, pre-trained models (i.e., BERT and ColBERT ) have the best OOD generalizability on unforeseen query type. For example, ColBERT trained (fine-tuned) on Person and tested on Location, the MRR@100 value even improves 5.6% compared with ColBERT trained and tested on Location. A possible explanation would be that pre-training on the huge text corpus can improve OOD generalization. It is consistent with the finding in [34], which indicates that pre-trained models are more robust to OOD examples on several NLP tasks. (3) Pre-trained models have shown great effectiveness under both the OOD and I.I.D. settings, and good robustness to OOD query types. Future works could design novel pre-training objectives tailored for IR that enhance ranking robustness. Analysis of OOD Generalizability on Unforeseen Corpus In this section, we analyze the empirical results on the OOD generalizability on unforeseen corpus. Similar to the used effectiveness evaluation metric in Section 3.4.1, we also adopt the MAP, NDCG@10, NDCG@20, P@10, P@20, MRR@10 and MRR@100 as the implementation of in Eq. (12) and (13). Table 6 shows the MAP, NDCG@10, NDCG@20, P@10, P@20, MRR@10 and MRR@100 performance of all the ranking models under both the OOD (i.e., the training and test corpora are different) and I.I.D. (i.e., the training and test corpora are the same) settings. Figure 5 shows the DR performance based on the MAP, NDCG, P and MRR, respectively. In the following, we first give an overall analysis of different ranking models. Then we analyze the OOD generalizability of traditional probabilistic ranking models, LTR models and neural ranking models, respectively. Recall that high values of DR attest to increased ranking robustness. 4.5.1 Overall analysis on all the ranking models. Firstly, we give an overall analysis on all the ranking models. We can observe that: (1) Under the I.I.D. setting (e.g., the training and test corpus is MQ2007), ColBERT generally performs the best (0.4759) followed by BERT (0.4656) and RankSVM (0.4601), in terms of MAP. (2) As show in Figure 5, almost all DR values are negative. It indicates that most ranking models are not able to well generalize to OOD examples. (3) Besides, the DR value with respect to MRR for unforeseen MS MARCO is much lower than that for unforeseen query types sampled from MS MARCO. A possible reason is that compared with different query types sampled from the same corpus, different corpora have greater differences among samples. Table 6. The performance of different ranking models with respect to unforeseen corpus. The "train" and "test" denote the training and test corpus for the ranking models, and the MAP, NDCG@10, NDCG@20, P@10, P@20, MRR@10 and MRR@100 performance are reported on the test set. Significant performance degradation with respect to the corresponding I.I.D. setting is denoted as '−' (p-value ≤ 0.05). 5. The OOD generalizability on unforeseen corpus in terms of DR (%) based on the MAP, NDCG, P, and MRR respectively. For each subfigure, the test corpus is described on the left and the training corpus is described above. Analysis on traditional probabilistic ranking models and LTR models. When we look at the traditional probabilistic ranking models and LTR models, we can find that: (1) Under the I.I.D. setting, BM25 performs better than QL on most corpus, and BM25 is a strong baseline which performs better than LTR models on Robust04. Under the OOD setting, QL is more robust to the unforeseen corpus than BM25. (2) LTR models show a good transfer effectiveness from MS MARCO to Robust04. For instance, the DR value based on MAP is close to 0 (i.e., -1.1%) when we train Prank on MS MARCO and then test it on Robust04. Besides, it is surprising that when we train RankSVM on MS MARCO and test it on Robust04, the DR value based on P@20 is positive (i.e., 2.7%). The reason might be that the distribution of hand-crafted features for Robust04 is similar to that for MS MARCO. (3) Prank shows a better OOD generalizability on unforeseen corpus than RankSVM and LambdaMART if we test them on Robust04 and MQ2007. For example, when we train Prank on MQ2007 and test it on Robust04, the DR value based on NDCG@20 is even positive (i.e., 8.6%), while LambdaMART achieves -13.2% DR under the same setting. One possible explanation is that Prank has simpler model architecture and less parameters compared with RankSVM and LambdaMART, which alleviates the problem of overfitting and increases the OOD generalizability. 4.5.3 Analysis on neural ranking models. When we look at the neural ranking models, we can see that: (1) In general, neural ranking models show the worst OOD generalizability on unforeseen corpus. The finding is consistent with that on unforeseen query type. For example, when we train Conv-KNRM on MQ2007 and test it on MS MARCO, the DR value based on MRR@100 is even -83.8%. (2) DRMM shows a good transfer effectiveness from Robust04 to both the MS MARCO and MQ2007. As shown in previous studies [29], DRMM have performed quite well on Robust04. In this way, DRMM trained on Robust04 may lean to generalize to other corpora. DEFENSIVE ABILITY AGAINST ADVERSARIAL OPERATIONS Deep neural networks have been found vulnerable to adversarial attack, where an imperceptible perturbation can trigger dramatic changes in the final result [3,101]. However, the vulnerability of neural ranking models remains under-explored. This poses a serious security risk on the practical search engines, where an imperceptible adversarial perturbation to the query/document can be sufficient to intentionally fool the neural ranking model to make wrong decisions, i.e., raise or lower the ranks of selected candidate documents. Therefore, in this section, we analyze the robustness of ranking models according to their ability to defend against adversarial operations, i.e., the defensive ability which has been formulated in Eq. (4). Specifically, we introduce two types of adversarial operations to measure the defensive ability, i.e., 1) The ability to defend against the query attack, and 2) The ability to defend against the document manipulation. Defensive Ability against Query Attack In this section, we introduce the definition and metric of the defensive ability against query attack. Definition of Defensive Ability against Query Attack . The defensive ability against query attack refers to that the ranking performance on the attacked queries. In a real-world scenario, the users' queries may be attacked unconsciously. For example, users may occasionally misspell or mistype a query keyword when performing a search. Such query typo is a longstanding real-world problem for IR, which has been extensively studied [31,67,103]. From the perspective of a user, if the search engine cannot tolerate the query typo, the user's satisfaction will remarkably drop. A reliable system that always produces acceptable retrieval performance, is more preferred than another system that fails on the occasional query typo. Specifically, we consider the effects of misspelled or mistyped queries by exploring several types of character-level and word-level edits. Formally, given a ranking model learned on { , d , y } =1 which are drawn from the training distribution G, we aim to evaluate its performance on the I.I.D. test queries attacked by a query attack function , i.e., ( ). Specifically, the defensive ability against query attack is defined as where , d and y denotes the query, the document list and the label from the I.I.D. test set, respectively. Metric of Defensive Ability against Query Attack . As for evaluation measure, we employ one automatic metric, namely, • DR evaluates the drop rate between the model performance on the original queries (i.e., ) and the ranking performance on the attacked queries (i.e., ( )), i.e., where and are defined as where the effectiveness evaluation metric can be defined in different ways, such as MRR@100, with respect to the specific experimental dataset. The ranking model would be more robust with a higher DR . Defensive Ability against Document Manipulation In this section, we introduce the definition and metric of the ability to defend against document manipulation. Definition of Defensive Ability against Document Manipulation. The defensive ability against document manipulation refers to that the ranking performance on the adversarial documents. In the Web search setting, many document authors are "ranking-incentivized" [26]. That is, they may introduce modifications to their documents, hoping to rank them higher for some queries by search engines. In this way, these modifications could be almost indiscernible by users and users will loose faith in the search system if they observe these rapid ranking changes. Thus, a ranking model should be robust to such manipulations for maintaining high-quality search results. Formally, given a ranking model learned on { , d , y } =1 which are drawn from the training distribution G, we aim to evaluate its performance on the I.I.D. test queries with the corresponding document list d attacked based on a document attack function , i.e., (d ). Specifically, the defensive ability against document manipulation is defined as where , d and y denotes the query, the document list and the label from the I.I.D. test set, respectively. Metric of Defensive Ability against Document Manipulation. As for evaluation measure, we employ Top Change (TC) and Kendall's-distance (KT) following the previous work [27]. Suppose that ( ) is a ranked document list with respect to a given query achieved by a ranking model. After the document manipulation on the document list d , a new ranked list ( ) ′ is obtained by the ranking model. The key idea to quantify robustness is to measure the distance between ( ) and ( ) ′ . Specifically, the lower the TC and KT value, the lower the "distance" between the two lists, i.e., the more robust the ranking model is. • TC indicates the similarity between the two ranked lists based on the highest ranked document, i.e., where is the set of unordered pairs of distinct documents in ( ) and ( ) ′ . is the size of the ranked list. ( ( ) , ( ) ′ ) denotes whether the document pair ( , ) is a discordant pair. A discordant pair is two documents whose relative ranking in one list is different than that in the other list. Specifically, ( ( ) , ( ) ′ ) is defined as, where ( ) ( ) and ( ) ′ ( ) are the rankings of the document in ( ) and ( ) ′ , respectively. Experimental Settings We first introduce our experimental settings, including datasets and implementation details. Datasets. The datasets used for evaluating the defensive ability are as follows. To evaluate the defensive ability against query attack, we conduct experiments on the MS MARCO dataset, which has been described in Section 4. We construct the novel training set by randomly sampling a quarter of queries from the original training set. We randomly sample 20% queries from the novel training set for validation. Besides, we directly use the original development set as the novel test set. To evaluate the defensive ability against document manipulation, we follow the previous work [27] to conduct experiments on the ASRC and ClueWeb09-B dataset. The detailed statistics of these datasets are shown in Table 7. • ASRC. Adversarial Search Collection (ASRC) 11 is an adversarial ranking dataset, which aims to analyze content-based ranking competitions so as to shed light on the strategic behavior of publishers [74]. The competition included 31 different repeated matches, each of which was with respect to a different TREC's ClueWeb09 query. The competition was run for eight rounds. Students are incentivized by course-grade rewards to manipulate their documents, in order to have them ranked higher in the next round. In the following, we will introduce our methods to simulate the adversarial query attack and document manipulation. • Implementation of Query Attack. The MS MARCO dataset is preprocessed in the same way as described in Section 4.3.2. We use the official top 100 ranked documents retrieved by QL. Then, to implement the query attack, we simulate the query attack at the character-level and word-level. For character-level query attack, we follow the previous works [42,70], which are inspired by psycholinguistic studies [19,75]. The psycholinguistic studies demonstrate that humans can comprehend text altered by jumbling internal characters, provided that the first and last characters of each word remain unperturbed. Specifically, we explore to perturb queries with four types of character-level edits: (1) Add, inserting a new lower-case character internally in a word; (2) Remove, deleting an internal character of a word; (3) Substitute: substituting an internal character for any letter; (4) Swap, swapping two adjacent internal characters of a word. For each query in the test set, we firstly randomly choose a word and then attack it using one randomly-chosen character-level edit. We denote such attacked queries as 1-char attacked queries and there are 25.32% Add, 24.96% Remove, 23.34% Substitute and 24.42% Swap in the final obtained 1-char attacked queries. Furthermore, we also randomly select one word from the 1-char attacked queries and then attack it using one randomly-chosen character-level edit [70]. We denote such attacked queries as 2-char attacked queries. We evaluate the performance of different ranking models on the original test queries, 1-char and 2-char attacked test queries respectively. For word-level query attack, we explore to perturb queries with three types of word-level edits: (1) Add, inserting a new lower-case word in a query; (2) Remove, deleting a word of a query; (3) Substitute: substituting a word with a random word. For each query in the test set, we firstly randomly choose a word and then attack it using one randomly-chosen word-level edit. • Implementation of Document Manipulation. We use the public dataset ASRC that was created as a result of an on-going ranking competition [74], to measure the document manipulation [27]. Specifically, the ranking competition involved 31 repeated matches that lasted for 8 rounds, where each match was with respect to a different query. Students in an IR course served as documents' authors. In the first round, in addition to the query itself, students were provided with an example relevant document, and were incentivized by bonus points to the course's grade to modify their documents so as to have them ranked as high as possible in the next round. Starting from the second round, students were presented with the ranking as well as the content of all documents submitted in the previous round in the match. To assure the fairness of the ranking competition, students had no prior knowledge of the ranking function and all data was anonymized. In this way, the ASRC dataset could simulate the document manipulation in the ranking competition. Since the ASRC dataset is too small to effectively train a ranking model, we firstly train the ranking models on the ClueWeb09-B, and then evaluate their defensive abilities on the ASRC [27]. Specifically, we randomly sample 75% queries from the ClueWeb09-B for training and leverage the remaining 25% queries for validation. For the ClueWeb09-B dataset, we use the QL model to retrieve the top 100 ranked documents to build an initial document list. Ranking Robustness to Query Attack In this section, we analyze the empirical results on the defensive ability against query attack. Specifically, we adopt the widely used metric for MS MARCO, i.e., MRR@100, as the implementation of the effectiveness evaluation metric in Eq. (17) and (18). Table 8 and Table 9 show the MRR@100 and DR performance of ranking models against the character-level and the word-level query attack, respectively. We first analyze the defensive ability of different ranking models against the character-level query attack. Then, we analyze the defensive ability of different ranking models against the world-level query attack. Recall that high values of DR attest to increased ranking robustness. 5.4.1 Analysis on the defensive ability against the character-level query attack. We first analyze the defensive ability against the character-level query attack. In the following, we first given an overall analysis of different ranking models. Then, we analyze traditional probabilistic ranking models, LTR models and neural ranking models, respectively. • Overall analysis on all the ranking models. Firstly, we given an overall analysis on all the ranking models against the query attack. We can observe that: (1) In the absence of any query attack, ColBERT performs the best (0.3360) followed by BERT (0.2798), LambdaMART (0.2710) and then BM25 (0.2612), in terms of MRR@100. However, even single-character attacks can be catastrophic, resulting in a significantly degraded performance of 26.1%, 27.6%, 13.2% and 23.3% in terms of DR , for ColBERT, BERT, LambdaMART, and BM25, respectively. (2) For all the ranking models, the MRR@100 performances on both 1-char and 2-char attacked queries are worse than that on the original queries. These results indicate that all the ranking models are not able to generalize to attacked queries quite well. (3) The absolute drop rate value between 2-char attacked queries and original queries are much higher than that between 1-char attacked queries and original queries (e.g., 46.4% v.s. 20.8% for RankSVM). By conducting further analysis, we find that the absolute drop rate value could be larger with more characters in a query attacked. Therefore, it is necessary to improve the robustness of ranking models against the query attack. (4) Overall, in terms of the DR between 1-char attacked and original queries, the relative robustness order of defending against query attack is DRMM < QL < BERT < ColBERT < BM25 < RankSVM < Prank < LambdaMART < Conv-KNRM < Duet < DSSM. • Analysis on traditional probabilistic ranking models and LTR models. When we look at the traditional probabilistic ranking models and LTR models, we find that: (1) Traditional probabilistic ranking models are less robust than LTR models under 1-char attack (e.g., -30.7% of QL v.s. -13.2% of LambdaMART ). Specifically, the adversarial edits might flip words either to a different word in the vocabulary or, more often, to the out-of-vocabulary token UNK. Consequently, adversarial edits can degrade ranking models by transforming the informative words in a query to UNK. For traditional probabilistic ranking models, they emphasize too much on exact matching signals between the query and the document, and treat each unique character combination differently, resulting in the vulnerability to query attack. (2) For the LTR models, LambdaMART is more robust than RankSVM and Prank, especially against the 2-char attack. One possible explanation is that as a listwise LTR method, LambdaMART can make use of the whole ranked document list, which can better reduce the effect of character-level adversarial attacks with more context information than the pairwise LTR method RankSVM and the pointwise LTR method Prank. • Analysis on neural ranking models. When we look at the neural ranking models, we find that: (1) DSSM is the most robust model against both the 1-char and 2-char query attack, but performs poorly on the original query set. The reason might be that DSSM utilizes a character-level n-gram based word hashing, which is more robust to misspelling problems than just treating each word as the basic semantic units. (2) Duet is the second most robust model against both the 1-char and 2-char query attack, while also performs poorly on the original query set. Specifically, Duet leverages both the local and distributed representations for text matching. In the distributed representation, an activation pattern that has some errors or other differences from past data can still be mapped to the query, using a similarity function. In this way, Duet is robust to noise and has the ability to generalize [60]. (3) Since DSSM and Duet have shown strong robustness, a promising direction to design a robust neural ranking model against the misspelling lies in applying the character-level operations. For instance, we could combine pre-training objectives with the n-gram word hashing layer to simultaneously achieve good ranking effectiveness and robustness. (4) DRMM is the least robust model against the 1-char attacked queries. The result indicates that the adversarial edits significantly degrade the DRMM model which directly uses GloVe [68] word embeddings, by transforming the informative words to UNK. Besides, for DRMM, the improvement of the absolute drop rate value with respect to 2-char queries over 1-char queries is only 3.5%. It is an interesting finding that while DRMM is susceptible to adversarial typos, it shows some robustness against the further attacks. (5) When we take a look at the pre-trained models (i.e., BERT and ColBERT ), the phenomenon is absolutely different with DRMM. For example, for ColBERT, the improvement of absolute drop rate value with respect to 2-char queries over 1-char queries is 25.1%, which is the highest among all the ranking models. These results indicate that the pre-trained models are less robust than other ranking models against the query attack. One possible explanation could be that such pre-trained models apply WordPiece [90] tokenization, where a word is broken down into more than one sub-words. In this way, an attacked word in a query may be decomposed into more than one attacked sub-words, which have a more significant impact on the performance. 5.4.2 Analysis on the defensive ability against the word-level query attack. We analyze the defensive ability against the word-level query attack. From the results, we can observe that: (1) Overall, for all the ranking models, the MRR@100 performance on the word-level query attack is worse than that on the original queries. The results indicate that all the ranking models are not able to generalize to word-level attacked queries quite well. (2) In terms of the DR between word-level attacked and original queries, the relative robustness order of defending against wordlevel query attack is QL < ColBERT < DRMM < BERT < BM25 < Prank < RankSVM < Conv-KNRM < LambdaMart < Duet < DSSM. (3) Traditional probabilistic ranking models are less robust than LTR models under word-level attack (e.g., -35.5% of QL v.s. -8.4% of LambdaMART ). Meanwhile, for the LTR models, LambdaMART is more robust than RankSVM and Prank. The reason is similar to what we have analyzed in Section 5.4.1. (4) It is surprising to find that DSSM and Duet are the most robust ranking models against the word-level query attack. In addition, the DR of Conv-KNRM is also relatively high (e.g., -11.0%). The three ranking models all apply the character-level n-gram operations. The results indicates that ranking models which contains a character-level operation will be more robust than those ranking models which treat each word as the basic semantic units. (5) When we take a look at pre-trained models (i.e., BERT and ColBERT), we could find they are not robust compared with other neural ranking models. For example, the DR of ColBERT is -33.9%, which is the highest among all the neural ranking models. One possible explanation could be that WordPiece [90] tokenization decompose an attacked word into more attacked sub-words, which have a more significant impact on the performance. Ranking Robustness under Document Manipulation In this section, we analyze the empirical results on the defensive ability against document manipulation. We first measure the average effectiveness of different ranking models and then show the robustness evaluation results of different models in terms of TC and KT. 5.5.1 Analysis on the effectiveness of ranking models. We first analyze the average effectiveness of ranking models on the ASRC dataset. Specifically, we use the mean average precision (MAP) [79] and normalized discounted cumulative gain at rank 5 (NDCG@5) [6] to evaluate ranking effectiveness [27]. The results are shown in Figure 6. From the results we observe that: (1) All the ranking models can generally achieve good effectiveness on the ASRC dataset. The high MAP and NDCG values can be attributed to the fact that most documents generated by the students were judged to be relevant (e.g., 1113 out of 1279 documents are relevant) [27]. (2) Overall, traditional probabilistic ranking models perform better than neural ranking models and LTR models. (3) Among all the neural ranking models, DSSM performs the best. (4) BERT performs second only to DSSM in neural ranking models. Specifically, we train the ranking models on the ClueWeb09-B dataset and then evaluate them on the ASRC dataset, i.e., under the OOD setting. As noted in Section 4, we have found that BERT shows greater robustness than other neural ranking models in terms of the OOD generalizability, which is quite consistent with the result on the ASRC. (5) Overall, the relative average effectiveness order in terms of MAP is DRMM < Conv-KNRM < ColBERT < Duet < BERT < LambdaMART < DSSM < Prank < QL = BM25 < RankSVM. 5.5.2 Analysis on the robustness of ranking models. Here, we analyze the robustness of ranking models against document manipulation on the ASRC dataset. The results are shown in From the results we observe that: (1) For the traditional probabilistic ranking models, QL and BM25 achieve the lowest TC and KT performance among all the ranking models, indicating that traditional probabilistic ranking models are the most robust to defend against document manipulations. In the generation process of the ASRC dataset, students were asked to modify relevant documents so as to have them ranked as high as possible in the next round. This suggests that traditional probabilistic ranking models have the lowest sensitivity to the difference between relevant documents. (2) For the LTR models, the ranked lists induced by RankSVM and Prank are more robust than those induced by LambdaMART. Since RankSVM and Prank is linear and LambdaMART is not, the variance of RankSVM and Prank is in general lower, and we saw that its ranking robustness is higher. Meanwhile, the ranked lists induced by Prank is less robust than those induced by RankSVM, indicating that pointwise learning objective may suffer more from the document manipulation compared with pairwise learning objective. (3) For the neural ranking models, they achieve comparable results with LTR models against document manipulation. The reason might be that, flipping original words in a document to new words for higher rank, both the statistical features and word embeddings change significantly, resulting in the performance drop. (4) Overall, the relative robustness order of defending against document manipulation (e.g., in terms of TC) is LambdaMART < Prank < DSSM < BERT = Duet < Conv-KNRM < ColBERT < RankSVM < DRMM < QL < BM25. RELATED WORK In this section, we briefly review the works related to our study, including the performance variance over different queries, the OOD generalizability and the adversarial operation. Performance Variance The performance variance over different queries has been studied as an early exploration of robustness in IR [11, 85-87, 99, 100, 104]. The key idea is to pay more attention to the difference on ranking performance, rather than the average effectivenss performance which is considered by most ranking models. Early works [85][86][87] mainly focused on the poorly performing queries of ranking systems. They evaluated the ranking robustness by proposing new metrics which emphasized the poorly performing queries. Beyond the widely-used average performance metrics such as the mean average precision (MAP) and the average of precision at rank 10 (P@10), Voorhees [85] proposed the percentage of queries with no relevant in the top 10 retrieved (%no) and the area underneath the MAP(X) vs. X curve to test the robustness of a ranking model. Besides, Voorhees [86] proposed the gMAP, which is a variant of the traditional MAP measure that used a geometric mean rather than an arithmetic mean to average individual query results, to evaluate the ranking robustness. The proposed gMAP showed promise of giving appropriate emphasis to poorly performing queries while being more stable at equal query set sizes. In addition, Voorhees [87] used the document from AQUAINT Corpus of English News Text to test poorly performing queries and summarized the results of the three-year run of the Robust track. Besides, some works [11,104] proposed the evaluation metrics to analyze the the performance variance over queries. For example, Collins-Thompson [11] proposed the R-Loss and Robustness Index to measure the robustness on the query expansion. R-Loss computed the averaged net loss of relevant documents in the retrieved document, due to the failure on query expansion. And Robustness Index was defined as the difference between the number of improved queries and the number of hurt queries. The improved/hurt queries refer to the queries whose performance is improved/hurt over the original model. Besides, Zighelnic and Kurland [104] proposed < to calculate the percentage of queries for which the retrieval performance of a query model is worse than that of the original query model. However, such robustness metrics were often defined separately from the effectiveness metric (e.g., MAP). There was a lack of a unified formulation to allow them to be evaluated in an integrated manner. Later, a series of methods were proposed based from the perspective of bias-variance decomposition in IR to solve the problem. Zhang et al. [99,100] analyzed the IR robustness from the perspective of bias-variance decomposition of IR evaluation. They proposed the variance of AP to analyze the robustness of ranking models in a unified framework with ranking effectiveness. Cormack and Grossman [13] quantified bias and variance of system rankings by treating rankings as embeddings in a Euclidean space. They then showed that shallow pooling has substantially higher bias and insubstantially lower variance than probability-proportional-to-size sampling. Recently, Shivaswamy and Chandrashekar [81] defined notions of bias and variance directly on pairwise ordering of items. They showed that ranking disagreements between true orderings and a ranking function could be decomposed into bias and variance components. This decomposition is similar to the squared loss and other losses that have been previously studied. OOD Generalizability The OOD generalizability refers to the model's ability to generalize to various new test sets. We first briefly review the works which study the OOD generalizability in Computer Vision (CV) and Natural language processing (NLP). Then we review the recent works in the IR field. Extensive prior works [32][33][34]48] have studied the OOD generalizability on CV and NLP. These works aimed to fairly measure the neural models' OOD generalizability in benchmark dataset of image/text classification. Hendrycks et al. [33] systematically studied seven robustness hypotheses on image classification and proposed three new robustness benchmarks to analyze them. The authors showed that using large models and synthetic data augmentation could improve robustness on real-world distribution shifts. Hendrycks et al. [34] systematically measured OOD generalization for seven NLP datasets by constructing a new robustness benchmark with realistic distribution shifts. By measuring OOD generalization and OOD detection of different neural models, they showed that pre-trained transformers are more effective at detecting OOD examples. Koh et al. [48] focused on the real world distribution shifts and summarized them into two types: domain generalization and subpopulation shift. The authors then proposed eight benchmark datasets in the wild to evaluate these distribution shifts. Recently, since deep learning methods became more prevalent to solve the ranking problems, many works began to study the problem of OOD generalizalibility in IR. Such works mainly focused on the cross domain adaption for neural ranking models. Cohen et al. [10] proposed cross domain regularization on the ranking models to improve their performance on unforeseen domains. They adopted the adversarial learning by using an adversarial discriminator and the gradient reversal layers [22] to train their neural ranking model on a small set of domains. The effectiveness of their proposed architecture was evaluated on cross domain question answering data. Long et al. [55] also leveraged the adversarial learning framework by devising a domain discriminator to solve the problem of domain adaptation. They evaluated the effectiveness of their proposed method on three domain transfer tasks, including cross-domain digits retrieval, image to image and image to videos transfers, on several benchmarks. Mao et al. [58] utilized the multi-layer joint kernelized mean distance to measure the distance between the target data and the source data. They measured the distance based on deep features extracted from the deep networks. The target data which was found by their method was then iteratively added to the training data. In ad-hoc retrieval, Yilmaz et al. [96] aggregated sentence-level evidence to fine-tune BERT models. The methods were proposed to solve the challenges of exceeding document length and document-level relevance judgments for BERT. By this way, the fine-tuned BERT models could capture cross-domain notions of relevance and can be directly used for ranking documents from other domains. Adversarial Operation Deep neural networks have been found vulnerable to adversarial operations [25,82]. Specifically, the key idea of adversarial operation is to find a minimal perturbation in the data that can maximize the risk of making wrong predictions. Early works have extensively studied the adversarial operation in CV [25,57,61,66]. Adversarial operation can be classified as white-box adversarial attacks and black-box adversarial attacks, which means that adversaries have full access to target models (e.g., model's architecture and parameters) or no knowledge about target models, respectively. Fast gradient sign method (FGSM) [25] and its variants (e.g., FGM [61] and PGD [57]) were classical white-box gradient-based attack methods. These methods added the noise to the whole image based on the gradient of loss. On the contrary, another popular approach Jacobian-based saliency map (JSMA) [12,66,89] only perturbed one pixel at a time. It chose the pixel with the highest saliency score, which was defined as the gradient of the target class multiplied by the sum of the gradients of other classes. As a result, JSMA produced adversarial examples which increased the probability of the target class while decreased others. While the above works focused on the gradient information of models, Naseer et al. [62] utilized the transferability of adversarial examples to train substitute models, in order to conduct a black-box attack. Another representative black-box attack was score-based method [9,51], which utilized the output score of model to conduct attack. Adversarial operations have also been conducted to the NLP field, including text classification [20,23,52,78], sentiment analysis [4,50,52,78] and natural language inference [4,59]. Different from CV where the image is represented as continuous data, the main problem of generating adversarial text in NLP is the discrete input space. To address the problem, Gong et al. [24] proposed to attack in the embedding space. They used FGSM to produce perturbations in word embedding and used nearest neighbor search to find the closest words. However, this approach treated all tokens as equally vulnerable and replace all tokens with their nearest neighbors, which led to non-sensical, word-salad outputs [92]. A number of works [38,39,52,77] utilized the ideas of JSMA (e.g., find important pixel) to solve this problem. For example, Jin et al. [39] first found the most important words by sorting all the original words in terms of their importance score. The importance score was calculated as the prediction change before and after deleting the word. Then the word was replaced by synonym which has the most similar semantic meaning with the original word. The candidate synonyms were selected to force the target model to make wrong predictions. This method was more efficient since it attacked the most importance words. Meanwhile, it was classified as a black-box method since it only required the prediction score of the model. In IR, early works on adversarial information retrieval mainly focused on identifying and addressing different types of spam [8]. Recently, Raifer et al. [74] studied the ranking competition on the web retrieval, which means that the document authors manipulate their documents intentionally to have them highly ranked. They constructed the ASRC dataset to simulate the ranking competition and used game theory to analyze the strategies employed by document authors. Following the above work, Goren et al. [27] defined the ranking robustness and analyzed the robustness of ranking functions based on the ASRC dataset. For the work related to the query attack, query spelling correction has been a lively research topic since the mid 2000's, especially in the NLP community [2,16]. The Microsoft Speller Challenge held in the year 2011 [88] also attracted much attention for the problem of query spelling correction. Hagen [31] presented a new large-scale collection of 54,772 queries with manually annotated spelling corrections. A recent work [103] proposed to train a more robust Dense Retriever and BERT re-ranker which are robust to typos in queries. Overall, the research area on the adversarial operation in IR is largely unexplored up till now, and more efforts are expected in this direction in the short future. CONCLUSION AND FUTURE WORK In this paper, we systematically analyzed the robustness of several representative neural ranking models against traditional probabilistic ranking models and LTR models. Specifically, we proposed three ways to define the robustness, i.e., the performance variance under I.I.D. settings, the OOD generalizability and the defensive ability against adversarial operations. The latter two definitions were further specialized into two different perspectives respectively. Overall, the results showed that neural ranking models are in general not robust as compared with other IR models in terms of 3 out of 5 robustness tasks. The exception is that the pre-trained ranking models achieve the best robustness from the perspective of the performance variance, while DSSM, Duet and Conv-KNRM model show robustness in terms of the defensive ability against query attacks. More research efforts are needed to develop robust neural ranking models for IR. Future research efforts could explore novel pre-training objectives and model architectures with character-level operations that enhance the robustness of ranking models. The analysis of the ranking robustness is a foundation for designing ideal ranking models in real world applications. We believe the way we study the robustness (definition and metric) as well as our findings would be beneficial to the IR community. In the future work, we would like to apply the findings to improve the robustness of existing ranking models. We would also try to design new robust neural ranking models based on the findings in this work. Moreover, it is valuable to define a unified formulation based on the different perspectives of the robustness to analyze the ranking models comprehensively.	2021-08-12T01:16:25.041Z	2021-08-11T00:00:00.000	{ "year": 2021, "sha1": "4dde5c87cd8eb425c13270b433f72a0d8dacbefc", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "de8ad480c0e98b5e37586e1e2370a188d3c369b8", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
204260113	pes2o/s2orc	v3-fos-license	EFFECTIVENESS OF CRUDE OIL DEGRADING FUNGI ISOLATED FROM PETROLEUM HYDROCARBON CONTAMINATED SOIL IN SIAK, RIAU Background: Biodegradation of petroleum hydrocarbon needs a specific technique called bioremediation to remove the environmental pollutants. Several indigenous microorganisms including fungi, bacteria, and actinomycetes are effective agents in degrading petroleum derivatives, aliphatic and polyaromatic hydrocarbons (PAHs). Objective: This research aimed to investigate indigenous fungi isolates from petroleum hydrocarbon contaminated soil in Siak which are capable to degrade hydrocarbon. Methods: The competence of indigenous fungi was isolated from a crude oil-contaminated soil which collected from one of oil-field in Siak, Riau. The effectiveness of isolates on the degradation crude oil was tested by culturing the isolates in Bushnell-Haas broth containing crude oil (5% v/v) for 16 days. A decrease in pH, change in optical density and amount of CO2 released were recorded to indirectly indicate the crude oil degradation by the fungi. To measure the percentage of crude oil biodegradation, gravimetric analysis was utilized. Results: The two colonies were selected and identified as Aspergillus sp LBKURCC151 and Penicillium sp LBKURCC153. The results showed that Aspergillus sp LBKURCC151 reached a higher level (61%) of biodegradation after 16 days under the optimum conditions in degrading total petroleum hydrocarbon than Penicillium sp LBKURCC153 (46%). Conclusion: These results indicated that Aspergillus sp LBKURCC151 and Penicillium sp LBKURCC153 are potential degraders for bioremediation in crude oil-contaminated area. INTRODUCTION Petroleum is one of the most important resources in today's industrial economy. However, oil sludge contaminant is an issue that every petroleum industry must face because mainly its quantity. Sludge oil contains different hydrocarbon as well as non hydrocarbon molecules. The toxicity of oil sludge contaminant can cause health effects. [1] Nowadays hydrocarbon removal is possible using different techniques. Although physical and chemical technique is mainly used, Bio-based systems offer interesting alternatives being an economic and environmentally friendly method. The specific technique used to degrade the environmental pollutants is called bioremediation. [2] Riau is one of Indonesia's province with large biodiversity can provide a huge amount of microorganisms isolate source. Indigenous fungi are suitable candidates to clean up the crude oil-contaminated soil. They are known to perform chemical modification and their apical growth mode enables them to reach inaccessible soil regions. These favorable properties are often associated with their ability to grow in environments with low nutrients concentrations, low humidity, and acidic pH. [3] There are several genus of Aspergillus sp, Fusarium sp and Penicillium sp that have good prospect for bioremediation application in Iraq. [4] Result of isolation and identification for indigenous potential microorganisms from bacterial and fungal group from oil contaminated soil located at Cepu East Java showed fungal isolates identified as A. fumigatus (FMC2) and A. niger (FMC6) have the potential to degrade hydrocarbon. [4,5] It would be interesting to find the potential indigenous fungi from Siak which are capable to degrade hydrocarbon. Soil and Crude Oil Samples Petroleum hydrocarboncontaminated samples were collected from one of Oil and Natural Gas Corporation oil-fields, Siak, Riau. Soil sample was collected at a depth of < 1 m from the surface of soil used a hand auger and placed into a sterile container. Crude oil, which was used to test capability of fungi isolates on biodegradation, was obtained from oil fields in Zamrud, Siak that produce Sumatra crude oil. Isolation of Strains from Contaminated Soils Fungi capable of degrading crude oil were isolated by using minimal medium of the following composition (in g L-1 distilled water) : KNO3, 1; K2HPO4, 1; NaCl, 0.1; MgSO4, 0.2; trace of CaCl2 0.1; FeCl3 0.02 g. The Minimal Medium (MM) chemicals were from Sigma Aldrich. Medium contained 250 mg L-1 chloramphenicol. A quantity of crude oilcontaminated soil was added into 50 mL sterilized MM containing 1 % (v/v) crude oil in an Erlenmeyer flask. Flask was shaken for 2 weeks at 150 rpm to homogenize the medium for microbial enrichment. [6] Crude oil-degrading fungi were isolated using dilution plate method. Sodium chloride 0.85 % was used as diluents for inoculums preparation. An aliquot of 1 mL enriched culture was inoculated into a sterile test tube containing 9.0 mL of the diluents. This gave 10 -1 dilution. Subsequently, sevenfold (10 -7 ) serial solutions were prepared from the 10 -1 dilution. 1 mL of dilution was poured on Potato Dextrose Agar (PDA) medium. Plates and media were incubated at 25 0 C in the dark. Selected colonies are transferred to appropriate media to allow fungal development. Stock cultures were maintained on the PDA slant, subcultured periodically and store at 4 0 C. Fungal Growth on Crude Oil All isolated fungi were tested for their capabilities to grow on crude Sumatera light oil as sole carbon source. Fungi were pre-grown in 9 cm diameter Petri dishes containing PDA Medium and incubated at room temperature in the dark for three days. Mycelium disks (5 mm diameter) were taken from the margin of the actively growing colonies, and inoculated in 9 cm plates containing Bushnell-Haas medium [7] added with 15 g L-1 agar, 0.1%(v/v) tween 80 and 5% (v/v) crude oil and without crude oil (control). Plates were inoculated in triplicates and incubated in the dark at room temperature and the mycelium growth were evaluated after 7 days. Two of the highest diameter grown fungal colonies were selected for further experiments. Identification of The Fungal Isolate Lacto Phenol Cotton Blue (LPCB) was employed for microscopic observation. The characteristics were compared with the standard description of "A Manual of Soil Fungi" [8]. Crude Oil Degradation Assay Two strains were selected from the previous experiment and tested for their capability to degrade crude oil in liquid culture by determining the following parameters. Fungi were inoculated as two agar plugs (5 mm diameter) to 98 mL Bushnell Haas Broth medium. This medium was then supplemented with 2 mL crude oil in 250 mL flask. The control flask had no organisms. Incubation was at room temperature (28-30°C) with constant shaking at 180 rev/min for 16 days and all cultures were performed in triplicate. To measure pH, pH of the fermented broth collected aseptically from each fungal isolated was determined using pH meter after 0, 4, 8, and 16 days of treatment. To measure Optical density determination, the optical density of the fermented broth from each fungal isolated was determined after 0, 4, 8, and 16 days of treatment at 620 nm using a spectrophotometer. To measure CO2 estimation, one mL of the fermented broth was taken after 4, 8, 12 and 16 days of treatment and titrated against 0.05 N NaOH solution. Two to three drops of phenolphthalein were used as the indicator and appearance of stable pink color was considered as the endpoint. The following formula can be used to calculate the amount of free CO2. [9] Free CO2 (g L-1) : To measure the percentage of biodegradation by crude oil, the extracted crude oil was analysed using gravimetric analysis. [10] After 16 days of time interval, all flasks were taken out and microbial activities were stopped by adding 1% 1N HCl. Culture broth was mixed with 10% (v/v) hexane in separating funnel and was shaken. The extracted oil was passed through anhydrous sodium sulphate to remove moisture. The hexane was evaporated on 600C water bath using rotary evaporator. The following formula can be used to calculate the percentage degradation of crude oil. [11] Where Wa : is weight of crude oil (initial) and Wb : is weight of crude oil (after treatment). RESULTS Two fungi strains were isolated from crude oil contaminated soil in siak Riau. The fungi were identified as Aspergillus sp LBKURCC151 (figure-1A) and Penicillium sp LBKURCC153 (figure-1B) based on the microscopic and macroscopic observation. The fungi were grown on PDA, and their cultural and morphological characteristics are summarized in Table 1. The two strains that displayed the highest diameter growth in previous experiment were studied for their capability to degrade crude oil in a liquid medium. The ability of the two selected fungal isolates in degrading crude oil were evaluated through three parameters: 1) the change in pH of fermented medium ( Figure 2); 2) the growth of fungi as detected by optical density (Figure 3); 3) CO2 released during degradation assay ( Figure 4). Furthermore, percentage of biodegradation was determined by gravimetric analysis in final fermented day ( Figure 5). Discoloration of fermented media showed in figure 6. DISCUSSION Aspergillus and Penicillium have been frequently reported as filamentous fungi which can remove hydrocarbons. [12] These fungi were able to grow and display highest diameter growths in Bushnell-Haas agar medium containing 5% (v/v) crude oil as sole carbon source, but with different efficiency compared to controls. These fungi isolates formed a fine and uniform mycelium compared to their growth in BHM agar without addition of 5% (v/v) crude oil. Figure 2 depicts the variations in the pH of medium during the fermented period and pH was found to be decreasing gradually from 7 -5.7 and 7-6.3 for Aspergillus sp LBKURCC151 and Penicillium sp LBKURCC153, respectively. The acidic environment might indicate the degradation of crude oil and a higher catabolic activity causes the pH to decrease within 16-day incubation period. [13] Aspergillus sp LBKURCC151 grew better in acidic condition than Penicillium sp LBKURCC153. Previous study also reported that several fungal isolates such Aspergillus niger, Trichoderma viride and Fusarium solani showed good growth in culture medium at pH 5.5. [14] Many species can metabolize over a wide pH range from highly acidic to extreme alkaline. Fungi have higher insensitivity to high hydrogen ion concentration and narrow pH range than bacteria. Decreasing pH levels probably is caused by organic acids production and other metabolic products by microbial degradation of hydrocarbon. Figure 3 shows the variations in the optical density which seems to be fluctuating change in optical density at 620 nm were recorded after 4, 8, 12 and 16 fermented days. The growth rate of each fungus had different maximum growth peaks. Aspergillus sp. LBKURCC151 had a maximum growth on the 8th day at 1.701 while Penicillium sp. LBKURCC153 had a maximum growth at 0.563. There were wide fluctuations in the growth of each fungus. The change in optical density could be attributed to the fact that they used the hydrocarbons as substrates for growth by probably releasing extracellular enzymes and acids which are capable of breaking down the recalcitrant hydrocarbon molecules, dismantling the long chains of hydrogen and carbon, thereby, converting petroleum into simpler forms or products that can be absorbed for the growth and nutrition of the fungi. This result was similar to previous research which shows that growth pattern of fungi in minimal salt had fluctuations. [15] Figure 4 shows an increase in CO2 released during the degradation of crude oil by these fungi. Release of carbon dioxide during the degradation can be used as an indication for activity of fungi in the growth media. Aspergillus sp LBKURCC151 showed more CO2 production than Penicillium sp LBKURCC153. In the previous study, Penicillium sp could decompose the substrate hydrocarbon and release more CO2 because of enzymes that were produced at a faster rate. Therefore this fungus can be utilized effectively as an agent of petrol degradation. [8] A large amount of CO2 was liberated in BHM with isolates than in control medium. Respiration of microorganisms occur very rapidly during the initial period of incubation when the lighter and more readily degraded fractions are degraded but slow down as the residue becomes more difficult to degrade on account of the increase of heavier fractions eg. aromatic compounds. The ability to analyses crude oil compounds into its components leads to oxidation of the carbon source in the crude oil components. Figure 5. Aspergillus sp LBKURCC151 and Penicillium sp LBKURCC153 were the fungi isolates demonstrating active ability to biodegrade crude oil in this study. There was a great difference between the fungi isolates that were inoculated in Bushnell-Haas broth media amended with 5% (v/v) crude oil compared to those inoculated in Bushnell-Haas broth media without 5% (v/v and Penicillium documbens (7.9%) after 3 weeks of incubation. [7] In earlier study also showed the same results with Aspergillus niger had highest percentage of hydrocarbons degradation. [11] Aspergillus species are the most efficient for utilizing hydrocarbons in crude oil compared to different genera. [9] In this study, two fungi isolates were able to grow in 5% (v/v) and have capability to degrade crude oil which showed discoloration of fermented media ( figure 6). Their capacity to remove hydrocarbon could be due to adaptation of these fungi to the pollutant composition, as well as to the enzymatic systems of the fungi. [16] CONCLUSION The data contained in this study show that two indigenous fungi isolates of crude oil contaminated soil have the potential in degrading petroleum hydrocarbon pollutants. This results confirmed by gravimetric analysis that have biodegradation percentage of Aspergillus sp LBKURCC151 and Penicillium sp. LBKURCC153 isolates reached 61% and 46%, respectively. To implement the isolates or their products, e.g., enzymes as bioremediating agents, through studies on optimal growth conditions, degradation mechanism and microorganisms consortium are necessary.	2019-09-26T09:08:50.479Z	2019-09-21T00:00:00.000	{ "year": 2019, "sha1": "a8d65854de2cb23180cad3c81fbe306568b3c302", "oa_license": null, "oa_url": "https://doi.org/10.32889/actabioina.v2i1.35", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "8470478f0bd2152b89a6c9c5bb0dfdda036a6cd3", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Chemistry" ] }
249543774	pes2o/s2orc	v3-fos-license	IL‐31 levels correlate with pruritus in patients with cholestatic and metabolic liver diseases and is farnesoid X receptor responsive in NASH Background and Aims: Pruritus is associated with multiple liver diseases, particularly those with cholestasis, but the mechanism remains incompletely understood. Our aim was to evaluate serum IL‐31 as a putative biomarker of pruritus in clinical trials of an farnesoid X receptor (FXR) agonist, cilofexor, in patients with NASH, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC). Approach and Results: Serum IL‐31 was measured in clinical studies of cilofexor in NASH, PSC, and PBC. In patients with PSC or PBC, baseline IL‐31 was elevated compared to patients with NASH and healthy volunteers (HVs). IL‐31 correlated with serum bile acids among patients with NASH, PBC, and PSC. Baseline IL‐31 levels in PSC and PBC were positively correlated with Visual Analog Scale for pruritus and 5‐D itch scores. In patients with NASH, cilofexor dose‐dependently increased IL‐31 from Week (W)1 to W24. In patients with NASH receiving cilofexor 100 mg, IL‐31 was higher in those with Grade 2–3 pruritus adverse events (AEs) than those with Grade 0–1 pruritus AEs. IL‐31 weakly correlated with C4 at baseline in patients with NASH, and among those receiving cilofexor 100 mg, changes in IL‐31 and C4 from baseline to W24 were negatively correlated. IL‐31 messenger RNA (mRNA) was elevated in hepatocytes from patients with PSC and NASH compared to HVs. In a humanized liver murine model, obeticholic acid increased IL‐31 mRNA expression in human hepatocytes and serum levels of human IL‐31. Conclusions: IL‐31 levels correlate with pruritus in patients with cholestatic disease and NASH, with FXR agonist therapy resulting in higher serum levels in the latter group. IL‐31 appears to derive in part from increased hepatocyte expression. These findings have therapeutic implications for patients with liver disease and pruritus. INTRODUCTION Pruritus is a poorly understood and debilitating feature of cholestatic liver diseases including primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). The pathobiology of cholestasis-associated pruritus remains incompletely understood, but elevated serum bile acid (BA) levels may play a prominent role. [1] Interestingly, although farnesoid X receptor (FXR) agonists effectively reduce BA levels in patients with PBC and PSC, increases in pruritus have been reported in patients treated with these therapies. [2][3][4][5] These observations suggest that other potential pruritogens (e.g., autotaxin, individual BA species, others) [6][7][8] may contribute both to the etiology of pruritus associated with cholestasis and FXR agonist therapy. FXR is an intracellular ligand-activated nuclear receptor expressed in intestinal epithelial cells and hepatocytes that functions as an enterohepatic regulator of BA homeostasis. [9] Activation of FXR by smallmolecule agonists, or its natural ligand BA, in intestinal epithelial cells results in elevated expression and secretion of fibroblast growth factor (FGF)-19 into the portal circulation. [9,10] FGF-19 binds to its cognate receptor, FGF receptor (FGFR) 4/β-Klotho receptor complex, on hepatocytes and transcriptionally represses CYP7A1, the rate-limiting enzyme for BA synthesis via a kinase mediated signaling cascade. [10] Changes in circulating FGF-19, or BA precursors like C4, are biomarkers for FXR activity. BA-derived steroidal FXR agonists such as obeticholic acid (OCA) or fully synthetic nonsteroidal FXR agonists are approved therapies for PBC or under clinical evaluation in patients with NASH and PSC, respectively. [11][12][13][14][15][16] Although pruritus is not typically associated with NASH, it is a common adverse event associated with FXR agonist therapy. [11,13,14,16] In the phase 3 trial of OCA in NASH, 51% of patients treated with OCA 25 mg reported pruritus and 9% discontinued the study due to treatment-related pruritus. [16] Nonsteroidal small-molecule FXR agonists have also been implicated in inducing pruritus, [13,14,17] suggest that pruritus is a class effect of FXR agonism. However, the pruritogen associated with FXR agonist treatment has not been identified. As described above, FXR agonists regulate the BA synthesis pathway by either directly activating hepatocyte FXR or through the FGF-19-FGFR 4/β-Klotho receptor axis. Given that patients with NASH treated with the engineered variant of FGF-19, aldafermin (previously NGM-282), have not reported pruritus, [18] we hypothesized that the pruritogen associated with FXR agonists may involve direct FXR agonism that is independent of FGF-19 signaling. IL-31 is a known pruritogen and an established drug target in atopic dermatitis. [19,20] IL-31 and its receptor heterodimers, IL-31 receptor A (IL-31RA) and oncostatin M β, mediate itch signaling through sensory neurons in the dorsal root ganglion. [21,22] Overexpression of IL-31 in rodents or injection of IL-31 in dogs, monkeys, or humans induces scratching behavior in animals and chronic itch sensation in humans. [19,[23][24][25] Nemolizumab, an anti-IL-31RA antibody, significantly reduces pruritus severity and the sensation of itch in patients with atopic dermatitis. [26,27] The link between IL-31 and pruritus in patients with liver disease is not well described. In a study of patients with PBC, serum IL-31 levels were positively correlated with platelet count and negatively correlated with the Fibrosis-4 index. [28] Pregnant women with intrahepatic cholestasis and pruritus have higher serum IL-31 levels compared to control patients. [29] However, the correlation of IL-31 with cholestatic itch and the underlying mechanisms associated with FXR agonist therapy remain unclear. The objective of this study was to evaluate serum IL-31 as a putative biomarker of pruritus in clinical trials of an FXR agonist in patients with NASH, PBC, and PSC. This study investigated IL-31 and associations between changes in serum IL-31 and pruritus. Patient populations for clinical serum samples Human serum samples obtained from five clinical studies of cilofexor were used in this analysis. Samples were taken from healthy volunteers (HVs; n = 60, baseline) who participated in a phase 1 study (NCT02654002) [30] and in phase 2 placebo-controlled studies of patients with noncirrhotic NASH (n = 140; NCT02854605), [11] cirrhotic NASH (n = 20; baseline samples from cohorts 7 and 8 in NCT02781584), noncirrhotic PSC (n = 52; NCT02943460), [12] and noncirrhotic PBC (n = 71; NCT02943447) ( Figure S1). All the subjects provided appropriate consent for participating in the clinical study, and the study was conducted in accordance with the ethics and/or institutional review committees. Commercially acquired liver biopsy samples for histology study Liver biopsy samples used for messenger RNA (mRNA) in situ hybridization (ISH) experiments were acquired commercially from HVs (TriStar Technology Group) and patients with histologically confirmed NASH (BioIVT) and PSC (Discovery of Life Sciences). Demographic information for these commercially acquired samples is provided in Table S1. Assays for serum cytokines Serum IL-31 and IL-4 were detected by Quanterix Simoa HD-1 analyzer, and single molecule array platforms were performed by Myriad RBM. Capture antibody conjugated paramagnetic beads were incubated with standards, samples, or controls and biotinylated detection antibodies. The beads were then washed and incubated with streptavidin-ß-galactosidase. After the final wash, the beads were loaded into the Simoa Disc with enzyme substrate, resorufin ßgalactopyranoside. The concentration of IL-31 or IL-4 in each sample is interpolated from a standard curve. The detection limit for IL-31 using the Quanterix platform was 0.06-0.089 pg/ml. The coefficient of variation for the same samples tested in different batches was 12% (interquartile range [IQR], 5, 25) with Spearman correlation of 0.98 (p < 0.001). Additional information on other commercially available enzyme-linked immunosorbent assay kits for detecting IL-31 and the Quanterix Simoa assay, including its sensitivity and specificity for samples in these experiments, is provided in the Supporting Materials. IL-31 mRNA expression and ISH Hepatic IL-31 (Thermo Fisher, assay ID: Hs01098710_m1) quantification was analyzed using a droplet digital polymerase chain reaction (ddPCR) assay in a QX200 ddPCR System (Bio-Rad). This was a duplex assay with a housekeeping TATA-box binding protein (TBP; Thermo Fisher, assay ID: Hs99999910_m1). The droplets were generated in a QX200 Droplet Generator (Bio-Rad) according to the manufacturer's instructions. The relative IL-31 expression was normalized to the TBP expression in the same well. IL-31 ISH was performed by a standard RNAscope platform described previously using a commercial vendor (ACDBio). [31] The IL-31 ISH utilized Fast Red Dye to visualize the positive signal, and all slides were counterstained with hematoxylin for nuclear staining. The nuclei were detected by VisioPharm AI Nuclear Detection deep learning algorithm (Vision PHARM Hoersholm Denmark) at the whole slide level. IL-31 positive nuclei were counted for each sample to calculate the percentage of IL-31 positive nuclei ( Figure S8). Chimeric mouse with humanized liver PXB mice with stable human hepatocyte engraftment in uPA severe combined immunodeficiency background were obtained from PhoenixBio. The human hepatocyte engraftment was assessed by the proportion of human albumin in the serum (> 90%). PXB mice were administered OCA (10 mg/kg, BID) for 18 days, and livers and plasma were collected 2 h after the last dose for measurement of IL-31 mRNA and protein. Statistical analysis Wilcoxon rank-sum test was used to assess differences between groups in continuous variables, including serum IL-31 levels according to the severity of pruritus (moderate to severe [Grade 2-3] vs. no or minor [Grade 0-1]) and treatment group (cilofexor 100 mg, 30 mg, vs. placebo). Associations between serum IL-31, BAs, and autotaxin levels and a Visual Analog Scale (VAS) for pruritus and 5-D itch scores were assessed using Spearman correlations (ρ). Information on imputation of missing biomarker data can be found in the Supporting Material. RESULTS Increased serum IL-31 levels in patients with liver disease IL-31 levels were measured in baseline serum samples of HVs and patients with cirrhotic NASH and noncirrhotic NASH, PBC, and PSC who participated in clinical trials of cilofexor. Compared to HVs, serum IL-31 levels were elevated in all groups of patients with liver disease. In NASH, median (IQR) serum IL-31 levels were higher among patients with cirrhosis (n = 20) than among noncirrhotic patients (n = 52) (0.3 pg/ml vs. 0.09 pg/ml, p < 0.001; Figure 1A). ( Figure 1A). Noncirrhotic patients with PBC and PSC had higher levels of IL-31 compared to patients with cirrhotic and noncirrhotic NASH (PBC 1.7 pg/ml [0.8, 5.5]) and PSC (2.7 pg/ml [0.8, 5.9], both p < 0.001 versus cirrhotic and noncirrhotic NASH). Serum IL-31 levels were significantly correlated with serum BA levels (ρ = 0.53, p < 0.001) in the overall NASH, PBC, and PSC population ( Figure 1B) but not with other biomarkers of liver injury and fibrosis such as alanine aminotransferase, aspartate aminotransferase, or Enhanced Liver Fibrosis score (data not shown), suggesting that increased serum IL-31 in patients with liver disease is predominantly related to the degree of cholestasis. Cilofexor increased serum IL-31 in patients with NASH and correlated with pruritus In the 24-week phase 2 study of cilofexor in noncirrhotic NASH (NCT02854605), 8 of 56 (14%), 1 of 56 (2%), and 0 of 28 patients receiving cilofexor 100 mg, cilofexor 30 mg, or placebo, respectively, had Grade 2-3 pruritus (Table 1). Several potential pruritogens were explored in the study, including changes in serum BA concentration and composition, autotaxin, and IL-31 levels. Baseline BA levels were not different between patients with Grade 2-3 pruritus compared to patients without pruritus at Weeks 1 or 4 (data not shown), and BA composition [11] or autotaxin levels were not associated with cilofexor treatment ( Figure S2). Cilofexor increased median serum IL-31 in a dose-dependent manner, with cilofexor 100 mg resulting in significantly higher serum IL-31 levels compared to placebo as early as the first week ( Table 3). Patients with Grade 2-3 pruritus had significantly higher serum IL-31 serum levels than those with Grade 0-1 pruritus at all postbaseline time points and as early as the first week: W1 (5.1-fold increase, p = 0.002), W4 (2.9-fold increase, p = 0.005), W8 (3.8-fold increase, p = 0.014), W12 (4.5-fold increase, p < 0.001), and W24 (7-fold increase, p = 0.01) ( Figure 2B, Table 3). These data indicate that cilofexor dose-dependently increased IL-31 levels in patients with NASH, with this increase associated with higher grades of pruritus. Cilofexor induced higher serum IL-31 in patients with NASH compared to HVs In the phase 1 study (NCT02654002), HVs were treated with cilofexor 30, 100, or 300 mg, and serum IL-31 or FGF-19 levels were assessed in predose and postdose (1-8 h) samples. A similar intensive sampling study was also performed in the study of cilofexor in noncirrhotic NASH. Cilofexor had similar target engagement in HVs and patients with NASH, as demonstrated by similarly increasing FGF-19 levels with 100 mg of cilofexor in HVs and patients with NASH (peak concentrations of 591.6 pg/ml and 829.7 pg/ml at 4 h postdose, respectively) ( Figure S3A). However, the same dose of cilofexor 100 mg led to higher serum levels of IL-31 in patients with noncirrhotic NASH compared to HVs. In HVs, serum IL-31 achieved a peak concentration of 0.19 pg/ml (0.1, 0.94), representing a 2.6-fold increase from predose levels ( Figure S3B). In contrast, the peak serum concentration of IL-31 in patients with noncirrhotic NASH was 13 pg/ml, a 23.6-fold increase from predose ( Figure 2C). Thus, induction of IL-31 secondary to cilofexor was more pronounced in patients with NASH than in HVs. Increased serum IL-31 in NASH is correlated with pharmacodynamic markers of FXR agonism FGF-19 and C4 are established pharmacodynamic biomarkers of FXR agonism and associated with BA synthesis. In serially collected serum samples from patients with NASH, 100 mg cilofexor increased both IL-31 and FGF-19 to peak concentrations at 4-6 h postdose ( Figure 2D). At W24, C4 levels were significantly lower in both the cilofexor 30 mg and 100 mg groups compared to placebo, as previously reported. [11] Although serum IL-31 was weakly correlated with C4 at baseline (ρ = -0.25, p = 0.068), changes in IL-31 and C4 from baseline to W24 in the cilofexor 100 mg group were significantly negatively correlated ( Figure 2E, ρ = −0.46, p < 0.001). The correlations between changes in serum IL-31 and pharmacodynamic markers of FXR agonism provide further evidence of an on-target effect of cilofexor. Cilofexor did not alter serum IL-31 levels in patients with PSC or PBC Serum IL-31 levels were assessed in two phase 2, placebo-controlled studies of cilofexor in PBC and PSC at baseline, W1, W4, and W12. In contrast to the increased serum IL-31 levels observed in patients with NASH (Figure 2A), cilofexor did not alter serum IL-31 in patients with PSC ( Figure 3A). Cilofexor did not exacerbate pruritus in patients with PSC with a numerically lower proportion of patients having Grade 2-3 pruritus compared to patients administered placebo. [12] Cilofexor also did not alter the VAS for pruritus or the 5-D Itch score in patients with PSC or PBC from W1 to W12 compared to placebo ( Figure S4). Associations between serum IL-31 and these patientreported outcomes were analyzed in patients with PSC and PBC at baseline. Serum IL-31 levels were (Table S2), confirming previous reports that autotaxin is associated with cholestatic itch. [7] In patients with PBC, treatment with cilofexor did not affect serum IL-31 levels in samples collected at W1, W4, and W12 compared to patients receiving placebo ( Figure 4A). At baseline, serum IL-31 was correlated with VAS (ρ = 0.39, p < 0.001) and 5-D Itch scores (ρ = 0.48, p < 0.001) ( Figure 4B,C). In serially collected serum samples collected postdose, 100 mg cilofexor mildly increased serum IL-31 levels from a median of 3 pg/ml at predose to 7.4 pg/ml (2.5-fold increase) at 5 h postdose ( Figure 4D). Cilofexor did not activate T helper 2 cells in patients with NASH and PSC We wanted to explore the cellular source of IL-31 in patients with liver disease. IL-31 is expressed and secreted by activated T helper 2 (Th2) cells, and Th2 cells contribute to elevated serum IL-31 in patients with atopic dermatitis. [19,32] IL-4 is a type II immune cytokine that increases with Th2 response. [33,34] In patients with NASH, cilofexor 100 mg did not increase serum IL-4 concentrations, whereas cilofexor 30 mg caused a numerical decrease in IL-4 levels that returned to baseline by W24 ( Figure S5A). The changes in serum IL-31 in patients with NASH treated with cilofexor were not significantly correlated with changes in serum IL-4 levels. Patients with PSC experienced a decrease in median IL-4 levels as soon as W1 that normalized to near (but less than) baseline levels by W12 (Figure S5B). Thus, cilofexor did not alter serum IL-4 levels in patients with NASH or PSC, suggesting that the increased serum IL-31 in patients with NASH treated with cilofexor is unlikely due to Th2 cell activation. IL-31 expression is elevated in hepatocytes from patients with NASH and PSC The correlation between serum IL-31 and C4/FGF-19 in patients with NASH treated with cilofexor suggested a direct effect of cilofexor on the primary FXR-expressing cells, hepatocytes, and/or intestinal epithelial cells. We investigated IL-31 expression by ISH in commercially procured samples of ileum and liver from HVs and patients with NASH and PSC and found IL-31-positive (IL-31 + ) cells only present in the liver ( Figure S7). In NASH and PSC, both nonparenchymal cells and hepatocytes stained positively for IL-31 in liver biopsies, whereas IL-31 + staining in liver samples from healthy donors was nearly absent ( Figure 5A). The number of IL-31 + cells were elevated in the liver biopsy samples from patients with NASH (0.74%) and PSC (0.85%) compared to HVs (0.18%) ( Figure 5D). The IL-31 + hepatocytes were located in both periductal and central venous areas, without a clear zonal distribution. OCA elevated IL-31 expression and secretion from human hepatocytes in PXB mouse We next evaluated the direct effect of the FXR agonist OCA (10 mg/kg once daily vs. vehicle for 18 days) on human hepatocytes in chimeric PXB mice. The Simoa assay was validated to detect human (not murine) IL-31 ( Figure S6). OCA increased IL-31 hepatic mRNA expression (7.8-fold higher than vehicle, p < 0.001) ( Figure 6A). The chimeric mice treated with OCA also Figure 6B). Hepatic IL-31 mRNA correlated with serum IL-31 levels in PXB mice treated with OCA (ρ = 0.61, p < 0.001) ( Figure 6C). The FXR agonist, OCA, therefore directly increased hepatocyte IL-31 gene expression and circulating human IL-31 in the humanized liver murine model. DISCUSSION Pruritus, a poorly understood and debilitating feature of cholestatic liver disease, is also commonly associated with treatment with OCA [2,3,15] and other nonsteroidal FXR agonists. [5,11,13,14] IL-31 is a known pruritogen whose blockade significantly reduces pruritus in patients with atopic dermatitis. [26,27] The present study demonstrated that circulating levels of IL-31 correlate with pharmacodynamic markers of FXR agonism in recent clinical trials of the FXR agonist cilofexor. In a clinical trial evaluating the nonsteroidal FXR agonist cilofexor as a treatment for NASH, the highest serum IL-31 levels were observed in patients who reported the most severe pruritus. Experimentally, the steroidal FXR agonist OCA directly increased circulating human IL-31 concentrations and hepatic IL-31 expression in a humanized liver murine model. Therefore, the present study provides a potential explanation for the somewhat perplexing observation of why both steroidal and nonsteroidal FXR agonists may cause pruritus despite concomitant reductions in levels of BAs. [2,5,11,[13][14][15]35] F I G U R E 6 Elevated hepatic IL-31 expression and plasma IL-31 in PXB mice treated by obeticholic acid (OCA). Chimeric mice treated with OCA had higher levels of IL-31 than vehicle controls in liver messenger RNA (mRNA) (A) and circulating (B). (C) The liver IL-31 mRNA and circulating IL-31 protein was significantly correlated. To the best of our knowledge, the cellular source of circulating IL-31 in liver diseases had not been previously reported. Dillon et al. first established IL-31 as a direct pruritogen experimentally and identified its highest expression in activated T helper cells. [19] In our study, serum IL-31 had a weak association with serum IL-4, but significantly correlated with circulating BA levels. This association suggested that the liver may represent a source of IL-31. Indeed, we observed that circulating IL-31 levels were elevated in patients with NASH, PSC, and PBC versus healthy controls with the highest levels found in the cholestatic disorders ( Figure 1A). ISH demonstrated nuclear localization of IL-31 RNA mostly in hepatocytes and in some nonparenchymal cells in NASH and PSC livers but not in healthy controls. Experimental FXR agonism with OCA in the humanized liver murine model demonstrated a significant increase in IL-31 gene expression and nearly a 10-fold increase in circulating human IL-31 protein. These findings suggest that FXR agonism in the liver is the primary determinant of IL-31 given the species differences in FXR found in this murine model where only the hepatocytes are human in origin. Other recent studies also point to an increase in circulating IL-31 with the development of experimental NASH. [36] Together these data provide evidence that the liver may represent a significant source of circulating IL-31 in clinical liver diseases. The complex causal mechanisms that contribute to pruritus led us to investigate other postulated pruritogens in the present study. Studies of BA and its receptor, Masrelated GPR family member X4 (MRGPRX4), and their relation to pruritus are under investigation. Deoxycholic acid (DCA), an unconjugated secondary BA, is among the most potent ligands for MRGPRX4. [37][38][39] However, despite reductions in total BA levels with cilofexor treatment in both NASH and PSC, relative levels of DCA, as well as the overall BA composition, did not change. [12,17] Experimentally, the addition of OCA to the PXB humanized mice did not alter the hepatic expression of MRGPRX4 (data not shown). However, this finding does not preclude a role for this receptor in the dorsal root ganglion in mediating cholestatic itch. Autotaxin, a secreted enzyme that generates lysophosphatidic acid, is elevated in patients with PBC with cholestatic itch. [7] Our study confirmed significant correlations between VAS and 5-D Itch scores with baseline serum autotaxin levels in patients with PBC and PSC (Table S2). However, our studies of cilofexor in NASH and PSC ( Figure S2) and a previous study of OCA in patients with PBC, [2] indicated that FXR agonism does not alter serum autotaxin levels. These findings suggest that the pruritus associated with FXR agonists may be distinct from the pruritus that originates from excess autotaxin activity. The notion that multiple molecular mediators may differentially contribute to a complex symptom such as pruritus is suggested by the discrepant findings in this study in which patients with NASH demonstrated induction of IL-31 with cilofexor, whereas patients with PBC and PSC showed no increase. In patients with PSC and PBC, baseline IL-31 levels correlated with pruritus, but levels of IL-31 were six to nine-fold higher in patients with PBC/PSC versus cirrhotic NASH at baseline. In the Phase 2 NASH study, cilofexor led to a 13-fold increase in IL-31 levels that were associated with the development of pruritus. These cilofexor-induced levels, however, were still below the levels seen in cholestatic liver disease at baseline prior to cilofexor treatment. In contrast, cilofexor did not increase IL-31 in both cholestatic diseases, suggesting a potential "ceiling" effect of this cytokine. Determining the relative contribution of IL-31 in mediating pruritus in these various liver diseases will likely require targeted therapies that interfere with IL-31 directly. Identification of IL-31 as a causative pruritogen in other liver diseases, most notably cholestatic disorders, could provide clinical benefit to patients suffering from intractable pruritus. The ileal BA transporter (IBAT) inhibitor, linerixibat, reduced cholestatic itch in patients with PBC with moderate-to-severe pruritus [40] ; another IBAT inhibitor, maralixibat, reduced serum BA levels and pruritus in patients with Alagille syndrome, an inherited cholestatic disease often characterized by severe pruritus. [41] Determining whether IL-31 levels decline in response to successful antipruritic interventions such as BA binding resins or IBAT inhibitors would provide further support for the role of IL-31 in mediating cholestatic pruritus. Therapeutics directly targeting IL-31 such as nemolizumab (an anti-IL-31RA antibody) and abrocitinib (a Janus kinase inhibitor, targeting IL-31 and other inflammatory pathways) have demonstrated efficacy in mitigating pruritus associated with atopic dermatitis. [26,27,42] In addition to their ability to ameliorate pruritus in patients with atopic dermatitis, these molecules also have a known safety profile. [26,27,42] Future randomized trials using clinically characterized anti-IL-31 blocking agents would provide a critical test of IL-31's role as a cytokine in mediating the pruritus associated with cholestatic diseases. In conclusion, clinical use of the FXR agonist cilofexor is associated with elevated serum IL-31 and pruritus severity in NASH, whereas baseline levels of IL-31 in PSC and PBC were associated with pruritus. FXR agonism in a humanized hepatic murine model directly induces IL-31, thereby demonstrating that the liver can be a source of this putative pruritogen in response to FXR agonists. These findings have therapeutic implications for the pruritus associated with FXR agonists and warrant exploration in other etiologies, especially in those patients with cholestatic disorders. FUNDING INFORMATION Funding for this project was provided by Gilead Sciences, Inc. Funding for this analysis was provided by Gilead Sciences, Inc. Medical writing support for the initial draft was provided by Gregory Suess, PhD, of AlphaScientia, LLC. CONFLICT OF INTEREST Andrew N. Billin owns stock in and is employed by Gilead. Christopher L. Bowlus consults for and received grants from Cymabay, GSK, and Mirum. He received grants from Intercept, Takeda, BMS, BiomX, Gilead, Novartis, and Pliant. Chuhan Chung owns stock in and is employed by Inipharm. He owns stock in Gilead. Jen-Chieh Chuang owns stock in and is employed by TLC Inc. Jun Xu owns stock in and is employed by Gilead. Keyur Patel consults for Gilead. He advises Intercept. Kris V. Kowdley consults for, is on the speakers' bureau for, and received grants from Gilead and Intercept. He consults for and received grants from Hightide, Mirum, NGM, and 89 Bio. He consults for Madrigal, PTG, and Genfit. He is on the speakers' bureau for AbbVie. He received grants from GSK, Pliant, Hanmi, Viking, and Pfizer. Lauri Diehl owns stock in and is employed by Gilead. Michael Trauner consults for, is on the speakers' bureau for, and received grants from Falk, Intercept, Gilead, and MSD. He consults for and received grants from Alberio. He consults for BiomX, Boehringer Ingelheim, Genfit, Janssen, Novartis, Shire, Phenex, and Regulus. He received grants from Cymabay, Takeda, Alnylam, Ultragenyx, and AbbVie. He is the coinventor of patents on the medical use of 24-norursodesoxycholic acid. Matt Peach owns stock in and is employed by Gilead. Mazen Noureddin owns stock in Anaetos, Chrownwell, Ciema, Rivus Pharma, and Viking. He advises and received grants from Gilead, Pfizer, and Madrigal. He advises 89BIO, Altimmune, cohBar, Cytodyn, Intercept, Novo Nordisk, Blade, EchoSens, Fractyl, NorthSea, Perspecturm, Terns, Siemens, and Roche. He received grants from Allergan, BMS, Galmed, Galectin, Genfit, Shire, Zydus, Conatus, Enanta, and Novartis. Mina Khoshdeli owns stock in and is employed by Gilead. Robert P. Myers owns stock in and is employed by The Liver Company. He receives stock from Gilead. Ryan S. Huss owns stock in and is employed by The Liver Company. He receives stock from Gilead. Wiley Minto owns stock in and is employed by Gilead. DATA AVAILABILITY STATEMENT Anonymized individual patient data will be shared upon request for research purposes dependent upon the nature of the request, the merit of the proposed research, the availability of the data, and its intended use. More information on data transparency from Gilead Sciences, Inc., can be found at https://www.gileadclin icaltrials.com/transparency-policy/.	2022-06-11T06:16:30.209Z	2022-06-10T00:00:00.000	{ "year": 2022, "sha1": "c7869d19c4a7fdb7b8512561637b5b22c231aeab", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "WoltersKluwer", "pdf_hash": "9b2ad17d3fcc44659d3d4a669b075b80a3be42fa", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
225703984	pes2o/s2orc	v3-fos-license	Stigma related to mental health issues – a study among adolescents in Sri Lanka One definition of stigma is that it is a “collection of negative attitudes, beliefs, thoughts, and behaviours that influence the individual or the general public to fear, reject, avoid, be prejudiced, and discriminate against people” (1). Stigma impedes treatment seeking, erodes self-esteem and limits the individual’s social network. Although stigma was described as a unitary characteristic in earlier studies, currently it is understood as being more complex and having many dimensions and attributes (2-4). Culture also influences the way stigma relating to mental illness is presented (5). Stigma related to mental health issues -a study among adolescents in Sri Lanka outcomes (13). Thus, reducing stigma and promoting early help seeking is essential in improving treatment results. Although stigma among adolescents has been studied amongst western populations, there are very few studies conducted amongst Asian populations (3,(14)(15)(16). According to the most recent population and housing census In Sri Lanka, adolescents (aged between 10-19 years, both male and female) account for approximately 16.2% of the population (17). While there are no major studies on this subject among adolescents, a study among undergraduates in early adulthood concluded that creating greater awareness about mental health issues improved help-seeking (18). Therefore, the objective of this study is to describe several attributes of stigma related to a selection of mental health issues amongst school going adolescents. Methods This cross-sectional study was conducted in the Sri Jayewardenepura educational zone in the Colombo District. Those in grades 9 and 10 were chosen for the study. The minimum sample size was calculated to be 844. Details of sample size calculation can be found in a previously published study (19). A multistage cluster sampling method was used as this education zone included several categories of schools, with different resource levels and different streams of study, and a total of 46 schools were selected for the study (19). Questions to assess stigma related to mental health issues, were adapted from the Attribution Questionnaire-C-8 (AQ-C-8), with the author's permission (20). This version of the questionnaire has been formulated for children and applied on adolescents between 10-18 years to assess for public stigma (20). The attribution questionnaire uses a collection of attributes about people with mental health issues (such as pity, shame, guilt, level of perceived danger, likelihood of helping or staying away from them). The selected attributes were self-scored by participants on a pre-coded 9 response Likert scale, with a higher score indicating that the given attributes were being more strongly endorsed by the respondent (i.e., score of 9 -strongly agree and a score of 1 -strongly disagree). The participants were asked to score these attributes in relation to four case vignettes. These vignettes were modelled on the Australian National Survey on Mental Health Literacy and Stigma 2011 (15). The four case vignettes used in the current study were: depression with suicidal ideation, social phobia, psychosis and diabetes mellitus. A vignette on a physical health condition (diabetes mellitus) was included for comparison. The content and face validity of the questionnaire was established using the Delphi method. A pilot study was also conducted to establish acceptance, comprehension and ease of administration (19). The data was analysed for frequency distribution, crosstabulation and median comparisons. A p value of 0.05 was used as the significance level. Ethics Consent and approvals have been obtained from the respective education offices of each zone and the principals of the selected schools. Ethical clearance for the study was obtained from the Ethics Review Committee (ERC) of the University of Colombo, Sri Lanka. The data collector visited the schools and obtained prior consent (informed and written) from the parents of students selected for the study. Information sheets about the study were also shared with the consent forms. Only those adolescents with parental consent were included in the study. Prior assent from participating adolescents was also obtained. Results Of the 1,500 students approached, 1,002 responded (response rate of 67%). There were 590 (58%) males in the sample with a mean age of 14 years (SD ± 0.94). An average monthly family income of more than LKR 20,000 was seen among 59.4% of the sample. Of the sample, 9.6% (n=97) responded positively to knowing a relative with mental health issues (family history), while 5.2% (n=52) responded positively to being treated for a mental health issue themselves (personal history). The central tendencies (mean and median scores), for the different stigma related attributes are listed in Table 1. A comparison of the median scores of each attribute between the different vignettes showed that there was a significant difference only for the attributes of blame, anger, willingness to help and preference to stay away (p<0.05) ( Table 2). The overall scores for anger and staying away were significantly higher (p<0.05) for psychosis compared to depression or social phobia ( Table 2). Comparison of median values in relation to family income (categorised as below and above LKR 20,000), the education level of the mother and father (categorised as above and below the General Certificate in Education -Ordinary Level exam) did not show significant difference in relation to any of the attributes. A family history or a personal history of being treated for a mental health issue in the past, was associated with a significantly higher median score for the attributes of avoidance, anger, and danger across all three mental health vignettes (p< 0.05) ( Table 3). A personal history was also significantly associated with lower median scores for pity and willingness to help (p< 0.05) ( Table 3). Stigma in relation to the different case vignettes In relation to all mental health case vignettes, there were higher scores for pity and helping the affected person and lower scores for danger, anger, and avoidance of the person, compared to the diabetes health vignette. Overall, this indicated that mental health issues were perceived as requiring more help, compared to those with diabetes. Interestingly, the responses also indicated that those with mental health issues were more likely to be offered help than those with diabetes, which was a good sign in relation to mental health issues. The responses indicate that this cohort is more likely to avoid those with psychosis than those with depression or social phobia. Although one would expect the attribution of danger to be associated with avoidance, this was not indicated in the current study. Previous studies have shown that those with psychotic disorders and/or substance misuse are more stigmatized compared to those with other mental health issues (21). In another study on stigma among adolescents, relative to depression, psychosis was associated with higher scores on the dimensions of 'dangerous/unpredictable'and 'social distance' (14). In the 2011 National Mental Health Literacy survey in Australia (youth component), perceptions of danger, unpredictability and a desire for social distance were generally higher for psychosis/ schizophrenia, than for other disorders (15). However, in the current study the scores indicate that there was also significantly more anger in relation to psychosis than other mental health vignettes. Therefore, it is possible that in this study the reported avoidance of those with psychosis may be more related to anger than danger. Stigma expressed by those who know a family member with illness, or with a personal history of mental health issues In the current study, knowing a relative with a mental health issue, or having a personal history of being treated for a mental health issue was associated with a significant increase in the scores for the attributes of anger, danger and avoidance across all three mental health case vignettes. A personal history of being treated for mental health was associated with significantly lower scores for pity and the likelihood of helping. Although previous evidence has reported that knowledge of mental health issues and exposure to them has been found to lower stigma, some research suggests that knowledge and contact may also increase certain attributes of stigma (7,20). The findings of the current study appear to support this latter view. The findings of this study suggest that it may not be mere exposure to mental health patients that reduce stigma. Factors such as the length of exposure, nature of experience (positive or negative experience), perception of treatment (beneficial and acceptable), and whether they recovered from their condition successfully, may all be important factors influencing stigma. A previous study reported that stigma was lower for an individual who suffered from major depression and recovered successfully after treatment, compared to an individual who was not treated (22). In another study undergraduates with a history of mental health treatment were assigned as room-mates to those with no mental health history. The stigma in the latter group stigma towards the former often increased, highlighting that naturalistic contact alone, if not structured appropriately, might be more harmful than helpful (23). Thus, it might be that in this study sample, those with family and personal history of mental health issues did not have positive experiences/ outcomes, which may have influenced the degree and nature of stigma they have towards those with mental health issues. Limitations The responses of the participants may not necessarily indicate how they would respond in reality. This has been described as an issue in vignette based studies (24). As some of the data was retrospective, recall bias as well as courtesy bias may have influenced the responses. It is also likely that there are more attributes and dimensions of stigma than the ones used in this study. As various studies have used multiple tools with different dimensions and attributes, comparisons between studies is also difficult. Conclusions The findings of this study suggest that adolescents in this cohort are more likely to feel pity and help in the cases of mental health issues, compared to the case of the given physical health issue. They were also more likely to avoid those with psychosis, than those with other mental health issues. Thus, interventions aimed at reducing stigma could make use of this sense of pity and tendency to help, to promote directing those with mental health issues towards appropriate services. However, strategies will also have to consider ways of changing perceptions about psychosis related disorders, as avoidance by peers may lead to poorer help seeking where early intervention is most needed. As seen in other studies, knowledge and exposure does not automatically reduce stigma towards those with mental health issues. In contrast, this study showed that merely knowing a person with mental health issues or having been treated for mental health issues can have a negative effect on stigma related attributes. As such, stigma mitigating programs may have to include aspects that go beyond mere exposure to those with mental health issues. A positive experience of the acceptability and benefits of treatment helping in recovery might be aspects to consider.	2020-06-25T09:09:37.757Z	2020-06-18T00:00:00.000	{ "year": 2020, "sha1": "32eacbbb6cfd47385f7808f6adfdb4e311fefc99", "oa_license": "CCBY", "oa_url": "http://sljpsyc.sljol.info/articles/10.4038/sljpsyc.v11i1.8243/galley/6094/download/", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "24f7aec8d45c86a46ee09e669d4a3554004e00c9", "s2fieldsofstudy": [ "Psychology" ], "extfieldsofstudy": [ "Psychology" ] }
119489898	pes2o/s2orc	v3-fos-license	On the hyperfine structure of the triplet $n^{3}S-$states of the four-electron atoms and ions Hyperfine structures of the triplet $n^3S-$states in the four-electron Be-atom(s) and Be-like ions are considered. It is shown that to determine the hyperfine structure splitting in such atomic systems one needs to know the triplet electron density at the central atomic nucleus $\rho_T(0)$. We have developed the procedure which allows allows one to determine such an electron density $\rho_T(0)$ for arbitrary four-electron atoms and ions. I. INTRODUCTION In this communication we develop the new ab-initio method which can be applied for accurate evaluation of the hyperfine structure splittings in the triplet 3 S−states of the fourelectron atoms and/or ions. As follows from experiments such triplet S(L = 0)−states have an interesting hyperfine structure. For simplicity, let us consider, the triplet 2 3 S−state of the four-electron beryllium atom(s) ( 7 Be, 9 Be and ∞ Be). In general, if F is the total electron-nuclear spin of the 2 3 S−state of an atom and I N is the spin of atomic nucleus (Be) and I N ≥ 1, then in experiments one can observe splitting of this state into a triplet of states. The total spin of these states equals F = I N + 1, I N and I N − 1, respectively. If I N = 1 2 , then we can see only a doublet of states with F = 1 2 and 3 2 . This method can also be used to determine the hyperfine structure splitting for an arbitrary bound (triplet) n 3 S−states in four-electron atoms and ions. This includes the triplet 2 3 S−state of the four-electron Be atoms (different isotopes). Our analyisis of the hyperfine structure of the four-electron atoms and ions is based on the generalization of the method developed earlier by Fermi [1] for the doublet n 2 S−states of three-electron Li-atom(s) and Li-like ions (see also [2]). First, we need to introduce the triplet electron density in a few-electron atom/ion. Formally, the triplet electron density is the spatial two-electron density distribution of the two atomic electrons which form one triplet pair. If we have a number of such pairs in atom/ion, then we need take into account all triplet electron pairs. Singlet electron pairs do not contribute to the triplet electron density. In reality, for accurate evaluations of the hyperfine structure splitting in the triplet states of four-electron atoms/ions one needs to know the triplet electron density at the central atomic nucleus which has non-zero electric charge Qe. The general definition of the electron density at the central atomic nucleus is written in , where i is the electron's index, while index A means the central atomic nucleus. For instance, for the singlet ground 1 1 S−state in the two-electron helium atom one finds ρ S (0) ≈ 1.8104293185013928, while for the triplet 2 3 S−state of the helium-3 atom we have ρ T (0) ≈ 1.31963500836957 [3]. The last numerical value leads to the following hyperfine structure splitting in the 2 3 S−state of the 3 He atom: ∆E hss = 6740.452154 MHz [3] (see also [4]). The corresponding experimental value is ∆E hss = 6739.701171(16) MHz [5]. However, such a definition of the electron density cannot be used for the triplet states in atoms/ions, if the total number of bound electrons exceeds two. The reason is obvious, since all atoms/ions with more than two bound electrons always have the shell electronic structure. This means that the internal electrons form a number of closed electron shells which have zero spin, i.e. singlet electron shells. The electrons from the outer-most shell(s) can interact with the nuclear spin I N , its numerical value it differs from zero. In general, this leads to the appearance of the hyperfine structure splitting in atoms and ions, if the total spin of outer-most electrons exceeds zero. This leads to the appearance of the hyperfine structure splitting in N e −electron atoms and ions, where N e ≥ 3. It is clear that in actual atoms and ions we have small interactions between electrons from internal and outermost electron shells, e.g., electronic correlations, spin-spin interactions, etc. As follows from this picture the analysis and numerical computations of the hyperfine structure splitting are significantly more complicated than for the two-electron helium atom. In the lowest-order approximation we need to define the triplet electron density at the atomic nucleus must be given in a different manner. An alternative definition of the triplet electron density at the atomic nucleus (A) can be written in the form (see, e.g., [1], [2], [6]) where δ(r iA ) is the electron-nucleus delta-function (the symbol A designates the atomic nucleus) and (σ z ) i is the σ z matrix of the i-th atomic electron, i.e. (σ z ) i α(i) = α(i) and (σ z ) i β(i) = −β(i) (see, e.g, [7], [8]). In Eq.(1) and everywhere below we assume that the wave function of the bound 2 3 S−state of the four-electron atom/ion has unit norm. As of the hyperfine structure splittings (see, e.g., [2]) which are in good agreement with the known experimental values [9]. By using this definition of the triplet electron density at the atomic nucleus, Eq.(1), we can write the following formula (Fermi-Segré formula (see, e.g., [7])) for the hyperfine structure splitting of the 2 3 S−states in the Be atom (see, e.g., [7]) where S e is the total electron spin of the atom, I N is the spin of the nucleus in those isotopes of the Be atom(s) for which \| I N \| = 0 and F is the total angular momentum operator F = L + S = S e + I N of the four-electron atom/ion. For the triplet S−states in the four-electron atoms/ions the vector-operator F = S e + I N can be considered as the total spin of the atom, i.e. the sum of the electron and nuclear spins. Also, in this formula .15267261 is the ratio of the proton and electron masses. The notation g e in Eq.(2) means the electron gyromagnetic ratio g e = -2.00223193043718 [10]. The factor g N for the 9 Be nucleus is Finally, the formula for the hyperfine structure splitting ∆E hf in the 2 3 S−state of the four-electron 7 Be and 9 Be atoms takes the form where the factor 6.579 683 920 61·10 9 (MHz/a.u.) has been used to re-calculate the ∆E hf below (see Eq.(4)) and Ψ is the wave function and E(< 0) is the total energy of the bound atomic state. This problem is considered in detail in the next Section. II. HAMILTONIAN AND BOUND STATE WAVE FUNCTIONS In the lowest-order approximation upon the fine-structure constant α we can consider the non-relativistic Schrödinger equation. The non-relativistic Hamiltonian H of an arbitrary four-electron atomic system (i.e. atom, or ion) is written in the form [7] 4). We also discuss a few isotopes of the Be-atom with the finite nuclear masses M A . In Eq.(4) and everywhere below in this study the subscript 5 denotes the atomic nucleus, while subscripts 1, 2, 3 and 4 stand for electrons. Note that the four-electron Be atom has two independent series of bound states: singlet states and triplet states. The multiplicities of these states equal 2 · 0 + 1 = 1 (singlet) and 2 · 1 + 1 = 3 (triplet). Below, we consider only the triplet bound states in the Be atom(s) and Be-like ions. To determine the bound state wave function of the Be atom in its 2 3 S−state we need to solve the corresponding Schrödinger equation for the bound state(s): HΨ = EΨ, where H is the Hamiltonian operator from Eq.(4), while E(< 0) is the total energy of the 2 3 S−state in the Be-atom. It is clear that the numerical value of E must be lower than the total energy of the ground 2 2 S−state of the three-electron Be + ion E ≈ -14.3247631764657 a.u. where the notation {r ij } designates the ten relative coordinates (electron-nuclear and electron-electron coordinates) in the four-electron Be atom, while the notation A e means the complete four-electron antisymmetrizer. The explicit formula for the A e operator is A e =ê −P 12 −P 13 −P 23 −P 14 −P 24 −P 34 +P 123 +P 132 +P 124 +P 142 +P 134 +P 143 +P 234 +P 243 −P 1234 −P 1243 −P 1324 −P 1342 −P 1423 −P 1432 +P 12P34 +P 13P24 +P 14P23 (6) Hereê is the identity permutation, whileP ij is the permutation of the spin and spatial coordinates of the i−th and j−th identical particles. Analogously, the notationsP ijk and P ijkl stand for the consequtive permutations of the spin and spatial coordinates of the three and four identical particles (electrons). In real calculations one needs to know the explicit expressions for the spatial projectors only. These spatial projectors can be obtained, e.g., by applying the A e operator to each component of the wave function in Eq. (5). At the second step we need to determine the scalar product (or spin integral) of the result and incident spin function. After the integration over all spin variables one finds the corresponding spatial projector. For instance, in the case of the first term in Eq.(5) we obtain the following spatial projector for the ψ−components of the total wave function P ψψ = 1 2 √ 6 (2ê + 2P 12 −P 13 −P 23 −P 14 −P 24 − 2P 34 − 2P 12P34 −P 123 −P 124 −P 132 −P 142 +P 134 +P 143 +P 234 +P 243 +P 1234 +P 1243 +P 1342 +P 1432 ) (7) Analogous formulas have been found [11] for two other spatial projectors P ψφ = P φψ and P φφ . These formulas for the P ψφ = P φψ and P φφ spatial projectors are significantly more complicated and they are not presented here (they can be found, e.g., in [11]). In actual bound state calculations we can always restrict ourselves to one spin function χ 11 (or one spin configuration) and use the formula, Eq.(7). The functions ψ(A; {r ij }) and φ(B; {r ij }) in Eq.(2) are the radial parts (or components) of the total wave function Ψ. For the bound states in various five-body systems these functions are approximated with the use of the KT-variational expansion written in ten-dimensional gaussoids [12]. Each of the spatial basis function in the KT expansion depends upon ten relative coordinates r ij only [12]. Here and everywhere below the notation r ij =\| r i −r j \|= r ji means the (ij)−relative coordinate, i.e. the scalar distance between the particles i and j (r i are the corresponding Cartesian coordinates of the i-th particle). For instance, for the where N A is the total number of basis function used in radial expansion, C k are the linear variational coefficients and P = P ψψ is the spatial projector defined by Eq. It is clear that few-body logic does not work well for actual atoms/ions with multi-shell electronic structure. This situation must be corrected in the future. III. HYPERFINE STRUCTURE SPLITTING Let us evaluate the hyperfine structure splitting for the triplet 2 3 S−state of the 9 Be atom. In our calculations we shall apply the numerical value of the electron density ρ T (0) at the atomic nucleus determined with the use of the formula where C is a normalization constant. Numerical computations of the overlap integrals between spin-functions included in Eq.(9) is significantly more complicated than for threeelectron atomic systems. Indeed, the total number of terms in the left-hand side wave function of the Eq.(9) equals 24 and each of these terms must be multiplied by four (number of the electron-nucleus delta-functions). This means that we have 96 terms which contribute to the numerical value of the triplet electron density at the central atomic nucleus ρ T (0). Formally, it is difficult to present here all details of analytical computations of the electron density ρ T (0). However, we can illustrate such computations by considering the two terms which can be found in Eq. (9). First, consider the term in Eq.(9) which contains the permutation operatorP 13 (see, Eq.(7). Action of this operator on the spin function χ (1) 11 = αβαα − βααα produces the function αβαα − ααβα. On the other hand, the explicit expression in the right-hand side of Eq.(9) takes the form where Ψ is the spatal part of the total wave function. The following integration over spin variables leads to the formula for the matrix elements which contains only integrals over spatial variables. Analogously, the term which include theP 12P34 permutation operator produces the result which leads to the following formula The explicit integration over electron spin variables of all other terms in Eq. (9) Note also that there are a few steps in our procedure which must be improved in the future computations. First, we need to improve the current accuracy of our wave functions. This means the better numerical accuracy of the wave functions constructed from the multidimensional (or ten-dimensional) gaussoids. It would be nice to use other basis sets of spatial functions in such calculations, since this can drastically improve the overall accuracy of the whole procedure. Second, in our current computations the second spin function χ (2) 11 = 2ααβα − βααα − αβαα is not used. Very likely, this also reduces our overall accuracy even further. Nevertheless, this study indicates clearly that direct computation of the hyperfine structure splitting of the bound triplet n 3 S−states of the four-electron atoms and ions are possible, since the corresponding analytical expression for the triplet electron density at the atomic nucleus has been derived. In the future our procedure will be modified to include two (or more) spin functions. V. ACKNOWLEDGMENTS This work was supported in part by the NSF through a grant for the Institute for Theoretical Atomic, Molecular, and Optical Physics (ITAMP) at Harvard University and the Smithsonian Astrophysical Observatory. Also, I wish to thank James Babb (ITAMP) and	2016-11-14T18:33:05.000Z	2016-11-09T00:00:00.000	{ "year": 2017, "sha1": "7f7d5279ebad9539f034058df3991cff19a75a5c", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1611.03126", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "7f7d5279ebad9539f034058df3991cff19a75a5c", "s2fieldsofstudy": [ "Physics", "Chemistry" ], "extfieldsofstudy": [ "Physics" ] }
38954025	pes2o/s2orc	v3-fos-license	Nurses in advanced roles : a review of acceptability in Portugal Objective: This paper focuses on the policy context for the deployment of nurses in advanced roles, with particular reference to Portugal. The health sector in Portugal, as in all countries, is labour intensive, and the scope to utilise nurses in more advanced roles is currently being debated. Methods: Mixed methods were used: an analysis of international data on the nursing workforce; an analysis of documents and media articles; interviews with key-informants; an online survey of managers, and a technical workshop with key-informants. Conclusions: The limited evidence base on nurses in advanced roles in Portugal is a constraint on progress, but it is not an excuse for inaction. Further research in Portugal on health professionals in innovative roles would assist in informing policy direction. There is the need to move forward with a fully informed policy dialogue, taking account of the current political, economic and health service realities of Portugal. Introduction This paper draws from work commissioned by the World Health Organisation, European Region and focuses on the policy context for the deployment of nurses in advanced roles, with particular reference to Portugal. Portugal initiated a phase of health sector reform of its National Health Service in the earlier part of this decade.Human resources issues were identified both as a focus for necessary reform, and as one area for potential improvement in health care delivery and health system effectiveness (1).The health sector in Portugal, as in all countries, is labour intensive, and the scope to utilise nurses and other non medical health professionals in more advanced roles was one of the issues that was identified during the reform process.Portugal is also one of several European countries currently facing a severe economic crisis which has impacted on public sector funding, employment and health care delivery (2) .This has both delayed implementation of some of the reform priorities, and raised the need for more urgent focus on methods of improving health care productivity and effectiveness. In Portugal, the composition of the health workforce has been described as an inefficient combination of resources that may lead to less than optimal productivity, and with access to certain services may be limited as a consequence (3) .Various stakeholders have advocated for the revision of the composition of the health workforce and of the scope of practice of professions such as Rev. Latino-Am.Enfermagem 2013 Jan.-Feb.;21(Spec):38-46. nursing, as a measure aimed at improving efficiency in the delivery of services (4)(5)(6) .One factor that have influenced these stakeholders is that Portugal has one of the lowest nurse to population and to physician ratios in develop countries, and one of the highest physician to population ratios.One argument in support of expanded scopes of practice for nurses in Portugal is that there are many successful international experiences that show that this can be done without negative effects on the quality of services or the safety of users (7)(8) . It is within this policy process of health sector cost containment, and ongoing debate about the respective roles of different health professionals in Portugal that the paper examines the scope and potential for using nurses in advanced roles in Portugal.The focus of this report is nurses working in advanced roles/ advanced practice.The "advanced practice nurse" is an umbrella term that that covers a number of nursing roles, the two main types being the nurse practitioner and the nurse specialist (sometimes referred to as clinical nurse specialist).In some countries advanced practice roles are registerable whilst in others the roles are not identified separately on the professional register (9) .One key factor in the role of advanced practice is the extent to which the nurse has the authority to legally prescribe drugs.Legislation to enable nurse prescribing is being implemented in a growing range of countries. Whilst nurses in advanced roles were implemented initially in North America, their use or potential is now observed in a range of countries in most regions of the world.An international study in 2010 reported that nurse practitioner/advanced practice education was available in 23 of 32 countries surveyed (10).There is a range of drivers that can provide the impetus for developing and introducing nurses in advanced roles, including skill shortages, the need to make more cost effective use of skills, the introduction of new services and technologies and changes in regulations and legislative reform (7)(8) . A review of the international evidence base on nurses in advanced roles does highlight that there is a growing body of evidence that examines the effectiveness of nurses developing their scope of practice into advanced roles (7)(8).Whilst much of the initial evidence emerged from research in North America, the UK and Australia, there has been a broadening out of the evidence base in recent years reflecting the increasing number of countries that are now supporting the use of nurses in advanced roles. The most robust evidence is in the form of systematic reviews and meta-analysis, of which there are relatively few examples (11) , but there are also a growing number of randomised controlled trials (RCT's) (12)(13) and some economic evaluations (14) .Differences in health systems contexts, current role delineations, data availability and evaluation methods constrain the development of general conclusions from the available international evidence base on the evaluation of advanced practice roles in nursing (7)(8) . Methods In order to meet the objectives of the analysis, a multipronged research strategy was developed, and five main elements are described in this paper: -An analysis of international data on the nursing workforce to "map" the nursing workforce profile of Portugal in the broader international context; -An analysis of recent official, scientific and professional documents, reports and media articles published in Portugal to provide specific country context; -Interviews with key-informants in Portugal; -An online survey of opinions of managers of primary care level services in Portugal; -A technical workshop with key-informants in Portugal. In view of the exploratory nature of the research, and the current political sensitivity of the issue in Portugal, strategies were selected which would provide both a firm evidence base drawing on international experience, to inform and contextualize the focus in Portugal, but which would also start engaging the range of stakeholders in Portugal in order to begin to map out the scope for any policy dialogue in the future.More specific details of the methods used in the study are provided below, as part of the reporting of results for each of the six aspects of the study. International comparison of nurse ratios One broad indicator of the availability of nursing staff is the nurse to population ratio.Annual data collated and published by the Organisation for Economic Co-operation and Development (OECD) gives a profile of the relative nurse to population ratio across the countries of the OECD, and some additional countries (15) .This comparison has to be undertaken with some caution because of differences in definition and methods of data collection but does prove some basis for analysis.OECD data (Figure 1) shows that there were about 9 practising nurses per 1 000 population on average across OECD countries in 2009.The number of nurses per capita was Nurses per 1000 (5) which noted that some categories of professionals, namely nurses and technicians may be underutilized, and which stated that some basic medical acts can safely be executed by adequately trained nurses and technicians; a background report on human resources for health, commissioned for the preparation of the new National Health Plan-2011-2016 (3) which stressed that "the low nurse/physician ratio indicates primarily a productivity and cost-effectiveness problem" and concluded that "there is scope for task shifting and delegation of tasks, particularly from physicians to nurses" ; and the recent report on "Reforming the hospital sector" commissioned by the Ministry of Health in Portugal (6) , which explicitly recommended "the attribution of new activities to nurses" (p108), to avoid using physicians for tasks which do not require their level of expertise; and a review of the Portuguese health system which noted that "One of the major challenges for the next 10 years, not yet translated into policy actions, is the redefinition of roles for health care professionals" (17). The search of media articles also showed that the topic of nurses in advanced roles has come increasingly to public attention in Portugal in recent times.Issues reported included various stakeholders stances on the possibility of legal changes to authorize nurses to prescribe medicines, broader issues related to health sector efficiency during the economic and financial crisis, and questions of the composition of the health workforce and of the distribution of roles among the various groups of professionals. Key informant interviews The Survey of first line managers The Key points emerging from the survey were that most respondents believed that discussion on advanced roles for nurses was relevant to the Portuguese context, and that there was specific scope for specific tasks to be undertaken by nurses.Most respondents (90%) agreed that the discussion of the development of nursing advanced roles in Portugal, in order to improve accessibility of health services is relevant, most (88% ) also agreed with the statement "Among the frequent tasks executed by physicians, there are some that could be executed by adequately trained nurses" (see Figure 3) Source: ACES survey Primary care was seen by 68% of respondents as the appropriate field to start experimenting with advanced roles, and 57% agreed that the expansion of nurse´s roles permitted the substitution of costly production inputs by less expensive ones (Figure 5).contraception and anti-tobacco, anti-pyretic in case of a high fever); one respondent stated that nurses should not only prescribe but also be authorized to make small adjustments of doses of medical prescriptions. The survey responses highlighted that, although there is some acceptance of the potential for expanded roles for nurses amongst some of the medical profession, there are concerns with regard to patient safety and quality of care.The nursing curriculum would need adjustments and professional regulation would have to be adapted, which would require collaboration between the Medical and Nursing Councils.In addition, the absence of financial and organizational incentives for nurses to take up more complex functions is also a potential constraint.Some respondents considered that the organizational conditions for effective teamwork are not all in place, which limits the possibility of expanding nurses' roles.Another constraint identified was the absence of information systems that track the activities of each professional and of guidelines and protocols that ensure that everyone knows what the other members of the team are supposed to do and actually do.This is perceived as a prerequisite to building mutual trust and developing informed policy making, a critical condition for the acceptance of the expansion of roles of nurses, and eventually of other professionals.Regarding the feasibility of expanding nurses' roles at present in Portugal, the consensus was that "the road is still long" before this may become fully possible. Workshop with key informants Among nurses and physicians there are very differing views, with some much in favor and others much against and all sorts of nuances in between.However there was a clearer consensus in saying that the current economic and financial crisis offered an opportunity to open the discussion on how services are delivered and how professionals can be used more efficiently.One The issue of the potential for nurses to have the right to prescribe was the most hotly debated.The consensus was that this needed to be regulated and that in principle, nurses should be able to prescribe medicines and order examinations and tests in the areas in which they are authorized to practice.All assumed that training would have to be adjusted in accordance to the knowledge requirements of these new activities. Finally, participants remarked that using evidence on successful international experiences was helpful, but that it was insufficient in itself.Evidence from national research in Portugal, which is in rather short supply at present, was also needed. The workshop showed that there was a need for clarification of the language used to discuss role expansion.Terms such as substitution, delegation, teams of peers, complementarity, role, tasks, competencies, scope of practice, come in the discussion, but they are not always used to describe the same issue, or understood in the same manner by all. To add to the confusion, there is no clear and operational definition of what is a medical task or a nursing task in the Portuguese legislation.Participants added that at ground level, professionals themselves had no clear understanding of what "belonged" to each professional group.Without such definitions, it may be more difficult to be precise about the contents of an expanded role, or which tasks could be delegated. Discussion The policy context for nurses in advanced roles The international review had highlighted that different countries are at different stages of development in terms of their use of nurses in advanced roles.The various elements of the study conducted in Portugal showed that there is no clear consensus on if, or how, any developments in advanced roles for nurses should be taken forward in the country.In this regard, the history of the introduction of nurse practitioners in the USA is of relevance.US was one of the first countries to develop such roles, almost 50 years ago.This chronology highlights the significance in the US of government funding as "pump priming" in the 1960s and 1970s, and the ongoing process of gaining legislative support through the 1970s, 1980s and 1990s (18) .Different countries will have different timetables and drivers.The key points that any country should consider in looking at the potential for using nurses in advanced roles will have to include (7): First, there is a need for stakeholders to agree on the need for advanced practice nurses.Key stakeholders will include representatives of the nursing and medical professions, ministries of health and education, employers and regulators.Several commentators noted the need to base this approach on mutual respect, and on developing a collaborative model of health care delivery Second, where principles of need have been agreed, there is then a requirement to ensure that the advanced role(s) have been defined, and the associated educational requirements have been identified. Third, issues of certification and regulation have to be determined.As summarised in this report, different countries have adopted different approaches.Some have developed a single national level approach to certification/licensure with separate national registration, whilst others have adopted a voluntary approach. Fourth, the career structure and payment system for advanced nurses has to be established, and the potential impacts on other professions, the organization and accessibility of services has to be assessed. Portugal is at the first stage of this possible process.As described in this report, there is a limited evidence base in the country about the potential impact of developing nurses in advanced roles.However, well documented international experience shows that this can be done effectively, whilst maintaining quality of care and with benefits for them and for providers as well. Conclusion The Figure 2 - Figure 2 -Nurse: Physician ratio, OECD countries c2009 second phase of the Portugal based assessment were 14 semi-structured face-to-face interviews, conducted between July and October of 2011 with keyinformants including representatives of medical and nursing councils, faculties and schools of medicine and nursing, hospital directors and professional associations.The main topics explored were t scope of practice of physicians and nurse, limitations and difficulties with the existing skill mix between physicians and nurses, and impact, opportunities and threats, of implementing advanced nursing in Portugal.The main finding was that opinions of key stakeholders are still divided in Portugal and there does not appear to exist a strong advocacy movement in favour of changes in the physician-nurse skill mix. key informant interviews were complemented by a survey of the opinions of first line managers of Groups of Health Centers (Agrupamentos de Centros de saúde-ACES).ACES were created in Portugal in 2009 with a mandate to improve coordination and efficiency of basic health services at local and sub-regional level through better planning and sharing of resources.There are 73 ACES, with a management structure consisting of an Executive Director, a Clinical Council (CC) and a Management Support Unit (UAG).The CC includes a family physician as president, a Public Health physician, a nurse and another health professional.ACES Executive Directors and Clinical Council members were targeted because they are at the front line of the delivery of services and they are well positioned to assess the relevance or not of the expansion of nurse´s roles in primary care, as well as its feasibility.A questionnaire of 10 closed (Likert scale) and two open questions was developed as an online survey.An email with the online survey link was sent to all the Executive Directors and members of Clinical Councils, using the Buchan J, Temido M, Fronteira I, Lapão L, Dussault G. mailing list of a training program to which the targeted population participated in 2009-2010 Of 365 emails sent (5x73 Group of Health Centers), 49 rebounded for being inactive and 101 valid answers were collected, corresponding to a 32% response rate. Figure 3 - Figure 3 -ACES response to question "Among the frequent tasks executed by physicians, some could be executed by trained nurses" (n=101) Figure 4 - Figure 4 -ACES response to question "The opportunities created by the expansion of the roles of nurses compensate for the risks" n=101) A technical workshop was organized on 10 February 2012 with the objective of obtaining initial responses from stakeholders, to the preliminary results of the study.The meeting was held under "Chatham House", whereby a report of the meeting was written, but all discussion was non attributable.There were 19 participants including managers of health services, officials of nursing and medical councils, and researchers.Much of the discussion concerned the contents of any expansion of roles of nurses.This question reflects on what would be the most appropriate skills-mix in health services to guarantee the quality of services and to better respond to demand, namely in terms of more efficient utilization of existing resources.It was argued that this discussion should take into account the specificities of the Portuguese health sector, e.g.how services are organized and financed.It was also deemed important to ensure that expanding the roles of nurses or of any other group would not have collateral negative effects on other professional groups. Figure 5 - Figure 5 -ACES response to question "The expansion of nurses' roles allows for the substitution of costly inputs by less costly inputs" n=101) limited evidence base on nurses in advanced roles in Portugal is a constraint on progress, but it is not an excuse for inaction.Further research in Portugal which looks at local experience with deploying health professionals in innovative and effective roles would assist in informing policy direction.Most importantly, there is the need to move forward with a fully informed policy dialogue, taking account of the current political, economic and health service realities of Portugal, which can explore in a structured and objective manner what is best for healthcare in the country, in terms of the overall health workforce profile.	2017-06-23T10:42:32.073Z	2013-02-01T00:00:00.000	{ "year": 2013, "sha1": "56656c95b2684f4aa22bd9255e2cb2eb8bb15533", "oa_license": "CCBY", "oa_url": "https://www.scielo.br/j/rlae/a/jhrfxQmmSzRMdHRFXvHZQMg/?format=pdf&lang=en", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "56656c95b2684f4aa22bd9255e2cb2eb8bb15533", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Political Science", "Medicine" ] }
11342973	pes2o/s2orc	v3-fos-license	The role of the breast cancer susceptibility gene 1 (BRCA1) in sporadic epithelial ovarian cancer Mutations within the BRCA1 tumor suppressor gene occur frequently in familial epithelial ovarian carcinomas but they are a rare event in the much more prevalent sporadic form of the disease. However, decreased BRCA1 expression occurs frequently in sporadic tumors, and the magnitude of this decrease has been correlated with increased disease progression. The near absence of somatic mutations consequently suggests that there are alternative mechanisms that may contribute to the observed loss of BRCA1 in sporadic tumors. Indeed, both allelic loss at the BRCA1 locus and epigenetic hypermethylation of the BRCA1 promoter play an important role in BRCA1 down-regulation; yet these mechanisms alone or in combination do not always account for the reduced BRCA1 expression. Alternatively, misregulation of specific upstream factors that control BRCA1 transcription may be a crucial means by which BRCA1 is lost. Therefore, determining how regulators of BRCA1 expression may be co-opted during sporadic ovarian tumorigenesis will lead to a better understanding of ovarian cancer etiology and it may help foster the future development of novel therapeutic strategies aimed at halting ovarian tumor progression. Introduction Epithelial ovarian cancer is the most lethal of all gynecological malignancies [1]. The poor survival associated with ovarian carcinoma is due, at least in part, to the fact that the disease is usually asymptomatic in its early stages. As a result, detection often occurs at a late, metastatic stage when the prognosis is poor. While the etiology of ovarian carcinogenesis is poorly understood, evidence from histopathological studies and recently developed mouse models of ovarian cancer progression suggest that the majority of the tumors originate from the ovarian surface epithelium (OSE), a simple cuboidal layer that covers the surface of the ovary [2][3][4][5]. It remains unclear at this time, however, if a predictable progression of molecular events within the OSE gives rise to a well-defined neoplastic pre-cursor that can be used to improve early detection and diagnosis. Changes in a number of genes, including p53, k-Ras, HER2/neu and c-Myc, have been implicated in ovarian carcinoma progression. However, none of these changes appear to occur in a stage-specific manner [6,7]. While global gene profiling approaches have recently identified a number of genes that are differentially expressed in epithelial ovarian cancer these alterations have not yet been be fully characterized with respect to stage, grade or functional importance [8][9][10]. Thus, to date, the most compelling target gene linked to the development of ovarian cancer continues to be the breast and ovarian cancer susceptibility gene 1 (BRCA1). The protein products of the BRCA1 gene regulate, at least in part, transcriptional activation, DNA repair, cell-cycle checkpoint control, and chromosomal re-modeling [11]. Such multi-faceted contributions to essential cellular functions imply a truly fundamental role for BRCA1 in normal development but they also confound our understanding of its role in tumorigenesis [12]. This confusion was initially compounded by the finding that complete BRCA1 ablation in transgenic mice blocks embryonic proliferation [13,14]. However, the subsequent generation of a targeted knockout in the mouse mammary epithelium did result in tumor formation, which is direct experimental evidence that BRCA1 can act as a tumor suppressor in a susceptible tissue [15]. While a BRCA1 knockout has not yet been targeted to the OSE there is compelling clinical evidence that the gene is also a tumor suppressor in the ovary. Drawing on parallels with the situation in the breast, this review will focus on the possible means by which a non-mutational suppression of BRCA1 can be achieved in highly prevalent, non-familial, sporadic epithelial ovarian carcinoma. BRCA1 in familial and sporadic tumors BRCA1 was originally isolated using positional cloning techniques and inactivating mutations were found in families with a high incidence of breast and ovarian cancer [16]. Specifically, germline alterations in the BRCA1 gene result in a predisposed likelihood of developing earlyonset breast and ovarian cancer with a dominant penetrance as high as 85% and 65% respectively [17]. Tumorigenicity only occurs in these familial BRCA1 heterozygotes if there is also a loss of the second wild type BRCA1 allele. The latter observation supports the notion that the original germline BRCA1 mutation acts recessively at the cellular level [18][19][20]. Although the presence of an inherited mutation in one BRCA1 allele continues to be one of the best-defined overall risk factors for the development of breast or ovarian cancer, these familial mutations, together with familial BRCA2 mutations, occur in less than 10% of all diagnosed cases [21,22]. The great majority of breast and ovarian carcinomas arise sporadically where inherited BRCA1 mutations do not occur. In addition, somatic BRCA1 mutations are virtually undetectable in sporadic breast cancers and they are extremely rare in sporadic ovarian cancers [19,[23][24][25][26]. Thus, at first glance, it would not be unreasonable to conclude that BRCA1 does not play a significant role in sporadic tumor development. However, mounting evidence suggests that alternative, non-mutational, mechanisms may suppress BRCA1 expression in these tumors [27]. The first group to conclude that BRCA1 may be important in sporadic tumor development observed a significant decrease in BRCA1 mRNA in high grade, invasive breast tumors [28]. The same investigators also demonstrated that a suppression of BRCA1 activity has functional consequences in cultured breast carcinoma cells [28]. Since then, a number of studies have confirmed the association between decreased BRCA1 mRNA and sporadic tumorigenesis in both the breast and the ovary [29][30][31]. Furthermore, immunohistochemical analyses of BRCA1 expression in sporadic breast and ovarian cancers revealed a significant reduction in BRCA1 protein [32,33]. This suppression of BRCA1 expression appears to be achieved through multiple means. For example, loss of heterozygosity (LOH) at the BRCA1 locus occurs in a significant proportion of sporadic of ovarian tumors [19,34]. Additionally, a comprehensive study by Russell et al. [35] found that 44% of the tumors had BRCA1 allelic loss, yet, strikingly, this event did not account for the loss of BRCA1 protein expression. Finally, 18% of the tumors exhibited a complete loss of BRCA1 protein in the absence of both LOH and allelic loss [35]. Taken together, these data indicate that epigenetic misregulation also contributes to the reduction of BRCA1 expression in sporadic tumors. Hypermethylation of the BRCA1 promoter Promoter hypermethylation is used during normal development to epigenetically downregulate gene expression in a tissue-specific manner. Methylation of the DNA occurs most frequently on the 5' cytosine residues within 5'-CpG-3' di-nucleotides, which often cluster together in CpG islands that can stretch for several kilobases [36]. In actively transcribed genes, CpG islands within regulatory regions are often unmethylated. In contrast, methylation at these sites represses transcription by altering chromatin structure such that the transcriptional machinery does not have proper access to functionally important regions of the promoter [37]. The proximal BRCA1 promoter lies within such a methylation-sensitive island and a developmentally inappropriate hypermethylation of the promoter does occur in some sporadic breast and ovarian tumors [38,39]. This hypermethylation may be functionally significant as it correlates with decreased BRCA1 mRNA [40]. An important question, yet to be answered, is whether such an abnormal promoter hypermethylation is the cause or consequence of an initial transcriptional repression [41]. Regardless, promoter hypermethylation has only been found in tumors where BRCA1 LOH has also occurred [40]. Thus, hypermethylation may serve as an epigenetic 'second hit' that inactivates the remaining wild type BRCA1 allele after LOH has occurred. While inappropriate promoter hypermethylation is very likely a powerful repressor of BRCA1 expression it is important to point out that it only occurs in a small subset of sporadic tumors [42,43]. Transcriptional regulation of BRCA1 The primary proximal BRCA1 promoter, which consists of less than 300 base pairs (bp), lies immediately upstream of the major breast-specific transcription start site located within the gene's first exon [44,45]. The regulation of this promoter is complex and a number of candidate regulatory sites have been identified and partially characterized (Fig 1). One of these elements, the positive regulatory region (PRR) at the 5' end of the promoter is both necessary and sufficient to maximally activate BRCA1 transcription [46]. Of particular interest, one putative regulatory site located within the PRR is a cyclic-AMP response element, which is capable of specifically binding to CREB, the cyclic-AMP response element binding protein [47]. Importantly, this element is a site of frequent hypermethylation in breast and ovarian tumors [48] and experimental methylation of this site decreases BRCA1 promoter activity in vitro [49]. Mutation of the CREB-binding con-sensus sequence within the context of an intact BRCA1 promoter [49] also causes a significant reduction in promoter activation in non-tumorigenic human OSE cells in culture [50]. Taken together, these findings indicate that the cAMP-response element is very likely an important positive regulator of BRCA1 expression in both the normal OSE and in tumor tissue. As an initial transcriptional repression is capable of contributing to the later hypermethylation of sensitive sites [41] misregulation of the transcriptional complex that binds to the cAMP-response element in the BRCA1 promoter could contribute to sporadic ovarian carcinoma development. Therefore, a careful examination of the signal transduction pathways that influence the activity and/or binding of transcriptional complexes to the cAMP-response element in the BRCA1 promoter may identify potentially important oncogenic events in sporadic ovarian carcinoma development. Schematic of the proximal BRCA1 promoter and the 8 functional sites that have been well characterized in breast cells It is conceivable that a number of the other transcriptional regulators of the BRCA1 promoter may also play a role in sporadic ovarian and breast carcinogenesis [27]. One such example is the GA-binding protein α/β (GABPα/β) which is a member of the ETS family of transcription factors [51]. GABPα/β specifically binds to three consecutive ETS factor-binding domains (described as the RIBS element) located immediately upstream of the CRE element in the positive response region (Fig 1) [52]. Transient overexpression of GABPα/β in breast carcinoma cells is able to stimulate BRCA1 promoter transactivation, thus demonstrating that it is potentially a direct positive regulator of BRCA1 expression [52]. Thus, loss of GABPα/β or lost responsiveness to GABPα/β-containing transcription factor complexes could result in decreased BRCA1 expression in sporadic tumors. Indeed, the promoter binding ability and transcriptional induction by GABPα/β is significantly reduced in mammary carcinoma cells. Comparatively, in the ovary the GABPα/β-binding RIBS element is highly active in normal OSE cells but not in ovarian carcinoma cells [50]. This intriguing observation suggests that the RIBS element, and perhaps aberrant function of the factors responsible for its regulation, may be specifically important in the repression of BRCA1 in sporadic ovarian tumors. Other DNA-binding proteins that may act on the RIBS element in the BRCA1 promoter are ID4 and ETS-2. ID4 is a recognized repressor of BRCA1 expression in both breast and ovarian carcinoma cell lines [53] that negatively regulates transcription by forming heterodimers with transcription factors through their helix-loop-helix domain [54]. ETS-2 is a member of the ETS family of transcription factors related to GABPα/β that also directly binds to the BRCA1 promoter in the RIBS domain [55]. Unlike GABPα/β, ETS-2 overexpression represses BRCA1 transcription which raises the possibility that multiple ETS factors may compete for binding within the RIBS domain such that upstream signaling pathways which differentially modulate the activation of these transcriptional complexes may be the critical regulators of BRCA1 expression. Conclusion Transcriptional suppression of the BRCA1 promoter occurs in sporadic ovarian carcinoma. Clearly, hypermethylation of the promoter plays an important role in this process. In addition, alterations in the transcription factor complexes that bind to the promoter also have a role to play, either upstream of hypermethylation or independent of it. Therefore, the future identification of critical regulators of these complexes will point the way to functionally important signaling pathways that are coopted during sporadic ovarian tumor formation and progression.	2018-04-03T02:20:58.541Z	2003-10-07T00:00:00.000	{ "year": 2003, "sha1": "425fa0a869e9b22c5677f1bb780550c2aa37555d", "oa_license": "CCBY", "oa_url": "https://rbej.biomedcentral.com/track/pdf/10.1186/1477-7827-1-72", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "67f2b386558a25f572b047dff65436d1ac7031ee", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
243155208	pes2o/s2orc	v3-fos-license	Internal Control: A Tool for Fraud Control in Deposit Money Banks in Nigeria The quest to curtail or eliminate frauds, errors and irregularities has made the institution of appropriate internal control inevitable in organization especially banks where the object of trade is money. The study therefore explored the use of internal control as a tool for fraud control in deposit money banks in Nigeria. Survey research design was adopted for the study with data collected using structured questionnaire from fifteen deposit money banks in Enugu metropolis. Result of hypothesis tested using Chi-Square (Fisher’s Exact Test) indicates that control environment, control activities and monitoring activities contribute significantly towards fraud control in an organization. The implication of the finding is that internal control is a vital the tool that protect organizations without which the organization must collapse due to fraud. One of the recommendations is that management should always ensure that strong and appropriate control activities are always in place to prevent financial leakages and promote accountability in the organization.. To that effect, there should also be appropriate segregation of duties to reduce a person’s opportunity to commit and conceal fraud or errors. Also, appropriate level of management should approve and authorize all transactions within a specified limit, and authorization should require dual signature. Also, there should be secured facilities and controlled access to computer programs and data files. multifaceted approach as organizations are affected by internal and external variables. Some variables from the external environment are beyond the control of the organization. Therefore, effectual internal control should be important parts of the responsibility of management at all levels to reduce risks (IFAC, 2012). Tunji (2013) in his research on effectiveness of internal control system as antidote for distress in banking industry in Nigeria noted that poor internal controls system in any organization will lead to the failure or suffering of the organization. Karagiorgos, Droalas and Dimou (2013) in a similar study discovered a close association of most bank failure to fraud. In the United States (US) alone, bank failures rose to 200 percent in 1980s partly due to fraud and mismanagement. In the year 2014, 14 banks failed in the US which is a reduction as compared to the same period in 2013 where 16 banks failed (Tumin, 2014). In Nigeria, bank distress have been associated with fraudulent practices as in the case of former Oceanic bank. In 2009, Central Bank of Nigeria (CBN) while using forensic accountants on 5 deposit money banks uncovered fraud which lead to the prosecution of the chief executive of Oceanic bank by Economic and Financial Crimes Commission (EFCC) and was sentenced to 18 months in prison following her conviction by the court. The rate and volume of fraud in Nigerian banking sector is high and has far reaching consequences as failure of a bank with wide spread of branches affects many people and economy hence the need to evaluate the contribution of internal control to fraud control in banks. Statement of the Problem The internal controls instituted by organizations are expected to prevent fraud and promote efficiency in the system. Equally expected from good internal control system is early detection of frauds, errors and irregularities. This is true as it provides security of assets, promote adherence to operational guideline/policy and procedure thus acting as whistle blowing mechanism amongst other functions. Globally, financial fraud is as old as mankind and in Nigeria, it get prominence 1930s during industrial and commercial bank distress. Fraud grew in Nigeria to the extent that Osisioma (2012) describes fraud as an industry that employs many people with apprentices. The volume of unethical financial actions and other unwholesome (corrupt) practices in Nigeria earned her the position of 149 th out of 180 countries assessed by Transparency International Corruption Index of 2020 in which she scored 25/100 (Transparency International, 2020). Nigerian banks recorded 37,817 cases and lost N15.15 billion in the year, 2018 despite the efforts of regulatory authorities . The Nigerian Deposit Insurance Corporation (NDIC) made this known in ThisDay Newspaper August 1, 2018. According to the report, the fraud and forgery incidences were carried out by both bank staff and non-bank individuals. These cases of fraud affect investors' confidence, as well as mobilization of savings by banks which would have been channeled into productive investments thus bringing about growth in the economy. If adequate savings are achieved it would make the banks solvent and profitable and build up the confidence of investors. It is in the light of the above that the study was carried out to ascertain the contribution of internal control to fraud control in banks in Nigeria. Research Objectives The primary aim of the study is to critically examine internal control as a tool for fraud control in deposit money banks in Nigeria. Specifically, the study want to: 1. Ascertain the extent control activities contribute towards fraud control in deposit money banks in Nigeria. 2. Establish the extent control environment helps in fraud control in deposit money banks in Nigeria. 3. Examine the contributions of monitoring activities towards fraud control in deposit money banks in Nigeria. Research Questions Based on the prior stated objectives of this research, the following research questions have been developed to be answered in due course of study: a. To what extent do control activities contribute towards fraud control in deposit money banks in Nigeria? b. To what extent has control environment helped in fraud control in deposit money banks in Nigeria? c. How do monitoring activities contribute towards fraud control in deposit money banks in Nigeria? Research Hypothesis The following hypotheses stated in their null form were tested in the work H o1 : No significant contribution from control activities towards fraud control in deposit money banks in Nigeria. H o2 : Control environment has no significant contribution towards fraud control in deposit money banks in Nigeria. H o3 : Monitoring activities have no significant contribution towards fraud control in deposit money banks in Nigeria. Scope of the Study The study was carried out on fifteen (15) REVIEW OF PREVIOUS WORKS Literature was reviewed in this order: conceptual review, theoretical framework, empirical review and research gap. Conceptual Review The study reviewed the concept of internal control, concept of fraud, control activities, control environment and monitoring activities. Internal Control The Institute of Chartered Accountants of England and Wales (ICAE & W) (1986) defines internal control as the all control, financial or otherwise, put in place by management of enterprise to ensure orderliness and efficiency in operation, while complying with management policy, securing the assets as well as complete and accurate records. It is therefore a management function covering financial and non financial aspects of the organization to ensures orderliness, compliance with laid down laws and order and security of all the assets and operations. Similar definition was given by Mayo (2006), as the measure taken by management of an organization to protect its resources against misuse, fraud, ineptitude; while making sure that correct record of accounts are kept and acting in line with the policy of the organization and performance assessment of divisions of the organization. Furthermore, Millechamp (2000), defines internal control system as autonomous review of systems of control and performance quality. It therefore emphasizes monitoring of laid down control measures in the organization and assessing the adequacy or otherwise of the system. From these definitions, internal control comprises the plan, procedures, methods and measures put in place in an organization to secure its resources, check the correctness of accounting of accounting data, thus promoting efficiency within the policy framework set by management. Thus, internal control can be viewed as all activities, processes and procedures put in place by management of an organization to engender smooth and efficient operation. Horngren (1990) gave two types of internal control namely: administrative controls ( comprising plans, methods and procedures that enhance management planning and control of operation) and financial controls (authorization of transactions, safeguard of assets and accuracy of accounting records which decreases wastes, intentional errors and fraud). Internal control has five components namely: control environment, control activities, monitoring processes, risk assessment (identifying, analyzing and responding to business risks which can be internal like errors and fraud or external like inflation exchange rate, interest rate etc) and information and communication system (processes and procedure for recording and reporting business transactions like purchases, sales, cash receipt and payment as well as ledger and journal entries) Objectives of Internal Control System According to the Committee of Sponsoring Organization (COSO) of the Treadway Commission (1999), these objectives are to: 1. Guarantee the effectiveness and efficiency of operations ( protection of asset inclusive) 2. Guarantee the dependability on financial reports presented 3. Ensure adherence to relevant laws and regulations and 4. Ensure the implementation of instructions and directives issued by board and management The Committee categorizes the above stated objectives of internal control system as a means to provide reasonable conviction that; a. Resources are safe and used for the purposes they were acquired. b. Business information are correct; and c. Laws and regulations are complied to by the employees. From the fore going, internal control is broad activity encompassing other aspects of the business like physical security, monitoring and so on. However, it does not encompass initiatives taken by the executives or management, like defining company strategies, setting objectives, taking management decisions, and handling risk or monitoring performance of different sections/units. Concept of fraud According to ICAN (2006), fraud consists of both the intentional misrepresentation in financial statements and use of trickery to get an undue benefit by the one or more persons, among management, employees, or third parties. This implies that fraud is criminal and carefully panned deceit of the users of the information for the eventual gain of the producer. Megis (2003) also refer to fraud as the misrepresentation by person (s) of a material fact known by the person(s) to be false or made with regard as to whether the fact is true with intent to deceive the other party such that the individual is injured or hurt.. It is also a type of irregularities involving the use of criminal deceit to attain an undeserved or illegitimate gain. In his own contribution, Archibong (1992), describes fraud as a premeditated and well planned tricky process or device undertaken by an individual or group of individuals, with the intention of attracting to individuals or group of individuals, ill-gotten benefits: monetary or otherwise, which would not have accrued in the absence of such deceitful act. Fraud may involve; manipulation, falsification or alternation of documents and records; recording transactions without substances; misappropriate of assets or theft; intentional misapplication of accounting policies; suppressing of transactions or omitting such transaction from records and willful misrepresentation of transactions or the entity's state of affairs (Agyemang, 2020). Fraud comes in different forms and magnitude. According to Biggs (2006), fraud could be classified into defalcation and manipulation while Megis (2003), also classified fraud into management fraud and employee fraud. Concept of Control Environment The control environment is a set of standards, processes, and structures upon which internal control across organization is based The top management of an organization establishes the guideline bearing in mind the importance of internal control. Control environment comprises the integrity and ethical values of the organization; the parameters enabling the top management to carry out its governance oversight responsibilities; the organizational structure and assignment of authority and responsibility; the process for attracting, developing, and retaining competent individuals; and the rigor around performance measures, incentives, rewards to drive accountability for performance, the way management assigns authority and responsibility, and organizes and develops its people (Ward & Smith, 2002) The resulting control environment has a pervasive impact on the overall system of internal control. It is the foundation for all other components of internal control as it provides discipline and structure. COSO (2013) Price Water House Coopers (PWC) (2012) indicated that, the control environment includes the governance and management functions and the attitudes, awareness, and actions of those charged with governance and management concerning the entity's internal control and its importance in the entity. Control environment, sets the tone of an institution and influences the control consciousness of its people. Control is also established at departmental or local control environment. Elements of the control environment include. a. The organizational structure of the institution; (The banks organogram defining the channel of communication and defined authorities and responsibilities). b. Management's philosophy and operating style. (formal or informal business strategy as well as aggressive or conservative operating philosophy c. The integrity, ethics, and competence of personnel. d. The external influences that affect the bank's operations and risk management practices (e.g., independent audits). e. The attention and route provided by the board of directors and its committees, especially the audit or risk management committees. f. The effectiveness of human resources policies and procedures. Jayne and Laura (2017) added that control environment set the tone of an organization, teach employees that right thing matters, setting expectations and appropriate behavior, addressing misconducts and other wrongdoings with commitment to competence by hiring the right staff and investment in employee education. Control activities The concept of control activities involves such activities as segregation of duties, authorization procedures and related responsibilities, adequate documentation and records, and protection of assets and records. In the light of the above, there are internal checks and balances as such there are approval limits and designated officer for such. Furthermore, no officer can start a transaction and handle it till the end. Proper recording is emphasized while adequate protection of assets and records from unauthorized access and use as well as against theft are put in place. It also encompasses approvals, verification and reconciliation occurring throughout the organization at all levels and in all functions (Layne & Laura, 2017) 2.1.5 Monitoring Activities These are used by management to ensure that controls are operating properly and that controls are modified in line with changes in assessment of risks which may reveal weaknesses in internal control. Usually, internal audit is used to ensure control or monitor control. This implies that even when there have been some measures of control put in places there must be in place a monitoring system to ensure compliance and early detection of breaches whenever they occur. The above could be achieved through continuous monitoring activities, intermittent evaluation or both. Theoretical Review: Differential opportunity Theory by Comer (1985) Comer put forward the theory based on the fact that every employee has the opportunity to commit fraud against the employer, against the supplier, against the customers of their employer, as well as, against third parties including government departments. Thus, the accessibility of the perpetrator to the accounts, assets, computer and premises of the organization, the skill possessed by the perpetrator to identify that such opportunity exist and to be used and the availability of adequate time to plan and execute the fraud are vital for fraud to occur. However, the opportunities is checked or regulated by the standard of internal control in place in the organization. The theory is appropriate for the study in that the internal controls put in place by organization aims at safe guard of assets, early detection of deviation from the organizational policies and procedures, effective and efficient running of the organization amongst others. In essence, internal control limits the occurrence of fraud. Empirical Review The interaction between internal control and fraud in banks has been attracting attention of scholars with a lot of empirical works yet without consensus. Some of those works were reviewed below. Haladu (2018) examined internal control measures for fraud prevention and detection in deposit money banks (DMBs) listed in Nigerian Stock Exchange (NSE). Proxies for internal control were control environment, risk assessment and control activities while discretional accruals were the proxies for fraud prevention and detections. The result of analysis revealed negative influence of internal control proxies on discretional accruals, however, risk assessment had insignificant. Furthermore, Oguda, Odhiambo and Byaruhanga (2015), verified the influence of internal control on fraud prevention and detection in Kakamenga County, Kenya, using purposive sampling method. Data were analysed using descriptive and inferential statistics. It was revealed that positive and significant relationship exists between internal control and fraud prevention and detection in district treasuries of Kakamenga County. In the same vein, Okonkwo and Ezegbu (2016), evaluated how internal control system in banks have facilitated in mitigating or preventing frauds in Nigerian banks. Using survey research design, findings include that internal control were not effective in checking frauds with branch managers as the major actors in fraudulent activities in Nigerian banks. In a similar study, Agyemang,(2020), assessed the effect of internal control on fraud prevention. Analysis of data collected through questionnaire using percentage suggests that good internal control and effective supervision and monitoring of internal control are capable of preventing and detecting fraud and fraudulent practices in Nigerian banks. Furthermore, Abiola and Oyewole (2013) explored the effect of internal control system on fraud detection in commercial banks in Nigeria. Results of data analysis using General Least Square, Correlation and Panel data analyses showed statistically significant relationship between employee training and fraud control amongst others Njoki (2016) in a explored internal audit functions and fraud detection among insurance companies operating in Kenya. He adopted a descriptive research designs and carried out census study of 41 insurance companies in Kenya. T-test result of analysis of primary data were collected using a structured established that there was a statistical relationship between Proactive Fraud Audit, Compliance to Policies, Risk Management, Control of Operation and Financial Reporting and fraud detection among insurance companies in Kenya. In a similar study, Adeniji (2004), in a case study of Power Holding Company of Nigeria examined the impact of internal audit on fraud detection and prevention. Data were collected using questionnaires and from company's financial statement. Chi-square result was suggestive that internal audit exhibits important function in public sector. Furthermore, Onyinlola (2010), examined the role of auditors in the detection, prevention and reporting of fraud in the manufacturing sector in Nigeria. Data obtained from 184 respondents on analysis revealed that the respondents placed very high expectation on auditors' duties on fraud prevention and detection which is in contrast to primary duties of an auditor. In their contribution, Samuel, Mohamed and Simon (2016), investigated the use of control activities in fraud control in financial institutions using a case of financial institutions in Meru town, Kenya. The study targeted a total of 106 respondents who included 20 managers and 86 supervisors of various financial institutions operating in Meru town, Kenya. The study used the questionnaire as a tool of collecting primary data and the hypotheses formulated were tested using chi-square statistical tool. The result of the findings established a significant relationship between control activities and fraud control. Chimeocha (2018) examined internal audit as an effective tool for fraud control in manufacturing organization using Michelle Laboratory as a case study. The study adopted a survey research design and primary data was collected using questionnaire. T-test result shows that internal audit has statistical significance association with fraud prevention in manufacturing organization. Summary of Literature Review/Gap Various literatures that were reviewed above and it was obvious that there was no uniformity in the approaches as well as variables adopted in their studies so far. Adeniji (2004), Njoki (2016) and Onyinlola (2010) examined the role of internal audit in fraud prevention and detection while Samuel, Mohammed and Simon (2016), investigated the use of internal control in fraud control but used only control activities in their study of banks in Meru Kenya. Similarly, Okonkwo and Ezegbu (2016), Oyewole (2013), and Haladu (2018) investigated internal control and fraud prevention and control but differed from the current study on the account of variables used to proxy for internal control. The study wants to ascertain the contribution of control activities, control environment and monitoring processes on fraud control in Nigerian banks. This is an expansion of the study and in a different environment; Enugu, Nigeria. METHODOLOGY The study adopted survey research design. The population of the study consists of 357 staff of banks that were within Enugu which comprise of 89 management staff (15 branch managers and 74 departmental managers) and 268 clerks. The study used questionnaire as a tool of collecting primary data. The questionnaire were designed in a way that the respondents were required to tick the appropriate answer according to their assessment based on modified four point Likert scale. The researcher prepared a list of questions based on the research objectives in order to solicit information from the target population; the questions were closed ended. The study adopted Taro Yamane formula in determining sample size of 286 from a population of 357. The sample was proportionately distributed among the 15 banks of study in line with their staff strength in the population of study. On reliability, the research adopted Cronbach approach which recommended that alpha value for each variable under study should not be less than 0.6 (Mohsen & Reg, 2011). The alpha value of 0.805 was derived by the use of Statistical Package for Social Sciences(SPSS). Face and content validity were adopted in validating the study. In analyzing the data, non-parametric statistical tools of Pearson Chi-Square was used. All the hypotheses were tested using the Fisher's Exact Test statistic at 5% level of significance. DATA PRESENTATION AND ANALYSIS This section deals with the presentation and analysis of the responses from the staff of 15 deposit money banks that were within Enugu Metropolis. The researchers administered 286 copies of the questionnaire of which 214 copies were returned, therefore 214 copies were found useful. Testing the Research Hypotheses In this section, the hypotheses earlier stated in this study in their null form were tested using Fisher's exact test. The critical or table value are compared with the computed P-value to decide whether to reject or accept a hypothesis. 4.3.1 Testing Hypothesis 1 H o1 : Control activities have no significant contribution towards fraud control in deposit money banks in Nigeria. N of Valid Cases 214 a. 8 cells (53.3%) have expected count less than 5. The minimum expected count is .12. b. The standardized statistic is -2.152. Source: Author's computation 2019 using SPSS Output file (Version 21.0) From table 4 above, the latter has 8 cells (53.3%) with expected count less than 5; giving minimum expected count of 0.12. With a significance level of 0.05, the hypothesis is tested using Fisher's exact test. Decision Rule: Since the test statistics, that is the P-values: 0.370 is greater than 0.05, we accept the alternative hypotheses stated implicitly. Therefore, it is concluded that Control activities have significant contribution towards fraud control in deposit money banks in Nigeria. Testing Hypothesis 2 H o2 : Control environment has no significant contribution towards fraud control in deposit money banks in Nigeria. With a significance level of 0.05, the hypothesis is tested using Fisher's exact test. Decision Rule: Since the test statistics, that is the P-values: 0.246 is greater than 0.05, we accept the alternative hypotheses stated implicitly. Therefore, it is concluded that control environment has significant contribution towards fraud control in deposit money banks in Nigeria. Testing Hypothesis 3 H o3 : Monitoring activities has no significant contribution towards fraud control in deposit money banks in Nigeria. 1%) have expected count less than 5. The minimum expected count is 0.2; therefore the Pearson Chi-Square will not be used as the value of the test statistic. With a significance level of 0.05, the hypothesis is tested using Fisher's Exact Test. Decision Rule: Since the test statistics, that is the P-values: 0. 431 is greater than 0.05, we accept the alternative hypotheses stated implicitly. Therefore, it is concluded that monitoring activities has significant contribution towards fraud control in deposit money banks in Nigeria. Discussion of Findings The primary aim of this study is to critically examine internal control as a tool for fraud control in deposit money banks in Nigeria. The following were revealed from analysis of date. Test of hypothesis 1 showed that control activities have significant contribution towards fraud control in deposit money banks in Nigeria. This is in agreement with the apriori expectation as it generally expected that good control activities should be able to keep fraud in check to a great extent. This finding is in agreement with the finding of Samuel, Mohamed and Simon (2016) and Haladu (2018) who established that significant relationship exist between Control Activities and fraud control. Effective control activities will ultimately reduce or remove completely fraud in deposit money banks as it entails provision of physical security to assets, as well as access code to computer data base thus preventing theft/fraud. Also, approvals and authorization of financial transactions are put in control by designating an officer for such and also tagging approval limits depending on the officer and organizational policy. However, this finding does not conform with that of Aruonoaghe and Ikyume (2013) who were in their work found a negative but non significant association between control activities and level of fraud prevention and detection. The above may not be unconnected with the volume of fraud going on in banks which most of time were unreported to avoid loss of confidence by the banking public. The result from hypothesis 2 revealed 0.284 significance which is greater than the significance level of signifance of 0.05. This simply implies that control environment has significant contribution towards fraud control in deposit money banks in Nigeria. This could be explained by the fact that control environment has all encompassing influence on the structure of business activities and how they are carried out, the way objectives are established, the way risks are assessed and fraud detected and addressed. Having such wide spread influence on various aspect of the organization, if properly handled/ controlled, would definitely have great contribution on fraud control. This finding agrees with Price Water House Coopers (PWC) (2012) which indicated that control environment is the foundation for all other components of internal control. The result of the chi-square test in hypothesis 3 revealed 0.284 significance value which is above 0.05 value set for alpha. Therefore, the hypothesis which states that monitoring activities has no significant contribution towards fraud control in deposit money banks in Nigeria is rejected. This is in agreement with the apriori expectation as good monitoring/supervision of internal control would help to ensure strict application of the measures hence better result. Internal control are usually well articulated and it is only on close supervision to ensure compliance that could enforce compliance. Summary of findings, Conclusion and Recommendations 5.1 Summary of Findings The study revealed the following: 1. Control activities have significant contribution towards fraud control in deposit money banks in Nigeria. 2. Control environment has significant contribution towards fraud control in deposit money banks in Nigeria. 3. Monitoring activities has significant contribution towards fraud control in deposit money banks in Nigeria. Conclusion From the research conducted, internal control can be used as an effective tool for fraud control in deposit money banks in Nigeria. Poor internal control would precipitate fraud, loss of funds and poor responsibility accounting. Growth and development of an organization lies on the quality of internal control put in place by the management. Recommendations Having conducted this research and analyzed the field data, the researcher recommends the following points, which if adhered to will positively bridge the gender gap. 1. Control activities have statistical association on level of fraud prevention and detection in deposit money banks in Nigeria, management should always ensure that strong and appropriate control activities are always in place to prevent financial leakages and promote accountability in the firm. There should also be appropriate segregation of duties to reduce a person's opportunity to commit fraud and conceal fraud or errors and an appropriate level of management should approve and authorize all transactions over a specified limit, and authorization should require dual signature. Also, there should be secure facilities and control access to computer programs and data files. 2. Control environment having significant statistical association with fraud control deposit money banks in Nigeria, then management should prepare a well-designed reporting system and conservative management philosophy and operating style should be adopted. External influences that affect the bank's operation and risk management should be avoided and also human resources policies and procedures should be made effective. 3. Finally, since result showed that monitoring activities has significant contribution towards fraud control in deposit money banks in Nigeria, management should ensure that internal and external audit functions are used fully as part of the organization's monitoring program and also, monitoring and follow up processes must be established to prevent fraudulent activities. Contributions to Knowledge This study has helped in showing how internal control components can be used to prevent fraud in banks in Nigeria. The study has also contributed to the enrichment of the literature on relevance of internal control system as a tool for fraud control in banks. It has also thrown more light to students, scholars and academics on the relationship between control environment, control activities, monitoring activities and fraud in banks. Area for Further Research Other researchers can look into internal control as a tool for fraud control in manufacturing companies in Nigeria. Risk assessment a tool for fraud control in deposit money banks in Nigeria.	2021-09-29T15:54:06.750Z	2021-07-01T00:00:00.000	{ "year": 2021, "sha1": "1ebeb020a6e3f3b2350a964da4726caea8850bbc", "oa_license": "CCBY", "oa_url": "https://iiste.org/Journals/index.php/EJBM/article/download/56773/58627", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "b9d6168ab6bc422a7347cba11caa4707318196f7", "s2fieldsofstudy": [ "Business", "Law" ], "extfieldsofstudy": [] }
245021751	pes2o/s2orc	v3-fos-license	The impact of the COVID-19 pandemic on child protective services caseworkers and administrators Background The COVID-19 pandemic has impacted children and young people experiencing child abuse and neglect. Child Protective Services (CPS) has played an important role in supporting children and families during the COVID-19 pandemic. Few studies to-date have evaluated the impact of the pandemic on CPS caseworkers and administrators in the United States. Objectives We conducted interviews to explore CPS caseworkers' and administrators' experiences working and serving families during the pandemic. Methods Participants were U.S.-based CPS caseworkers and administrators. We conducted semi-structured virtual interviews with participants and used an inductive thematic analysis approach. Results We conducted 37 interviews. Participants discussed how the COVID-19 pandemic has changed the way they conduct investigations and provide services to families in the CPS system. Several services were adapted to occur virtually, providing challenges and unique opportunities. Participants also described the personal barriers they faced during the pandemic, including working remotely, experiencing burnout, and challenges obtaining personal protective equipment. Finally, participants shared creative solutions they engaged in to support children and families during the COVID-19 pandemic, including expanding collaborations with other community-based organizations. Discussion This study suggests the important role that CPS has played during the pandemic and challenges individual CPS workers felt, in terms of both experiencing burnout and difficulty obtaining personalized protective equipment. Inclusion of the CPS system in emergency preparedness planning for future pandemics or natural disasters will ensure continuation of these vital services. Introduction Child abuse and neglect (CAN) is a pervasive public health problem, impacting one in seven children (Wildeman et al., 2014). As experiencing CAN deeply impacts the health and wellbeing of children and families (Griffith, 2020), it is vital that we identify and mitigate the sources of violence against children today to ensure the future wellbeing of our society. The COVID-19 pandemic has caused global unrest and economic instability experienced as worsening health morbidity and mortality, poverty, and stress (Kharas, 2020;Perry et al., 2021). While there is limited precedent for an international crisis equal to the magnitude of the COVID-19 pandemic, previous disasters have demonstrated increased stress, changes in routine, closing of schools, and an overall decline in community resources, all of which have been identified as risk factors for CAN (Zahran, Shelley, Peek, 2009). Past work has documented increases in physical abuse after natural disasters and during times of economic disruption (Berger et al., 2011;Keenan et al., 2004;Seddighi et al., 2021). Reports from early in the COVID-19 pandemic demonstrated a decrease in reports of suspected CAN by 20-70% during the Spring of 2020 as compared to the same period in 2019 (Campbell, 2020). In addition, national data indicated a decrease in the total number of emergency department visits for CAN in 2020 compared to 2019, but also a noted an increase in hospitalizations due to CAN (Swedo et al., 2020). There is some concern that this decrease reflects a lack of interaction between children and mandated reporters, rather than a true reduction in CAN, particularly in light of increased hospitalizations (Font, 2021;Nguyen, 2021). These findings combined with concerns that the pervasive stress experienced by parents during the pandemic may increase CAN have inspired a plethora of research surrounding the impact of the pandemic on children experiencing CAN (Cappa & Jijon, 2021). However, less work has considered the role of the child protective services (CPS) system in the context of a pandemic. The role of a CPS worker is multifaceted and includes investigating reports of possible CAN, providing ongoing services to families requiring assistance in safely caring for their children, arranging for children to live with relatives or foster families when they are not safe at home, and organizing reunification, adoption, or other living arrangements for youth leaving foster care (Child Welfare Information Gateway, 2020). More than 3.5 million children are involved in an investigation or response from CPS yearly, and an estimated 1.9 million children receive preventative services (defined as services, often educational, aimed toward supporting parents and caregivers) from the CPS system (U.S. Department of Health &amp et al., 2021). CPS workers are an important part of the healthcare and human services workforce, functioning as first responders to many suspected CAN cases. As such, they are subject to burnout and exposure to vicarious trauma (work-related trauma often experienced by these workers due to their repeated exposures to victims of violence; Font, 2021; Tavormina & Clossey, 2017) which even prior to the COVID-19 pandemic correlated with a high turnover rate among CPS workers (Leake et al., 2017). Recent research has described how healthcare workers have experienced stress, emotional exhaustion, and burnout during the COVID-19 pandemic (Mehta et al., 2021;Nelson & Kaminsky, 2020). One source of this stress for frontline workers was concern for personal health and access to personal protective equipment (PPE) (Nyashanu et al., 2020;Restauri & Sheridan, 2020). Understanding the obstacles faced by members of the CPS system during this pivotal moment can shed light on the needs of workers supporting our nation's most vulnerable children and families. Additionally, addressing these difficulties while highlighting the resilience exhibited by CPS frontline workers and administrators can help combat burnout while ensuring sustainability of the CPS workforce during the COVID-19 pandemic and future crises. Therefore, in this study we conducted interviews with US-based CPS caseworkers and administrators to explore: 1) their experiences during the COVID-19 pandemic; and 2) how their agencies responded during the pandemic to support CPS workers and families involved in the CPS system. Study design and study team Data for this article are taken from a larger qualitative study examining the impact of the COVID-19 pandemic on children and families, specifically its effect on family violence and related service provision from the perspectives of intimate partner violence (IPV) advocates, IPV administrators, and CPS caseworkers and administrators. Our study team comprised a multi-disciplinary group representing multiple academic institutions, Futures Without Violence, the American Academy of Pediatrics, and the Centers for Disease Control and Prevention's (CDC) Division of Violence Prevention. This diverse team allowed for multidisciplinary discussion, continuous evaluation of sources of potential bias, and data triangulation from multiple expert perspectives (Patton, 2015). The IPV aspects of the larger study centered on the experiences of IPV survivors and their families during the pandemic, the role of IPV advocates and agencies in supporting families, and practice and policy implications emerging during the pandemic. We examined these questions through interviews with IPV advocates and administrators; results from these data are available elsewhere (Garcia et al., 2021;Ragavan et al., 2021). The CPS components of this study, which are described in this article, focused on CPS workers' and administrators' descriptions of their experiences during the COVID-19 pandemic (both personally and professionally) and their perceptions of how their work was impacted by the pandemic. We used a descriptive qualitative approach for this study. Descriptive qualitative research is defined as "research designed to produce a low-inference description of a phenomenon" (Kahlke, 2014). Descriptive qualitative research is particularly useful for hypothesis generation in the context of new or unprecedented phenomenon, such as the COVID-19 pandemic. Descriptive qualitative research relies on an inductive approach, with the majority of codes emerging from the data. We chose this approach to amplify the voices of CPS caseworkers and administrators and hear about their experiences during this unprecedented global health emergency. The University of Pittsburgh Institutional Review Board approved this study and deemed it exempt. Participants Study inclusion criteria included: 1) identifying as a CPS caseworker or administrator in the United States; 2) being 18 years of age or older; and 3) being able to complete the interview in English. CPS caseworkers were defined as those working directly with families; administrators were those with supervisory roles. Development of the interview guide The interview guides explored workers' perceptions of the COVID-19 pandemic's impact on CPS, on the families they serve, their organization's response or adaptations made, and challenges that arose. Questions focused on: 1) how CPS staff and agencies adapted in response to the pandemic; 2) the experiences of CPS caseworkers and administrators during pandemic; and 3) how CPS supported children and families during the pandemic. Separate interview guides were developed by the research team for caseworkers and administrators, though similar themes were addressed in both. Prior to using the interview guides, we pilot tested them with 1 CPS caseworker and 1 administrator and made adjustments based on their feedback. Additional questions were added during data collection based on emerging codes, such as asking about specific supports needed by frontline workers. Online supplemental materials include the questions asked during interviews. Training of qualitative interviewers Interviews were conducted by 4 of the authors (VR, LR, CH, KP). Prior to starting data collection, the 4 interviewers attended a qualitative methods training facilitated by an expert in qualitative research (JC). This training included content on how to conduct interviews and strategies for managing challenging situations during interviews; we also reviewed the interview guide. The interviewers then practiced conducting interviews on each other and received feedback from the study team. Transcripts from the first few interviews for each interviewer were critically reviewed by qualitative researchers on the team (JC, MR), with feedback provided prior to beginning the next round of interviews. Ongoing training was provided throughout the data collection process. Participant recruitment Participant recruitment occurred through announcements on listservs and word-of-mouth. We started by using the networks of a senior child abuse pediatrician (RB), as well as sending out emails through state listservs. Invitations were distributed widely across the US and territories. Eligible participants emailed or called the study team to set up an interview. Some direct recruitment, via email, was performed to increase responses. We also used snowball sampling, where administrators were asked if they knew caseworkers who would be interested in participating in the study. Data collection Study team members trained and experienced in qualitative interviewing conducted virtual interviews through a private videoconference. An information script was reviewed prior to the interview and participants provided verbal agreement to participate, as required by our IRB for an exempt study. Interviews were conducted confidentially and all identifying information was removed during transcription. Interviews were audio recorded, conducted in English, and lasted approximately 60 min. The interviewer obtained demographic information including race/ethnicity, gender identity, region of the country, and number of years working at the agency. After completing the interview, participants were offered a $30 electronic gift card. Interviews continued until thematic saturation was reached, or when no new themes emerged (Guest et al., 2006). Data transcription and analysis For coding and analysis, the research team used an inductive, thematic-analysis approach, developing codes as they emerged from the data (Braun & Clarke, 2006). To support the organization and management of codes, audio recordings were transcribed verbatim and uploaded into the Dedoose software program (version 7.5.16;Dedoose, 2016). Two coders independently coded each transcript line by line, actively developing a codebook containing individual codes and their definitions (Braun & Clarke, 2006;Patton, 2015). A consensus coder then reviewed the coding of each transcript and noted any discrepancies. An example of a discrepancy which was changed based on consensus coding was adding a new code "effect of COVID on CPS agency" to distinguish impact on agency from direct impact on staff. The entire coding team met weekly to resolve discrepancies through methodically reviewing each discrepancy and discussing until consensus was reached. The full team met monthly to review emerging codes, make any necessary changes to the interview guide, and consolidate codes into themes. To further triangulate our emerging data, we held monthly meetings with a group of stakeholders affiliated with national and regional violence prevention and victim services organizations around the US. Emerging codes and themes were shared with these stakeholders and feedback was elicited to help shape the results based on their personal and professional experiences. Results We conducted interviews from July 2020-January 2021 with 21 CPS caseworkers and 16 administrators working in 12 US states. The majority of respondents (81%) identified as female. Other demographic characteristics are found in Table 1. Five themes emerged from the data: 1) impact of the COVID-19 pandemic on conducting investigations; 2) impact of the COVID-19 pandemic on provision of ongoing child protective services; 3) opportunities and challenges for CPS staff working remotely during the COVID-19 pandemic; 4) CPS staff experiencing burnout and challenges obtaining personal protective equipment (PPE); and 5) new ways CPS agencies and staff supported children and families during the pandemic. Representative quotations are provided throughout the results section; Table 2 provides a summary of the results. Theme 1: impact of the COVID-19 pandemic on conducting investigations The majority of participants reported that CPS continued to adhere to pre-pandemic investigation guidelines, conducting cases that reached investigation threshold. An administrator noted, "in terms of what is safe, what is unsafe, what meets criteria for abuse and what doesn't, and what meets criteria for neglect and what does not, [the pandemic] has changed nothing." A caseworker stated: "if we have safety concerns for kids, we still have to go into the home and see the home, see the family, talk with the kids." (P34) Further, the threshold for removal of children from the home was unchanged according to interviewed CPS staff. A CPS caseworker noted: "if a child is at imminent risk of harm, then we have to [remove them] … I don't care if the kid's COVID positive, we have to make that decision… I think when we talk about the threshold of removal, it hasn't so much changed the way we actually do it and the way we determine imminent risk." (P14). However, most participants did note modifications to the investigation process. A caseworker noted: "[we have had to get] more creative about trying to really limit our time inside of people's houses, by going out on walks with kids to do interviews or talking with them out in the front yard." (P34) Some organizations continued to require caseworkers to conduct an in-person home visit with each investigation, while others incorporated virtual visits and continually adjusted their requirements based on COVID-19 severity in their region. Other organizations had dedicated field teams that conduct in-person assessments, while the rest of the workers remained remote. As an administrator shared: "[we implemented] a system where we had identified field workers who did not carry a caseload, but essentially made all the field contacts for staff at home…so our staff at home were technically doing the investigations and directing the investigations, and we had limited staff who were actually making field contact with our families." (P20). Privacy concerns and challenges with virtual service provision were highlighted by most participants. At times with high community-based COVID-19 prevalence, or if a family was confirmed COVID-19 positive, caseworkers described conducting a virtual home walk-through, with a subsequent in-person home visit when it was safe to do so. One caseworker shared: "even during those virtual check-ins and face-to-faces, I need to be talking with all of the adults in the household, I need to be seeing the kids…to check-in with the kids privately. They'll do a swivel of the room to show that they're alone." (P34) An administrator discussed privacy concerns while interviewing children, noting: "behind a phone or behind a camera…the perpetrators could be close or near [by]. It doesn't allow Table 1 Demographic characteristics of CPS frontline workers and administrators (total n = 37). Region Participants ( us to see things by the eye as much as we want to…It's not givin' us that thorough look that we would otherwise need or want when we're out in the field." (P22) A caseworker shared a similar perspective: "it was easier when we had the field team workers 'cause that way, they were able to still somewhat see them face-to-face…It was always nice to have someone there personally to see what we're missing 'cause parents can hide stuff." (P15). In an attempt to preserve confidentiality and fidelity of interviews with clients, the majority of caseworkers reported conducting separate, private virtual interviews with children when in-person visits were not feasible. For younger children, several challenges to this practice were noted. A caseworker shared: "for those kids who are verbal, but easily get distracted, kids three, four, five years old, you really have to put in a lot of effort to interview and get their attention and get all the information you can." (P17) That caseworker noted that they prefer to talk to children "off-site at a neutral spot away from parents. It does take that stress off of kids." (P17) Participants regularly noted challenges finding such sites due to COVID-19 physical distancing regulations. A caseworker explained: "community meeting spots have been taken away." (P17). Theme 2: impact of the COVID-19 pandemic on provision of ongoing child protective services Ongoing services-those implemented when there is a perceived need to mitigate future risk in a family for whom the initial investigation is completed-primarily switched to virtual. Participants described the challenges they experienced with virtual service provision. An administrator shared: "initially, the biggest change…was the pausing of face-to-face contact with clients…Pausing faceto-face contact and switching everything to telephone and teleconference we did immediately." (P30) A caseworker shared how virtual service provision changed in-person visitations: "[prior to COVID-19 we] were supposed to be seeing kids in custody once a month in the home…Right now we have permission to do all virtual. I don't go into the home unless it's absolutely necessary or an emergency." (P33). Most participants also shared challenges arising from placing children in foster homes, noting that many foster homes put their licensures on hold. One administrator relayed: "we don't really have a whole lot of options if we have a child that needs to come into [foster] care; they need to be removed from their parents…we don't have a whole lot of options that we used to have before." (P25) These barriers were compounded if a child had been exposed to COVID-19. As one caseworker noted: "no foster home wants a kid who's got COVID. No foster home wants a kid who's been exposed to COVID, because they don't wanna put themselves or their families at risk." (P34) While some workers stated they had not seen a reduction in foster family availability, this caseworker noted: "for kids [who] have either been exposed to COVID or for kids that have COVID, it's almost impossible to find placement for those kiddos." Visitation between parents and children in foster care became increasingly challenging to coordinate. Participants described how the indoor locations where visitations had once taken place were often inaccessible due to COVID-19 restrictions, which led workers to arrange visitation outdoors. In late 2020, once the weather started to change, it became harder to find private, safe locations for visitation. One administrator commented: "for a period, we ceased all visitation. Several months later we were able to then reinitiate visitation…Providing PPE to biological parents, letting them-can you hug, can you not hug?…it's a lot to pay attention to." (P28) Some agency visitation rules were based on the ages of children. An administrator noted specific rules for children under age five: "[they] can still see parents face-to-face because of that young age vulnerability bonding. Kids five and older have to do televisits" (CPS • Some CPS agencies proactively reached out to families • Caseworkers more readily connected families to supports within the community • There was an increase in requests for basic resources, as well as technologic supports • New COVID-specific support systems were created in some counties VR 16) based on the COVID severity in their region. Another administrator described challenges related to bonding during these physically-distanced meetings: "parents have to wear full PPE in order to have a visit with their baby and how effective is that bonding with their baby when the baby can't even see the face of their parent?" (P29). While there were many challenges experienced by CPS workers and their clients during this timeframe, participants did relay some opportunities arising from adaptations made during the pandemic. Importantly, many caseworkers noted improved family engagement during the pandemic due to the transition to virtual service provision. One caseworker explained: "[previously] parents would have issues, especially coming to say court hearings or family partnership meetings, and they'd have to leave work or miss work. The families that are still working, if they're from home, it's easier for them to just hop online, click a button, and they've joined the meeting." (P21) One administrator explained the shift that occurred, stating "we're in a government building, so previously, any parent essentially with a warrant, which is a lot of our parents, would refuse to come to meetings because they could get arrested here. Now, they're showing up to these meetings because they're via phone. They don't have to get here with public transportation. They're not getting arrested because they have active warrants. They're actually making more progress faster on their treatment plans because of the virtual world we're in now." (P24) Overall, as one caseworker remarked: "while there were definitely a lot of benefits pre-COVID to being in court in-person and family partnership meetings in-person…I think what we're noticing a lot is we have less no-shows, more engagement." (P21). Theme 3: opportunities and challenges for CPS staff working remotely during the COVID-19 pandemic The majority of participants reported working from home since the onset of the pandemic, at least part-time. This allowed for increased productivity and efficiency when connecting with coworkers and clients virtually. One caseworker commented: "[I] found that I have more time to support families with the introduction of telehealth. I would actually see my [clients] more virtually throughout the month than I was when I was seeing them in person." (P14) An administrator commented: "caseworkers really are feeling a better work-life balance with being able to work remote. They're not having long commutes. They can throw a load of laundry in between meetings or whatever. We've heard multiple times that, despite the stress the pandemic is causing, they do feel like there's a better work-life balance." (P31). Some CPS administrators stated their organizations were able to provide new supports for workers at home during this time. Many participants noted that they appreciated working from home and felt supported by their department during the transition period. As one administrator explained, "child protection has been extremely flexible with allowing us to make our schedules work with our families' needs at the time." (P27) While some caseworkers faced technologic challenges such as inadequate connectivity, they were often able to overcome these issues with the help of their department. As one caseworker noted, "it's like we've found some rhythms to make things easier for us…and the department has given us cellphones and laptops to work from home. Their technical support has boosted significantly." (P24). Just as CPS agencies shifted to operating virtually, so too did other organizations and operations such as the court system, allowing for increased efficiency. One caseworker noted: "[pre-pandemic] if I had a 9:00 a.m. hearing and then a 10:30 a.m. hearing in person, I'd be in court my whole morning through lunch. Just for two court hearings. Now that we're virtual, I can literally plan work in between my court hearings now because I know when my court hearing's actually gonna occur." (P26) Within their own organization both administrators and caseworkers noted "[there was] more supervision and…continued check-ins…There's more trainings and more workshops and the participation has…[increased] too." (P16). While pandemic adaptations created some opportunities, some CPS staff felt that they accomplished more working from the office and noted it was difficult to balance their working time at home while caring for family members. One administrator noted that their staff were facing challenges similar to those of the families they serve: "we've had some staff have to go off on family medical [leave] 'cause of childcare. They have children who are at home in school and so they're saying I have to focus on my children." (P7). Theme 4: CPS staff experiencing burnout and challenges obtaining PPE during the COVID-19 pandemic Participants shared many new sources of stress caused by the COVID-19 pandemic. Obtaining PPE, and the delay therein, was a significant challenge for many departments. This led to workers feeling unsupported by their agencies. As one caseworker noted: "[initially] there just wasn't a whole lot of support…as far as acknowledging what was going on and trying to get us-I think it was two or three months into the pandemic and lockdowns before we got PPE gear." (P34) This caseworker added "[I was] literally stitching pillowcases together to make my own masks because I was having to go out. I know a lot of essential workers were in those kinds of positions." (P34) As CPS caseworkers continued to perform their duties throughout the pandemic, many described feeling under-recognized as frontline workers. One caseworker noted: "[there has not been sufficient acknowledgment of-we're also still out there on the front line, going out and doing all this stuff for kids." (P34) An administrator reported: "[caseworkers are] very rarely recognized as being the ones that are out on the frontlines. Our agency hasn't stopped in any way, shape, or form since this pandemic started. The law requires us to see children when we need to see children. There's no loopholes with that. We were given the N95 masks to wear in case we're near somebody who has COVID. We don't really have any protections or guarantees or anything throughout all of this. We're the ones that are in the middle of trying to help these people who are financially ruined by this pandemic." (P13) Some caseworkers felt so overwhelmed during the pandemic that they were not able to give their clients the support they knew they required: "[I don't] have the time to go above and beyond and try to provide those extra services, even though I've no doubt they'll be helpful." (P26). Implementation of additional supports for caseworkers was important in combatting burnout. These ranged from technical support to mental health services, as well as encouragement for workers to utilize pre-existing services like an employee assistance program. One administrator reported: "[I would send] reminders to staff to utilize our employee assistance, if they need it." (P31) Administrators recognized that this was a challenging, isolating time for workers, noting: "[we have] caseworkers who are trying to homeschool their kids, and work. All the challenges that all of America are facing. It takes an emotional toll, especially on top of a job that's emotional and taxing anyway." (P31) Administrators noted that there was a pre-existing issue of job retention within CPS which was exacerbated by the pandemic. Per one administrator: "one of the most profound elements is the unbelievable drop in the number of investigators once COVID hit… We are under 700 investigators right now. We sometimes are up between 750 and 800… This is a time where we have a lot of people leaving child protection regardless, based on the age of our employees and the time of year." (P28) Thus, some agencies held a variety of trainings for caseworkers, including mindfulness, self-care and interviewing tips while being mindful of COVID-19 restrictions. One administrator reported: "[we are] doing a mindfulness training…We have an actual worker from another county that's presenting it for us... I'll be honest, a lot of our staff are in counseling right now. Their own personal counseling because they had to." (P22). Theme 5: new ways CPS supported children and families during COVID-19 During the pandemic, especially during the height of shelter-in-place orders, many people reported feeling isolated. Some agencies proactively reached out to clients during the pandemic to ensure their needs were being met. One administrator reported: "[we looked at] all the data, all the cases within the last six months…[Caseworkers] called [clients]… saying we just want to know do you need anything. Letting them know it's not that we had any complaints or anything, but we just wanted to see if they were okay and if they needed anything." (P7). Agencies utilized existing resources and developed new strategies to meet the needs of families. One administrator stated: "we tried to guide [families] to the services that are in the community. We have put in services into place as far as in-home services, or parent aid services, or child health support…much more freely since the pandemic. It might be a case where an in-home provider, you really needed to justify it a year ago…whereas now we're saying, 'Let's put it in and try it and see if it's at all beneficial.'" (P30) Another administrator reported: "[we are sending] out not just investigators, but…child welfare specialists who can connect with things like housing, food pantry, mental health services, and make really important connections." (P28). Basic resources such as food and technology were some of the items most commonly requested by families. As one administrator reported: "[we] provided more hard service supports than we probably normally do. Gift cards for groceries, that kind of thing." (P31) Financially, a caseworker from one county reported: "[there is an] additional funding resource available…called COVID emergency assistance. It's basically an application that the family can fill out [to] receive assistance for…rental, or gas, food, those sorts of things." (P21) Additionally, during the pandemic many families became increasingly reliant on technology, and some counties were able to provide smartphones to families in need. An administrator noted: "each worker [was] probably accessing 10 to 12 phones each, and we have 40 field CPSW staff in our office. We were getting 5 to 10 iPhones delivered daily" (P30) which they then handed out to families. Participants discussed developing collaborative relationships with other community-based organizations to help support families. One caseworker noted: "[we also have to] think about the parents, how are we supporting them? With our role, it's pretty limited, so… what can we do as an agency to connect with other agencies to fully come up with really good resources for these families." (P23) Another caseworker shared: "[our county created] a care portal. Basically, if a family has a request for say car seats, or a gas card, we can submit a request through this portal, and it essentially goes out to any business or church that is participating in the program." (P21) Some organizations also reached out to members of the community for additional support. One administrator noted: "guidance counselors were having no work to do, so we were asking them to go knock on the door and not go in the house necessarily but knock on the door to see if they could engage kids." (P32) This was helpful, because as this administrator explained "our agency can't do it alone. Before they report an [educational] neglect case to us, we ask them now to have a community response team meeting." (P32). Discussion To our knowledge, this is one of the first studies to describe the perspectives of CPS workers in the US regarding the impact of the COVID-19 pandemic on themselves and the families they serve. Participants highlighted challenges in virtual service provision, while also noting how engaging with families virtually provided flexibility and opportunity for both families and CPS workers. Virtual visits (telemedicine) became more ubiquitous across healthcare settings after the start of the COVID-19 pandemic and have been shown to increase patient's access to healthcare (Koonin et al., 2020;Mann et al., 2020). Our study builds on prior work in the healthcare space by showing that virtual service provision similarly offers families involved with CPS greater opportunity to engage with services. This is particularly critical because one of the reasons that children may remain in the child welfare system is because of challenges parents may face juggling CPS-mandated services with their employment schedules, thereby criminalizing poverty (Keegan Eamon & Kopels, 2004;Roberts, 2002). However, virtual CPS visits also created privacy concerns, as many participants noted challenges guaranteeing confidentiality during visits. Similar concerns have been noted in studies examining virtual mental health therapy (Jentsch & Schnock, 2020;Ragavan et al., 2020). Additionally, virtual family visitation was highlighted by participants as an adaptation made during the pandemic which had potentially harmful impacts on families and parent-child bonding. For that reason, organizations advocating for children and families, such as the American Academy of Pediatrics have released guidance recommending in-person visits continue whenever possible (American Academy of Pediatrics, 2021). While research prior to the pandemic demonstrated that CPS workers experience high levels of burnout, compassion fatigue and vicarious trauma, the CPS caseworkers in the study noted that the pandemic compounded their baseline stress (Tavormina & Clossey, 2017). These findings are similar to studies conducted with healthcare workers, which have found high levels of burnout due to the COVID-19 pandemic (Denning et al., 2021;Mehta et al., 2021;Raudenská et al., 2020). Caseworkers furthermore noted that they felt overwhelmed and stretched beyond their capacities, and verbalized their awareness that this likely had negative repercussions for their clients. Studies have found that it likely negatively impacts child outcomes as well (Denneet al., E. Denne M. Stevenson T, 2019). In the face of numerous stressors, the one most cited by participants was feeling ignored and unrecognized as frontline workers specifically as it relates to PPE prioritization. Lack of transparency about PPE shortages, panic around lack of PPE, and unclear PPE guidance created compounding stress for healthcare workers (Nyashanu et al., 2020;O'Sullivan, 2020). Our study suggests that CPS workers were also not provided adequate PPE, and experienced delays in obtaining PPE, which contributed to stress surrounding contracting or spreading the virus to clients as well as their families. Additionally, it exacerbated their feelings of neglect within the health and human services system as a whole, as they felt that they were operating as frontline workers while not being adequately prepared for the challenges that this provided. While CPS workers were prioritized in Phase 1a for COVID-19 vaccine by the Advisory Council on Immunization Practices, many participants did not feel like they were being prioritized and struggled with a lack of PPE (Centers for Disease Control and Prevention, 2021). Despite the many challenges associated with the COVID-19 pandemic, there were also several silver linings from a professional standpoint including improved efficiency when working remotely, new ways of connecting with coworkers, and opportunities surrounding virtual trainings. Our findings parallel those of a qualitative study regarding the adaptations that one state made to their CPS training program during the pandemic-finding that leadership was able to provide tangible support for their staff by encouraging selfcare, promoting work flexibility and actively listening to their employees (Schwab-Reese et al., 2020). Given the high burnout and turnover rate for CPS workers (Leake et al., 2017;McFadden et al., 2018) these changes could be part of a post-pandemic work-life balance set up for CPS agencies looking to retain workers longer. In addition to finding innovative solutions to support their own professional wellbeing, CPS workers reported that they were able to support families in new ways, such as proactively reaching out to clients and innovatively utilizing existing resources. In particular, participants highlighted the importance of strengthening collaborations with trusted community organizations providing other needed services. Historically the child welfare system has faced challenges coordinating with other community-based resources, due to communication barriers, lack of resources and competing primary foci between CPS and the collaborating agencies (Darlington et al., 2004). Building partnerships and strengthening relationship among CPS, schools and community organizations (e.g., summer camps, victim services agencies, faith-based groups) might be a potential approach to providing resources and supports for children and families in the CPS system. We recognize several limitations to this study. Despite extensive, nationwide outreach attempts by the study team, there was a concentration of responses from agencies in the Northeast and Midwest and fewer to no caseworkers and administrators were interviewed from the West Coast and the southern United States. A majority of the participants identified as non-Hispanic white and women, which, while not representative of the United States at large, is representative of CPS caseworkers nationwide (65% identify as non-Hispanic white, 77% as women (Careers, 2021) Zippia.com. We faced challenges getting approvals from some states for CPS staff to participate in this study, which was different from our experiencing interviewing IPV advocates and administrators as part of the larger study. Some CPS caseworkers were not comfortable with recording conversations, despite assurances of privacy and confidentiality per IRB protocol. As a result of the challenges, there is likely selection bias in terms of who participated in this study. Another limitation was that we interviewed CPS employees rather than the families they serve. Families and CPS workers may perceive the same situations and events in different ways. We also did not limit our study to experienced CPS employees, although we believe this is a strength as it allowed us to elucidate a full range of experiences. Additionally, while not necessarily a limitation, we recognize that few participants discussed the specific needs of communities of color, particularly Black and American Indian or Alaskan Native (AIAN) persons who are over-represented in the CPS system due to histories of racism that have perpetrated cycles of state-sanctioned violence and family separation (Palusci & Botash, 2021;Roberts, 2002). Furthermore, families of color (in the cited study, Black and Hispanic/Latino), as compared with non-Hispanic white parents, reported feeling like the CPS system and processes were less understandable (Cleveland & Quas, 2020). The COVID-19 pandemic, which, due to structural racism, disproportionately impacts Black, AIAN, and Hispanic/Latino communities, likely compounded these pre-existing disparities (Dorn et al., 2020). Interviewees' minimal conversation around racism is potentially expected, as we did not explicit ask about experiences of communities of color (instead used the broader term marginalized communities; supplemental materials). Additionally, studies have shown that white people often avoid discussing issues of race and class, engaging in so-called "strategic colorblindness" (Apfelbaum & Sommers, 2008). Further research is needed to better understand why the intersectional pandemics of racism, COVID-19, and CAN did not emerge in this study. Conclusion and implications The results from this study set the foundation for research, policy and practice recommendations. Future quantitative research conducted with larger groups of CPS employees and families impacted by the CPS system may be helpful for triangulating some of the findings from this exploratory qualitative study. From a policy and practice perspective, it is likely that CPS caseworkers will have an increased number of clients to support in the aftermath of the COVID-19 pandemic, with physical distancing measures ceasing, and society returning to "normal." There may also be more families requiring services given the deleterious effects of the pandemic. Resources and support are needed for families in the CPS system and for agencies supporting them, including continued opportunity to engage with private virtual services. Additionally, mitigating burnout among CPS workers through flexible schedules, mental health support, virtual trainings, and social connections are important steps to sustain the CPS workforce. This work also provides guidance for future pandemics or natural disasters. Disaster planning should consider the impact of children and families in the CPS system and ensure safe ways to continue visitation. During future pandemics, clearly identifying CPS workers as front-line workers will help ensure their access to PPE and other important resources. Encouraging communication among CPS systems in different regions and states will be important to share how innovative solutions to common challenges were successfully implemented. Funding This study was funded by a cooperative agreement from the Centers for Disease Control and Prevention (NU38OT000282). The findings and conclusions in this manuscript are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the American Academy of Pediatrics. Declaration of competing interest The authors do not have any conflicts of interest to disclose. The findings and conclusions in this manuscript are those of the author (s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention.	2021-12-12T14:40:00.764Z	2021-12-01T00:00:00.000	{ "year": 2021, "sha1": "dd63ef89ccb746f2847bdb03893d1d9dbd608237", "oa_license": null, "oa_url": "https://doi.org/10.1016/j.chiabu.2021.105431", "oa_status": "BRONZE", "pdf_src": "PubMedCentral", "pdf_hash": "ce8433c81b4e1a7c71d4f6db335fe6049a51ea6f", "s2fieldsofstudy": [ "Psychology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
225103228	pes2o/s2orc	v3-fos-license	Scalable Federated Learning over Passive Optical Networks Two-step aggregation is introduced to facilitate scalable federated learning (SFL) over passive optical networks (PONs). Results reveal that the SFL keeps the required PON upstream bandwidth constant regardless of the number of involved clients, while bringing ~10% learning accuracy improvement. Introduction Federated learning (FL) is a privacy-preserving paradigm of distributed learning, where clients (e.g., mobile terminals, user devices) can collaboratively learn a shared model while keeping all the data locally [1]. During the training process of FL, each client needs to periodically transmit its local model parameters to the centralized parameter server (CPS), where a set of global model parameters are updated according to aggregation strategies such as the federated averaging algorithm (FedAvg) [1]. Then, the CPS sends the global model parameters to each client to update its local model for a new round of training. One challenge here is that the amount of training traffic generated by the model updates can be huge, particularly for the upstream which often has limited bandwidth compared to the downstream. For example, the training traffic for a convolutional neural network (CNN) is up to tens of Mbits [2]. Considering hundreds of or thousands of FL clients or even more, the upstream bandwidth consumption could be huge. To achieve optimal learning performance, FL often needs many rounds, where each round involves a client selection process to chose a certain number of clients to participate in the training process. FL can be done both synchronously and asynchronously. The synchronous training requests that the model updates to be carried out within a fixed period so that the CPS can conduct aggregation timely. The synchronous training typically runs more efficiently than the asynchronous one [1] but needs to make sure the local model to be updated within the required time. Passive optical network (PON) is one of the most promising access technologies to support various residential broadband and mobile services for end users due to its high capacity and energy efficiency [3]. Furthermore, the quality of experience (QoE) for end users with these services can be improved by using emerging machine learning technologies. For services that have privacy concerns, federated learning is a promising approach for QoE improvement. When running synchronized FL training over PONs, a significant challenge is how to efficiently transmit a large amount of traffic under the required synchronization time over PONs to ensure the learning performance. In this regard, our previous work [4] proposed a bandwidth slicing mechanism for PONs to involve a sufficient number of clients within a threshold of synchronization time. The mechanism reserves a certain amount of bandwidth for each FL task, where we found when the involved number of clients increases, the reserved bandwidth becomes the constraint and the learning accuracy may be greatly affected. In this paper, going beyond [4], we propose two-step aggregation method to facilitate scalable federated learning (SFL) running over PONs, in which the local model parameters are firstly aggregated at optical network units (ONUs) and then further aggregated by the CPS at the central office. In such a way, the required upstream bandwidth for model parameters transmission becomes constant, regardless of the number of clients. Our results reveal that even with limited reserved upstream bandwidth, the number of clients that satisfy synchronization time requirements could be sufficiently large, achieving good learning accuracy. Figure 1 illustrates the classical and scalable FL over PONs, in which n ONUs are connected with the OLT that is associated with the CPS (i.e., OLT/CPS) at the central office. Each ONU can be co-located with a base station (BS) or an access point (AP), where m mobile devices (e.g., mobile phones) can access wireless networks. These mobile devices (i.e., clients) can work together to train a shared model (e.g., CNN model) for machine learning assisted services (e.g., object recognition) while keeping all the data within the mobile devices. The training is implemented in a round-based manner. At the beginning of the !" round, the CPS selects a certain number of clients to join the FL task and distributes to them a global model, which is parameterized by # ! . After receiving # ! , the client_i,j (i.e., the !" client belonging to the !" ONU; i ∈ [1, ]; ∈ [1, ]) runs local model training with the global model # ! and local data ( $,& ). This results in a local model at the !" round and the parameters can be updated by using stochastic gradient descent (SGD), Scalable FL over PONs where is the learning rate, and ∇ $,& ( $,& ! ) is the gradient of loss function for the !" client belonging to the !" ONU at the !" round. After completing the local training, if selected the model updates $,& !'( from the client_i,j are sent to the associated ONU by accessing the BS and further forwarded to the OLT/CPS. As shown in Fig.1 (a), in the classical case each ONU just forwards the received local model parameters to the OLT/CPS. After gathering updates from all clients, the CPS aggregates local models and updates the global model. Here we employ FedAvg [1]. samples for the client_i,j and is the total number of data samples for all the involved clients. The data volume of all clients' model parameters may be huge, causing a high load on the PON upstream, especially when a large number of clients are involved in the FL task. The proposed SFL introduces a middle-layer aggregation as shown in Fig. 1 ]. An application protocol interface (API) may be needed to implement this function for ONU management and control. Typically, medium access control (MAC) protocol reserves some bytes for quality of service in control overhead [5,6], which can be utilized to indicate the model update traffic in Layer 2 and facilitate AF at the ONUs for the SFL. Thanks to the two-step aggregation, the data that needs to be transmitted in the PON upstream can be reduced significantly. After gathering aggregated updates from the ONUs, the CPS updates the global model [2] is used to evaluate the performance of the proposed SFL over PONs. The dataset of FEMNIST [2] is used for model training, where a CNN model with two 5x5 convolution layers is employed and the FedAvg [1] is chosen for parameter aggregation at the CPS. The data volume for the CNN model update is 26.416 Mbits per client. Hyperparameters (e.g., learning rate, batch size) are set following [2]. Simulations are performed on a workstation with a 10-cores CPU, 64 GB of RAM, 2 TB of storage, and Ubuntu 18.04 LTS. A single FL task is implemented over a time-division multiplexing PON with 16 ONUs, where each ONU/BS can connect up to 20 clients. The distance between the OLT and ONUs is set to 20 km. In each round, the CPS randomly selects clients to be involved in the training. Performance evaluation A home-made simulator that is developed based on LEAF and Tensorflow One which is set to 2 seconds. $,& depends on the data size, computation resource of clients, and hyperparameters of the at the ONU $ also needs to be considered in addition to $,& / and $,& 0 . And, the clients that belong to the same ONU have the same one-round synchronization time. To save training time, the threshold of the one-round synchronization time is set to 25 seconds, and the client_i,j with $,& > 25 seconds (i.e., straggler ) is not able to join the global model updating. Figure 2(a) shows the upstream bandwidth for one FL task per round in the classical FL (i.e., benchmark) and the proposed SFL. It can be seen that the communication cost saving of the SFL compared to the benchmark is linearly increased as a function of the number of the selected clients (N) for training. When N=48, such a saving is already 66.7% while it continues to increase to 87.5% when N increases to 128. It indicates that with the number of clients growing, the communication delay in PON of the benchmark increases whereas the proposed FL remains the same. As a result, for the benchmark the number of selected clients that can join the global model updating (i.e., involved clients) may drop due to the required synchronization time threshold. However, it does not happen in the proposed FL. This is shown in Fig. 2(b), where the number of the involved clients for the benchmark is much smaller than the SFL and fluctuates between 1 and 20 for both N = 48 and 128. In contrast, the upstream traffic in PON of the proposed SFL is constant and almost all the clients satisfy the synchronization time requirements and can be involved for global model updates, as shown in Fig. 2(b). Since the learning accuracy often depends on the number of involved clients, i.e., the more clients contribute to the training the better learning accuracy can be achieved, it can be expected that the SFL outperforms the benchmark in terms of learning accuracy. The results in Fig. 2(c) clearly reveal that, when N = 128, the learning accuracy for the benchmark is about 0.77, while that for SFL is up to 0.85, leading to an increase of 10%. Conclusion This paper proposes a scalable federated learning (SFL) approach over PONs, which introduces two-step aggregation, namely local model parameters are first aggregated at ONUs and then further aggregated at the OLT by the CPS, to keep the amount of the upstream traffic constant regardless of the number of the involved FL clients. Its advantages on the PON upstream bandwidth saving compared to the classical FL become more obvious when the number of the involved clients increases, leading to higher learning accuracy. Besides, the proposed SFL also potentially improve privacy as the aggregated local models transmitted in PON make the individual local model for each client difficult to interpret.	2020-10-30T01:01:09.748Z	2020-10-29T00:00:00.000	{ "year": 2020, "sha1": "ca5a609bc22a1a687e0405e480d9cf473ad6ff00", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "ca5a609bc22a1a687e0405e480d9cf473ad6ff00", "s2fieldsofstudy": [ "Engineering", "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
21726031	pes2o/s2orc	v3-fos-license	Hepatotoxicity Implies chemical-driven liver damage induced by certain medicinal and other chemical agents There are increasing evidences that free radicals and reactive oxygen species play a crucial role in the various steps that initiate and regulate the progression of liver diseases. Oxidative stress in hepatotoxicity resulting from increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant defence mechanism are responsible for nephrotoxicity. So, pharmacological studies in this work were done to evaluate: presence of protective effects of an antioxidant Hesperedine on carbon tetrachlorideinduced hepatic toxicity and nephro-toxicity, to evaluate its effects on oxidants and antioxidants parameters and to evaluate its effect on kidney and liver functions and histo-pathological changes. Liver enzymes level AST and ALT: was increased significantly in rats treated with CCl4 but decreased significantly in rats treated with antioxidant HDN (100 mg/ kg/ day) and in rats treated with antioxidant HDN (200 mg/ kg/ day). In comparison between antioxidant treated rats groups liver enzymes level was decreased significantly in rats treated with antioxidant HDN (200 mg/ kg/ day) than in rats treated with antioxidant HDN (100 mg/ kg/ day). Serum creatinine level: was increased insignificantly in rats treated with CCl4 but decreased insignificantly in rats treated with antioxidant HDN (100 mg/ kg/ day) and in rats treated with antioxidant HDN (200 mg/ kg/ day). In comparison between antioxidant treated rats groups liver enzymes level was decreased insignificantly in rats treated with antioxidant HDN (200 mg/ kg/ day) than in rats treated with antioxidant HDN (100 mg/ kg/ day). So, we recommend uses of antioxidant Hesperedine as it has a valuable role in improvement of liver functions and as a prophylactic of hepatic and renal tissues against toxicity achieved by free radicals. INTRODUCTION Hepato-toxicity Implies chemical-driven liver damage. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ. Other chemical agents, such as those used in laboratories and industries, *Corresponding Author Email: sayyed_2006@yahoo.com natural chemicals (e.g., microcystins) and herbal remedies can also induce hepatotoxicity. Chemicals that cause liver injury are called hepatotoxins. Chemicals often cause subclinical injury to liver which manifests only as abnormal liver enzyme tests. Drug-induced liver injury is responsible for 5% of all hospital admissions and 50% of all acute liver failures. More than 75 % of cases of idiosyncratic drug reactions result in liver transplantation or death (Ostapowicz et al., 2002;McNally and Peter, 2006). Mitochondria are prominent targets for the hepatotoxicity of many drugs. Dysfunction of these vital cell organelles results in impairment of energy metabolism and an intracellular oxidant stress with excessive formation of reactive oxygen species and peroxy-nitrite. Induction of cytochrome P450 isoenzymes such as CYP2E1 also promotes oxidant stress and cell injury, once hepatocellular function is impaired, accumulation of bile acids causes additional stress and cytotoxicity. Cell injury, gut-derived endotoxin or a combination of both also activate Kupffer cells and recruit neutrophils into the liver. Although responsible for removal of cell debris and part of the host-defense system, under certain circumstances these inflammatory cells initiate additional liver injury (Jaeschke et al., 2002). Drug-induced liver diseases mimic all forms of acute and chronic hepatobiliary diseases. However, the predominant clinical presentation resembles acute icteric hepatitis or cholestatic liver disease. The former is the more serious and often has a 10% mortality rate, regardless of the causative drug, . Acute icteric hepatitis is accompanied by markedly elevated serum transaminase levels and a minimal increase in the level of alkaline phosphatase. Coagulopathy and encephalopathy are present in more severe cases. Cholestatic disease (which is also referred to as cholestatic hepatitis) is not usually life threatening; it presents with jaundice, pruritus, and marked increases in alkaline phosphatase levels, as well as mild increases in alanine aminotransferase (ALT) levels. Mixed injury patterns with intermediate to marked increases in ALT and alkaline phosphatase levels can resemble atypical hepatitis or granulomatus hepatitis, . Biochemical markers (e.g. alanine transferase, alkaline phosphatase and bilirubin) are often used to indicate liver damage. Liver injury is defined as a rise in either (a) ALT level more than three times of upper limit of normal (ULN), (b) ALP level more than twice ULN, or (c) total bilirubin level more than twice ULN when associated with increased ALT or ALP, (Bénichou, 1990 andMumoli et al., 2006). Oxidative stress in hepatotoxicity, resulting from increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses, actively contributes to excessive tissue remodeling, (Ismail and Pinzani, 2009). Indeed, oxidative stress, presumably by favoring mitochondrial permeability transition, is able to promote hepatocyte death (necrotic and/or apoptotic). In some of clinically relevant conditions, generation of ROS within hepatocytes may represent a consequence of an altered metabolic state (like in NAFLD and NASH) or of ethanol metabolism (as in ASH), with ROS being generated mainly by mitochondrial electron transport chain or through the involvement of selected cytochrome P450 Bakheet et al. 145 isoforms like cytochrome P2E1 (CYP2E1), (Tilg and Hotamisligil, 2006). Glutathione (GSH) is a critical cellular antioxidant. After GSH depletion with buthionine sulfoximine (BSO), the toxicity of ethanol, iron, arachidonic acid and acetaminophen was strikingly enhanced, (Chen et al., 1997;Chen and Cederbaum, 1998;Sakurai and Cederbaum, 1998;Wu and Cederbaum, 1999). Cytochrome P4502E1 (CYP2E1), the ethanolinducible form, metabolizes and activates many toxicologically important substrates, including ethanol, carbon tetrachloride, acetaminophen, and Nnitrosodimethylamine, to more toxic products, (Guengerich et al., 1990;Koop, 1992). CYP2E1dependent ethanol metabolism produces oxidative stress through generation of reactive oxygen species (ROS), a possible mechanism by which ethanol is hepatotoxic, (Dianzani, 1985 andBondy, 1992). Induction of cytochrome P4502E1 by ethanol is a central pathway by which ethanol generates oxidative stress, and in the intragastric model of ethanol feeding a prominent induction of CYP2E1 occurs along with significant alcohol liver injury, (Morimoto et al., 1994;Nanji et al., 1994). Immunochemical studies indicate that the cellular site of covalent binding correlates with the toxicity, (Roberts et al., 1991 andHart et al., 1995). Recent work shows that nitrated tyrosine occurs in hepatic centrilobular cells. These adducts colocalize in cells containing the acetaminophen-protein adducts, (Hinson et al., 2000). Peroxynitrite, a highly reactive nitrating and oxidizing species formed by the rapid reaction of nitric oxide (NO) and superoxide, produces nitrated tyrosine, (Pryor and Squadrito, 1995;Beckman, 1996). Carbon tetrachloride is a colourless liquid, non flammable, and is heavier than air, (Etim et al., 2008). Consequently, it has been widely used as a fire extinguisher being useful for fighting fires near electrical equipment because it does not conduct electricity, (The World Book Encyclopedia, 1992). Carbon tetrachloride is very toxic and because of this, most of its uses in households and industries have been suspended, (Etim et al., 2008). Consequently, little is known about the early effects of this organic solvent in vivo, particularly on mitochondrial function. It has been shown recently in a murine model of liver fibrosis that chronic administration of CCl4 for 6 weeks led to mitochondrial DNA (mtDNA) alterations, reduced glutathione (GSH) depletion and decreased aconitase activity , overexpression of Bcl-2 reduced liver fibrosis for the first 3 weeks of treatment by protecting hepatocytes against mitochondrial damage, but subsequently failed to prevent fibrosis with the persistence of the aggression. CCl4 is activated by cytochrome P450 (CYP) 2E1, and very marginally by other CYPs (CYP2B and CYP3A), to form the trichloromethyl (CCl3 . ) free radical, which can react with oxygen to produce the trichloromethyl peroxy radical (CCl3OO . ). Both radicals are highly reactive species that may covalently bind to macromolecules to form nucleic acid, protein and lipid adducts. However, the evidence for such interactions with liver DNA in vivo is limited, (Recknagel et al., 1989 andWeber et al., 2003). In this study, we used an in vivo model to explore the very early toxic events, particularly regarding mitochondria, occurring after CCl4 administration. Inhibition of CCl4 activation by the CYP2E1 inhibitor diethyldithiocarbamate (DDTC) and impairment of CCl4induced lipid peroxidation by antioxidants allowed us to establish a direct link between lipid peroxidation and mitochondrial alterations. Antibiotics, commonly used aminoglycosides, are nephrotoxic agents. Their nephrotoxicity is mainly attributed to induction of OS and depletion of antioxidat enzyme activities in kidney. Inducible nitric oxide synthase, nuclear factor kappa-B, nitogen-activated protein kinase (iNOS/NFκB/p38MAPK respectively) pathway, OS taking place in this axis, is involved in gentamicin-induced nephrotoxicity, (Tugcu et al., 2006 andOzbek et al., 2009). The protective effect of anti oxidants and reactive oxygen scavenger agents against gentamicin-induced nephrotoxicity. Antineoplastic agents are commonly used for the treatment of metastatic cancers. Some of these are nephrotoxic, (Ozbek et al., 2010 andManiu et al., 2011). Excess ROS production and depressed antioxidant defence mechanism are responsible for nephrotoxicity. Cisplatin is the well-known and commonly used antineoplastic and nephrotoxic agent. Other nephrotoxic anticancer agents are carboplatin, methotrexate, doxorubicin, cyclosporine, and adriamycin. Immunosuppressant such as sirolimus and cyclosporine leads to nephrotoxicity via OS, (Giustarini et al., 2009). In this era, analgesics, especially paracetamol and acetaminophen (APAP), and nonsteroidal antiinflammatory drugs (NSAIDs) are widely used throught the world. Paracetamol and APAP are nephrotoxic drugs. Several in vitro and in vivo studies showed that analgesics nephrotoxicity is caused by increased ROS in kidney, (Zhao et al, 2011) showed the increased ROS, nitric oxide, and MDA levels, together with depleted glutathione (GSH) concentration in the kidney of rats. However, rhein, Chinese herb, can attenuate APAPinduced nephrotoxicity in a dose-dependent manner, (Zhao et al., 2011). Some studies showed a significant increase in MDA and decreases in GSHPx, CAT, and SOD activities in APAP treated rat kidneys. These findings support the induction of OS in rat kidney by APAP. Significant beneficial changes were noted in serum and tissue OS indicators in rats treated with strong antioxidant pineal hormone melatonin and curcumin, , reported increased OS and TNF-alpha production in rat tissues, (Ghosh et al., 2010), reported that diclofenac (NSAID) leads to nephrotoxicity by increasing intrarenal ROS in rat kidney, and antioxidant, Nacetylcysteine, prevents kidney damage, (Efrati et al., 2007). GSH is able to regenerate the most important antioxidants, Vitamins C and E, back to their active forms; it can reduce tocopherol radical of Vitamin E directly, or indirectly, via reduction of semidehydroascorbate to ascorbate. The capacity of glutathione to regenerate the most important antioxidants is linked with the redox state of the glutathione disulphide-glutathione couple (GSSG/2GSH), (Pastore et al., 2003). Hesperidin is a flavanone glycoside named after the term 'Hesperidium', referring to citrus fruits which are the main source of hesperidin. Hesperidin and its aglycone are common dietary flavonoids due to being large compounds of citrus fruits (alongside naringenin) and especially the peels and pericarp, (Kanes et al., 1993). There are inhibitory effects of hesperitin on two intestinal transporters, the OATP2B1 (Organic Acid Transporting Polypeptide 2B1) transporter and MRP2 (Multidrug Resistance Protein 2). OATP2B1 appears to be acutely inhibited with supplementation of hesperidin, whereas low doses of hesperidin over a few weeks appear to downregulate the MRP2 transporter .It is notable to know that the OATPs play a fundamental role in the transport of drugs across the cell membrane, particularly in the liver and kidney. In the liver, OATPs are expressed on the basolateral membrane of hepatocytes, transporting compounds into the hepatocyte for biotransformation (Price et al., 2006). A 0.079% hesperidin suspension given to rats for eight weeks is able to increase the overall exposure (147%) and peak concentration (138%) to the drug pravastatin, (Shirasaka et al., 2013) which is thought to be due to inhibition of the transport protein known as Multi-drug Resistance Protein 2 (MRP2) that mediate pravastatin efflux into the intestines after absorption, (Tamai, 2012) . There appear to be antioxidant effects in the brain where hesperidin reduces the increase in lipid peroxidation during cognitive damange, but this appears to be indirect through nitric oxide signalling (inhibition) rather than a direct antioxidant effect, (Olivenza et al., 2000;McEwen, 2001;Alexaki et al., 2004 andTakeda et al., 2008). Damage of DNA is reduced by hesperidin (Sahu et al. ,2013).Hesperidin intake in diabetic rats appears to significantly but not fully reduce levels of the (vascular endothelial growth factors )VEGF and PKCβ (Protein kinase cβ), and it is thought that the reduction in signalling (from VEGF towards PKCβ) causes a protective effect on the retinal membrane and reduces the progression of diabetic retinopathy, (Donnelly et al., 2004;Liu et al., 2008;Wang et al., 2010 andKumar et al., 2012). MATERIALS AND METHODS This study was conducted on Thirty two male albino rats. Animals were obtained from the animal house of faculty of medicine, Al-Azhar University. Their weight ranged between 160-200 grams each at the beginning of the experiment. Rats were housed in four groups with 8 rats each in clean capacious macrolane cages under standard laboratory conditions, including good aerated room with suitable temperature (25±5°C), maintained at good light, standard rodent food and water were available. CCL 4 : El-Naser Pharmaceuticals chemical company, Egypt Hesperidine (HDN): Sigma, Aldrich. -Serum Creatinine determination kits: Diamond., USA. In the present study, the animals were divided into the following groups. Each group consisted of 8 rats: Group 1: These animals received a vehicle for HDN (i.e. CarboxyMethylCellulose) by oral route for eight days and on 8 th day, they were administered the subcutaneous injection of olive oil . ( Tirkey , 2005 ) Group II: These animals received vehicle for 10 days and were challenged with CCl4 2 ml/kg/s.c. (40% v/v in olive oil) on 8th day (Mandal and Sinha, 2002) Group III: These rats received only HDN 100 mg/kg/p.o. daily for 10 days CCl4+ HDN (100): Rats received HDN continuously for 8 days. On eight day just after HDN treatment they received CCl4 2ml/kg/s.c in olive oil. HDN was further continued for 2 more days. (Tirkey, 2005) Group IV: These rats received only HDN 200 mg/kg/p.o. daily for 10 days CCl4+ HDN (200): Rats received HDN continuously for 8 days. On eight day just after HDN treatment they received CCl4 2ml/kg/s.c in olive oil. HDN was further continued for 2 more days. (Tirkey, 2005) Forty-eight hours after the last CCl4 injection, rats were sacrificed and blood samples were collected, centrifuged and the serum from each animal was kept in epindorff tubes in the deep freezer at (-20°C) until analyzed for liver functions. RESULTS AND DISCUSSION In the present study, induction of acute hepatic toxicity and nephrotoxicity in Wistar male albino rats was done by s.c injection of CCl4 2 ml/kg/s.c. (40% v/v in olive oil)for a single dose of which is a well characterized model for acute hepatic toxicity and nephrotoxicity has been extensively performed and revealed microscopically in the liver as extensive damage ,very severe vaculation, inflammatory cells infilteration, Irregular architecture (damaged sinusoids, rows and disintegrated central vein) and degenerated nuclei and in the kidney as vaculation, degenerated nuclei, obliteration of the tubules ,inflammatory cell infilteration and disruption of the lattice nature of the cells and damaged cell membranes. (Table 1 and figure 1 to 8) These results are in agreement with the results obtained by Al-Qarawi et al, 2004 who reported the hitopathological changes in acute hepatic toxicity, Montilla et al. 1990 who proved CCl4 hepatotoxicity by LD50 of CCl4, the modification of Nembutal-induced sleep, the action on bile flow, serum transaminase and hepatic fatty acids levels and a histopathological study of liver tissue. Kodama andOguchi, 1990 andPrakash et al. 2008, have also obtained similar results to our study on the effect of CCl4 on hepatic and kidney architecture. Abdel Moneim and Mahmoud, 2013, who reported that CCl4 induces nephrotoxicity which can be detected by estimation of oxidation and antioxidation components plus histopathological changes. Khan et al. 2009, noticed glomerular degeneration, tubular brush border loss, tubular dilatation, necrosis of epithelium and interstitial oedema in CCl4 treated rats. The results of the present study are in disagreement with the results obtained by Zimmerman et al. 1983 as they found an increased frequency of glomerulosclerosis, tubulointerstitial alterations and reduced renal mass only on long-term CCl4 administration in rats. CCl4 not only initiates lipid peroxidation but also reduces tissue GSH and SOD activities, and this depletion may result from -less vaculation in the tubules than in group III (HDN100) -More viability of the cells than in group III (HDN100). -More regular tubules than in group III (HDN100). -Intact Bowman capsule -It is so close to normal group oxidative modification of these proteins, (Augustyniak et al., 2005). CCl4 intoxication can lead to alteration in gene expression and depletion of SOD and catalase levels in kidney and heart. Oxidative stress causes depletion of intracellular GSH, leading to serious consequences. CCl4-induced early signs of hepatotoxicity could be mediated through two different mechanisms involving or not lipid peroxidation. Lipid peroxidation triggered mtDNA degradation and mitochondrial dysfunction but not other CCl4-induced deleterious events such as hepatocyte swelling, abnormal expression of heme oxygenase 1(HO-1) and heat shock protein (Hsp70), and reduction of CYP2E1 mRNA levels, (Szymonik-Lesiuk et al., 2003). Kidney tissue has great affinity for CCl4 because of the predominant presence of the cytochrome p450 in the cortex. Previous reports suggest that CCl4 generates free radicals with the implication of pathological environment by damaging the integrity of cell membranes, elevating thiobarbituric acid reactive substances (TBARS) level with subsequent necrosis and affecting physical parameters of kidney such as urinary and serum profile (Sahreen et al., 2011). In the present study, CCl4 induces a severe hepatic damage as represented by markedly elevated levels of ALT and AST. These results are in agreement with the studies Alam et al. 2000; Mousa et al. 2004 andPrakash et al. 2008 who proved that administration of CCl4 causes hepatotoxicity detected by increased levels of ALT and AST. Usually, the extent of hepatic damage is assessed by the increased level of cytoplasmic enzymes (ALT and AST), thus leads to leakage of large quantities of enzymes into the blood circulation. This was associated by massive centrilobular necrosis, ballooning degeneration and cellular infiltration of the liver, (Shankar et al., 2008).In response to hepatocellular injury initiated by the biotransformation of CCl4 to reactive radicals, "activated" Kupffer cells in liver respond by releasing increased amounts of active oxygen species and other bioactive agents,n ( El-Sisi et al., 1993) these products include conjugated dienes, lipid hydroperoxides, malonaldehyde-like substances, and other short-chain hydrocarbons, (Tom et al.,1984). Reduced glutathione (GSH) is a major endogenous antioxidant which counterbalances free radical mediated damage. It is well known that GSH is involved in the protection of normal cell structure and function by maintaining the redox homeostasis, quenching of free radicals and by participating in detoxification reactions, (Pushpakiran et al., 2004). Superoxide dismutase (SOD) an enzyme that catalyzes the dismutation of superoxide (O2−) into oxygen and hydrogen peroxide. Thus, it is an important antioxidant defense in nearly all cells exposed to oxygen, (Shahid et al., 2012). The results of the present study showed that, subcutaneous injection of CCl4 lead to decreased hepatic reduced glutathione (GSH) level, superoxide dismutase (SOD) level and increased Malondialdehyde (MDA) level. These results are in agreement with the studies of Kang et al. 2001 who noticed that CCl4 causes decreased hepatic reduced glutathione (GSH) level, superoxide dismutase (SOD) level. Manjrekar et al. 2008 who noticed that CCl4 causes decreased hepatic reduced glutathione (GSH) level and increased Malondialdehyde (MDA) level. Siu-Po and Kam-Ming, 1996 who noticed that CCl4 causes decreased hepatic reduced glutathione (GSH) level. Pereira -Filho et al. 2008 claimed that hepatic malondialdehyde (MDA) levels were also highly significantly increased in CCl4 treated group, showing an increased oxidative stress compared to control group. The increased MDA level suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals as described above and confirmed by (George et al, 2002;Loki and Rajamohan, 2003and Rajesh and Latha, 2004. The results of this present study are in disagreement with Stryjecka-Zimmer. et al , 2003 who claimed that change in antioxidant enzyme activities may be relevant to the ability of the liver and other investigated organs to cope with oxidative stress during CCl4 poisoning No statistically significant changes in SOD and glutathione peroxidase (GPX) activities were observed in the liver after CCl4 administration. Oxidative stress in hepatotoxicity, resulting from increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses, actively contributes to excessive tissue remodeling (Ismail and Pinzani, 2009).ROS and other reactive mediators such as 4-hydroxynonenal (HNE) can be generated outside PMNLs, being released either by activated inflammatory cells or deriving from hepatocytes damaged by the specific etiological agent or conditions (Duffield et al., 2005). Indeed, oxidative stress, presumably by favoring mitochondrial permeability transition, is able to promote hepatocyte death (necrotic and/or apoptotic). In some of clinically relevant conditions, generation of ROS within hepatocytes may represent a consequence of an altered metabolic state (like in NAFLD and NASH), with ROS being generated mainly by mitochondrial electron transport chain or through the involvement of selected cytochrome P450 isoforms like cytochrome P2E1 (CYP2E1), (Tilg and Hotamisligil, 2006). Case control studies and various documented case reports increasingly establish that hydrocarbon solvents produce renal diseases in humans, (Ruprah et al, 1985). Bakheet et al. 153 To assess renal affection by detection of renal functions: Serum samples were assayed for serum creatinine, (Bhattacharya et al., 2005). The results of the present study showed that insignificant increase of Serum Creatinine in CCl4 intoxicated group.The results of the present study are in agreement with the studies of Zimmerman et al , 1983 who did not report any rise in kidney functions levels even after chronic treatment of CCl4 in nephrectomized rats, Ogawa et al. 1992 found an increased frequency of glomerulosclerosis and tubulointerstitial alterations in rats with reduced renal mass on CCl4 administration thereby indicating nephrotoxicity only on long-term CCl4 administration in rats. The results of the present study are in disagreement with Olagunjua et al. 2009 who noticed increase in kidney function in CCl4-induced nephrotoxicity and Stephen et al. 2007 who reported that the nephrotoxicity can be detected by kidney functions tests. Renal sources for ROS are activated macrophages, vascular cells, and various glomerular cells. ROS may affect cells of the host organism, especially at sites of inflammation in addition to playing a role in the defense system against other agents. This effect plays a role in a variety of renal diseases such as glomerulonephritis and tubulointerstitial nephritis which can contribute to proteinuria and other conditions (Ichikawa et al., 1994). The presence of inflammation is well documented factor influencing the development of oxidative stress in dialysis patients (Samouilidou and Grapsa, 2003). However the pathology related with renal function failure that is stimulated by CCl4 remains controversial. As kidneys have an affinity against CCl4, and as they contain predominantly, cytochrome p450 in the cortex, it is very possible that CCl4 contributes a lot to nephrotoxicity, (Ogeturk et al., 2005). The results of the present study showed that oral administration of hesperedine (100mg/kg) and (200mg/kg) significantly decrease the ALT and AST in CCl4-treated rat and in the group of the dose 200mg/kg produces more decrease in ALT and AST. The results of the present study are in agreement with the study done by Ahmad et al. 2012 who proved that hesperedine ameliorates the hepatotoxicity-induced by acetaminophen, and this was detected by decrease in ALT and AST not only that but also he noticed that the acuity of toxicity is decreased gradually by increasing the dose of hesperedine similar to our results. Balakrishan and Menon, 2007 reported that administration of hesperedine to nicotine treated rats at different doses decreases these enzymes significantly but in dose-dependent manner. Anandan and Ramaswamy, 2012 reported protective effects of hesperidin (HDN 100 mg/kg) for 14 days against gentamicin (GEN 100 mg/kg) induced hepatoxicity for 8 days detected by decrease in ALT and AST. Park et al. 2012 reported that protective effects of hesperidin+ Curdlan (HDN + CDN 100 mg/kg) for 7 days against γ-radiation induced hepatoxicity. AST and ALT are the aminotransferase in liver cells. They are cytoplasmic in nature, but upon liver injury large quantities of these enzymes enter into the circulatory system due to altered permeability of membrane, centrilobular necrosis, degeneration, and reduced performance status of the liver. So the elevated serum ALT and AST are the most sensitive biomarkers used in the diagnosis of liver diseases (Pari andKumar, 2002 andGao et al., 2012). CCl4 induced a severe hepatic damage as represented by markedly elevated levels of ALT and AST coupled with a marked hepatic oxidative stress (Tirkey et al., 2005). Oxidative stress in hepatotoxicity, resulting from increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses, actively contributes to excessive tissue remodelling (Ismail and Pinzani, 2009). Hesperidin in combination with diosmin, shows a marked protective effect against inflammatory disorders, both in vivo and in vitro, possibly through a mechanism involving an inhibition of eicosanoid synthesis and/or antioxidant free radical scavenger activity (Jean and Bodinier, 1994). The results of the present study showed that oral administration of Hesperedine (100mg/kg) causes insignificant decrease in Malondialdehyde (MDA) and insignificant increased hepatic reduced glutathione (GSH) and superoxide dismutase (SOD) levels. These results of the present study are in agreement with the study done by Tirkey et al. 2005 who proved that oral administration of Hesperedine (100mg/kg) causes insignificant decrease in Malondialdehyde (MDA) and insignificant increased hepatic reduced glutathione (GSH) level and superoxide dismutase (SOD) level. The results of the present study are in disagreement with the study done by Park et al. 2010 who observed protective effects of hesperidin+ Curdlan (HDN+CDN 100 mg/kg) for 7 days against γ-radiation induced hepatoxicity, through significant decrease in Malondialdehyde (MDA) and significant increased hepatic reduced glutathione (GSH) level and superoxide dismutase (SOD) level. Anandan and Ramaswamy, 2012 observed protective effects of hesperidin but in gentamycine-induced hepatoxicity, this was detected by significant decrease in Malondialdehyde (MDA) and significant increased hepatic reduced glutathione (GSH) level and superoxide dismutase (SOD) level. The present study showed that oral administration of Hesperedine (200mg/kg) causes significant decrease in Malondialdehyde (MDA) and significant increase hepatic reduced glutathione (GSH) level and superoxide dismutase (SOD) level. These results are in agreement with the study done by (Xiao-min et al. 2011) who reported significant decrease in Malondialdehyde (MDA) and significant increased hepatic reduced glutathione (GSH) level and superoxide dismutase (SOD) level by studying the protective effect of Hesperidin on hepatotoxicity induced by cisplatin. Wei and Jun, 2010 posted that HDN had protective effects on CCl4-induced chemical liver injury. It was possibly related to removal of free radicals and inhibition of lipid peroxidation. HDN(250 and 500 mg/kg) could reduce the levels of MDA and significant increased hepatic superoxide dismutase (SOD) level. Wei and Jun, 2010 also observed certain cytokines as IL-1 and TNF are inhibited by HDN (250 and 500 mg/kg) through decreasing mRNA expression. Xiao-min et al, 2011 reported that administration of hesperidin (300mg/kg p.o.) for 7 consecutive days had a remarkable protective effect on hepatotoxicity induced by cisplatin (5mg/kg, intraperitoneally for 5 consecutive days from the third day of hesperedine administration). The protective effect of hesperidin was possibly related to removal of free radicals and inhibition of lipid peroxidation produced by cisplatin intoxication. HDN (300mg/kg) could reduce the levels of MDA, significant increased hepatic superoxide dismutase (SOD) level and significant increased GSH. Shrivastava, 2011 noticed that administration of hesperidin (HDN) (100mg/kg p.o.), for 7 days had a remarkable protective effect on Cardiotoxicity induced by single intraperitoneal injection of cyclophosphamide CP (200mg/kg body weight). The protective effect of hesperidin was possibly related to removal of free radicals and inhibition of lipid peroxidation produced by cyclophosphamide intoxication .HDN (100mg/kg) could reduce the levels of MDA, significant decreased LDH, CPK, ALT and AST. Also (Tirkey et al., 2005 andPradeep et al., 2008) obtained similar results to our study on the effect of hesperedine on oxidants and antioxidants parameters. Ko et al. 1995 reported that certain natural extracts containing antioxidants protect against the CCl4-induced increased lipid peroxide levels and impairment in hepatic GSH status.Hepatic malondialdehyde (MDA) levels were also highly significantly increased in CCl4 treated group, showing an increased oxidative stress compared to control group. The increased MDA level suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals as described by (Pereira-Filho et al., 2008) and confirmed by . Glutathione is an important intracellular antioxidant that also plays a role in the detoxification and elimination of potential carcinogens and toxins. Studies in animals have found that glutathione synthesis and tissue glutathione levels are significantly lower in aged animals than in younger animals, leading to decreased ability of aged animals to respond to oxidative stress or toxin exposure (Hagen et al., 2000). Superoxide dismutase (SOD) catalyzes the destruction of the O 2free radical. CCl4 challenge significantly decreased the levels of SOD and catalase in liver, by alteration in gene expression and depletion of SOD and catalase levels (Stryjecka-Zimmer et al, 2003). Antioxidants are agents that inhibit or neutralize potentially harmful elements known as free radicals (Zielinska el al., 2001;Galati and O'brien, 2004). Flavonoids are naturally occurring polyphenolic compounds in plants that are thought to have positive effects on human health (Wahsha and Al-Jassabi, 2009). HDN administration ameliorates the increased level of lipid peroxidation after CCl4 treatment, able to show improvement in the levels of endogenous antioxidant enzymes SOD and Improvement of hepatic GSH levels in HDN-treated rats in comparison to CCl4 intoxicated rats, thereby this demonstrates the antioxidant effect of HDN (Tirkey et al, 2005). Flavonoids are known to operate via direct scavenging of Reactive Oxygen Species (ROS), chelation of redox active transition metal ions, inhibition of enzymes involved in ROS production, regeneration of endogenous antioxidants (Fitzgeorge et al., 1994;Zielinska et al., 2001). It was found that Hesperidin has an important antioxidant activity in humans, it enhances the integrity of the blood vessels and it is found in great quantity in citrus fruits (lemons and oranges) (Tripoli et al., 2007). Hesperidin and Silymarin are polyphenolic compounds which play an important role as antioxidants; they can directly quench free radicals, inhibit the enzymes of oxygen reduction pathways and also prevent the sequestration of transient metal actions (Chatterjee et al., 1999;Berker et al., 2007). The radical scavenging power of flavonoids is thought to be related to their structure. Flavonoids in general, scavenge oxidizing radicals preferentially via their B-ring catechol; in particular the ortho-dihydroxy structure in the B ring gives a higher stability during the formation of aroxyl radicals and participation in electron dislocation. The presence of the 3' and 5' OH functions together give a maximum radical scavenging potential: this property is found in both Silymarin and Hesperidin, (Markham, 1982;Joshi et al., 2005;Andersen and Markham, 2006). The results of the present study showed that oral administration of hesperedine (100mg/kg) and (200mg/kg) significantly improves hepatic architecture microscopically in dose-dependent manner as the group of hesperedine administration (100mg/kg) shows slight improvement while the group of hesperedine administration (200mg/kg) shows no difference with control normal group. This result is in agreement with the study done by (Balakrishan et al, 2007) who observed that administration of hesperedine to nicotine treated rats at different doses improves hepatic architecture significantly in dose-dependent manner even in high doses ,he doesnot observe any morphological changes compared to normal. Ahmad et al, 2012 observed that Hesperidin alleviates acetaminophen induced toxicity in Bakheet et al. 155 dose-dependent manner and in high doses he doesnot observe any morphological changes compared to normal.Also Bentli et al. 2013 obtained similar results to our study on the effect of hesperedine on hepatic architecture. Our data showed that oral administration of hesperedine (100mg/kg) and (200mg/kg) significantly improves renal architecture microscopically in dosedependent manner as the group of hesperedine administration (100mg/kg) showed slight improvement while the group of hesperedine administration (200mg/kg) showed no difference with control normal group. This result is in agreement with the histopathological study done by Anandan and Subramanian, 2012, who proved renal protective effect of hesperidin on gentamicin-induced acute nephrotoxicity. Balakrishan et al, 2006 administration of hesperedine to nicotine treated rats at different doses improves renal architecture significantly but in dosedependent manner, and in high doses he doesnot observe any morphological changes compared to normal. Ahmad et al. 2012 observed that Hesperidin alleviates acetaminophen induced toxicity in dosedependent manner and in high doses he doesnot observe any morphological changes compared to normal. Sahu et al. 2013 who reported that Hesperidin attenuates cisplatin-induced acute renal injury by decreasing oxidative stress, inflammation and DNA damage.	2017-11-02T01:37:00.665Z	2015-01-01T00:00:00.000	{ "year": 2015, "sha1": "0556c58eefb0c5efa8c4f118c17ace2149321ac3", "oa_license": null, "oa_url": "https://doi.org/10.21694/2380-5706.15001", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "0556c58eefb0c5efa8c4f118c17ace2149321ac3", "s2fieldsofstudy": [ "Biology", "Medicine", "Chemistry" ], "extfieldsofstudy": [] }
4475396	pes2o/s2orc	v3-fos-license	Biphasic voltage‐dependent inactivation of human NaV1.3, 1.6 and 1.7 Na+ channels expressed in rodent insulin‐secreting cells Key points Na+ current inactivation is biphasic in insulin‐secreting cells, proceeding with two voltage dependences that are half‐maximal at ∼−100 mV and −60 mV. Inactivation of voltage‐gated Na+ (NaV) channels occurs at ∼30 mV more negative voltages in insulin‐secreting Ins1 and primary β‐cells than in HEK, CHO or glucagon‐secreting αTC1‐6 cells. The difference in inactivation between Ins1 and non‐β‐cells persists in the inside‐out patch configuration, discounting an involvement of a diffusible factor. In Ins1 cells and primary β‐cells, but not in HEK cells, inactivation of a single NaV subtype is biphasic and follows two voltage dependences separated by 30–40 mV. We propose that NaV channels adopt different inactivation behaviours depending on the local membrane environment. Abstract Pancreatic β‐cells are equipped with voltage‐gated Na+ channels that undergo biphasic voltage‐dependent steady‐state inactivation. A small Na+ current component (10–15%) inactivates over physiological membrane potentials and contributes to action potential firing. However, the major Na+ channel component is completely inactivated at −90 to −80 mV and is therefore inactive in the β‐cell. It has been proposed that the biphasic inactivation reflects the contribution of different NaV α‐subunits. We tested this possibility by expression of TTX‐resistant variants of the NaV subunits found in β‐cells (NaV1.3, NaV1.6 and NaV1.7) in insulin‐secreting Ins1 cells and in non‐β‐cells (including HEK and CHO cells). We found that all NaV subunits inactivated at 20–30 mV more negative membrane potentials in Ins1 cells than in HEK or CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. NaV1.7 inactivated at 15‐20 mV more negative membrane potentials than NaV1.3 and NaV1.6 in Ins1 cells but this small difference is insufficient to solely explain the biphasic inactivation in Ins1 cells. In Ins1 cells, but never in the other cell types, widely different components of NaV inactivation (separated by 30 mV) were also observed following expression of a single type of NaV α‐subunit. The more positive component exhibited a voltage dependence of inactivation similar to that found in HEK and CHO cells. We propose that biphasic NaV inactivation in insulin‐secreting cells reflects insertion of channels in membrane domains that differ with regard to lipid and/or membrane protein composition. Introduction Voltage-gated Na + (Na V ) channels are expressed in nearly all electrically excitable cells where they play a key role in action potential initiation and generation (Hille, 2001). Na V channels exhibit a dual dependence on voltage: depolarization results in both rapid activation and a slower time-dependent inactivation. During inactivation, the Na V channels enter a non-conducting state. Reversal of the 'inactivated' state requires hyperpolarization of the membrane, the extent of which can vary according to cell type and Na V subtype (Catterall et al. 2017). The balance between activation and inactivation results in a 'sodium window current' that determines cellular excitability. Na V channels consist of a pore-forming α-subunit, which can form heterodimers or heterotrimers with auxiliary β-subunits that modify their gating properties (Calhoun & Isom, 2014;Kruger & Isom, 2016). Insulin-secreting β-cells are equipped with Na V channels and express Na V 1.3, Na V 1.6 and Na V 1.7 α-subunits that are encoded by Scn3a, Scn8a and Scn9a genes, respectively. Moreover, they principally express Scn1b, which encodes the β 1 -subunit (Benner et al. 2014;Adriaenssens et al. 2016;DiGruccio et al. 2016). In mouse and rat (but not in human) β-cells, Na V channels exhibit an unusual voltage dependence of inactivation (Hiriart & Matteson, 1988;Lou et al. 2003;Braun et al. 2008;Zhang et al. 2014). In most mouse β-cells, inactivation proceeds at unphysiologically negative membrane potentials such that no Na + voltagegated currents remain activatable at membrane potentials above −70 mV. In β-cells, full Na V reactivation requires membrane potentials as negative as ß−120 mV. This is 40-50 mV more negative than the most repolarized membrane potential of the β-cell. As a result, most Na V channels are 'locked' in the non-conducting inactivated state (Plant, 1988;Gopel et al. 1999). However, more recently it was reported that inactivation in β-cells is biphasic and consists of an additional small Na + current component (10-15% of the total Na + current) that persists at physiologically relevant membrane potentials in one-third of the β-cells (Vignali et al. 2006;Zhang et al. 2014). It has been proposed that the two components of inactivation reflect different Na + channel subtypes (Vignali et al. 2006;Zhang et al. 2014). Indeed, we have shown that whereas Na V 1.7 gives rise to the component inactivating at hyperpolarized voltages, Na V 1.3 accounts for the component inactivating over more physiological membrane potentials (Zhang et al. 2014). This would suggest that Na V 1.7 channels in β-cells inactivate at 40 mV more negative membrane potentials than in other cells and that β-cells contain a factor modulating Na V 1.7 channels in a subtype-specific fashion. Here we have compared the inactivation properties of different Na V channel subtypes when expressed in insulin-secreting cells and in HEK, CHO and glucagonsecreting αTC1-6 cells. To isolate the expressed current, we generated a tetrodotoxin (TTX)-resistant form of the channels and blocked endogenous channels by inclusion of TTX in the bath medium. Our data confirm that Na V 1.7 currents do indeed inactivate at more hyperpolarized membrane potentials than Na V 1.3 and Na V 1.6 in Ins1 cells but that the difference is small and insufficient to explain the biphasic inactivation observed in pancreatic β-cells. Intriguingly, expression of a single Na V subtype gives rise to currents that undergo biphasic inactivation over distinct and widely separated membrane potential ranges in individual Ins1 cells but never in the other cell types. We propose a model that accounts for the α-subunit-independent biphasic inactivation of Na V channels in β-cells. The rat insulinoma cell line Ins1 832/13 (referred to as Ins1) was provided by J. Lang (Université de Bordeaux, France) and cultured in RPMI supplemented with 11 mM glucose (also 5 mM and 25 mM glucose in the indicated experiments), 10% fetal calf serum, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol and 100 U ml −1 penicillin and 100 μg ml −1 streptomycin. In specified experiments Ins1 cells were cultured for 48 h with 10 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St Louis, MO, USA) or 100 μM diazoxide (Sigma-Aldrich); or for 24 h with the insulin receptor antagonist S961 (Sigma-Aldrich). The human embryonic kidney cell line (AD-293; referred to as HEK cells from here on) was obtained from Agilent Technologies (Santa Clara, CA, USA), the mouse pancreatic α-cell line (αTC1-6) was obtained from ATCC (Manassas, VA, USA), the Chinese hamster ovary cell line (CHO) was obtained from European Collection of Authenticated Cell Cultures (Salisbury, UK). All cell lines were cultured according to the providers' protocols. Transfection of plasmids and small interfering RNA (siRNA) duplexes was performed using Lipofectamine R 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's guidelines. Cells plated on 35 mm dishes were co-transfected with 1.5 μg of DNA encoding Na V 1.3, Na V 1.6 and Na V 1.7 α-subunits and 50 ng of DNA encoding β-subunits (as specified) and assayed 24-48 h after transfection. GFP fluorescence was used to select for transfected cells, which subsequently were tested for channel expression by whole-cell patch-clamp recording techniques. siRNA-mediated knockdown experiments were performed in Ins1 cells. siRNA duplexes against rat Scn3b and scrambled negative control (OriGene Technologies, Inc.) were applied at a final concentration of 60 nM. For efficient knockdown, the cells were transfected on day 1 and day 3 and used for experiments on day 4. The efficiency of knockdown was assessed by qPCR. RNA isolation and quantitative RT-PCR RNA was isolated using a combination of TRI reagent and Ambion PureLink RNA Mini Kit (Thermo Fisher Scientific). On-column DNase treatment was performed to eliminate genomic DNA contamination. cDNA was synthesized using the High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). Real-time qPCR was performed using SYBR Green detection and gene specific QuantiTect Primer Assays (Qiagen, Hileden, Germany). Relative expression was calculated using the C t method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and peptidylprolyl isomerase A (PPIA) were used as reference genes. J Physiol 596.9 The effect of acute changes in the extracellular glucose concentration were measured in the perforated-patch configuration, as previously described (De Marinis et al. 2010). Perforation was achieved using amphotericin B (Sigma-Aldrich), present in the intracellular solution at 0.4 mg ml −1 final concentration. The intracellular pipette solution was composed of (mM): 76 Cs 2 SO 4 , 10 NaCL, 10 KCl, 1 MgCl 2 and 5 Hepes, adjusted to pH 7.15 with CsOH. The extracellular solution used in the whole-cell configuration was also used for perforated patch clamp measurements at a final glucose concentration of 1 or 20 mM. For cell-attached experiments, the pipette solution had an extracellular composition of (mM): 118 NaCl, 20 tetraethylammonium-Cl, 5.6 KCl, 1.2 MgCl 2 , 5 HEPES, 2 CoCl 2 and 5 D-glucose, adjusted to pH 7.4 with NaOH. During seal formation, the cells were immersed in standard extracellular medium containing (mM): 137 NaCl, 5.6 KCl, 10 Hepes (pH 7.4 using NaOH), 1.1 MgCl 2 and 2.6 CaCl 2 . Once the seal was formed, the high K + extracellular solution was perfused, consisting of (mM): 125 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 EGTA, 10 Hepes and 5 D-glucose, adjusted to pH 7.4 with KOH to depolarize the cell to ß0 mV (Nernst potential) to allow accurate control of the membrane potential. After recordings were made in the cell-attached configuration, an extracellular solution with an intracellular ion composition was perfused, ready for patch excision into an inside-out configuration. The extracellular medium used for the inside-out patch experiments was identical to the high-K + solution specified above except that pH was adjusted to 7.2 and 3 mM Mg-ATP was added. All electrophysiological experiments were performed at 34°C. For 'patch cramming' experiments, Ins1 and HEK cells were cultured in droplets of their respective culture media in the same 35 mm dish. The same solutions that were used for cell-attached and inside-out configurations were then applied. However, upon excision of the membrane, the electrode was 'crammed' into a neighbouring HEK cell followed by subsequent measurements of voltage-dependent activation and inactivation (Kramer, 1990). A standard two-pulse protocol was used to assess inactivation. The pulse protocol consisted of a 50 ms conditioning pulse of potentials varying from −150 to 0 mV (in increments of 5 mV) followed by a 5 ms test pulse to 0 mV. The peak inward current (I) produced after each depolarization to 0 mV was measured and normalized to the maximum Na + current amplitude (I max ). The data are presented as h Ý (= I/I max ), which was plotted against the conditioning voltage. For each cell, the data points were approximated to a single or a double Boltzmann function to determine the half-maximal inactivation (V h ) and the slope factor (k). The most appropriate fit to the data (single or double Boltzmann) was determined using the Akaike information criterion using Origin software (OriginLab Corp., Northampton, MA, USA). Inactivation curves were fitted to a single Boltzmann function when the fit to a double Boltzmann function resulted in either two V h values with a difference of less than 15 mV or a V h composed of less than 15% of the total current. Steady-state activation was assessed by applying a depolarizing pulse, between −70 and +60 mV (in increments of 10 mV) for 20 ms, from a holding potential of −150 mV. The peak inward current (I) produced after depolarization to each voltage step was measured and used to calculate the Na + conductance (G) by the relation where V is the membrane potential and V r is the estimated reversal potential, calculated using the Nernst equation from the extra-and intracellular Na + concentrations in the media (Gonoi & Hille, 1987). The sigmoidal curve produced was fitted to a Boltzmann function, which was used to determine the half-maximal activation (V h ) and the slope factor (k). Data analysis All data are given as mean values ± SEM of the indicated number of experiments (n). Statistical significances were calculated using Student's t test or ANOVA (for multiple comparisons, as appropriate). Characterization of TTX-resistant Na + channels To further explore the role of the different Na + channel α-subunits and their contribution to voltage dependence of inactivation, it was important to isolate the current from individual Na V channel α-subunits. As there are currently no reliable α-subunit-specific Na + blockers, we generated TTX-resistant α-subunits by site-directed mutagenesis (see Methods) and expressed them in clonal β-cells and HEK cells. Figure 2A and B shows Na + currents recorded from non-transfected Ins1 and HEK cells during a voltage-clamp depolarization to 0 mV. All untransfected Ins1 cells contained TTX-sensitive voltage-gated Na + currents (Na V currents; n = 21). By contrast, none of the HEK cells contained any Na V currents. After transfection of the cells with mutant Na V 1.7, large TTX-resistant Na V currents were observed in both Ins1 and HEK cells ( Fig. 2C and D). Similar data were obtained when cells were transfected with TTX-resistant Na V 1.3 and Na V 1.6 or wild-type Na V 1.5 -a naturally TTX-resistant channel (data not shown). It was ascertained in HEK cells that making the Na V 1.3, Na V 1.6 and Na V 1.7 channels TTX resistant did not affect voltage dependence of inactivation (not shown). From here on the channels made resistant to TTX will be referred to simply as Na V 1.7, Na V 1.3 and Na V 1.6. Inactivation of Na V 1.3 and Na V 1.7 expressed in Ins1 cells We expressed Na V 1.3 or Na V 1.7 in Ins1 cells and determined their voltage dependence of activation and inactivation, which were described by fitting single Boltzmann functions to the data points (Table 1). The two types of Na V channel α-subunit exhibited rather different inactivation behaviours, and V h averaged −76 ± 2 (n = 34) and −92 ± 2 mV (n = 47) for Na V 1.3 and Na V 1.7, respectively (Fig. 3A). However, it is noticeable that the inactivation of Na V 1.7 in Ins1 cells shows some slight deviation from a single Boltzmann function (arrow). Although the inactivation of Na V 1.3 appears to be monophasic, inactivation in individual cells was clearly best described using a double Boltzmann fit to the data. This aspect will be addressed further below (Fig. 11). We have previously proposed that biphasic inactivation of the Na + current in primary β-cells reflects the expression of Na V 1.3 and Na V 1.7 α-subunits and that the two subunits underlie the positive and negative inactivation components, respectively. We modelled Na + current inactivation behaviour in a cell containing 15% Na V 1.3 and 85% Na V 1.7 (the observed relative contribution of the positive and negative inactivation components in primary β-cells (Zhang et al. 2014). As shown in Fig. 3C, the resulting inactivation curve thus obtained exhibits a clear shoulder at conditioning membrane potentials of −70 to −40 mV (arrow). We conclude that expression of different α-subunits may contribute to the biphasic inactivation observed in primary β-cells. However, as will be explained below, this is not the only contributing factor. Comparison of Na V 1.3, Na V 1.5, Na V 1.6 and Na V 1. inactivation in Ins1 and HEK cells The inactivation of Na V 1.7 in Ins1 cells (V h = −93 mV) is more negative than what has been reported previously in HEK cells and in neurones (Herzog et al. 2003;Eberhardt et al. 2014). We next systematically compared inactivation of Na V 1.3, Na V 1.5, Na V 1.6 and Na V 1.7 co-expressed with β 1 and β 2 in Ins1 and HEK cells ( Fig. 4A-D). We found that for all α-subunits, inactivation (expressed as V h ) occurred at membrane potentials 20-30 mV more negative in Ins1 cells than in HEK cells or CHO cells and that inactivation of Na V 1.7 occurred at 10-20 mV more negative membrane potentials than Na V 1.3 and Na V 1.6 regardless of the cell type (Table 1). Inactivation properties of both Na V 1.3 and Na V 1.7 expressed in CHO cells were similar to that observed J Physiol 596.9 in HEK cells ( Fig. 5A and B and Table 1). In the glucagon-secreting cell line αTC1-6, inactivation of both Na V 1.3 and Na V 1.7 was more similar to that found in HEK and CHO cells than in the insulin-secreting cells ( Fig. 5A and B and Table 1). Impact of β-subunits on Na V inactivation The Na V channel β-subunits modulate inactivation of Na V α-subunits (Cummins et al. 2001;Calhoun & Isom, 2014). Moreover, as the β-subunits are prone to cell-specific post-translational modulation, their effect currents in Ins1 and HEK cells A, endogenous Na V current from Ins1 cells in response to a step depolarization from −150 to 0 mV in the absence (black; control) and presence of TTX (red; TTX treated). B, no endogenous Na V currents were evoked in HEK cells by a step depolarization from −150 to 0 mV. C, Na V currents evoked in response to a step depolarization from −150 to 0 mV in Ins1 cells co-transfected with mutant TTX-resistant Na V 1.7 and β 1 -and β 2 -subunits in the absence (black; control) and presence of TTX (red; TTX treated). The reduction of the peak current induced by TTX reflects block of endogenous Na V current. D, same as in C but expressed in HEK cells. [Colour figure can be viewed at wileyonlinelibrary.com] on Na V inactivation may be cell specific, i.e. the same β-subunit can have different effects on Na V inactivation in different cell types (Isom et al. 1992, Moran et al. 2000Meadows & Isom, 2005). To test whether modulation of β-subunits is the mechanism by which Na V currents in Ins1 cells inactivate at more hyperpolarized potentials compared to other cells types, siRNA knockdown was used to ablate β-subunit expression in Ins1 cells. The only β-subunit expressed endogenously in Ins1 cells is the β 3 -subunit (Fig. 6A). At the mRNA level, 71% knockdown of Scn3b was achieved with no compensatory increase in other β-subunits (i.e. Scn1b, 2b or 4b). Down-regulation of Scn3b only marginally affected the inactivation of the end-ogenous Na + current in Ins1 cells (Fig. 6B, Table 2). The inactivation of the endogenous Na V current was clearly biphasic but this was not affected by down-regulating the β 3 -subunit. We also compared the inactivation of the expressed human Na V 1.3 and Na V 1.7 when co-expressed with β 3 (rather than β 1 and β 2 , as in previous experiments) in Ins1 cells ( Fig. 6C and D). In both cases, overexpression of the β 3 -subunit made biphasic inactivation more apparent (arrows). Finally, we compared Na V 1.3 and Na V 1.7 inactivation when co-expressed with or without β 3 in HEK cells. Whereas the inactivation of Na V 1.7 was shifted by 4 mV Na v 1.7 in Ins1 cells A, voltage dependence of inactivation (h Ý ) of Na V 1.7 (black; n = 47) and Na V 1.3 (red; n = 34) when the α-subunit is co-expressed with β 1 -and β 2 -subunits in Ins1 cells. The curves represent a single Boltzmann fit to the data. Note slight deviation from inactivation at membrane potentials between −70 and −40 mV in Ins1 cells expressing Na V 1.7 (arrow). B, voltage dependence of Na V 1.7 (black; n = 39) and Na V 1.3 (red; n = 31) current activation (G/G max ) in Ins1 cells. The curves represent a single Boltzmann fit to the data. C, the black curve represents a model of β-cell Na V current inactivation behaviour composed of 15% Na V 1.3 and 85% Na V 1. towards more negative voltages in the presence of β 3 , inactivation of Na V 1.3 was shifted 11 mV towards more depolarized membrane potentials ( Fig. 6E and F). It was ascertained that HEK cells do not express any endogenous β-subunits (not shown). Collectively, these data suggest that the widely different inactivation behaviours of Na V 1.3 and Na V 1.7 when expressed in Ins1 and HEK cells cannot be attributed to any of the differential modulation of β-subunits in a cell-specific manner, expression of a different endogenous β-subunit complement in Ins1 cells or the α-subunits forming heterodimers with different β-subunits (in which case overexpression of β 3 should have made biphasic inactivation less apparent). Rather the data suggest that the hyperpolarized inactivation observed in β-cells is due to a direct effect on the Na V channel α-subunit. Moreover, we conclude that the biphasic inactivation of the endogenous Na + current in Ins1 cells need not reflect the presence of multiple α-subunits as biphasic inactivation was also observed in the experiments involving expression of Na V 1.3 and Na V 1.7 alone. Negative Na V inactivation in Ins1 cells is not due to cytosolic diffusible factor We hypothesized that the inactivation of Na V 1.3 and Na V 1.7 is shifted towards more negative membrane potentials in Ins1 cells (and mouse β-cells) compared to other cells (dorsal root ganglion neurones, HEK, CHO, αTC1-6) because of the presence of a cytoplasmic factor in β-cells that is not present in other cells. To explore this possibility, Na V 1.3 and Na V 1.7 were expressed in Ins1 cells and CHO cells and inactivation was measured in cell-attached and subsequently in inside-out . Voltage dependence of inactivation of Na V 1.7, Na V 1.3, Na V 1.6 and Na V 1.5 currents in Ins1 cells compared to HEK cells A, voltage dependence of Na V 1.7 current inactivation (h Ý ) when the α-subunit is co-expressed with β 1 -and β 2 -subunits in Ins1 (same data as in Fig. 3A: dashed curve) and HEK cells (red; n = 9). The curve represents a single Boltzmann fit to the data. B, as in A but for Na V 1.3 (dashed curve same data as in Fig. 3A; red, n = 7). C, as in A but for Na V 1.6 (black, n = 35; red, n = 10). D, as in A but for Na V 1.5 (black, n = 9; red, n = 10). See also patch configuration (to promote rapid 'wash-out' of any attached modulators). These experiments required channel expression high enough to allow measurements of macroscopic ('whole-cell') currents in cell-attached patches ( Fig. 7A and B). Figure 7C shows inactivation curves for Na V 1.3 currents recorded in cell-attached patches and at various times after excision and formation of inside-out patches in Ins1 cells. In the cell-attached configuration, V h for Na V 1.7 was −92 ± 3 mV (n = 23) and −74 ± 4 mV (n = 15) in Ins1 and CHO cells, respectively. The corresponding values for Na V 1.3 were −69 ± 2 (n = 18) and −56 ± 5 mV (n = 6) in Ins1 and CHO cells, respectively. We acknowledge that these values are not identical to those measured in the whole-cell configuration. In the cell-attached experiments, the membrane potential of the cell was assumed to be 0 mV when the cells were immersed in the high-[K + ] o medium, but it is possible that a slight voltage difference remains. This idea is supported by the observation that following excision of the patches, there was an immediate ß10 mV shift in inactivation in the hyperpolarizing direction for both Na V 1.3 and Na V 1.7 expressed in either cell type. In the cell-attached patches, inactivation of Na V 1.3 was invariably monophasic. However, the distribution at which Na V 1.3 inactivated in the cell-attached configuration appears to have two distinct components. Figure 7F shows the normalized cumulative distribution of V h measured in 18 membrane patches (each represented by a single point) in Ins1 cells expressing Na V 1.3. The continuous curve represents a double Boltzmann fit to data points with V h values of −74 mV and −58 mV. Five of the 18 patches (28%) had V h values more positive than −60 mV. For Na V 1.7 (black squares and line), the cumulative distribution for 23 patches was essentially monophasic with a V h of −93 mV with only two patches (9%) showing a V h at membrane potentials more positive than −65 mV. If there is a diffusible modulator of Na V inactivation present in β-cells that shifts inactivation, then patch excision would result in a positive shift of inactivation (i.e. towards that observed in CHO cells). As already remarked, patch excision resulted in an immediate ß10 mV shift of inactivation towards more hyperpolarized membrane potentials for both Na V 1.3 and Na V 1.7 and for both Ins1 and CHO cells. Following patch excision there was then a time-dependent additional and parallel negative shift of V h in both cell types until V h eventually settled at −120 mV in Ins1 cells and −100 mV in CHO cells (Fig. 7D). Similar time-and cell-dependent changes were observed for Na V 1.3 channels (Fig. 7E). If diffusible factors modulating Na V current inactivation were present in Ins1, then the curves would have been expected to converge. In addition, there was a time-dependent decrease in current amplitude. We attribute this to rundown of channel activity, a process observed for many types of channels (Becq, 1996). Importantly, the shift in inactivation was nearly maximal 2 min after patch excision when effects on current kinetics were moderate (compare red and blue traces in Fig. 7A and B). Activation of the Na V 1.3 and Na V 1.7 channels underwent similar time-dependent changes in the hyperpolarizing direct following patch excision (not shown). We also tested the alternative possibility that inactivation is more negative in Ins1 cells because something present in all other cells is missing in these cells. This was tested by an approach similar to that in currents in αTC1-6 and CHO cells A, voltage dependence of inactivation of Na V 1.7 currents when the α-subunit is co-expressed with β 1 -and β 2 -subunits in αTC1-6 (red; n = 10) and CHO (black; n = 10) cells. The curve represents a single Boltzmann fit to the data and the grey dashed line represents the inactivation of Na V 1.7 expressed in Ins1 cells (shown in Fig. 3). B, same as in A, but experiments were conducted with Na V 1.3 in αTC1-6 (red; n = 9) and CHO cells (black; n = 13). See also Table 1 The curves represent a double Boltzmann fit to the data. C, voltage dependence of Na V 1.7 current inactivation when the α-subunit is co-expressed with β 3 -subunit (black; n = 9) in Ins1 cells. Dashed red curve represents voltage dependence of Na V 1.7 current inactivation when the α-subunit is co-expressed with β 1 -and β 2 -subunits in Ins1 cells (data shown in Fig. 3). The curves represent a double Boltzmann fit to the data. Arrow indicates a component of inactivation occurring at positive membrane potentials. D, as in C but for Na V 1.3 (black; n = 11). E, voltage dependence of Na V 1.7 current inactivation when the α-subunit is co-expressed with β 3 -subunit (black; n = 9) or GFP alone (red; n = 12) in HEK cells. The curves represent a single Boltzmann fit to the data. F, as in E, but for Na V 1.3 (black, n = 12; red; n = 10). V h and k value for the respective conditions are given in Table 2. Na v 1.7 in cell-attached and inside-out configuration A, Na V 1.7 currents when the α-subunit is co-expressed with β 1 and β 2 in Ins1 cells in response to a step depolarization from −150 to 0 mV in cell-attached (CA), inside-out (IO) and 2 and 5 min after excision. B, same as in A, but with Na V 1.3 currents in Ins1 cells. C, voltage dependence of inactivation of Na V 1.3 currents shown in B (same colour code). D, summarized voltage dependence of inactivation of Na V 1.7 currents in Ins1 (black) and CHO cells (red) of indicated number of patches/cells (n). E, same as in D but for Na V 1.3. F, cumulative distribution of Na V 1.3 (red) and Na V 1.7 (black) V h recorded in cell-attached patches on Ins1 cells. Data have been normalized (n/ࢣn) to the total number of patches (n = 18 patches for Na V 1.3 and n = 23 patches for Na V 1.7 but instead of leaving the patch in the bath medium after excision, the patch was 'crammed' (Kramer, 1990) into a neighbouring HEK cell (Fig. 8A). However, as shown in Fig. 8B and C, the time-dependent shift of V h towards more negative membrane potentials persisted and was as pronounced after cramming the electrode into a HEK cell as when the patch was left in the bath medium; inactivation still underwent a 25 mV shift towards more negative membrane potentials 5-10 min after patch excision and cramming into the neighbouring HEK cell (Fig. 8D). Cytoplasmic domains of Na V 1.3 and Na V 1.7 do not confer different inactivation behaviours The data of Figs 7 and 8, suggest that the differences in inactivation of Na V channels between Ins1 and HEK and CHO cells is unlikely to be attributable to freely diffusible factors present in the cytosol of either cell type. However, we acknowledge that the change in inactivation may result from firmer interaction between the Na V α-subunits and another factor (protein) at the time they are inserted into the plasmalemma. The schematic diagram in Fig. 9A shows the topology of the Na V 1.7 channels and highlights the areas of divergence in the amino acid sequence between Na V 1.3 and Na V 1.7. As indicated, the greatest differences are found in the N-terminus, the C-terminus and the cytoplasmic loops L1 and L2. We hypothesized that the sequence variations contribute both to differences in inactivation between (i) Na V 1.3 and Na V 1.7 and (ii) Ins1 and HEK cells. We acknowledge that this assumes that the differences in the intracellular/cytoplasmic milieu are transduced via Figure 9. Na V 1.7-Na V 1.3 chimeras expressed in Ins1 and HEK cells A, schematic representation of the structure of the Na V 1.7 channel. Highlighted in red are the areas of divergence in the amino acid sequence to Na V 1.3. Sequence alignment was performed using the Clustal Omega program (www.uniprot.org/align/), using the NCBI reference sequences NP 002968.1 and NP 008853.3 for human Na V 1.7 and Na V 1.3, respectively. B, voltage dependence of inactivation of N-terminal chimera currents when the α-subunit is co-expressed with β 1 -and β 2 -subunits in Ins1 (black; n = 9) and HEK cells (red; n = 6); and control TTX-resistant Na V 1.7 currents when the α-subunit is co-expressed with β 1 -and β 2 -subunits in Ins1 (dashed black line; n = 37) and HEK cells (dashed red line; n = 6). C, as in B but for L1L2 chimera currents (black, n = 15; red, n = 9) with the same controls as in B. D, as in B but for L3 chimera currents (black, n = 16; red, n = 11) with the same controls as in B. E, as in B but for C-terminal chimera currents (black, n = 7; red, n = 8) with the same controls as in B. V h and k values are given in Table 3. [Colour figure can be viewed at wileyonlinelibrary.com] J Physiol 596.9 the cytoplasmic domains of the channels. It follows from these premises that substituting the cytoplasmic domains of Na V 1.7 for Na V 1.3 will reduce the differences in V h between the two α-subunits (i.e. change V h from −92 mV to −76 mV in Ins1 cells; Table 1). We generated chimeric channels based on the Na V 1.7 backbone by replacing the N-terminal domain, L1 and L2, the C-terminal domain or L3 sequences with the corresponding sequences of Na V 1.3 and analysed their properties in Ins1 (black) and HEK cells (red) (Fig. 9B-E: see also Table 3). For comparison, the inactivation curves for wild-type Na V 1.7 in Ins1 and HEK cells are also shown (dashed black and red lines). None of the substitutions significantly affected Na V 1.7 inactivation. Taken together with the data of Figs 7 and 8, it seems unlikely that the negative shift is caused by an intracellular factor interacting with the cytoplasmic domains of the Na V 1.7 channels. Biphasic inactivation of endogenous Na V currents in Ins1 cells We examined the expression of Na V channel α-subunits in Ins1 cells and found that these cells express high levels of Scn3a (Na V 1.3) and low levels of Scn2a (Na V 1.2) and Scn8a (Na V 1.6) (Fig. 10A). We measured the inactivation of the endogenous Na V currents, and the average inactivation curve (n = 24) is shown in Fig. 10B. We found that whereas inactivation was monophasic in 50% of the cells (12 of 24 cells) with a V h of −88 ± 2 mV (Fig. 10C), it was clearly biphasic in the remaining cells (n = 12; Fig. 10D). In this subgroup of cells, the major component (comprising 71 ± 6%) was similar to that in cells with monophasic inactivation and had a V h of −87 ± 3 mV. In addition, there was a smaller component that accounted for 29 ± 6% of the total with a V h of −43 ± 2 mV. This characteristic inactivation appears to be unique to β-cells because αTC1-6 cells, which also express Na V 1.2, Na V 1.3, Na V 1.6 and Na V 1.7, displayed only a single component of inactivation with a V h of −55 ± 4 mV (n = 6; Fig. 10E and F). Although we could not rule out that the expression of α-subunits other than Na V 1.3 explains the biphasic inactivation behaviour in Ins1 cells, we think that this is unlikely given the very low expression of Scn2a and Scn8a and we instead hypothesize that Na V 1.3 channels expressed in the same cell may undergo inactivation with different voltage dependences. Figure 11A shows inactivation in four different Ins1 cells in which inactivation of expressed Na V 1.3 channels was clearly biphasic with values of V h ranging between −86 and −37 mV. Such cells accounted for 70% of all cells tested. In these 26 cells (of a total of 34 cells), the negative and positive components of inactivation contributed 57 ± 5% and 43 ± 5% of the total current, respectively. The corresponding values of V h were −87 ± 2 and −55 ± 2 mV (Table 4). In addition, there were cells in which inactivation was monophasic (n = 8; Table 4): in most of these cells, inactivation occurred at negative voltages (V h = −90 mV) (Fig. 11B) but in two cells inactivation was instead at positive voltages (V h = −50 mV) (Fig. 11C). Similarly, in cells expressing Na V 1.6, 25 cells out of a total of 35 cells exhibited biphasic inactivation (Table 4). For Na V 1.7, biphasic inactivation was observed in 9 out of 47 cells (P < 0.001 by χ 2 vs. both Na V 1.3 and Na V 1.6). None of the α-subunits showed biphasic inactivation when expressed in HEK, CHO or αTC1-6 cells. It is worth noticing that the fraction of cells showing biphasic Na + current inactivation was not increased by substitution of cytoplasmic Na V 1.7 for Na V 1.3 domains (Table 3). Figure 11D shows the relationship between Na + current density and V h of the negative and positive components fitted to a linear regression with r 2 values of 0.04 and 0.01, respectively. It is clear that there is no impact of current density on V h . However, there are clearly two distinct groups that inactivate at distinct membrane potentials, separated by approximately 30 mV. Interestingly, cells that displayed monophasic inactivation fall into either of these distinct groups. Similar data were obtained for Na V 1.6 and Na V 1.7 channels expressed in Ins1 cells ( Fig. 11E and F). Modulation of inactivation Protein kinase C (PKC) phosphorylation of Na V 1.7 exhibits a depolarizing effect on the voltage dependence of inactivation in some (Tan et al. 2014) but not all cell types (Vijayaragavan et al. 2004). Moreover, Na V 1.3 and Na V 1.6 contain a putative PKC phosphorylation site in the same L3 region. We therefore tested whether there was acute activation or inhibition by PKC of Na V 1.7 inactivation by application of PMA (10 nM) or the inhibitor BIM23056 (100 nM). However, neither compound affected Na V 1.7 inactivation ( Fig. 12A and Table 5). Down-regulation of PKC (by long-term exposure to PMA) was likewise without effect on inactivation of endogenous Na V currents in Ins1 cells (Fig. 12B) and it did not affect the biphasic pattern of inactivation. Consistent with earlier reports in the rat insulinoma RINm5F (Rorsman et al. 1986), chronic exposure to PMA reduced the amplitude of the Na V current (not shown). Na V current inactivation has been reported to be regulated by the intracellular ATP concentration (Zou et al. 2013) suggestive of metabolic regulation. We therefore tested the effect of metabolic regulation by culturing cells at 5, 11 or 25 mM glucose for 48 h. No effects on the inactivation of endogenous Na V currents was observed (Fig. 12C). We also tested the acute effects of elevating glucose from 1 to 20 mM on Na V 1.3 and Na V 1.7 currents, 2 preparations). B, voltage dependence of inactivation of endogenous Ins1 Na V currents, average of all cells (n = 24). Curve represents a double Boltzmann fit to the data. C, as in B but for cells with Na V inactivation best fitted to a single Boltzmann function (i.e. had a monophasic inactivation; n = 12). D, as in C but for cells where inactivation was best fitted to a double Boltzmann function (i.e. had a biphasic inactivation; n = 12). E, relative mRNA expression of Na V channel α-subunits in αTC1-6 cells (n = 3 preparations). F, voltage dependence of inactivation of endogenous αTC1-6 Na V currents (n = 6). Na v 1.6 Na v 1.7 currents in Ins1 cells A, examples of biphasic inactivation of Na V 1.3 currents when the α-subunit is co-expressed with β 1 and β 2 -subunits in four different Ins1 cells (nos 1-4). The tables next to the curves show V h and k values for the components inactivating at negative (−) and more positive (+) membrane potentials. The curves represent a double Boltzmann fit to the data. B, average voltage dependence of inactivation of Na V 1.3 currents in Ins1 that were best described with a single Boltzmann fit to the data and had a V h <−70 mV (n = 6). C, average voltage dependence of inactivation of Na V 1.3 current in Ins1 cells that were best described with a single Boltzmann fit to the data that had a V h >−70 mV (n = 2). D, relationship between monophasic, biphasic negative (−ve) and biphasic positive (+ve) V h values and the peak Na + current density of Na V 1.3 in Ins1 cells. The lines represent linear regression fits to the data. The r 2 values are given next to the respective fit. E and F, same as in D but for Na V 1.6 (E) and Na V 1.7 (F). (2) (2) A B D C E F Figure 12. Modulation of Na V inactivation in Ins1 cells A, voltage dependence of inactivation of Na V 1.7 α-subunit co-expressed with β 1 -and β 2 -subunits in control (red; n = 5), 100 nM BIM23056 treated (black; n = 5) and 10 nM PMA treated (grey; n = 4) Ins1 cells. The curves represent a single Boltzmann fit to the data. B, voltage dependence of inactivation of endogenous Na V currents in control Ins1 cells (red; n = 4) and Ins1 cells chronically (48 h) treated with 10 nM PMA (black; n = 11). The curves represent a double Boltzmann fit to the data. C, voltage dependence of inactivation of endogenous Na V currents in Ins1 cells in response to a 48 h chronic incubation in 5 mM (black; n = 22), 11 mM (red; n = 22) and 25 mM (grey; n = 18) glucose. D, same as in A, but for control (red; n = 5) and 50 μM PIP 2 diC8 treated (black; n = 5) Ins1 cells. E, summarized voltage dependence of inactivation of Na V 1.3 currents in Ins1 (red, grey and black) and HEK cells (blue) of indicated number of cells (n) at increasing concentrations of extracellular Co 2+ (0.2, 2 and 10 mM). The lines represent linear regression fits to the data. The r 2 values are given next to the respective fit. F, same as in E but for the voltage dependence of activation of Na V 1.3 currents in Ins1 (red) and HEK cells (blue). [Colour figure can be viewed at wileyonlinelibrary.com] J Physiol 596.9 Table 3. Na V 1.7-Na V 1.3 chimeras expressed in Ins1 and HEK cells but no effects on inactivation were observed over 10 min in perforated patch recordings (not shown). Insulin-secreting cells will be exposed to high concentrations of insulin (a biologically very active molecule). We tested the potential long-term role of insulin signalling by treating Ins1 cells expressing Na V 1.7 with K ATP channel activator diazoxide (100 μM for 48 h) or a receptor antagonist S961 (1 μM for 24 h) (Schaffer et al. 2008) to inhibit its release or its action, respectively. Again, no impact on Na V 1.7 inactivation was observed. It has been proposed that β-cells contain higher levels of PIP 2 than other cells and that this, via modulation of the ATP sensitivity, explains how K ATP channels remain active in intact β-cells (Baukrowitz et al. 1998;Shyng & Nichols, 1998). We therefore considered the possibility that differences in PIP 2 content could underlie the differences in Na V 1.7 inactivation in Ins1 and HEK cells but inclusion of PIP 2 diC8 (50 μM) into the pipette solution was without effect on Na V 1.7 inactivation (Fig. 12D). Likewise, neomycin (50 μM), which would shield the negative charges of PIP 2 (MacGregor et al. 2002;Bista et al. 2015), was without effect on Na V 1.7 expressed in Ins1 cells (not shown). Thus, differences in PIP 2 concentrations in Ins1 and HEK cells is unlikely to cause of the different Na V current inactivation properties. Next we tested whether there was a difference in surface charge between Ins1 and HEK cells that might influence Na V channel inactivation. One major source of negative charge is due to glycosylation of extracellular Na V channel domains that consist of the carbohydrate derivative N-acetylneuraminic acid or sialic acid (Ednie & Bennett, 2012). To explore differences in surface charge we generated inactivation curves for Na V 1.3 in the presence of 0.2, 2 and 10 mM extracellular Co 2+ in Ins1 and HEK cells Table 5 Biphasic inactivation of Na V 1.3 in primary β-cells Finally we expressed Na V 1.3 in primary mouse β-cells. Although the transfection rate was low, large TTX-resistant Na V 1.3 currents were observed in three β-cells (Fig. 13A). The average current amplitude was −1 ± 0.1 nA. These currents underwent voltage-dependent inactivation with a V h of −88 mV. However, in two of the three cells, there was biphasic inactivation with a small (43% and 22%) component that inactivated at −95 and −103 mV and a larger (57% and 78%) component that inactivated at −73 mV in both cells. We have previously reported that ablation of Scn3a leads to the loss of Na V current components that inactivate at positive membrane potentials (Zhang et al. 2014). We have now reanalysed these data and expressed current amplitudes in absolute (in pA) rather than relative (normalized to maximum current) terms. Figure 13B shows Na V currents recorded in control (Scn3a +/− ) and knockout Scn3a −/− β-cells. In agreement with the previous conclusion, ablation of Scn3a was associated with the loss of the Na V current inactivating at positive membrane potentials (Fig. 13B, shaded area). However, there was also a large reduction (ß200 pA) of Na V current at negative membrane potentials. Figure 13C shows the net Na V 1.3 current isolated by subtracting the currents in Scn3a −/− from those measured in Scn3a +/− mice. The net current shows biphasic inactivation and can be described as the sum of two Boltzmann functions with values of V h of −86 and −57 mV that comprised 76% and 24%, respectively. These values are close to those observed for the isolated Na V 1.3 current and similar to the two components of inactivation observed in Ins1 cells expressing Na V 1.3 (Table 4). Discussion We have compared the inactivation of Na V channels in insulin-secreting Ins1 and three other non-β-cell types (HEK, CHO and αTC1-6 cells). To this end, we generated mutant TTX-resistant Na V channels, which allowed us to isolate the expressed channels by blocking the endogenous Na V channels with TTX. J Physiol 596.9 Na V channels inactivate at negative membrane potentials in Ins1 cells We showed that Na V channels inactivate at ß30 mV more negative membrane potentials when expressed in Ins1 cells compared to what is seen in HEK cells and dorsal root ganglion neurones (Herzog et al. 2003;Eberhardt et al. 2014). We also confirmed that inactivation of Na V 1.7 occurs at more negative membrane potentials than Na V 1.3, Na V 1.5 and Na V 1.6 when these channels are expressed in Ins1 cells. Thus, although the Na V α-subunit expressed makes a significant difference to voltage dependence of inactivation, it appears that there is something special about Ins1 cells (echoing what is observed in primary β-cells) that shifts inactivation to functionally irrelevant membrane potentials. Individual Na V subtypes exhibit two distinct inactivation behaviours In >75% of the experiments with Na V 1.3, two distinct components of inactivation (each accounting for -on average -ß50% of the total current) separated by ß30 mV were observed. Biphasic inactivation was also observed for Na V 1.7 (in 20% of experiments) and Na V 1.6 (60%). Importantly, biphasic inactivation was not observed when these channels were instead expressed in HEK, CHO or αTC1-6 cells. It should also be noted that the voltage dependence for the component inactivating at more depolarized membrane potentials is very similar to that found in HEK or CHO cells (in which biphasic inactivation was never observed). In cells that showed monophasic inactivation, it proceeded at either positive or negative voltages but not in between. Likewise, when the measurements were done in cell-attached patches, inactivation again tended to proceed at either negative or positive voltages but was never biphasic. It appears that the data obtained in Ins1 cells can be extended to primary β-cells and the net current that can be isolated by subtracting the currents recorded from the Na V 1.3-deficient cells from control cells likewise exhibited a biphasic voltage dependence of inactivation. Thus, it appears that although different α-subunits contribute to the biphasic inactivation, there is also an additional cell-specific modulation whereby a single α-subunit may exhibit two distinct voltage dependences of inactivation. Negative inactivation does not result from a diffusible factor We explored the possibility that inactivation at negative membrane potentials reflects the interaction between the Na V α-subunits and an intracellular diffusible factor. This was done by recording macroscopic Na V currents in membrane patches before and after patch excision, the rationale being that wash-out of any such factor would be more efficient in the inside-out configuration than in the whole-cell configuration. If such an intracellular factor shifting the inactivation of Na V currents towards a more negative membrane potential exists in β-cells, then we would expect patch excision to be associated with a change in inactivation towards more positive voltages. However, such a jump was not observed. By contrast, in both Ins1 and CHO cells for Na V 1.3 and Na V 1.7 channels alike, patch excision resulted in a time-dependent shift of inactivation towards more negative membrane potentials. Why this negative shift occurs is not immediately clear but has been observed by others and might reflect the time-dependent loss of charged molecules that affect the transmembrane voltage sensed by the inactivation particle(s) (Cachelin et al. 1983;Jo & Bean, 2014). However, in the context of the current study, the key observation here is the absence of even a transient shift towards more depolarized membrane potentials. Thus, we argue that there is no diffusible modulator of channel modulation. The fact that substituting the cytoplasmic domains of Na V 1.7 for Na V 1.3 is also without effect on the voltage dependence of inactivation also suggests that the chief determinant of Na V current inactivation lies within the transmembrane domains. We can discount the possibility that β-subunits account for the negative inactivation of Na V in insulin-secreting cells as (1) Ins1 cells and primary mouse β-cells express different β-subunits (β 1 and β 3 , respectively) and yet show the same negative inactivation in both cell types; (2) expressing Na V with β 3 instead of β 1 and β 2 has only a small (ß5 mV) effects on channel inactivation; and (3) knockdown of β-subunits in Ins1 cells yielded the same effect on inactivation as control, suggesting that the β-subunit is not modulated in a cell-specific manner to modulate channel gating. Biphasic inactivation in Ins1 cells: possible interpretation of the data An explanation of these data would have to account both for the fact that inactivation of all studied Na V subtypes is shifted by 20-30 mV in Ins1 cells relative to that seen in other cell types (HEK, CHO, etc.) and the finding that biphasic Na + current inactivation is observed also when a single Na V subtype is expressed. We considered the possibility that inactivation of the Na V currents is more negative in Ins1 cells than in HEK cells as a result of surface charge effects due to differential sialylation of the channels and/or membrane. We tested this using increasing concentrations of extracellular Co 2+ . Hypothetically, Na V channels expressed in Ins1 cells might carry more negative charges than channels expressed in HEK cells (for example due to increased sialylation of the extracellular domains). Accordingly, the voltage difference sensed by the inactivation gate would be reduced and this might explain the shift in inactivation towards more negative voltages. If this were the case, then increasing the divalent cationic strength would be expected to produce a In non-β-cells, the plasma membrane is uniform in terms of impact on Na V current inactivation and the relationship between membrane potential and the fraction of activatable Na V channels (h Ý ). However, β-cells contain specialized domains that differ from the rest of the plasma membrane. Na V channels within these domains undergo inactivation at more negative membrane potentials whereas Na V channels outside these regions inactivate with the same voltage dependence as the corresponding Na V channels when expressed in non-β-cells. Thus, the Ins1 cell will contain two electrophysiologically distinct populations of Na V channels (❶ and ❷), accounting for the biphasic Na V current inactivation. [Colour figure can be viewed at wileyonlinelibrary.com] J Physiol 596.9 greater shift in Ins1 cells (because there are more negative charges to shield). However, we think that this possibility can be discarded because varying the extracellular cationic strength had the same effects in both cell types and both negative and positive components of inactivation in Ins1 cells. Moreover, the voltage dependence of activation was superimposable in Ins1 and HEK cells and was invariably monophasic in both cell types. For both inactivation and activation, there was the same 20-25 mV shift in gating for a 50-fold increase in extracellular Co 2+ . Altogether, these observations make it less likely that the widely different voltage dependences of inactivation in Ins1 and HEK cells can be attributed to variable surface charge effects. Rather, we favour the idea that the differences between Ins1 and other cell types is likely to reside within the plasma membrane itself and does not involve cytoplasmic factors. The plasma membrane lipidome is extremely complex and contains >2000 lipid subtypes (Galbiati et al. 2001;Simons & Ehehalt, 2002). The heterogeneous distribution of lipids within the plasma membrane is not uniform but shows considerable regional differences. Indeed, the plasma membrane has been described as a 'patchwork of different lipid environments' (Edidin, 1993). Perhaps the best known example of such lipid aggregates is the lipid rafts that are enriched in cholesterol. There are many examples of lipid microdomains and cholesterol affecting ion channel function (Dart, 2010). For example, in β-cells, cholesterol depletion causes a hyperpolarizing shift in the inactivation curve of K V 2.1 channels (Xia et al. 2004). We therefore propose that the lipidome of the β-cell plasma membrane is different from that of HEK and most other cells and that the β-cell membrane contains specialized lipid domains that shift inactivation into very negative membrane potentials. Na + channels outside these domains behave exactly as in other cell types. Indeed, in cells showing biphasic Na V current inactivation, the positive component shows essentially the same voltage dependence as in non-β-cells (see schematic representation in Fig. 14). The true nature of these specialized domains remains to be established but that such domains exist is suggested by the cell-attached measurements in which a small part of the cell (1 μm 2 ) is isolated and where channel inactivation was invariably monophasic but with V h values varying between −80 and −55 mV. We speculate that this is because all channels within an individual patch reside in a uniform membrane environment, which may differ between patches. It is interesting that the 'inactivation curve' for Na V 1.3 channels reconstructed from the cumulative distribution of V h in cell-attached patches was biphasic (with V h values of −75 and −55 mV) and resembled that which can be recorded from individual cells expressing this α-subunit. For Na V 1.7, the cumulative distribution was monophasic with a V h of ß−90 mV. Notably, 10% of the patches had V h values ß30 mV more positive. This is in agreement with the whole-cell data indicating biphasic inactivation in <20% of the cells expressing Na V 1.7. The idea that negative and positive inactivation results from insertion of the α-subunits into membrane domains of different composition might seem in conflict with the observation that the fraction of Na V channels inactivating at negative and positive membrane potentials varied for Na V 1.3, Na V 1.6 and Na V 1.7. However, it is possible that the intracellular trafficking and the association of Na V channels with distinct membrane domains is α-subunit-dependent. The idea that Na V channel inactivation is influenced by the lipid composition of the plasma membrane also raises the interesting possibility that differences in diet may explain why the same Na V α-subunit inactivates at widely different voltages in rodent, dog, pig and human β-cells (Rorsman & Ashcroft, 2018). Harnessing the properties of Na V channels in β-cells for therapeutic use? It is clear that inactivation of Na V channels in rodent β-cells is special in that it proceeds at membrane potentials 20-30 mV more negative than in other cell types. In the case of Na V 1.7 channels, this effect may be particularly dramatic in as far as all channels will have undergone inactivation at the physiological membrane potentials (−70 mV and above). If the negative shift of inactivation in β-cells could be harnessed (i.e. by topical/local application of agents modifying the membrane), it may provide a novel means of regulating Na + channel activity. Na V 1.7 channels play an important role in pain perception (Cox et al. 2006;Dib-Hajj et al. 2013). If the inactivation of the Na V 1.7 channels in nociceptive neurons became more like in β-cells, then the channels would inactivate and thus be unavailable for action potential propagation, which may represent a novel means of pain relief, especially in disease states associated with Na V 1.7 hyperexcitability (Dib-Hajj et al. 2008).	2018-04-03T01:34:43.486Z	2018-03-30T00:00:00.000	{ "year": 2018, "sha1": "5e314d7e905282da917c50225381225a40063d61", "oa_license": "CCBY", "oa_url": "https://physoc.onlinelibrary.wiley.com/doi/pdfdirect/10.1113/JP275587", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "5e314d7e905282da917c50225381225a40063d61", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
31917231	pes2o/s2orc	v3-fos-license	Disappearing portal venous gas in acute pancreatitis and small bowel ischemia We report an usual case of hepatic portal venous gas (HPVG) in the setting of acute pancreatitis and small bowel ischemia. Interestingly, the HPVG disappeared within 2 hours of the original computed tomography scan, despite the patient having small bowel ischemia. The patient had a complicated clinical course, dying 62 days postadmission. This case highlights that HPVG in setting of acute pancreatitis and small bowel ischemia has a very high morbidity and mortality, requiring early detection and aggressive surgical management. Introduction Acute pancreatitis affects an estimated 20,000 people annually in the UK [1], and it accounts for the most gastrointestinalrelated hospital admissions in the United States [2]. A study conducted in Wales (1999e2010) observed that the incidence of acute pancreatitis is increasing annually by approximately 2.7% each year [3]. Rates are also increasing internationally. In the "Japan National Survey," incidence increased from 12.1 to 15.4 per 100,000 between 1987 and 1998 [4]; meanwhile, in the Netherlands, the incidence has increased from 13.2 to 14.7 per 100,000 over the time period 2000e2005 [2]. Reported overall mortality rates vary from 4% to 10%, increasing substantially to 15% to 30% for severe cases [1]. Complications of acute pancreatitis can be either local or systemic events. The Revised Atlanta Classification aims to standardize the reporting of severity of acute pancreatitis to ensure consensus and standardized treatment protocols [6]. Alternatively, acute pancreatitis can also be subdivided into interstitial edematous pancreatitis and necrotizing pancreatitis [ in patients with necrotizing pancreatitis, but usually has a benign course [7]. Alternatively, hepatoportal venous gas (HPVG) is a rarer complication and more ominous finding [8]. It usually indicates concurrent mesenteric ischemia and is associated with substantial complications [8]. Case report A 64-year-old gentleman was referred to our hospital from another institution with a diagnosis of acute pancreatitis (Imrie/Glassgow Score ¼ 6). Underlying biliary pathology was the suspected causative factor. Initial computed tomography (CT) scan of the abdomen demonstrated extensive edematous changes involving the entire pancreas with a significant volume of HPVG ( Fig. 1). Concern regarding one segment of small bowel was raised. The patient proceeded to have a CT mesenteric angiogram within 2 hours of the original CT scan which showed a poorly enhancing segment of small bowel that was concerning for venous thrombosis (Fig. 2). Interestingly, there was a significant reduction in the volume of HPVG (Fig. 3). Despite this, the patient's clinical condition disimproved and the decision to proceed for emergency laparotomy immediately was decided. At laparotomy, resection of a nonviable ischemic segment of small bowel (60 cm) was performed. Omental saponification was also evident (Fig. 4). The patient had a prolonged intensive care admission (45 days) for the management of multiorgan dysfunction including acute kidney injury and adult respiratory distress syndrome before being discharged to the ward. The patient was readmitted to the intensive care unit 72 hours later due to overwhelming sepsis. Repeat imaging confirmed large peripancreatic necrotic collections (Fig. 5), and several interventional radiology drainages were performed. Despite escalation in both antimicrobials, antifungal and inotropic support the patient's clinical condition deteriorated and he died on the 62nd day of admission. Discussion HPVG is a worrisome radiological finding [7]. It typically indicates an underlying life-threatening gastrointestinal pathology such as ischemia and/or inflammation, which frequently requires emergency surgical intervention [9]. Historically, the presence of HPVG has been associated with mortality rates >75% [10], but in contemporary times, mortality is 30%e40% [9]. Traditionally, the presence of HPVG mandated explorative laparotomy, but with improvements in radiological investigations (helical multislice CT), HPVG is now attributed to other benign causes that can be managed conservatively [8]. The underlying pathogenesis of HPVG in pancreatitis is not fully understood. There are several mechanisms speculated regarding its etiology. It is hypothesized that pancreatic enzymes could directly damage the intestinal wall or alternatively the presence of intestinal ischemia allows air to translocate from the bowel lumen into the portal venous system [8,10]. Alternatively, in the case of sepsis, gas producing bacteria such as Escherichia coli may enter into the intestinal wall through mucosal defects and become the source of the HPVG [8,10]. There are some sparse reports of management of HPVG without surgical intervention and some rare incidents of disappearing HPVG. Niki et al. describe a case of disappearing HPVG in a 64-year-old woman with severe lower abdominal pain. The pain resolved quickly, and no underlying pathology was evident on radiological imaging. Repeat imaging showed complete resolution of HPVG within 18 hours of the initial CT scan. She was later diagnosed with terminal ileitis at colonoscopy [11]. Martin-Smith et al. reported a case of extensive pancreatic necrosis with HPVG, splenic vein thrombosis, and pneumatosis coli of the cecum and ascending colon. The patient was managed conservatively. Repeat CT scan 5 days later showed progression of pancreatic necrosis but with complete resolution of the HPVG. By day 12, the patient was asymptomatic, diet was reintroduced, and biochemical markers were all improving [12]. However, Park et al. [8] reported two cases of acute pancreatitis with intestinal ischemia and HPVG that had very poor outcomes. Our case is quite unusual as the HPVG rapidly disappeared (within 2 hours) despite the patient having small bowel ischemia. However, we still advocate that exploratory laparotomy was necessary, especially when imaging raised the possibility of nonviable bowel and when there was a significant deterioration in patient's clinical status. A recent study from Korea observed a lower mortality rate (40%) for patients undergoing prompt emergency laparotomy, compared with those who did not undergo surgery (100% mortality) when imaging predicted HPVG was unlikely to resolve with conservative management [13]. Key indicators for prompt surgery included the presence of intraperitoneal free air or HPVG with nonviable bowel on CT imaging [13]. The presence of HPVG in acute pancreatitis is a poor prognostic sign, requiring early detection combined with aggressive (surgical) management, especially if associated with mesenteric ischemia. It indicates a complicated disease course and is associated with high morbidity and mortality. r e f e r e n c e s	2018-04-03T06:16:07.607Z	2017-02-27T00:00:00.000	{ "year": 2017, "sha1": "39a5519e8895a77cf0fd7b0545c340e3dc2f7c0c", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.radcr.2017.01.006", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "39a5519e8895a77cf0fd7b0545c340e3dc2f7c0c", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
221911963	pes2o/s2orc	v3-fos-license	Identification and characterization of deschloro-chlorothricin obtained from a large natural product library targeting aurora A kinase in multiple myeloma Multiple myeloma (MM) is a devastating disease with low survival rates worldwide. The mean lifetime of patients may be extendable with new drug alternatives. Aurora A kinase (AURKA) is crucial in oncogenesis, because its overexpression or amplification may incline the development of various types of cancer, including MM. Therefore, inhibitors of AURKA are innovative and promising targets. Natural compounds always represented a valuable resource for anticancer drug development. In the present study, based on virtual drug screening of more than 48,000 natural compounds, the antibiotic deschloro-chlorotricin (DCCT) has been identified to bind to AURKA with even higher binding affinity (free bindung energy: −12.25 kcal/mol) than the known AURKA inhibitor, alisertib (free binding energy: −11.25 kcal/mol). The in silico studies have been verified in vitro by using microscale thermophoresis. DCCT inhibited MM cell lines (KMS-11, L-363, RPMI-8226, MOLP-8, OPM-2, NCI-H929) with IC50 values in a range from 0.01 to 0.12 μM. Furthermore, DCCT downregulated AURKA protein expression, induced G2/M cell cycle arrest and disturbed the cellular microtubule network as determined by Western blotting, flow cytometry, and fluorescence microscopy. Thus, DCCT may be a promising lead structure for further derivatization and the development of specific AURKA inhibitors in MM therapy. Introduction Multiple myeloma (MM) is a critical plasma cell proliferative disorder characterized by the accumulation of malignant plasma cells in the bone marrow, bone lesions and immunodeficiency causing 1% of all cancers, and 10% of hematological malignancies [1 -3]. Based on the Surveillance, Epidemiology, and End Results (SEER) Program data from 2010 to 2016 published by National Cancer Institute (NCI), 5-year relative survival of patients with myeloma is 53.9% [4], which may be increased by newly developed drugs. Multidrug resistance (MDR), a phenomenon where tumors gain resistance towards structurally and functionally diverse drugs at the same time, is a substantial obstacle leading to treatment failure in cancer therapy, including MM [5,6]. Therefore, novel treatment regimens targeting innovative proteins or signaling pathways involved in drug resistance may have the potential to overcome drug resistance and to extend patients' survival time for individuals with MM. Aurora kinases (AURKs) including aurora kinases A, B, and C are members of the serine/threonine kinase family and are key players involved in genetic stability during cell division [7]. They have significant roles in oncogenesis, i.e. their overexpression or amplification are involved in tumorigenesis of lung cancer, colorectal carcinoma, and melanoma. Moreover, AURK inhibitors (AKIs) prohibiting the occurrence of radio-and chemo-resistance are innovative and promising drug candidates in cancer [8,9]. However, only a small number of AKIs have been investigated in clinical trials due to their frequently high toxicity towards healthy cells [10,11]. In this regard, nature as a unique source for novel chemical scaffolds may represent a valuable repository of selective and effective compounds with few side effects. To exemplify, 113 out of 136 new chemical entities (83%) excluding non-biologicals and vaccines in the field of anticancer drugs were either natural products, natural product derivatives or natural product mimics in the time frame covered from 1981 to 2014 [12]. Therefore, natural compounds/products may provide ample opportunities for the discovery of novel drug leads, which may enable the design of prospective (semi)synthetic derivatives with improved pharmacological features such as AKIs. Aurora A kinase (AURKA) is a centrosomal kinase with fundamental roles during mitosis including centrosome maturation, nuclear envelope breakdown, mitotic entry, centrosome separation, spindle pole formation, and spindle checkpoint activity [13,14]. Overexpression of AURKA has been announced in numerous cancer types, such as laryngeal, ovarian, breast cancers [15]. Furthermore, the association of AURKA with cell proliferation, epithelial-mesenchymal transition, metastasis, chemoresistance, and self-renewal capacity of cancer stem cells have been previously reviewed [9,16]. MM is characterized by a genetic imbalance with various chromosomal deformities [17]. AURKA sets in centrosomes in the early S phase and during mitosis an AURKA fraction correlates with spindle microtubules proximal to the spindle poles [3]. AURKA has a role in cytokinesis and is activated by phosphorylation during G2/M phase transition in the cell cycle. Impaired AURKA may incline aneuploidy characteristics of tumors [3,18]. AURKA upregulation is associated with centrosome amplification and worse prognosis in MM [19][20][21]. Inhibition of AURKA expression in MM cells inclined apoptosis through abrogation of G2/M cell cycle progression [20,21]. Taken together, inhibition of AURKA may be a convincing strategy in MM therapy. In the present research, we screened over 48,000 compounds from a natural product library of the ZINC database. We aimed to discover natural drug candidates as AURKA inhibitors in MM by the combined use of both in silico and in vitro approaches. Furthermore, the elucidation of modes of action of identified candidate molecules in MM cells was also planned. Within the context of the present research, our findings may stimulate the derivatization of lead compounds for the development of prospective (semi)synthetic compounds or combination regimens in MM targeting AURKA. Cell lines MM cell lines comprising KMS-11, MOLP-8, NCI-H929, L-363, RPMI-8226, OPM-2 were originated from the L e i b n i z I n s t i t u t e D S M Z -G e r m a n C o l l e c t i o n o f Microorganisms and Cell Cultures. The cells were provided by Dr. Ellen Leich (University of Würzburg) and Dr. Manik, Chatterjee (University of Würzburg) [22]. Parts of this research were carried out using the supercomputer Mogon and advisory services offered by Johannes Gutenberg Universiy Mainz (hpc.uni-mainz.de), which is a member of the Alliance for High Performance Computing in Rhineland-Palatinate (www.ahrp.info) and the Gauss Alliance e.V. Molecular docking with AutoDock 1.5.6 If virtual screening results with a binding affinity of ≤ −10.2 kcal/mol were achieved, the corresponding compounds were further taken to the molecular docking with AutoDock 4.2.6 (The Scripps Research Institute, CA, USA) [26]. AutoDockTools 1.5.6 was performed to prepare the files for the molecular docking. Protein and ligand files were converted to PDBQT (Protein Data Bank Partial Charge and Atom Type) files. The grid boxes were created as mentioned above in the virtual screening section around the amino acids of AURKA involved in ligand binding according to the literature [25]. Lamarckian Algorithm, calculating 250 runs and 25,000,000 energy evaluations for each cycle, was used in docking analysis, as previously decribed [27]. Docking log (dlg) files, containing the lowest binding, the mean binding affinity and the predicted inhibition constant (pKi), provided essential knowledge about docking results. Interacting amino acids (via hydrogen bonding and hydrophobic interactions) with the AURKA were identified using AutoDockTools. Visual Molecular Dynamics 1.9.3 (VMD) was used for docking visualizations (http://www.ks.uiuc.edu/Research/ vmd/) [28]. As in the virtual screening, parts of this work were conducted using the supercomputer Mogon and advisory services offered by Johannes Gutenberg University Mainz (hpc.uni-mainz.de). Based on the virtual screening and molecular docking results, 9 identified candidate compounds were provided by MicroCombiChem GmbH (Wiesbaden, Germany) for in vitro verification of the in silico results. Cytotoxicity assay The cytotoxicity of the tested compounds has been studied by use of resazurin reduction assay [29,30]. The assay depends on the reduction of resazurin to resarufin by viable cells. Nonviable cells do not exhibit a blue staining due to losing their metabolic capacity. Briefly, 1 × 10 4 cells in a total volume of 100 μL were seeded in 96-well cell culture plate. The cells were incubated with various concentrations of the regarding compound to get a total volume of 200 μL/well for 72 h. Then, 0.01% of resazurin (Sigma-Aldrich, Germany) diluted in double-distilled water (ddH 2 O) was added (20 μL/well) and incubated for another 4 h. Infinite M2000 ProTM plate reader (Tecan, Germany) was used to measure the fluorescence using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each assay was independently performed thrice with six parallel replicates each. Dose response curves of each cell were formed using GraphPad Prism® v6.0 software (GraphPad Software Inc., San Diego, CA, USA). The 50% inhibition concentrations (IC 50 ) were calculated by nonlinear regression using Microsoft Excel. Microscale thermophoresis (MST) Microscale thermophoresis (MST) was performed for assessment of the interaction between compound (5) and AURKA (Sigma-Aldrich, Germany). The method is performed as previously described [31,32]. The AURKA protein was labeled using Monolith Protein Labeling Kit RED-NHS 2nd Generation (MO-L011, NanoTemper Technologies GmbH, Munich, Germany) according to manufacturer's instructions. The AURKA protein concentration used was 400 nm, it was titrated against different concentrations of compound (5). Analysis buffer used includes; 50 mM Tris buffer pH 7.0, 150 mM NaCl, 10 mM MgCl 2 and 0.05% Tween 20. Samples of interaction were filled into Monolith NT.115 standard capillaries (MO-K022, NanoTemper Technologies GmbH, Munich, Germany). Monolith NT.115 instrument (NanoTemper Technologies) was used for fluorescent signal measurement. Test was performed using 60% LED power and 40 MST power. For analysis, we used MO.Affinity analysis software (Nano Temper Technologies) to generate of fitting curve of interaction and calculation of dissociation constant (Kd). Analysis of cell cycle distribution by flow cytometry The MOLP-8 cells (1 × 10 6 cells/well) were seeded into 6well plates and treated with ranging concentrations (0.5 × IC 50 , IC 50 , 2 × IC 50 and 4 × IC 50 ) of compound (5) for 24 h. The cells were collected, washed with phosphate buffered saline (PBS) and fixed with 96% ice-cold ethanol. Following the fixation of the cells, they were washed with PBS again, dissolved in PBS and stained with propidium iodide (PI, Sigma-Aldrich) at a final concentration of 50 μg/mL for 15 min at room temperature in the dark. The BD Accuri™ C6 Flow cytometer (Becton-Dickinson, Heidelberg, Germany) was used to perform cell cycle analyses at 488 nm excitation wavelength, and emission was measured by a 610/20 nm band pass filter. A total number of 1 × 10 4 cells were counted for each experiment. All experiments were performed at least thrice [33]. Imaging of structure and dynamics of the microtubule cytoskeleton by fluorescence misroscopy U2OS-GFP -α-tubulin cells (5 × 10 5 /well) were seeded into 6well plates, each including a sterile ibi Treat μ-slide (ibidi, Germany). The cells were enabled to attach overnight, treated with 10 or 25 μM of compound (5) or DMSO (solvent control) and incubated at 37°C for 24 h. Then, the cells were rinsed with PBS and fixed by 4% p-formaldehyde at room temperatuere for 30 min. Subsequently, the cells were washed with PBS and stained for 5 min with 1 μM of 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Darmstadt, Germany), followed by washing with PBS again and mounting. Fluorescence imaging was performed by using 470 nm excitation and 525 nm emission for GFP and 447 nm emission for DAPI of EVOS digital inverted microscope (Life Technologies). Each experiment was done at least thrice and representative images were selected [34]. Protein extraction MOLP-8 cells (3 × 10 6 ) were seeded in six-well plates and treated with various concentrations (0.01, 0.5, 1, 5, 10 μM) of compound (5) for 24 h. The cells were then washed with PBS and transferred into 1.5 ml Eppendorf tubes. M-PER® Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Germany) with protease inhibitor (1:100) was used for protein purification by shaking for 30 min at 4°C. Then, cell lysates were centrifuged at 14,000×g for 15 min at 4°C and the supernatants were shifted to the clean tubes. Total protein concentrations were calculated using a NanoDrop 1000 spectrophotometer (Thermo Scientific) [35]. SDS PAGE and Western blot analysis Thirty mg/mL of the protein fraction was taken and 5% βmercaptoethanol including SDS-loading dye was added following by heating at 95°C for 10 min. Subsequent to the denaturation process, the proteins were loaded onto 10% SDS-polyacrylamide gels. Then, a western blotting apparatus was used to transfer the proteins on a PVDF membrane (Roti® PVDF, pore size 0.45 μm, Carl Roth GmbH, Karlsruhe, Germany) Then, the membrane was blocked with blocking buffer (5% BSA in Tris-buffered saline Tween 20 (TBST)) for 1 h at room temperature and then incubated with primary antibody [Aurora A/AIK antibody rabbit polyclonal antibody (1:1000, Cell Signaling Technology, Frankfurt, Germany)] overnight at 4°C. Then, HRP-linked anti-rabbit IgG (1:2000, Cell Signaling) was incubated with the membranes for 1 h. Luminata Classico HRP Western Blot substrate (Merck Millipore, Schwalbach, Germany) was used for the detection step and membranes were visualized by using Alpha Innotech FluorChem Q system (Biozym, Oldendorf, Germany) [32,36]. Virtual screening and molecular docking Using more than 48,000 natural compounds from the ZINC database, we identified the top 105 compounds with a binding affinity equal to or less than −10.2 kcal/mol by virtual screening. Subsequently, defined docking calculations of these 105 compounds were performed by covering the residues involved in hydrogen bonds and hydrophobic interactions of AURKA and its selective inhibitor SAR156497 in the literature [25]. Based on the molecular docking results, the top 9 compounds unraveled higher binding affinities than that of alisertib (MLN8237, Milennium Pharmaceuticals, Inc., Cambridge, MA), which is a highly selective AURKA small-molecule inhibitor developed for the treatment of malignancies [37,38] (Table 1). All 9 compounds bound to the same pharmacophore as SAR156497 and alisertib in defined docking approach and their binding affinities were ranged from −14.41 to −11.53 kcal/mol. Remarkably, all 9 compounds bound with higher affinity to AURKA than alisertib ( Table 1). Four of them displayed only hydrophobic interactions, whereas the other five compounds formed both hydrogen bonds and hydrophobic interactions. In the context of in silico assessments, we elicited these 9 compounds for in vitro verification of computational results. Cytotoxicity Preliminary screening was performed to determine cytotoxic drug candidates among the top 9 compounds by the evaluation of growth inhibition of various MM cells at a fixed concentration of 10 μM of each test compound. Although the cytotoxic responses of ZINC000252515584 (compound 5, a secondary metabolite termed "deschloro-chlorothricin" (DCCT) isolated from Streptomyces antibioticus. [23,39]) and ZINC000077262838 were remarkable on numerous MM cells at a fixed concentration of 10 μM (Table 2), we focused only on ZINC000252515584 (deschloro-chlorothricin, DCCT) ( Fig. 1) within the context of our research and further took it to the comprehensive cytotoxicity studies at ranging concentrations on more MM cells, because it was the only compound interacting with AURKA based on the microscale thermophoresis. DCCT was tested in numerous MM cells and dose-response curves were created (Fig. 2). It demonstrated 50% cell viability inhibition in multiple myeloma cells KMS-11, L-363, MOLP-8, NCI-H929, OPM-2 and RPMI-8226 at concentrations of 0.12 ± 0.01 μM, 0.05 ± < 0.01 μM, 0.01 ± 0.01 μM, 0.08 ± 0.01 μM, 0.07 ± < 0.01 μM, and 0.06 ± < 0.01 μM, respectively. Generally, these results indicated that MM cells were quite sensitive to DCCT. Particularly, MOLP-8 cells were the most sensitive cells among the other studied MM cells and were futher investigated to assess the mode of action of DCCT in these cells. Microscale thermophoresis The in vitro binding of DCCT to AURKA was verified with microscale thermophoresis. Different concentrations of DCCT ranging from 0.02 to 400 μM (1:1 dilution) were titrated against constant concentration of AURKA (400 nm). MST result showed a concentration-dependent fluorescence of AURKA against DCCT (Fig. 3). This suggest strong binding of the ligand DCCT to AURKA with a K d value of 8. Influence of deschloro-chlorothricin (DCCT) on microtubules We treated U2OS cells, which express an α-tubulin-GFP fusion protein with various concentrations of DCCT to evaluate its influence on the cellular microtubule network. Figure 5 illustrates the dose-dependent effect of DCCT on the microtubule network. In non-treated cells, the microtubules continuously distributed throughout the cytoplasm and constituted an intracellular network apart from the nuclear region. The mass of the microtubule network in U2OS cells reduced in particular at the cell periphery if treated with DCCT. Their brightness and thickness decreased dose-dependently compared to non-treated cells, all pointing out the inhibitory effect of DCCT on microtubule formation (Fig. 5). Western blot analysis The impact of DCCT on the AURKA expression was evaluated by Western blot analysis. DCCT remarkably downregulated AURKA expression in a dose-dependent manner in DCCT-treated cells (Fig. 6). Fig. 1 a The chemical structure of the compound (5) Virtual screening and molecular docking In silico virtual screening and molecular docking were performed to predict probable ligands interacting with AURKA, which may provide potential drug candidates as promising AURKA inhibitors in cancer therapy. Nature as a unique resource and represents a great number of substances with potent bioactivities and presumably fewer side effects against cancer. Thus, screening a great number of natural compounds in the ZINC database targeting AURKs may result in the discovery of natural drug candidates and the construction of a natural library, which may provide lead structures or possibilities for the development of prospective (semi)synthetic drugs or combination interventions for the treatment of MM. Furthermore, computational approaches may enable the identification of repurposable drug candidates by saving time and reducing the costs of medications until reaching the market, all highlighting the prominence of in silico approaches [40][41][42][43]. DCCT was among the top 9 compounds with the highest binding affinity to AURKA based on in silico analyses, which was subsequently shown to be the only compound interacting with AURKA by microscale thermophoresis. Molecular docking analysis demonstrated its interactions with AURKA (Fig. 1b) (Fig. 1c). Besides, an interaction of DCCT with vital amino acids in the binding pocket, enabled the understanding of the binding mode of the compounds, which may provide a model for prospective drug design for AURKA inhibitors. These amino acids are involved in the crystalized structure of the complex of AURKA and its inhibitor SAR156497 showing the interaction and include Val 147, Lys 162, Leu 178, Leu 194, Glu 211, Tyr 212, Ala 213, Leu 263, Ala 273, Asp 274 [25]. In addition to having a higher binding affinity to AURKA than alisertib, all findings pointed out the potential of DCCT as an novel AURKA inhibitor candidate. Cytotoxicity A great number of natural compounds were reported to be active in vitro at concentrations of about 1 to 50 μM, and 15 μM was assigned as the target concentration generally used in most of the dose calculations [44]. On the other hand, the mean IC 50 of 9 common chemotherapeutics was reported to be as low as 0.48 μM based on the simple analysis of data gained from the National Cancer Institute [44]. In the present study, we reduced the cut-off point to 10 μM. The top 9 compounds were tested at this concentration and only the compounds with an inhibitory effect ≥50% were regarded as highly cytotoxic against multiple myeloma cells. Two out of 9 c o m p o u n d s ( Z I N C 0 0 0 2 5 2 5 1 5 5 8 4 ( D C C T ) a n d ZINC000077262838) were identified as promising natural substances, among which only DCCT was further examined for the evaluation of detailed cytotoxicity on various MM cells and underlying molecular mechanisms due to interaction with AURKA. Fig. 3 Binding of deschlorochlorothricin to aurora kinase A as measured by MST. Experiment was performed with 60% LED power and 40% MST power. The curve shows the difference in the bound and unbound state of the aurora kinase A in presence of deschloro-chlorothricin. A fit was performed according to the law of mass action DCCT displayed remarkable cytotoxicity on MM cell lines with extremely low IC 50 values, which were even less than those of an AURKA inhibitor and clinical drug alisertib. To exemplify, a previous study indicated that alisertib inhibited cell viability on OMP-2 and RPMI-8226 cells with IC 50 values of 4.37 and 10.32 μM, respectively [3], which were even higher than those of DCCT in our study, emphasizing the potential of the compound as promising natural drug candidate in MM interventions. Microscale thermophoresis The outcomes of in silico virtual screening and molecular docking studies were validated by MST binding assay with purified in vitro. As assumed, the MST signals differed between the bound and unbound proteins, suggesting the interaction of the DCCT with AURKA. Cell cycle distribution The expression and activity of all aurora kinases increase in mitosis. AURKA acts as a mitotic centrosomal kinase involved in chromosome maturation and regulation of G2/M phase transition [45]. During the proliferation of normal cells, AURKA is activated by phosphorylation during G2 to M phase transition in the cell cycle [18]. If the AURKA activity in tumor cells was disrupted, the accumulation of cells in G2/ M phase and delayed mitotic entry were observed [46]. In another study, the inhibition of AURKA resulted in abrogation of G2/M cell cycle progression in MM cells [21]. Likewise, deschloro-chlorothricin induced G2/M arrest, as previously indicated for AURKA inhibitors in many reports [47][48][49][50]. Influence of deschloro-chlorothricin (DCCT) on microtubules The U2OS cells expressing α-tubulin-GFP were treated with DCCT to reveal the impact on the microtubule cytoskeleton by using digital inverted microscopy. Indeed, an unfavorable effect of DCCT was observed on microtubules, since the number of filaments as well as the intensity of tubulin staining reduced in dose-dependent manner. The aurora kinases participate in cell cycle progression, mostly during the G2/M phases. AURKA is located on the poles of the mitotic spindle as well as the centrosomes and moves to centromeres during mitosis [13,51,52]. During mitosis, maintaining balanced chromosome segregation holds vital importance for the construction of a bipolar mitotic spindle. Because inadequacy in the spindle bipolar architecture may result in abnormal chromosome segregation and genetic instability in cancer cells. Microtubule-created forces have essential roles in the assembly of the mitotic bipolar spindle [53,54]. These forces are associated with the dynamic microtubule properties and produced by the directional motion of motor proteins along microtubules [55]. Within the context of these findings, aurora A organizes centrosome assembly and stability, as well as nucleation and polymerization of centrosomal microtubules assigning a role in microtubule organization [56][57][58][59] and inhibitors may disrupt microtubule formation leading the repression of abnormal proliferation of cancerous cells. Therefore, DCCT inhibiting AURKA activity and thus, leading abnormal microtubule formation may be a promising drug candidate targeting AURKA in cancer therapy. Western blot analysis AURKA enters the centrosome early in G2 and has been involved in the activation of CDK1/cyclin B on the centrosome [60]. Activated AURKA successively phosphorylating various centrosomal proteins, functions in centrosome maturation and mitotic spindle formation. The AURKA gene is usually overexpressed in cancer including MM [61,62] and 6 Western blot analysis of aurora kinase A in deschlorochlorothricin-treated MOLP-8 cells. The cells were incubated with 0.01, 0.5, 1, 5, 10 μM concentrations of deschlorochlorothricin for 24 h. Then, total protein was extracted and Western blotting was performed. The chart displays the change in the protein expression after normalization to GAPDH as mean ± SD for three independent experiments. Asterisks (***) indicated statistically significant downregulation by Student's t test (p < 0.001) compared to DMSOnontreated (control) cells its amplification results in chromosome segregation anomalies related to malignant transformation both in vitro and in vivo [60,63]. Moreover, the upregulation of AURKA inclined chemoresistance in breast cancer and ovarian cancer cells [16,64]. In other studies, the suppression of AURKA expression enhanced paclitaxel-induced apoptosis in numerous cancer cells including kidney and breast cancer cell lines [65,66]. Taken together, significant downregulation of AURKA by DCCT may provide additional strategies to combat MM, because it may either prohibit tumorigenesis through inhibiting chromosome segregation anomalies or improve the sensitivity of cancer cells to chemotherapeutics in the clinic. Further studies are required to emphasize the importance of the downregulation of aurora A in the chemosensitization of MM. Conclusion In the present study, compound (5) (DCCT), a natural compound from the ZINC database, was identified as AURKA inhibitor based on in silico virtual screening and molecular docking studies, and the activity was experimentally validated in MM cells. DCCT was tested for the first time against several MM cells with a focus on determining its mechanism of action. Integration of computational and in vitro approaches enabled the experimental validation of in silico outcomes. DCCT remarkably inhibited the growth of MM cells, among which MOLP-8 cells were the most sensitive ones to the DCCT. These effects were related to the cell cycle arrest of the G2/M phase, the suppression of microtubule formation and the inhibition of AURKA activity and expression. The research not only renders a better understanding of how DCCT acts as antitumor agent on MM cells, but also provides information about the possible role of this drug in the treatment of MM. We introduced DCCT as a promising anticancer drug candidate worth further development. Funding Open Access funding enabled and organized by Projekt DEAL. We are grateful for a postdoc stipend given to N.Ö. by the Germany Academic Exchange Service (DAAD) (Funding program/-ID: Research Grants-Short-Term Grants, 2019 (57440917)) for a research stay at Johannes Gutenberg University, Mainz, Germany. The authors gratefully acknowledge the computing time granted on the supercomputer Mogon at Johannes Gutenberg University Mainz (hpc.uni-mainz.de). Compliance with ethical standards Conflict of interest EF and AK are employees of MicroCombiChem, Wiesbaden, Germany. All other 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. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. /All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. /All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent For this type of study, formal consent is not required. Informed consent was obtained from all individual participants included in the study. 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To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.	2020-09-26T13:37:51.692Z	2020-09-25T00:00:00.000	{ "year": 2020, "sha1": "a678c02d9e0ef24aa97b2c1efde1ab333931c02f", "oa_license": "CCBY", "oa_url": "https://europepmc.org/articles/pmc8551148?pdf=render", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "09d8ec453159191f2815dbde38df0310c056be61", "s2fieldsofstudy": [ "Medicine", "Chemistry" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
119086137	pes2o/s2orc	v3-fos-license	Towards a determination of the spectrum of QCD using a space-time lattice Progress by the Lattice Hadron Physics Collaboration in determining the baryon and meson resonance spectrum of QCD using Monte Carlo methods with space-time lattices is described. The extraction of excited-state energies necessitates the evaluation of correlation matrices of sets of operators, and the importance of extended three-quark operators to capture both the radial and orbital structures of baryons is emphasized. The use of both quark-field smearing and link-field smearing in the operators is essential for reducing the couplings of the operators to the high-frequency modes and for reducing statistical noise in the correlators. The extraction of nine energy levels in a given symmetry channel is demonstrated, and identifying the continuum spin quantum numbers of the levels is discussed. Introduction A charge from the late Nathan Isgur to use Monte Carlo methods to extract the spectrum of hadron resonances and hadronic properties resulted in the formation of the Lattice Hadron Physics Collaboration (LHPC) in the year 2000. As part of a national collaboration of lattice QCD theorists, the LHPC acquired funding from several sources, including the DOE's Scientific Discovery through Advanced Computing initiative, to build large computing clusters at JLab, Fermilab, and Brookhaven, as well as to develop the software to carry out the needed large-scale computations. The LHPC has several broad goals: to compute the spectrum of QCD from first principles, to investigate hadron structure by computing form factors, structure functions, and other matrix elements, and to study hadron-hadron interactions. This talk focuses solely on our efforts to determine the spectrum of QCD. Extracting the spectrum of resonances in QCD is a big challenge. The determination of excited-state energies requires the use of correlation matrices of sets of operators. The masses and widths of unstable hadrons (resonances) must be deduced from the energies of single-particle and multihadron stationary states in a finite-sized box. This necessitates the use of multi-hadron operators in the correlation matrices, and the computations must be performed in full QCD at realistically-light quark masses. For these reasons, the computation of the QCD spectrum is indeed a long-term project. This talk is a brief status report of our efforts. I first describe in Sec. 2 how excited-state energies are extracted in our Monte Carlo calculations, and discuss issues related to unstable resonances. Our construction of baryon operators is then outlined in Sec. 3 (see Ref. 1 for further details), and in Sec. 4, the importance of using smeared quark and gluon fields is demonstrated. (see also Ref. 2). The first-time extraction of nine nucleon energy levels is also presented in this section. Concluding remarks are made and future work is outlined in the concluding Sec. 5. Excited states and resonances In the path integral formulation of quantum field theory with imaginary time, stationary-state energies are extracted from the asymptotic decay rates of temporal correlations of the field operators. If Φ(t) is a Heisenbergpicture operator which annihilates the hadron of interest at time t, then its evolution Φ(t) = e Ht Φ(0)e −Ht , where H is the Hamiltonian, can be used to show that in a finite-sized box, where {\|n } is the complete set of discrete eigenvectors of H. We assume the existence of a transfer matrix, and temporal boundary conditions have been ignored for illustrative purposes. One can then extract A 1 and E 1 −E 0 as t → ∞, assuming 0\|Φ(0)\|0 = 0 and 0\|Φ(0)\|1 = 0. A convenient visual tool for demonstrating energy-level extraction is the so-called "effective mass" defined by m eff (t) = ln[C(t)/C(t + a t )], where t is time and a t is the temporal lattice spacing. The effective mass tends to the actual mass (or energy) of the ground state as t becomes large, signalled by a plateau in the effective mass. At smaller times before this plateau is observed, the effective mass varies due to contributions from other states in the spectrum. The correlation C(t) is estimated with some statistical uncertainty since the Monte Carlo method is used, and usually, the ratio of the noise to the signal increases with temporal separation t. Hence, judiciously-chosen operators having reduced couplings with contaminating higher-lying states are important in order to observe a plateau in the effective mass before noise swamps the signal. Key ingredients in constructing such operators are the use of smeared quark and gluon fields, the incorporation of spatially-extended assemblages of the fields, and the use of sets of different operators to exploit improvements from variational methods. Many of the hadron states we wish to study are unstable resonances. Our computations are done out of necessity in a box of finite volume with periodic boundary conditions. Hence, the momenta of the particles we study are quantized, so all states are discrete in our computations. Thus, we can only determine the discrete energy spectrum of stationary states in a periodic box, which are admixtures of single hadrons and multi-hadron states. Resonance masses and widths must somehow be deduced from the finite-box spectrum 5,6,7,8 . Once the masses of the stable single particle Table 1. Continuum limit spin identification: the number n J Λ of times that the Λ irrep. of the octahedral point group O h occurs in the (reducible) subduction of the J irrep. of SU (2). The numbers for G 1u , G 2u , Hu are the same as for G 1g , G 2g , Hg, respectively. states have been determined, the placement and pattern of their scattering states are known approximately, and the dependences of their energies on the volume are roughly known. Resonances show up as extra states with little volume dependence. Our initial goal is simply to ferret out these resonances, not to pin down their properties to high precision. Operator construction Our approach to constructing hadron operators is to directly combine the physical characteristics of hadrons with the symmetries of the lattice regularization of QCD used in simulations. For baryons at rest, our operators are formed using group-theoretical projections onto the irreducible representations (irreps) of the O h symmetry group of a three-dimensional cubic lattice. There are four two-dimensional irreps G 1g , G 1u , G 2g , G 2u and two four-dimensional representations H g and H u . The continuum-limit spins J of our states must be deduced by examining degeneracy patterns across the different O h irreps (see Table 1). For example, a J P = 1 2 + state will show up in the G 1g channel without degenerate partners in the other channels, and a J P = 3 2 + state will show up in the H g channel without degenerate partners in the other channels. Four of the six polarizations of a J P = 5 2 + state show up as a level in the H g channel, and the other two will occur as a degenerate partner in the G 2g channel, whereas three degenerate levels, one in each of the three G 1g , G 2g , H g channels, may indicate a single J P = 7 2 + state (or the accidental degeneracy of a spin-1 2 and a 5 2 state). Baryons are expected to be rather large objects, so local operators will not suffice. Our approach to constructing spatially-extended operators is to use covariant displacements of the quark fields along the links of the lattice. Displacements in different directions are used to build up the appropriate orbital structure, and displacements of different lengths can build up the needed radial structure. All our three-quark baryon operators are superpositions of gauge-invariant, translationally-invariant terms of the form where A, B, C indicate quark flavor, a, b, c are color indices, α, β, γ are Dirac spin indices,ψ indicates a smeared quark field, andD (p) j denotes the smeared p-link covariant displacement operator in the j-th direction. The smearing of the quark and gauge field will be discussed later. There are six different spatial orientations that we use, shown in Fig 1. The singlydisplaced operators are meant to mock up a diquark-quark coupling, and the doubly-displaced and triply-displaced operators are chosen since they favor the ∆-flux and Y -flux configurations, respectively. Next, the Φ ABC αβγ,ijk are combined into elemental operators B F a (t) having the appropriate flavor structure characterized by isospin, strangeness, etc. We work in the m u = m d (equal u and d quark masses) approximation, and thus, require that the elemental operators have definite isospin, that is, they satisfy appropriate commutation relations with the isospin operators τ 3 , τ + , τ − . Since we plan to compute full correlation matrices, we need not be concerned with forming operators according to an SU (3) flavor symmetry. Maple code which manipulates Grassmann fields was used to identify maximal sets of linearly independent elemental operators. The operators used are shown in Table 2, and the numbers of such independent operators are listed in this table as well. The final step in our operator construction is to apply group-theoretical projections to obtain operators which transform irreducibly under all lattice rotation and reflection symmetries: where Λ refers to an is a Λ representation matrix corresponding to group element R, and U R is the quantum operator which implements the symmetry operations. The projections in Eq. (5) are carried out using computer software written in the Maple 9 symbolic manipulation language. Field smearing and operator pruning For single-site (local) hadron operators, it is well known that the use of spatially-smeared quark fields is crucial. For extended baryon operators, one expects quark-field smearing to be equally important, but the relevance and interplay of link-field smearing is less well known. Thus, we decided that a systematic study of both quark-field and link-variable smearing was warranted. The link variables were smeared U →Ũ using the analytic stout link method of Ref. 10. There are two tunable parameters, the number of iterations n ρ and the staple weight ρ. For the quark-field, we employed gauge-covariant Gaussian smearing where∆ denotes the smeared three-dimensional gauge-covariant Laplacian. The two parameters to tune in this smearing procedure are the smearing radius σ s and the integer number of iterations n σ . For our tests of the efficacy of quark-field and gauge-link smearing, correlators were computed for three particular nucleon operators: a singlesite operator O SS in the G 1g irreducible representation of the cubic point group, a singly-displaced operator O SD with a particular choice of each Dirac index, and a triply-displaced-T operator O T DT with a specific choice of each Dirac index. Our findings are summarized in Fig. 2. The top row shows that applying only quark-field smearing to the three selected nucleon operators significantly reduces couplings to higher-lying states, but the dis- placed operators remain excessively noisy. The second row illustrates that including only link-field smearing substantially reduces the noise, but does not appreciably alter the effective masses themselves. The bottom row shows dramatic improvement from reduced couplings to excited states and dramatically reduced noise when both quark-field and link-field smearing is applied, especially for the extended operators. The "best" quark-field smearing parameters n σ and σ were determined by requiring that the effective mass for the three chosen operators reach a plateau as close to the source as possible. The gauge-link smearing parameters were tuned so as to minimize the noise in the effective masses. One interesting point we also learned was that the preferred link-smearing parameters determined from the static quark-antiquark potential produced the smallest error in the extended baryon operators. The computation of correlation matrices using hundreds of operators is not feasible, so it is necessary to "prune" out unnecessary operators. The first step in this pruning is to examine the effective masses of the diagonal elements of the correlation matrices to identify and eliminate noisy operators. Keeping only operators with small statistical uncertainties yields a set of about forty to fifty operators in each symmetry channel. We then computed the correlation matrix of this reduced set of operators, examining whether further reductions to the operator sets could be made without increased contamination in the principal effective masses. These computations are still ongoing, but preliminary results are shown in Fig. 3. This figure shows that it is possible to extract at least nine levels in a given symmetry channel, a feat which has never before been accomplished. Demonstrating that this number of energy levels can be reliably extracted is an important milestone in our long-term project. Conclusion We have outlined a program to study the resonance spectrum in lattice QCD. The use of the variational method and the need to isolate several energy levels in each channel require a sufficiently broad basis of operators. Having developed suitable group-theory methods to project operators onto the irreducible representations of the cubic group, and having examined the efficacy of both quark-and gauge-link-smearing, we are now identifying a more limited set of operators that we will employ in a large-scale study of the hadron spectrum. Our methods are applicable not only to baryons, but also to mesons, tetra-quark and pentaquark systems, and to states with excited glue. Only by performing such a program can we hope to identify the states of QCD, and in particular their spins and parities, in the continuum limit. Ultimately, when quark loops are included at realistically light quark masses, multi-hadron (baryon-meson) operators must be included in our correlation matrices, and finite-volume techniques will need to be employed to ferret out the baryon resonances from uninteresting scattering states. We are currently exploring different ways of building such operators.	2017-09-27T09:59:42.954Z	2006-01-23T00:00:00.000	{ "year": 2006, "sha1": "1c8fb7a3777d3b4f506c40c8254e703a27fb5be6", "oa_license": null, "oa_url": "http://arxiv.org/pdf/hep-lat/0601029", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "1c8fb7a3777d3b4f506c40c8254e703a27fb5be6", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
245592827	pes2o/s2orc	v3-fos-license	USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity Nrf2 is an antioxidant transcriptional activator in many types of cells, and its dysfunction plays key roles in a variety of human disorders, including Parkinson's disease (PD). PD is characterized by the selective loss of dopaminergic neurons in PD-affected brain regions. Dopamine treatment of neuronal cells stimulates the production of reactive oxygen species (ROS) and increases ROS-dependent neuronal apoptosis. In this study, we found that the ubiquitin-specific protease 10 (USP10) protein reduces dopamine-induced ROS production of neuronal cells and ROS-dependent apoptosis by stimulating the antioxidant activity of Nrf2. USP10 interacted with the Nrf2 activator p62, increased the phosphorylation of p62, increased the interaction of p62 with the Nrf2 inhibitor Keap1, and stimulated Nrf2 antioxidant transcriptional activity. In addition, USP10 augmented dopamine-induced Nrf2 translation. Taken together, these results indicate that USP10 is a key regulator of Nrf2 antioxidant activity in neuronal cells and suggest that USP10 activators are promising therapeutic agents for oxidative stress–related diseases, including PD. Reactive oxygen species (ROS) play a physiological and pathological role in various types of cells and tissues (1). For instance, intracellular ROS function as signal transduction molecules in various biological processes. However, their increased production has been implicated in many pathological conditions, such as neurodegenerative diseases, cardiovascular diseases, and cancer (2). Therefore, the amount of intracellular ROS is strictly regulated by several antioxidant systems. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an antioxidant factor that stimulates the transcription of a number of antioxidant genes, such as NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase-1 (HO-1), under basal and various oxidative stress conditions (3,4). Nrf2 activity is negatively and positively regulated by Kelch-like ECHassociated protein (Keap1) and p62 (SQSTM1), respectively. Prior to oxidative stress, the level of Nrf2 protein is low, since Nrf2 is ubiquitinated and continuously degraded by the proteasome (5). Nrf2 ubiquitination is regulated by interaction with Keap1, which acts as an Nrf2 adaptor to the ubiquitin ligase complex containing Cullin-3. However, during oxidative stress, three mechanisms enhance Nrf2 activity. First, oxidants induce cysteine modification of Keap1, which decreases Keap1's affinity for Nrf2 (5). Second, an oxidant induces the phosphorylation of serine 349 of p62 (pp62-S349), enhancing the interaction of p62 with Keap1 and releasing Nrf2 from Keap1 (6). Third, oxidants enhance the translation of Nrf2, which increases the amount of Nrf2 protein (7)(8)(9). Thus, Nrf2 activity is tightly regulated under both basal and oxidative stress conditions. Dysfunction of the Nrf2/Keap1/p62 system has been associated with many neurodegenerative disorders, including Parkinson's disease (PD) (10). PD is characterized by the degeneration and death of dopaminergic neurons, which have cell bodies in the substantia nigra and axons projecting to the caudate nucleus and putamen (striatum) (11). While dopamine is a key neurotransmitter secreted by neurons that signal other neurons, accumulating evidence suggests that dopamineinduced ROS production plays a key role in the selective cell death of dopaminergic neurons in PD (12). For instance, dopamine treatment of neuronal cells stimulates ROS production and induces ROS-dependent apoptosis (13). Ubiquitin-specific protease 10 (USP10) is a deubiquitinase ubiquitously expressed in many cell types, including neurons, and it augments antioxidant activity in cultured cells (14,15). For instance, USP10 depletion in cultured nonneuronal cells treated with arsenite has been shown to increase ROS production and enhance ROS-induced apoptosis (16). In the present study, we found that USP10 inhibits the dopamine-induced ROS production and ROS-dependent apoptosis in neuronal cells by activating Nrf2-mediated antioxidant gene expression. USP10 depletion increases dopamine-induced apoptosis of neuronal cells Dopamine stimulation of neurons induces ROS production, which has been shown to play a key role in cell death of dopaminergic neurons in PD (17). USP10 has been shown to inhibit ROS production and ROS-dependent apoptosis in nonneuronal cells (16). We therefore investigated whether or not USP10 plays a role in dopamine-induced neuronal cell death. SH-SY5Y is derived from neuroblastoma and has a phenotype similar to that of dopaminergic neurons (18). We reduced the expression of USP10 protein of SH-SY5Y using small interfering RNA (siRNA) targeting USP10 (siUSP10, USP10-KD). Western blotting detected a reduced expression of USP10 protein in USP10-KD cells (Fig. 1A). These USP10-KD cells were treated with 0.1 to 0.4 mM dopamine for 12 h, and then their cell viability was analyzed by measuring the cellular dehydrogenase activity. Dopamine treatment A, SH-SY5Y cells were transfected with USP10-siRNA (USP10-1, 2, or 3) or control (NT) using Lipofectamine RNAiMAX. Whole cell lysates prepared from transfected cells were characterized by Western blotting with anti-USP10 and anti-β-actin antibodies. B, SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. The cells were then treated with 0.1 to 0.4 mM dopamine or DMSO for 12 h. The cells were treated with CCK-8 solution for 1 h. Culture supernatant was prepared from the transfected cells, and the absorbance (485 nm) of the culture supernatant was measured with an absorption meter (TriStar LB 941). The ratio of absorbance of cells to that of control siRNA (NT) treated with DMSO was presented as the mean and standard deviation (SD) of three samples. The significance of the differences was assessed by a one-way ANOVA, followed by Tukey's multiple comparison test. p < 0.01; p < 0.001; **p < 0.0001. C and D, USP10-KD (siUSP10-1 or 2) and control (siNT) cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h, and cells were then stained with anticleavage caspase-3 (red) and Hoechst 33258 (blue). The staining was evaluated with a fluorescence microscope. The ratio of cells with cleaved caspase-3 relative to total cells (measured by number of nuclei) was presented as the mean and SD of five samples in (D). The significance of the difference in Figure 1D was assessed by Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. p < 0.05; p < 0.01; p < 0.001. Scale bar is 10 μm. ANOVA, analysis of variance; NS, Not significant; USP10, ubiquitin-specific protease 10. dose-dependently reduced the viability of USP10-KD cells, and the reduction in the number of cells was greater than that in USP10-WT cells (Fig. 1B). Cleaved caspase-3 is a marker of apoptosis. Anticleaved caspase-3 staining detected apoptosis of USP10-KD cells treated with dopamine for 4 and 8 h, and the degree was greater than that in the control cells (siNT) (Fig. 1, C and D). These results showed that USP10 suppresses dopamineinduced apoptosis of SH-SY5Y cells. Depletion of USP10 increases the production of ROS Dopamine-induced ROS production in neurons is a key factor in dopamine-induced apoptosis (2,19). Therefore, we next investigated whether or not ROS play a role in dopamineinduced cell death of USP10-KD cells. ROS measurement using the ROS detector fluorescent probe CM-H2DCFDA showed that USP10-KD enhanced ROS production in SH-SY5Y cells (Fig. 2, A and B). Furthermore, the USP10-KDenhanced cell death was attenuated by N-acetylcysteine (NAC), a precursor of the ROS scavenger glutathione (Fig. 2C). These results indicated that USP10-KD increases dopamineinduced cell death by enhancing the ROS production in SH-SY5Y cells. Hydrogen peroxide (H 2 O 2 ) is an endogenous ROS that is upregulated under many oxidative stress conditions. We investigated whether or not USP10 regulates H 2 O 2 -induced cytotoxicity in SH-SY5Y cells (Fig. 2D). H 2 O 2 treatment for 12 h induced a dose-dependent decrease in the viability of SH-SY5Y cells (siNT), which was further enhanced by USP10-KD (siUSP10). These results showed that USP10 regulates cell death in SH-SY5Y cells under two distinct oxidative stress conditions. . USP10-KD augments dopamine-induced ROS production and ROS-dependent cell death. A and B, SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 5 μM CM-H2DCFDA (Green), an ROS-sensitive fluorescence dye, for 30 min. Transfected cells were then treated with 0.4 mM dopamine or DMSO for 6 h. The fluorescence intensity was measured. The ratio of fluorescence intensity of cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. p < 0.05; **p < 0.0001. Scale bar is 10 μm. Bright-field observations of cells were also presented. C, SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. Cells were pretreated with 500 μM N-acetyl cysteine 30 min before dopamine treatment and further treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h. Cell viability was evaluated by measuring the absorbance (485 nm) of culture medium with an absorbance meter (TriStar LB 941). The ratio of absorbance obtained from cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. p < 0.0001. D, USP10-KD (siUSP10-2) and control (siNT) cells were treated with 50 to 200 μM H 2 O 2 for 12 h. Cell viability was measured with the CCK-8 kit. The absorbance obtained from cells was normalized to that of the control (siNT) treated with DMSO, and the ratio was presented as the mean and SD from three samples. p < 0.01; **p < 0.0001. NS, not significant; USP10, ubiquitin-specific protease 10. stress and attenuates oxidative stress-induced cellular damage. In the basal state, Nrf2 is continuously ubiquitinated and degraded by the ubiquitin-proteasome system. Such Nrf2 ubiquitination is stimulated by its interaction with Keap1, an Nrf2 adaptor to the cullin-3 ubiquitin ligase complex. However, exposure to oxidants stimulates the release of Nrf2 from Keap1 mainly by two mechanisms. First, the oxidant modifies the cysteine residues of Keap1, which reduces the interaction affinity of Keap1 and Nrf2. Second, the oxidant activates the Keap1 inhibitor p62 by phosphorylating the serine residue of p62 at position 349 (pp62-S349) (6,20). pp62-S349 then interacts strongly with Keap1 and stimulates the release of Nrf2 from Keap1 (6). Based on this information, we investigated whether or not USP10 regulates the Nrf2/Keap1/p62 antioxidant system in dopamine-induced ROS-dependent apoptosis in SH-SY5Y cells (Fig. 3). Dopamine treatment of USP10-WT cells (siNT) increased the levels of Nrf2 and the Nrf2-inducible gene HO-1, Whole cell lysates prepared from these cells were characterized by Western blotting using the indicated antibodies. D, SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 h. Lysates of nuclear and cytoplasmic fractions were prepared from transfected cells and characterized by Western blotting using the indicated antibodies. α-tubulin and HDAC1 were used as protein markers localized in the cytoplasm and nucleus, respectively. USP10, ubiquitin-specific protease 10. Nrf2 regulation by USP10 but the increases were reduced by USP10-KD (siUSP10) (Fig. 3A). In addition, dopamine treatment increased the amount of high-molecular-weight p62 bands (HMW-p62s) in USP10-WT cells, but the increase was attenuated by USP10-KD. Dopamine treatment of USP10-WT cells decreased the amount of Keap1 monomer and increased the amount of HMW-Keap1s. In contrast to Nrf2 and p62, USP10-KD did not decrease the amounts of Keap1 monomer or HMW-Keap1s with or without dopamine treatment, instead increasing the amount of Keap1 monomer without dopamine treatment (Fig. 3A). The molecular weights of HMW-p62s and HMW-Keap1s suggested that HMW-p62s and HMW-Keap1s were likely to be p62 multimers and Keap1 multimers, respectively. These findings suggested that the downregulation of Nrf2 and p62 and upregulation of Keap1 by USP10-KD were responsible for the increased dopamine-induced ROS production and ROS-dependent apoptosis of USP10-KD cells. It is noteworthy that unlike the dopamine-treated cells, the amount of Nrf2 in USP10-KD cells without dopamine treatment was higher than that in USP10-WT cells (Fig. 3). This result suggests that USP10 reduces the expression of Nrf2 in cells without dopamine treatment. Next, we examined how dopamine induces HMW-p62 and HMW-Keap1 in SH-SY5Y cells. Ascorbic acid is a watersoluble antioxidant. Treatment with ascorbic acid decreased the amounts of HMW-p62 and HMW-Keap1 in dopaminetreated SH-SY5Y cells (Fig. 3B). Thus, the oxidative activity of dopamine is required for the induction of HMW-p62 and HMW in dopamine-treated cells. However, in contrast to dopamine, H 2 O 2 treatment did not induce HMW-p62 and HMW-Keap1 in SH-SY5Y cells (Fig. 3C). Taken together, these results suggested that dopamine induces HMW-p62 and HMW-Keap1 in an oxidant-dependent and dopamine-specific mechanism. Oxidative stress induces the translocation of Nrf2 from the cytoplasm to the nucleus, and nuclear Nrf2 activates the transcription of antioxidant genes. Four-hour dopamine treatment of USP10-WT cells increased the amount of nuclear Nrf2, but the increase was attenuated by USP10-KD (Fig. 3D). These results showed that USP10-KD reduced the amount of nuclear Nrf2 in dopamine-stimulated cells, thereby increasing ROS production. Depletion of either Nrf2 or p62 augments dopamine toxicity on neuronal cells After dopamine treatment, USP10-KD in SH-SY5Y cells decreased the amounts of Nrf2 and p62 proteins (Fig. 3) and simultaneously enhanced dopamine-induced apoptosis ( Fig. 1). Given that Nrf2 and p62 inhibit oxidant-induced cell death of various cell types, we next examined whether or not Nrf2 and p62 regulate the dopamine toxicity of SH-SY5Y cells. Nrf2-KD significantly enhanced the dopamine-induced SH-SY5Y cell death (Fig. 4, A-C). Western blotting showed that Nrf2-KD reduced the amount of p62 in dopamine-treated cells (Fig. 4A). These results are consistent with the fact that Nrf2 stimulates p62 transcription (4). In contrast, Nrf2-KD had little effect on the amount of Keap1 and USP10 in dopamine-treated cells (Fig. 4B). Like Nrf2-KD, p62-KD enhanced cell death and the enhancement was comparable to USP10-KD (Fig. 4, D and E). p62-KD decreased the amount of Nrf2 and Nrf2-induced gene (HO-1) but increased the amount of Keap1 monomer. It should be noted that p62-KD had little effect on the amount of USP10, suggesting that USP10 is an upstream regulator of p62 in dopamine-induced apoptosis of SH-SY5Y cells. Taken together, these results support the finding that USP10 inhibits the ROS-dependent apoptosis of SH-SY5Y cells by promoting Nrf2 activity. Keap1-KD attenuates USP10-KD-augmentation of dopamine-induced cell death The data above indicated that USP10 attenuates dopamineinduced neuronal death by enhancing the antioxidant activity of Nrf2. In that case, USP10-KD-augmented dopamine-induced cell death should be reduced by inactivation of the Nrf2 inhibitor Keap1. To test this hypothesis, we reduced the Keap1 expression in USP10-KD cells (Fig. 5A). Western blotting showed that Keap1-KD reduced the amount of Keap1 with and without dopamine treatment (Fig. 5A). USP10-KD decreased the amount of Nrf2 and an Nrf2-inducible gene HO-1 in dopamine-treated cells, but the decreases were attenuated by Keap1-KD. Keap1-KD significantly reduced USP10-KDenhanced dopamine toxicity against SH-SY5Y cells (Fig. 5B). Taken together, these results indicate that USP10 suppresses dopamine-induced SH-SY5Y cell death by stimulating the antioxidant activity of Nrf2, which is inhibited by Keap1. PERK, a kinase of Nrf2, is not involved in the reduction of antioxidant activity of Nrf2 by USP10-KD. PERK is a kinase that phosphorylates Nrf2 under various stress conditions (21,22). The phosphorylation of Nrf2 by PERK enhances the antioxidant activity of Nrf2 by promoting its dissociation from Keap1 (21). We examined whether dopamine activates the kinase activity of PERK in SH-SY5Y cells by measuring the phosphorylation of PERK, a substrate of PERK (pPERK), by Western blotting (Fig. 6A). Dopamine treatment induced an upward shift in the mobility of some PERKs, and this shift was abolished by the PERK inhibitor GSK260414 (23). Thapsigargin, an activator of the kinase activity of PERK, also induced the upward migration of some PERKs in Western blotting. These results suggest that PERK with an upward mobility shift is phosphorylated PERK and that dopamine stimulates the autophosphorylation of PERK by PERK. Dopamine treatment increased the amounts of Nrf2 and the Nrf2-induced gene HO-1, and the increase in HO-1, but not in Nrf2, was partially reduced by a PERK inhibitor, suggesting that PERK enhances dopamine-induced Nrf2 activity to HO-1. Next, we examined whether USP10 regulates the phosphorylation of PERK (pPERK) (Fig. 6B). USP10-KD slightly increased the amount of pPERK in SH-SY5Y cells treated with dopamine for 4 and 8 h. These results suggest that PERK is not Nrf2 regulation by USP10 involved in the Nrf2 activation by USP10 in dopamine-treated SH-SY5Y cells. USP10-KD does not reduce the amount of Nrf2 mRNA in neuronal cells The amount of Nrf2 protein is mainly regulated by proteasome-mediated degradation. To investigate how USP10-KD decreases the Nrf2 protein level in dopaminetreated cells, USP10-KD cells were treated with either a proteasome inhibitor (MG-132) or an autophagy inhibitor (BafA1) (Fig. 7A). Treatment with MG-132 but not BafA1 significantly increased the Nrf2 level in USP10-WT cells with and without dopamine treatment, suggesting that the Nrf2 protein is degraded by proteasome with and without dopamine treatment. MG-132 significantly increased the amount Culture medium was prepared from transfected cells, and the absorbance (485 nm) of culture medium was measured by an absorbance meter (TriStar LB 941). The ratio of absorbance of the cells relative to that of the control siRNA (siNT) treated with DMSO was presented as the mean and SD from three samples. p < 0.0001. NS, not significant. D, SH-SY5Y cells were transfected with p62-siRNA (p62-1 or 3) or control (NT) using Lipofectamine RNAiMAX. Cells were then treated with 0.4 mM dopamine or DMSO for 4 h or 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. E, SH-SY5Y cells were transfected with p62-siRNA (sip62-1 or 2), USP10-siRNA (siUSP10-1 or 2) and the control (siNT) using Lipofectamine RNAiMAX. Cells were then treated with 0.4 mM dopamine or DMSO for 12 h and with CCK-8 solution for 1 h, and the cell viability was measured with the CCK-8 kit by measuring the absorbance (485 nm) of culture supernatant. The ratio of the absorbance of cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. p < 0.0001. Nrf2 regulation by USP10 of Nrf2 in USP10-KD cells without dopamine treatment, but the increase with dopamine treatment was small and much less than that in USP10-WT cells. These results suggest that the degradation of Nrf2 protein by proteasome only partially explains the reduction in Nrf2 in dopamine-treated USP10-KD cells. To understand the mechanism by which USP10 increases the amount of Nrf2 protein without inhibiting its degradation, we examined whether or not USP10-KD regulates the mRNA levels of Nrf2 and three Nrf2 target genes, NQO1, GSTM1, and p62. Dopamine treatment increased the levels of two Nrf2 inducible mRNAs (NQO1 and p62) in USP10-WT cells, but their levels were decreased by USP10-KD (Fig. 7B). Regarding GSTM1, USP10-KD decreased the mRNA level of GSTM1 with and without dopamine treatment, suggesting that USP10 stimulates Nrf2 activity even without dopamine treatment. These results indicated that USP10-KD reduces dopaminestimulated Nrf2 transcriptional activity. Importantly, in contrast to the other genes tested, the Nrf2 mRNA level was not reduced by USP10-KD, even though dopamine increased the level of Nrf2 mRNA. Given that the proteasome inhibitor MG-132 only partially reversed the decrease in Nrf2 protein in dopamine-treated USP10-KD cells (Fig. 7A), these results suggest that USP10-KD reduces the translation of Nrf2 in dopamine-treated cells. USP10-KD suppresses global translation in neuronal cells The data above suggested that USP10-KD decreases the dopamine-induced translation of Nrf2 in SH-SY5Y cells (Figs. 3 and 7). Next, we examined whether or not USP10 affects global translation in SH-SY5Y cells using a puromycin incorporation assay. Puromycin is incorporated into newly synthesized proteins, and its incorporation (new protein synthesis) is detected by antipuromycin antibody. SH-SY5Y cells were treated with dopamine and then further treated with puromycin for 10 min. Cell lysates were characterized by antipuromycin antibody. USP10-KD slightly increased new protein synthesis without dopamine treatment but significantly repressed protein synthesis with dopamine treatment (Fig. 8A). These results indicated that USP10-KD suppresses global translation in dopamine-treated SH-SY5Y cells. Under various stress conditions including oxidative stress, protein synthesis (translation) from many mRNAs is repressed, reducing the amount of misfolded proteins and protecting cells from the toxicity of misfolded proteins, thereby promoting recovery from stress. Stress granules (SGs) are a key mechanism associated with translational repression under various stress conditions (24). SGs are stress-inducible cytoplasmic RNA granules that contain many translationally inactivated mRNAs and translation-initiating factors (25). USP10 is localized in SGs under various stress conditions and positively or negatively regulates SG formation, depending on the type of stress and cell (16,26). To understand the role of USP10 in Nrf2 translation, we examined the SG formation in USP10-KD cells. G3BP1 and TIA1 are markers of SG. Dopamine treatment induced SG formation in USP10-KD cells, and the level was much higher than that in USP10-WT cells (Fig. 8, B and C). USP10 was localized in G3BP1-SG in dopaminetreated USP10-WT cells (Fig. 8, D and E). This finding is consistent with the localization of USP10 in G3BP1-SG in nonneuronal cells under various stress conditions (16,26). These results suggest that the increase in SG formation by USP10-KD in dopamine-treated cells is associated with the suppression of global translation in dopamine-treated USP10-KD cells. Ribosomes are a component of the translation machinery. The repression of translation by various stresses is accompanied by the ubiquitination of several ribosomal proteins. USP10 is known to deubiquitinate ribosomal proteins, such as Nrf2 regulation by USP10 ribosomal protein S2 (RPS2), ribosomal protein S3 (RPS3), and ribosomal protein S10 (RPS10), and inhibit their degradation in lysosomes (27). Therefore, we examined whether USP10 regulates the ubiquitination of RPS2, RPS3, and RPS10 in SH-SY5Y cells treated with dopamine ( Fig. 9). The amounts of ubiquitinated-RPS2 (ub-RSP2), ub-RPS3, and ub-RPS10 were increased in USP10-KD cells before dopamine treatment, while the amounts of nonubiquitinated-RPS2/RPS3/RPS10 were unchanged. Treatment with dopamine for 4 h in both USP10-KD and USP10-WT cells increased the amount of ub-RPS2 and ub-RPS3. In addition, the treatment from 4 to 8 h decreased the amount of ub-RPS2 and ub-RPS3. On the other hand, the amount of ub-RPS10 was decreased by dopamine treatment. These results suggest that increased ub-RPS2, ub-RPS3, and/or ub-RPS10 levels may be involved in the repression of translation in dopamine-treated USP10-KD cells. USP10-KD reduces the amount of phosphorylated p62 Phosphorylated p62 at serine 349 (pp62-S349) activates Nrf2 much more strongly than unphosphorylated p62 (6). We therefore investigated whether or not USP10 affects the amount of pp62-S349 in SH-SY5Y cells (Fig. 10A). An immunoprecipitation analysis showed that USP10-KD with and without dopamine treatment decreased the amount of pp62-S349 and simultaneously reduced the interaction of p62 with Keap1. p62 interacted with USP10 in cells without dopamine treatment, and the interaction was slightly reduced by dopamine treatment. These results suggested that USP10 interacts with p62 to increase the amount of pp62-S349 and that pp62-S349 increased by USP10 reduces the degradation of Nrf2 in dopamine-treated cells. pp62-S349 has been shown to be degraded by autophagy (28). In contrast to the immunoprecipitation analysis, Western blotting failed to detect pp62-S349 in SH-SY5Y with or without dopamine treatment, but pp62-S349 in SH-SY5Y cells was detected in BafA1-treated cells, and the amount was increased by dopamine treatment (Fig. 10B). In contrast to USP10-WT cells, little pp62-S349 was detected in BafA1/ dopamine-treated USP10-KD cells. These results suggested that USP10-KD reduces the amount of pp62-S349, and the reduction is not mediated by autophagic degradation of pp62-S349. USP10-KD reduces p62-body formation in neuronal cells Oxidative stress induces p62 and pp62-S349 to form a protein condensation called a p62-body, and the p62-body colocalizes with Keap1 and induces degradation of both Keap1 and p62 (29)(30)(31). We therefore investigated whether or not USP10 controls the formation of p62-body (Fig. 11). Dopamine treatment induced the formation of p62-body, p62/ Keap1-body, and p62/pp62-body in USP10-WT cells, but these condensate formations were reduced in USP10-KD cells (Fig. 11, A and B). These results suggest that USP10 increases the amount of p62/Keap1-body formation in dopaminetreated cells, thereby attenuating the Keap1 inhibitory activity of Nrf2. p62 interacted with USP10 in SH-SY5Y cells (Fig. 10A), but USP10 hardly colocalized with the p62-body in cells treated with dopamine (Fig. 11, C and D). These results suggested that protein-protein interaction between p62 and USP10 is not necessary for the formation of p62-bodies in dopamine-treated cells. Phosphorylated p62 attenuates the USP10-KD-augmentation of dopamine-induced cell death The reduced amount of pp62-S349 in USP10-KD cells was correlated with dopamine-induced cell death (Figs. 1 and 10). To assess the protective role of pp62-S349 in dopamineinduced cell death, we established SH-SY5Y cells that express a phosphorylation-mimic p62 mutant (p62-S349E, Ser to Glu mutation at position 349 of p62), phosphorylationdefective p62 mutant (p62-S349A, Ser to Ala mutation at position 349 of p62), or p62-WT. Anti-p62 antibody detected comparable amounts of p62-S349E, p62-S349A, and p62-WT in SH-SY5Y cells, and these levels were much higher than those of endogenous p62 in vector-transfected cells (Fig. 12A). A high amount of pp62-S349 was detected in p62-S349Etransfected cells, whereas only small amounts were detected in p62-WT or p62-S349A cells. p62-S349E increased the amount of Nrf2 equivalently in both USP10-KD and USP10-WT cells without dopamine treatment, and these increases were barely Nrf2 regulation by USP10 detectable in p62-WT and p62-S349A. Dopamine treatment increased the amount of Nrf2 in USP10-WT cells expressing p62-WT and p62-S349A, and the amount was comparable to that in USP10-WT cells expressing p62-S349E. The amount of Nrf2 in dopamine-treated USP10-KD cells expressing three p62 was less than that in USP10-WT cells. In addition, p62-S349E increased the amount of Nrf2-induced gene NQO1 in both USP10-KD and USP10-WT cells with or without dopamine treatment, and these increases were barely detectable in p62-WT and p62-S349A. We next examined the activities of three p62 proteins to dopamine toxicity to SH-SY5Y cells. The overexpression of p62-S349E in dopamine-treated USP10-KD cells prominently increased cell viability and reduced apoptosis, but these effects were not observed by p62-S349A or p62-WT (Fig. 12, B and C). These results indicated that Nrf2 activation by p62-S349E in USP10-KD cells reduced dopamine toxicity to USP10-KD cells. It should be noted that the dopamine-induced toxicity to USP10-KD cells expressing p62-S349E was still higher than that of USP10-WT cells expressing p62-S349E (Fig. 12B). These results suggest that USP10 has a different activity from increasing p62 phosphorylation (pp62-S349) in suppressing dopamine-induced cell death. Discussion The antioxidant activity of Nrf2/Keap1/p62 system plays an important protective role in various oxidative stresses in neurons. In the present study, we found that USP10 in neuronal cells reduces dopamine-induced ROS production and ROS-dependent apoptosis by stimulating antioxidant activity of Nrf2. These results indicate that USP10 is a key player in protecting neurons from oxidative stress-induced apoptosis by stimulating Nrf2 antioxidant activity. Dopamine has been reported to induce not only apoptosis but also cell death by autophagy in neuronal cells (32). Therefore, USP10 may be involved in not only apoptosis but also autophagic cell death in neurons. We found that USP10 stimulates Nrf2 activity in neuronal cells through two mechanisms (Fig. 12D). First, USP10 promotes the dopamine-induced global translation including Nrf2 in SH-SY5Y cells (Figs. 7 and 8). In this regard, USP10 has been shown to deubiquitinate several ribosomal proteins and inhibit their lysosome-mediated degradation (27). We also found that USP10-KD increases the ubiquitination of RPS2, RPS3, and RPS10 in SH-SY5Y cells treated with and without dopamine. Therefore, USP10-KD-induced ribosome dysfunction may inhibit global translation in dopamine-treated cells. Further analyses are needed to understand how USP10 controls the translation of Nrf2 under oxidative stress conditions. As a second mechanism for activating Nrf2, USP10 increased the amount of pp62-S349 in neuronal cells, and this pp62-S349 then activated the Nrf2-dependent antioxidant activity (Figs. 10-12). The increase in p62 phosphorylation by USP10 (pp62-S349) can be explained by several mechanisms. For instance, USP10 may stimulate p62 kinase or inhibit p62phosphatase. Understanding the mechanism by which USP10 regulates Nrf2 activity is important for deepening our understanding of Nrf2-dependent antioxidant activity. Unlike the dopamine-treated cells, the amount of Nrf2 in USP10-KD cells treated with dopamine was higher than that in USP10-WT cells (Figs. 3, A and D and 7A). This result suggests that USP10 reduces the expression of Nrf2 in cells without dopamine treatment. In the puromycin incorporation assay, USP10-KD slightly increased global protein synthesis in SH-SY5Y cells. Therefore, USP10 may decrease the translation of Nrf2 in SH-SY5Y cells without dopamine treatment Accumulating evidence suggests that the dysfunction of Nrf2 and/or p62 promotes the development of PD (33,34). α-Synuclein is a causative factor of familial and sporadic PD. Cytoplasmic α-synuclein-positive aggregates in PD neurons, called Lewy bodies, are the hallmark pathology of PD patients (35). Interestingly, USP10 and p62 have been shown to localize to Lewy bodies in PD neurons (14,36). Furthermore, the coexpression of USP10 and α-synuclein in cultured cells treated with a proteasome inhibitor induces cytoplasmic α-synuclein/USP10/p62-positive aggregates that resemble Lewy bodies (14). These studies suggest that USP10 and p62 play a role in two characteristic pathologies in PD neurons: α-synuclein aggregation and inhibition of ROS-dependent neuronal apoptosis. It is important to clarify how these two activities of USP10 and p62 work together in the PD development process. The expression of Nrf2 and USP10 in the brains of PD patients has been studied separately. According to an immunohistochemical study by Ramsey et al., Nrf2 is localized in the nuclei of neurons in the substantia nigra of PD patients, whereas it is expressed in the cytoplasm in normal human controls (37). These results suggest that high oxidative stress in PD patients induces the activation of Nrf2, but the activation is not sufficient to inhibit oxidative stress-induced neuronal toxicity. On the other hand, it has been reported that USP10 protein levels in the amygdala of PD patients are slightly increased compared with controls (14). Therefore, it will be important to clarify how USP10 regulates neuronal Nrf2 activity in the pathogenesis of PD. The Nrf2/Keap1/p62 antioxidant system has been shown to play important roles in a variety of diseases, including neurodegenerative diseases and cancer, making it an attractive therapeutic target for the treatment of these diseases. USP10 was found to be a key regulator of the Nrf2/Keap1/p62 system and neuronal apoptosis. Thus, USP10 activity against the Nrf2/ Keap1/p62 system is also a promising drug target for oxidative stress-associated diseases, including PD. Experimental procedures Cell lines and culture condition SH-SY5Y is a human neuroblastoma cell line (18), and cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (11965092; Thermo Fisher Scientific) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 50% Opti-MEM (Thermo Fisher Scientific), 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and MEM Nonessential AA solution. To generate retroviral vectors expressing p62, we used the Platinum-E (Plat-E) retroviral packaging cell line expressing gag-pol and envelope proteins of Moloney murine leukemia virus, which were cultured in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, Figures 6B and 9 were performed simultaneously as one experiment. Therefore, the data for β-actin and USP10 shown in Figure 6B are also represented in Figure 9. USP10, ubiquitin-specific protease 10. Non-essential AA solution. Reagents and antibodies The following reagents were purchased from the indicated Plasmids Retroviral expression plasmids for p62 and p62-mutants have been described previously (6). Transfected cells were treated with 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h, and culture medium was prepared from CCK-8-treated cells. The absorbance (485 nm) of culture medium was then measured with an absorbance meter (TriStar LB 941). The ratio of absorbance obtained from cells relative to that of the control cells (V) transfected with siNT (NT) treated with DMSO was presented as the mean and SD from three samples. *p < 0.0001. C, the indicated SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) by Lipofectamine RNAiMAX. Transfected cells were treated with either 0.4 mM dopamine or DMSO for 8 h, and then the cells were stained with anti-cleaved caspase-3 and Hoechst 33258. The staining was evaluated by a fluorescence microscope. The ratio of cells with cleaved caspase-3 staining relative to total cells measured by the number of nuclei was presented as the mean and SD of seven samples in (C). The significance of the differences was assessed by Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. p < 0.05; p < 0.01; **p < 0.0001. D, the current model of USP10 regulation of Nrf2dependent antioxidant activity in neuronal cells. USP10 activates the antioxidant activity of Nrf2 through two mechanisms. First, USP10 promotes phosphorylation of p62 (pp62-S349) via an unknown molecule (X), such as a phosphatase or kinase. Second, USP10 promotes the dopamine-induced translation of Nrf2 through inhibition of SG formation and/or deubiquitination of ribosomal proteins. NS, Not significant; USP10, ubiquitin-specific protease 10. The puromycin incorporation assay To detect newly synthesized proteins, SH-SY5Y cells are incubated with puromycin at a final concentration of 10 μg/ml for 10 min before harvesting the cells. To detect puromycin incorporated into the proteins, cell lysates were characterized by Western blotting with antipuromycin antibody. Establishment of SH-SY5Y cells overexpressing p62 mutants To establish a retrovirus expressing p62-WT or p62 mutant (p62-S349E, p62-S349A), Plat-E cells were transfected with the retrovirus vector expressing p62-WT or p62 mutant using FuGENE6, according to the manufacturer's instructions. At 48 h after transfection, the culture supernatant containing viruses was collected and infected to SH-SY5Y cells in the presence of 8 μg/ml polybrene. At 24 h after infection, cells were cultured in medium containing 1 μg/ml puromycin. The expression of p62 or p62 mutants (p62-S349E and p62-S349A) in SH-SY5Y cells was measured by Western blotting. Immunofluorescent assay SH-SY5Y cells were cultured on glass coverslips in 6-well plates and transfected with siRNA using Lipofectamine RNAiMAX reagent. Cells were fixed with 4% paraformaldehyde in PBS for 20 min, followed by permeabilization and blocking in blocking buffer (0.1% Triton X-100 and 0.2% gelatin dissolved in PBS) for 30 min. The cells were then treated with the primary antibodies diluted in blocking buffer for 60 min and further incubated with the secondary antibody of Alexa488-or Alexa594-conjugated goat anti-mouse, Alexa488conjugated goat anti-rabbit, Alexa488-or Alexa594conjugated donkey anti-rabbit, or Alexa594-conjugated goat anti-guinea pig antibody (A11029, A11032, A11034, A21206, A21207, A11076; Thermo Fisher Scientific) diluted in blocking buffer for 60 min. Cell nuclei were stained with Hoechst 33258. The samples were mounted in ProLong Glass Antifade Mountant (P36984; Thermo Fisher Scientific), and the stainings from three fields (>80 cells) were analyzed with a fluorescence microscope (BZ-X810; KEYENCE) and the FIJI ImageJ software program (https://imagej.net). p62-positive, p62/Keap1-positive, and pp62-S349/p62-positive condensates with a size >0.5 μm 2 were then evaluated as p62-body, p62/ Keap1-body, and p62/pp62-body, respectively. Cells containing more than two p62 bodies were evaluated as cells with a p62body. The proportions (%) of cells with a p62/Keap1-body, p62-body, or p62/pp62-body relative to total cells were presented as the mean and SD. To measure SG formation, stainings of three fields (>80 cells) were analyzed by a microscope and the FIJI ImageJ software program, and G3BP1/TIA1double-positive condensates with a size >0.25 μm 2 were evaluated as SG. The proportions (%) of cells with SG relative to total cells were presented as the mean and SD Coimmunoprecipitation assay SH-SY5Y cells were cultured in a 100-mm-dish (430167; Corning) and transfected with siRNA (USP10-siRNA) using Lipofectamine RNAiMAX reagent. Cells were lysed with icecold NP-40 lysis buffer (1% Nonidet P-40, 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin), and lysates were precleaned by protein G-sepharose (17-0618-01; Cytiva) for 60 min at 4 C. The precleaned lysates were treated with rabbit anti-p62 antibody or normal rabbit IgG. Immune complexes were precipitated by protein G-sepharose beads for 2 h at 4 C. The beads were then washed and boiled in SDS lysis buffer, and the proteins released from the beads were subjected to Western blotting. ROS detection assay Cells (1.5 × 10 4 ) were seeded in 96-well glass plates (Matsunami Glass Ind., Ltd). The next day, the cells were transfected with siRNA using Lipofectamine RNAiMAX. At 48 h after transfection, the cells were treated with 5 μM CM-H2DCFDA (General Oxidative Stress Indicator; C6827; Thermo Fisher Scientific) for 30 min in PBS treated with 0.5 mM MgCl2, 0.9 mM CaCl2, and 0.4% FBS. The cells were then treated with dopamine with complete medium for 6 h. The mean fluorescence intensity (MFI) of CM-H2DCFDA was analyzed by fluorescence microscopy, and the amount of ROS in the cells was measured by subtracting the MFI of noncellular regions from the MFI of cells using the FIJI ImageJ software program. Cellular and noncellular regions were distinguished by bright field microscopy. All experiments were performed in a phenol red-free medium. Cell viability assay Cell viability was measured using a Cell Counting Kit-8 (CCK-8) (DOJINDO). SH-SY5Y cells were cultured on 96-well plates for 24 h. Cells were transfected with siRNA (USP10-siRNA, p62-siRNA, Nrf2-siRNA, Keap1-siRNA, or the control siRNA) using Lipofectamine RNAiMAX. At 48 h after transfection, cells were treated with 0.1 to 0.4 mM dopamine for 12 h and then with the CCK-8 kit containing WST-8 in fresh medium for 1 h, and the absorbance (485 nm) of culture medium was measured using a TriStar LB 941 Microplate Reader (Berthold). Quantification of mRNA Total RNA was extracted from SH-SY5Y cells using an RNA extraction kit (NucleoSpin RNA; TAKARA) according to the manufacturer's protocol. Total RNA (5 ng) was subjected to reverse transcription and polymerase chain reaction (RT-PCR) reaction using a One Step TB Green PrimeScript RT-PCR Kit II (TAKARA) and a primer set. Real-time RT-PCR was performed using a Thermal Cycler Dice Real Time System (TAKARA). The NQO1, GSTM1, p62, and Nrf2 mRNA levels were normalized according to the β-actin mRNA level. The sequences of the primers used for real time RT-PCR were as follows: NQO1 forward is ccctgcgaactttcagtatcc, and reverse is ctttcagaatggcagggactc; GSTM1 forward is tgcccatgatactggggta, and reverse is gccactggcttctgtcataat; p62 forward is cacctgtctgagggcttctc, and reverse is cacactctccccaacgttct; Nrf2 forward is cggtatgcaacaggacattg, and reverse is agaggatgctgctgaaggaa, β-actin forward is gacaggatgcagaaggagatcac, and reverse is gtcatactcctgcttgctgatcc. Statistical analyses Data were analyzed with a one-way analysis of variance (one-way ANOVA) followed by Tukey's multiple comparisons test using the Prism8 software program (GraphPad). The data in Figures 1D and 12C did not meet the requirement of a oneway ANOVA that the standard deviations of all samples are comparable, as assessed by the Brown-Forsythe test. Therefore, the significances of the difference in Figures 1D and 12C were calculated by Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. Data availability All data are contained within the manuscript.	2021-11-26T16:24:16.755Z	2021-11-01T00:00:00.000	{ "year": 2021, "sha1": "8c7c83269296fafab18b2e604a2bd2b65faf36fe", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/article/S0021925821012576/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "d35e7854a4f34ea5fae6899b8c7db7a4a4acea21", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Medicine" ] }
237753860	pes2o/s2orc	v3-fos-license	Hidden spontaneous polarisation in the chalcohalide photovoltaic absorber Sn2SbS2I3 Perovskite-inspired materials aim to replicate the optoelectronic performance of lead-halide perovskites, while eliminating issues with stability and toxicity. Chalcohalides of group IV/V elements have attracted attention due to enhanced stability provided by stronger metal-chalcogen bonds, alongside compositional flexibility and ns2 lone pair cations – a performance-defining feature of halide perovskites. Following the experimental report of solution-grown tin-antimony sulfoiodide (Sn2SbS2I3) solar cells, with power conversion efficiencies above 4%, we assess the structural and electronic properties of this emerging photovoltaic material. We find that the reported centrosymmetric Cmcm crystal structure represents an average over multiple polar Cmc21 configurations. The instability is confirmed through a combination of lattice dynamics and molecular dynamics simulations. We predict a large spontaneous polarisation of 37 μC cm−2 that could be active for electron–hole separation in operating solar cells. We further assess the radiative efficiency limit of this material, calculating ηmax > 30% for film thicknesses t > 0.5 μm. Perovskite-inspired materials aim to replicate the optoelectronic performance of lead-halide perovskites, while eliminating issues with stability and toxicity. Chalcohalides of group IV/V elements have attracted attention due to enhanced stability provided by stronger metal-chalcogen bonds, alongside compositional flexibility and ns 2 lone pair cations -a performance-defining feature of halide perovskites. Following the experimental report of solution-grown tin-antimony sulfoiodide (Sn 2 SbS 2 I 3 ) solar cells, with power conversion efficiencies above 4%, we assess the structural and electronic properties of this emerging photovoltaic material. We find that the reported centrosymmetric Cmcm crystal structure represents an average over multiple polar Cmc2 1 configurations. The instability is confirmed through a combination of lattice dynamics and molecular dynamics simulations. We predict a large spontaneous polarisation of 37 lC cm À2 that could be active for electron-hole separation in operating solar cells. We further assess the radiative efficiency limit of this material, calculating g max 4 30% for film thicknesses t 4 0.5 lm. The photovoltaic performance of lead-halide perovskites has spurred major research efforts toward the discovery of 'perovskiteinspired materials' (PIMs). [1][2][3] Through the use of lead-free and stable alternative materials, PIMs aim to replicate the ability of halide perovskites to combine high optoelectronic performance with low-cost solution processing methods, while overcoming their infamous stability and toxicity drawbacks. The applications of PIMs are not solely limited to solar cell devices. In fact, these materials have seen successful implementation in a wide range of optoelectronic applications, such as light-emitting diodes, photocatalysts, radiation detectors, thin film transistors and memristors. 1 A defining feature of halide perovskites is the combination of a lone-pair cation with a halide anion which can produce, inter alia, dispersive valence and conduction bands, defect tolerance, and strong dielectric screening. 1,4 On the other hand, the soft metal-halide bonding contributes to the poor chemical and thermal stabilities of these materials. 5 Chalcohalide PIMs offer a route around this issue, demonstrating remarkably higher air and water stability -due to the increased strength of metalchalcogen bonding 4,5 -while retaining the ns 2 cation-halide anion combination. These materials have already demonstrated promising efficiencies (44% 1,6,7 ) alongside low-cost fabrication methods, representing a fertile area for stable, non-toxic highperformance solar cells. 8 Further increases in power-conversion efficiencies (PCEs) will be required to achieve commercial viability. 5 Here we focus on tin antimony sulfoiodide (Sn 2 SbS 2 I 3 ). Olivier-Fourcade et al. 9 reported the preparation and structural characterisation of Sn 2 SbS 2 I 3 in 1980, determining an orthorhombic Cmcm space group using X-ray diffraction measurements. This work was followed up with single-crystal X-ray analysis by the same research group in 1984. 10 Later, Dolgikh 11 prepared both Sn 2 SbS 2 I 3 and the isostructural, isoelectronic Pb 2 SbS 2 I 3 compound and investigated the optical dielectric response. Then in 1990, Starosta et al. 12 prepared both antimony sulfoiodide compounds and used photoconductivity measurements to determine optical band gaps of 1.5 and 2.0 eV for the Sn and Pb-based materials, respectively. They also discussed the seemingly anomalous trend of increased band gap with chemical substitution of heavier elements. In a major breakthrough, Sn 2 SbS 2 I 3 was recently reported to exhibit a photovoltaic efficiency exceeding 4% in the first experimental device fabrication. 4 Nie et al. used a low-cost single-step deposition method and reported good stability under conditions of high temperature, humidity and illumination. The achievement of a power conversion efficiency exceeding that first reported for methylammonium lead-iodide (MAPI) 13 is promising. While Sn 2 SbS 2 I 3 has been known for several decades, 9 it is now at the forefront of potential defect-tolerant PIMs for optoelectronic applications. In this work, we characterise the physical properties of Sn 2 SbS 2 I 3 using ab initio methods in order to understand the atomistic origins of impressive PV performance, and to assess the efficiency potential going forward. Computational methods We employ a combination of methods to probe the static and dynamic crystal structure of Sn 2 SbS 2 I 3 , in addition to its electronic and optical properties. All base calculations were performed using Density Functional Theory (DFT) within periodic boundary conditions through the Vienna Ab Initio Simulation Package (VASP). [14][15][16][17] After testing several DFT exchange-correlation functionals (Section S2.2, ESI †), the optB86b-vdW 18 functional was chosen for geometry optimisation, yielding the closest agreement with experiment for the c/a lattice parameter ratio. The ability of this dispersion-corrected functional to incorporate van der Waals interactions in solids has been well demonstrated, yielding accurate predictions of lattice parameters in lone-pair materials. [19][20][21][22] To calculate relative formation energies and the ferroelectric-switching barrier with accuracy beyond DFT, the Random Phase Approximation (RPA) to the correlation energy was employed, using electronic wavefunctions calculated with the HSE06 23 screened hybrid DFT functional. 24,25 The HSE06 functional, with full inclusion of spin-orbit coupling effects, was also used for calculations of optical and electronic behaviourhaving been demonstrated to yield accurate predictions of band gaps in semiconductor materials. 26,27 Comprehensive details of the computational implementation are provided in Section S1 of the ESI, † and all calculation data and analyses are provided in an online repository at doi.org/10.5281/zenodo.4683140. Structural analysis Geometry optimisations were performed for Sn 2 SbS 2 I 3 in both Cmcm and Cmc2 1 space groups, using the entries on the Materials Project repository 28 as the starting points. The relaxed crystal structures and unit cell dimensions are provided in Fig. 1 and Table 1, respectively. The Cmcm structure comprises infinite chains of (Sn 2 S 2 I 2 ) n along the a direction, tightly-packed along the b direction to form layers in the ab plane, with antimony and iodine atoms located between layers, yielding the overall (Sn 2 SbS 2 I 3 ) n stoichiometry. The (Sn 2 S 2 I 2 ) n chains are formed from face-sharing SnS 3 I 2 pyramids, comprising a parallelepid base of S 2 I 2 and an apical sulfur atom. In fact, this (Sn 2 S 2 I 2 ) n structural motif matches the 1D chain structures of the AChX (A = Bi, Sb; Ch = S, Se; X = Br, I) ns 2 -cation chalcohalide family. [29][30][31][32] Moving to the Cmc2 1 crystal structure, the coordination environments of Sb and (to a lesser extent) Sn shift to produce connected chains of the formula unit (Sn 2 SbS 2 I 3 ) n along the a direction, as the Sb atoms attach to the (Sn 2 S 2 I 2 ) n layers. The a and b cell lengths are similar for both polymorphs, with the greatest difference occurring along the c direction. Much of the structural behaviour in this system is governed by the lone-pair activities of Sb(III) and Sn(II) cations. While the antimony cations are found to exhibit significant localisation of the ns 2 electrons, only minimal distortion from spherical symmetry is witnessed for the Sn(II) lone-pair, due to a preferential alignment and thus enhanced interaction with anion p states (Section S3, ESI †). In both polymorphs, the Sb lone-pair is directed toward halide-bordered voids; either along the interchain gap (b direction) for Cmcm symmetry or toward the interchain gap (c direction) for Cmc2 1 -with more pronounced localisation visible in the Cmc2 1 case (Figs. S6-S8, ESI †). Indeed, this dynamic stereochemical activity of the Sb lone-pair is one of the primary driving factors behind the formation of the distorted, lower-symmetry Cmc2 1 polymorph -through a second-order Jahn-Teller instability, often observed in lone-pair chalcogenides. 22,[33][34][35] While all experimental works have reported the Cmcm crystal structure for Sn 2 SbS 2 I 3 , 9,10,12 Ibanez et al. 10 noted that assignment of Sb to an 8f Wyckoff position (i.e. the Sb Wyckoff site in Cmc2 1 symmetry) with 50% occupancy, as opposed to the 4c site for Cmcm, gave a significant reduction in R-factor -a measure of agreement between the structure model and diffraction data 36 -from 0.105 to 0.066. Moreover, both Olivier-Fourcade et al. 9 and Ibanez et al. 10 observed large Debye-Waller (B) displacement factors for the Sb and Sn sites in Sn 2 SbS 2 I 3 (i.e. the site positions which differ most between Cmcm and Cmc2 1 structures), even at temperatures as low as T = 173 K, alongside large anisotropy in the atomic displacement ellipsoids. Further structural and lone-pair analysis, with direct comparison to experiment (Fig. S4, ESI †), is provided in Sections S2 and S3 (ESI †). Thermodynamic & dynamic stability To ensure a high level of accuracy in the calculated phase stabilities, the Random Phase Approximation (RPA) to the correlation energy was employed. This beyond-DFT method has been demonstrated to yield predictions in excellent agreement with experimental results for the relative formation energies of structural polymorphs. 1,38,39 With this method, the lower-symmetry Cmc2 1 phase was predicted to be the thermodynamically-favoured polymorph, with a formation energy 35.8 meV per atom below Cmcm. Further evidence of Cmcm instability was obtained by computing the phonon dispersions of both Sn 2 SbS 2 I 3 polymorphsshown in Fig. 2. Imaginary harmonic modes in the phonon dispersion (i.e. those with negative frequencies; o o 0) indicate the presence of atomic displacements which lower the system energy. Two strong imaginary modes are witnessed in the Cmcm dispersion, demonstrating dynamic structural instability for this polymorph. Using the ISODISTORT 40,41 package to visualise the imaginary-mode phonon eigenvectors, we confirm that these energy-lowering distortions correspond to different, equivalent Cmcm -Cmc2 1 structural transitions (Fig. S5, ESI †). In contrast, no imaginary modes are observed for the Cmc2 1 polymorph. Thus we find the Cmc2 1 polymorph to exhibit both thermodynamic and dynamic stability, with converse behaviour for the Cmcm phase. We propose that experimental reports of centrosymmetric Cmcm Sn 2 SbS 2 I 3 are the result of macroscopic averaging over locally non-centrosymmetric Cmc2 1 configurations. Similar to other ns 2 cation materials, 22,29,33 this polar phase behaviour is driven by a second-order (pseudo) Jahn-Teller instability, in which the off-centring of the Sb(III) ions leads to enhanced bonding interactions between the sp-hybridised Sb ns 2 lone pair and the anion p states (yielding a small degree of Sb p character at 5 eV below the valence band maximum (VBM); Fig. 5a). Spontaneous lattice polarisation In the Cmc2 1 ground-state structure, the lack of inversion symmetry results in a spontaneous lattice polarisation DP = 37.0 mC cm À2 (calculated within the Berry phase formalism of the Modern Theory of Polarisation). 42 The strong polarity places Sn 2 SbS 2 I 3 next to the likes of ferroelectric oxide perovskites such as BaTiO 3 (B27 mC cm À2 ) 43 and KNbO 3 (B30 mC cm À2 ), well above that of MAPbI 3 ('MAPI')(4.4 mC cm À2 ) 44 and the archetypal 'photoferroic' SbSI (11 mC cm À2 ). 29,45 The mirror (m) and c-glide planes of the Cmc2 1 space group result in zero polarisation in Fig. 2 Simulated harmonic phonon dispersions of Sn 2 SbS 2 I 3 in the Cmcm (a) and Cmc2 1 (b) crystal structures, alongside vertical plots of the atomprojected phonon density of states. Generated using ThermoPlotter. 37 Brillouin zone path shown in Section S10a (ESI †). the ab plane, with all electronic polarisation directed along the c axis, corresponding to the shift in Sb position indicated by the arrows in Fig. 1 and Fig. S5 (ESI †). We identify substantial mixed ionic-covalent bonding character in this system, reminiscent of that found in ns 2 -cation metal halides. 1,32,46 Significant covalency is indicated by the cross-band-gap hybridisation of both cation p orbitals with both anion p orbitals, demonstrated by their overlap in both the conduction and valence bands (Fig. 5 and Fig. S13, S22, ESI †). Further evidence of enhanced orbital overlap is derived from the reduced cation-anion distances, relative to their ionic radii, save for the Sb-I pair (Table S1, ESI †). The resulting mixed ionic-covalent bonding gives rise to strong lattice polarisation, with large, anisotropic Born effective charges ( Table 2) and dielectric tensor (e x,y,z = [51.3, 18.2, 22.4]). 1,32,47,48 To calculate the barrier to polarisation switching, the Nudged Elastic Band (NEB) 50 method was employed to map out the potential energy surface (PES) along the minimum-energy path between equivalent Cmc2 1 configurations (Fig. 3). We find that the Cmcm phase corresponds to the transition state between Cmc2 1 configurations, with no local stability around this saddle point on the PES. Consequently, the energetic barrier to ferroelectric switching corresponds to the relative energy of the Cmcm and Cmc2 1 polymorphs; DE = 35.8 meV per atom. This value gives rise to a moderate coercive fieldcalculated as 750 kV cm À1 for a single-crystal ferroelectric domain, using Landau theory (eqn (S1), ESI †). 51 While the actual value will likely be at least an order of magnitude below this, due to a number of effects including domain formation, it places this material in a range intermediate between the weakly-polar leadhalide perovskites 52 and the stronger oxide perovskites, 51 as might be expected for a lone-pair chalcohalide material. To demonstrate the stability of lattice polarisation at finite temperatures, we performed Molecular Dynamics (MD) simulations for Sn 2 SbS 2 I 3 within an NVT ensemble at temperatures of 300 K and 500 K (Section S8, ESI †). Upon distortion from Cmcm to Cmc2 1 , the two nearest-neighbour S atoms for Sb become inequivalent, forming short (in the polarisation direction) and long Sb-S bonds ( Fig. 1 and Fig. S25, ESI †), allowing the polarisation dynamics to be visualised through the Sb-S bond lengths. As shown in Fig. 4, no appreciable swapping of the Sb-S bonds (corresponding to polarisation switching) is observed for the room-temperature MD runs, within the simulation timescale. On the other hand, transient hopping of Sb atoms occurs during the T = 500 K runs, as the material approaches a phase transition to higher-symmetry Cmcm (at which point the Sb-S bonds become equivalent and the probability densities merge; Fig. S25, ESI †), indicating a significant decrease in both the strength and stability of lattice polarisation at elevated temperatures. These results confirm the persistence of polar Table 2 Mean Born effective charge tensors (Z Ã ii ; \|e\|) for each atomic species in Sn 2 SbS 2 I 3 , calculated using the optB86b-vdW DFT functional. a The Born effective charge is a measure of the relationship between polarisation and atomic displacement, and is greater for the Cmcm phase due to structural instability. 47 Fig. 1), calculated using the Nudged Elastic Band method. 50 Filled circles represent calculated data points and the solid line is a spline fit. X axis given in units of mass-weighted displacement. distortions at room temperature and dynamic fluctuations at 500 K. We cannot comment on the size of the polar domains that would be formed, however. The presence of this previously-hidden polar distortion in Sn 2 SbS 2 I 3 poses several exciting prospects. In terms of PV applications, spontaneous polarisation can produce open-circuit voltages above the electronic band gap, via the Bulk Photovoltaic Effect, potentially allowing efficiencies above the standard limit for a single-junction solar cell. [53][54][55][56] Moreover, the combination of ferroelectric behaviour and spin-orbit coupling could permit switchable spin texture for 'ferroelectric Rashba semiconductor' applications, 57 while the effect on longitudinal optical (LO) phonons could favour polaron formation. 58 Electronic structure The electronic band structure of Cmc2 1 Sn 2 SbS 2 I 3 is shown in Fig. 4. The band gap is direct, occurring at the Y high-symmetry k-point -which corresponds to maximum antiphase interactions along the conventional a crystal direction (i.e. along the (Sn 2 SbS 2 I 3 ) n chains in Fig. 1). The fundamental energy gap is calculated as E g = 1.08 eV, placing it in the ideal range for a photovoltaic absorber material, with a 'detailed-balance' efficiency limit of 32.5%. 62 As illustrated by the orbital-projected density of states and band-edge charge densities in Fig. 5a and Fig. S13, S14 (ESI †), the conduction band minimum (CBM) arises from Sb p-I p interactions, while the VBM is comprised of antibonding interactions between the Sn 5s 2 lone pair and both anion p states (Fig. 6). Notably, this electronic structure allows the explanation of the reported 'anomalous' trend in band gap energies within the A 2 SbS 2 I 3 (A = Sn, Pb) isostructure family, for which Starosta et al. 12 found a gap 0.5 eV larger for the Pb-based compound. While band gaps of conventional semiconductors tend to decrease upon substitution of heavier elements, due to increased orbital energies, we witness a strong contradiction to this trend in this material class. This behaviour occurs due to the relativistic contraction of the Pb 6s orbitals, so that the Sn 5s states are in fact higher in energy, 63 thus enhancing the anti-bonding interaction of the ns 2 lone-pair with the anion p states at the VBM (Fig. 6). 64 The result is a more disperse, higher-energy VBM with a reduced energy gap. See Section S6 (ESI †) for a discussion of the effects of spin-orbit interactions and the electronic structure of the Cmcm polymorph. The optical absorption spectrum, shown in Fig. 4, exhibits a weak onset at the fundamental electronic band gap E g = 1.08 eV. There are two primary origins of this behaviour. Firstly, we find a low electronic degeneracy at the band extrema ( Fig. 5a and Fig. S16, S17, ESI †), itself a consequence of the low crystal symmetry, which rapidly increases with many more interband transitions available at E Z 1.4 eV. Another contributing factor is a weak transition dipole moment between the VBM and CBM electronic states (Fig. S15-S17, ESI †) -a consequence of both symmetry restriction and low spatial overlap. In the centrosymmetric Cmcm structure, the even (gerade) parity of both the VBM and CBM wavefunctions about the crystal inversion centre results in a formally symmetry-forbidden transition at the direct gap (as the electric dipole operator is of odd parity, thus yielding an overall zero optical transition matrix element; Section S6, ESI †). For the Cmc2 1 ground-state polymorph, this symmetry selection rule is broken by the shift in Sb positions (removing the inversion symmetry) as well as spin-orbit splitting, however these effects represent a relatively minor perturbation to the electronic structure (DE g,Cmcm vs. Cmc21 = 0.03 eV; Fig. S11, ESI †). Consequently, while the direct VBM -CBM transition is no longer formally forbidden in the Cmc2 1 structure, the optical transition matrix element remains weak due to symmetry restraints, compounded by a spatial separation of the VBM and CBM states (Fig. S14, ESI †). The combination of a rapidly increasing joint density of states This behaviour, we propose, is a likely cause of the apparent mismatch between the calculated fundamental band gap (E g = 1.08 eV) and that measured by optical spectroscopy (E Opt,Exp = 1.41 eV). While the slow onset of absorption renders the unambiguous determination of an optical gap difficult, Tauc-plot fitting of the calculated absorption within the 1-1.8 eV range gives a best linear fit (with R 2 = 0.93) for an optical gap in the range 1.4-1.5 eV (Fig. S18, ESI †). Indeed, this plot closely resembles that measured by Nie et al., 4 who also reported a large Urbach energy of 464 meV -which may in part be an artefact of the slow absorption onset. To quantify the efficiency potential of this material as a photovoltaic (PV) absorber, based on the calculated electronic and optical properties, the maximum PV efficiency (Z max ) as a function of film thickness was calculated using both the Spectroscopic Limited Maximum Efficiency (SLME) 60 and Blank et al. 61 metrics (Fig. 5c). As a consequence of the weak absorption onset, the attainable efficiency shows a strong dependence on film thickness, however this may be combatted through optimisation of the surface scattering properties. Using a Lambertian scattering surface, maximum efficiencies Z max 4 30% are achieved at thicknesses t 4 0.5 mm, demonstrating the potential application of this material class in high-performance earth-abundant solar cells. Importantly, this model assumes the radiative limit (i.e. internal quantum luminescence efficiency Q i = 1), and so the presence of non-radiative recombination (Q i o 1) will act to reduce the achievable efficiency and lead to a distinct optimal thickness in the range 0.5-5 mm (Fig. S19, ESI †). Potential for defect tolerance One of the primary origins of non-radiative electron-hole recombination is defects in the bulk crystal and at interfaces. By introducing electronic states within the bandgap, defects can facilitate carrier trapping and annihilation, thus reducing the open-circuit voltage (V oc ) and photovoltaic efficiency. [65][66][67] A primary driving factor behind the surge in research interest for ns 2 -cation PIMs is their potential to exhibit defect tolerancefacilitating high efficiencies despite low-cost solution synthesis. 1,68,69 Sn 2 SbS 2 I 3 exhibits several material properties which are known to contribute to defect tolerance. Firstly, we find the cation s 2 and anion p orbitals interact to produce a valence band maximum of anti-bonding character. This bonding behaviour is illustrated by the Crystal Orbital Hamiltonian Population 70,71 (COHP) analysis shown in Fig. 6, which decomposes the electronic density of states into regions of bonding and anti-bonding orbital interactions. The cation ns 2 -anion p anti-bonding interaction produces a high energy VBM, with an ionisation potential of 5.06 eV (Fig. S9, ESI †) -less than that of MAPI (5.70 eV), 72 SbSI (5.37 eV) 30 and Sb 2 Se 3 (5.13 eV), 35 and slightly larger than that of FaSnI 3 (4.88 eV) 73 -favouring the formation of shallow acceptor defects which are innocuous to PV performance. The substantial mixed ionic-covalent character and lattice polarisability in Sn 2 SbS 2 I 3 , discussed previously, results in a strong dielectric screening (e x,y,z = [51.3, 18.2, 22.4]) that will limit the electrostatic interactions between defects and charge carriers, thus reducing the probability of carrier capture and trap-mediated recombination. 1,32 One-dimensional atomic chain structures, exhibited by this (Fig. 1) and related materials such as Sb 2 Se 3 , 35 BiOI 32 and SbSI, 29,30 can yield benign grain boundaries, greatly reducing charge-carrier recombination in polycrystalline absorber materials. 32,74 The small electronic band gap (E g B 1.1 eV), wide conduction and valence bands, and relatively small electron effective mass (m e = 0.29) also favour defect-tolerant behaviour in this material. 1 Indeed, the presence of moderate defect tolerance is partially suggested by the impressive PV efficiency (Z 4 4%) and photoluminescence lifetimes (47 ns) obtained by Nie et al. 4 in the first experimental device fabrication for this material. In conclusion, we present a theoretical characterisation of the Sn 2 SbS 2 I 3 photovoltaic absorber. While experimental investigations have reported a non-polar, centrosymmetric Cmcm crystal structure, we propose that this in fact represents a macroscopic average over multiple Cmc2 1 configurations. Crucially, this leads to the prediction of ferroelectricity, with promising implications for high-efficiency photovoltaic operation and other technological applications. Through ab initio calculation of the electronic and optical properties, we identify an ideal electronic band gap for a photovoltaic absorber (E g = 1.08 eV), with power-conversion efficiencies Z max 4 30% at the radiative limit. These features, alongside several properties related to 'defect tolerance', present a promising outlook for the potential application of both this material and other unexplored members of the A 2 BCh 2 X 3 class. Considering only isoelectronic, earth-abundant and nontoxic substituents, there are in fact 36 possible elemental combinations for the quaternary group IV/V chalcohalide family (A = Sn, Ge; B = Sb, Bi; Ch = O, S, Se; X = I, Br, Cl) which may be synthesisable -the majority of which have not yet been investigated. ‡ Thus the performance potential in this system opens a zoo of compositional permutations for solution-processed ferroelectric and optoelectronic devices. Conflicts of interest There are no conflicts to declare.	2021-08-27T17:07:39.866Z	2021-07-05T00:00:00.000	{ "year": 2021, "sha1": "b63de5ebfe42654ee18301d0e3e2dd82c499c10a", "oa_license": "CCBY", "oa_url": "https://pubs.rsc.org/en/content/articlepdf/2021/mh/d1mh00764e", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "7e007b2b0e15d4737ce79625e344acc0e37c8991", "s2fieldsofstudy": [ "Materials Science", "Physics" ], "extfieldsofstudy": [ "Medicine", "Materials Science" ] }
235965924	pes2o/s2orc	v3-fos-license	Co-Creative Problem Solving to Support Rapid Learning of Systems Knowledge Towards High-Tech Innovations: A Longitudinal Case Study : This article explores co-creative problem solving to support rapid learning of systems knowledge in the concept phase towards innovation. We introduce the term co-creative problem solving to describe the act of collective creation between systems engineers and stakeholders during problem solving. The context of this research is a mature Norwegian industry accustomed to efﬁciency and risk aversion, challenged by late validation of systems design due to poor utilization of systems knowledge. We have explored co-creation between systems engineers and stakeholders such as project managers, business developers, and subject-matter experts through a longitudinal in-depth industry case in the energy domain. The primary outcome is insights into how co-creative problem solving supports rapid learning of systems knowledge in the industry case. We propose a method building on the ﬁndings from the research results to support systems engineers in similar contexts facing similar challenges. Introduction Co-creation was coined by Prahalad and Ramaswamy in 2004 [1], focusing on the concept of co-creation between enterprises and consumers with the purpose of value creation and innovation [2][3][4][5]. The use of co-creation in the marketing domain has grown significantly in the last decade [6] and proliferated to other domains such as design, focusing on collective creativity between designers and stakeholders [7][8][9][10][11]. The value of co-creation in systems engineering is less explored. This article seeks to extend cocreation in systems engineering [12], focusing on co-creation between systems engineers and stakeholders such as project managers, business developers, and subject-matter experts. Inspired by Sanders and Stappers [9], we introduce the term co-creative problem solving to describe the act of collective creativity between systems engineers and stakeholders during problem solving in the concept phase of systems development. People, organizations, and technical functionality contribute to increasing complexity in today's high-tech systems. Checkland and Wilson described such sociotechnical problems as real-world problems and introduced the Soft Systems Methodology (SSM) to address this [13][14][15][16][17]. Jackson [18] introduced Critical Systems Thinking to combine systems approaches and cope with various forms of complexity. We define systems knowledge as knowledge of the system [19] over its life cycle and this underpins the research in systems theory [20]. Due to the ambiguous and uncertain nature of the concept phase, systems engineers should strive for rapid learning of systems knowledge and early validation of systems design. Collective creativity in problem and solution exploration using cocreative workshops [21,22] and creative problem-solving teams [23,24] has been shown to be suitable for this purpose. Co-creation and similar human-centric approaches aim to support the creation of significant innovations in collaboration with customers and users [11,[25][26][27]. We distinguish between incremental and significant innovations, the latter meaning solutions beyond the ordinary (The Boderc research project [28], conducted through the Embedded Systems Institute in Eindhoven, the Netherlands, inspired this terminology). Mature organizations often rely on consolidation and incremental innovation to grow, while significant innovations are much harder to create [29,30]. A challenge for mature companies is that their approaches are often more suitable for incremental innovation than significant innovation. The neglect of the importance of collaboration and creativity in systems development is a typical challenge identified among engineers in the energy domain [31]. West [32] claimed that creativity is relatively easy as most engineers are highly creative. However, transforming creative ideas into innovative concepts is more difficult due to "resistance to change and structural and cultural barriers" [32]. In this article, we seek to bridge these barriers and explore how co-creative problem solving may support the creation of significant innovation in a mature Norwegian high-tech company. The research presented in this article is part of a research collaboration project with four Norwegian high-tech industry partners and two academic partners in systems engineering and systems oriented design, respectively. In our former research, we identified that the industry needs to support rapid learning of systems knowledge through concept exploration, early validation, and knowledge transfer in the concept phase [33]. Furthermore, we explored the use of co-creative methods and tools [21,22,[34][35][36][37][38] to support the Norwegian high-tech industry in achieving their needs. We concluded on eight success criteria inspired by systems and design theories for a new way of working. In this article, we apply the success criteria as a guide to explore and analyze the usefulness of co-creative problem solving in an industrial setting. For two years, we have interacted with a development team at one of the industry partners. The industry partner is a large-size global engineering, procurement, construction, and installation (EPCI) supplier with about four decades of EPCI experience in the energy domain. A company accustomed to a business management system based on typical (hard) systems engineering focusing on efficiency and risk aversion. The company is currently developing an innovative renewable energy system for the ocean space named Deep Purple. The innovation is leading the company's transition toward sustainable energy production systems. Deep Purple is an innovative large-scale, complex system of systems, building on the company's excellence in subsea technology. However, the innovation also requires the company to gain new knowledge as they enter a new domain in the renewable market. They need new ways of working to support rapid learning and early validation. Our research has explored the usefulness of co-creative problem solving and gained insights into applying this way of working in the industrial setting. This article aims at answering the following research questions: RQ1: How may co-creative problem solving support the systems engineers in rapid learning and early validation? RQ2: How may the systems engineers apply co-creative problem solving in an industrial setting? RQ3: What may be the main challenges for the systems engineers to adopt co-creative problem solving as a new way of working? The primary outcome is insights into how co-creative problem solving supports rapid learning and early validation in the industry case. We propose a method building on the findings from the research results to support systems engineers in similar contexts facing similar challenges. This article contributes to the body of knowledge in two ways: (1) adding academic rigor to collaborative and creative ways of working in systems engineering, and (2) proposing an industrial-relevant method for systems engineers to apply co-creative problem solving. We apply the notion of rigor and relevance as described by Ivarsson & Gorschek [39]. We structure the article as follows. Firstly, Section 2 provides literature on systems and design practices that have inspired this research. Section 3 introduces the method that evolved in this research, and Section 4 describes the research design. The first stage of that research produces insight into how co-creative problem solving may support systems engineers in the concept phase towards high-tech innovations, aiming to add to the academic rigor (Section 5). The second stage of that research realizes and applies a method for co-creative problem solving in the industry case, aiming to contribute to the industrial relevance (Section 6). Section 7 discusses our findings before concluding in Section 8. Literature Review This section reviews the literature on systems and design practices that have inspired this research. We also include literature on creativity practices in engineering and general theory on creativity. Co-creation stems from the enterprise and marketing domain, describing the cocreation between consumers and enterprises as part of the value creation process [1]. In participatory design, Sanders and Stappers [9] described co-creation as collective creativity in any form. They further narrowed this term into co-design and described this as "the creativity of designers and people not trained in design working together in the design development process", with non-designers typically being users or customers [40]. They emphasized the need to view users and customers as partners rather than subjects in the front end of product development. Contemporary design thinking, as practiced by the Innovation Design Engineering Organization (IDEO, Cambridge, MA, USA), the Stanford Design School (Stanford, CA, USA), and the International Business Machines Cooperation (IBM, Armonk, NY, USA), is by many seen as the recipe for innovations [10]. Design thinking practices co-creation using techniques such as rapid prototyping [41]. Design thinking focuses on empathizing with users to create innovative products or services, providing a more extraordinary user experience. IDEO advocated this mindset to create innovative new products and services and transform creative people and organizations [25]. Jones [7] discussed various types of co-creation and identified possibilities for improving design co-creation methods. He highlighted the importance of continuity and investment in this way of working to provide insight into complex problems. Jones called for a systemic design framework to enable practitioners to select and modify the various co-creation methods. Various literature within systems engineering described the need for collective and creative approaches to creating new products and systems. Pugh [42,43] highlighted the importance of group work and creativity in concept generation and evaluation (the Pugh matrix) as early as the 1980s. Sage & Armstrong [44] proposed collaborative and creative methods for systems synthesis, such as brainstorming [45] and the morphological box approach [46]. Lippert and Cloutier [47] described an extended use of TRIZ [48] to support systems engineers in creating innovations within digital systems engineering. White [49] proposed a practical methodology for complex adaptive systems engineering (CASE) to improve traditional systems engineering in sociotechnical systems engineering. His methodology covered organizational and team aspects, such as the need for brainstorming approaches and user experimentation. The methodology aimed to be an iterative and adaptive way of working for the team to operate on the "edge of chaos" effectively [49]. Looking towards systems architecting, Maier and Rechtin [50] described a great systems architect as being as skilled as an engineer and as creative as an artist. Sillito [51] emphasized the need for an analytic, inventive, and creative process in systems architecting to thoroughly understand the problem and solution domain and create suitable architectures. He distinguished between architecting and architectural modeling, both activities interacting and creating a system fit for purpose. The main objective of the systems architect is to understand how the system behaves and communicate this to others effectively [51]. Muller [52] described facilitating workshops as one of the tools of the Systems 2021, 9,42 4 of 20 systems architect. Workshops enable crossfertilizing and sharing systems insight such as in product specification, design, or business strategy. To conduct effective workshops, Muller proposed timeboxing and multi-views in iteration [53]. Multi-views use a CAFCR [54] framework to view the system from the customer, application, functional, conceptual, and realization perspective iteratively as the concept matures. Timeboxing is to set an appropriate timebox to achieve this within the duration of the workshop. McFadzean [55,56] supports the importance of multiple perspectives in the engineering of innovative products and systems. She called for a more vigorous use of creativity techniques in engineering and proposed a framework for creative problem-solving teams. The framework enables teams to select appropriate techniques based on their level of experience and need. McFadzean [23,24] further elaborated on the role of a neutral facilitator in such teams. The facilitator needs to address soft issues within the team to establish trust and deal with conflicts. She also described the effectiveness of such teams to depend on their experiences in using creative problem-solving techniques. According to McFadzean, some people may find creative problem-solving techniques uncomfortable. This discomfort can reduce the effectiveness of such techniques. In the worst case, people may not participate at all. Using groups to stimulate creativity is also supported by Paulus and Nijstad [57]. They found ideas produced by a group more innovative in a productive group setting, considering factors such as trust, attitude, and the number of participants. They proposed the usage of a facilitator and a leader to cope with unproductive team dynamics. The group should be diverse enough to provide knowledge on the subject but not too diverse as this may cause misunderstandings and conflicts. A diverse group enables a change of perspective and stimulates activation of long-term memory, resulting in more innovative ideas. Paulus et al. also found positive effects of combining individual ideation with group ideation to avoid participants biased by each other's opinions and ideas. Dorst and Cross [58] and Dorst [59] described the importance of coevolving on the problem and solution space by going back and forth on problem and solution exploration to support creative designs. De Bono [60] emphasized the importance of exploring the problem landscape thoroughly to gain insight instead of jumping to a solution. Furthermore, De Bono [60] described lateral thinking as a way of thinking to explore the problem space and stimulate creativity. Without a thorough understanding of the problem and solution spaces, engineers and designers are likely to jump to solutions and develop systems not fit for purpose. Bonnema, Veenvliet, and Broenink [61] (p. 9) identified solution focus as a challenge among engineers in development teams and claimed that "many engineers think in solutions". Daly, Yilmaz, Christian, et al. [62] found that the generation of multiple concepts was crucial in creating innovations to avoid such fixation. Furthermore, Murray, Studer, Daly, et al. [63] emphasized the importance of problem exploration perspectives to create innovative designs. A Method for Co-Creative Problem Solving This section introduces the method that we realized and applied in the industry case. Figure 1 illustrates the method, composed of a three-stage process and a timeline. The threestage process evolved as we explored sessions in the industry case and bears similarities to best practices for conducting effective meetings such as [64]. Systems and design practices focusing on collective creativity in problem and solution exploration (Section 2) inspired the timeline. The method distinguishes from an expert-led co-creation workshop in primarily two ways:  it supports the use of inexperienced facilitators to conduct the sessions, as the systems engineers in the industry case lacked such experience;  it emphasizes the use of brief sessions to fit within a busy workday for the participants in the industry case. In Stage 1, the problem owner and the facilitator gain an understanding of the problem. They identify the need of the session and decide on the scope. The primary outcome of this stage is a mutual understanding of the problem owner's need for the session's outcome. In Stage 2, the problem owner and the facilitator identify the purpose of the session and choose relevant participants. They plan the timeline and select specific techniques fitting the purpose and the participants. The primary outcome of this stage is the timeline, including specific tasks and the choice of participants. In Stage 3, the problem owner and the facilitator conduct the session in collaboration. The facilitator facilitates the participants through the tasks in the timeline while the problem owner leads the session. The primary outcome of the session is rapid learning and early validation. The timeline in Figure 1 consists of five main tasks for a structured problem and solution exploration. It is the combination and order of the tasks that are important in its cocreative problem-solving capabilities. The timeline aims to create a collective understanding and shared ownership for the participants regarding both the problem and solution space. Furthermore, the timeline combines individual and group exercises to prevent blocking and to enable the participants to build on each other's perspectives. Task #1 Purpose informs the purpose of the session to get the participant to aim towards the problem identified in Stage 1. In Task #2 Individual exercise, each participant reflects upon the problem and provides their views using simple artifacts, such as sticky notes. Furthermore, in Task #3 Consensus, the facilitator facilitates discussion of these views in plenum and adapts the session's problem description accordingly to share ownership. In Task #4, Creative problem solving, the facilitator facilitates the participants in a creative problem-solving technique to support the participants in challenging systems boundaries and exploring multiple perspectives. Finally, in Task #5 Sum-up, the facilitator sums up the session and connects to the problem description. This task verifies that the solution exploration fits the session's problem and stimulates reflection among the participants. The method distinguishes from an expert-led co-creation workshop in primarily two ways: • it supports the use of inexperienced facilitators to conduct the sessions, as the systems engineers in the industry case lacked such experience; • it emphasizes the use of brief sessions to fit within a busy workday for the participants in the industry case. In Stage 1, the problem owner and the facilitator gain an understanding of the problem. They identify the need of the session and decide on the scope. The primary outcome of this stage is a mutual understanding of the problem owner's need for the session's outcome. In Stage 2, the problem owner and the facilitator identify the purpose of the session and choose relevant participants. They plan the timeline and select specific techniques fitting the purpose and the participants. The primary outcome of this stage is the timeline, including specific tasks and the choice of participants. In Stage 3, the problem owner and the facilitator conduct the session in collaboration. The facilitator facilitates the participants through the tasks in the timeline while the problem owner leads the session. The primary outcome of the session is rapid learning and early validation. The timeline in Figure 1 consists of five main tasks for a structured problem and solution exploration. It is the combination and order of the tasks that are important in its co-creative problem-solving capabilities. The timeline aims to create a collective understanding and shared ownership for the participants regarding both the problem and solution space. Furthermore, the timeline combines individual and group exercises to prevent blocking and to enable the participants to build on each other's perspectives. Task #1 Purpose informs the purpose of the session to get the participant to aim towards the problem identified in Stage 1. In Task #2 Individual exercise, each participant reflects upon the problem and provides their views using simple artifacts, such as sticky notes. Furthermore, in Task #3 Consensus, the facilitator facilitates discussion of these views in plenum and adapts the session's problem description accordingly to share ownership. In Task #4, Creative problem solving, the facilitator facilitates the participants in a creative problem-solving technique to support the participants in challenging systems boundaries and exploring multiple perspectives. Finally, in Task #5 Sum-up, the facilitator sums up the session and connects to the problem description. This task verifies that the solution exploration fits the session's problem and stimulates reflection among the participants. Research Approach Action research forms the basis of this research and allows the researcher to acquire knowledge of real-world problems and improve [65]. In this research, we conducted a longitudinal study of about two years within the development team in the concept phase of developing the Deep Purple system at the industry partner. Deep Purple is a complex system of systems leading the company's transition toward sustainable energy production systems. Thus, we selected the participants in the development team as we consider Deep Purple as a representative industry case to explore co-creative problem solving. The main author actively engaged with the team in the concept phase of developing the Deep Purple system. The participants represent both genders and hold formal education in engineering and systems engineering disciplines. Table 1 shows the profile of the team members, including years of relevant work experience, which reflect the participants' age. Figure 2 illustrates the research design. The first rectangle shows our previous work [33], where we identified the industry needs and the success criteria ( Table 2). The two following rectangles of Figure 2 show the two stages of research presented in this article. In Stage 1, we explored and analyzed nine sessions to gain insight into how co-creative approaches supported the systems engineers in the industry case to achieve the success criteria. In Stage 2, we used the insights gained from Stage 1 to synthesize and apply a method to support the systems engineers in co-creative problem solving. Systems 2021, 9, x FOR PEER REVIEW 6 of 21 Research Approach Action research forms the basis of this research and allows the researcher to acquire knowledge of real-world problems and improve [65]. In this research, we conducted a longitudinal study of about two years within the development team in the concept phase of developing the Deep Purple system at the industry partner. Deep Purple is a complex system of systems leading the company's transition toward sustainable energy production systems. Thus, we selected the participants in the development team as we consider Deep Purple as a representative industry case to explore co-creative problem solving. The main author actively engaged with the team in the concept phase of developing the Deep Purple system. The participants represent both genders and hold formal education in engineering and systems engineering disciplines. Table 1 shows the profile of the team members, including years of relevant work experience, which reflect the participants' age. Figure 2 illustrates the research design. The first rectangle shows our previous work [33], where we identified the industry needs and the success criteria ( Table 2). The two following rectangles of Figure 2 show the two stages of research presented in this article. In Stage 1, we explored and analyzed nine sessions to gain insight into how co-creative approaches supported the systems engineers in the industry case to achieve the success criteria. In Stage 2, we used the insights gained from Stage 1 to synthesize and apply a method to support the systems engineers in co-creative problem solving. Methodology in Stage 1-Exploring Co-Creative Approaches in the Industry Case We planned sessions guided by the formerly identified success criteria ( Table 2) combined with inspiration from design and systems practices, including creativity practices in engineering and general theory on creativity. We adapted to the team's daily challenges during concept development and planned for sessions when a team member had a specific problem and needed to gather stakeholders and discuss. The researchers and team members facilitated the sessions, neither holding any previous facilitation skills. We did not aim for one session to fulfill all criteria; instead, we used the criteria to gain insight into how co-creative approaches could support the team to achieve the criteria. Focusing on customer Systems oriented design 7 Enabling creativity Systems thinking, participatory design 8 Focusing on user Participatory design, systems oriented design, design thinking 1 The positioning reflects the authors' opinions of the main academic field(s) to support the given criterion based on the findings from the former research results. During the sessions in Stage 1, we conducted participant observations and passive observations. After the sessions, we conducted informal interviews, focus groups, and questionnaires. The questionnaire included a set of statements derived from the success criteria using a five-point Likert scale [66]. The scale ranged from strongly disagree, disagree, neutral, agree, to strongly agree. The questionnaire also included open-ended questions for participants to report on benefits and concerns. The Likert scale responses were analyzed using a Net Promoter Score (NPS) [67,68]. Promoters reply strongly agree, while the detractors reply neutral, disagree, or strongly disagree. Agree is neither promoter nor detractor and hence left out of the NPS score. Appendix A provides the questionnaire results, including NPS. Table 3 provides an overview of the sessions conducted in Stage 1, the context of the sessions, the number of participants and respondents to the questionnaire, and corresponding methods for data collection. We collected data during the planning of the sessions, during the sessions, and from the participants after the sessions using questionnaires and focus groups. To analyze the sessions, we found inspiration in the framework proposed by Midgley, Cavana, Brocklesby, et al. [69] for evaluating systemic problem structuring methods. The framework focuses on "the use of a particular method (or set of methods) in a context for particular purposes, giving rise to outcomes". We view the method as the proposed method (Section 3), the context as the industry case, the purposes as the sessions, and the outcome as the goal to support rapid learning of systems knowledge guided by the success criteria (Table 2). While analyzing the sessions in the industry case, we found it beneficial to differentiate between the session's applicability and usability to distinguish between factors impacted by the industry case (the context) and the success criteria (the outcome). Applicability describes how well the session was conducted in the industry case, including planning and structure, while usability describes the session's ability to achieve the formerly identified success criteria. We categorized the notes into applicability and usability and used the success criteria to code [70] the notes on usability. An example of an interview-note made in S3 and coded with SC3 is "Good suggestions which put the technical solutions into a wider perspective. This will help us to introduce our ideas and solutions to the stakeholders". Assigning the code SC3 "showing business potential" to this note describes the session's ability to view the concept from a business perspective. Three main capabilities indicating support for rapid learning of systems knowledge in the sessions emerged from analyzing the coded notes on usability. From analyzing the notes on applicability, we identified three impacting factors for applying useful sessions. We made a qualitative assessment of the coded notes for each session and scored each capability in each session using a five-point scale from 1 (very low) to 5 (very high). Sessions providing very low support for the capability scored 1, while sessions providing very high support for the capability scored 5. Methodology in Stage 2-Realizing and Applying a Method for Co-Creative Problem Solving We integrated the findings from Stage 1 into a method to support co-creative problem solving in the industry case. We passively observed the development team for an initial evaluation while applying the method in two sessions, Session A and B. After the sessions, we conducted informal interviews with the facilitator and the problem owner. Table 4 provides details on the application of the method in the two sessions. Validity of Data This research primarily builds on qualitative and participative research methods. While this approach is valuable to gain an in-depth understanding of industry challenges in their relevant context [65,68,[71][72][73], there is a risk of researcher bias and challenges in the generalization of the research results. To ensure the trustworthiness of our findings and reduce the risk of researcher bias, we used Maxwell's eight-point checklist for qualitative research [74]. We actively engaged with the systems engineers in the industry case through a longitudinal study to gain an in-depth understanding of their challenges and potential solutions. We conducted nine co-creative sessions with systems engineers and stakeholders to ensure rich data collection from multiple contexts. Furthermore, we triangulated data collection using various methods to analyze the results from multiple perspectives. Results from Stage 1-Exploring Co-Creative Sessions in the Industry Case This section describes the main results from exploring and analyzing nine sessions in the industry case using the research methodology presented in Section 4.1. Table 5 provides an overview of the techniques and artifacts explored in the sessions. In Sessions 2 to 8, we applied brainstorming [45], brainwriting [75], and Gigamapping [76] for ideation. In addition, we used other techniques to stimulate creativity, such as coevolving on problem and solutions spaces [58,77], combining individual and group exercises [57], and challenging operational scenarios of concepts through adding or removing constraints [78]. We also explored CAFCR [54], focusing on connecting desired systems qualities to the systems realization and identifying gaps and trade-offs. We explored the use Systems 2021, 9, 42 9 of 20 of a playful warm-up exercise inspired by design thinking in Session 5. Furthermore, we used large paper plots, visualizations, markers, sticky notes, flip overs, and a whiteboard. Table 6 shows an overview of the number of qualitative field notes from the sessions, including data from observations, interviews, questionnaires, and reflections. The observation notes include field notes made during the session. The interview notes include the notes made during informal interviews, focus groups, and open-ended questions in the questionnaire after the sessions. Furthermore, the questionnaire notes include the most significant promoters and detractors of the statements in the questionnaire (Appendix A). Finally, the reflective notes include reflections made by the researchers after the sessions. The two last rows show the number of notes on the applicability and usability of the sessions, respectively. Applicability describes how well the session was conducted, including planning and structure, while usability describes the session's ability to achieve the formerly identified success criteria (Table 2). Analysing the Sessions' Applicability We identified three main impacting factors for applying useful sessions from analyzing the notes on applicability. These are the understanding of the upfront problem and need, session length, and session structure. The subsequent sections elaborate on these factors. Understanding of the Upfront Problem and Need Throughout the sessions, we identified the importance of two roles: the facilitator and the problem owner. The problem owner is the person responsible for the progress of the problem and has an incentive to take on this role. In Session 1, the facilitator took time to discuss with problem owners upfront and carried out a session that satisfied the problem owners' expectations. In Sessions 2 and 4, the problem owner was also acting as facilitator. While this worked well in Session 4, we observed Session 2 suffering from a schedule overrun. A neutral facilitator applying appropriate timeboxes could have prevented this overrun, both during planning and execution. In Session 4, we posed the following questions to the problem owner and facilitator during planning: Q1. What does the problem owner want to get out of the session (purpose)? Q2. Who should participate (background and knowledge)? Q3. What is the appropriate duration of the session? The questions were based on findings from Sessions 1 to 3 and inspiration from best practices for effective meetings, such as [64]. Q1 immediately arose when we planned for Session 3. When the problem owner and facilitator aligned on the problem and need, Q2 followed when selecting techniques. From Sessions 5 to 7, we divided the roles of the problem owner and the facilitator more clearly and used the questions (Q1-3) during planning. These sessions provided a useful outcome to the problem owners. In Session 8, the facilitator and the problem owner used little time to discuss the problem upfront. During the session, it became clear that the planned exercises did not match the need of the problem owner. This session did not play according to the planned agenda, and the outcome was poor. Session Length We found that the session length depended on session purpose and commitment from the participants. We found the participants perceived longer sessions as a waste of time in a busy workday. In the sessions with subject-matter experts from other parts of the organization, we observed reluctance to attend more extended sessions due to practical reasons such as lack of cost accounts. In Session 4, we planned for only one hour, as all participants were subject-matter experts from other parts of the organization. The problem owner was satisfied with the outcome. However, some of the participants reported concerns about too little time. In Session 9, we planned for a full day as the scope of the session was quite large. Likewise, in this session, two participants were subject-matter experts from other parts of the organization. At the last minute, the subject-matter experts canceled the session and replaced it with a two-hour skype meeting. The subject-matter experts were not dedicated to support the innovation and lacked a cost account to attend a full-day session. Sessions 3 to 7 were around 2 h, and we found these sessions to provide a useful outcome within an acceptable timeframe. Session Structure In Session 1, the participants showed little responsibility to keep to the scheduled time. The facilitator often cut off discussions between the participants to keep to the Systems 2021, 9, 42 11 of 20 scheduled time. There was minimum use of techniques to engage the participants during the discussions. After lunch, the participants were tired and started to wear out. The session could not go through the planned agenda within the scheduled time slot, even though it had a 1-day duration. Session 2 suffered from a schedule overrun. In this session, the participants were engaged using artifacts such as sticky notes, guide questions, and visualizations. The problem owner planned for a session length of about three hours. However, it ended up as three subsessions with a total length of 11 h. For Sessions 3 to 9, we aimed to activate the participants and conduct structured sessions using engaging techniques and artifacts and shorter lengths. We conducted sessions according to the following structure: • The problem owner introduces the problem or possibility, good to put visuals or statements up on the wall (in Session 3, this was a specific sentence in a research application form, in Session 5, these were system qualities of the subsea storage concept). • The participants use sticky notes in an individual exercise to reflect upon the problem. • Each of the participants present their sticky notes and puts them up on the wall for discussion. • Creative problem-solving technique, choose the appropriate technique for purpose and participants. • Sum up by discussing main findings, if relevant make an action list. We chose timeboxes (Timeboxes set an appropriate length of a task that enables the participants to produce about 80% complete. The reasoning behind this is that 80% is often achievable quickly, while the last 20% is considerably more challenging to achieve and not worth the effort in sessions [53]) fitting to the minimum time achievable for each activity, considering the appropriate session length. The core of the session, being techniques for creative problem solving, required a significant amount of the time. In Session 5, we planned for 60 min for this; the facilitator chose to stop after 45 min due to saturation. In Session 7, we used around 30 min. From Session 3 onward, we found this way of planning agendas to be effective. The feedback and the observations show that the participants appreciated using timeboxes to push them through the activities. Analysing the Sessions' Usability To analyze the notes on usability, we coded the notes according to the success criteria in Table 2. Table 7 shows the number of coded notes in Sessions 1 to 9 for each success criterion. Three main capabilities indicating support for rapid learning of systems knowledge emerged from analyzing the coded notes. The capabilities are problem and solution exploration, collective creation, and creative thinking. To gain further insight into how the sessions supported these capabilities, we scored the usefulness of each session using a five-point scale. Sessions providing very low support for the capability scored 1, while sessions providing very high support for the capability scored 5. Figure 3 provides the scoring results. The subsequent sections elaborate on these scorings. Grasping complexity - Showing business potential 1 Sharing knowledge 2 7 2 7 3 1 10 1 -5 Visualizing 1 1 1 -2 ---1 6 Focusing on customer - Enabling creativity --3 -3 5 1 -2 8 Focusing on user 1 1 1 1 ----2 3 tential 1 1 2 3 -1 ---4 Sharing knowledge 2 7 2 7 3 1 10 1 -5 Visualizing 1 1 1 -2 ---1 6 Focusing on customer - Enabling creativity --3 -3 5 1 -2 8 Focusing on user 1 1 1 1 ----2 Figure 3. Scoring how the sessions achieve the main capabilities. Problem and Solution Exploration We define problem and solution exploration as exploring both the problem and the solution space in the session, considering enabling technology, business case, customer needs, and user needs. In Sessions 1 and 2, we observed the participants primarily focusing on solution exploration and less on problem exploration. In Sessions 3 to 9, we planned for a structured agenda and found this supported the participants in reflecting on and discussing both the problem and solution exploration. Sessions 3, 4, and 6 scored high on problem and solution exploration, while Session 7 scored medium. In these sessions, we observed the problem owner strive to select participants holding the relevant knowledge. In Session 4, the context of the session was to explore enabling technology for a specific concept that could impact the company's strategy for future applications. We observed the problem owner selecting subject-matter experts holding the knowledge of the enabling technology to validate the current concept early and explore options. In Session 6, we observed the problem owner include business developers to explore possibilities for an early phase pilot project. Further, in Session 5, we observed the problem owner gain awareness of the lack of operational knowledge. As a result, the problem owner included the subject-matter experts holding this knowledge in Session 9. Collective Creation We define collective creation as exploring or maturing an idea or concept in the session with a high degree of uncertainty and complexity. Typically, this requires knowledge Problem and Solution Exploration We define problem and solution exploration as exploring both the problem and the solution space in the session, considering enabling technology, business case, customer needs, and user needs. In Sessions 1 and 2, we observed the participants primarily focusing on solution exploration and less on problem exploration. In Sessions 3 to 9, we planned for a structured agenda and found this supported the participants in reflecting on and discussing both the problem and solution exploration. Sessions 3, 4, and 6 scored high on problem and solution exploration, while Session 7 scored medium. In these sessions, we observed the problem owner strive to select participants holding the relevant knowledge. In Session 4, the context of the session was to explore enabling technology for a specific concept that could impact the company's strategy for future applications. We observed the problem owner selecting subject-matter experts holding the knowledge of the enabling technology to validate the current concept early and explore options. In Session 6, we observed the problem owner include business developers to explore possibilities for an early phase pilot project. Further, in Session 5, we observed the problem owner gain awareness of the lack of operational knowledge. As a result, the problem owner included the subject-matter experts holding this knowledge in Session 9. Collective Creation We define collective creation as exploring or maturing an idea or concept in the session with a high degree of uncertainty and complexity. Typically, this requires knowledge sharing and striving for early failures. In Sessions 1 and 2, we observed techniques supporting analytical thinking and less exploration. In Session 2, several participants found it challenging to discuss the concept, reporting reasons such as high uncertainty and lack of knowledge. From Sessions 3 to 9, we planned sessions to support the participants in collective creation using techniques to cope with complexity, such as Gigamapping, CAFCR, and ideation. Sessions 3 to 7 scored high and very high on achieving collective creation. In these sessions, we found the participants appreciated the possibility of sharing their knowledge and perspectives on early phase concepts using techniques such as free-format Gigamapping. In Session 3, the participants reported "rapid identification of possibilities", indicating that the session supported the team in early failures. However, we also reflected after Session 4 that "failing early or perhaps being comfortable admitting that we try to fail is difficult to achieve", indicating that failure is considered harmful in this industry, which focuses on high quality and risk reduction. We found most participants appreciated the use of creative exercises in the sessions and that participants more experienced using such techniques also appeared to be more confident using them. This confidence became clear in Session 3, where the participants engaged in Gigamapping without further instructions from the facilitator. However, we also observed participants reluctant to engage in creative exercises. Creative Thinking We define creative thinking as exploring multiple perspectives and challenging perceived boundaries during problem and solution exploration. In Session 1, we observed a lack of techniques to stimulate creative thinking, such as visualization, engaging techniques, and timeboxes. From Sessions 2 to 9, we made use of techniques and visualizations to support creative thinking. Sessions 3, 5, and 6 scored high or very high on creative thinking. In these sessions, we found the participants to appreciate the creative artifacts reporting "visual", "creative", "brainstorming", and "sticky notes" as benefits. In Session 6, we found the use of brainwriting combined with group discussion especially beneficial. This combination enabled the participants to think for themselves before discussing their views. Session 6 resulted in 52 ideas in one hour. The participants claimed that the ideas held a higher level of innovation than assumed from a "normal" meeting in the debrief. Session 9 planned to include creative exercises; however, the subject-matter experts replaced this session with a two-hour "normal" meeting. The problem owner stated in the debrief of the meeting: "This was no creative session; the creativity had happened before the meeting". Furthermore, the problem owner stated that he/she missed the possibility to interact with the subject-matter experts in creative exercises to gain a deeper systems insight. Results from Stage 2-Realizing and Applying a Method in the Industry Case This section describes the main results from realizing and applying a method in the industry case using the research methodology presented in Section 4.2. We integrated the capabilities and impacting factors identified in Stage 1 into a method. Furthermore, we applied the method in two sessions (Sessions A and B) in the industry case for an initial evaluation. In Sessions A and B, we passively observed the systems engineers applying the proposed method. After the sessions, we conducted informal interviews with the facilitator and the problem owner. In both sessions, we found the two first stages of the method, understanding of the problem and need and session planning, supported the problem owner to reflect and discuss the problem before conducting the session. Further, splitting the roles of the problem owner and the facilitator forced the problem owner to discuss and reflect on the problem in collaboration with the facilitator before conducting the session. The problem owner enjoyed the possibility to reflect and discuss the problem before the session. The facilitator enjoyed being involved and gaining ownership of the problem. In Session A, both the problem owner and facilitator found the method provided valuable structure to the session. The structure pressured the participants to share perspectives and explore problems and solutions in a brief time. The timeboxing avoided the participants getting lost in detailed discussions. However, we observed during planning that they failed to plan for an individual exercise and did not include this in the session. In Session B, we observed that the problem owner and the facilitator had difficulties planning for a creative problemsolving technique fitting to the context of the session. They ended up planning to conduct a creative technique on the go. The session was highly talkative, the problem was unclear, and the facilitator could not conduct a creative technique on the fly. We found that both the facilitator and the problem owner appreciated the method during the debrief. However, both missed using a creative problem-solving technique and would plan to use one next time. The facilitator stated that he/she found it challenging to apply a creative exercise as the purpose of the session was unclear. As the facilitator said during the debriefing of Session B, "without creativity techniques, it will only be a lot of talks". Discussion This section answers the research questions and compares and contrasts the results with existing work in the body of knowledge. The end of this section provides contributions to theory and implications for practice. RQ1: How may co-creative problem solving support the systems engineers in rapid learning and early validation? Guided by eight success criteria, we planned and conducted nine sessions in the industry case to gain insight into how to realize a method for co-creative problem solving. We identified three main capabilities for a new method: problem and solution exploration, collective creation, and creative thinking. We scored the sessions to gain further insights into how the sessions supported the capabilities and argue that the identified capabilities may support the systems engineers in rapid learning and early validation towards high-tech innovations. Figure 4 illustrates the primary connections between the success criteria on the left and the capabilities on the right. The solid lines illustrate a strong connection between the success criteria and the capabilities, while the dotted lines illustrate a weaker connection. method during the debrief. However, both missed using a creative problem-solving technique and would plan to use one next time. The facilitator stated that he/she found it challenging to apply a creative exercise as the purpose of the session was unclear. As the facilitator said during the debriefing of Session B, "without creativity techniques, it will only be a lot of talks". Discussion This section answers the research questions and compares and contrasts the results with existing work in the body of knowledge. The end of this section provides contributions to theory and implications for practice. RQ1: How may co-creative problem solving support the systems engineers in rapid learning and early validation? Guided by eight success criteria, we planned and conducted nine sessions in the industry case to gain insight into how to realize a method for co-creative problem solving. We identified three main capabilities for a new method: problem and solution exploration, collective creation, and creative thinking. We scored the sessions to gain further insights into how the sessions supported the capabilities and argue that the identified capabilities may support the systems engineers in rapid learning and early validation towards high-tech innovations. Figure 4 illustrates the primary connections between the success criteria on the left and the capabilities on the right. The solid lines illustrate a strong connection between the success criteria and the capabilities, while the dotted lines illustrate a weaker connection. The importance of a proper problem and solution exploration towards innovation is well supported in literature [55,56,58,60,62,63,77]. Bonnema, Veenvliet, and Broenink [61] (p. 9) identified solution focus as a challenge among engineers in development teams and claimed that "many engineers think in solutions". We identified a timeline to support a structured problem and solution exploration and thus prevent such fixation. The timeline supports a proper problem exploration in the first half and solution exploration in the latter. Furthermore, we found it important that the participants held the required knowledge to conduct problem and solution exploration. Such knowledge typically includes insight into customer needs by focusing on the customer, systems operational context by focusing on the user, and enabling technology and business case by showing business potential. Co-creation and co-design focus on collective creativity in value creation [1] and design development [9]. Systems-centric approaches such as systems architecting and systems engineering emphasize collaboration, creativity, multiple perspectives in concept generation [42,43], and systems development [44,50,51,54]. To support collective creation, we aimed for knowledge sharing of early phase concepts and strived for early failure by sharing multiple perspectives using techniques such as CAFCR [54]. We also applied techniques to support the participants in grasping complexity, such as Gigamapping [76]. Furthermore, we applied a variety of means to enable creativity and support creative thinking. Human-centric approaches, such as design thinking, emphasize creativity to explore innovative concepts [25]. Firstly, the sessions applied engaging and creative techniques, such as brainwriting [75]. Secondly, the sessions applied time constraints using timeboxes [53] to push the participants to engage and create. Thirdly, the timeline combines individual and group exercises to ensure all participants share their perspectives and build on each other's ideas and thoughts. Paulus et al. [57] emphasized the importance of combining individual and group exercises to enhance creativity. Finally, the sessions applied visualizations to stimulate creativity, either in the form of collective creation of sketches or preparing visualizations before the session. Kerzner, Goodwin, Dykes, et al. [79] highlighted the importance of visualization in creative workshops. RQ2: How may the systems engineers apply co-creative problem solving in an industrial setting? Building on the findings from exploring the nine sessions, we realized a method consisting of a three-stage process and the timeline to support the systems engineers to achieve the capabilities in the industry case. The three stages are an understanding of the upfront problem and need, session planning, and the actual session, while the timeline combines five main tasks for a structured problem and solution exploration. Furthermore, the timeline aims to create a collective understanding and shared ownership of both the problem and solution space. The method splits the roles of the problem owner and the facilitator. Paulus et al. [57] and Muller [52] described a similar approach for creative groups and workshops. The problem owner holds the system's insight and need for progress on the problem and has the incentive to take on this role. The facilitator leans on the problem owner for support during the session to share knowledge of the problem. Furthermore, the method makes use of brief sessions using inexperienced facilitators. The systems engineers in the industry case lacked facilitation experience, and shorter sessions are often easier to facilitate than full-day workshops. Using internal facilitators may also enable free discussions without confidentiality issues and avoids expensive facilitators who do not hold the necessary systems knowledge. Facilitation skills are not necessarily that common among engineers. However, this may vary in different organizations and cultures. We foresee the need for more experienced facilitators when including external and unfamiliar stakeholders such as customers. Conducting brief sessions with a few familiar participants may provide un-trained facilitators with facilitation experiences in a safe environment. While exploring sessions, we experienced stakeholders reluctant to attend more extended sessions due to practical reasons such as lack of cost account and busy workdays. Aiming to cope with such practicalities, we strived to balance an acceptable session length in the industry case with the time needed to gain valuable insight through the timeline. We found sessions with a duration of about two hours to provide valuable outcomes when planned well, including about six participants familiar with the session context. Sessions including several participants unfamiliar with the session context may require a longer time. RQ3: What may be the main challenges for the systems engineers to adopt co-creative problem solving as a new way of working? After realizing the method, we applied it in the industry case in two sessions, Sessions A and B, for an initial evaluation. In Session A, we found the method supported the systems engineers in planning and conducting a structured problem-solving session with stakeholders. In Session B, we observed the facilitator and the problem owner failing in the upfront planning, resulting in a session with participants mainly talking. In both sessions, the facilitator and the problem owner strived to include participants holding the needed knowledge for early validation of essential aspects such as business case, value proposition, and enabling technology. Due to the context of the industry case, we could not include external stakeholders, such as customers. We would have liked to include external stakeholders to evaluate how the method supports systems knowledge through co-creation with beneficiaries such as customers or users. The problem owner and the facilitator need to consider the necessity and possibility of customer inclusion during planning. When observing the systems engineers applying the method without interference from the researchers, we found that they neglected creative thinking during planning. In Session A, they did not include the individual exercise. In Session B, they failed to plan for a creative problem-solving exercise. The problem owner and the facilitator appreciated the method's ability to plan and structure the session. They stated that they missed using a creative exercise in debrief of Session B. McFadzean found the usability of a method focusing on collective creativity in engineering teams dependent on the participants' willingness and ability to engage in such activities [23,24]. This way of working is unfamiliar to some people and challenges their comfort zones and their perception of acceptable ways of working. Setting session rules or informing participants upfront of what creative problem-solving techniques are may support people to feel less uneasy and prepare for engaging in this new way of working. While exploring the sessions, we observed that once the participants familiarized themselves with creative problem-solving techniques, most seemed to approve and appreciate this way of working. McFadzean [23,24] proposed a framework to ease the selection of techniques based on needs and level of experience. We propose to make use of McFadzean's framework [56] as a guide during session planning. By gradually building experience in using co-creative problem solving, we expect the effectiveness of the method to increase. Jones [7] emphasized the importance of continuity and investment in this way of working to provide insight into complex problems. Following the recommendations by Jones, we propose a systemic use of the method to enhance co-creation between systems engineers and stakeholders and support a long-term outcome. Contributions to Theory This article contributes to the body of knowledge by extending co-creation in systems engineering and proposes an industrial-relevant method for systems engineering to apply co-creative problem solving. We identify and define three main capabilities for co-creation in systems engineering to support rapid learning of systems knowledge in a context of socio-technical complexity. By exploring and scoring co-creative sessions in the industry case, we gain insights into achieving the capabilities in an industrial setting. We map the formerly identified success criteria to the capabilities and propose that these criteria can be means for future validation. Implications for Practice The proposed method indicates partial support for the systems engineers in an indepth understanding of the problem and a structured problem and solution exploration. In the context of a mature Norwegian high-tech industry accustomed to efficiency and risk aversion, we foresee the method supporting this industry in exchanging knowledge and perspectives leading to rapid learning of systems knowledge and early validation of systems design. Thus, the method offers a step towards innovations that create value for beneficiaries such as customers, users, and businesses. Conclusions Guided by eight success criteria, this article explores co-creative problem solving through nine sessions in a real industry case. Furthermore, this article identifies and defines three main capabilities to provide rapid learning of systems knowledge in the concept phase towards innovations. These capabilities are problem and solution exploration, collective creation, and creative thinking. Building on the findings from exploring the nine sessions, we propose a method to support systems engineers using a three-stage process and a timeline for conducting co-creative sessions to achieve the capabilities. Our findings from an initial evaluation imply that the method partly supported the systems engineers in a structured problem and solution exploration focusing on multiple perspectives and shared ownership. Furthermore, our findings imply that the main challenges in applying the method are the systems engineers' experience in using creative problem-solving techniques and the willingness to engage in such activities. Previous research on co-creation includes value co-creation in the marketing domain and co-creation in other domains such as design, focusing on collective creativity between designers and stakeholders (co-design). This article aims to extend co-creation in systems engineering to support systems engineers in rapid learning of systems knowledge to cope with complexity in systems development. Collaboration and creativity have been found to be essential in systems development, such as during concept evaluation and systems synthesis. However, co-creation in systems engineering is less explored. This article looks towards systems and design practices and proposes a co-creative method between systems engineers and stakeholders. The method enables the exchange of knowledge and perspectives and offers a step towards innovations that create value for beneficiaries such as customers, users, and businesses. Limitations and Future Research Directions Action research forms the basis of this research in a longitudinal study of about two years within a development team in the concept phase of an innovative complex system of systems. Action research and similar participatory approaches are applied in research on systems engineering to gain an in-depth understanding of industry challenges and improve. However, these research approaches increase the risk of researcher bias and challenge the generalization of research results. We strived for valid research results by focusing on triangulation and rich data collection. We focused our research primarily on one industry case. Hence, we cannot claim that our findings nor the proposed method fit other contexts and needs than described in this article. Due to the context of the industry case, we could not include external stakeholders in the initial evaluation of the proposed method. We propose further research to evaluate how the method support rapid learning of systems knowledge by including external stakeholders such as customers or users. Based on our research, the company established a new work process for their innovation projects, including the proposed method. The willingness to adopt the method in the industry case indicates that the systems engineers found the method valuable and aim to continue to use it. However, we need further research over a longer time to fully evaluate how such a method supports the systems engineers to achieve the identified capabilities. We propose to continue the use of the success criteria as means for future validation. Acknowledgments: The authors are grateful to the people in the Deep Purple team for being our guinea pigs in this research. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Table A1 provides an overview of the statements and the NPS scores in the survey used in S1 to S5. The leftmost column shows the success criteria (SC) that the statements derive from. The rightmost columns indicate the most significant promoters and detractors in bold (NPS ≥ 3, NPS ≤ −3).	2021-07-17T13:13:37.544Z	2021-06-11T00:00:00.000	{ "year": 2021, "sha1": "74f689ded9b9242c4359c4ee9c766927bd232e21", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2079-8954/9/2/42/pdf", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "a1213d3562cc486a8d0a36d63701d48d412095da", "s2fieldsofstudy": [ "Engineering", "Education" ], "extfieldsofstudy": [ "Computer Science" ] }
256726968	pes2o/s2orc	v3-fos-license	An interaction-driven many-particle quantum heat engine and its universal behavior A quantum heat engine (QHE) based on the interaction driving of a many-particle working medium is introduced. The cycle alternates isochoric heating and cooling strokes with both interaction-driven processes that are simultaneously isochoric and isentropic. When the working substance is confined in a tight waveguide, the efficiency of the cycle becomes universal at low temperatures and governed by the ratio of velocities of a Luttinger liquid. We demonstrate the performance of the engine with an interacting Bose gas as a working medium and show that the average work per particle is maximum at criticality. We further discuss a work outcoupling mechanism based on the dependence of the interaction strength on the external spin degrees of freedom. INTRODUCTION Universality plays a crucial role in thermodynamics, as emphasized by the description of engines, that transform heat and other resources into work. The working substance can often be described as an ideal gas and the control of interparticle interactions or the equation of state is generally beyond reach. As a result, the role of the working substance and the presence of many-particle effects are secondary in paradigmatic cycles (Carnot, Otto, etc.). 1 In the quantum domain, this state of affairs should be revisited as suggested by recent works on many-particle quantum thermodynamics. The quantum statistics of the working substance can substantially affect the performance of a quantum engine. 2 The need to consider a many-particle thermodynamic cycle arises naturally in an effort to scale-up thermodynamic devices [3][4][5][6] and has prompted the identification of optimal confining potentials 7 as well as the design of superadiabatic protocols, [8][9][10][11] first proposed in a single-particle setting. [12][13][14] The realization of superadiabatic strokes with ultracold atoms using a Fermi gas as a working substance has been reported. 10,11,15 Intrinsically manyparticle effects in the working substance, such as the divergence of energy fluctuations near a second-order phase transition 16 and many-body localization, 17 can prove advantageous. Many-particle thermodynamics can lead to a quantum-enhanced behavior, whereby a single many-particle cycle can outperform an ensemble of independent single-particle machines. 8,18 Quantum technologies have also uncovered avenues to design thermodynamic cycles. Traditionally, interactions between particles are generally considered to be "fixed by Nature" in condensed matter. However, a variety of techniques makes possible to modify interparticle interactions in different quantum platforms. A paradigmatic example is the use of Feschbach and confinementinduced resonances in ultracold atoms. 19 The engineering of interparticle interactions is similarly at reach in digital quantum simulation in trapped ions, Rydberg atoms, and superconducting qubits. 20 We introduce a thermodynamic cycle that exploits the manyparticle nature of the working substance and thus have no singleparticle counterpart. It consists of four isochoric strokes, alternating heating and cooling with a modulation of the interparticle interactions. Using Luttinger liquid theory, the efficiency of the cycle is shown to be universal in the low-temperature regime of a one-dimensional (1D) working substance, i.e. solely depending on the sound velocities at the states A and B (Fig. 1). The universal efficiency of the cycle is also shown to characterize the regime of strong interactions in the working substance, in which the spectrum is approximately scale invariant. When an interacting Bose gas is used as such, the average work output per particle is maximum at criticality. RESULTS Interaction-driven thermodynamic cycle Consider a quantum heat engine (QHE) with a working substance consisting of a low-dimensional ultracold gas tightly confined in a waveguide. 25,26 Ultracold gases have previously been explored in quantum cycles where work is done via expansion and compression processes, both in the non-interacting 7,27 and interacting regimes. 8,[28][29][30][31][32] We propose the implementation of a quantum cycle consisting of four isochoric strokes, in which heating and cooling strokes are alternated with isentropic interaction-driven processes. In the latter, work is done onto and by the working substance by increasing and decreasing the interatomic interaction strength, respectively. This work can be transferred to other degrees of freedom as we shall discuss below. The working substance consists of N particles with interparticle interactions parameterized by the interaction strength c. Both the particle number N and the system size L are kept constant throughout the cycle and any equilibrium point is parameterized by a point ðc; TÞ indexed by the temperature T and the interaction strength c. Specifically the interaction-driven quantum cycle, shown in Fig. 1 for the 1D Lieb-Linger gas, 21,22 involves the following strokes: (1) Interaction ramp-up isentrope (A ! B): The working substance is initially in the thermal state A parameterized by ðc A ; T A Þ and decoupled from any heat reservoir. Under unitary evolution, the interaction strength is enhanced to the value c B and the final state is non-thermal. (2) Hot isochore (B ! C): Keeping c B constant, the working substance is put in contact with the hot reservoir at temperature T C and reaches the equilibrium state ðc B ; T C Þ. (3) Interaction ramp-down isentrope (C ! D): The working substance is decoupled from the hot reservoir and performs work adiabatically while the interaction strength decreases from c B to c A , reaching a non-thermal state. (4) Cold isochore (D ! A): The working substance is put in contact with the cold reservoir with temperature T A keeping the interaction strength constant until it reaches the thermal state ðc A ; T A Þ. The work extracted from the heat engine is given by W ¼ W 3 À W 1 ¼ Q 2 À Q 4 while the efficiency of the heat engine reads Interacting Bose gas as a working substance Consider as a working substance an ultracold interacting Bose gas tightly confined in a waveguide, 33,34 as realized in the laboratory. [35][36][37] The effective Hamiltonian for N particles is that of the Lieb-Liniger model: 21,22 where x j' ¼ x j À x ' , with 2m ¼¼ 1. The spectral properties of the Hamiltonian of Eq. 2 can be found using coordinate Bethe ansatz. 21,22 We consider a box-like trap 38,39 where any energy eigenvalue can be written as E ¼ P i k 2 i in terms of the ordered quasimomenta 0<k 1 <k 2 < Á Á Á <k N . The latter are the (Bethe) roots fk i g of the coupled algebraic equations determined by the sequence of quantum numbers fI i g with i ¼ 1; 2; Á Á Á ; N. As a function of c and T, the 1D Bose gas exhibits a rich phase diagram. We first consider the regime of strong interactions and use a Taylor series expansion in 1=c. For a given set of quantum numbers I n ¼ fI ðnÞ i g, the corresponding energy eigenvalue takes the form where the interaction-dependent factor λ c reads The spectrum of a strongly interacting Bose gas is thus characterized by eigenvalues with scale-invariant behavior, i.e., ϵ n ðcÞ=ϵ n ðc 0 Þ ¼ λ c =λ 0 c . This scale invariance in the spectrum allows us to assign an effective temperature along the isentropes; see Supplementary Information Section I. In this regime, the work output is thus set by W ¼ Q 2 À Q 4 ¼ ½1 À ðλ cA =λ cB ÞQ 2 and the efficiency becomes independent of temperature of the heat reservoirs, as we show in Supplementary Information. Being the spectrum scaleinvariant, the efficiency can be expressed in terms of the scaling factor. 40 This feature is as well shared by the well-known harmonic Otto cycle. 41 This is a consequence of the fact that both the cycles involve the driving of an isolated quantum system with a scaleinvariant energy spectrum. However, the interaction-driven cycle generally involves a change of density of states which can be regarded as the change of the generalized exclusion statistics in an ideal gas; 42 also see Supplementary Information Sections II and III. Moreover, the scale invariant character of the energy spectrum in the strongly interacting regime is also present for multicomponent Bose and Fermi gases, preserving the universal efficiency given by Eq. 6. For weaker interactions, we resort to a numerically-exact solution of Eqs. 3 for finite particle number N. We enumerate all the possible sets I n of quantum numbers for low-energy states and solve Eqs. 3 numerically for given c. With the resulting quasimomenta fk n;1 ; k n;2 ; Á Á Á g, and the corresponding energy eigenvalues ϵ n ¼ P i k 2 n;i , the probability that the Bose gas at temperature T is found with energy ϵ n is set by the Boltzmann weights p n ¼ e Àϵn=T = P m e Àϵm=T (with k B ¼ 1). The equilibrium energy of the states A and C is set by thermal averages of the form hEi ¼ P n p n ϵ n that in turn yield the expressions for Q 2 and Q 4 . Here, a proper cutoff of the possible sets fI n g can be determined by p n =p G ( 1, where p G is the probability for the working substance to be in the ground state. The numerical results for the efficiency η and output work W are shown in Fig. 2 as a function of the interaction strength. For fixed values of T C and T A , the maximum work is studied as a function of c A while keeping c B constant (Fig. 2). The efficiency is well reproduced by Eq. 6 at strong coupling, that captures the monotonic decay with increasing interaction strength. The efficiency is found to be essentially independent of the temperature in the strong interaction regime, whereas the work output is governed by the temperature and interaction strength. Universal efficiency at low temperature In the thermodynamic limit (where N and L ! 1 with n ¼ N=L being kept constant), the equilibrium state of the 1D Bose gas is Fig. 1 Interaction-driven quantum cycle. The working substance is driven through four sequential isochoric strokes alternating heating and cooling processes at different temperature T with isentropes in which the interparticle interaction strength c is ramped up and down. The color coding indicates the mean energy across the T À c plane. The dashed lines correspond to the thermal entropy calculated from the thermodynamic Bethe ansatz equation of the Lieb-Liniger gas. [21][22][23][24] The effective temperature assigned along the isentropes results from scale invariance in the energy spectrum, that holds for strong coupling and the Luttinger liquid region; see Section "Universal efficiency at low temperature" and the Supplementary Information Section I [45][46][47][48][49] in which the free energy density reads where E 0 is the energy density of the ground state, v s is the sound velocity which depends on particle density n and interaction c. The entropy density s can be obtained as the derivative of free energy, s ¼ À∂F =∂T ¼ πT=3v s . The expression for the heat absorbed and released are respectively given by energy differences where s i ¼ πT i =3v i s and v i s with v B s ¼ v C s and v A s ¼ v D s denote the entropy density and the sound velocity of the state i, respectively. Using the fact that the strokes (1) and (3) are isentropes, it follows that where T B and T D are the temperatures at states B and D, respectively. As a result, the efficiency and work output are given by where κ ¼ T A =T C . Since T A < T B < T C , we have 0 < κ < ξ < 1. This efficiency (11) is universal regardless the microscopic physics of the working substance with a low-energy TLL description. The work output for fixed T A and T C is maximized at ξ ¼ ξ c as shown in Supplementary Information Section IV, As the TLL theory describes the universal low-energy behavior of 1D many-body systems, Eqs. 11-13 provide a universal description of the efficiency and work of QHE with a 1D interacting working substance at low temperatures, which are applicable to any cycle in which work and heat exchange occurs in different strokes. In particular, in the strongly interacting regime, the sound velocity of 1D Bose gases is given by v s ' 2πnð1 À 4nc À1 þ 12n 2 c À2 Þ. 24 In this regime, the result 11 thus reduces to Eq. 6. On the other hand, in the weak interaction , 24 and thus the efficiency reaches the asymptotic value indicating an enhancement of the performance with respect to the strongly interacting case. Quantum critical region We next focus on the performance of an interaction-driven QHE across the phase diagram of the 1D Bose gas and characterized the role of quantum criticality. 50 The 1D Bose gas displays a rich critical behavior as a function of the temperature T and chemical potential μ (Fig. 3). In the region of μ=T ( 0, i.e. the mean distance between atoms is much larger than the thermal wave length λ th ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi 2π 2 =ðmk B TÞ p , and the system behaves as a classical gas (CG). A quantum critical region (QC) emerges between two critical temperatures (see the white dashed lines in Fig. 3c) fanning out from the critical point μ c ¼ 0. In this region, the energy gap closes universally as Δ $ jμ À μ c j zν , in terms of the correlation length and dynamic critical exponents ν ¼ 1=2 and z ¼ 2. The TLL region characterized by a phononic spectrum is found with μ>0 and T below the right critical temperature. For fixed cycle parameters (c A , c B , T A , and T C ), we study the performance of the engine across the QC region by changing n. We numerically calculate the efficiency η and average work W=N by using the TBA equation 16, and show that near the QC region W=N has a maximum value; see Fig. 3. We set c A ¼ 1, c B ¼ 3, T A ¼ 1, and T C ¼ 5 for the heat engine and let the density n increase from 0:1 to 23. The red, green, and blue dashed lines in Fig. 3 correspond to the densities n ' 0:3, 1:4, and 3:0, respectively. In order to understand the maximum of W=N, we also plot these three engine cycles (A ! B ! C ! D ! A) in the phase diagram of specific heat in the T À μ plane; see Fig. 3 (c). The performance of the cycle is optimal at the critical region (green dashed line). Specifically, when the stroke A ! B is near the TLL boundary and the stroke C ! D is in the critical region, the average work output per particle is maximized. In this scenario, the two adiabatic processes (A ! B and C ! D) pass through zones in which the change in the energy dispersion is maximized, as shown in Fig. 3c. Qualitatively, the process A ! B passes though the region where the specific heat is maximum, whereas the C ! D evolves through the region with lowest specific heat. In the adiabatic process, the particle number N and entropy S are all fixed. Figure 3d shows the work for the different cycles, where the work for each adiabatic process is given by W ¼ R c final c initial ∂E=∂c ð Þdc. Even if the process is not rigorously adiabatic, 51 this maximum work output still holds, as further discussed in Supplementary Information Section IV. DISCUSSION The modulation of the interaction strength c is associated with the performance of work, that has recently been investigated for different working substances. [52][53][54] An experimentally-realizable work outcoupling mechanism can be engineered when the coupling strength depends on the configuration of other degrees of freedom. This is analogous to the standard Carnot or Otto cycles where the working substance is confined, e.g., in a box-like trap. 41 The latter is endowed with a dynamical degree of freedom that is assumed to be slow (massive) so that in the spirit of the Born-Oppenheimer approximation it can be replaced by a parameter. Similarly, the modulation of the coupling strength in an interaction-driven cycle can be associated with the coupling to external degrees of freedom. As an instance, consider the choice of an interacting SU(2) 1D spinor Fermi gas in a tight waveguide as a working substance. The Hamiltonian can be effectively mapped to the Lieb-Liniger model (see ref. 55 and Supplementary Information Section V), with a coupling strength c ¼ cðhŜ j ÁŜ ' iÞ that depends on the spinor degrees of freedom, 55,56 an approximation corroborated by the exact solution in a broad range of parameters. 57 Another example relies on the use of confinement-induced resonances 19,58 that govern the scattering properties of a quasi-1D working substance in a waveguide. The transverse frequency ω ? of the latter directly determines the interaction strength c, i.e., c ¼ cðω ? Þ, and the role of ω ? parallels that of the trap parameters in the conventional Carnot and Otto cycles. The possibility of extracting work from quench dynamics was recently studied. 59,60 Finally, we note that an interaction-driven cycle can also be used to describe QHEs in which the interaction-driven strokes are substituted by processes involving the transmutation of the particle quantum exchange statistics, e.g., a change of the statistical parameter of the working substance. The Hamiltonian of Eq. 2 can be used to describe 1D anyons with pair-wise contact interactions with coupling strengthc and statistical parameter θ characterizing the exchange statistics, smoothly interpolating between bosons and fermions. 61,62 The spectral properties of this Lieb-Liniger anyons can be mapped to a bosonic Lieb-Liniger model given by Eq. 2 with coupling strength c ¼c= cosðθ=2Þ. 61,62 The modulation of c can be achieved by the control of the particle statistics, tuning θ as proposed in ref. 63 In summary, we have proposed an experimental realization of an interaction-driven QHE that has no single-particle counterpart: it alternates heating and cooling strokes with processes that are both isochoric and isentropic and in which work is done onto or by the working substance by changing in the interatomic interaction strength. This cycle can be realized with a Bose gas in a tight waveguide as a working substance. Using Luttinger liquid theory, the engine efficiency has been shown to be universal in the low temperature limit, and set by the ratio of the sound velocities in the interaction-driven strokes. The optimal work can be achieved by changing the ratio of the sound velocity, e.g., by tuning the interaction strength. An analysis of the engine performance across the phase diagram of the Bose gas indicates that quantum criticality maximizes the efficiency of the cycle. Our proposal can be extended to Carnot-like interaction-driven cycles in which work and heat are simultaneously exchanged in each stroke. Exploiting effects beyond adiabatic limit may lead to a quantum-enhanced performance. 8,18 The use of non-thermal reservoirs [64][65][66] and quantum measurements 67,68 constitutes another interesting prospect. Our results identify confined Bose gases as an ideal platform for the engineering of scalable manyparticle quantum thermodynamic devices. METHODS In the Yang-Yang thermodynamics of the 1D Bose gas, the pressure is given by where the "dressed energy" εðkÞ is determined by thermodynamic Bethe Anstatz (TBA) equation Z 2c c 2 þ ðk À qÞ 2 1 þ e ÀεðqÞ=T dq: The particle density n and the entropy density s can be derived from the thermodynamic relations in terms of which the internal energy density reads E ¼ Àp þ μn þ Ts. Both interaction-driven strokes are considered to be adiabatic. As a result, the heat absorbed during the hot isochore stroke (B ! C) is given by Q 2 ¼ L½Eðc B ; T C Þ À Eðc B ; T B Þ, while the heat released during the cold isochore (D ! A) equals Q 4 ¼ L½Eðc A ; T D Þ À Eðc A ; T A Þ. Here T B and T D can be determined in terms of the isentropic strokes, in which sðc A ; T A Þ ¼ sðc B ; T B Þ and sðc B ; T C Þ ¼ sðc A ; T D Þ, where T A and T C are the temperature of the cold and hot reservoir, respectively. The efficiency and work can then be obtained by numerically solving the TBA equation. This article was previously published as a preprint. 69 Fig. 3 Performance across the phase diagram. a Efficiency η as a function of the density n. b Average work output W=N as a function of density n. c Phase diagram of a Bose gas given by a contour plot of the specific heat. The three different polygons delineate interaction-driven thermodynamic cycles in different regimes of the phase diagram corresponding to the three dashed lines in the other panels. The equilibrium states A and C are chosen with fixed c and T for the different cycles. The efficiency saturates at 0:42 given by Eq. 14 in the TLL regime. (d) The energy derivative ∂E=∂c per particle as a function of the interaction strength c. The areas of these three polygons correspond to the works per particle W=N for the three cycles in figure (c), respectively Y.-Y. Chen et al.	2023-02-10T14:04:47.872Z	2019-10-17T00:00:00.000	{ "year": 2019, "sha1": "3fa514d0b4a1b2fc5909c167cc3accbb77002603", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41534-019-0204-5.pdf", "oa_status": "GOLD", "pdf_src": "SpringerNature", "pdf_hash": "3fa514d0b4a1b2fc5909c167cc3accbb77002603", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [] }
250225889	pes2o/s2orc	v3-fos-license	Low-Protein Infant Formula and Obesity Risk Infant formulas have been designed to mimic human milk for infants who cannot be breastfed. The overall goal is to establish similar functional outcomes to assure optimal growth, development, maturation of the immune system, and programming of the metabolic system. However, after decades of improving infant formula, growth patterns and body composition development are still different in formula-fed infants compared to breastfed infants, which could contribute to an increased risk of obesity among formula-fed infants. It has been hypothesized that the lower protein concentration of breast milk compared to infant formula influences infants’ growth and body composition. Thus, several trials in formula-fed infants with different protein intake levels have been performed to test this hypothesis. In this review, we discuss the current evidence on low-protein infant formula and obesity risk, including future perspectives and implications. Background Obesity in children and adolescents is associated with increases in blood pressure and blood glucose levels [1], rates of premature death [2], and the risk of coronary heart disease in adulthood [3]. Moreover, childhood obesity often tracks into adulthood [4], which, on its own, increases the risk of morbidity and mortality [5]. Therefore, childhood overweight and obesity can be considered major public health concerns. Infant feeding may play a pivotal role in the risk of obesity in later life. The strongest and most consistent association for a protective, long-term effect is documented for breastfeeding, which is recommended for the prevention of obesity, cardiovascular diseases, and hypertension, as well as for the reduction of serum cholesterol in adulthood. Could Lower Protein Intake Be a Mediator in the Reduction of Obesity? The mechanism behind the protective effect of breastfeeding is not fully understood. Apart from potential demographic differences, several mechanisms may account for the lower obesity risk in breastfed infants compared to formula-fed infants, such as differences in appetite regulation, early growth patterns, circulating leptin, and the gut microbiome. Another candidate mechanism is early-life protein intake, which will be the focus of this article. The protein concentration in breast milk decreases over the weeks of lactation, while the protein concentration of infant formulas remains constant ( Figure 1). It has been shown During the last decade, systematic reviews have been conducted to assess the longterm health effects of different protein intake levels [7][8][9]. These reviews show conflicting results regarding the beneficial effects of reduced protein intake early in life on obesity risk. The general conclusion is we need more data to provide a conclusive answer. Accordingly, we performed a multicenter double-blinded trial, the ProtEUs study, in which the effects of a modified low-protein infant formula were assessed for both shortterm [10] and long-term [11] growth and body composition. The ProtEUs study showed that the use of this infant formula with a reduced protein content of 20% was safe and supports adequate growth and body composition up until the age of 6 months [10]. Up until the age of 2 years, no differences in outcomes were found between the intervention and the control group, except for a temporal lower fat-free mass index at 4 months of age [11]. In addition and in contrast to the original hypothesis, a reduced protein intake during the first months of life did not affect glucose homeostasis or the insulin-like growth factor (IGF) system in formula-fed infants at the age of 4 months [12]. Since the anabolic hormones insulin and insulin-like growth factor-1 (IGF-1) are responsive to fluctuations in protein intake [13] and have growth-promoting properties [14,15], we expected to find lower insulin and IGF-1 levels in the modified low-protein group. However, there were no significant differences in both hormones between the two formula groups. In line with these results, no association between the IGF-1 level at 4 months and anthropometric outcomes until the age of 2 years was found. So, our conclusion was that up until the age of 2 years, lowering protein intake during the first 6 months of life to values of around 9.2 g/day, when compared to 11.4 g/day, did not affect body composition. However, a lower protein intake during infancy could still be beneficial. For instance, renal function may be positively affected by a lower amount of protein consumed by the infant [16]. It has been shown that excessive protein intake is a potential risk factor for the acceleration of renal function deterioration in children with mild chronic renal insufficiency [17]. The European Childhood Obesity Project (CHOP) study found that kidney size was increased in infants aged 6 months who were fed a higher-protein During the last decade, systematic reviews have been conducted to assess the longterm health effects of different protein intake levels [7][8][9]. These reviews show conflicting results regarding the beneficial effects of reduced protein intake early in life on obesity risk. The general conclusion is we need more data to provide a conclusive answer. Accordingly, we performed a multicenter double-blinded trial, the ProtEUs study, in which the effects of a modified low-protein infant formula were assessed for both shortterm [10] and long-term [11] growth and body composition. The ProtEUs study showed that the use of this infant formula with a reduced protein content of 20% was safe and supports adequate growth and body composition up until the age of 6 months [10]. Up until the age of 2 years, no differences in outcomes were found between the intervention and the control group, except for a temporal lower fat-free mass index at 4 months of age [11]. In addition and in contrast to the original hypothesis, a reduced protein intake during the first months of life did not affect glucose homeostasis or the insulin-like growth factor (IGF) system in formula-fed infants at the age of 4 months [12]. Since the anabolic hormones insulin and insulin-like growth factor-1 (IGF-1) are responsive to fluctuations in protein intake [13] and have growth-promoting properties [14,15], we expected to find lower insulin and IGF-1 levels in the modified low-protein group. However, there were no significant differences in both hormones between the two formula groups. In line with these results, no association between the IGF-1 level at 4 months and anthropometric outcomes until the age of 2 years was found. So, our conclusion was that up until the age of 2 years, lowering protein intake during the first 6 months of life to values of around 9.2 g/day, when compared to 11.4 g/day, did not affect body composition. However, a lower protein intake during infancy could still be beneficial. For instance, renal function may be positively affected by a lower amount of protein consumed by the infant [16]. It has been shown that excessive protein intake is a potential risk factor for the acceleration of renal function deterioration in children with mild chronic renal insufficiency [17]. The European Childhood Obesity Project (CHOP) study found that kidney size was increased in infants aged 6 months who were fed a higher-protein infant formula compared to lower-protein formula or breastfeeding [18]. As infants' kidneys are still developing, given the potential adverse effects of a high protein intake on renal function deterioration, the European Food Safety Authority (EFSA) states that a low-protein intake during the first months of life is desirable. In addition, a high-protein intake early in life leads to increased urea production, which can impair the water balance of infants [19]. Given that lowering the protein intake reduces urea production [10], less protein intake via infant formula could also be beneficial in relation to this concern. It needs to be noted that our study only addressed the effects after two years, so the effects of this nutritional intervention may appear in later childhood or even in early adolescence. Therefore, long-term follow-up of children participating in clinical trials assessing the effects of a low-protein infant formula is needed [20]. A possible explanation of the lack of effect on body composition could be that only very high protein intake increases the risk of obesity [21][22][23][24] (Figure 2). Hence, a threshold of early protein intake and its adverse effect on obesity risk might be possible. This threshold of protein concentration present in infant formula could be between 2.7 (higher-protein infant formula used in the Early Protein and Obesity in Childhood (EPOCH) study; no effect on obesity risk) and 4.4 g/100 kcal (higher-protein infant formula used in the CHOP study; negative effect on obesity risk), based on the available studies ( Figure 2). In addition to the physiological benefits, and from a completely different angle, a protein reduction in infant formula may positively affect the carbon footprint (CFP), a relative measurement of the amount of CO 2 released in the environment during the life cycle of a product or activity, since food production, especially of dairy products, affects the environment in many ways, and dairy, meat, and eggs account for 83% of the greenhouse gas (GHG) emissions from the average EU diet [25,26]. Furthermore, since milk prices are based on the value of solids in the milk, with protein as one of the most expensive components of cow's milk [26], it is plausible that a decrease in protein needs for the production of infant formula reduces both the cost price and the consumer price for infant formula. [23], EPOCH [27], and ProtEUs [10] during the first 12 months of life (from 6 to 12 months, formula-fed infants in the ProtEUs were fed with standard follow-on formula). The protein concentrations in breast milk were adapted from a systematic review and meta-analysis of the nutrient content of term breast milk [28]. Growth in Breastfed and Formula-Fed Infants Even by lowering the protein levels, differences in growth, weight gain, and body composition are still found when comparing formula-fed infants to breastfed infants. In the ProtEUs study, the total fat mass, total fat-free mass, and fat-free mass index (FFMI) were significantly higher in both formula groups versus breastfed infants until the age of 6 months [10]. At the age of 2, significant differences were found in body composition [23], EPOCH [27], and ProtEUs [10] during the first 12 months of life (from 6 to 12 months, formula-fed infants in the ProtEUs were fed with standard follow-on formula). The protein concentrations in breast milk were adapted from a systematic review and meta-analysis of the nutrient content of term breast milk [28]. Growth in Breastfed and Formula-Fed Infants Even by lowering the protein levels, differences in growth, weight gain, and body composition are still found when comparing formula-fed infants to breastfed infants. In the ProtEUs study, the total fat mass, total fat-free mass, and fat-free mass index (FFMI) were significantly higher in both formula groups versus breastfed infants until the age of 6 months [10]. At the age of 2, significant differences were found in body composition between the children fed with low-protein infant formula and the breastfed reference group. In contrast, feeding children a standard infant formula did not result in body composition differences compared to breastfed children [11]. Contrary to these findings, the high-protein group included in the CHOP study had a significantly higher body fat percentage, fat mass index (FMI), and FFMI than the low-protein group at the age of 2 years. Furthermore, the breastfed infants in the CHOP study had the highest body fat percentage compared to both formula groups at that age [29]. The fat mass and fat-free mass (% of body weight) were similar in the formula and breastfed groups included in the EPOCH study at the ages of 1 and 3 years [27]. The mechanisms through which increased protein intake may affect growth and body composition remain to be clarified. As mentioned before, the anabolic hormones insulin and IGF-1 are responsive to fluctuations in protein intake [13] and have growth-stimulating properties [14,15]. Although no short-term associations among insulin, glucose, the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), and body composition have been described during the first year of life; these associations were seen at 2 years of age [12]. This may suggest that the reduction in insulin sensitivity early in life affects body composition at a later age rather than the opposite. There is conflicting evidence comparing IGF-1 levels in formula-fed infants with different protein intake levels [12,27,30], and the association between IGF-1 and (the quality of) growth. The different findings highlight the complexity of the effect of IGF-1 on growth and body composition and the role of protein intake on IGF-1: both protein quantity and quality may be relevant to consider in this respect. The binding proteins (IGF-BP1, BP2, and BP3) and insulin sensitivity may play an important role in growth and body composition trajectories as formula-fed infants had lower insulin sensitivity compared to breastfed infants [12]. The higher growth rates and differences in the body composition of infants fed with low-protein infant formula compared to breastfed infants call for the use of infant formulas with an even lower protein content than 1.7 g protein per 100 kcal. Recently, it was shown that an infant formula with a protein amount of only 1.43 g/100 kcal resulted in a significantly lower weight gain rate than that of formula containing 1.9 g or 2.18 g protein/100 kcal during the first 4 months of life [31]. Although the investigators reported adequate growth, no broad conclusions can be reached regarding the safety of this verylow-protein infant formula. Importantly, the study formulas were not isoenergetic, and no data are yet available on the subjects' body composition, volume intake, and blood parameters. Thus, before we can lower the protein intake to values closer to those found in human milk, more studies are needed on safety and efficacy. The Study Formula In general, it is crucial that both the investigational and the control formula are similar in every respect except for the component of interest. However, numerous reports show that infants can compensate for reduced (macronutrient) intake by increasing volume intake [27,32,33]. This may be assessed with food questionnaires, but the low accuracy and precision of food questionnaires are significant limitations. Unintentional inaccurate measurements and the over-dispensing of infant formula powder by the caregivers [34] are potential additional limitations. Therefore, additional techniques or methods are needed to measure the intake of infant formula (fluid intake) more precisely, for instance, by using stable isotopes [35]. However, this technique can be used for the measurement of fluid only. Therefore, this method cannot be used when, e.g., in addition to infant formula, complementary feeding is introduced. Within studies investigating the effect of protein quantity or quality of infant formulas, the introduction of any other ingredients or additional modifications to experimental formulas, other than quantitative protein, could affect the obtained results [33,36]. As a consequence, findings cannot be attributed solely to differences in protein intake in such circumstances. Using a control product with a protein level and quality that is based on the standard infant formula currently on the market makes it possible to compare the outcome of the intervention group with the result of a control group that reflects the current situation. This makes it easier to evaluate the clinical relevance of the study and its impact. Still, adequate potency is required in studies investigating one component of an experimental formula. On the other hand, designing an experimental study, developing a study formula, and conducting a clinical study requires at least 6 years before these results are available to the public. Adding several ingredients with a supposed beneficial effect on health outcomes may speed up improvements to infant formula, despite that one does not exactly know which ingredient is responsible for what effect. Different Study Designs in Large Studies Addressing Protein Intake in Early Life Different conclusions have been drawn after investigating the effect of protein intake early in life and its effect on later obesity risk. However, no effects of early protein intake on obesity risk in adults are available yet. The CHOP study found an effect of higher protein intake provided by infant formula during the first 12 months (Figure 2) of life on growth, body composition, and obesity risk until the age of 6. The EPOCH study, with a similar intervention period as the CHOP study, did not find an effect of different protein intake on growth and body composition until the age of 5. As argued before, this could be due to the amount of protein intake during early life (Figure 2). At age 2, the CHOP, EPOCH, and ProtEUs studies describe different effects of protein intake on growth and body composition [10,11,22,24,27]. Whereas CHOP and EPOCH used formulas that did not differ substantially in quality, the ProtEUs study used a different approach. In the ProtEUs study, the amino acid content was based upon individually determined amino acid requirements, which led to a completely different amino acid profile. Despite this change, no effect on body composition nor BMI was observed. However, the ProtEUs study demonstrated that an infant formula with a modified amino acid composition and much lower protein intake during infancy generated adequate weight gain rates. The start of the intervention might also be of importance. In general, the effects of nutritional interventions, if present, are likely to be larger the sooner the infant is exposed to the intervention. However, formula feeding may interfere with the optimal feeding choice: breastfeeding. The EPOCH study recruited infants within a week after birth (Figure 2, EPOCH [27]). Infants included in the CHOP study and the ProtEUs study were enrolled during the first 8 and 6 weeks of life, respectively, to ensure the parents were fully supported to breastfeed their infant (instead of starting with infant formula in an early phase). Cooperation with obstetrics departments provides the opportunity to inform parents about the possibility of participating in nutritional research as soon as possible after birth. However, home births are common in the Netherlands (14% of births in 2019 [37]) compared to other European countries such as Germany, France, Britain, and Belgium. Therefore, in addition, informing (future) parents in the prenatal phase would result in an earlier inclusion of infants into a nutritional study. Protein Quality Originally, protein formulas are derived from cow's milk with an adapted whey-tocasein ratio. Despite this adaptation, amino acid concentrations differ substantially from that of breast milk. For healthy term-born infants, up until the age of 6 months, the assumption is that human milk from a well-nourished mother can be regarded as providing optimal intake for the infant. These data can guide the composition of infant formula. However, compositional similarity is not an adequate determinant nor indicator of the safety and differences in nutritional adequacy/bioavailability and digestibility, and the matrix complexity must be taken into account. Therefore, the nutritional requirements of formula-fed infants should be defined on the basis of experimental studies. For amino acids or protein, the nitrogen balance method is used to investigate the difference between nitrogen intake and the amount excreted in urine, feces, skin, and miscellaneous losses, such as breath and sweat. Since it is considered unacceptable to maintain infants and children on either deficient or excessive intake, alternative methods were needed. The availability of isotopically labeled tracers made it possible to determine the metabolic fate of a labeled amino acid at varying dietary intake levels. An approach to estimating indispensable amino acid requirements using amino acid oxidation is the indirect amino acid oxidation (IAAO) technique. The advantages of the IAAO method are the short adaptation time of the study diet (1-3 days), and no dietary restrictions on the intake of the test amino as a result of the fact that the requirement of one amino acid is determined by the oxidation of another amino acid. This makes it possible to study all possible dietary levels of essential amino acids. Within the ProtEUs study, a customized blend of essential amino acids was used in the investigational group. The amino acid composition present in this infant formula was based on the outcomes of clinical trials conducted in healthy term formula-fed infants using the IAAO technique. However, we showed that adapting both the protein quantity and quality of infant formula did not affect infants' growth or body composition. Growth Adequacy Evaluation and Safety Mostly, modifications or the addition of an ingredient new to infant formulas are driven by the manufacturer's desire to produce a formula that mimics the advantages of breastfeeding when provided to the child. The safety of new infant formulas will need to be judged against two controls: the previous version of the formula without the added ingredient and human milk. Overall, in clinical trials, infant growth is used as a safety parameter. The growth of infants fed lower-protein infant formula is compared with that of standard infant formula and breastfed controls. It is difficult to decide whether the growth of breastfed or standard formula-fed infants is the reference growth in these studies. The World Health Organization (WHO) describes the growth of carefully selected and exclusively breastfed infants as the reference growth for infants until the age of 6 months. However, assessing the safety of lower-protein infant formula by comparing the growth of infants fed this proposed formula to the growth of breastfed infants has both regulatory and research issues. Clinical studies that assess the effects of infant formulas with new ingredients or compositions are difficult to design as infants cannot be randomized to consume formula or human milk. It is therefore difficult to sort out which of the factors of breastfeeding are due to nutritional components and which are accounted for by social and psychological factors. Obviously, randomized trials assigning infants to breastfeed or formula-feed are not ethically feasible. The American Academy of Pediatrics (AAP) concluded that the weight gain rate is the single most valuable component of the clinical evaluation of infant formula [38] and recommended that growth studies conducted for the purpose of assessing new infant formulas should be able to detect a difference of 3.0 g of weight gain per day between the investigational infant formula group and the control group (standard infant formula) during the first 3 to 17 weeks of life [38,39]. However, the higher weight gain rate in formula-fed infants during the first months of life compared to breastfed infants might be the cause of higher weight-for-length, BMI, and obesity risk in later life. Therefore, it might be desirable to lower the weight gain rate of formula-fed infants. The evaluation of infants' growth using anthropometric data converted to z scores using the WHO Child Growth Standards [40] makes it possible to assess whether infants' growth is within a normal range. It must be taken into account that these growth curves are based on healthy breastfed infants with different growth characteristics because of their widely diverse cultural backgrounds. Furthermore, the infants were exclusively or predominantly breastfed for at least 4 months only. Improving Infant Formulas Infant formulas have been designed to mimic human milk for infants who cannot be breastfed. The overall goal is to establish similar functional outcomes to assure optimal growth, development, maturation of the immune system, and programming of the metabolic system. The potential advantages of breastfeeding are the reasons behind many of the proposed addition of ingredients to infant formulas. However, after decades of improving infant formula, growth pattern [41] and body composition development [42] are still different in formula-fed infants compared to breastfed infants, which could contribute to an increased risk of obesity among formula-fed infants. In 2017, the EFSA concluded that follow-on formula with a protein content of at least 1.6 g/100 kcal from intact cow's or goat's milk protein, otherwise complying with the requirements of relevant EU legislation, is safe and suitable for healthy infants living in Europe [43]. Our findings call for the use of infant formulas with an even lower protein content than 1.7 g protein per 100 kcal. In line with follow-on formula, it is likely that a protein content of at least 1.8 g/100 kcal of infant formula will also be lowered in the upcoming years. Based on the protein concentrations present in breast milk ( Figure 1) and to better approach the protein needs of formula-fed infants, it has been proposed that different formulas could be created for the first half year of life with different protein concentrations ( Figure 3) [44]. This staging concept could achieve growth and metabolic outcomes more similar to that of breastfed infants. However, additional studies on amino acid requirements in formula-fed infants need to be performed at different ages. rope [43]. Our findings call for the use of infant formulas with an even lower protein content than 1.7 g protein per 100 kcal. In line with follow-on formula, it is likely that a protein content of at least 1.8 g g/100 kcal of infant formula will also be lowered in the upcoming years. Based on the protein concentrations present in breast milk ( Figure 1) and to better approach the protein needs of formula-fed infants, it has been proposed that different formulas could be created for the first half year of life with different protein concentrations ( Figure 3) [44]. This staging concept could achieve growth and metabolic outcomes more similar to that of breastfed infants. However, additional studies on amino acid requirements in formula-fed infants need to be performed at different ages. From the introduction of the first infant formula onwards, milk protein and its processes have been optimized to provide the most optimal amino acid profiles in infant formula. However, based on requirement studies [45][46][47][48][49][50], there is still no intact protein to provide the right composition of essential amino acids. Therefore, the addition of free amino acids is unavoidable, which has its disadvantages. The use of alternative proteins for infant formula, such as egg protein or plant protein, could be considered but need adequate testing for safety and efficacy. Plant Protein-Based Infant Formula Many studies have shown the big impact of the food industry on the environment. Animal-based food production generally has a bigger climate impact than plant-based From the introduction of the first infant formula onwards, milk protein and its processes have been optimized to provide the most optimal amino acid profiles in infant formula. However, based on requirement studies [45][46][47][48][49][50], there is still no intact protein to provide the right composition of essential amino acids. Therefore, the addition of free amino acids is unavoidable, which has its disadvantages. The use of alternative proteins for infant formula, such as egg protein or plant protein, could be considered but need adequate testing for safety and efficacy. Plant Protein-Based Infant Formula Many studies have shown the big impact of the food industry on the environment. Animal-based food production generally has a bigger climate impact than plant-based foods due to higher emissions from its production, manure management, and enteric fermentation [51][52][53]. Although the options for replacing animal protein with plant protein in an adult diet are abundant, in infant formula, these options are very limited. An important reason for this is the high quality of protein present in bovine milk, along with high digestibility [54]. In contrast, plant proteins are of low quality and frequently lack one or more essential amino acids. Moreover, they usually have low digestibility. An example of a promising plant protein for infant formula that warrants further investigation is isolated quinoa proteins (IQPs) as the protein quality is relatively high. Moreover, during its protein isolation process, unfavorable compounds are mostly removed, ensuring that the final product can comply with the maximum residue concentrations allowed. More research is needed before the introduction of IQP in infant formulas is considered, but it has several promising features that warrant further investigation [55]. The replacement of animal proteins with plant proteins involves a lot of critical aspects in order to optimize the effect of processing on product quality. For instance, the heating step needs improvement. In addition, the protein isolates obtained from different plants have a different color than the conventional protein sources used in infant formula. Moreover, the taste of plant products differs a lot from the currently used milk sources. Both parents and infants need to accept these changes and get used to the taste and appearance of the formula. A practical aspect that needs to be taken into account is the certainty of plant product availability. A stable, long-lasting supply of both the agricultural company and the protein manufacturer must be secured. Promising Interventions and Preventive Strategies It has been suggested that subgroups could benefit from a lower-protein intake during the first months of their lives. An example of such a subgroup is infants born to overweight or obese mothers since they are at increased risk of being overweight later in life [56][57][58][59][60][61][62]. A reason for this could be the accelerated growth during the first year of life of infants born to obese mothers [63] since rapid weight gain in infancy is associated with later overweight and obesity [64,65]. Some years ago, it was already shown that an infant formula with a lower protein content slows weight gain in infants aged 3 to 12 months of overweight and obese mothers [66]. To investigate if low-protein infant formula has the potential to reduce the risk of future obesity in this group of infants, we are establishing a large multicenter long-term randomized controlled trial in infants at risk of adiposity, e.g., infants born to overweight or obese mothers. Within this trial, we will investigate the effects of a new infant formula with a protein level closer to breastmilk on growth and body composition. Moreover, the protein used in this investigational infant formula will be partly derived from plants, and the formula will be composed of a high level of intact protein (only 10-20% will be derived from free amino acids). As mentioned before, the transition from animal-based protein to plant-based protein aims to have a positive outcome on the environment, which has been mentioned before. By feeding infants this formula, we not only expect to improve growth and body composition development in formula-fed infants but also to positively influence the climate impact of the food industry. It seems that preventive strategies for reducing childhood obesity should focus on maternal BMI rather than on pregnancy complications such as gestational diabetes and gestational hypertensive disorders [67]. Therefore, behavioral interventions in an overweight or obese pregnant woman might improve clinical outcomes in the child. However, there is conflicting evidence as to whether diet and physical activity in these women would be effective and reduce the incidence of large-for-gestational-age infants, thereby affecting the risk of childhood obesity [68][69][70][71]. Large, high-quality studies including long-term follow-up are warranted to answer these research questions. The adverse effects of a maternal diet with a high glycemic index increase the glucose transfer to the fetus, and, consequently, fetal growth and adiposity [72,73], particularly among overweight or obese pregnant women who are more likely to consume diets rich in carbohydrates and to have impaired glucose tolerance [74]. Recently it has been shown that among overweight and obese women and their children, a higher maternal earlypregnancy dietary glycemic index was associated with increases in childhood BMI and fat accumulation [75]. Therefore, targeting the dietary glycemic index among overweight and obese pregnant women may be a promising tool to help prevent childhood adiposity in the future. The Importance of Breastfeeding Support We can adjust and further improve infant formula, but we will not be able to mimic breast milk or the art of breastfeeding because of its complexity and its effect on infant physiology, including reducing the risk of obesity, among other benefits [76]. Breast milk is the gold standard of infant nutrition and provides a mixture of unique components required for the optimal development of the infant. Breast milk is beneficial for short-and long-term health of both infants and their mothers. Therefore, the support and promotion of breastfeeding by healthcare professionals are of paramount importance. Based on the current evidence, it is necessary to execute new trials evaluating infant formulas with improved protein quality together with further reductions in protein content. In addition, further research should study possible underlying mechanisms of early protein intake on later health and the most optimal protein source for infant formula for optimal growth and body composition.	2022-07-03T15:08:32.530Z	2022-06-30T00:00:00.000	{ "year": 2022, "sha1": "51f043387e525ce951a8a9b0566d84eaf9e273d7", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2072-6643/14/13/2728/pdf?version=1656579779", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "c00763d4d456b951fafb3b9d68e98e663202928f", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
7374416	pes2o/s2orc	v3-fos-license	Application of stereotactic biopsy for diagnosing intracranial lesions in patients with AIDS in China Abstract Rationale: The aim of the study was to evaluate stereotactic biopsy for diagnosing intracranial lesions in patients with AIDS. Patient concerns: Seven AIDS patients with an intracranial lesion who underwent stereotactic biopsy were included in this retrospective study (4 males and 3 females, 15 to 49 years old). The patients’ disease history ranged from 1 month to 1 year. The samples were examined by hematoxylin-eosin (HE) staining and immunohistochemical examination. Diagnoses, interventions and outcomes: All patients were successfully sampled, and the histological results showed inflammation in 4 cases, toxoplasma gondii infection in 1 case, astrocytoma in 1 case, and abscess in 1 case. The clinical diagnosis included toxoplasma encephalitis (TE) in 2 cases, cryptococcus encephalitis in 2 cases, cytomegalovirus (CMV) encephalitis in 2 case, tubercular abscess in 1 case, astrocytoma in 1 case, and co-infection of TE with Cryptococcus infection in 1 patient. The clinical diagnosis was made according to the plasma and cerebrospinal fluid (CSF) laboratory testing, the imaging data and the histological findings. The diagnostic yield was 100%, and the post-operation morbidity was 14.3% (1/7) with an asymptomatic haemorrhage and seizure in 1 case. There was no operation-related mortality. Patients were followed up for 6 months to 6 years; 1 case fully recovered, 4 cases significantly improved in symptoms, and 2 died. Lessons: Stereotactic biopsy is a safe and effective way of diagnosing intracranial lesions in patient with AIDS. It is helpful for the differential diagnosis and for choosing a suitable therapy. Due to the broad spectrum of nervous system abnormalities in AIDS, histological findings are very valuable. However, histology is not a unique tool for making a definite diagnosis, whereas the combination of molecular pathology and stereotactic biopsy should play a more important role in the future. Introduction AIDS (acquired immune deficiency syndrome), a chronic systemic disease, is caused by HIV (human immunodeficiency virus). It remains a major global public health issue. The WHO (World Health Organization) report shows that 2.1 (1.8-2.4) million people are newly infected with HIV, and 1.1 (0.94-1.3) million people died from HIV-related causes globally in 2015. Approximately 36.7 (34.0-39.8) million people were living with HIV at the end of 2015. [1] It is clear that the central nervous system (CNS) is a primary target for HIV. [2] The immunodeficiency caused by the virus can lead to a plethora of other opportunistic cerebral infections as well as neoplasia. [3] CNS involvement presents as the first clinical symptom of AIDS in ∼10% to 20% of patients. [4] Many (40-60%) patients develop neurological abnormalities during the disease course. [5] Moreover, 75% to 80% of all AIDS patients show neuropathological changes in the CNS upon brain autopsy, [6] and multiple pathologies are found in 17% of cases. [7] AIDS-related nervous system abnormalities can be classified into the following 5 types: HIV-related primary infections, such as aseptic meningitis and encephalitis; opportunistic infections of the nervous system, such as cytomegalovirus (CMV) encephalomyelitis, toxoplasma encephalitis (TE), progressive multifocal leukoencephalopathy (PML), herpes viral encephalitis, Cryptococcal meningitis, and Mycobacterium tuberculosis infection; neoplasms, commonly non-Hodgkin's lymphoma (NHL) and occasionally Kaposi's sarcoma; cerebrovascular diseases and peripheral neuropathy. [4] With the rapid development and broad use of medical imaging technology, most AIDS-related brain lesions can be found at an early stage. However, they cannot provide an adequate diagnosis. Usually, a histopathological examination is still needed for choosing a suitable treatment. Brain biopsy is indicated in the absence of clinical and radiologic improvement after empiric therapy. [5] Stereotactic biopsy has been considered with a high diagnostic yield and safety for diagnosing intracranial lesions. [8,9] There are no previous reports on stereotactic biopsy performed in AIDS patients with intracranial lesions in China. In this study, 7 AIDS patients with intracranial lesions who underwent stereotactic biopsy were retrospectively analysed. General information and symptoms From November 2010 to January 2016, 7 AIDS patients with intracranial lesions underwent stereotactic biopsy. There were 4 males and 3 females who were 15 to 49 (mean: 36) years old. The disease history ranged from 1 month to 1 year. Informed consent was collected in advance of biopsy from every patient and their family members. The clinical symptoms included headache, dizziness, limb weakness, fever and cough, dysphagia, vision loss, and epilepsy (Table 1). Preoperative computed tomography (CT) and magnetic resonance imaging (MRI) scanning were performed in all cases. After 3 weeks of empiric treatment, neither clinical symptoms nor imaging improvement were found. Lesion site and size The lesions were located in the cerebellum in 3 cases, in the temporal lobe in 3 cases, in the frontal lobe in 2 cases, in the parietal lobe in 1 case, in the occipital lobe in 1 case, and in the basal ganglia in 2 cases, and there were multiple intracranial lesions in 6 patients ( Table 1, Fig. 1). The lesion sizes varied from 0.2 cmÂ0.3 cmÂ0.5 cm to 2 cmÂ5 cmÂ7 cm. Laboratory tests The toxoplasma IgM and IgG and cytomegalovirus IgM and IgG in blood serum; cytomegalovirus DNA (deoxyribonucleic acid) and Cryptococcus antigen in cerebrospinal fluid (CSF); and routine CSF were examined ( Table 2). Surgical procedure A stereotactic frame (Leksell G type, Sweden) was installed in the patients under local anesthesia; 3.0 T MRI scans (layer thickness: 2 mm) was taken, and T1 enhancement (when enhanced nodularity could be found in the lesions) or T2 FLAIR was used to measured and calculate the coordinates of the intracranial lesions. [10] The trajectory was selected according to which vascular and functional areas of the brain could be avoided. Biopsy specimens were collected from 2 different lesions (in 1 case) or from only a single lesion when there were multiple lesions with similar imaging characteristics. Due to the infectious nature of AIDS, special care was taken to protect the operating room staff. During surgery, the surgeon put on a face mask, protective glasses, disposable surgical gowns, and double gloves. A scalp incision and burr hole were made under local anesthesia. After the dura was opened, an arc was installed, and the coordinates were checked. A side-cut aspirating biopsy needle (with a diameter of 2.5 mm, Leksell, Sweden) was used to obtain tissue specimens from 4 directions. Each specimen was ∼2 Â 2 Â 10 mm in size and fixed in 10% formalin. The samples were examined with hematoxylin-eosin (HE) staining and periodic acid-Schiff (PAS) staining as well as immunohistochemical study with monoclonal antibodies. After the operation, the patients remained in the neurosurgical department for overnight observation. The respiration, heart rate, blood pressure, pulse, pupil response, and patient awareness were monitored. Usually prophylactic anti-epilepsy treatment was given. A head CT scan was performed on the day following operation. Histological result and clinical diagnosis All patients were successfully sampled, and the histological results included inflammation in 4 cases, toxoplasma gondii infection in 1 case, astrocytoma (WHO II) in 1 case, and abscess in 1 case. Taking the histological results, laboratory tests, imaging findings, and clinical data together, the clinical diagnosis included TE in 2 cases, Cryptococcus encephalitis in 2 cases, CMV encephalitis in 2 case, tubercular abscess in 1 case, and astrocytoma in 1 case (WHO II). Co-infection with Cryptococcus encephalitis was found in 1 patient with TE. In cases 3 and 4, the clinical diagnosis was directly obtained from histological results (Fig. 2). The diagnostic yield was 100% (Table 3). Complications Intraoperative biopsy-related cerebral hemorrhage in the sampling area was documented in 1 patient, who also had an early Therapeutic results Based on the clinical diagnosis obtained from the biopsy, the therapies were adjusted in 6 out of 7 patients. Therapies were not adjusted in the patient who was diagnosed with astrocytoma (WHO II), refused further treatment, and died 2 months later. One patient died 1 month later with a diagnosis of Cytomegalovirus encephalitis; his CD4 level was only 1/microliter, and his death was not directly related to the biopsy. Another 5 patients were followed up for 2 to 6 years; 1 patient fully recovered (Fig. 1), and the remaining 4 patients improved significantly. The median survival time was longer than 28 months (range from 24 to 72 months, Table 3). Discussion Although CT and MRI scanners can give us the accurate position of intracranial lesions, they lack the specificity required to secure a firm etiological diagnosis. [11] We searched the published English literature about stereotactic biopsy in AIDS. Databases, including PubMed, EbscoHost, and Ovid, were searched for the keywords "biopsy," "brain," "HIV," "AIDS," and variations of these words. Only human studies published from 1985 to 2016 were considered. Studies were included if they reported original research data on the diagnostic rate of brain stereotactic biopsy in HIV/AIDS patients and the final histopathological diagnosis from brain stereotactic biopsy. Studies were excluded if they met any of the following criteria: a patient population <10; a study population already included in another study; a patient cohort retrospectively selected based on a certain diagnosis, such as PML; the samples were collected by another form of biopsy, such as craniotomy; incomplete data; and the manuscript was a commentary, technical note or review. A total of 19 studies were included for review [3,5,10,[12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] (Table 4). The average positive biopsy rate, complication rate, and hemorrhage morbidity and mortality were 80.00% to 100% (mean 90.98%, 746/820), 0% to 20.00% (mean 5.93%, 37/624), 0% to 18.18% (mean 3.84%, 24/624), and 0% to 9.09% (mean 2.08%, 13/624), respectively. The spectrum of neurologic disease that complicates HIV infection is extremely broad. Similarly, we also analyzed the diagnostic results in the literature and found the following: in 17 (Table 5). Obviously, the most common intracranial lesions included lymphoma, PML, and TE. However, some other papers showed a different pattern. [5,13,15,17,18,22,23] We had a similar result in our study; the clinical diagnosis included TE in 2 cases (28.6%), Cryptococcus encephalitis in 2 cases (28.6%), CMV encephalitis in 2 cases (28.6%), astrocytoma in 1 case (14.3%), and tubercular abscess in 1 case (14.3%); no lymphoma cases were observed. To explain the differences in the proportions of HIV-related focal brain lesion-causing disorders, several possibilities are considered. Ammassari [28] compared the years following the introduction of highly active antiretroviral therapy (HAART) with the pre-HAART era for trends in the proportions of HIVrelated focal brain lesion-causing disorders. They found that the major diagnoses in the 281 patients were toxoplasmic encephalitis (36.4%), primary CNS lymphoma (26.7%), progressive multifocal leukoencephalopathy (18.2%), and focal HIV encephalopathy (5.0%). They concluded that since the introduction of HAART, the incidence of toxoplasmic encephalitis has decreased or stabilized, whereas CNS lymphoma has dramatically declined. According to our observation, lymphoma usually Table 4 Literature of stereotactic biopsy in AIDS patients (1). To improve the diagnostic yield rates and reduce the morbidity, significant attention has been paid to the procedure, including surgical planning to determine the biopsy trajectory, imaging technique, and target choice. Non-necrotic areas or lesions with marked enhancement components are more suitable, whereas the mostly central part of the lesions is necrotic, and the biopsy positive rate may be low. The common postoperative complications of stereotactic biopsy were hemorrhage, edema, epilepsy, infections, and more, whereas the most frequent complication was hemorrhage. [10,29] In this study, 1 patient had an early sampling area with bleeding and a seizure. To reduce the possibility of post-operation hemorrhage, the preoperative platelet count should be more than 100,000/mL, [30] and careful surgical planning should be performed to avoid the vessels within the trajectory. [31] Postoperative CT scans should be regularly performed to find the hemorrhage as early as possible. Stereotactic MRI guided biopsy is a minimally invasive procedure with low morbidity and high diagnostic accuracy for diagnosing and grading brain lesions. The diagnostic accuracy of stereotactic biopsy can be further enhanced by the careful interpretation of neuroradiological and clinical information. Our results differ from previous reports in that the study results were divided into histological results and clinical diagnoses. It is easy to discriminate between tumors and infections, but it is usually very hard to histologically discriminate between different infections types contributing to brain lesions in HIV patients. Very few cases have typical histological characteristics for making a definite pathologic diagnosis. In our study, only 2 of 7 cases were clinically diagnosed from direct histological findings. In other cases, serum and CSF laboratory testing, imaging data (single or multiple lesions, with or without edema or enhancement, etc.), and empirical or diagnostic treatment results were incorporated with histological results to make a final, correct clinical diagnosis. If the patient's symptoms, images, and/ or biochemical test results improved, we could confirm the correctness of the diagnosis. Conclusion The stereotactic biopsy of cerebral lesions is an extremely safe, effective procedure for evaluating intracranial lesions in AIDS patients, establishing a tissue-based diagnosis of CNS lesions, and developing suitable treatments in AIDS patients, and this approach has an acceptable risk/benefit ratio. However, in some cases, the histological diagnosis should be integrated with other information to make a correct clinical diagnosis. On the other hand, the development and application of molecular pathology to diagnose intracranial AIDS lesions may improve the future diagnostic yield of stereotactic biopsies. [32,33,34]	2018-04-03T04:13:55.239Z	2016-12-01T00:00:00.000	{ "year": 2016, "sha1": "57e1673dd006d8b13af62d1860b5185258bd3c7b", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1097/md.0000000000005526", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "57e1673dd006d8b13af62d1860b5185258bd3c7b", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
9281245	pes2o/s2orc	v3-fos-license	Characterization of eleven monosomic alien addition lines added from Gossypium anomalum to Gossypium hirsutum using improved GISH and SSR markers Background Gossypium anomalum (BB genome) possesses the desirable characteristics of drought tolerance, resistance to diseases and insect pests, and the potential for high quality fibers. However, it is difficult to transfer the genes associated with these desirable traits into cultivated cotton (G. hirsutum, AADD genome). Monosomic alien addition lines (MAALs) can be used as a bridge to transfer desired genes from wild species into G. hirsutum. In cotton, however, the high number and smaller size of the chromosomes has resulted in difficulties in discriminating chromosomes from wild species in cultivated cotton background, the development of cotton MAALs has lagged far behind many other crops. To date, no set of G. hirsutum-G. anomalum MAALs was reported. Here the amphiploid (AADDBB genome) derived from G. hirsutum × G. anomalum was used to generate a set of G. hirsutum-G. anomalum MAALs through a combination of consecutive backcrossing, genomic in situ hybridization (GISH), morphological survey and microsatellite marker identification. Results We improved the GISH technique used in our previous research by using a mixture of two probes from G. anomalum and G. herbaceum (AA genome). The results indicate that a ratio of 4:3 (G. anomalum : G. herbaceum) is the most suitable for discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. Using this improved GISH technique, 108 MAAL individuals were isolated. Next, 170 G. hirsutum- and G. anomalum-specific codominant markers were obtained and employed for characterization of these MAAL individuals. Finally, eleven out of 13 MAALs were identified. Unfortunately, we were unable to isolate Chrs. 1Ba and 5Ba due to their very low incidences in backcrossing generation, as these remained in a condition of multiple additions. Conclusions The characterized lines can be employed as bridges for the transfer of desired genes from G. anomalum into G. hirsutum, as well as for gene assignment, isolation of chromosome-specific probes, development of chromosome-specific “paints” for fluorochrome-labeled DNA fragments, physical mapping, and selective isolation and mapping of cDNAs/genes for a particular G. anomalum chromosome. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0913-2) contains supplementary material, which is available to authorized users. Background Cotton is the leading natural textile fiber crop in the world. Approximately 5 % of the world's arable land is used for cotton planting, generating about $630.6 billion in 2011 [1]. Cotton belongs to the Gossypium genus of Malvaceae, which contains five tetraploid species (2n = 4× = 52, AADD genome) and approximately 45 diploid species (eight genomes from A to G and K, 2n = 2× = 26) [2]. Upland cotton (G. hirsutum) is the most widely cultivated species and its production accounts for over 95% of the world's cotton production [3]. During the development of its cultivars, cotton has been subjected to long-term artificial selection, which narrowed its genetic base and gave rise to several difficulties in breeding. Cotton breeders face a scarcity of genetically diverse resources, therefore expanding the genetic base of cotton cultivars is imperative. Wild or untapped species have many excellent characteristics and contain abundant desirable genes, which have yet to be unlocked by prebreeding. G. anomalum (2n = 2× = 26, BB genome) which is native to Africa, mainly Angola and Namibia [2], has the favorable characteristics of drought tolerance and resistance to diseases (cotton wilt, angular leaf spot) and insect pests (springtails, aphids): more importantly, it also possesses genes with the potential to produce high quality fibers (good fiber strength and Fig. 1 Genomic in situ hybridization of the putative alien chromosomes of G. anomalum in the G. hirsutum background using two G. herbaceum and G. anomalum probes. Genomic DNA from G. anomalum and G. herbaceum was labeled with digoxigenin-11-dUTP and Bio-16-dUTP by nick translation, respectively. Chromosomes of the At-subgenome of G. hirsutum were cross-hybridized with both the G. anomalum and G. herbaceum probes and produced white signals and chromosomes of the Dt-subgenome of G. hirsutum were stained with 4′,6-diamidino-2-phenylindole (DAPI) and produced blue signals. Chromosomes from G. anomalum were hybridized with G. anomalum probe and produced red signals. a mitotic chromosome spread of the 52 chromosomes of G. hirsutum. b mitotic chromosome spread of the 26 chromosomes of G. anomalum. c-l mitotic chromosome spread showing the 52 G. hirsutum (white and blue) chromosomes and three (c), two (d), and one (e, f, g, h, i, j, k and l) individual chromosomes of G. anomalum (red), respectively. Scale bar = 5μm fineness) [4] and cytoplasmic male sterility [5][6][7]. However, it is difficult to transfer these desirable genes into cultivated cotton through conventional breeding methods due to the isolation of wild species from cultivated species, which limits chromosome pairing and genetic recombination. Monosomic alien addition lines (MAALs) contain only one alien chromosome in addition to the receptor background chromosomes. MAALs can be used as a bridge to transfer desired genes from wild species into G. hirsutum [8]. Over the past two decades, MAALs have been widely available for numerous crops [9], and these can be used for effectively identifying favorable genes in wild species, allowing for more accurate and faster transfer of such genes to create introgression lines, the effect of specific alien chromosomes to be examined, homeologies with chromosomes of cultivated species to be compared [10,11], and physical maps of specific chromosomes to be constructed [12]. In cotton, however, the high number and smaller size of the chromosomes has resulted in difficulties in discriminating chromosomes from wild species in cultivated cotton background, therefore the development of cotton MAALs has lagged far behind many other crops. No set of cotton MAALs was reported until cotton molecular genetic maps were constructed and a genomic in situ hybridization (GISH) technique for cotton was developed. Previously, only one complete set of G. hirsutum-G. australe MAALs had been developed using simple sequence repeat (SSR) markers and GISH [9,13,14]. Two G. hirsutum-G. somalense MAALs and several G. hirsutum-G. sturtianum MAALs have also been obtained [11,15]. In this study, the G. hirsutum-G. anomalum hexaploid was used as a maternal parent in the continuous backcrossing with upland cotton (recipient parent, G. hirsutum acc. TM-1), and eleven MAALs were isolated using GISH and SSR markers. These MAALs may be useful for mining and transferring favorable genes from G. anomalum into G. hirsutum on a genome-wide scale, mapping genes on chromosomes, analyzing genome structure and evolution, and micro-cloning for chromosomespecific library construction. Results Alien chromosomes from G. anomalum in G. hirsutum were examined by the improved GISH The GISH technique used in our previous research was improved as follows. Genomic DNA extracted from G. anomalum and G. herbaceum was labeled with digoxigenin-11-dUTP and Bio-16-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation, respectively. The labeled DNA was mixed at a variety of ratios for GISH analysis using chromosomes from the mitotic metaphases as target templates. The results indicate that a ratio of 4:3 is the most suitable for discrimination of chromosomes from G. anomalum and the Atsubgenome of G. hirsutum. At this ratio the chromosomes from G. anomalum only hybridized with the G. anomalum probe to produce a red signal, while chromosomes of the At-subgenome of G. hirsutum cross-hybridized with both the G. anomalum and G. herbaceum probes to produce a white signal and chromosomes of the Dt-subgenome of G. hirsutum were stained with 4' ,6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics), producing a blue color. Therefore, the GISH technique has been improved and can be further used to differentiate chromosomes from G. anomalum and the At-subgenome of G. hirsutum (Fig. 1). Note: a GDC genetic distance coverage (cM); b Genetic distance (cM) between two adjacent markers on a chromosome; c Percentage of chromosome covered by markers (%) Progenies of the pentaploid of (G. hirsutum × G. anomalum) × G. hirsutum backcrossed with G. hirsutum were subjected to GISH to determine the number of alien chromosomes transferred from G. anomalum to G. hirsutum using visible fluorescent hybridization signals. Thirty eight individuals of the BC 1 population were examined by GISH analysis (Additional file 1: Table S1). The analysis demonstrated that 27 (71.05 %) carried 2 to 6 alien chromosomes, and 6 (15.79 %) carried 7 to 9 alien chromosomes. Only two (5.26 %) individuals carried one chromosome, 6B a and 13B a of G. anomalum, resepctively. One (2.63 %) plant had no alien chromosomes and the final two (5.26 %) plants had 13 alien chromosomes from G. anomalum ( Fig. 1; Table 1). A total of 290 individuals from the BC 2 generation were further analyzed by GISH. The results indicated that 106 (36.55 %) individuals had one alien chromosome of G. anomalum and 121 (41.72 %) had no alien chromosomes in the G. hirsutum background. 50 (17.24 %) and 10 (3.45 %) individuals carried two and three alien chromosomes, respectively, and another 1 (0.34 %) carried four alien chromosomes. The results demonstrated that most of the BC 2 individuals carried 0-1 alien chromosomes, and only a small number contained multiple alien chromosomes ( Fig. 1; Table 1). Screening of a set of putative G. anomalum chromosomespecific SSR primer pairs During the evolution of Gossypium, chromosomal translocations occurred between genomes A 1 , A 2 , and B 1 , while genome D remained relatively stable [16]. Numerous recent reports also show that translocations occurred between chromosomes in the At-subgenome of the tetraploids [17], while no large structural variation was found in the Dt-subgenome. Therefore, we only selected SSR primers from the Dt -subgenome of the tetraploid cotton linkage map to screen putative G. anomalum chromosome-specific SSR primer pairs. Of Then, based on the tetraploid cotton linkage map constructed by our institute [17], the above 452 codominant markers were located, and of these, 170 wellamplified and evenly distributed codominant markers within an interval of 10 cM were finally selected for use in genotyping the entire BC 1 F 1 and BC 2 F 1 population. The 170 codominant markers were distributed on the Dt-subgenome chromosomes, ranging from 10 to 18 markers per chromosome, with coverage of 80.9-100.0 % and a density of 6.7-15.0 cM of each chromosome ( Table 2; Fig. 2). The G. anomalum-specific SSR markers could be used to track and identify the alien chromosomes from G. anomalum in G. hirsutum. Identity of alien chromosomes from G. anomalum as discriminated by SSR analysis Table 1). Two MAALs were not found, MAAL-1B a and MAAL-5B a ; therefore Chrs. 1B a and 5B a were not isolated and remained as multiple addition lines. During the development of MAALs, Chr. 10B a appeared most frequently, with an incidence of 16.87 %, followed by 15.89 % for 4B a , 12.47 % for 6B a , and 9.29 % for 12B a . Chrs. 5B a and 9B a showed very low incidences of 2.69 % and 2.44 % (Table 1). Morphological traits of MAALs Morphological data were gathered during the cotton growing stage. The results shown in Tables 3, 4 and 5 indicate that the eleven MAALs differed from one another and also differed from their parents in terms of their morphological traits, such as plant type, leaf shape, size of flower and boll (Figs. 5 and 6; Tables 3, 4 and 5). Most of these MAALs grew slower than the recipient, TM-1. We found that MAAL-8B a leaves had a very dark green 4G a (b, c and d) and 6G a to 13G a (e, f, g, h, i, j, k and l), respectively. Scale bar = 5μm color. We also observed that MAAL-7B a , MAAL-12B a and MAAL-13B a had relatively bigger leaves, while MAAL-8B a , MAAL-9B a and MAAL-10B a had relatively smaller leaves than the other lines (Fig. 5b). In addition, MAAL-6B a , MAAL-10B a , MAAL-11B a and MAAL-12B a had relatively larger flowers than the others. Only MAAL-7B a showed petal spots and MAAL-6B a had very light brown fibers, indicating that genes for petal spots and light brown fibers are located on chromosomes 7B a and 6B a (Figs. 5a and 6d), respectively. MAAL-2B a and MAAL-12B a had relatively longer bolls and MAAL-7B a had the widest boll diameter, while MAAL-8B a had the shortest bolls and MAAL-10B a had the smallest boll diameter (Fig. 6c). MAAL-6B a , MAAL-7B a and MAAL-9B a had a relatively larger boll weight, while MAAL-8B a , MAAL-10B a and MAAL-11B a had a relatively smaller boll weight than the others (Table 4). We found that MAAL-7B a had longer fibers than the others (Fig.6d) Discussion MAALs are powerful tools in crop breeding since they can be used to produce alien translocation and substitution lines, to study interspecific relationships, and to construct single chromosome libraries. They can also be used in gene mining, gene assignment, gene expression pattern analysis, gene function analysis, physical gene mapping, isolation of chromosome-specific probes, selective isolation and mapping of cDNA/gene of a particular chromosome. Numerous reports have shown that the development of MAALs has been successfully achieved in many crops such as wheat [18][19][20][21], rice [22] tomato [23], potato [24], cucumber [25], tobacco [26], oat [12], sugar beet [27,28], and rapeseed [29,30]. MAALs have played and are playing important roles in numerous types of plant genomic research. The development of MAALs in Gossypium began as early as the 1980s but greatly lagged behind other crops due to the large number (2n = 52) and small size of chromosomes, which led to difficulty in accurately discriminating each chromosome, therefore, little progress has been made in cotton. So far only one set of MAALs has been completed [9], and this work benefited from advances in the development of GISH and molecular markers in cotton. However, in this study, due to the very close relationship between chromosomes of the At-subgenome in G. hirsutum and those in G. anomalum often leading to cross-hybridization in GISH, we had to first improve the GISH technique by adjusting the ratio of the two different probes used. We tried five different combinations and found that the ratio of 4:3 was more suitable than any others for the discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. Therefore, using a combination of the improved GISH methodology, G. anomalum chromosome-specific SSR molecular markers and conventional morphological survey, eleven MAALs were isolated and characterized, and two remain to be isolated from multiple addition states by further backcrossing. Several previous reports showed that G. anomalum contains the favorable characteristics of drought tolerance and resistance to diseases (cotton Verticillium wilt, angular leaf spot) and insect pests (springtails, aphids); and more importantly, it also possesses genes with the Fig. 4 A set of G. anomalum-specific SSR markers were used to identify individual alien chromosomes of G. anomalum in G. hirsutum. a-k the G. anomalum-specific amplicons were obtained using 11 individual chromosome-specific primer pairs for markers; NAU5421, BNL2443, NAU7579, NAU3677, dPL0492, BNL2597, BNL3383, NAU4881, NAU9520, dPL0379, and dPL0864. The chromosomes correspond to D 2 to D 4 and D 6 to D 13 in cultivated tetraploid cotton. P1, G. hirsutum; P2, G. anomalum; F1, the hexaploid of G. hirsutum and G. anomalum; 1-11 show that each of these plants possesses a single different individual chromosome from G. anomalum, corresponding to 2B a to 4B a , and 6B a to 13B a . M, molecular size marker (50 bp ladder). Arrows (red) indicate chromosome-specific markers for G. anomalum potential to produce high quality fibers (good fiber strength and fineness) [4] and cytoplasmic male sterility [5][6][7]. Our previous reports also demonstrated that using G. anomalum as a donor parent and G. hirsutum as a recipient parent, a series of introgression lines with longer, stronger and finer fibers has been developed [31]. Shen et al. [32] mapped QTLs on Chr. 7 affecting fiber length in an F 2 population derived from G. anomalum introgression line 7235 crossed with TM-1. However, in this study, we investigated some agronomic traits of MAALs and observed that most MAALs had poor performances in fiber quality or fiber yield components, implying that the added alien chromosomes had negative effects on most agronomic traits (Tables 4 and 6; Fig. 6). For example, the bolls of all MAALs were lighter than those of the recipient TM-1; and the fibers of all six MAALs were shorter than TM-1 (the fiber properties of the other five MAALs were not measured due to a lack of fiber samples). The resultant phenomena may be caused by linkage drag, which means that there were very close linkages between favorable and unfavorable genes on the same chromosome, even though the fibers of some MAALs were found to be stronger than those of TM-1. Therefore, to enhance the transfer of desirable genes and eliminate undesirable genes from G. anomalum, it is necessary to break the linkage drags to promote chromosome recombination between G. hirsutum and G. anomalum. The development of chromosome translocation lines or introgression lines may be an alternative choice based on the MAALs. We deeply believe that these MAALs of G. hirsutum-G. anomalum would be a powerful tool for systematically transferring desirable genes chromosome by chromosome from G. anomalum into G. hirsutum, as well as for gene mining, gene assignment, gene function analysis, gene physical mapping, isolation of chromosome-specific probes, selective isolation and mapping of cDNAs for a particular chromosome, and genomic research. Conclusions From this study, we draw two conclusions. (1) The GISH technique used in our previous research has been improved by using a mixture of two probes at a ratio of 4:3 (G. anomalum and G. herbaceum) to avoid cross-hybridization caused by the very close relationship between chromosomes from G. anomalum and the At-subgenome of G. hirsutum, which can be suitable for recognizing alien chromosomes of G. anomalum in G. hirsutum background. (2) Eleven out of 13 potential MAALs were isolated, which would be used, at the chromosome level, for effectively identifying favorable genes in G. anomalum, allowing for more accurate and faster transfer of such genes to create introgression lines, the effect of specific alien chromosomes to be examined, homeologies with chromosomes of cultivated species to be compared, and physical maps of specific chromosomes to be constructed. Plant materials In 2012, the amphiploid (allohexaploid) (2n = 6× = 78, AADDBB genome) (previously obtained in our institue) derived from the doubled triploid hybrid of G. hirsutum (2n = 4× = 52, AADD genome) × G. anomalum (2n = 2× = 26, BB genome, obtained from Cotton Research Institute of Chinese Academy of Agricultural Sciences) was backcrossed as a maternal parent with G. hirsutum acc TM-1, the genetic standard line of upland cotton. In 2013, two pentaploid individuals were obtained at Pailou Experimental Station of Nanjing Agricultural University (PES/NJAU) and used as both paternal and maternal parents in the backcross with TM-1. The BC 1 seeds obtained were planted in plastic cups with sterilized soil and incubated in the phytotron at Nanjing Agricultural Scheme for developing the monosomic alien addition lines The interspecific hexaploid was backcrossed with Gossypium hirsutum acc TM-1 (obtained from the Southern Plains Agricultural Research Center, USDA-ARS) to produce the pentaploid (2n = 5× = 65, AADDB genome), then the pentaploid progenies were further consecutively backcrossed with TM-1 to generate backcross progenies (BC 1 and BC 2 ). GISH was used to characterize alien chromosomes in all backcross progenies from the BC 1 generation. When more than one alien chromosome was added from G. anomalum, the progenies were further backcrossed with TM-1 to produce monosomic alien addition lines. If only one alien chromosome was added to the background of Upland cotton, the progenies were further examined using chromosomespecific SSR markers of G. anomalum to determine the identity of the added chromosome. G. anomalum, TM-1, BC 1 , and BC 2 chromosome preparation Cotton seeds were cultivated in an incubator at 29°C and their root tips were cut off when they grew to 3 cm long (seedling plant). The tips were immersed in 25 μg/ ml cycloheximide at room temperature for 2 h to accumulate metaphase cells and then transferred to Carnoy I Finally, slides containing at least 20 well-spread somatic chromosomes at mitotic metaphase were prepared and stored at -70°C overnight. Genomic in situ hybridization (GISH) Due to the very close relationships that exist between chromosomes of the B genome in G. anomalum and those of the At subgenome in G. hirsutum, two probes were employed here to avoid cross-hybridization between these chromosomes. Genomic DNA extracted from G. anomalum and G. herbaceum (2n = 2× = 26, AA genome) were labeled with digoxigenin-11-dUTP and Bio-16-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation, respectively. The probe fragment size was between 200-500 bp. Fluorescence in situ hybridization was carried out as described by [33] and [9] with some modifications. The mixing ratio of DNA probes from G. anomalum and G. herbaceum were adjusted to five different ratios, 2:1, 4:3, 1:1, 2:3, and 1:2, to determine the optimal ratio for discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. DNA extraction and G. anomalum-specific primer screening Genomic DNA was extracted from young leaves of the two parents, G. anomalum and G. hirsutum acc. TM-1, the interspecific hexaploid, the pentaploid, and the BC 1 and BC 2 individuals using the method described by [34] with some modifications. A total of 2,168 pairs of SSR primers were selected from the high density genetic linkage map of Sea island and Upland cotton constructed in our institute [17] and employed to screen G. anomalumspecific primers. PCR reactions were performed and their amplified products were separated by PAGE, as described by [35,36]. The G. anomalum-specific marker primers obtained were further used to characterize each chromosome from G. anomalum. MAAL nomenclature Thirteen G. hirsutum-G. anomalum MAALs were named MAAL-1B a to MAAL-13B a , according to the method described by [9], in which B represents the B genome of G. anomalum and 'a' refers to the initial letter of anomalum. The chromosome numbers 1 to 13 in the B genome of G. anomalum correspond to the homoeologous chromosomes in the Dt-subgenome of tetraploid cotton. Investigation of agronomic traits of monosomic alien addition line At the point of transition from the vegetative to the reproductive stage, the shape and size of fully expanded leaves from the same position in TM-1, G. anomalum, hexaploid and MAAL plants were investigated. Floral morphological traits from these MAALs were investigated in the flowering period. The size of cotton bolls at 35 days post-anthesis was also measured by vernier caliper. Finally, the hundred-seed weight, ginning outturn and single boll weight of the matured bolls were investigated. All the data were analyzed using the SPSS software version 18.0. Additional file Additional file 1: Table S1.	2017-08-03T01:39:53.213Z	2016-10-07T00:00:00.000	{ "year": 2016, "sha1": "3f3b26f0e5ee320ebac35fe318ad58b07428a8d3", "oa_license": "CCBY", "oa_url": "https://bmcplantbiol.biomedcentral.com/track/pdf/10.1186/s12870-016-0913-2", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "4fbc30ce5d743bd70b88aa2f4464a1d6eac4a5ef", "s2fieldsofstudy": [ "Biology", "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
5071073	pes2o/s2orc	v3-fos-license	Channel-aware adaptive receivers for linearly precoded MIMO-OFDM systems with imperfect CSIT Within the context of linearly precoded MIMO-OFDM (combination of multiple antenna techniques with multicarrier transmission schemes such as orthogonal frequency division multiplexing) systems with multiple-stream transmission, maximum likelihood detection (MLD) has been shown to offer large performance gains when compared to an all-linear setup (i.e., linear transmitter/receiver) when either perfect or imperfect channel state information at the transmitter (CSIT) is available. Unfortunately, these gains come at the cost of a higher complexity. In particular, the increase in computational cost is more significant when the receiver is designed to operate with soft information and even more dramatic when, in order to optimise error rate performance, iterative decoding is allowed. In order to exploit the best features of each detection technique, this paper proposes a method to selectively choose the detection strategy (ML or linear) for each individual subcarrier as a function of the instantaneous channel conditions and CSIT accuracy. Numerical results show that a cautious and selective use of ML detection substantially reduces complexity while still reaping most of the performance advantage. Introduction The combination of multiple antenna techniques (socalled MIMO) with multicarrier transmission schemes such as orthogonal frequency division multiplexing (OFDM), the so-called MIMO-OFDM architecture, is now at the heart of most state-of-the-art wireless systems and future standards [1][2][3].In this context, techniques that exploit the availability of channel state information at the transmitter (CSIT) have been intensively researched (see [4,5] for a review).It is well known that the capacityachieving strategy when perfect CSIT is available is to pre-cancel interference among simultaneously transmitted streams, a scheme usually referred to as dirty paper coding (DPC) [6]; however, its high computational cost motivates the need for simpler strategies.Palomar et al., in their landmark paper [7], introduced a framework for the optimisation of MIMO-OFDM systems with CSIT based on linear processing at the transmitter and receiver.The proposed scheme defines transmit and receiver filters that are based on the singular value decomposition (SVD) of the whitened channel matrix and performs a distribution of the available power among the different transmit modes using waterfilling in accordance with various performance metrics.Further insight on this architecture was provided in [8,9], where the diversity order performance was analysed for single-and multiple-stream configurations (i.e.spatial division multiplexing beamforming).These studies showed that such schemes lose diversity when increasing the number of transmitted streams as performance becomes dominated by the worst employed spatial transmission mode.Very recently, it has been shown in [10] that full diversity can be restored by incorporating a linear transformation at transmission spreading the symbols to be transmitted over the available spatial modes.Unfortunately, this diversity advantage comes at the cost of having to rely on joint maximum likelihood detection (MLD) at the receiver. Most of these results assumed perfect CSIT, which is a rather optimistic hypothesis in practical deployments.Channel feedback delay and quantization noise are typical impairments affecting the quality of CSIT, whose effects should be accounted for.To this end, [11] incorporated channel knowledge imperfections in the design http://jwcn.eurasipjournals.com/content/2013/1/240 of a linear transmitter/receiver architecture that, as the CSIT approaches perfection, converges towards the solution of [7].A related work by Sengul et al. [12] proposes a codebook construction methodology based on a Lloyd quantizer design that aims at the improvement of the robustness against imperfect CSIT in linearly precoded bit-interleaved coded modulation (BICM) systems while still relying on linear filters at the receiver side.In [13], precoding strategies combined with forward error correction were considered but again limiting the context to that of linear detectors.Remarkably, it should be noted that, under imperfect CSIT, the optimisation of error rate metrics requires MLD-based reception. The use of MLD in combination with linear precoding has been extensively studied in [14] and [15] under perfect and imperfect CSIT, showing that large reductions in the bit/packet error rate (BER/PER) are possible at the cost of an increased receiver complexity as even smart implementations (i.e.sphere decoding [16]) are computationally demanding at low signal-to-noise ratios (SNRs), where practical systems usually operate [17].To address this downside, this paper proposes the selective and careful application of MLD only under very specific conditions, which depend on the specific channel realisation and CSIT accuracy, while otherwise relying on linear detection.The introduced technique is shown to be effective with various architectures, namely hard-, softand iteratively decoded receivers.This scheme is specially appropriate for scenarios where the channel and/or CSIT accuracy may vary widely from packet to packet.As an illustrative example of this type of scenario, this paper considers wireless local area networks (WLANs) based on the IEEE 802.11n standard [18], whose multipleaccess policy based on carrier sense multiple access with collision avoidance (CSMA/CA) causes substantial variations in the accuracy of the available channel information at the transmitter.It is worth mentioning that a related idea, but in the context of MIMO systems without CSIT and restricted to the 2 × 2 MIMO setup, was introduced in [19], where the detection strategy selection was based on the condition number of the channel correlation matrix resulting in the utilisation of linear and ML detection for well-conditioned and ill-conditioned channels, respectively. The rest of the paper is organised as follows: Section 2 introduces the system model under consideration including a description of the assumptions regarding the channel model and CSIT accuracy.Section 3 begins by reviewing the two classic detectors at hand, minimum mean square error (MMSE) and MLD, within the context of the considered scenario and subsequently introduces the channel-aware adaptive detector.In Section 4, the adaptive detector concept is revisited within the framework of soft and iterative detection strategies.Numerical results are presented in Section 5 illustrating the benefits of the channel-aware adaptive detector.Finally, the main outcomes of this work are recapped in Section 6. This introduction concludes with a brief notational remark.Vectors and matrices are denoted by bold lower case and bold upper case letters, respectively.The superscript (•) H denotes the complex transpose (Hermitian) of the corresponding variable.The symbol I k denotes the k-dimensional identity matrix, whereas D(x) is used to represent a (block) diagonal matrix having x at its main (block) diagonal and [A] i,j serves to indicate the (i, j)-element of matrix A. System model A MIMO-OFDM architecture is considered where the transmitter and receiver are equipped with N T and N R antennas, respectively, which are capable of simultaneously transmitting N s ≤ min(N T , N R ) data streams.The available system bandwidth is exploited by means of N c subcarriers out of which N d are used to carry data and N p are destined to pilot signals and guard bands. Transmitter processing Following the usual processing steps of BICM systems, incoming information packets are first channel encoded and possibly punctured to satisfy prescribed rate constraints, and the resulting bits are then distributed among N s streams corresponding to different spatial branches.On each spatial branch, bits are interleaved and mapped onto modulation symbols drawn from an Mary modulation alphabet, resulting in the set of symbol streams s 1 , • • • , s N s .Each spatial stream is then organised into segments of N d symbols that will eventually become OFDM symbols (with the addition of pilot/null subcarriers) (Figure 1). The mapping from information to transmit symbols (i.e.precoding) on subcarrier q at discrete time instant n is carried out as where W[q, n], with dimensions N T × N s , represents the precoding matrix and s with s i [q, n] denoting the symbol corresponding to the ith stream to be transmitted on the qth subcarrier at time instant n.Finally, the precoded symbols are supplied to an OFDM modulator consisting of an IFFT plus the addition of a cyclic prefix (CP). Channel modelling The channel between an arbitrary pair of Tx and Rx antennas is assumed to be frequency-selective with a scenario-dependent power delay profile common to all Tx-Rx pairs.Let us denote by H[q, n] the matrix representing the channel frequency response on the qth subcarrier http://jwcn.eurasipjournals.com/content/2013/1/240 Figure 1 Transmitter block diagram for linearly precoded system. where an arbitrary entry h i,j [q, n] (1 ≤ i ≤ N R , 1 ≤ j ≤ N T ) corresponds to the frequency response on subcarrier q of the channel linking Tx antenna j and Rx antenna i. It is assumed that the entries of H[q, n] are uncorrelated a (i.e.Tx/Rx antennas are sufficiently spaced).Channel estimation at the receiver is considered accurate enough so as to render any CSI error at the Rx side (CSIR) negligible.In contrast, CSIT is considered to be imperfect in such a way that [11] where H[q, n] represents the channel mean known at the transmitter (estimated channel), [q, n] denotes the channel estimation noise whose entries are CN (0, 1), and ρ[n] ∈ [0, 1] can be a packet-dependent random variable effectively modelling the CSIT accuracy for the current packet, which is also known at the receiver side. Reception equation Figure 2 depicts a generic receiver based on hard decoding.Reception begins with the standard OFDM demodulation consisting of the CP removal and FFT processing.Perfect Tx/Rx synchronisation and a CP length exceeding the duration of the channel impulse response are assumed, thus guaranteeing that consecutive OFDM symbols do not suffer from inter-block interference.Under these conditions, the detection procedure works on an OFDM symbol basis, thus allowing us to drop the timerelated index n from subsequent equations (e.g.s[q, n] → s[q] ).The received baseband samples on subcarrier q, denoted by T with r i [q] representing the received sample on the ith receive antenna, for an arbitrary OFDM symbol are given by where A[q] = H[q] W[q], and the N R × 1 vector υ[q] corresponds to the noise samples affecting the qth subcarrier, which are assumed to be i.i.d. and drawn from a zero-mean complex Gaussian distribution with variance σ 2 v .It is assumed that, on average, each subcarrier has unit energy available to transmit N s symbols and that the channel frequency response is normalised so that the average signal-to-noise ratio per subcarrier can be defined as Channel-aware robust detection In [11] a linear architecture is proposed able to robustly cope with CSIT imperfections.In particular, a practical (uncoded/hard decoded) BER minimisation approach consists of a precoding filter defined by where U[q] has as columns the eigenvectors of Rq = is the power allocation matrix whose coefficients can be found optimising a prescribed objective metric [7].The matrix C is a (subcarrier-independent) unitary transform that spreads the incoming symbols among the different spatial modes.It has been recently shown in [10] that choosing C to be the product of a unitary transform (e.g.Fourier, Hadamard) and a constellation rotation, in the form of a diagonal matrix with different phase factors, maximises diversity and leads to optimum performance in terms of BER. Linear and non-linear detectors Given the received samples in (4), the optimum linear MMSE receive filter is given by [11] allowing (hard) symbol estimates to be obtained as This detector is also BER optimal when two conditions are met: 1.The CSIT is perfect.2. The rotation matrix C in ( 5) is diagonal. Under these two conditions, the overall precoderchannnel-detector chain is perfectly diagonalized, thus allowing symbol-by-symbol detection without any performance penalty.Nevertheless, note that condition 2, despite simplifying the detection procedure, inherently induces a diversity loss as it implies that, in loaded setups (N s = min{N T , N R }), some of the symbols are transmitted on weak spatial modes that will dominate the error performance [8].Very recently, [14] and [15] have studied what gains are achieved when one or both of these conditions are not fulfilled and the receiver linearity constraint is neglected, thus allowing the application of MLD.In this case, symbol estimates are given by Results in [14,15] show that, in fully loaded configurations, ( 8) is very advantageous over (7) in terms of BER, although this comes at the cost of an increased receiver complexity, even when employing efficient implementations such as sphere decoding.Regardless of the detection method, either MMSE or ML, estimated symbols are then demodulated, and the corresponding bits, subsequently de-interleaved, (spatially) de-parsed and finally supplied to a Viterbi decoder to obtain an estimate of the transmitted packet. Adaptive detector Assuming that the receiver has knowledge of the precoding matrix used by the transmitter b , a detection strategy decision can be made based on the instantaneous channel realisation and specific CSIT accuracy.To this end, let the linear receiver form the overall processing matrix, where the diagonal terms correspond to the eigenvalues of Rq and form the set of accessible spatial modes available for transmission.There is an intimate connection between the two conditions guaranteeing the optimality of MMSE detection and the structure of ( 9): • When conditions 1 and 2 hold, it is obvious that β i,j [q] = 0 ∀i, j and MMSE detection is optimum.• When condition 1 holds and condition 2 does not hold, the magnitude of the interfering terms β i,j [q] depends on the conditioning of H[q].If the matrix is well conditioned, MMSE will perform well, but if it is not, MLD will result in a significant advantage.• When condition 1 does not hold and condition 2 holds, it will depend on the actual realisation of parameter ρ.If ρ 1, the overall processing matrix will be virtually diagonalized, making MMSE detection optimal.In contrast, the further away ρ is from 1, the more significant interfering terms β i,j [q] will become, thus requiring MLD for acceptable performance.• When conditions 1 and 2 do not hold, the detection strategy selection will depend on both the channel matrix conditioning and the specific CSIT accuracy. To find a decision criterion able to determine the most appropriate detection strategy in light of the instantaneous conditions of the system, let us define SNR i [q] as Similarly, the signal-to-interference plus noise ratio (SINR) can be defined as Clearly, on those subcarriers where SNR i [q] SINR i [q], there is a strong indication of significant interfering terms (either because of poor channel conditioning and/or mismatched transmit and receive filters due to imperfect CSIT) that would favour the application of MLD.With this observation in mind, Algorithm 1 can be used to decide which detection scheme should be selected on the qth subcarrier.To this end, the algorithm evaluates expressions (10) and (11) on each subcarrier for each http://jwcn.eurasipjournals.com/content/2013/1/240spatial mode and, utilising the mapping F : N s → , derives two metrics, one for the overall SNR and another one for the overall SINR, on which the detection decision for that subcarrier will be based.Potential mapping functions to be used are the mean, minimum or maximum of the incoming N s -long vector.Parameter α ∈ [0, 1] in Algorithm 1 is used to allow a certain amount of interference to be present yet still relying on MMSE detection.It effectively acts as a complexity-performance trading knob: as α → 0, the more interference is allowed and the more often the system relies on MMSE detection (lower complexity and poorer performance than with ML).Two important remarks are in place: • The subcarrier-based nature of the algorithm is to be emphasised.Most likely, for a given packet, some of the subcarriers will be linearly detected while others will require the use of MLD.• Sphere detection-based MLD usually starts the search procedure using the zero forcing (ZF) solution as the centre of the sphere.However, if an estimate of the noise power σ 2 is available, centering the search around the MMSE solution is computationally advantageous [20]; thus, the computation of ( 6), even in the case of eventually relying on MLD, still plays a role. Algorithm 1 Detection decision procedure. Detection on subcarrier q using ML.else Detection on subcarrier q using MMSE.end if Iterative soft detection Despite the importance of hard decoding in its own right, most practical deployments are based on the use of softbased decoding principles.Consequently, it is important to consider the performance of the proposed adaptive detection scheme when the component detectors extract soft information, typically in the form of log-likelihood ratios (LLRs), from the received samples.Furthermore, soft detectors are often able to operate iteratively following turbo receiver design principles.In this case, the MIMO detector and channel decoder exchange (soft) information back and forth with the corresponding LLRs becoming more reliable at each iteration [21].The next subsections describe two popular soft-based detection schemes, one based on MLD and another one based on MMSE, and the iterative extension within the context of the considered setup. MLD-based soft detection For the case of ML detection based on the sphere decoder, the authors in [21] introduced the list sphere decoder (LSD) that not only renders the most likely (hard) estimate s[q] ML but also provides a list of the closest candidate points to the ML solution.This list enables the derivation of soft information in the form of LLR for each bit.To this end, the transformation s[q] = M (b) is defined as the modulation mapping to arrive at symbol vector T where Making use of the max-log approximation, the LLR for a given bit b p (belonging to an arbitrary subcarrier q and stream i) can be approximated by [21] [q] being the N cand group candidates for which the Euclidean distance r[q] −A[q] s[q] 2 is smallest. MMSE-based soft detection In order to derive soft estimates from the MMSE-detected samples, the procedure described in [22] is adapted to the situation at hand.To this end, let us define the post-MMSE receive filter SNR for an arbitrary subcarrier q and stream i as (13) http://jwcn.eurasipjournals.com/content/2013/1/240 Based on [22], the LLR for the in-phase bit on the pth position of the symbol stream i is given by where D I,p is given by the mappings defined in [22] (14 to 18).LLRs for the bits in quadrature are computed using an analogous procedure. Note that the extraction of the soft information is much more involved in the case of MLD-based processing, even when employing the lower-complexity LSD, than for the MMSE-based detector.As shown in Figure 3 and ignoring for now the iterative processing (shaded region), the resulting LLR streams L D1 1 , • • • , L D1 N s , whose entries are given by L ML i or L MMSE i depending on the detection procedure employed for the corresponding subcarrier, are subsequently de-segmented, de-interleaved, de-parsed and de-punctured to form the coded LLR stream that is finally supplied to a Viterbi decoder to yield the estimated information bits. Channel-aware iterative soft detection Further performance improvements in the form of lower error rates can be achieved if the detector and channel decoder are allowed to exchange information, specially when the detector is based on ML detection principles [21].In fact, it has been observed that when detection relies on linear processing techniques such as MMSE, the benefits of iterative reception become rather marginal [23].Consequently, in this work, the application of iterative processing is limited to those cases where MLD has been selected as the preferred detection strategy. As shown in Figure 3, each (subcarrier-based) detector operates in accordance with Algorithm 1 to decide which detection strategy, MLD or MMSE, should be used and computes the corresponding LLR for each bit using (12) or (14), respectively.The de-segmentation process is then in charge of collecting the LLR values computed for the successive OFDM symbols forming a packet/stream resulting in the LLR streams L D1 1 , • • • , L D1 N s .These LLRs, after subtracting any a priori knowledge available from previous iterations, give rise to the extrinsic information L E1 1 , • • • , L E1 N s , which after suitable de-interleaving and spatial de-parsing, results in the input stream to the maximum a posteriori (MAP) decoder (L A2 1 ).The MAP decoder has a double output: on one hand, an estimate of the information symbols, and on the other hand, a refined version of the input LLRs.This latter output, L D2 1 , after subtracting already known information (L A2 1 ), results in the extrinsic information L E2 1 to be fed back to the detection stage.To this end, signal L E2 1 is suitably parsed and interleaved resulting in the sequences L A1 1 , forming the a priori information for the next turbo iteration.Note that only the LLRs corresponding to those subcarriers that have been detected using MLD are fed back to the detector (denoted in Figure 3 by ) while no information is fed back to the MMSE-detected subcarriers. Simulation setup The simulation environment has been defined in accordance with specifications from the IEEE 802.11n architectures [18], considering a setup with N T = N R = 4 antennas transmitting N s = 4 streams.The system operates on a bandwidth of B = 20 MHz using N c = 64 subcarriers out of which N d = 52 are used for data transmission and the rest are devoted to pilot signalling and guard bands.For all simulations, transmission modes with either quadrature phase shift keying (QPSK) or 16-QAM modulation and a 1/2-rate convolutional channel coder with generator polynomials g [133, 171] 8 have been employed.Full cyclic prefix is used in order to guarantee the avoidance of interference among successively transmitted OFDM symbols.Power allocation matrices are computed according to the ARITH-MSE criterion in [7] for hard decoding and uniform power allocation for soft/iterative decoding d .Two different spatial spreading matrices have been considered, C = I N s (no spreading) and C = N s (full spreading), with I N s and N s denoting the identity and rotated Walsh-Hadamard matrices of dimension N s , respectively. Interestingly, IEEE 802.11-based systems are a representative scenario where the CSIT accuracy may (widely) vary over a short time frame.This is due to the channel contention mechanism that, based on CSMA/CA, causes the time span between the reception of channel-related feedback at Tx and its utilisation to fluctuate on a packet basis and, moreover, to make it heavily dependent on the number of active users in the system.Note that when users enter or exit the system, the average delay in using the acquired CSIT for the rest of the active users is likely to vary, thus effectively implying a degradation or improvement in the CSIT accuracy.For the results shown here, the channel, generated following the specifications in [24], is assumed to remain static over the duration of a packet and vary independently from packet to packet (block fading). Without loss of generality, it is assumed in (3) that ρ = 1 − ϕ where ϕ is a random variable with a gamma probability density function (μ, 1) truncated to the interval [0, 1].In particular, the results here are presented for μ = 0.015, μ = 0.37 and μ = 1.31, which lead to average values of ρ = 0.99, ρ = 0.8 and ρ = 0.5, respectively.Figure 4 depicts histograms for 2,500 realisations (i.e.packets) of the resulting ρ parameter for the three different values of μ.It can clearly be appreciated that, indeed, a value of μ = 0.015 results in a very accurate CSIT for most of the frames, whereas when μ = 0.37 or μ = 1.31, the corresponding ρ values clearly suggest that a significant proportion of packets are transmitted with a rather imperfect CSIT.Note that the generation of ρ based on the gamma distribution allows the modelling of various network operating conditions just by adjusting a single parameter (μ).Algorithm 1 parameters for the hard-decoded setup have been chosen to be α ∈ {0.25, 0.5, 0.75} while F(x) = min(x), and for the softdecoded setup, α ∈ {0.75, 0.85, 0.95} and F(x) = min(x).Lastly, for the iterative decoding configuration, Algorithm 1 is configured with F(x) = min(x) and α = 0.975.The different choice of α for the hard, soft and iterative decoding is due to the very different SNR levels each of these configurations operates in that affects what can be regarded as a tolerable level of interference or not.The use of other mapping functions such as F(x) = max(x) or F(x) = mean(x) has also been tested, but the corresponding results do not differ substantially from the ones presented next using F(x) = min(x). Hard-decoded results Figure 5 presents results for the non-spread setup.The left plot shows the PER performance when very accurate CSIT is available, and as expected under these conditions, the linear receiver in (6) virtually diagonalises the overall processing chain, thus making linear detection nearly optimal.Only for those very few cases where CSIT error is significant, MLD provides an advantage over MMSE, a situation leading to the rather small gain observed between the performance of both fixed detectors.The performance for the adaptive detector following Algorithm 1 remains unaltered regardless of the value of α.The top-right plot in the same figure shows the percentage of utilisation of each detector for the particular case of α = 0.75, and remarkably, Algorithm 1 overwhelmingly chooses the linear approach for any SNR level.When CSIT quality diminishes, differences among the PER performance of the various detectors appear, as shown in the central plot in Figure 5.There are two noticeable facts regarding the performance of MLD: firstly, it now clearly outperforms linear detection, and secondly, it improves upon the results obtained using very accurate CSIT.This somewhat counterintuitive effect, already observed and discussed in [15], is caused by the absence of spatial spreading: the mismatch between Tx and Rx due to a lousy CSIT causes significant interfering terms to appear in (9) that effectively act as a form of spreading that the MLD can exploit, resulting in a 2-dB gain over linear processing.The adaptive detector, depending on the value of α, exhibits various degrees of performance.For the particular choice of α = 0.75, it basically achieves optimum performance while, as shown on the bottom-right plot of Figure 5, it only triggers the use of MLD for about 40% to 50% of the detector invocations with the rest of the times relying on linear processing. Results in Figure 6 correspond to a setup employing rotated Walsh-Hadamard spatial spreading.In this case, either with accurate or inaccurate CSIT, MLD clearly outperforms linear detection by up to 4 dB (left and central plots in Figure 6).The adaptive detector is seen to yield different error rates depending on the chosen α.For the particular choice of α = 0.75, the attained PER is within 0.5 dB of the optimal solution (fixed MLD) while requiring only the use of MLD for about 40% of the detector invocations when CSIT is accurate and for around 60% when this is rather inaccurate. Soft-decoded results For the soft-decoded case, only non-spread configurations are considered (C = I N s ) as it has been shown in [14,25] that, in soft-based BICM systems, spatial spreading leads to a PER performance degradation.PER performance and complexity results are shown in Figure 7 for the three considered levels of CSIT accuracy when employing softbased decoding.Focusing on the PER performance, it can be observed that the more inaccurate the CSIT is, the bigger the difference between MLD and MMSE detection becomes.In particular, all detectors achieve similar performance for μ = 0.015 (top-left plot in Figure 7) whereas for μ = 0.37, and even more markedly when μ = 1.31, the performance gap between ML and MMSE is somewhere between 2 and 3 dB for the typically relevant range of PER values (10 −3 to 10 −1 ).The adaptive detector behaves as expected: for near-perfect CSIT, similar PER performance was obtained independent of α (α ∈ {0.75, 0.85, 0.95}), while under imperfect CSIT, the performance (and complexity) of the adaptive detector is greatly influenced by the particular choice of α.The bottom plots in Figure 7 represent the percentages of detector utilisation when employing Algorithm 1 with α = 0.95 for the three values of μ.Notice that large values of α such as this one strive for performance optimisation.As anticipated, when CSIT is nearly perfect, the adaptive detector relies almost exclusively on MMSE detection.In contrast, as CSIT accuracy degrades, the adaptive detector tends to favour the more frequent use of MLD to the point that for very poor-quality CSIT information, the adaptive detector almost invariably triggers the use of MLD.Nevertheless, note that, although not shown here and as in the hard-decoded scenario, a lower value of α would lead to significant computational savings (more frequent use of MMSE for μ = 0.37 or μ = 1.31) at the cost of a slight degradation in PER performance. Iteratively decoded results Figure 8 shows results when iterative decoding is applied.For this scenario, the system was configured to use 16-QAM modulation, no spatial spreading was in use (C = I N s ) and the adaptive detector's threshold was set to α = 0.975.Results are shown for two different degrees of statistical CSIT quality, μ = 0.015 and μ = 1.31, corresponding to very accurate and rather inaccurate channel knowledge, respectively.The first remarkable point to note is that, as noted in previous results, when the transmitter can rely on a good channel estimate (μ = 0.015), there are no very large differences between the (non-adaptive) ML and MMSE soft detectors, with only a marginal benefit for the ML-based receiver as shown in the left plot in Figure 8.When iterative detection is allowed e , the ML detector is able to somewhat improve performance over its non-iterative counterpart.Nonetheless, it is remarkable that the SNR gap between the low-complexity (non-iterative) MMSE-based soft detector and the much more complex iterative ML receiver is less than 1 dB.When the detection strategy on each subcarrier is selected according to Algorithm 1, it can be clearly observed that for the vast majority of cases, MMSE detection is selected (see top-right plot in Figure 8) with ML resulting in the chosen strategy only for those few cases where there was a considerable mismatch between the CSIT and the true channel.Consequently, the performance of the adaptive detector lies in the narrow ground between that of the fixed receivers.As shown in the figure, the adaptive detector was tested with and without the possibility of iterating, and as with the fixed ML decoder, it was observed that iterating resulted in a rather marginal improvement.Note that as mentioned in Section 4.3, when using the channel-aware iterative detector, information feedback is only conducted for those subcarriers employing ML detection.When the statistical CSIT quality is considerably degraded (μ = 1.31), results are significantly different.As it can be observed in the middle plot of Figure 8, and in line with results in the previous subsection, noniterative ML detection provides a gain of nearly 3 dB with respect to MMSE.Furthermore, when the ML receiver is allowed to iterate, a further 1-to 1.5-dB gain is achieved.When using channel-aware detection, it is observed that Algorithm 1, either with or without iterations, rightly chooses to rely on ML detection owing to the rather large interfering terms the receiver observes when evaluating (10) and (11). An important remark It is worth emphasising that the merits of the adaptive detector should be valued by globally appreciating the results on each of the considered scenarios (hard, soft and iterative decoding): a single configuration for each type of receiver (α = 0.75, F(x) = min(x) for hard decoding, α = 0.95, F(x) = min(x) for soft decoding and α = 0.975, F(x) = min(x) for iterative decoding) leads to a strategy able to attain virtually optimum PER performance while potentially offering a very significant complexity reduction with respect to the full use of MLD.In other words, Algorithm 1 provides the receiver with the capability of distinguishing when MLD will be effective and when MMSE will suffice.This scheme can therefore be very attractive in those scenarios where the quality of CSIT may vary over time such as it occurs in today's WLAN environments depending on the number of users in the system or changes in the environment.Furthermore, note that the parameter α acts as a performance/complexity trading knob, thus enabling the reconfiguration of the system as a function of, for instance, the available battery power or required processing latency. Conclusions This paper has proposed an adaptive detection technique that allows the receiver of a MIMO-OFDM linearly precoded system to toggle between the use of MMSE and MLD depending on the CSIT accuracy and/or channel conditioning.The introduced technique works on a persubcarrier basis and is compatible with different receiver architectures, namely hard, soft and iterative decoding schemes.Numerical results have shown that regardless of the receiver setup, the adaptive detector is able to distinguish the system conditions that allow MLD to boost performance from those where the much simpler MMSE detector would perform (near) optimally. Endnotes a The incorporation of antenna correlation effects to the current system model is trivial; however, it unnecessarily complicates notation without providing any further insight or significantly altering numerical results. b This is a realistic assumption since the receiver should be aware of the last CSI information sent to the transmitter.Alternatively, if the transmitter sends pilot symbols through the precoder (and channel), the receiver can also deduct the precoding filter used in transmission. c To simplify the notation, the subcarrier and stream indices are skipped when referring to the bits. d The (possibly iterative) utilisation of soft information at the receiver suggests using capacity-based measures to optimise the power allocation.Unfortunately, under imperfect CSIT, no closed-form solutions are available and power allocation solutions require convex numerical optimisation procedures (see [5] for a detailed discussion on this issue).http://jwcn.eurasipjournals.com/content/2013/1/240e For the results shown here, only two iterations between detector and decoder were allowed as it was observed that further iterations did not bring along any significant performance benefit. Figure 2 Figure 2 Receiver block diagram with hard detection. where the characters B p,+1 and B p,−1 represent the sets of 2 N b −1 bit vectors whose p th position is a '+1' or '−1' , respectively.Moderate values of M and/or N s make the sets B p,+1 and B p,−1 extremely large, making the search in(12) computationally challenging.To address this issue, the LSD limits the search to the sets Bp,+1 = B p,+1 ∩ C and Bp,−1 = B p,−1 ∩ C where C is the set containing the bit vectors corresponding to the N cand candidates closer, in a Euclidean sense, to the received samples, i.e.C = b 1 , . . ., b N cand where b Figure 5 Figure 5 PER and complexity for setup without spatial spreading (C = I Ns ) using hard decoding.QPSK modulation. Figure 6 Figure 6 PER and complexity for setup with rotated Walsh-Hadamard spatial spreading (C = Ns ) using hard decoding.QPSK modulation.	2018-01-23T22:38:59.635Z	2013-10-07T00:00:00.000	{ "year": 2013, "sha1": "1754079aba95b665282917183dfea44ca2619a1f", "oa_license": "CCBY", "oa_url": "https://jwcn-eurasipjournals.springeropen.com/counter/pdf/10.1186/1687-1499-2013-240", "oa_status": "GOLD", "pdf_src": "SpringerNature", "pdf_hash": "6f1a45cbeb7778c7b0870dbd7e32714e37a883b8", "s2fieldsofstudy": [ "Business", "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
59031408	pes2o/s2orc	v3-fos-license	Broad-band spectral energy distribution of 3000 Angstroem break quasars from the Sloan Digital Sky Survey The Sloan Digital Sky Survey (SDSS) discovered a few unusual quasars with a characteristic break in the continuum around 3000 A that neither shows the typical structure of broad absorption line (BAL) troughs nor is explained by typical intrinsic dust reddening. We used the method of Kohonen self-organising maps for a systematical search for quasars with such properties in the SDSS spectra archive. We constructed a sample of 23 quasars classified as 3000 A break quasars and two comparison samples of quasars with similar properties, to some extent, but also showing typical BAL features. We computed ensemble-averaged broad-band SEDs based on archival data from SDSS, GALEX, 2MASS, UKIDSS, and WISE. The SEDs were corrected for intrinsic dust absorption by the comparison with the average SED of normal quasars. The de-reddened arithmetic median composite SED of the 3000 A break quasars is found to be indistinguishable from that of the unusual BAL quasars. We conclude that 3000 A break quasars are most likely extreme versions of BAL quasars. Assuming that the intrinsic SED of the continuum source is represented by the quasar composite SED, the 3000 A break quasars tend to be intrinsically more luminous than ordinary quasars. (Abstract modified to match the arXiv format.) Introduction In the past two decades, huge spectroscopic surveys have produced a tremendous increase in the database of known quasars. This exciting evolution also led to the discovery of a variety of quasar spectra that dramatically differ from the standard spectral energy distribution (SED) of active galactic nuclei (AGN) and confirmed the existence of populations of exotic and hitherto unknown quasar types. These rare peculiar objects possibly represent links to special evolutionary stages and provide a laboratory for studying the processes of potential importance for the cosmic structure evolution, such as feedback mechanisms between accretion activity and star formation. With its efficient quasar selection technique and huge number of highquality spectra, the Sloan Digital Sky Survey (SDSS; York et al. 2000), is uniquely qualified to significantly increase the database of unusual quasars. Based on early SDSS data, Hall et al. (2002a) discussed in detail the spectra of about 20 unusual quasars that are characterised by unusual broad absorption line (BAL) properties, in some cases combined with heavily reddened continua. The present paper focuses on unusual SDSS spectra of the type of two objects referred to as "mysterious objects" by Hall et al. (2002a), J010540.75-003313.9 and J220445.27+003141.8. Their characterising spectral features are a lack of both substantial typical emission lines and obvious absorption troughs in combination with a drop-off around 3000 Å 1 , which appears too steep to be caused by dust reddening with reddening laws taken as typical of quasars. Following Jiang et al. (2013), we call this feature "3000 Å break" throughout the present paper. Both two prototypical 3000 Å break quasars show associated Mg ii λλ2796.3, 2803.5 absorption, are unresolved FIRST radio sources, and are much more luminous than any galaxy. Regardless of their physical interpretation, these objects almost certainly represent quasar types that are extremely rare in presently available samples. The most convincing explanation to date is unusually broad structures from line absorption with broad smooth outflow velocities, probably in combination with moderate dust reddening Green 2006). Quasar broad absorption lines (BALs), usually seen as blueshifted troughs, are naturally explained as footprints of powerful, sub-relativistic (up to ∼ 0.2c) outflows (Foltz et al. 1983;Weymann et al. 1991), probably related to radiation-driven winds from the accretion disc (Murray & Chiang 1998;Elvis H. Meusinger et al.: 3000 Å break quasars 2000;Everett et al. 2002) and are thus the most obvious manifestation of matter accelerated by the central engine of AGNs (Fabian 2012;Arav et al. 2013). BAL quasars are usually classified into those showing broad absorption lines from only highionisation species, such as C iv and N v (HiBALs), and those showing in addition also absorption from low-ionisation species, such as Mg ii and Al iii (LoBALs). The comparison of samples of LoBAL and HiBAL quasars showed that the former have substantially redder ultraviolet (UV) continua and stronger UV Fe ii and Fe iii emission (Weymann et al. 1991). A rare subclass of the LoBAL quasars are the FeLoBAL quasars showing low-ionisation BALs from metastable excited states of Fe ii and Fe iii. The catalogued FeLoBAL quasars constitute only ∼ 2% of the BAL quasars and ∼ 0.3% of the total quasar population, where the rate of incidence measured strongly depends on the selection criteria and the true FeLoBAL quasar fraction may be considerably higher (e.g., Trump et al. 2006;Urrutia et al. 2009;Dai et al. 2012;Lucy et al. 2014). Unlike HiBAL quasars, LoBALs may not be explained by orientation alone (e.g., Ghosh & Punsly 2007;Montenegro-Montes et al. 2009;Zhang et al. 2010). BAL variability studies of FeLoBALs suggest that the outflowing optically thick gas resides on a range of scales Filiz Ak et al. 2012;Vivek et al. 2012;Capellupo et al. 2013;McGraw et al. 2015). From the analysis of spectral variability in 28 BAL quasars, Zhang et al. (2015) conclude that short distances from the central engine are particularly characteristic of FeLoBALs with overlapping absorption troughs, perhaps including matter in a rotating disk wind. Rotationally dominated outflows may also produce redshifted absorption seen in a small fraction of BAL quasars, though infall of relatively dense gas at radii as small as several hundred Schwarzschild radii provides an alternative explanation (Hall et al. 2013). There is positive evidence that FeLoBAL quasars may represent an evolutionary stage of the quasar phenomenon when a dusty obscuring cocoon is being expelled by a powerful quasardriven wind (Voit et al. 1993;Egami et al. 1996;Becker et al. 1997;Canalizo & Stockton 2001;Lípari et al. 2009;Dai et al. 2012;Farrah et al. 2007Farrah et al. , 2012Morabito et al. 2011;. From a sample of 31 FeLoBAL quasars at 0.8 < z < 1, selected on the basis of their rest-frame UV spectral properties, Farrah et al. (2012) concluded that the radiatively driven outflows from FeLoBAL quasars act to curtail obscured star formation in the host galaxies to less than ∼ 25% of the total infrared (IR) luminosity. Studies of BAL quasar samples dominated by HiBALs (Willott et al. 2003;Gallagher et al. 2007;Cao Orrjales et al. 2012;Pu 2015) indicate that BAL quasars are not significantly different from non-BAL quasars in terms of their IR to UV spectral energy distribution (SED) and their IR luminosities. A similar result was published for LoBAL quasars by Lazarova et al. (2012). However, this finding does not necessarily mean that LoBAL quasars are not related to a rapid transition state from a ultra-luminous infrared galaxy (ULIRG) phase to a more quiescent phase of non-BAL quasars. Lazarova et al.(2012) argue that it could be that the majority of the LoBAL quasars in their sample could have already passed through the short phase of quenching the star formation rate to the normal level. Hall et al. (2002a) quoted only two other objects with spectra that are probably similar to the two prototypical 3000 Å quasars, namely the low-z quasars FBQS 1503+2330 (z = 0.40) and FBQS 1055+3124 (z = 0.49) from the FIRST Bright Quasar Survey (White et al. 2000). Another four quasars with similar spectra were discovered by Plotkin et al. (2008Plotkin et al. ( , 2010 and independently by Meusinger et al. (2012;hereafter Paper I). All these objects were targeted for spectroscopy because they were detected as radio sources. The first similar object that was selected neither as a radio source nor by the SDSS colour selection was J134246.24+284027.5 from the Tautenburg-Calar Alto variability and (zero-) proper motion survey (Meusinger et al. 2005). The small sample of these sources, enlarged by the similar object J085502.20+280219.6, was presented in Paper I. The first aim of the present study was to increase the sample of quasars with properties characteristic of 3000 Å break quasars. Rather than trying to perform an unbiased search, our approach is focused on selecting a larger sample from the SDSS spectra archive. The tremendous amount of data produced by the SDSS requires efficient methods for browsing the huge archive. We developed the software tool ASPECT to produce large Kohonen Self-Organising Maps (SOMs) for up to one million SDSS spectra (in der Au et al. 2012). In previous studies, we computed SOMs for the quasar spectra from the SDSS data release 7 (DR7; Abazajian et al. 2009) to select unusual quasars. A resulting sample of about 1000 unusual quasars of different types was published and discussed in Paper I, a large set of 365 Kohonen-selected weak-line quasars was analysed by Meusinger & Balafkan (2014). Thereafter we applied the SOM technique also to galaxies, stars, and unknowns in the SDSS DR7, and to the quasar spectra in the SDSS DR10 (Ahn et al. 2014) with the main aim to search for further 3000 Å break quasar candidates. The present sample combines objects from Paper I and newly discovered objects from SDSS DR7 and DR10. Section 2 gives an overview of the quasar selection. Our main goal is to construct a composite broad-band SED of 3000 Å break quasars from the extreme UV to the mid IR and to compare it with the SEDs of ordinary quasars and of unusual BAL quasars. The SEDs are the subject of Sect. 3. We combined photometric data from GALEX, SDSS, 2MASS, UKIDSS, WISE, Spitzer, and Herschel with the main aim to search for differences between the SEDs of different types. A particular question is the role of internal reddening at the quasar redshift. In Sect. 4, we discuss three scenarios for interpreting the 3000 Å break. Finally, the results are summarised in Sect. 5. Individual descriptions and SEDs for the quasars are given in two Appendixes. Sample selection In Paper I, we presented a sample of nine quasars with properties similar to J010540.75-003313.9 and J220445.27+003141.8 from Hall et al. (2002a) in combination with 11 possibly related quasars that may represent links between that type and other odd quasar types such as unusual BAL quasars. With the only exception of J134246.24+284027.5 from Meusinger et al. (2005), all quasars were selected from a systematic search for spectroscopic outliers among the objects from SDSS Data Release 7 (DR7; Abazajian et al. 2009) classified by the spectroscopic pipeline as quasars. While the spectroscopic pipeline of the SDSS works accurately and efficiently for the vast majority of the spectra, it tends to fail in case of very unusual ones. We developed the software package ASPECT, which is able to project a huge number of spectra into a sorted topological map (in der Au et al. 2012). The approach is based on the Kohonen method (Kohonen 2001) of self-organising maps (SOMs). This is an artificial network algorithm that uses unsupervised learning to produce a twodimensional mapping of higher-dimensional input data (spectra), where the spectra are sorted ('clustered') by their relative similarity. Because of its clustering properties, the resulting SOM provides an efficient tool for selecting certain spec-tral types or spectroscopic outliers. In Paper I, we computed 37 SOMs for about 10 5 SDSS quasars binned in narrow redshift intervals. The final selection of unusual quasar spectra was based on a combination of the clustering power of the SOMs with their In the present study, we aimed to create a larger sample of 3000 Å break quasars by a systematic search in the SDSS spectra archive. The selection method is basically the same as in Paper I. We continued computing SOMs for different sets with large numbers of spectra. First, in addition to the objects classified as quasars, we computed SOMs for other spectral classes from the SDSS DR7: ∼ 4 · 10 4 stars (40 SOMs), ∼ 8 · 10 5 galaxies (160 SOMs), and ∼ 10 4 unclassified objects (spectral class = unknown) with sufficiently high signal-to-noise ratios (5 SOMs). 2 In a second step, we clustered ∼ 3·10 5 quasar spectra from SDSS DR10 (Ahn et al. 2014). All SOMs were inspected by eye to se-lect candidates for 3000 Å break quasars and odd BAL quasars. After examining all selected spectra individually and rejecting contaminants (see below), a sample of 30 quasars was selected from this new search. The final sample for the present study was compiled from the newly selected quasars in combination with 34 quasars from Paper I. Following Paper I, we defined three subsamples: (A) Objects with optical-to-UV spectra similar to J010540.75-003313.9 and J220445.27+003141.8. The characteristic spectral features are: (a) the lack of both substantial typical quasar emission lines (except broad Fe ii emission and [O ii] 3730 in some cases) and obvious BAL troughs, (b) a typical blue continuum at restframe wavelengths > ∼ 3000 Å, (c) a continuum drop-off at around 3000 Å that appears too steep to be caused by dust reddening (see Hall et al. 2002a for a more detailed description). The sample contains 23 quasars, including the nine objects from the upper part of Table 8 in Paper I. These objects are referred to as 3000 Å break quasars throughout this paper. (B) Fifteen quasars with spectra that resemble those in sample A to some degree but not in all aspects. The sample includes the objects from the lower part of Table 8 in Paper I. (C) A comparison sample of 26 BAL quasars with extreme BALs and probably strong dust reddening, including the objects shown in Fig. 11 of Paper I. Some quasars from samples B and C show a pronounced peak in the spectrum around 3000 Å. Nevertheless, they were not classified as sample A objects because the break can be clearly attributed to LoBALs. The most extreme objects of this type J173049.10+585059.5 (#46) and J094317.59+541705.1 (#52) have already been discussed in Paper I. The SDSS and BOSS spectra of the two prototypical sample A objects from Hall et al. (2002a), J010540.75-003313.9 and J220445.27+003141.8, are shown in Fig. 1. The time lag between the two spectra corresponds to 4.3 yr in the rest frame for both quasars. Quasars are variable on time scales of months to years (Kelly et al. 2009;MacLeod et al. 2010) and 3000 Å break quasars are also known to be variable (Meusinger et al. 2005(Meusinger et al. , 2011. It is thus not surprising that the two spectra are not fully identical. However, the variability properties of the two quasars appear to be different (see Sect. 4.1). The quasars from the three samples are listed in Table 1. Remarks on single objects are given in Appendix A. The optical spectra are shown on the left-hand side of Fig. B.1 in Appendix B (see Sect. 3.2.3). The redshifts z of the new quasars were estimated as described in Paper I. The process requires detailed analysis of each spectrum individually. For most quasars in samples A and B, emission lines could be identified (80% and 53%, respectively). On the other hand, it is in the nature of the un-usual BAL quasars in sample C that for most objects z could be measured from absorption lines only (73%). Nevertheless, we feel that redshifts should be correct to within a few percent for the vast majority. Quasars with uncertain redshift estimates were flagged in Table 1. In Fig. 2, we compare the redshifts from the present study with those from the SDSS spectroscopic pipeline as provided by the SDSS DR12 explorer page. To guide the eye, the 1:1 relation (dashed line) is over-plotted. Although the pipeline is efficient and accurate for the overwhelming majority of sources, there is a substantial danger of misinterpretations in the case of unusual spectra like those from the present study. It has long been known that BAL quasars mimic high-z quasars (Appenzeller et al. 2005). In fact, 41% of the SDSS quasars in sample A were targeted as high-z quasars (SDSS target flag PrimTarget = target qso hiz) and the 3000 Å break in the spectrum was obviously mismatched by the pipeline with the depression of the continuum shortwards of the Lyman α line due to either the Lyman forest or the Lyman break for many of the quasars from Table 1. Others were wrongly identified as galaxies at relatively small z. The redshift distributions are shown in Fig. 3. The distributions are similar for samples A and B with mean values z = 1.457 and 1.54, respectively. At such redshifts the Mg ii 2800Å line is in the middle of the SDSS and BOSS spectra so that the continuum at either side of the break is clearly visible. Sample C is weighted towards higher redshifts (z ≈ 2.2) where the optical spectrum is completely dominated by the intrinsic UV part where the absorption lines of FeLoBAL quasars are concentrated. Uncertain classifications and possible contamination There is a certain danger in the selection process of wrongly classifying peculiar stellar types as 3000 Å break quasars. This holds in particular for peculiar DQ white dwarfs (DQ WDs; Pelletier et al. 1986;Koester & Knist 2006;Hall & Maxwell 2008) with their characteristic absorption troughs resulting from from sequences of Swan bands of the C 2 molecules. The absorption troughs can be deep, widely overlapping, and sometimes apparently distorted (Kowalsky 2010). In combination with a blue continuum at longer wavelengths, the abrupt decline in the spectrum around λ ≈ 6000 Å towards shorter wavelengths makes such spectra similar, in some respects, to 3000 Å break quasars at z ≈ 1. At the top of Fig. 4, the SDSS spectra of four peculiar DQ WDs are shown to illustrate the effect. The bandheads of the usually strongest sequences of C 2 bands (Tanabashi et al. 2007) were over-plotted as vertical red dashed lines and those from the red CN bands (Davis et al. 1987) as magenta lines. The downscaled sky spectrum is over-plotted in green at the bottom of each panel. J133359.83+001655.0 is a rare peculiar cool magnetic DQ (DQpec) with broad molecular bands (Schmidt et al. 2003;Vornanen et al. 2013). J095051.70+122758.5 looks quite similar, with pronounced band heads at roughly 5600Å, 5200Å, and 4700Å, and is obviously a newly discovered peculiar DQ. 3 J124739.04+064604.5 and J180302.57+232043.3, also classified as peculiar DQs (Kilic et al. 2010;Limoges et al. 2013;Kleinman et al. 2013), look similar to the former two spectra, but display a declining flux density towards the shortest wavelengths. All four stars have significant proper motions between 200 and 400 mas yr .1 . The interpretation of the spectra in the bottom row of Fig. 4 is much more uncertain. None of them was found in the SIMBAD database 4 . The spectra show a break at ∼ 6200Å that could be interpreted as a redshifted 3000 Å break of a quasar. As an alternative, it can be identified with the bandhead of the reddest of the five marked C 2 troughs in C stars, where all other other Swan bands are, however, only weakly indicated, if at all. It is very unusual for DQs that the ∼ 4700Å trough is missing, while the ∼ 6200Å trough is so strong. The proper motions are essentially zero for J235115.56+385452.6 (9 ± 13 mas yr −1 ) and J234334.52+141244.4 (0 ± 0 mas yr −1 ), not measured for J215619.83+112609.8, and only marginally significant for J025529.12+325816.9 (34 ± 12 mas yr −1 ). On the other hand, a stellar origin is indicated by the presence of stellar absorption lines at z = 0 indicated by the blue dotted vertical lines (from left to right: Ca iiλλ 3935,3970, Mg i λ 5175, Na i λλ 5892,5898, Hα, and the Ca ii infrared triplet). Though a definitive classification remains unclear, we conclude that the first three, J025529.12+325816.9, J235115.56+385452.6, J234334.52+141244.4 are most likely stars, perhaps peculiar carbon stars. The spectrum of J215619.83+112609.8 is simply too noisy. We emphasise that we paid particular attention in the selection of 3000 Å break quasar candidates to minimise the risk of contamination by objects with such uncertain classification as for those in the bottom row of Fig. 4. We checked our final sample from Table 1 also against the catalogue of optically selected BL Lac objects from SDSS-DR7 . Only one source, J075437.85+422115.3, was classified there as a high-confidence BL Lac candidate. Observational data The present study takes advantage of existing archival data. The major surveys exploited here, apart from the SDSS, are the Two Micron All-Sky Survey (2MASS; Skrutskie et al. 2006), the UKIRT Infrared Deep Sky Survey (UKIDSS; Lawrence et al. 2007;Hambly et al. 2008) in the near infrared (NIR), the Wide-Field Infrared Survey Explorer (WISE; Wright et al. 2010) in NIR and mid infrared (MIR), the Faint Images of the Radio Sky at Twenty Centimeters (FIRST; White et al. 2000), and the data products from from the Galaxy Evolution Explorer (GALEX; Morrissey et al. 2007) in the near and far ultraviolet (NUV, FUV). The VizieR Service at CDS Strasbourg 5 was used to extract the data from the SDSS Photometric Catalog, Release 7 (Abazajian et al. 2009), the GALEX-DR5 (GR5) sources from AIS and MIS (Bianchi et al. 2011), and the UKIDSS-DR9 Large Area Surveys (Lawrence et al. 2007). The NASA/IPAC Infrared Science Archive (IRSA) 6 was used to identify the SDSS sources in the 2MASS All-Sky Point Source Catalogue and in the AllWISE Source Catalog from WISE. IRSA was also used to search for counterparts in the IRAS Faint Source Catalogue (Moshir et al. 1992) in the far infrared (FIR) and in the catalogues from the Spitzer Space Observatory (Werner et al. 2004) in MIR and FIR. The Herschel Stripe 82 Survey (Viero 2014) was checked for sub-mm detections using VizieR. No source was detected by IRAS, one source by Herschel, and only a few 5 http://vizier.u-strasbg.fr/viz-bin/VizieR 6 http://irsa.ipac.caltech.edu/applications/Gator/index.html sources by Spitzer. A summary of the number of detections in the various photometric bands is given in Table 2. Magnitudes were transformed into fluxes and the fluxes were corrected for Galactic foreground extinction using E(B−V) from Schlafly & Finkbeiner (2011) 7 . Figure 5 shows the observed UV, optical, and IR fluxes of the quasars from our sample after foreground extinction correction. Also plotted are the flux limits of the corresponding major surveys. The GALEX limits were adopted from Bianchi et al. (2007) for GALEX/SDSS matched sources with photometric errors < 0.3 mag, but the limits are a function of the field position and can thus be lower. We used the Mikulski Archive for Space Telescopes (MAST) 8 to select the faintest GALEX sources in fields of 10 arcmin size around our quasars. In nearly all fields, GALEX sources were found that are several tenths of a magnitude fainter than the above-mentioned limits. The survey limits given for the four WISE bands are point source sensitivities in unconfused regions on the ecliptic 9 . The WISE sensitivity improves towards the ecliptic poles due to denser coverage and lower zodiacal background. For illustration purposes, we over-plotted in Fig. 5 the prototypical 3000 Å break quasar J220445.27+003141.8. Provided that the SED of our quasars does not increase much more strongly towards the FIR than for J220445.27+003141.8, the IRAS Faint Source Catalogue (Moshir et al. 1992) is not deep enough to contain the brightest quasars from our sample. On the other hand, a substantial part of our sample is above the NUV limit, but not above the FUV limit. Nevertheless, upper GALEX limits provide useful information on the shape of the SED at the shortest wavelengths. The SED of J220445.27+003141.8 shifted towards fainter fluxes by 1.5 dex is indicated by the dotted curve. It demonstrates that even the faintest quasars should be detected by UKIDSS and in the WISE bands w1 and w2 if their SED is similar to that of J220445.27+003141.8. (Helfand et al. 2015). The fields of the remaining 7 http://irsa.ipac.caltech.edu/applications/DUST 8 https://galex.stsci.edu/GR6/ 9 http://wise2.ipac.caltech.edu/docs/release/allsky/ 36 objects were inspected on the FIRST image cutouts 10 to search for possible sources slightly below the hard catalogue limit of 1 mJy. Only one additional radio source was found, J115436.60+030006.3 in addition to J134246.24+284027.5, which was already known as a sub-mJy source. The FIRST radio fluxes were not directly involved in the study of the SED, but were used to estimate the fraction of radio-loud quasars in our samples (Sect. 3.5). The optical spectra were downloaded from the SDSS DR12 (Alam et al. 2015), the spectrum of J134246.24+284027.5 is from Meusinger et al. (2005). For 33 quasars, BOSS spectra are available that have a wider wavelength coverage than the spectra from the SDSS spectrograph. If a quasar had both a SDSS and a BOSS spectrum, we always downloaded the latter. All spectra were corrected for Galactic foreground extinction, again using E(B − V) from Schlafly & Finkbeiner (2011). Fitting the photometric data In general, the wavelength coverage in the IR is too poor to fit model SEDs to the observational data in a same way as done, for examplem by Farrah et al. (2012) and Lazarova et al. (2012). Instead we restricted our analysis of the individual quasar SEDs to a qualitative comparison with the composite SED of normal quasars. We assumed that the SED is composed of an UV/optical part and an IR part, roughly separated by the 1µm inflection. According to the AGN standard model, the accretion disk is responsible for the UV and optical continuum in the big blue bump (BBB), whereas the IR bump is attributed to the putative dusty torus and a possible starburst component in the quasar host galaxy. Shang et al. (2011) constructed composite (median) SEDs both for radio-quiet and radio-loud quasars from high-quality single-epoch multi-wavelength data for 85 optically bright nonblazar quasars. Although there is a large variation from quasar to quasar, these composite SEDs are considered representative for UV-optically bright quasars. Accordingly we used the Shang et al. composite to construct two components of the SED starting with the BBB. Figure 6a shows the corresponding part from the 1 µm inflection to the Lyman break. The black solid curve is the composite for the radio-quiet quasars, the black dashed curve is for the radio-loud ones. The SED derived from broadband photometry should be compared with a smoothed version of the composite spectrum, shown here in magenta for only the radio-quiet quasars. The blue curve is the composite SED derived from SDSS and GALEX photometric data (Trammell et al. 2007), and it extends to shorter wavelengths than in Shang et al. (2011). The green curve is the composite from SDSS and 2MASS photometry (Labita et al. 2008), the red one is the NIR spectral template from Glikman et al. (2006). For comparison, the cyan curve is the continuum of a simple multi-temperature blackbody accretion disk model (Shakura & Sunyaev 1973) with a disk temperature parameter T * = 9 · 10 4 K roughly corrected for effects that have been considered by more elaborated disk models (see Meusinger & Weiss 2013). To construct the blue component of the SED, i.e. BBB plus emission lines, we used a smoothed version of the radio-quiet Fig. 6b, together with the entire composite spectrum. Intrinsic dust reddening The general shape of the spectra of 3000 Å break quasars suggests that reddening by dust may play a role. In general, thermal dust emission of quasars in the infrared exhibits substantial dust masses. The dust is assumed to be concentrated in a toruslike configuration around the central engine (Antonucci et al. 1993) in a circum-nuclear starburst or distributed on a galaxywide scale over the host galaxy. There are tentative arguments 11 The composite for radio-quiet quasars was used because the radio loudness of our sources is generally low, though the percentage of FIRST radio sources is relatively high (see Sect. 3.5). supporting the view that quasars spend a substantial part of their lifetimes in a dust-enshrouded environment, especially in young evolutionary stages (e.g., Sanders et al. 1988;Hopkins et al. 2004). FeLoBAL quasars are invariably reddened AGNs with high IR luminosities (e.g., Farrah et al. 2007Farrah et al. , 2012. Excess reddening by foreground dusty absorbers seem to be rare (Fynbo et al. 2013), though there are convincing detections in a few cases. Here we simply assume that the dust is located at the redshift of the quasars and that the blue component of the quasar SED may need to be corrected for intrinsic reddening, while reddening of the IR component is negligible in our context. There has been a long-standing debate over the reddening law for quasars. The lack of a strong λ2175 extinction bump and of a substantial curvature of the UV continuum led to the assumption of only modest reddening (e.g., Pitman et al. 2000;Cheng et al 1991). From the analysis of a large sample of SDSS quasars, Richards et al. (2003) concluded that the reddening law in quasars at z < 2.2 can be described by the reddening curve from the Small Magellanic Cloud (SMC), but not by the reddening in the Large Magellanic Cloud or the Milky Way (MW) galaxy. A similar conclusion was drawn by Hopkins et al. (2004). The SMC extinction is characterised by a stronger increase in the reddening in the UV compared to the MW. Gaskell et al. (2004) analysed a large sample of AGNs and concluded that the UV extinction curves are very flat, probably caused by a relative lack of small dust grains in the environment of the quasar. Similarly, "grey" extinction curves were also predicted for ultra-luminous infrared galaxies (ULIRGs) at z ∼ 1 and were attributed to the lack of small grains as predicted by supernova dust models (Shimizu et al. 2011). Here we estimated the intrinsic reddening by comparing the individual quasar SED with the reddened composite from Fig. 6b. First the BBB component was reddened, and then the sum of the two components of the quasar composite SED was fitted to the observed quasar SED. The SMC reddening curve (Pei 1992) was adopted, but the Gaskell curve was used for comparison as well. The fitting procedure has two free parameters, the colour excess E(B − V) and the relative shift between the BBB and the IR component. Owing to the highly peculiar SEDs, the procedure is subject to substantial uncertainties. Because of the 3000 Å break in the spectra from subsamples A and B and the usually strong effect of BALs shortwards of 3000 Å for sample C, the fit has to be restricted essentially to the spectral range between the 1µm deflection and ∼3000 Å. The spectra of at least some quasars from our sample exhibit strong broad iron emission features in the optical and in the UV that complicate the comparison with the standard quasar SED. Stronger-than-usual iron emission can lead to underestimating the amount of reddening. In principle, contamination by host galaxy light provides another potential source of uncertainty. However, because of the relatively high luminosities of our quasars (see Sect. 3.4.2; Paper I), we assume that the host contribution should be negligible on average. Individual quasar broad-band SEDs The individual SEDs for all quasars are shown in Fig. B.1. The observed optical spectra, expressed by log F λ (λ), are given on the left-hand side. The panels on the right-hand side show the broad-band SEDs log νF ν (log ν) in the quasar's rest frame. More detailed information on Fig. B.1 is given in Appendix B. Sample-averaged SEDs The comparison of single quasar SEDs with a composite spectrum should be considered with care. Shang et al. (2011) caution that there is a large variation in their individual quasar SEDs. Given the effects of absorption by dust and gas, an even larger object-to-object variation is expected for the unusual quasars from our sample. Temporal flux variability adds a further complication, especially at short wavelengths, i.e. when connecting SDSS with GALEX data taken at different epochs. These difficulties are substantially reduced in a statistical approach where ensemble averaged SEDs are used. We normalised the extinction-corrected fluxes for each single quasar in such a way that the normal quasar composite SED from Fig. 6b is best matched both around 4000 Å and around the 1 µm inflection. The normalised fluxes are plotted as green squares in Fig. 8 for the samples A to C in the first three rows from top. SMC extinction correction was applied on the lefthand side, and the Gaskell extinction curve was used at the righthand side. The symbols from Fig. 2 with error bars represent arithmetic median values and standard deviations in frequency bins of 0.1 dex width. The normal quasar composite is overplotted in black. The same symbols were used in the panels at the bottom of Fig. 8 to compare the medians from the three samples with each other. The symbols were interconnected just to guide the eye. The following inferences are made from Fig. 8. In most cases, the decline at log ν[Hz] > ∼ 15, defining the 3000 Å break quasars from sample A, does not disappear after the correction for intrinsic dust reddening. Exceptions are J160827.08+075811.5 (# 6), J111541.01+263328.6 (# 11), and perhaps J161836.09+153313.5 (# 12) for SMC dust. We caution, however, that the reddening correction might be over-estimated for these spectra. The SMC dust-corrected data of sample A seem to indicate a slight change of the slope of the average SED at highest frequencies. However, given the small number of data points and the huge scatter, this trend should not be considered significant. The effect is much weaker for the data corrected with the Gaskell reddening law. Surprisingly, there are no significant differences between the averaged SEDs of the three samples. In particular, the average SED of the unusual BAL quasars from sample C shows the same trend in decreasing flux at log ν > ∼ 15 as the 3000 Å break quasars from sample A. The ensembleaveraged SEDs of the three samples seem to be indistinguishable from each other and from the SED of normal quasars. We only notice a trend towards slightly lower MIR fluxes or alternatively slightly higher UV fluxes for our quasars. Infrared luminosity Quasars are known to be invariably luminous in the IR with luminosities comparable to ULIRGs with L IR > 10 12 L . Haas et al. (2003) derived 3 -1000 µm IR luminosities for a sample of 64 Palomar-Green quasars. All of their quasars at 0.5 < ∼ z < ∼ 2 detected in the IR have L IR > 10 12 L with L IR > 10 13 L for the majority. Farrah et al. (2012) investigated the optical to FIR SEDs of a sample of reddened FeLoBAL quasars with 0.8 < z < 1.8 and found 1 -1000 µm IR luminosities at rest-frame L IR > 10 12 L with L IR > 10 13 L for one third of the sample. Under the assumption of isotropy, the IR luminosity is L IR = 4π d 2 L f ν,IR , where d L is the luminosity distance and f ν,IR is the flux F ν integrated over the IR spectral range. To estimate the IR Fig. 8. Composite SEDs from photometric data corrected for Galactic foreground reddening and intrinsic dust reddening (green filled squares) for the quasars in samples A, B, and C by adopting SMC reddening (left) and Gaskell reddening (right), respectively: median values (symbols as in Fig. 2) and standard deviations (vertical error bars). The normal quasar composite from Fig. 6 is over-plotted in black. luminosities for the quasars from our sample, we simply integrated the best matching quasar composite from 1 -1000 µm in the rest frame. The results are listed in Table 1. With the exception of J140025-012957 at z = 0.585, all quasars have L IR > 10 12 L . In line with Farrah et al. (2012), we found that 39% and 30% of the quasars from samples A and B, respectively, have L IR > 10 13 L , whereas the percentage is higher (77%) for sample C. The L IR histograms are similar for all three samples. The mean luminosities and standard deviations are L IR = 1.9 ± 2.8, 1.1 ± 1.6, and 1.7 ± 1.2 10 13 L for samples A, B, and C. Monochromatic 3000 Å luminosity We computed monochromatic luminosities at 3000 Å rest frame as L 3000 = 4π d 2 L f ν,3000 where f ν,3000 = ν F ν at λ = 3000Å. As for the IR luminosities, the flux was estimated from the bestmatching normal quasar composite. That means, the 3000 Å break is assumed to be caused by absorption by dust or gas and is corrected for in that way. In other words, it is implicitly assumed that the intrinsic emission is represented by the SED of normal quasars. This is in principle correct for the reddened BAL quasars in sample C, but it is only a working hypothesis for samples A and B (see Sect. 4). Figure 9 displays the comparison of those 3000 Å luminosities with the L 3000 given by Shen et al. (2011) for the quasars from the SDSS DR7 quasar catalogue (Schneider et al. 2010). 12 Independent of the redshift, the corrected 3000 Å luminosities for the quasars from our samples tend to be higher than those of typical SDSS quasars. Fig. 9. Monochromatic 3000 Å luminosities, L 3000 , versus redshift for 80 847 quasars from the Shen catalogue (small black dots) and for the present samples. Symbols as in Fig. 2. Figure 10 shows the distribution of the ratios of the IR luminosity to the monochromatic luminosity at 3000 Å for samples A, B, and C. The histograms are similar for all three samples, except for a trend towards lower ratios in sample C. The mean ratios are 1.73 ± 0.51, 1.60 ± 0.39, and 1.37 ± 0.59 for samples A, B, and C, in good agreement with the ratio L IR / 3000 ≈ 1.8 derived from the composite spectrum for ordinary radio-quiet quasars. 12 The catalogue from Shen et al. (2011) gives L 3000 only for z < ∼ 2.25. Fig. 10. Histograms of the ratio of the IR luminosity L IR to the 3000 Å monochromatic luminosity L 3000 for samples A, B, and C. To illustrate the effect of the absorption correction, the lefthand panel of Fig. 11 shows the comparison of L 3000 from our study with the monochromatic 3000 Å luminosities given by Shen et al. (2011), where we corrected the latter for the intrinsic dust reddening with the values of E(B − V) from Table 1. The scatter around the 1:1 relation (dotted line) reflects that the dust reddening correction procedure corrects only for a part of the decline of the SED at λ < ∼ 3000 Å. It is thus not surprising that the scatter is greatest for quasars from sample C with their strong absorption troughs. The largest differences are measured for J094317.59+541705.1 (# 51) and J173049.10+585059.5 (# 46), two extreme FeLoBAL quasars where the continuum drops rapidly at λ < ∼ 3000 Å to a level close to the detection threshold down to the shortest wavelengths covered by the optical spectra (see also Urrutia et al. 2009;Hall et al. 2002a;Paper I). In the middle panel of Fig. 11, the IR luminosity is plotted versus the (uncorrected) 3000 Å luminosity from Shen et al. (2011). For comparison we over-plotted those FeLoBAL quasars from Farrah et al. (2012) for which L 3000 is available in the Shen catalogue (black symbols). The dotted line in this diagram represents the quasar composite spectrum. The right-hand panel of Fig. 11 shows L IR versus L 3000 . In agreement with Figs. 8 and 10, the majority of our quasars are fitted by the relation for normal quasars with a trend towards either slightly lower L IR or slightly higher L 3000 for some quasars, in particular from sample C. Fig. 11. Left: Monochromatic luminosity L 3000 derived here from the fit of the AGN composite SED to the extinction-corrected photometric data compared with the values from the Shen catalogue after correcting for the same amount of extinction. Symbols as in Fig. 2. Middle: Infrared luminosity L IR versus the monochromatic 3000 Å luminosity L 3000 from the Shen catalogue without extinction corrections. The black plus signs are for the quasars from Farrah et al. (2012). Right: Infrared luminosity L IR versus the extinction-corrected luminosity L 3000 from the present study. Radio loudness Thirty quasars (47%) from our sample were detected by FIRST, 33 quasars are below the FIRST level, and one quasar, J005041.59+143755.9, is not in the FIRST area. The percentage of FIRST detected quasars decreases from 74% for sample A to 60% for sample B and 16% for sample C. It was shown in Paper I that the radio detection fraction strongly correlates with the degree of peculiarity of the spectrum and that this trend can be fully explained as a selection effect. Of the 22 SDSS quasars in sample A, 16 (73%) were selected for SDSS spectroscopy because they were detected as FIRST radio sources. The ratio of the number of quasars targeted as FIRST sources to the number of quasars targeted not as such is 2, compared to 0.01 for the normal quasar population in the SDSS DR7 quasar catalogue (see Paper I). Quasars are usually classified as radio-loud based on the radio-to-optical flux ratio. We use the K-corrected ratio R = F 5GHz /F 2500Å of the 5 GHz radio flux density to the 2500 Å UV flux density in the quasar rest frame as a measure of radio loudness (Stocke et al. 1992). The 5 GHz flux is extrapolated from the FIRST 1.4 GHz flux adopting a power law F ∝ ν α R with α R = −0.5. The UV flux density at 2500 Å is taken from the best-matching SED after correction for intrinsic reddening. That is, the R parameter is corrected not only for intrinsic dust reddening but also for the strong BAL absorption effects. The results are listed in the last column of Table 1, where upper limits are given for FIRST non-detections assuming an upper limit of 0.7 mJy for the detection on the FIRST image cutouts (see Sect. 3.1). The value R = 10 is commonly used to distinguish radioloud and radio-quiet quasars (e.g., Stocke et al. 1992;Francis et al. 1993;Urry & Padovani 1995;Richards et al. 2011), although this value is to some degree rather arbitrary (e.g., Falcke et al. 1996;Wang et al. 2006). In general, the distribution of the radio loudness of quasars appears to be bimodal with about 10% being radio loud (Kellermann et al. 1989;White et al. 2000;Ivezić et al. 2002). The mean value of the radio-loudness parameter for the 63 quasars from Table 1 in the FIRST area isR = 5.6. Only three quasars (5%) have 10 < R < 100. This range of the radio-loudness parameter is sometimes called radio-intermediate (Falcke et al. 1996;Wang et al. 2006). All three quasars belong to samples A and B. Extreme overlapping absorption troughs The photometric data of the prototypical quasar J010540.75 − 003313.9 indicate enhanced FIR emission (Fig. 7). For the vast majority of our objects, however, the FIR is not covered by our photometric data, and no conclusion can be drawn on a possible starburst component. From MIR to UV, the SED is dominated by the AGN. The results from the previous section demonstrate that the SED of 3000 Å break quasars from MIR to NUV is indistinguishable from the SED of unusual BAL quasars. All three quasar samples have SEDs that are similar to that one of normal quasars. This agrees with previous studies (see Sect. 1) showing that BAL quasars are not significantly different from non-BAL quasars in terms of their MIR-UV SED and IR luminosities. The similarity of the mean SEDs of samples A, B, and C from MIR to FUV supports the explanation of 3000 Å quasars as LoBAL quasars. It seems reasonable to assume that the main difference between samples A and C is unusually wide, overlapping absorption troughs for sample A quasars. Green (2006) proposes a warm (ionised), probably filamentary relativistic wind that is smooth in velocity space with a profile "more like a shallow bowl than a trough". The proposed absorption structure is then not identical to typical BALs, but its velocity profile is broader and smoother. Single absorbers with high outflow velocities are indicated for some objects from our sample. For example, the absorber system at z abs = 1.158 in the z em = 1.359 quasar J161836.09+153313.5 (# 12) with a relative velocity ∼ 0.07c (Sect. A) makes it plausible that high-velocity BALs contribute to the absorption. The onset of a broad absorption structure at about 3000 Å (towards shorter wavelengths) is likely due to overlapping absorption troughs from Mg ii and Fe ii, possibly in Fig. 12. Spectra of six 3000 Å break quasars from sample A, de-reddened with the reddening law from Jiang et al. (2013;red). The observed spectra (after correction for Galactic foreground extinction) are shown in black, the SDSS quasar composite (Vanden Berk et al. 2001) in blue. combination with iron emission at λ > ∼ 3000 Å. It is well known that LoBAL quasars have enhanced Fe II emission line blends, and it is evident that this is also the case for many quasars from the comparison sample C. Blends by UV iron emission lines are likely to contribute also to the observed continuum structures in the spectra from sample A, but this is much less evident. The understanding of their UV spectra would certainly benefit from a detailed quantitative analysis of the iron emission, but this is clearly beyond the scope of the present paper. Figure 1 indicates spectral variability for both J010540.75-003313.9 and J220445.27+003141.8 (Sect 2.1). The difference between the two spectra of J220445.27+003141.8 shows the characteristic variability behaviour of the quasar continuum, namely stronger variability at shorter wavelengths (Wilhite et al. 2005;Meusinger et al. 2011). The addition of BALs can strongly modulate the wavelength dependence of quasar variability, and in fact, BAL trough variability has been observed in a number of cases and references therein; Sect. 1). The difference of the two spectra of J010540.75-003313.9 at λ obs < ∼ 5400Å is clearly different from that of J220445.27+003141.8 and might hint at variable absorption troughs. If true, the observed spectral variability of J010540.75-003313.9 further sup-ports the interpretation of the spectral shape at λ < 3000 Å as due to unusually broad absorption line structures. Reddening with a steeper than SMC extinction curve Although most of the statistical studies on dust reddening of quasars suggest an extinction curve like in the SMC or flatter (Sect. 3.2.2), reddening curves steeper than in the SMC were suggested for some samples or individual quasars Zhou et al. 2006;Fynbo et al. 2013;Jiang et al. 2013;Leighly et al. 2014). Presuming that the continuum of the dereddened broad-band SED is similar to that of the average quasar SED, Jiang et al. (2013) have recently derived a reddening curve for the local 3000 Å break quasar IRAS 14026+4341 that steeply rises towards short wavelengths at λ < 3000 Å, which is very different from the reddening usually observed in nearby galaxies. These authors argue that such an unusual reddening law can be caused by the speculative assumption of an exotic dust grain size distribution that lacks large grains. Is the spectral shape of the 3000 Å break quasars from sample A explained by the Jiang reddening curve? Figure 12 shows six representative examples. The quasars # 10, 13, 15, and 18 clearly indicate absorption structures shortwards of the Mg ii line that are better matched by a more or less flat trough or bowl than by the Jiang reddening curve. In the case of J215950.30+124718.4 (# 15), the de-reddened spectrum displays characteristic absorption structures from Fe ii. J145045.56+461504.2 (# 18) is obviously over-corrected below 2200 Å. The de-reddened spectrum of J160827.08+075811.5 (# 6) resembles a weak-line quasar, but with an unusual broad emission structure shortwards of Mg ii. One might speculate that this feature is an extremely broad wing of the Mg ii (see also Hall et al. 2002a), but this would require an unrealistically low systemic redshift of z em = 1.13. 13 The structure in the de-reddened spectra therefore remains unexplained. The same applies to J010540.75-003313.9 (# 5). We conclude that the reddening curve derived by Jiang et al. (2013) for IRAS 14026+4341 does not provide a reasonable explanation of the characteristic features of the 3000 Å break quasars from our sample. There are two arguments that IRAS 14026+4341 is different from our 3000 Å break quasars. First, spectropolarimetric observations of IRAS 14026+4341 revealed high polarisation that rises rapidly towards the blue, peaks at ∼ 3000 Å (rest-frame), and remains nearly constant at shorter wavelengths (Hines et al. 2001). However, spectropolarimetric observations of the two prototypical 3000 Å quasars J010540.75-003313.9 and J220445.27+003141.8 showed that their polarisation is very weak (DiPompeo et al. 2011). Second, IRAS 14026+4341 is brighter in the MIR and FIR than typical quasars (Fig. 13), but the quasars from our sample are not (see Figs. 8 and 11). Truncated accretion disks Finally, we briefly address the question of whether the 3000 Å break can be explained by an accretion disk with a strongly suppressed innermost part. An interesting candidate is the extremely X-ray weak BAL quasar PG 0043+039 whose UV spectrum peaks at ∼ 2500Å and shows a set of broad humps that can be explained as cyclotron line emission from a strongly magnetised hot plasma close to the supermassive black hole (SMBH; Kollatschny et al. 2015a,b). Fig. 14. BOSS spectrum of J220445.27+003141.8 (black) compared with the spectra of an accretion disks with a temperature parameter T * = 9 · 10 5 K (Meusinger & Weiss 2013) and an inner hole of radius r in from 3 to 38 (red curves, top to bottom) in logarithmic steps of 0.1 dex, arbitrarily normalised at 3900Å. A natural reason for a distortion of the accretion flow could be an inspiralling secondary SMBH in a close supermassive binary black hole (SMBBH; Gültekin & Miller 2012). The SMBHs in a galaxy merger are expected to sink into the potential well of the remnant via dynamical friction and to form a SMBBH (Begelman et al. 1980). The first luminous, spatially resolved binary quasar that clearly inhabits an ongoing galaxy merger was presented by Green et al. (2010). The first FeLoBAL quasar in a relatively small separation binary was discovered by Gregg et al. (2002). At a small separation and a sufficiently large ratio of the mass of the secondary SMBH to the binary mass, al- most the entire region inside of the orbit of the secondary will be dynamically unstable. The major effect will be an inner hole in the accretion disk (AD) and thus a substantial suppression of the emission at short wavelengths, leaving a red SED (Gültekin & Miller 2012). Direct observational evidence of close SMBBHs is sparse, but there are promising candidates (e.g., Valtonen et al. 2011;Dotti et al. 2012;Graham et al. 2015). A circum-binary AD with a central hole was recently proposed to explain the optical-to-UV continuum of the nearby quasar Mrk 231 (Yan et al. 2015). Its optical and UV spectrum shows a maximum at ∼ 4000 Å followed by a decline at a shorter wavelength that is, however, much shallower than for the 3000 Å break quasars. We computed spectra of model ADs assuming the Shakura & Sunyaev (1973) approximation for a set of disk temperature parameters with the inner radius as a free parameter. As expected, the decline in the observed spectra is much steeper than predicted by the model (Fig. 14). Further peculiarities In a few cases, peculiarities in the spectra of very unusual quasars might stem from the superimposition with other objects on the line of sight (Paper I). J091613.59+292106.2 (# 4) is the only quasar in sample A where the decrease in the SDSS spectrum shortwards of the peak turns into an increase at short wavelengths. In Paper I, we argued that this feature is perhaps due to reddening by Galactic dust 14 at the redshift of the quasar. However, the agreement between the de-reddened spectrum and the quasar composite is rather poor at λ obs < ∼ 4400 Å (Fig. 15, top). Alternatively, the observed spectrum might be the superimposition of the spectra of two quasars at approximately the same redshift (Fig. 15, bottom). The part at λ obs < ∼ 4600 is well matched by the unreddened SDSS quasar composite and might thus represent an ordinary quasar. The residual from subtracting this component from the observed spectrum of J091613.59+292106.2 is similar to a typical 3000 Å break quasar spectrum. Summary and conclusions The 3000 Å break quasars constitute a rare type of luminous AGNs. The defining spectral features are a usual blue continuum at λ > ∼ 3000 Å, in combination with a drop-off around 3000 Å that does not clearly show the typical structure of broad absorption lines, and a lack of substantial typical quasar emission lines. The prototypes are J010540.75-003313.9 and J220445.27+003141.8 from Hall et al. (2002a). We searched for 3000 Å break quasars in the spectra database of the SDSS DR7 and DR10 using a number of large Kohonen self-organising maps. We compiled a sample of 23 quasars of the type of 3000 Å break quasars (sample A). In addition, we compiled both a comparison sample of other unusual quasars that is dominated by LoBAL quasars with overlapping absorption BAL troughs or with many absorption lines (sample C) and a sample with quasars showing properties between those of samples A and C (sample B). Particular attention was paid to avoiding contamination by unusual DQ white dwarfs that were found to mimic the spectra of 3000 Å break quasars. We constructed broad-band SEDs for all 64 quasars using archive data from GALEX, 2MASS, UKIDSS, WISE, Spitzer, and Herschel, in addition to the photometric and spectroscopic data from the SDSS. We corrected for dust reddening at the redshift of the quasars by the comparison with the mean broad-band SED of normal quasars for standard assumptions on the reddening law (SMC reddening and alternatively a flat reddening curve). The derived mean values of the colour excess E(B−V) ≈ 0.1−0.2 mag are higher than for normal quasars. However, as for the entire SDSS quasar population, strongly reddened quasars with E(B − V) > 0.4 mag are very rare. Provided the intrinsic SED is similar to the standard quasar SED, the quasars from all three samples are intrinsically relatively bright in the UV. The absorption-corrected monochromatic luminosities at 3000 Å are, on average, higher than for normal quasars at the same redshift. At wavelengths > ∼ 3000 Å, the de-reddened arithmetic median composite SEDs from MIR to NUV are very similar for the three samples and are indistinguishable from the SED of normal quasars. The 3000 Å break in the continuum does not vanish after de-reddening with standard reddening laws. The similarity of the broad-band SEDs of samples A, B, and C supports the interpretation of 3000 Å break quasars as extreme versions of LoBAL quasars. We conclude that 3000 Å break quasars are most likely extreme versions of LoBAL quasars. A definitive proof could be provided by high-resolution spectra with sufficiently high signal-to-noise ratio and/or by further multi-epoch spectroscopy. In either case, it is quite possible that individual blue-shifted absorption features might be separately identified. The percentage of FIRST-detected radio sources (74%, 60%, and 16% for samples A, B, and C, respectively) is much higher than for the entire population of SDSS quasars. This is most likely a selection bias caused by the fact that the majority of the quasars with spectra similar to 3000 Å break quasars were targeted by the SDSS, not because of their UV properties but because they were detected as radio sources. If this is true, it seems likely that a much larger population of such quasars exists that can be found by a dedicated search program focused on the UV properties when making use of the photometric data from IR surveys in combination with proper motion data from ESA's Gaia mission to reduce stellar contamination. (11) J111541.01+263328.6 The (noisy) SDSS spectrum displays two absorption troughs from Mg ii and Al iii at z abs = 1.327. Identification of the double emission peak at the red edge of the 6500Å trough with Mg ii λλ 2796.3, 2803.5 yields z em = 1.355. Emission redshift uncertain. (12) J161836.09+153313.5 Another source discovered by Plotkin et al. 2010 and independently in Paper I. In earlier SDSS data releases identified as high-z quasar due to a confusion of the peak with Lyman α. Carballo et al. (2008) give a revised value z = 1.322 based on the identification of the peak with Mg ii. SDSS spectrum similar to J160827.08+075811.5 (# 6). If the narrow emission line at λ obs ≈ 8800Å is [O ii] λ3730, the systemic redshift is z em = 1.359 and the peak of the spectrum is at shorter wavelengths than Mg ii. The system of narrow Mg ii and Fe ii absorbers at z abs = 1.158 make it plausible that high-velocity BALs contribute to the absorption. (13) J130941.35+112540.1 Another source discovered by Plotkin et al. 2010 and independently in Paper I. Similar to J134246.24+284027.5 (# 8), though the break is at the blue side of the Mg ii absorption. Figure B.1 displays the individual SEDs for all quasars from our samples A, B, and C in the same order as in Table 1. On the lefthand side, the observed optical spectra expressed by log F λ (λ) are shown in black, over-plotted with a slightly smoothed version in red. Each panel is labelled with the running number from Table 1, the name, and the redshifts estimated from emission and/or absorption features. For comparison, the SDSS quasar composite spectrum from Vanden Berk et al. (2001) is overplotted in blue. The vertical solid green lines above the quasar spectrum indicate the positions of the strongest emission lines in the composite, the dotted lines indicate typical weaker emission lines. The vertical green lines below the quasar spectrum indicate the positions of strong absorption lines seen in the spectra of BAL quasars (taken from Table 1 in Hall et al. 2002a). The panels on the right-hand side of Fig. B.1 show the broadband SEDs as log νF ν (log ν) in the quasar's rest frame. The transformation into the rest frame is based on the emission redshift, if available, and on the absorption redshift in all other cases. The original fluxes are plotted as red squares with error bars without correction for intrinsic dust reddening. For the majority of the data points, the error bars are smaller than the symbol size. Upper limits (for the data from GALEX and WISE) are indicated by downward arrows. FIRST radio sources are labelled as such in the lower left-hand corner of each panel. For quasars measured in the NIR bands J, H, and K both by 2MASS and UKIDSS, only the UKIDSS fluxes were plotted. The blue squares are the fluxes corrected for intrinsic SMC reddening, the magenta squares for Gaskell dust. The data points are interconnected by dotted lines in order to guide the eye. The SMC extinction-corrected optical spectra are over-plotted in cyan. The vertical yellow stripes indicate the positions of the silicate absorption troughs in the MIR, the vertical green lines mark the positions of the PAH emission features.	2016-01-04T15:45:40.000Z	2016-01-04T00:00:00.000	{ "year": 2016, "sha1": "77b6013b9db3efa58a3981e10033a0539d38255d", "oa_license": null, "oa_url": "https://www.aanda.org/articles/aa/pdf/2016/03/aa27277-15.pdf", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "77b6013b9db3efa58a3981e10033a0539d38255d", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
532340	pes2o/s2orc	v3-fos-license	Diabetes in hospital deliveries in public and private health system hospitals OBJETIVE: To analyze trends of diagnoses of diabetes mellitus in hospital deliveries. METHODS: Transversal study analyzing data on hospital deliveries for pregnant women living in Ribeirao Preto, SP, from 1998 to 2007. The data on diabetes mellitus in pregnancy were obtained from the Faculdade de Medicina de Ribeirao Preto of the Universidade de São Paulo Hospital Data Processing Center. The data analyzed were: age group, type of delivery (vaginal or cesarean), length of hospitalization and type of care: public (SUS) or private (private and supplemented health care). RESULTS: There was a 3.9 fold increase in the proportion of deliveries with a record of diabetes in relation to the total number of births (p = 0.01). This increase was of 4.5 times in deliveries in the public health care system (p = 0.01) and 3 times in private care. An increase in the presence of diabetes was observed in all age groups, proportionally larger in lower age groups. The frequency of cesarean delivery in pregnancies which recorded diabetes fell from 64.5% in 1998/1999 to 39.8% in 2006/2007 in the public system; in the private system the frequency remained over 90%. CONCLUSIONS: The presence of diabetes mellitus in hospital deliveries increased throughout the two year periods, despite a fall in the overall number of deliveries and an increase in the number of women of childbearing age living in Ribeirao Preto. This trend means that not only diagnosis and treatment, but also pre-pregnancy interventions which may reverse it are called for. DESCRIPTORS: Diabetes, Gestational. Delivery, Obstetric. Cesarean Section. Hospitals, Private. Hospitals, Public. 2 Diabetes in hospital deliveries Teixeira CRS et al At the moment, an increase in the prevalence of diabetes has been observed in almost all countries. In Brazil, a population-based study carried out in the state capitals between 1986-1988, with a population of 30-69 year olds, showed a prevalence of 7.6% for diabetes and, in the state of Sao Paulo, this was 9.7%.12 More recent regional studies show higher rates; of 15.0% in the city of Ribeirao Preto, SP, Southeastern Brazil in 2005-2007,14 and of 13.5%, in Sao Carlos, SP, in 2008.4 With increased prevalence in the general population, it is possible that this increase is also occurring among pregnant women. Various studies report the growing trend of gestational and pre-gestational diabetes mellitus (GDM),7,8,11 which is linked to higher risk of morbidity and maternal and perinatal mortality.2,6,18,21 In the USA, the growing trend for the presence of GDM in hospital deliveries, in all age groups has been described for the period 1994-2004, with a marked increase in type 2 diabetes.11 A study which evaluated the frequency of diabetes type 1 (DM1), type 2 (DM2) and GDM in 261 diabetic pregnant women in Ribeiaão Preto, SP, between 1992 and 1999, found GDM to be the prominent condition, present in 53.2% of the women.13 The aim of this study was to analyze trends for diagnosing diabetes mellitus in hospital deliveries. INTRODUCTION International Classifi cation of Diseases (ICD-10).a The types of diabetes were not analyzed, as the reliability of this information on the hospital admission form was limited or it was not included. The following were considered independent variables: age group, how the costs of hospitalization were covered (SUS and other hospitals), type of delivery and mean duration of the hospitalization in days. Hospitalizations classifi ed as not covered by the SUS included private admissions, with payment made directly to the doctor or the establishment, and those which were pre-paid, such as health insurance plans, group medicine, cooperatives, partnerships or self-management.With regards to the type of delivery, they were classifi ed according to the resolution, these being: vaginal (normal or forceps) or surgical (cesarean). For the descriptive study of the variables, the Statistical Package for the Social Sciences (SPSS), version 17.0 was used.In the results, the data are shown grouped in two-year-periods, for less variability and better visualization. To test the theory of variation in the rates of DM throughout the period studied, a regression model was used in which the number of women with DM mentioned in each two year period is considered to follow Poisson distribution, being the mean of the product between the total number of pregnancies in the two year period and the respective DM rate in hospital deliveries.In order to control the effects of overdispersion of data, the model included a random effect with normal distribution. 1The rate of DM mentioned in delivery was considered to be related to the time by a logarithmic link function so that, when testing the signifi cance of the coeffi cient associated with time, the null hypothesis of homogeneity of these rates over time is tested.Thus, coeffi cients which enabled the stratifi cation of rates by age group were included in this function.The level of signifi cance adopted was 0.05.SAS/STAT, version 9.2 were used for this analysis. The research project was approved by the Ethics Committee of the Centro de Saúde Escola of the Faculdade de Medicina, Ribeirao Preto, Universidade de São Paulo (Protocol nº 51/2011). RESULTS Between 1998 and 2007, by age group, type of care, mean duration of hospitalization and type of delivery, it was observed that the total number of births tended to decrease over the two-year-periods, this was proportionally more accentuated in lower age groups.In contrast, the frequency of births in the over 35 age group increased from 9.5% in the two-year-period 1998-1999 to 12.7% in the 2006-2007 period.The deliveries were predominantly fi nanced by the SUS (605 of the total).In the period in question, the frequency of cesareans was 30.2% in the SUS and 86.9% in the other hospitals. The mean duration of hospitalization for cesareans was 2.8 days in the SUS and 1.6 days in the other hospitals; for vaginal deliveries, the mean stays were 1.9 and 1.2 days respectively (Table 1). With regards to the presence of diagnoses of DM in hospital deliveries (Table 2), a 3.4 fold increase was 90%.The mean length of hospitalizations for deliveries with the presence of diabetes was 3.0 days in vaginal births and 3.8 days in cesarean births for those treated by the SUS and 1.7 and 2.0 days, respectively, in those treated in the other hospitals. Table 3 shows the percentages of births with a record of diabetes according to age group and how the treatment was paid for.The proportion of diabetes was more frequent and increasing in the 35 and over age group (p = 0.01).However, even in the 15 to 24 age group, the presence of diabetes was signifi cant, with a growing frequency (p < 0.01) in a greater proportion to that of other age groups.In both public and private health care the frequency of diabetes in deliveries was signifi cant in all age groups (SUS: 15-24 and 24-35, p < 0.01 and 35 and over, p = 0.01; other hospitals: p = 0.01 in all age groups; 15-24 and 25-34, p < 0.01 and 35 and over, p = 0.01).Both the proportion of diabetes and the trend to increase were more accentuated in deliveries treated by the SUS than those in the other hospitals, as can be seen in Figures 1 and 2. DISCUSSION With regards to diagnoses of diabetes in deliveries for residents in Ribeirao Preto, it was observed that there was a 3.9 fold increase in the 1998 to 2007 period.International population studies have also shown trends of increases in the presence of diabetes in pregnancy. 2,7,8,11These findings reinforce the theory that the increase in diabetes described in the population of Ribeirao Preto 14 may also be observed in the presence of DM in women resident in the city giving birth. Thus, the data show an increasing tendency for the presence of DM in deliveries throughout the two-year-periods, in spite of the trend for the total number of births becoming reduced and the increase in the number of women of childbearing age living in Ribeirao Preto. As the prevalence of DM increases with age, 11,14 the fact that there has been an increase in the number of births to women in higher age groups may contribute to the increased presence of DM in pregnant women. More advanced maternal age, defi ned as aged 35 and above on the due date, has become more common. 5ccording to the results obtained in Ribeirao Preto, the proportion of deliveries in this age group increased from 9.5% in the two-year-period 1998-1999 to 12.7% in the 2006-2007 period. In this study, the frequency of DM diagnosis in hospital discharges for deliveries in Ribeirao Preto was 2.1% in the period in question, 0.8% in 1998-1999 and 3.2% in 2006-2007.In deliveries in the SUS, the frequency of diabetes was higher than in other hospitals, a difference that became even more accentuated in the latter two-year-periods and was twice as high in the 2006/2007 period (4.0%versus 2.0%).This fi nding may be due to the fact that the majority of referral services for high risk pregnancies belong to the SUS. 13 International data show that DM is diagnosed in approximately 7% of pregnancies, with a variation of 1% to 14%, depending on the population studied and the diagnostic tests used. 10,11 is not possible to detail the criteria used to diagnose diabetes in this study, as the data were obtained from hospital admission forms from diverse hospitals, with codes for birth and associated morbidities.It is probable that some cases of pre-diabetes, which would now be classifi es as DM by the criteria currently in rigor, 17,19 were not categorized as such.Thus, the frequency of DM in hospital deliveries in Ribeirao Preto may be underestimated. With regards to the age group of women with DM in delivery, it was observed that there was an increase in all age groups, but more so among the youngest.Various studies have presented similar results, highlighting concerns about increasing levels of diabetes in young women, a group which was thought to be at lower risk. 9,11In the 15-24 age group, during this period, there was a 6.4 fold increase in the proportion of records of diabetes in hospital births.Health, resolution in 1998, b increasing remuneration for normal delivery and establishing a maximum payment limit for cesareans per hospital, may have contributed to the drop in cesarean rates in the SUS. With regards the mean duration of hospitalization, the mean stay during the period in question varied from 2.9 to 3.5 days for births with DM, greater than for those in which DM was not present, which varied from 1.7 to 2.1 days, irrespective of the type of delivery.The mean stay for births in the SUS is longer, whether cesarean or vaginal, with or without the presence of DM.The longer stay in the SUS may refl ect diverse situations, such as; treating a higher number of women with complications in their pregnancies, the health care professional who attended the birth, in general, not being the same one as for pre-natal care, and even for questions of management.Such aspects merit more specifi c study and do not form part of this study's scope. Another aspect to be considered relates to improvements in the quality of hospital records during the period studied.In Brazil, in the last two decades, there has been increasing interest in the systematic use of health care system databases as a tool in creating health care policies and in planning and managing health care services. 3e data of this study highlight the trend for DM to be present in increasing numbers of deliveries, especially in the younger age groups, and in deliveries carried out by both the public and private health care services.Faced with this situation, the importance of thinking about preventing diabetes mellitus before pregnancy, in all age groups, stands out. Bearing in mind the risks of perinatal and maternal morbidity associated with diabetes, it's increasing signifi cance in pregnancy calls not only for its identifi cation and treatment, but also for pre-pregnancy interventions so that this trend can be reversed in pregnant women. Table 1 . Deliveries according to age group, care, type of delivery and mean duration of hospitalization.Ribeirao Preto, SP, SoutheasternBrazil, 1998-2007.observed in the number of deliveries over the period.This increase occurred in all age groups, being most accentuated in the lower age groups.Deliveries with the presence of DM were predominantly in public (SUS) health care in all of the two-year-periods.In the period from 1998 to 2007, the frequency of cesareans for pregnant women with DM was 49.7% in those treated by the SUS and 93.2% in the other hospitals; a reduction was observed in the proportion of cesarean deliveries in pregnant women with diabetes treated by the SUS (64.5% in 1998/1999 to 39.8% in 2006/2007), while the proportion in the other hospitals remained above Table 2 . Deliveries for women with diabetes mellitus according to age group, care, type of delivery and mean duration of hospitalization.Ribeirao Preto, SP, SoutheasternBrazil, 1998-2007. Table 3 . Deliveries for women with diabetes mellitus according to age group and care.Ribeirao Preto, SP, SoutheasternBrazil, 1998-2007. a According to the Poisson regression model Figure 1.Proportion of hospital deliveries with record of diabetes according to age group and type of care between the two-year periods.Ribeirao Preto, SP, Southeastern Brazil, 1998-2007.Time -Private (Supplemented and private health care) Time -Public (SUS) The decrease observed in the proportion of cesarean deliveries with DM attended by the SUS is noteworthy, varying from 64.5% in 1998/1999 to 49.7% in 2006/2007, in contrast with the other hospitals, where levels remained stable and above 90%.The Ministry of Figure 2. Proportion (%) of total hospital deliveries with record of diabetes by age group between the two-year-periods.Ribeirao Preto, SP, Southeastern Brazil, 1998-2007.	2017-05-29T15:55:39.880Z	2013-06-01T00:00:00.000	{ "year": 2013, "sha1": "181a0381b68e3b96efa68c5015d29d224a2ab482", "oa_license": "CCBY", "oa_url": "https://www.scielo.br/j/rsp/a/YVvFWXJ9V8yScVd3RM3qp8y/?format=pdf&lang=pt", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "181a0381b68e3b96efa68c5015d29d224a2ab482", "s2fieldsofstudy": [ "Medicine", "Political Science" ], "extfieldsofstudy": [ "Medicine" ] }
7716010	pes2o/s2orc	v3-fos-license	System-level determinants of immunization coverage disparities among health districts in Burkina Faso: a multiple case study Background Despite rapid and tangible progress in vaccine coverage and in premature mortality rates registered in sub-Saharan Africa, inequities to access remain firmly entrenched, large pockets of low vaccination coverage persist, and coverage often varies considerably across regions, districts, and health facilities' areas of responsibility. This paper focuses on system-related factors that can explain disparities in immunization coverage among districts in Burkina Faso. Methods A multiple-case study was conducted of six districts representative of different immunization trends and overall performance. A participative process that involved local experts and key actors led to a focus on key factors that could possibly determine the efficiency and efficacy of district vaccination services: occurrence of disease outbreaks and immunization days, overall district management performance, resources available for vaccination services, and institutional elements. The methodology, geared toward reconstructing the evolution of vaccine services performance from 2000 to 2006, is based on data from documents and from individual and group interviews in each of the six health districts. The process of interpreting results brought together the field personnel and the research team. Results The districts that perform best are those that assemble a set of favourable conditions. However, the leadership of the district medical officer (DMO) appears to be the main conduit and the rallying point for these conditions. Typically, strong leadership that is recognized by the field teams ensures smooth operation of the vaccination services, promotes the emergence of new initiatives and offers some protection against risks related to outbreaks of epidemics or supplementary activities that can hinder routine functioning. The same is true for the ability of nurse managers and their teams to cope with new situations (epidemics, shortages of certain stocks). Conclusion The discourse on factors that determine the performance or breakdown of local health care systems in lower and middle income countries remains largely concentrated on technocratic and financial considerations, targeting institutional reforms, availability of resources, or accessibility of health services. The leadership role of those responsible for the district, and more broadly, of those we label "the human factor", in the performance of local health care systems is mentioned only marginally. This study shows that strong and committed leadership promotes an effective mobilization of teams and creates the conditions for good performance in districts, even when they have only limited access to supports provided by external partners. Abstract in French See the full article online for a translation of this abstract in French. Background Large-scale mobilization of the international community has helped improve immunization coverage and reduce vaccine-preventable mortality [1]. Progress has been rapid and tangible [2], particularly in sub-Saharan Africa, where national programs have greatly benefited from measures to reinforce the capacity for intervention [3]. However, inequities to access remain significant, large pockets of low vaccination coverage persist, and coverage varies considerably across regions [4], districts [3], and health facilities' catchment areas [5]. In Burkina Faso, the most recent national survey of vaccination coverage showed a 41 percentage point disparity (31%-72%) between health regions with the lowest and highest complete vaccine coverage rates and a 35 percentage point disparity (58%-93%) for diphtheria, tetanus, polio and pertussis vaccine (DTPP3). Heterogeneity is also found at the district level, where coverage can vary considerably among and even within districts. There were gaps of more than 50 percentage points between the extremes of the districts in Burkina Faso and an average gap of 28 percentage points between districts within regions. This paper focuses on district-level factors that can explain these disparities. Thus far, much less attention has been paid to district-level factors than to "micro-level" factors that might determine the propensity to have children vaccinated, either in relation to the demand side (characteristics of families, mothers and children [6]), or the supply side (characteristics of services provided locally). For example, studies in Africa, southeast Asia, and South America [7][8][9][10][11][12][13][14][15] have shown that immunization services' utilization is related to the acceptability, accessibility, quality, and affordability of the services provided by the health facilities and front line staff. Low vaccination coverage has been associated with lack of continuity in services (vaccine shortages, staff absenteeism, and irregularly held immunization sessions and outreach activities), poor accessibility (charges for vaccines or cards, excessive travel distance, long waiting time, and language barriers), unsuitable immunization sessions (insufficient numbers, inconvenient sessions, inappropriate schedules, and late arrival of personnel), and dissatisfaction with providers' attitudes (unfriendly behaviours, limited information transmitted to mothers, and lack of compassion/concern about the child's health). Beyond these locally determined influences, we know little about why some districts perform better than others. Indirect evidence suggests district performance is directly related to the availability of resources required for regular supplies [15], proper functioning of the cold chain [16], and service continuity. One survey suggests that territories' vaccine coverage improves as the density of health facilities increases [17]. High turnover of senior management staff [18], restricted staff mobility [13], poor inter-sectoral collaboration [19], and faulty service organization were presumed to be related to non-performing districts. Health districts' vaccine coverage performance has also been associated with their reactions to events requiring mobilization of local capacities that could divert health workers from routine activities. These events include disease outbreaks and immunization days (IDs), about which contradictory effects have been reported [5,6,13,20,21]. Finally, health personnel motivation and attitudes [19,22] and management leadership have been identified as factors affecting the sustainability and quality of health and immunization programs [9,13,19,22,23]. Suspected managerial breakdowns in the districts are also a key focus of the Reaching Every District (RED) approach proposed by WHO to improve vaccine coverage in lowcoverage areas. The RED approach targets five immunization functions: regular outreach services; supportive supervision; community links with service delivery; monitoring and use of data for action; and improved management capacities [3,24]. Others also recommend developing new strategies to improve the performance of vaccination activities [25] and training all mid-level immunization program managers in supervisory techniques and management [26]. Can performance gaps between districts and process inefficiencies at the district level be explained ultimately by outside contingencies, poor choice of intervention strategies, inappropriate organizational modalities, or suboptimal resource allocation? The answers are not clear and, to our knowledge, no systematic approaches have been undertaken to identify the determinants of these disparities. Most of the literature is based on fragmented and limited evidence and examines factors associated with vaccine coverage in only one district of a country. This paper presents the results of a study exploring district-related factors that may account for variations in district vaccine coverage in Burkina Faso. Six districts with contrasting outcomes participated in this study. Discussions with decision makers allowed us to preselect a number of district-related factors seen as potentially influential. Based on the literature review, the research team then translated these factors into seven research hypotheses. The first four, which are focused on resources, were that, all else being equal, immunization coverage should be higher or moving forward in districts where: 1. donor-supported projects provide resources for routine vaccination activities; 2. the creation of new health posts has improved service accessibility; 3. health posts meet the staffing standards; 4. there is no discontinuity in supplies, nor cold chain failures. The remaining three hypotheses refer to circumstances that are management-focused: 5. the management has introduced immunization strategies to complement the usual EPI-recommended activities; 6. the team copes appropriately with events such as outbreaks and IDs that could disrupt routine activities; 7. the District Medical Officer (DMO) demonstrates a high level of dynamism and commitment. Design The study was based on a multiple-case study design [27]. As the organizing force for all immunization activities, the district was the main unit of analysis, each district being a case. The convenience sample was intended to illustrate the diversity of evolutions in vaccination coverage. Six contrasting cases were selected: three showing increasing rates of DTPP3 and measles coverage between 2000 and 2005, two whose rates stagnated during that time frame, and one recording decreasing coverage. The cases belong to six different health regions and represent a variety of economic and socio-political conditions. Data collection The cases were investigated by reviewing documents, consulting a wide array of key informants and local actors, and drawing on secondary data. Based on discussions with stakeholders and relevant literature, the research team identified potentially influential district-related factors that were subsequently reviewed and discussed with local actors. Two focus groups were held for this purpose in each district, one with the chief nurses of the primary health centres (PHCs) and another with the district medical office (DO) staff. Data related to district resources, activities, and service coverage were gathered from: i) the health information system; ii) documentary sources, such as various Ministry of Health departments' statistical reports and districts' action plans and activity reports; and iii) interviews with key informants (10-15 per district) such as DO staff, health personnel, and certain individuals who had served at the district and had since been reassigned. The indicators of vaccination coverage and the activity statistics regarding the utilization of various front line services (childbirth, antenatal visits, and curative visits) come from health statistics collated by district teams using health centres' activity registers. Documentary sources and interviews with district personnel allowed us to reconstruct the history of the presence of technical and financial partners (projects or activities financed by aid agencies or NGOs), of local vaccination strategies, or of meningitis epidemics (for further details see Table 1). Analysis The analyses used a qualitative approach based on a "pattern matching" system to compare the situation encountered against theoretical propositions derived from the hypotheses [27]. The analyses consisted of three phases. First, a qualitative time-series analysis was carried out in each case. Information from a variety of data sources was organized to highlight the temporal changes and unforeseen events observed over the dependent and independent variables' evolution. We examined each district's 2000-2005 trends in immunization coverage for DTPP3 and measles. We then compared historical trends for each independent variable with trends in DTPP3 and measles immunization coverage. In order to obtain a general overview of the evolution of the utilisation of front line services by the population, the analysis of the evolution of vaccination coverage was compared with that of antenatal consultations and of childbirths that occurred in the health facilities. The research team interpreted these trends, using mural graphs that summarized, for each case, the various data series and key events that occurred during the observation period. Simplified versions of these graphs are presented in the Results section. In the second phase, cases were cross-analyzed, taking each hypothesis individually. This approach fed into the BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 interpretations by means of a replication process aimed at maximizing internal validity. Rather than literal replication, we favoured theoretical replication [27] to see whether predicted patterns were found across and within cases (whether, for example, repeated events-e.g. the replacement of a DMO, followed by the replacement of that replacement-would allow us to test a hypothesis several times during the observation period). The final phase involved returning to the field to discuss the results with the vaccination teams who participated in the group interviews. One or two field visits per district were carried out. These discussions allowed us to validate and refine our interpretation. Results The first subsection below presents results from the single-case analyses. For each district (assigned fictitious names), trends in immunization coverage are compared with trends in those factors and events that are hypothesized to have influenced immunization coverage during the period of observation. Trends in the utilization rates BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 of antenatal consultations and outpatient visits are also considered as indicators of primary health care activity. The second subsection examines the results across the six cases from the perspective of the initial hypotheses. Drawing upon focus group discussions (FGDs), the third subsection introduces the key elements and arguments pertaining to DMO leadership. Case studies Case I: Koya district Time-series analysis: In Koya, DTPP3 coverage increased from 78% (2000) to 94% (2005), maintaining immunization performances above the regional mean throughout the six-year period (with a comparative advantage of 10 to 26 percentage points). The evolution of coverage for both antigens is illustrated in the upper part of Figure 1, with their trends paralleling that of antenatal consultations. The lower part of Figure 1 illustrates historical trends and main events of the period for some key indicators accounting for the independent variables. The district experienced outbreaks of measles (2001), meningitis (2003), then measles again (2004), responding to each effectively with a campaign. The study period saw improved geographic access to the district's PHCs (from an average > 7 km to < 6 km) due to an increase in their numbers, from 23 to 30. In the first three years, only 10% to 13% of health centres met staffing standards, but this proportion increased steadily, reaching 60% in 2005. The district experienced regular disruptions in logistics, cold chain, and vaccine supplies. In 2001 and 2003, all PHCs were affected by a cold chain failure. However, the support of a few technical and financial partners (TFPs), who provided refrigerators, improved cold chain functionality. Between 2000 and 2005, two medical officers were in office, for three years each (Figure1). Both maintained the immunization strategy introduced before 2000 that included registering foreign children in border villages and, in agricultural and farmers' valleys, attracting people at markets, in order to control targeted foreign populations. FGDs -main results: In focus groups, local actors attributed Koya's consistently strong immunization performance to the district's tradition of social mobilization, going back to the EPI's establishment. Such mobilization benefits from the attendance of the nomadic populations' chiefs and the village chiefs at immunization sessions to enlist the villagers' participation, and from the support of the high commissioner and previous DMOs. Mobilization not only makes the population more responsive to immunization, but also sets the groundwork for taking immunization seriously, which helps sustain such strategies. The focus groups considered the current DMO, in office since 2003, to be less committed and apparently resting on his predecessors' laurels. BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 ):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 mothers' participation in immunization sessions. In 2003, support from financial partners and a major contribution of supplies and equipment enabled more PHCs to ensure the cold chain (78% in 2004 and 91% in 2005), reestablished the supply of syringes, and supported supervision. FGDs -main results: With a shift in DMO leadership, the immunization coverage trend reversed direction. Local actors decried the former DMO's lack of transparency in managing resources, his abuse of authority, and his reliance on political connections to deflect criticisms. The district's action plan was not transmitted to the team, nor were consultation meetings held with PHC chiefs or the DO team. The report of the Immunization Cluster Survey carried out in Burkina Faso in 2003 was not shared with the collaborators. The working environment had deteriorated; there were protests calling for his removal, and BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 Measles coverage fluctuated in the first three years, then steadily increased to 81%. The district's performance remained below the regional average throughout the six-year period; however, the gap narrowed over time and immunization rates reached the regional average by 2005 ( Figure 4). Utilization rates for antenatal care and outpatient visits also rose throughout the period, with a steeper incline starting in 2003. In 2003, the DMO prioritized immunization, reinforcing the enhanced outreach strategy through an initiative specially geared to this district's low-density, scattered, and mobile population. Health personnel went door-to-door to identify children and vaccinate them according to their age bracket and immunization status. However FGDs -main results: A new DMO appointed in 2002, noting weak immunization performances, was able to mobilize the team in response. Records indicate more frequent DMO visits to PHCs after his arrival, as well as regular supervisions and statutory meetings. He developed an action plan and introduced a bonus for immunization agents. The DMO's commitment and the resources injected into immunization starting in 2003 are reflected in an increase in coverage. While an undeniable improvement was observed, the extent of the increase is questionable. Local actors share the authors' concern about the validity of three-digit immunization numbers; however, because this district, being on the border, attracts users from outside the country, coverage rates as compiled by the information systems may indeed surpass 100%. , who had been active and productive, assertive and appreciative BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 (sending appreciation letters to team members who did good work). During his mandate, team members had been assigned clear tasks. Financial partners' substantial support positively affected immunization performance; some encouraged social mobilization for immunization through a program that provided food to mothers who brought their children to be vaccinated. According to local actors, this program's interruption in 2004 had a demobilizing impact on mothers and likely contributed to the steady fall in immunization performance. Case VI: Boka district Time-series analysis: Boka is in an accessible region with moderate seasonal population flows. This district registered high immunization performance over the whole period under observation, consistently over the national BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 Reinforced outreach strategy average (DTPP3 and measles coverage above 85% and 70%, respectively, since 2001) ( Figure 6). Antenatal care and outpatient visits utilization rates were also consistently increasing. BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 The district has enjoyed the support of partners which, although not numerous, have been quite stable in the district and have supported immunization activities. FGDs -main results: In 2003 and 2004, this district attained almost complete coverage for both antigens. The district attracts clients from neighbouring districts, thus explaining the fact that coverage levels calculated on the basis of activity statistics exceed 100%. In focus groups, local actors considered the commitment and effectiveness of the latest DMO to be the determining factor. He introduced the child census and, most importantly, ensured rigorous follow-up of the enhanced outreach strategy, which included regular updating of the census register and strict adherence to the village visitation program. He also conducted four supervisions per year and regularly called BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 Census of children PHC chiefs on his cell phone to verify the observance of activities; when a planned visit was not respected, the nurse was required to prepare a narrative report. Local actors also noted how the DMO made use of TFPs' financial support for immunization to extend services and implement new strategies such as the children census. (1) Donor-supported projects: TFPs generally make it possible for districts to have essential resources for immunization such as vehicles and motorbikes for outreach visits, supplies, and a suitable cold chain. Some provide very specific support, as in the distribution of food in Dié which district officials consider to be a powerful inducement for women to attend vaccination sessions. However, our cross-case analyses show no unequivocal relation between the presence of a project or of external partners and district coverage performance. Even when TFPs provide significant support, performance does not appear to improve, except in districts where the DMO provides strong leadership and gives vaccination a high priority (as in Dié). Conversely, the presence of fewer TFPs does not seem to be a limiting factor in Bougou and Koya, where the EPI is a priority and DMO leadership is well established, and where everything unfolds as if the national resources (from the State, equity capital, and the Health Development Support Program) made it possible to provide adequate services. Interviews and observations in the Koya district also suggest that the TFPs' interest in funding activities in general, and those of the EPI in particular, might be related to how much collaboration there is between the local teams and foreign partners, and consequently also, to some extent, to the DMO's leadership. Cross-case analysis (2) Geographic access: Several stakeholders expected the reduction in PHCs' catchment area radius to have a measurable positive impact on vaccine coverage, particularly for DTPP3, which requires several visits. The average radius is 9 km; in our sample, district radius ranged from 15 to 5 km. In Koya, where seven new centres were opened, and in Dara, where the catchment radius was substantially reduced (from 15 to 12 km), improved vaccine coverage might be explained by investment in new health facilities. However, in Dara, this increase can also be attributed to reinforced immunization strategies and the priority given to the EPI by district authorities. Prioritization by an effective leadership also explains the higher than national average immunization coverage in Boka, despite its radius being greater than the national average. (3) Staffing standards: In Burkina Faso, the percentage of PHCs in a given district that meet the Ministry's staffing standard is used to estimate unmet needs. A full complement of staff enables districts to effectively implement vaccination activities such as outreach visits, react better to epidemics, and organize vaccination campaigns. This is why our respondents stressed the importance of this factor in explaining performance variations. In fact, however, our results did not support a clear association between staff levels and district performance over the years covered by the observation. In Dié we noted a steady decrease in vaccine coverage even though 90% of PHCs were fully staffed, whereas in Mandé vaccine coverage expanded while as little as 20% of PHCs had the required staff. It was the same in Koya, which, despite the low number of PHCs that met staffing norms, maintained high and steadily increasing levels of coverage between 2000 and 2003. The significantly improved human resources availability as of 2004 also had no apparent impact on vaccine coverage, which seemed to be holding steady. (4) Logistics, cold chain failures: A significant improvement in logistics translates clearly, as in Mandé and Bougou, into improved vaccine coverage. In both cases, nonetheless, these improvements can also be attributed to district authorities' reinforcement of immunization strategies and TFPs' injection of resources into the EPI. Conversely, while all Koya's PHCs experienced cold chain failures in 2001 and 2003 that resulted in coverage setbacks, their amplitude was effectively contained through extended social mobilization. (5) Local vaccination strategies: Beyond national immunization strategies, the vaccination performance of certain districts can be linked to their commitment to immunization and to how well they adapt their strategies to the district's context. These adaptations emerge either in response to an awareness of poor vaccine coverage that requires a specific response, or from the recognition that certain national approaches are not appropriate for the local context. They can take different forms: enhanced outreach strategies, social mobilization, monitoring or inventorying of vaccination targets. In Koya, social mobilization explains the steady, high coverage maintained despite adverse factors such as epidemics, frequent cold chain breakdowns, vaccine shortages, or staffing shortfalls. In this district, all activity indicators related to primary health care services and population coverage are at high levels, which helps to explain this sustained social mobilization. Moreover, in Bougou and Dara, the designation of the EPI as an action priority and the consequent development of new, context-adapted approaches are strongly linked with improvements in vaccination coverage. In both cases, the original idea and the implementation of these processes can be attributed to DMO leadership. BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 However, these had no measurable impact on those districts' annual vaccine coverage, and districts reacted to the outbreaks with campaigns or by intensifying vaccination activities targeting those specific diseases. (b) Immunization days: Several respondents worried that IDs not only diverted the districts' attention and resources, but also demotivated populations, particularly in their "door-to-door" activities that differ from those used in routine vaccination activities, which promote encounters between health personnel and members of the population at village gathering points. They saw this as a source of confusion for mothers about where to bring their children for routine immunization in the future. Other field staff are not bothered by the IDs, for which sessions are programmed in advance and inserted into their activity programs. In practice, neither IDs nor vaccination campaigns seem to have any measurable impact on the performance of routine vaccination services, even though we counted 9 and 10 campaigns in one year in Mandé and Boka, respectively. (7) DMO leadership. An important result of our participatory approach for data analysis and validation was uncovering the key role of the human factor in explaining the levels, progression, and trend reversals in districts' immunization coverage. In the two districts-Mandé, in the first years of observation, and Dié-where the DMO's presence, motivation, charisma, and collaboration were judged to be poor, there was a considerable decline not only in vaccination performance, but also, significantly, in other indicators used to monitor primary care activities. Both cases reported poorly motivated staff, low levels of collaboration between the district teams and the PHCs' chief nurses, and no strategic direction defined by the district team, nor action priorities, nor targets for available resources to be implemented in the district. Conversely, districts whose leadership was considered to be strong and of high quality, such as Boka, displayed consistently high levels of immunization coverage. In the singular case of Koya, the observed consistently high levels appear to be attributable not so much to individual leadership, but more to the collective commitment to immunization of several community actors and officials (former DMOs included). In Mandé, Dara, and Bougou, changes in leadership appeared to reverse negative trends, inducing growth in coverage and in other activity indicators. Evidence suggests that, in Boka and Dara, the DMO promoted locally-adapted initiatives (a child census and a door-to-door strategy, respectively) in contexts characterized by large mobile populations. These two cases suggest that strong leadership helps create the conditions that facilitate expanding coverage. In both districts, a change in leadership ultimately improved the synergy with local TFPs and resulted in expanded donor-based support for the districts' action plans. This, in turn, broadened the DMOs' latitude to reinforce or expand immunization strategies. These cases also illustrate the DMOs' human resources management skills. In Boka, the DMO was able, through rigorous supervision and follow-up, to secure his team's commitment to sustaining the child census and to ensure smooth operation in a comparatively less accessible district. In Dara, the observed extended immunization coverage reflected the DMO's ability to obtain the greatest output from a level of staffing considered largely insufficient to the district's needs. In Mandé, the intensive efforts of nine and seven ID campaigns in 2004 and 2005, respectively, were handled appropriately by the district manager with no apparent impact on routine activities. In Koya, strong social mobilization appeared to counteract the consequences of supply shortages and logistic failures; despite cold chain failures that affected all PHCs in two different years, routine functioning was not impaired. Discussion Recent studies have advanced our understanding of system-related sources of disparities in coverage among countries, and of macro-level effects that are potentially attributable to human resources allocation [17], vaccine prices [6,28], decentralization [20], institutional performance [6], or aid received, whether technical or financial [2,3,28,29]. Given health districts' growing autonomy, this study is an attempt to contribute, with earlier studies [5,6,19], to better-developed factual bases to explain performance variations in vaccine coverage among districts of the same country, i.e., among territorial entities in comparable political, economic, and institutional environments. This process is all the more interesting because vaccine coverage is particularly sensitive to local health care systems' performance and constitutes a relevant marker of efficacy and good operation [15]. Four of the hypotheses refer to the potential impact of resource allocation on vaccination services efficacy and vaccine coverage progression. First, the results of our study show no unequivocal relation between the presence of a project or of external partners and the performance of districts in terms of coverage. TFPs, whether external aid organizations or cooperation projects, are currently present in nearly all the districts. However, their number, the scope of their interventions, and the types of support they provide vary considerably. Several comparative studies have demonstrated the impact on vaccine coverage of countries' access to transnational initiatives or to various forms of development aid [2,3,28,29]. Second, vaccine coverage is sensitive to district logistics, and, in particular, the cold chain. This is not surprising, given the extent to which immunization activities are contingent upon the BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 continuous availability of the vaccines. It confirms what has been largely demonstrated [15,16], including in Burkina Faso [8]. Third, immunization coverage did not change much in districts where geographic access improved during the period of observation. As in most prevention services, there is evidence that vaccination demand is sensitive to the efforts consumers must expend to receive the services, and vaccine coverage is closely linked with geographic accessibility [7,9,10,30]. While this study does not allow us to draw clear conclusions, we nevertheless believe this should not call into question the necessity of maintaining, as a matter of common sense, strategies for developing primary health care resources in both heavily populated and relatively remote areas, as well as outreach programs to cover geographically dispersed populations. Fourth, the level of staffing in the districts' health facilities was not a key determinant of the districts' performance. The results of our study do not correspond with the expectations of decision makers and field staff, who, as mentioned, tend to consider that inadequate staffing levels are an important constraint on activities. They are also inconsistent with the results of a large comparative study [17]. However, the results did not surprise the research team, for whom it is clear that the link between human resources availability and health system effectiveness in Burkina Faso is very tenuous at both the macro and micro levels [31,32]. Two hypotheses deal with the districts' ability to cope with destabilizing situations. Seasonal epidemics and IDs habitually mobilize an important part of the districts' resources and require considerable exertion, and it has been suggested they might negatively affect routine vaccination activities. On the whole, districts seemed to adapt well and were able to adjust their vaccination activities. The seasonal epidemics did not show a tangible impact. These results tally with those of the comparative analysis of 82 countries carried out by Gauri and Khaleghian [6]. This conclusion must nevertheless be qualified because examination of the evolution of coverage at the national level shows that national performance in vaccination may be sensitive to country-wide epidemics. It may be that districts' coping capacity is limited and dependent on national directives and on the scale of the epidemics, such that they are able to adapt when faced with episodes of low or medium scale. Finally, while the study introduces factual data into the current controversy on the potentially negative impacts of IDs on routine activities, it does not resolve it. Contrary to what has been reported in India [21] and Pakistan [13], for example, neither IDs nor vaccination campaigns seem to have any measurable impact on the performance of routine vaccination services. The core finding of our study is the primordial role of the DMO's leadership in strengthening vaccine coverage per-formance. Our starting hypothesis, according to which the DMO's dynamism and commitment could positively influence the overall performance of vaccination teams and services, is verified. We also found that a strong and committed leadership promotes effective mobilization of teams and creates the conditions for good district performance, even when these districts have only limited access to support from external partners. In Burkina Faso, leadership skills are not a criterion for a DMO's appointment, nor are they fostered as a part of an institutionalized supervision of the DMO under the health region's responsibility. The choice of DMO is made at the central level without clear, standardized criteria. As a consequence, a newly appointed DMO might have little experience or technical knowledge of management, and may scarcely be interested in public health matters. Supervision and training that should be assured by the regional medical office is generally lacking. Our results suggest that leadership skills should receive more attention when a DMO appointment is considered, as well as throughout a DMO's mandate, through adequate support and supervision. Some studies show that immunization services and, more generally, the performance of health districts are linked to the professional and ethical practices [5,19,33,34], commitment, efforts, and motivation of health personnel [13,19,22]. Deficiencies in these qualities arise largely from poor managerial skills and inadequate leadership of the health districts [35]. However, the role of the human factor in local health care system performance remains largely unexplored; it is virtually absent in the technical and administrative institutional discourse and is usually totally obscured by decision makers and development agencies [35]. Preferred strategies such as the RED approach refer to them only indirectly, either in terms of improving governance [3,24] or strengthening the management capacities of mid-level managers [26]. Even if things seem to be slowly progressing, the discourse around factors that determine the performance or breakdown of local health care systems in lower and middle income countries (LMICs) remains largely concentrated on technocratic and financial considerations, targeting institutional reforms, resource availability, or health services accessibility. Initially, the study was not planned to be an in-depth analysis of DMO leadership and neither the specification of study variables nor the analysis could have been based on a leadership framework developed a priori. It is therefore difficult to ascertain precisely and measurably the leadership qualities of the different DMOs who served in the six districts during the period under consideration, and this is definitely a limitation of our study. BMC International Health and Human Rights 2009, 9(Suppl 1):S15 http://www.biomedcentral.com/1472-698X/9/S1/S15 Empirically, from interviews with the field teams, the idea emerged that certain qualities of the DMO could play a key role in the performance of vaccination teams and services. These qualities are presented in Table 1. Given the limitations mentioned earlier, this list is provided for illustration purposes only, with no assumptions regarding its validity outside the context of this study. More in-depth studies are required to identify clearly the key elements of leadership in the context of managing district teams and to document the impacts, still not well understood, of the human factor on district performance. Because of the relatively exploratory character of our approach and its setting in the reality of Burkina Faso, one limitation of the study is the extent to which the results may be generalized. Also, the local context and the participative process led us to concentrate on a relatively limited number of exogenous and endogenous factors to explain differences observed in the degree and progression of coverage in only six districts. Large-scale studies might make it possible to explore further the different mechanisms of causality and the means by which external environments, the human factor, available resources, and institutional elements determine the efficiency and efficacy of district vaccination services. Conclusions The key to success appears to reside in the districts' ability to assemble a set of favourable conditions in which the human factor might play a major role. But the importance of leadership should not overshadow the fact that bringing together these favourable conditions and, particularly, implementing initiatives that are dis-trict-specific and adapted to their realities, such as the enhanced outreach strategy or child census, require at least a minimal amount of financial and technical resources. Our results indicate that a district can get these resources either from redirecting the funding priorities for its action plan and reallocating its own resources (supplied by the central authorities), or else from TFPs. Our observations suggest, in particular, that a change of team and new district leadership could, as was recently seen in The Gambia, provide the impulse needed to create a more collaborative dynamic with local TFPs [35] and encourage them to provide even greater support to the district's action plans. Two decades ago, when the primary health care model was becoming widely adopted, Simmonds [22] advocated for devoting more substantial efforts to strengthening leadership capacities and setting up appropriate incentive systems. These recommendations, too often ignored in the implementation of decentralization, remain very relevant today. List of abbreviations used DMO -District medical officer; DO -District medical office; RED -Reaching Every District; PHC -Primary health centres; DTPP3 -Diphtheria, tetanus, polio and pertussis vaccine; FGDs -Focus group discussions; ID -Immunization day; TFPs -Technical and financial partners; WFP -World Food Program; EPI -Expanded Program on Immunization; LMICs -Lower and middle income countries. Qualities most often mentioned by field staff during focus groups were: (1) Exercising authority: "leadership" authority, personality, charisma, ability "to keep on top of things" (2) Managing teams: taking care to transmit and share information, listening, holding regular meetings, motivating staff, encouraging staff participation in decision-making (3) Ability to create a good working environment (4) Professionalism, voluntarism: able to analyze situations; volunteering; able to innovate and look for new solutions; undertaking new approaches; responding well to unanticipated situations; able to have his decisions recognized at the central and regional levels (5) Diligence: being always present in the district (6) Transparency in the management of resources	2014-10-01T00:00:00.000Z	0001-01-01T00:00:00.000	{ "year": 2009, "sha1": "a52203d867cc2edf359d6493f99e2a533124e695", "oa_license": "CCBY", "oa_url": "https://bmcinthealthhumrights.biomedcentral.com/track/pdf/10.1186/1472-698X-9-S1-S15", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a52203d867cc2edf359d6493f99e2a533124e695", "s2fieldsofstudy": [ "Medicine", "Political Science" ], "extfieldsofstudy": [ "Medicine" ] }
117768160	pes2o/s2orc	v3-fos-license	Self-affine Manifolds This paper studies closed 3-manifolds which are the attractors of a system of finitely many affine contractions that tile $\mathbb{R}^3$. Such attractors are called self-affine tiles. Effective characterization and recognition theorems for these 3-manifolds as well as theoretical generalizations of these results to higher dimensions are established. The methods developed build a bridge linking geometric topology with iterated function systems and their attractors. A method to model self-affine tiles by simple iterative systems is developed in order to study their topology. The model is functorial in the sense that there is an easily computable map that induces isomorphisms between the natural subdivisions of the attractor of the model and the self-affine tile. It has many beneficial qualities including ease of computation allowing one to determine topological properties of the attractor of the model such as connectedness and whether it is a manifold. The induced map between the attractor of the model and the self-affine tile is a quotient map and can be checked in certain cases to be monotone or cell-like. Deep theorems from geometric topology are applied to characterize and develop algorithms to recognize when a self-affine tile is a topological or generalized manifold in all dimensions. These new tools are used to check that several self-affine tiles in the literature are 3-balls. An example of a wild 3-dimensional self-affine tile is given whose boundary is a topological 2-sphere but which is not itself a 3-ball. The paper describes how any 3-dimensional handlebody can be given the structure of a self-affine 3-manifold. It is conjectured that every self-affine tile which is a manifold is a handlebody. 1. Introduction 1 2. Main results: models, the tiling complex, and self-affine manifolds 4 3. Self-affine Z n -tiles and their models 10 4. Properties of monotone models 14 5. Self-affine Z n -tiles whose boundaries are manifolds 17 6. Ideal tiles 20 7. Self-affine Z n -tiles that are homeomorphic to a ball 22 8. Proofs for the examples 26 References 31 Introduction A great deal of work in the literature has concentrated on tilings of R n whose tiles are defined by a finite collection of contractions. One of the most prevalent examples are tilings by self-affine tiles where the contractions are affine translates of a single linear contraction. A long-standing open question is whether there exists a closed 3-manifold which is a nontrivial self-affine tile, a so-called self-affine 3-manifold. To settle this question in the affirmative the current paper effectively characterizes and recognizes self-affine 3-manifolds and gives theoretical generalizations of these results to higher dimensions. The methods developed in this paper build a bridge linking Date: February 14, 2014. The first author was supported by the Simons Foundation Collaboration Grant #246221. The second author was supported by project I1136 funded by the Agence National de la Recherche and the Austrian Science Fund, and by project W1230 funded by the Austrian Science Fund. two previously unrelated areas of mathematics: geometric topology on the one side and iterated function systems and their attractors on the other side. Much work is devoted to how a subset of the Euclidean plane can admit a tiling by self-affine tiles. In the planar case, the topology of these tiles has been studied thoroughly. Much less is known about the topology of self-affine tiles of Euclidean 3-space. In particular it has been an open question as to which (if any) 3-manifolds admit a nontrivial self-affine tiling of R 3 . A number of examples have appeared in the literature which were conjectured to be self-affine tilings of R 3 by 3-balls. In the current paper we address these questions by describing an often effective method of determining that a given 3-dimensional self-affine tile is a tamely embedded 3-manifold. The method gives affirmative answers for the previously conjectured examples, and is also used to give examples of 3-dimensional self-affine tiles which are handlebodies of higher genus. Examples are also given of self-affine tiles in R 3 whose boundaries are wildly embedded surfaces and thus are not 3-manifolds. Our method also has potential to allow effective computations in higher dimensions. The proofs of our results require a careful formulation of the problem in terms of a certain type of algebro-geometric complexes which are used to approximate the tile and allow for arbitrarily fine computations due to their recursive structure. Deep tools of geometric topology developed by Cannon and Edwards are then used to determine the homeomorphism type of the boundary and check if it is a tamely embedded surface in the case of dimension 3. We offer Conjectures 1 and 2 stating that every self-affine manifold is homeomorphic to a handlebody. The study of self-affine tiles and their tiling properties goes back to the work of Thurston [50] and Kenyon [30]. In the 1990s Lagarias and Wang [34,35,36] proved fundamental properties of self-affine tiles. Wang [52] surveys these early results on tiles like tiling properties, Hausdorff dimension of the boundary and relations to wavelets. By now there exists a vast literature on self-affine tiles. The topics of research include their geometric, topological, and fractal properties, characterization problems, relations to number systems, and wavelet theory (see e.g. [1,6,12,23,26,37,48]). The topology of (mainly planar) self-affine tiles is the topic of considerable study (cf. for instance [3,25,27,31,35]). In particular, the case where the self-affine tile is a 2-manifold (i.e., a closed disk) has been well understood from early on (cf. [3,5,38,39,40]). The question of determining the topology of higher dimensional self-affine tiles arose naturally; in particular, if they could be manifolds in a nontrivial way and if there is a method to recognize whether a self-affine tile (or its boundary) is a manifold (see Gelbrich [24] who first raised this question in 1996, and more recently [2,4,17,38]). We will call a 3-dimensional self-affine tile which is a topological 3-manifold with boundary a self-affine 3-manifold. Numerous specific examples of nontrivial self-affine tiles were conjectured in the literature to be topological 3-dimensional balls (see for instance [4,24]). However, until this point, no example of a nontrivial self-affine 3-manifold has been exhibited (in [41] self-affine tiles that are n-dimensional parallelepipeds are characterized). In the current article we give conditions that characterize self-affine 3-manifolds. We go on to give an effective algorithm to decide whether a 3-dimensional self-affine tile is a manifold with boundary. Finally we apply our algorithm to conjectured examples and show that they are topological 3-dimensional balls. During the course of our inquiry we show that many of our results generalize to the n-dimensional case. 1.1. Classical conjectures and solutions. We start with the exact definition of the fundamental objects studied in the present paper. Definition 1.1 (Self-affine tile). Let A be an expanding n × n integer matrix, that is, a matrix each of whose eigenvalues has modulus strictly greater than one. Let D ⊂ Z n be a complete set of residue class representatives of Z n /AZ n , called the digit set. We define the self-affine tile T = T (A, D) as the unique nonempty compact set satisfying (1.2) AT = T + D. If the self-affine tile T tiles R n with respect to the lattice Z n we say that T induces a self-affine tiling and call T a self-affine Z n -tile. The self-affine tile T is well-defined because it is the unique solution of the iterated function system {ϕ d \| d ∈ D} with ϕ d (x) = A −1 (x + d) (cf. Hutchinson [29] and note that there is a norm Theorem 5. 15. For n ≥ 6 let T be a self-affine Z n -tile which admits a monotone model M . Assume that almost all boundary stars of M are cellular and ∂M is a manifold. If T satisfies the disjoint disks property then ∂T is an (n − 1)-manifold. Theorem 7.22 allows us to algorithmically recognize which n-dimensional tiles are tame n-balls. Theorem 7.22. Let T be a self-affine Z n -tile. If ∂T is an (n − 1)-sphere in R n and each loop in the in-out graph (which is always finite) I contains a node N such that (i) ∂N ∼ = S n−1 , (ii) ∂N \ ∂T is simply connected, then ∂T is locally spherical and thus tame. Consequently T is homeomorphic to D n . Given that a self-affine manifold tiles itself by arbitrarily small copies of itself, it seems that its topology cannot be very complicated. We offer the following stronger conjecture: Conjecture 2. Every self-affine n-manifold is homeomorphic to an n-dimensional handlebody. 2. Main results: models, the tiling complex, and self-affine manifolds In this section we present our main results. The underlying idea behind our theory is to "model" a given self-affine Z n -tile T by a set M ⊂ R n that tiles R n by Z n -translates and retains as many topological properties of T as possible. To understand the topology of T it is then sufficient to study M . We give ways that allow to construct M as a finite simplicial complex. This strategy will enable us to derive various new results on topological properties of self-affine Z n -tiles. We will need the following notations. Let M ⊂ R n be a compact set that is the closure of its interior and whose boundary has µ(∂M ) = 0, where µ denotes the n-dimensional Lebesgue measure. If the Z n -translates of M cover R n with disjoint interiors we say that M is a Z n -tile. Note that a Z n -tile always has µ(M ) = 1. As a self-affine Z n -tile T is equal to the closure of its interior and µ(∂T ) = 0 (cf. [52, Theorem 2.1]), T is a Z n -tile. Recall that a function f : R n → R n is called Z n -equivariant if f (x + z) = f (x) + z holds for each x ∈ R n , z ∈ Z n . If, in addition, we have f (0) = 0 then f (z) = z holds for each z ∈ Z n . 2.1. Models for self-affine Z n -tiles. We start with a fundamental definition. If F is clear from the context, we will write M instead of (M, F ) for a model. Note that F is not assumed to be contracting in Definition 2.1. Thus in general, M is not uniquely defined by the set equation (2.2). Let (M, F ) be a model for the self-affine tile T = T (A, D). To transfer topological information from M to T we will make extensive use of the canonical quotient map Q : R n → R n defined by (F (k) denotes the k-th iterate of F ). The following result will be proved in Theorem 3.8. Note that that any subspace X ⊂ M has its canonical quotient Q(X) under this map. In particular, Theorem 2.4 shows that T and ∂T are the canonical quotients of M and ∂M , respectively. 2.2. Neighbors, cells, and the tiling complex. Let M be a Z n -tile. In all what follows, the intersection of M with subsets of its Z n -translates will play an important role. We consider sets of n-tuples of integers to be coordinates for unoriented "cells" in a "tiling complex" defined below and accordingly introduce the notation We extend the range of · M to complexes, i.e., to sets C of sets in Z n by setting If M is the self-affine Z n -tile T , we often omit the subscript T and write S instead of S T . Definition 2.6 (Neighbor structure and tiling complex). The neighbor structure of a Z n -tile M is the set which reflects the underlying intersection structure of the tiling induced by M and will also be considered a formal simplicial complex. The faces of S ∈ K(M ) are the elements of A face of S ∈ K(M ) is proper if it is not equal to S. We consider the set K(M ) of cells along with the notion of faces induced by F M (S) to be the tiling complex for M . Moreover, we set for the set of all i-cells of the tiling complex K(M ). Given a simplex S ∈ K(M ) we define the operator that is, the set of maximal proper faces of S in K(M ). We think of δS M as a form of simplicial boundary of S M . The operator δ can be extended to a collection C of i-cells by setting We intuit the sets S as formal unoriented simplices and S M as their (dual) geometric realizations. Note that it can happen that S is a proper face of S but that S M = S M . 2.3. Monotone models. We refine the notion of model so that it preserves the neighbor structure of the modeled self-affine Z n -tile T . Proposition 4.1 shows that for a model M having the same neighbor structure 1 as T , the canonical quotient map Q preserves intersections: Q S M = S for each S ⊂ Z n . The following notion of monotone model enables us to say more about the point preimages of the canonical quotient map Q. Recall that a quotient map is monotone if point preimages are connected. We state the following result, which follows immediately from Proposition 4.3 (i). Theorem 2.10. If M is a monotone model for the self-affine Z n -tile T then the restriction is a monotone quotient map for each S ∈ K(T ). We saw in Theorem 2.4 that Q∂M = ∂T holds for a model M of the self-affine Z n -tile T . If M is even a monotone model, this follows from Theorem 2.10 because ∂M = ∂ {0} M = δ{0} M and ∂T = ∂ {0} = δ{0} . Under certain conditions Q behaves nicely also for boundaries of cells S with \|S\| ≥ 2. To make this precise let M be a Z n -tile and set for the set of all i-cells of K(M ) containing 0. Assume that the (dual) geometric realization i 0 carries the subspace topology inherited from R n and, for i ≥ 2, denote by ∂ i = ∂ i,M (we omit the index M as it is clear from the context) the boundary relative to K(M ) i 0 M . The following definition contains a property of K(M ) i 0 that allows us to derive results on the behaviour of Q with respect to these boundary operators. We can show the following result which is proved at the end of Section 4.1. 2.4. Self-affine tiles and manifolds. In Section 5.1 we will state a criterion for a self-affine Z 2 -tile to be homeomorphic to a closed disk. In view of the Jordan Curve Theorem this is equivalent to ∂T being homeomorphic to a circle. Criteria of that kind have been published before (see e.g. [5,39]). We shall prove analogs of these results for boundaries of self-affine Z 3 -tiles that are homeomorphic to a closed surface. To treat surfaces of positive genus we need to exclude pathological point preimages of the restriction of Q to ∂M . We thus define the following property of point preimages. Definition 2.13 (Semi-contractible). Let M be a monotone model for a self-affine Z 3 -tile with ∂M being a closed surface and let Q be the associated quotient map. We say that M is semi-contractible if each point preimage of Q\| ∂M is contained in a contractible neighborhood (e.g. a disk). We note that a model with spherical boundary is semi-contractible. Theorem 2.14. Let T be a self-affine Z 3 -tile with connected interior which admits a semi-contractible monotone model M whose boundary is the closed surface S. Then the following assertions hold. (i) ∂T is homeomorphic to S. (ii) Under the restriction Q\| ∂M preimages of points do not path separate ∂M . We immediately obtain the following consequence. Corollary 2.15. Let T be a self-affine Z 3 -tile with connected interior which admits a monotone model M with 2-sphere boundary. Then ∂T is homeomorphic to the 2-sphere and point preimages of the quotient map Q\| ∂M do not path separate. Theorem 2.14 can be extended to finite unions of Z 3 -translates of T (see Proposition 5.9). The next theorem shows that under certain conditions ∂T = s∈Z n \{0} {0, s} admits a natural CW-structure defined by the intersections S . Recall that a set is degenerate if it contains fewer than 2 points. Theorem 2.16. Let T be a self-affine Z 3 -tile with connected interior which admits a semicontractible combinatorial monotone model M whose boundary is the closed surface S. Let S ∈ K(T ) be nondegenerate. If S M is a closed topological manifold or a ball then its canonical quotient S is either homeomorphic to S M or degenerate. The proofs of Theorems 2.14 and 2.16 are contained in Section 5.2 where we also state an easy criterion for checking whether the interior of a self-affine Z n -tile is connected (see Lemma 5.10). It is important to note that the homeomorphisms asserted in these theorems are usually not Q since Q is not necessarily injective. However, one can use Q to construct this homeomorphism with the help of Moore's decomposition theorem (see Proposition 5.2 and [43,44]). Theorem 2.16 requires S to contain at least 2 elements. Under the given conditions we cannot expect T = {0} to be homeomorphic to a 3-ball even if the same is true for M . Indeed, in Section 8.2 we give an example of a monotone model which is homeomorphic to a 3-ball but whose underlying self-affine Z 3 -tile is wild and indeed not even simply connected, even though its boundary can be shown to be a sphere by applying Corollary 2.15. An image of this example is depicted on the right hand side of Figure 1. It is the self-affine Z 3 -tile T = T (A, D) with A = diag (9,9,9) whose digits set is visualized on the left hand side of Figure 1 (see Section 8.2 for an exact definition). In studying this example we shall prove the following result. Theorem 2.17. There exists a self-affine Z 3 -tile whose boundary is a 2-sphere, but which is not homeomorphic to a 3-ball (a self-affine wild crumpled cube). We are also able to prove generalizations of Theorem 2.14 to higher dimensions and give criteria for boundaries of self-affine Z n -tiles to be (n − 1)-manifolds. Since their statements require more notation we postpone it to Section 5.3. In Section 7 we deal with self-affine Z n -tiles that are homeomorphic to closed balls. For n = 3 a combination of Theorem 2.14 and a result of Bing [7] yields the following characterization result. Theorem 2.18. Let T be a self-affine Z 3 -tile whose interior is connected and 1-ULC. Then T is a self-affine 3-manifold if and only if it admits a monotone model with a boundary that is a closed 2-manifold. Using a result of Cannon [8] we will give an algorithmic criterion that allows to check whether a self-affine Z n -tile with spherical boundary is homeomorphic to a closed ball. The exact statement of this result, which requires the definition of the in-out graph, can be found in Theorem 7.22. Together with Theorem 2.14 we shall use this result to give nontrivial examples of Z 3 -tiles that are homeomorphic to closed 3-balls and other 3-manifolds (see Section 2.7). 2.5. Subdivision. Let T = T (A, D) be a self-affine Z n -tile and (M, F ) a model for T satisfying K(T ) = K(M ). We now describe a generalization of the set equations (1.2) and (2.2) to the cells S and S M for S ∈ K(T ), respectively. We start with the following definition. Definition 2.19 (Subdivision operator). Let T = T (A, D) be a self-affine Z n -tile. Then the subdivision operator P is given by where D S denotes the set of functions from S to D. It is unnecessary to define a similar operator for the model M since K(T ) = K(M ) and F (z) = Az holds for each z ∈ Z n which implies that P (S) = {(p + F )(S) ∈ K(M ) \| p ∈ D S }. As which we shall prove in Theorems 3.13 and 3.21. In Lemma 6.1 we will see that P behaves nicely with respect to the "simplicial boundary operator" δ defined in (2.7) and (2.8), in particular, (2.21) δP (S) = P (δS). 2.6. Ideal tiles. Up to this point it is unclear how one could construct nontrivial models for a given self-affine Z n -tile. We now give necessary conditions that define an ideal tile for a self-affine Z n -tile which allows to construct a monotone model for that tile. (ii) For each S ∈ K(Z) the set S Z is connected and homeomorphic to P (S) Z . (iii) For each S ∈ K(Z) each homeomorphism from δS Z to δP (S) Z extends to a homeomorphism from S Z to P (S) Z . Note that since Z is a Z n -tile one only needs to check the above hypotheses for all S containing 0. Condition (i) can be checked algorithmically by using so-called boundary and vertex graphs (cf. e.g. [46] and see Remark 3.15). In most cases ideal tiles are chosen to be polyhedra. Then Conditions (ii) and (iii) can be checked by direct inspection for n = 3. For higher dimensions, in Section 6.2 we show how to check (ii) by techniques from algebraic topology in certain cases. Lemma 6.2 shows that (iii) is true for large classes of topological spaces. Theorem 2.23. Let Z be an ideal tile for a self-affine Z n -tile T and u ∈ int(Z) then there is a homeomorphism F such that (Z − u, F ) is a monotone model for T . Examples of ideal tiles are given in Figures 3 and 5. 2.7. Examples. We now illustrate our theory by examples of self-affine Z 3 -tiles. In Section 2.7.1 we give a first example of a tile that is homeomorphic to a 3-ball. Section 2.7.2 is devoted to a tile that was already studied in 1996 by Gelbrich [24]. We can now show that it is homeomorphic to a 3-ball. Finally, in Section 2.7.3 we state an existence result for a self-affine Z 3 -tile whose boundary is a surface of genus g for each g ∈ N. 2.7.1. A self-affine Z 3 -tile that is homeomorphic to a 3-ball. Let and let T ⊂ R 3 be the unique nonempty compact set satisfying AT = T + D. As A is expanding and D is a complete set of residue class representatives of Z 3 /AZ 3 the set T is a self-affine tile. Moreover, [36, Corollary 6.2] yields that T tiles R 3 by Z 3 -translates making T a self-affine Z 3 -tile. An image of T is depicted in Figure 2. We shall prove the following result. Figure 2. A self-affine Z n -tile that is homeomorphic to a closed ball. Theorem 2.25. Let T be the self-affine Z 3 -tile defined by the set equation AT = T + D with A and D given as in (2.24). T is homeomorphic to a closed 3-dimensional ball. Moreover, we are able to establish the following topological characterization result for the cells of T (the finite graphs Γ 2 , Γ 3 , and Γ 4 are defined in Remark 3.15 and constructed explicitly in Section 8). Proposition 2.26. Let T be the self-affine Z 3 -tile defined by AT = T + D with A and D defined as in (2.24), and let S ⊂ Z 3 with 0 ∈ S be given. • If \|S\| = 2 then S ∼ = D 2 if S is a node of the graph Γ 2 and S = ∅ otherwise. In Section 8.1 will give detailed proofs of these results. Again it is easy to check that T , which is depicted in Figure 3, is a self-affine Z 3 -tile. In his paper, Gelbrich asked whether T is homeomorphic to a closed 3-dimensional ball. We are now able to answer his question in the affirmative. 2.7.3. Self-affine Z 3 -tiles whose boundaries are surfaces of positive genus. We give the following result on boundary surfaces of positive genera (see also Section 8.4). Proposition 2.29. For each genus g ∈ N there is a self-affine Z 3 -tile T whose boundary is a surface of genus g. Although we do not want to go into details, we mention that it is possible to show that T is a self-similar 3-manifold by adapting the ball-checking algorithm provided in Section 7 (see in particular Remark 7.24). The fundamental neighborhoods can be chosen to be cubes here. Thus each 3-dimensional handlebody is homeomorphic to a Z 3 -tile. Self-affine Z n -tiles and their models In this section we extend standard notations and results on self-affine Z n -tiles and show how much of these results remains true for models. Before we start we equip R n with a norm \|\| · \|\| such that the associated operator norm (also denoted by \|\| · \|\|) satisfies As A is expanding such a choice is possible (cf. e.g. [35, Section 3]). 3.1. Basic properties of the canonical quotient map. Let T = T (A, D) be a self-affine Z n -tile and (M, F ) a model for it. We now give some auxiliary results on models and study properties of the canonical quotient map Q : R n → R n defined in (2.3). Our first aim is to associate with a given model M a sequence of models which converges to the tile T . For this reason define the set Proof. Let k ∈ N, x ∈ R n , and z ∈ Z n . The Z n -equivariance of A −1 F implies that We now are in a position to prove the following convergence result for models. The limit of a sequence of models in its statement is taken with respect to the product of the Hausdorff metric and the metric of uniform convergence. Iterating this for k times (q 0 is the identity) and setting Since D is a complete set of residue class representatives of Z n /AZ n , D k is a complete set of residue class representatives of Z n /A k Z n . Thus, as M is a Z n -tile, equation (3.6) implies that also M k is a Z n -tile. Next we show that (M k , F k ) satisfies the set equation (2.2). Indeed, as F k q k = q k F holds by the definition of q k and F k , we have As Lemma 3.3 implies that To prove the convergence result let D(·, ·) be the Hausdorff metric. For each ε > 0 we may choose k ∈ N in a way that A −k T and A −k M are contained in a ball of diameter ε around the origin. Using (3.6) and the k-th iterate of the set equation (1.2), we gain As \|\|A\|\| > 1 and A −1 F is continuous and Z n -equivariant, the maps A −k (A −1 F )A k uniformly converge to the identity. Thus F k → A uniformly for k → ∞ and the proof is finished. We mention that the sequence (M k ) of models yields approximations not only of the tile T , but also of the dynamical system defined by its set equation (see e.g. Theorem 3.21). The following result contains Theorem 2.4 as well as some properties of Q. Theorem 3.8. Let (M, F ) be a model for the self-affine Z n -tile T . Then the canonical quotient map Q = lim k→∞ q k is continuous, Z n -equivariant, and satisfies the following properties. Proof. We first show that (q k (x)) k≥0 is a Cauchy sequence for each x ∈ R n . Observe that by continuity and Z n -equivariance of A −1 F there is an absolute constant c > 0 such that By (3.1) we have \|\|A −1 \|\| < 1 and, by the triangle inequality and a geometric series consideration Thus (q k (x)) k≥0 is Cauchy and, hence, converges for each x ∈ R n . This defines the function Q for each x ∈ R n . As the convergence in (3.9) is uniform in x we conclude that Q is continuous. Z n -equivariance of Q follows from Z n -equivariance of q k (see Lemma 3.3), and (i) is an immediate consequence of the convergence statement in Proposition 3.4. To prove (ii) we have to show that for each ε > 0 there is k 0 ∈ N such that To prove this let ε > 0 be arbitrary and choose k 0 in a way that (3.7) holds for k ≥ k 0 . First we note that, since M k and T are Z n -tiles, we have As M k is a Z n -tile this implies that there is an element y ∈ ∂M k with \|\|x − y\|\| < ε. By analogous reasoning, for each x ∈ ∂M k there exists y ∈ ∂T with \|\|x − y\|\| < ε. This proves (3.10) and, hence, also (ii). The following lemma will be needed in some computations. Proof. The first identity follows immediately from the definition of Q. The second one follows as 3.2. The generalized set equation. Let T = T (A, D) be a self-affine Z n -tile. Using the subdivision operator P from Definition 2.19 we will now extend the standard notion of set equation (1.2) to intersections S (see also [46] where this is done by using so-called boundary graphs). To this end we need "powers" of P which we define inductively by Theorem 3.13 (The generalized set equation). Let T be a self-affine Z n -tile and S ∈ K(T ). Then As (T, A) trivially is a model of T having the same neighboring structure as T , Theorem 3.13 is a special case of the first equality in Theorem 3.21. Thus we refrain from proving Theorem 3.13 here. Remark 3.15. For S ∈ K(T ) with 0 ∈ S equation (3.14) yields S = S ∈P (S) A −1 S . For each S occurring on the right hand side of this equation choose a fixed element s ∈ S . Then it becomes and each shifted set S − s is an element of K(T ) containing 0. To each S ∈ K(T ) with 0 ∈ S we associate an indeterminate X S whose range of values is the space of nonempty compact subsets of R n . Using (3.16) we define the (finite) collection of set equations which defines a graph directed iterated function system whose unique solution is given by X S = S for each S ∈ K(T ) with 0 ∈ S. Note that \|S\| = \|S \| holds for each S ∈ P (S). Thus, following [46], for each i ≥ 1 we define the graph Γ i as follows. The set of nodes of Γ i is given by K(T ) i 0 . Moreover, there is an edge from S to S if and only if S ∈ P (S). Using these graphs, the equations in (3.17) can be written as The algorithmic construction of the graphs Γ i is detailed in [46] (see also [20,Chapter 3] for basic definitions and results on graph directed iterated function systems). Figures 6 and 7 in Section 8.1 contain an example of Γ 2 and Γ 3 , respectively. 3.3. Walks. Let D k be as in (3.5). Iterating the set equation (1.2) for T yields that Note that each term is a subdivision of the previous one, i.e., the tile T can be subdivided finer and finer by the collections and, equivalently, the set of collections (D k ) k≥0 admits natural bonding maps that define an inverse system. We will call each "thread" (d k ) k≥0 of this system a "walk" in T . Thus each walk corresponds to a nested sequence of subtiles of T . More generally, the set equation in (3.14) gives coordinates for S with S ∈ K(T ) in a similar way. Indeed, it induces a sequence of subdivisions In analogy to the last paragraph we say that a "walk" in S is a sequence (S k ) where S k ∈ P (k) (S) so that S k+1 is in P (S k ) or rather S k ∈ P −1 (S k+1 ). According to (3.14) this walk yields the nested sequence (A −k S k ) k≥0 whose intersection contains a single element of S . As D is a complete set of cosets of Z n /AZ n , one can check that S = S implies P (S) ∩ P (S ) = ∅. For this reason there is again an "ancestor" function R from P (S) to the set of finite subsets of Z n such that P = R −1 , i.e., R(S ) = S if S ∈ P (S). This motivates the following precise definition of walks. Definition 3.18 (Walks). For S ∈ K(T ), the set W (S) of walks in S is the inverse limit of the sequence (P (k) (S)) with bonding map R : P (k) (S) → P (k−1) (S). Let π k : W (S) → P (k) (S) be the canonical projection and define w k = A −k π k (w) for a walk w and C k = A −k π k (C) for a set of walks C. Walks are coordinates for a nested collection of sets. The set W (S) can also be described as the set of infinite walks in a finite graph (see e.g. [46]); this motivates the terminology "walk". Recall that the image of P (k) is contained in K(T ) for each k by definition. Thus for a walk w, the sequence (w k ) is a nested sequence of nonempty compact sets. The diameter of w k approaches zero (since A −1 is a contraction) and consequently defines a unique limit point. is a continuous map of the Cantor set W (S) to S . If C is a set of walks, we will use the notation In particular, set W = W ({0}) and ∂W = s∈Z n \{0} W ({0, s}). Theorem 3.13 implies 3.4. The generalized set equation and walks in a model. There are many similarities between a self-affine Z n -tile and its model, particularly if they have the same neighbor structure. We obtain the following analog of Theorem 3.13 for models. Recall that M k is defined in (3.2). Then for every S ∈ K(T ) and every k ∈ N, Together with the set equation (2.2) for M this yields Since K(T ) = K(M ), this subdivision is again governed by the function P and we arrive at Iterating this for k times proves the first equality. To prove the second one recall that Let (M, F ) be a model of a self-affine Z n -tile T satisfying K(M ) = K(T ). For S ∈ K(M ) to a walk w ∈ W (S) we associate the nested collection and, for a set C ⊂ W (S), we define We denote which may contain more than one point as F −1 is not necessarily a contraction. This is an important difference between a self-affine Z n -tile and its model. However, as K(M ) = K(T ), the definition of a walk assures that w M cannot be empty. Again, this definition extends to sets C of walks by setting C M = k≥1 C M,k . Using this notation we obtain Properties of monotone models In this section we investigate mapping properties of the canonical quotient map Q under the condition that (M, F ) is a monotone model for a self-affine Z n -tile T . (i) The sequence S M k converges to S in the Hausdorff metric D for each S ∈ K(T ). Proof. To prove (i) let ε > 0 be arbitrary. Since A is expanding and both T and M are compact we may choose k 0 ∈ N in a way that A −k T and A −k M are contained in a ball of diameter ε around the origin for each k ≥ k 0 . Using Theorems 3.13 and 3.21 we get To prove (iii), using Lemma 3.12 and (ii) we derive We will now use the sets w M to study preimages of Q. Proof. As the sets w M and w T , w ∈ C, are compact it suffices to check the first equivalence for each finite subset C of C by the finite intersection property for compact sets. Assume that w∈C w T = ∅. As w∈C w k k∈N is a nested sequence of compact sets this is equivalent to w∈C w k = ∅ for each k ∈ N. Because K(M ) = K(T ), this is in turn equivalent to w∈C w M,k = ∅ for each k ∈ N. As the sequence w∈C w M,k k∈N is a nested sequence of compact sets, this is finally equivalent to w∈C w M = ∅, proving the first equivalence. As for the second one note that the limit point w T is a singleton for each w ∈ C T . Thus C T contains more than one element if and only if there exist w, w ∈ C having disjoint limit points which is equivalent to w∈C w M = ∅. Recall that a quotient is monotone if point preimages are connected. By Lemma 4.2 all the sets w M in the union on the right share a common point. It therefore remains to prove that w M is connected for each w ∈ W (S). However, since M is a monotone model for T , the cell S M is connected for each S ⊂ Z n . Thus the set w M is a nested union of the connected sets w M,k and therefore itself connected. To prove (ii) first observe that Choose x ∈ δS . Then, by Proposition 4.1 (iii) we gain and everything runs exactly as in (i). In view of (3.11), (iii) is an immediate consequence of (ii). Theorem 2.10 is a consequence of Proposition 4.3 (i). Just note that all decompositions are accomplished by the restrictions Q\| S M of Q. We now turn to the proof of Theorem 2.12 which clarifies the effect of the canonical quotient map Q on boundaries of cells. Proof of Theorem 2.12. Let S ∈ K(T ) i 0 , i ≥ 2, be arbitrary but fixed. To prove (i) let x ∈ ∂ i S be given. Then, since K(T ) i 0 is a finite complex and cells are closed in To prove (ii) let x ∈ Q∂ i S M be given. By the same argument as in 4.2. Cell-like maps. In order to prove our higher dimensional results in Section 5.3 we need to make sure that Q is a cellular mapping in the following sense. if there is a sequence (C i ) i≥1 of n-cells such that C i+1 ⊂ int(C i ) and K = i≥1 C i . A space X is cell-like if there is an embedding ι of X in a manifold M such that ι(X) is cellular in M. A mapping is cellular or cell-like if its point preimages are cellular or cell-like, respectively. A simple diagonalization argument gives the following lemma. To formulate the result on the cellularity of Q we need one more definition. One checks that ∂ -star k (y) is the closed star of y in the complex ∂M induced by the sets A −k S M . The following lemma contains a more convenient representation for boundary stars. Proof. The lemma follows if we prove that for each w ∈ ∂W with y ∈ w M,k there exists w ∈ ∂W with w M,k = w M,k and Qy = w T . To construct w , let S i = π i (w) for 1 ≤ i ≤ k. Inductively, assume that for 1 ≤ i < j a choice of S i ∈ P (S i−1 ) has been made such that y ∈ (F −1 ) (i) S i M . By the generalized set equation for models in (3.22), we may choose S j ∈ P (S j−1 ) such that y ∈ (F −1 ) (j) S j M . Then w = (S i ) satisfies π k (w ) = π k (w) and, hence, w M,k = w M,k . Moreover, we have y ∈ w M by the definition of w M and Qy ∈ w T by Proposition 4.1 (iii). is the intersection of a nested sequence. Suppose that P M ∩ ∂ 2 P M,j = ∅ for some j ≥ 0. Since (P M,k ) is a nested sequence containing P M , we have that P M ∩ ∂ 2 P M,k = ∅ for each k ≥ j. As ∂W M,k covers ∂M for each fixed k by the set equation for S M in (3.22), there is a walk w ∈ ∂W such that Qw M = x but P M ∩ w M,k = ∅ holds for each k ≥ j. Thus P M ∩ w M = ∅, which implies that there is some w ∈ P satisfying w M ∩ w M = ∅. However, by Lemma 4.2 this yields that Qw M = w T = w T = x, a contradiction. Thus for each k ≥ j we have P M ⊂ int(P M,k ) (where the interior is taken relative to ∂M ) and we may choose a properly nested subsequence of (P M,k ). In view of Lemma 4.5 it remains to prove that P M,k is cellular for large k. To this end let y ∈ (Q\| ∂M ) −1 (x). Then Lemma 4.7 implies that and P M,k is cellular for large k by the assumption that almost all boundary stars are cellular. 5. Self-affine Z n -tiles whose boundaries are manifolds 5.1. The planar case. We start this section with an easy criterion for a self-affine Z 2 -tile T ⊂ R 2 to be homeomorphic to a closed disk D 2 . Theorem 5.1. A self-affine Z 2 -tile T admits a monotone model which is homeomorphic to D 2 if and only if T is homeomorphic to D 2 . Proof. If T admits a monotone model which is homeomorphic to D 2 , Proposition 4.3 (iii) implies that ∂T is a monotone quotient of S 1 and thus is either a singleton or homeomorphic to S 1 . As T is the closure of its interior, ∂T cannot be a singleton and Jordan's Curve Theorem implies that T ∼ = D 2 . For the converse just observe that (T, A) is a monotone model for T (it is not hard to check that S is always connected for a self-affine tile T which is homeomorphic to D 2 ). It is known that a Z 2 -tile M which is a closed disk has either 6 or 8 "neighbors" (i.e., K(M ) 5.2. Surface boundaries of self-affine Z 3 -tiles. In this section we will prove our results on self-affine Z 3 -tiles with surface boundary stated in Theorems 2.14 and 2.16. To this end we will use the following result on monotone upper semi-continuous decompositions of 2-manifolds that was proved by Roberts and Steenrod [44] generalizing a theorem of Moore [43]. Proposition 5.2 (cf. [44,Theorem 1]). Let S be a compact 2-manifold without boundary and let G be a monotone upper semi-continuous decomposition of S. If G contains at least 2 elements and H 1 (g; Z 2 ) = 0 for each g ∈ G, then G is homeomorphic to S. The following easy lemma, which will be needed on several occasions, starts a list of preparatory results. Lemma 5.3. Let M ⊂ R n be compact and equal to the closure of its interior. Assume that U is a nonempty bounded component of R n \ M . Then int(M + a) ⊂ U holds for each a ∈ R n . Proof. As int(M ) = ∅ the result is true for a = 0, and we may assume that a = 0. If the assertion was wrong we had ∂(U + a) ⊂ M + a ⊂ U which is absurd for a bounded set U . We will now use the Mayer-Vietoris-sequence forČech cohomology groups (see [42, p. 67]) in order to prove our result. Since A, B are closed subsets of R n a part of the Mayer-Vietoris sequence reads As A and B satisfy the conditions of [ Inserting this in (5.7) yields that the sequence · · · → 0 → Z → 0 → · · · is exact, which is absurd. This yields the desired contradiction and the result is proved. We are now in a position to give the proof of Theorems 2.14 and 2.16. Proof of Theorem 2.14. As M is a monotone model for T , by Theorem 3.8 the canonical quotient map Q in (2.3) is well-defined and maps M onto T . In view of Proposition 5.2 to prove (i) it suffices to prove that for each x ∈ ∂T the preimage (Q\| ∂M ) −1 (x) is connected and satisfies H 1 ((Q\| ∂M ) −1 (x); Z 2 ) = 0 (note that ∂T is not a singleton as T is the closure of its interior). Connectivity of (Q\| ∂M ) −1 (x) is a consequence of Proposition 4.3 (iii). Since M is semi-contractible, to prove H 1 ((Q\| ∂M ) −1 (x); Z 2 ) = 0 it is sufficient to show (ii). To prove (ii) assume on the contrary that (Q\| ∂M ) −1 (x) path separates ∂M between two elements u, v ∈ ∂M . We first observe that being the continuous image of the locally connected continuum ∂M (by Theorem 3.8 (ii)), the set ∂T is a locally connected continuum. Moreover, as int(T ) is connected, Proposition 5.6 implies that ∂T has no cut point. Thus ∂T \ {x} is arcwise connected (see e.g. [32, §52, II, Theorem 16]) and we may connect Q(u) and Q(v) by a path in ∂T \ {x}. Since (Q\| ∂M ) −1 (y) is connected for each y ∈ ∂T it is easy to see that the preimage of a continuum is a continuum. In particular, (Q\| ∂M ) −1 ( ) is a continuum which contains u and v and is disjoint from (Q\| ∂M ) −1 (x). Thus u and v are ε-chain connected in (Q\| ∂M ) −1 ( ) with an ε that is so small that (in view of the fact that ∂M is a locally arcwise connected continuum) we can construct an arc that connects u and v in ∂M avoiding (Q\| ∂M ) −1 (x). This contradicts our assumption. As mentioned in the introduction, there is a version of Theorem 2.14 for finite unions of Z 3 -translates of T . Before we make this precise, for a Z n -tile M we set Proof of Theorem 2.16. Let S ∈ K(T ) be nondegenerate and assume, without loss of generality, that 0 ∈ S. Assume that S M is a closed manifold or a ball inside of the surface ∂M ∼ = S. Thus S M is either a closed surface, a closed disk, a circle, an arc, or a point. We have to show that S has the required properties. To this end we will use the fact that S = Q S M is a monotone quotient of S M by Proposition 4.3 (i). We first dispose off the easy cases. If S M is a point, observing that the monotone quotient of a point is a point, we gain that S = Q S M is a point. Similarly, if S M is an arc, the fact that the monotone quotient of an arc is either an arc or a point yields that S is an arc or a point. The case where S M is a circle is also settled because it is well-known that the monotone quotient of a circle is a circle or a point. Finally, if S M is a closed surface, we have S M = S as a closed surface has no proper subsurface. Thus Theorem 2.14 (i) yields that S ∼ = S. It remains to consider the case where S M is a closed disk. Since M is combinatorial, this implies that \|S\| = 2 and ∂ 2 S M = δS M . Thus, as K(M ) Now assume that Q∂ 2 S M is a circle. As the monotone image of a disk cannot be a circle (see e.g. [13, I, §4, Exercise 9]; observe that a disk is unicoherent but a circle is not) Q S M \Q∂ 2 S M = S \ Q∂ 2 S M = ∅. Since M is combinatorial, Theorem 2.12 (i) implies that ∂ 2 S ⊂ Q∂ 2 S M , hence, S \ ∂ 2 S = ∅. As S is a proper subset of ∂T (otherwise we get a contradiction to Lemma 5.3 as above) we conclude that ∂ 2 S is a subset of the circle Q∂ 2 S M that separates ∂T ∼ = S. This implies that ∂ 2 S = Q∂ 2 S M . Recall that a circle in a closed surface is nulhomotopic if and only if it bounds two components, at least one of which is a disk. Since ∂ 2 S M bounds S M , ∂ 2 S M is nulhomotopic, and thus its canonical image ∂ 2 S is a nulhomotopic circle in the surface ∂T which must itself bound a disk. Any component of the complement of ∂ 2 S M which is a disk must map to a complementary component of ∂ 2 S which is a disk under Q. Thus, as S M is a disk, also S = Q S M is a disk. To check that int(T ) is connected, the following easy criterion is often applicable (cf. [2, Proposition 13.1]). .5) is a nested union of open sets each of which is, by induction, connected. Manifolds and the disjoint disks property. In the present section we present generalizations of Theorem 2.14 to higher dimensions. In this setting we are able to give a checkable criterion for the boundary of a self-affine Z n -tile T to be a generalized manifold. To make sure that ∂T is actually a manifold (for dimension n ≥ 6), according to the work of Cannon [10], one has to assume the disjoint disks property. Definition 5.11 (Generalized n-manifold; see e.g. [10]). A space X is a generalized n-manifold if it has the following properties. • X is a Euclidean neighborhood retract (ENR), i.e., for some integer n it embeds in R n as a retract of an open subset of R n . • X is a homology n-manifold, i.e., H * (X, X \ {x}; Z) ∼ = H * (R n , R n \ {0}; Z) for each x ∈ X. A generalized n-manifold is called resolvable if it is a proper cell-like upper semi-continuous decomposition of an n-manifold. Theorem 5.12. Let T be a self-affine Z n -tile which admits a monotone model M . Assume that almost all boundary stars of M are cellular and ∂M is a manifold. Then ∂T is a generalized (n − 1)-manifold with Q\| ∂M : ∂M → ∂T a cellular quotient map from the manifold ∂M . In other words, ∂M is a cell-like resolution of the generalized manifold ∂T . Proof. By assumption, M satisfies the conditions of Proposition 4.9 and thus Q\| ∂M : ∂M → ∂T is a cellular quotient map. Now recall that Lacher [33, (11.2) Corollary] implies that a cell-like image of a compact manifold is an ENR, and [16,Proposition 8.5.1] states that every n-dimensional resolvable space is an n-dimensional homology manifold. This implies the result. Remark 5.13. A priori there are infinitely many boundary stars to check for cellularity. However, as will be explained in Remark 7.21, it suffices to check only one representative of finitely many "equivalence classes" of boundary stars. To make the step from a generalized manifold to a topological manifold, we need the well-known disjoint disks property (cf. [9]). Definition 5.14 (Disjoint disks property). A metric space (X, d) has the disjoint disks property if for every pair of maps g 1 , g 2 : D 2 → X and every ε > 0 there exist maps g 1 , g 2 : D 2 → X such that max{d(g 1 , g 1 ), d(g 2 , g 2 )} < ε and g 1 (D 2 ) ∩ g 2 (D 2 ) = ∅. Generalizing Theorem 2.14 this allows us to state a result on self-affine Z n -tiles of dimension ≥ 6 whose boundary is a manifold. Theorem 5.15. For n ≥ 6 let T be a self-affine Z n -tile which admits a monotone model M . Assume that almost all boundary stars of M are cellular and ∂M is a manifold. If T satisfies the disjoint disks property then ∂T is an (n − 1)-manifold. For dimensions less than 5 the disjoint disks property is not suited to detect manifolds, so we cannot use it for boundaries of self-affine tiles of dimension n < 6. In Daverman and Repovš [15] alternatives for the disjoint disks property for 3-manifolds are proposed; for 4-manifolds no such alternatives seem to be known so far. 6. Ideal tiles 6.1. Ideal tiles and monotone models. The first aim of this section is to prove Theorem 2.23 which states that each ideal tile is a monotone model up to translation. To this end we need to prove (2.21) which is contained in the following lemma. Proof. Since D is a complete set of residue classes of Z n /AZ n we have We proceed with the proof of the theorem. . Note that by compactness of M , S ∈ C for ≥ 0 implies that S is finite. Now assume that the homeomorphism f has been defined Z n -equivariantly on C −1 M , that is, Choose a maximal set E of pairwise inequivalent elements of C \ C −1 , that is, E contains precisely one element of S + Z n for each S ∈ C \ C −1 . Let S ∈ E . By definition, δS is a union of cells of C −1 . The induction hypothesis yields that each cell in δS M is mapped homeomorphically to the according cell in Lemma 6.2. Let X be a contractible union of cones over spheres which pairwise intersect only at single points on their boundaries. The boundary of X is the union of the bases of the cones which comprise it. Then X has the property that any self-homeomorphism of its boundary can be extended to a self-homeomorphism of X. Proof. Let C i be the cones which comprise X. Suppose ψ : ∂X → ∂X is a homeomorphism. Then, by considering cut points in the boundary, we see that ψ \| ∂Ci is a homeomorphism onto ∂C i for some unique choice of i . Coning the map ψ \| ∂Ci , we obtain an extension homeomorphism ψ \| Ci : C i → C i satisfyingψ \| ∂Ci = ψ \| ∂Ci . By again considering cut points on the boundary, we see that the map i → i is a permutation. Since the C i only meet at single boundary points, the mapsψ \| int(Ci) have disjoint images. Thus the homeomorphismsψ \| Ci patch together to form the desired homeomorphismψ. 6.2. Checking whether a simplicial complex is a ball. Let T be a self-affine Z n -tile. In Theorem 2.23 we require that Z is an ideal tile for T . Particularly for low dimensions n, Condition (ii) of Definition 2.22 can be checked easily by direct inspection. However, in higher dimensions this may be tricky and we are forced to use a systematic approach based on methods from classical algebraic topology. Such an approach is discussed in the present section. Indeed, for many instances, the ideal tile Z can be chosen to be a triangulable complex such that each nonempty set S Z is a closed ball that is the closure of a single cell of this complex (see e.g. the examples discussed in Sections 8.1 and 8.2). We shall discuss how one can check Property (ii) of Definition 2.22 in this case. In particular, we have to check that P (S) Z is a ball of the same dimension as S Z . By translation invariance it suffices to check this for all S ∈ K(Z) containing 0. The intersections S Z as well as P (S) Z can be nonempty only for finitely many of these sets since Z as well as P ({0}) Z is compact. Thus there are only finitely many instances to check. By definition, P (S) Z is a triangulable complex made up of finitely many triangulable complexes of the form S Z . We have to check that this simplicial complex is the underlying set of a ball. As we will need this later on for different complexes, we now switch to a general setting and describe how to check whether a given simplicial complex is the underlying set of a ball. In particular, let K be a simplicial complex and σ be one of its cells. Recall that the star of σ in K, denoted by st(σ) is given by the set of all simplices in K that have σ as a face. Moreover, the link of σ in K, denoted by lk(σ) is given by where cl(X) is the smallest subcomplex of K that contains each simplex in X. As we first have to check that K is a manifold, we need the following result (cf. [16, Theorem 8.10.2]; note that this is a consequence of Cannon's Double Suspension Theorem, see [9]). Lemma 6.3. A simplicial complex K is the underlying set of a topological n-manifold if and only if, for each k-simplex σ ∈ K, lk(σ) has the homology of S n−k−1 (i.e., H i (lk(σ); Z) = 0 for i = n − k − 1 and H n−k−1 (lk(σ); Z) = Z) and, for each vertex v ∈ K, lk(v) is simply connected. We now give an outline on how to check that K is a ball. For all classical theorems from algebraic topology we are using here, we refer the reader e.g. to Hatcher [28]: (i) we need to check that the simplicial complex K is a manifold. By Lemma 6.3 it suffices to calculate homology groups and the fundamental group of certain links. This can be done by using the Mayer-Vietoris-Sequence and the Seifert-van Kampen Theorem, respectively. (ii) We check that K has trivial homology groups and trivial fundamental group. Again we use the Mayer-Vietoris-Sequence and the Seifert-van Kampen Theorem. (iii) The Theorems of Hurewicz and Whitehead now imply that K is homotopy equivalent to a ball. (iv) The generalized Poincaré Theorem proved by Smale [47], Freedman [21], and Perelman (see [11]) yields that K is a ball. Summing up, checking that K is a ball of appropriate dimension is achieved by calculating homology groups and fundamental groups of simplicial complexes. 7. Self-affine Z n -tiles that are homeomorphic to a ball Let T be a self-affine Z n -tile. In Theorems 2.14 and 5.15 we gave criteria for ∂T to be homeomorphic to a manifold. In the present section we shall assume that ∂T is homeomorphic to S n−1 and give criteria under which this implies that the tile T itself is homeomorphic to the closed n-dimensional disk D n , i.e., that ∂T is tamely embedded in R n . 7.1. Cannon's criterion and fundamental neighborhoods. We start with some terminology. 1-LCC). A set X ⊂ R n is said to be 1-LCC if for each ε > 0 there is a δ > 0 such that each loop of diameter less than δ in R n \ X can be contracted to a point in a subset of R n \ X of diameter less than ε. Definition 7.2 (Locally spherical). An (n − 1)-sphere S ⊂ R n is said to be locally spherical if each p ∈ S has a neighborhood basis {U m \| m ∈ N} such that ∂U m ∼ = S n−1 and ∂U m \ S is simply connected. If n = 3 the simple connectivity of ∂U m \ S is equivalent to the fact that ∂U m ∩ S is connected. Cannon's criterion now reads as follows. Proposition 7.3 (cf. [8,5.1 Theorem]). If S is an (n − 1)-sphere in R n that is locally spherical then S is 1-LCC. We combine this criterion with the following result. Proposition 7.4. If an (n − 1)-sphere S in R n is 1-LCC then S is tamely embedded. Bing [7] proved this result for n = 3, for n = 4 it is proved by Freedman and Quinn [22], and for n ≥ 5 it is due to Daverman (see [16,Theorem 7.6.5]). We will use the next corollary, which is just a combination of Propositions 7.3 and 7.4. Corollary 7.5. If S is an (n − 1)-sphere in R n that is locally spherical then S is tamely embedded in R n . The self-affine structure of T produces natural candidates for neighborhood bases. To make this more precise we first recall from (5.8) that [S] = s∈S (T + s) for S ⊂ Z n . Definition 7.6 (Fundamental neighborhood). Let T be a self-affine Z n -tile and let S ⊂ Z n be given in a way that The following lemma is an immediate consequence of this definition. Lemma 7.8. Let T be a self-affine Z n -tile. The set A = {A −k L \| L a fundamental neighborhood } forms a basis for the topology of R n . In particular, each x ∈ ∂T admits a neighborhood basis made up of elements of A. If N ∈ A is given in a way that A k N is a fundamental neighborhood we say that N is of level k and write level(N ) = k. Lemma 7.8 implies that the set contains a neighborhood basis for each x ∈ ∂T . Our aim is to provide an algorithm that allows to check whether this neighborhood basis can always be chosen in a way that it meets the conditions of Corollary 7.5. To this matter we define the following equivalence relation on B. Definition 7.9 (Equivalent neighborhoods). Let T be a self-affine Z n -tile, let N 1 , N 2 ∈ B be given and set k i = level(N i ) for i ∈ {1, 2}. If there exists u ∈ Z n such that we say that N 1 is equivalent to N 2 . In this case we write N 1 ∼ N 2 . We need the following result. Let S = {s (s) \| s ∈ S)}. If S satisfies the maximality condition in (7.7) we are done, if not, successively add elements of Z d to S until it satisfies this condition. Since Z n is discrete and T is compact at most finitely many elements can be added. 7.2. The in-out graph. Using the relation "Parents" we can define an infinite directed graph I whose nodes are the elements of B and whose edges are defined by The following lemma shows that the Parent relation depends only on the equivalence class of the edges N with respect to the equivalence relation defined in Definition 7.9. We will show that N 2 := A −k2 (A k1 N 1 − u) satisfies the requirements of our lemma. It is clear that N 2 ∈ A with level(N 2 ) = k 2 + 1. Moreover, as N 1 ⊂ N 1 equation (7.12) implies that N 2 ⊂ N 2 . It remains to show that N 2 ∈ B and N 1 ∼ N 2 . To this matter observe that (7.13) yields From this we get the desired properties. Definition 7.14 (In-out graph). For N ∈ B denote by N the equivalence class of N with respect to the equivalence relation "∼". The in-out graph is a directed graph I which is defined as follows. • The nodes of I are the equivalence classes {N \| N ∈ B}. • There is a directed edge N → N in I if there is an edge N → N in I. Lemma 7.15. The in-out graph I is finite. Proof. Choose some order on Z n , set and let D k be defined as in (3.5). For each finite set Y ⊂ Z n define the functions where u ∈ Y is chosen to be minimal with respect to this order. Let N = A −k [S] be an element of . Moreover, as {s, s + v} = ∅ holds for each "neighbor" v ∈ N we get Now pick u ∈ S minimal with respect to the above order of Z d . Then Moreover, as Subtracting u this yields From (7.18) and (7.19) we see that each equivalence class N is completely characterized by the sets α(S), β(S), γ(S). As these sets are contained in the finite set S + N − u, the estimate in (7.17) implies that they are contained in the ball of radius 4 diam(T ) around the origin. Thus there are only finitely many choices for these sets. Using Lemma 7.11 we can provide the following algorithm to calculate I. Proposition 7.20. The in-out graph I can be constructed by the following finite recurrence process. Recurrence start: The equivalence class N of each fundamental neighborhood N contained in B is a node of I. Recurrence step: Suppose that N is a node of I. For all N satisfying N ∈ Parents(N ) the node N together with the edge N → N belong to I. End of recurrence: Iterate until no new nodes occur in a recurrence step. Proof. Let R be the graph constructed by this recurrence process. Obviously, each node of R is also a node of I. Suppose that there is a node N of I that is not a node of R. Choose N ∈ B in a way that level(N ) is minimal with this property. Let N ∈ Parents(N ). Then N ∈ R by the choice of N . The recurrence step above now implies together with Lemma 7.11 that N is a node of I, a contradiction. Thus R and I have the same set of nodes. Since the edges are defined in the same way, the result follows. Remark 7.21. Let (M, F ) be a monotone model for a self-affine Z n -tile T = T (A, D). Although (F −1 ) (k) [S] M , S ⊂ Z n , do not necessarily form a basis for the topology of R n , by the same arguments as above, a finite in-out graph I M can be constructed also for M . Since two boundary stars B 1 and B 2 are homeomorphic (i.e., equivalent) if A k1 B 1 = A k2 B 2 + u, it suffices to check cellularity of boundary stars only for one representative of each equivalence class. The finiteness of I M immediately implies that there are only finitely many such equivalence classes to check. In order to check cellularity of a given boundary star, the methods outlined in Section 6.2 can be used. 7.3. Results on self-affine balls. We can now prove the following theorem. Theorem 7.22. Let T be a self-affine Z n -tile. If ∂T is an (n − 1)-sphere in R n and each loop in the in-out graph I contains a node N such that (i) ∂N ∼ = S n−1 , (ii) ∂N \ ∂T is simply connected, then ∂T is locally spherical and thus tame. Consequently T is homeomorphic to D n . Proof. Let \|I\| be the number of nodes in I and assume that k > \|I\|. Let N ∈ B be a neighborhood of an element x ∈ ∂T with level(N ) = k and let N → N k−1 → · · · → N 1 → N 0 be a walk in the graph I. The associated walk in I is N → N k−1 → · · · → N 1 → N 0 . As k > \|I\| the first \|I\| edges of this walk contain a loop. Thus, by assumption, there is ∈ {k − \|I\|, . . . , k} such that ∂N \ ∂T is simply connected and ∂N ∼ = S n−1 . As k was arbitrary and ∈ {k − \|I\|, . . . , k}, we constructed an arbitrarily small neighborhood N of x that satisfies the properties of Corollary 7.5. Since x ∈ ∂T was arbitrary, this proves the result. For n = 3 we can simplify this by using the remark after Definition 7.2. Corollary 7.23. Let T be a self-affine Z 3 -tile. If ∂T is a 2-sphere in R 3 and each loop in the in-out graph I contains a node N such that (i) ∂N ∼ = S 2 , (ii) ∂N ∩ ∂T is connected. Then ∂T is locally spherical and thus tame. Consequently T is homeomorphic to D 3 . Remark 7.24. It seems that the neighbor basis B leads to satisfactory results if the self-affine Z n -tile T has only face-neighbors (i.e. neighbors that intersect T is an (n − 1)-dimensional set; see Section 8.1). However, as Cannon's criterion is necessary and sufficient, for tiles that are homeomorphic to balls such neighborhood bases always exist. In Section 8.3 we consider a tile with "degenerate" neighbors. To show that this tile is homeomorphic to a ball, we will change the fundamental neighborhoods slightly. As being locally spherical is a local property, the results of the present section can be adapted to check whether T is homeomorphic to other manifolds with boundary (see Section 8.4). Proofs for the examples We now provide the proofs for the examples given in Section 2.7 and construct a self-affine crumpled cube in order to prove Theorem 2.17. In particular, concerning the example in Section 2.7.1 we work out detailed proofs of Theorem 2.25 and Proposition 2.26. After that, in Section 8.2 we give a proof of Theorem 2.17. In Section 8.3 we sketch the proof of Theorem 2.28 and finally, Section 8.4 is devoted to self-affine Z 3 -tiles that have a surface of positive genus as boundary. 8.1. A self-affine Z 3 -tile that is homeomorphic to a 3-ball. Let A and D be given as in (2.24) and consider the tile T = T (A, D) defined by this data. Our aim is to prove Theorem 2.25 and Proposition 2.26 using Corollary 2.15, Theorem 2.16, and Corollary 7.23. Sphere checking. To prove that ∂T is homeomorphic to the sphere S 2 we have to establish a monotone model that satisfies the conditions of Corollary 2.15. In view of Theorem 2.23 we start with setting up an ideal tile Z of T . By inspecting the neighbor structure K(T ) of the tile T it turns out that choosing Z to be equal to the prism spanned by the vectors (0, 1, 0) t , (1, 5 4 , 0) t , ( 3 2 , 1 2 , 1) t is a good candidate. The prism Z and Z 1 = P ({0}) Z = A −1 (Z + D) are shown in Figure 5. It is easy to see that Z + Z 3 forms a Z 3 -tile of R 3 having connected interior. Thus it suffices to check Items (i), (ii), and (iii) of Definition 2.22 to make sure that Z is an ideal tile for T . In order to verify Item (i) we have to check which of the sets S , S Z are nonempty. By translation invariance, we can confine ourselves to sets S and S Z with 0 ∈ S. To characterize all nonempty cells S with 0 ∈ S, by Remark 3.15 it suffices to construct the graphs Γ i (i ≥ 0). This can be done by standard algorithms (see e.g. [46]). Indeed, in our example we get the Graphs Γ 2 and Γ 3 depicted in Figures 6 and 7, respectively. Moreover, the nodes of Γ 4 are contained in Table 1 and Γ i is empty for i ≥ 5. By inspecting the nodes of these graphs we know all sets S ⊂ Z 3 containing 0 that correspond to a nonempty intersection S . On the other hand, the nonempty intersections S Z with 0 ∈ S can easily be determined as Z is an explicitly given prism in R 3 . Comparing the collection of nonempty sets S and S Z we obtain that K(T ) = K(Z) and Item (i) is verified. Here a node a1b1c1 a2b2c2 corresponds to the intersection T ∩ (T + (a 1 , b 1 , c 1 ) t ) ∩ (T + (a 2 , b 2 , c 2 ) t ). To check Item (ii) we need to make sure that S Z and P (S) Z are connected and that S Z ∼ = P (S) Z holds for each S ⊂ Z 3 . Since Z and P ({0}) Z are explicitly given polyhedra (see Figure 5) it is a routine calculation to check (ii). Indeed it is easy to see that all the nonempty sets S Z and P (S) Z are balls of dimension 4 − \|S\|. To check (iii) we observe that Z is combinatorial, i.e., for each S ⊂ Z 3 with \|S\| = 1, i ≥ 2 we have δS Z = ∂ i S Z and δP (S) Z = ∂ i P (S) Z . Thus each homeomorphism between the spheres Table 2. The translates s corresponding to the subtiles A −4 (T + s) lying in the interior of T . δS Z and δP (S) Z extends to a homeomorphism between the balls S Z and P (S) Z and (iii) is shown. Summing up we proved that the prism Z is an ideal tile for T . Thus, by Theorem 2.23 there is u ∈ Z 3 such that M = Z − u is a monotone model for T . As ∂M is a sphere, this monotone model is semi-contractible in the sense of Definition 2.13. Thus to apply Corollary 2.15 it remains to check that int(T ) is connected. To this end we use Lemma 5.10. To construct the set E with the required properties, note that a subtile A −k (T + s) of T (s ∈ Z 3 ) is contained in the interior of T if and only if each of its "neighbors" A −k (T + s + v) (v ∈ N with N as in (7.16)) is a subtile of T . The tile T has 4 4 subtiles of the shape A −4 (T + s) (s ∈ Z 3 ). Examining their neighbors, from this collection we select those which are contained in int(T ). It turns out that these are the 30 subtiles A −4 (T + s) with s ∈ I 4 given by Table 2. Now set E := s∈I4 A −4 (T + s). As A −4 (T + s) is connected for each s ∈ I 4 , we check that E is connected by showing that {A −4 (T + s) \| s ∈ I 4 } forms a chain. In other words, define a graph C whose nodes are the elements of I 4 . There is an edge between s 1 and s 2 if and only if We have to show that C is a connected graph. As we know that (8.1) holds if and only if s 1 −s 2 ∈ N it is easy to set up this graph and to verify it is connected. It is now straightforward to show that E ∩ ϕ d (E) = ∅ for each d ∈ D. Applying Lemma 5.10 we conclude that int(T ) is connected. Summing up, we may invoke Corollary 2.15 to T and have thus proved the following result. Proposition 8.2. Let T be the self-affine Z 3 -tile defined by AT = T + D with A and D as in (2.24). Then ∂T is homeomorphic to the sphere S 2 . Recall that each nonempty S M is a ball of dimension 4 − \|S\|. Moreover, from each node in the graphs Γ 2 and Γ 3 there lead away infinitely many different infinite walks. Thus the sets S with S being a node of these graphs, contain infinitely many points. Moreover, the sets S with S being a node of Γ 4 are single points. Thus, since we already saw that Z and, hence, M , is combinatorial, Theorem 2.16 implies Proposition 2.26. Ball checking. In order prove that T is homeomorphic to a ball we want to apply Corollary 7.23. To this matter we have to construct the in-out graph I which can be done by the algorithm proposed in Proposition 7.20. In the present example there exist 24 fundamental neighborhoods in B, one for each node of Γ 4 (see Table 1). As these lie in pairwise different equivalence classes (in the sense of Definition 7.9), the recurrence starts with 24 nodes. After eight recurrence steps we arrive at the in-out graph I which has 2888 nodes. We now have to verify Conditions (i) and (ii) of Corollary 7.23 to prove that T is homeomorphic to a ball. As Γ 5 is empty and each node of Γ 3 is a subset of a node of Γ 4 , each set S ⊂ Z 3 with the property S = ∅ and S ∪ {s} = ∅ for all s ∈ Z 3 \ S has exactly 4 elements. Therefore, each fundamental neighborhood can be written as [S] + u, with S ∈ Γ 4 and u ∈ Z n and, hence, each node N of I is of the form N = A −k ([S] + u), with k ∈ N, u ∈ Z n , and S ∈ Γ 4 . This implies that N is homeomorphic to [S] for some S ∈ Γ 4 and checking Condition (i) of Corollary 7.23 amounts to checking whether ∂[S] ∼ = S 2 holds for each of the 24 nodes of Γ 4 . To prove that ∂[S] ∼ = S 2 we may use Proposition 5.9. To check the conditions of this proposition, it remains to check the following items for each S ∈ Γ 4 : As [S] M is a union of four prisms one can check (a) by direct inspection or standard methods (see Section 6.2). To check (b) observe that T tiles R 3 by Z 3 -translates. Thus the definition of the fundamental neighborhood implies that the singleton S is contained in the interior of [S] and, hence, there is a small open B ball centered in S that is contained in int([S]). As int(T ) is connected and B contains inner points of T + s for each s ∈ S, the interior of [S] is connected. As the tiling T + Z 3 is locally finite, also (c) can be checked combinatorially by using the connectivity of int(T ). Condition (ii) of Corollary 7.23 has to be checked for each of the 2888 nodes of I. We explain how this is done for a given node of I. Let N be a node of I and consider ∂N ∩ ∂T . Suppose N = A −k ([S] + u), then there exist S 1 , . . . , S m ⊂ Z n such that As S i is connected for each i ∈ {1, . . . , m}, this set is connected if {S 1 , . . . , S m } forms a chain. In other words, define a graph C(N ) whose nodes are the sets S 1 , . . . , S m and there is an undirected edge between S i and S j if and only if S i ∩ S j = S i ∪ S j = ∅. All the information required to construct this graph is contained in Proposition 2.26. The set ∂N ∩ ∂T is connected if and only if C(N ) is a connected finite graph. We checked connectivity for each node of I with the aid of a Mathematica program. It turns out that in each walk of length 2 of I there is at least one node satisfying (ii). Summing up, in each loop of I there is at least one node satisfying the conditions of Corollary 7.23. This proves that T is homeomorphic to a closed ball and Theorem 2.25 is established. (i) ∂T is homeomorphic to S 2 . (ii) T is not homeomorphic to a ball. We only sketch the proof and omit routine calculations. Sketch of the proof. To prove (i) we will apply Corollary 2.15. To this matter we first construct a monotone model. By Theorem 2.23 it suffices to come up with an ideal tile for T . Set Z = [0, 1] 3 . To show that Z is an ideal tile we need to verify conditions (i), (ii) and (iii) of Definition 2.22 (int(Z) is obviously connected). To check that K(Z) = K(T ) we have to describe which set S Z with 0 ∈ S is nonempty. As Z is the unit cube, this is an easy task. In order to characterize the nonempty sets S we can construct the graphs Γ i as we did in Section 8.1 (indeed, in the present situation it would even be possible to check this directly). Comparing the two characterizations one sees that K(Z) = K(T ). As Z is a cube and P ({0}) Z is the complex depicted on the left hand side of Figure 1, it is easy to verify conditions (ii) and (iii) of Definition 2.22 and we have shown that Z is an ideal tile. Thus, Theorem 2.23 shows that T admits a monotone model (M, F ) with ∂M ∼ = S 2 . It remains to show that int(T ) is connected. In view of Lemma 5.10 we construct a connected set E ⊂ int(T ) with the property that E ∩ ϕ d (E) = ∅ for each d ∈ D. It is easy to see that the midpoint of the cube ϕ d (Z) is an element of int(T ). For each face of ϕ d (Z) that is also contained in another cube ϕ d (Z) connect the midpoint of ϕ d (Z) to the midpoint of this face by an arc that is contained in int(T ). Call the union of all these arcs Y d and set E = d∈D Y d . One easily checks that E has the required properties. To prove (ii) we proceed as in the classical proof for Alexander's Horned Sphere and show that the complement R 3 \ T is not simply connected since we cannot homotope out a loop that surrounds one of the "horns" of T (see e.g. [28, Example 2B.2, page 170ff]). As for Condition (i) it is again a matter of calculating the graphs Γ i (i ≥ 0) by known algorithms and compare the results with the intersection structure of the polyhedron Z. As for Condition (ii) it turns out that four of the intersections {0, s} Z are the union of two disks intersecting in a single point so that we are not in the situation covered by Section 6.2. Nevertheless, Condition (ii) can be checked easily by direct inspection and Condition (iii) follows from Lemma 6.2. Thus Z is an ideal tile for T and, hence, Theorem 2.23 implies that there is a monotone model (M, F ) for T whose boundary is homeomorphic to S 2 . To apply Theorem 2.14 it therefore remains to check that int(T ) is connected. This is again done with the help of Lemma 5.10. Summing up we obtain that Gelbrich's tile T satisfies ∂T ∼ = S 2 . The self-affine Z 3 -tile T = T (A, D) is depicted in Figure 4. From these pictures it is plausible to assume that T is a solid torus. Using Theorem 2.14 we can prove the following result. To construct this self-affine torus T , starting with a 6 × 6 × 6 cube, we cut out a hole and -to compensate for the digits killed by digging this hole -we added a "half" handle on the top and on the bottom of the cube. To construct boundary surfaces of genus g, we have to dig g holes and to add g such "half" handles on the top and on the bottom. Since, seeing the case g = 1 above, this construction is quite obvious and we omit the details for the proof of Proposition 2.29.	2015-11-09T16:06:05.000Z	2014-02-12T00:00:00.000	{ "year": 2014, "sha1": "37a1b51865145b16266c119457357cccd285a335", "oa_license": "elsevier-specific: oa user license", "oa_url": "https://doi.org/10.1016/j.aim.2015.11.022", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "37a1b51865145b16266c119457357cccd285a335", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
15793613	pes2o/s2orc	v3-fos-license	Weyl group orbit functions in image processing We deal with the Fourier-like analysis of functions on discrete grids in two-dimensional simplexes using $C-$ and $E-$ Weyl group orbit functions. For these cases we present the convolution theorem. We provide an example of application of image processing using the $C-$ functions and the convolutions for spatial filtering of the treated image. Introduction The development of information technologies has inspired also the development of the information compression, the most famous part of which is the image and video compression. The compression is based on the information structure in order to optimize compression speed, compression rate and the possible losses of information during the compression. Development of the theory of orbit functions opens a space for their use in the processing of the information sampled on grids in simplexes and polyhedra in n−dimensional space. These functions can be used for decomposition of any discrete values on the grids to orthogonal series. The density of grid points is controlled by a suitable choice of parameter. Moreover, we can glue together more simplexes and study the information carried in the grid in this ensemble. In this paper, we focus on the simplest non-trivial case of utilization of orbit functions in two dimensions. It corresponds to a two-dimensional digital image processing. In comparison with the most widespread method for image processing -Fourier analysis, i.e., the decomposition into exponential series in two perpendicular directions, we decompose discrete functions on points of the grid in a number of orbit functions without the division into several directions. Our approach is a generalization of discrete Fourier and cosine transform. In this paper, we summarize the properties of C− and E−orbit functions connected with Weyl groups of simple Lie algebras A 2 , C 2 and G 2 . These functions are a generalization of the classical cosine and exponential function and they act in fundamental domains of the Lie algebras. In these domains we introduce a discrete grid on which it is possible to define discrete C− and E− orbit transform. For an illustrative example of analysis and image processing we split a square image into two triangles and we effectuate corresponding C−orbit transform. The paper is organized as follows. Section 2 summarize some known facts about the spatial filtering using a convolution. In Section 3 we remind basic notations from the theory of Weyl group orbit functions. In particular, we describe the discrete transforms based on finite families of orbit functions in Subsection 3.3. In section 4 we define C− and E−orbit convolution and we formulate the orbit convolutions theorems. Finally, in Subsection 4.2 we provide examples of image processing using C−orbit functions. We include two appendices with technical details for the orbit transforms. Spatial filtering A variety of filters play an important role in image processing, in image improving and in detail recognition. For example, the spatial filtering uses convolution of functions which is performed via Fourier transform as a multiplication of the Fourier images. Fourier analysis is based on the decomposition of brightness values in each digitized image points along the rows and columns into Fourier series. The Fourier transform is then processed. The inverse discrete Fourier transform shows processing of digital images. This way we can highlight some features of the image -remove the noise or enhance blur edges. The whole process is described in several papers, for an overview see for example [1,2]. For image compression JPEG the discrete cosine transforms are used. They are of four types and the convolution via multiplications in these cases is more complicated, it combines cosine and sine discrete transform except the discrete cosine transform of type II. The simplest filtering technique is the averaging the light intensities at points. Intensity of each new pixel is the mean value of the intensities of the 8 neighboring pixels and the pixel itself in the original image. Other filters use the intensities of neighboring pixels multiplied by different relative weights and the pixel is assigned by a mean value of 9 intensities. Other filters take into account a number of other surrounding pixels, 25 pixels together with the center. Intensities in 9 or 25 pixel can be expressed as 3 × 3 or 5 × 5 matrix. Averaging over neighboring pixels is mathematically expressed by the convolution of the original intensity matrix with 3 × 3 or 5 × 5 matrix, so-called convolution kernel. The elements of this matrix are the weights assigned to the corresponding pixel in the area according to the desired filter type. For the treatment of pixel intensities on the edge we need to extend a line above and below the picture and a column on the left and the right in the 3 × 3 matrix case. In the case of 5 × 5 matrix we need to add to each side two columns and two rows. Filters mentioned above are called linear spatial filters. Their application to a digital image creates a new image using a linear combination of brightness values in the surrounding pixels. The intensities of the digital image in each pixel are defined by the matrix f (m, n). If we want to apply a filter comprising eight neighboring pixels with different weights, we construct the 3 × 3 weights matrix New digital image has the intensity in each pixel given by a matrix F (m, n) and their values are This corresponds to the sum of all the values of the 3 × 3 matrix we get as a pointwise multiplication of the filter 3 × 3 matrix cut around the filtered pixel. Mathematically, it is a discrete convolution For defining the orbit convolutions we proceed in a similar way as for the discrete cosine transform DCT II, where for two functions f and g it is defined and for cosine transform F c the following relation holds [3] F Weyl group orbit functions 3.1. Weyl groups and affine Weyl groups. We consider the simple Lie algebras of rank two, namely A 2 , C 2 and G 2 . Each of them is described by its set of simple roots ∆ = {α 1 , α 2 } ∈ R 2 . In the case of A 2 , the roots are of the same length, for C 2 and G 2 we distinguish so-called short root and long root. We use the standard normalization α, α = 2 for the long roots. Coroots are defined as α ∨ = 2α/ α, α . Moreover, we define the weights ω i and coweights ω ∨ i , which are dual to root and coroots in the sense α ∨ i , ω j = α i , ω ∨ j = δ ij . The weight lattice P is defined as all integer combinations of weights. We denote the reflections with respect to the hyperplanes orthogonal to the simple roots by r 1 and r 2 , i.e., They generate a Weyl group corresponding to each Lie algebra. The action of W on the set of simple roots gives a root system W (∆) in R 2 . It contains a unique highest root ξ = m 1 α 1 + m 2 α 2 , where the coefficients m 1,2 are called the marks. Analogously, a root system W (∆ ∨ ) is obtained from the action of W on the set of coroots, its highest root is denoted by η = m ∨ 1 α ∨ 1 + m ∨ 2 α ∨ 2 , the coefficients are called the dual marks. Let r ξ denote the reflection with respect to the hyperplane orthogonal to ξ and we define r 0 by The affine Weyl group W aff is generated by {r 0 , r 1 , r 2 }. Its fundamental domain is a connected subset of R 2 such that it contains exactly one point of each affine Weyl group orbit. It can be chosen [4] as the convex hull of the points 0, The root systems and the fundamental domains of affine Weyl group of A 2 , C 2 and G 2 are depicted in Figure 1. The even Weyl group W e is defined as W e = {w ∈ W \| det(w) = 1}. Its fundamental domain is F e = F ∪ r i (int F ), where r i is a simple reflection and int F denotes the interior of F [5]. Corresponding dual even affine Weyl group is denotedŴ aff e and its fundamental domain is given by The functions are invariant with respect to the affine Weyl group, therefore, we can consider x ∈ F only. The family of S−functions is defined for every x ∈ R 2 and λ ∈ P as They are antiinvariant with respect to W aff , moreover, they vanish on the boundary of the fundamental domain. We can consider x ∈ int F . Finally, the E−orbit functions are defined for every x ∈ R 2 and λ ∈ P as They are invariant with respect to the even affine Weyl group, we restrict them on F e . For Weyl groups with two different lengths of root in their root system other families of orbit functions can be defined. For more details see [9,10]. In this paper we consider convolution based on the C− and E−functions, S−function convolution does not differ significantly from the C−function case. 3.3. Discrete orthogonality and orbit transform. The method of discretization of orbit functions was described in detail in the papers [4,5]. The general idea is the following: In the fundamental domain we define a finite grid of points F M , where M is an integer of our choice which allows us to control the density of the grid. A discrete scalar product of functions is then defined using this points. We describe a finite family of orbit functions which are pairwise orthogonal with respect to this scalar product by defining a grid of parameters labeling the functions. Finally, we give the explicit orthogonality relations. Appendix 6.1 summarize details about the choice of the grids. We consider a space of discrete functions sampled on the points of F M with a scalar product defined for each pair of functions f, g as The weight function ε(x) is given by the order of the Weyl orbit of x, ε(x) = \|W \| \| stab W (x)\| . The set of parameters Λ M gives us a finite family of orbit functions which are pairwise orthogonal with respect to the scalar product (1). For every λ, λ ∈ Λ M it holds that where the coefficient h ∨ λ is the order of the stabilizer of λ, c is determinant of the Cartan matrix of the corresponding Weyl group and \|W \| is its order. The values of \|W \|, c, ε(x) and h ∨ λ are listed in Appendix 6.2. The discrete orthogonality allows us to perform a Fourier like transform, called C−orbit transform. We consider a function f sampled on the points of F M . We can interpolate it by a sum of C−functions where we require f (x) = I M (x) for every x ∈ F M . Therefore, the coefficients F λ are equal to In the case of E−orbit functions we consider the grids F e M and Λ e M . The scalar product is defined as The weight function ε e (x) is given by the order of the even Weyl orbit of x, where the coefficient h e∨ λ is the order of the stabilizer of λ and \|W e \| is the order of the even Weyl group.. The values of \|W e \|, ε e (x) and h e∨ λ are listed in Appendix 6.2. The E−orbit transform is provided as follows. We consider a function f sampled on the points of F e M . We can interpolate it by a sum of E−functions where we require f (x) = I e M (x) for every x ∈ F e M . Therefore, the coefficients F λ are equal to 4. Orbit convolution 4.1. Orbit convolution theorem. The main aim of this work is to define a discrete orbit functions convolution, i.e., a mapping of two functions sampled on F M which respects a relation analogous to the classical convolution theorem. Such definition comes naturally from the orbit functions discretization theory. The C-orbit convolution is for every pair of discrete functions f, g and u ∈ F M defined as Such a convolution is well defined, the shifts in the convolution kernel g respect the symmetry of the Weyl group of A 2 . We can write the C−orbit convolution theorem. where F λ and G λ are the C−orbit transforms of f and g given by (3). Its proof is straightforward, it uses the relations (4) and the following formula for the product of an orbit function with the complex conjugate of an orbit function with the same label but different argument: Analogously, the E-orbit convolution is defined for discrete functions f, g sampled on F e M and u ∈ F e M as The E−orbit convolution theorem is then the following. Theorem 4.2. Let f, g be any functions defined on the points of F e M and u ∈ F e M . Then where F λ and G λ are the E−orbit transforms of f and g given by (7). 4.2. Examples of image filtering. For the purpose of demonstrating the differences between the orbit convolution and convolution on R 2 we take an artificial image of a hexagon. Three of spatial filters are presented: a mean filter, often used for image denoising; a sharpen filter which is useful for contrast enhancing; and a simple edge detecting filter which suppresses the monotonic (in the sense of pixel brightness) parts of an image. In R 2 these filters are described by matrices: The filters are constructed to be as similar to the filters used for orbit convolution as possible. There are some restrictions for the orbit convolution coming from its definition, the most significant is the summation over all reflections of the convolution kernel. This property is unpleasant, since it does not give us the possibility to apply changes in a single direction, i.e., detecting only horizontal edges. For this reason we cannot use all convolution kernels we can use for image filtering in R 2 . When developing a spatial filter for orbit convolution from kernel for filtering in R 2 we have to take the formula (9) into account. Many filters are supposed to preserve the average value of brightness in the image. In the frequency domain the related value is situated in the point (0, 0). The normalization of the filter is done by dividing the weighted sum of kernel points by coefficients ε(x). There is also a second level of normalization, arising from the summation over all Weyl reflections of a point, the filter is divided by the number of reflections. Some filters, mostly the ones based on differences, have the weighted sum equal to zero, thus not requiring any normalization. There are two major restrictions for the orbit convolution kernels: the reflection of the kernel, which disables filtering in a single direction, and the placement of the kernel center. For the convolution on R 2 the kernel center is located in the middle point of the kernel, for orbit convolution the center is in the point (0, 0). This brings further restriction, the filter cannot count with all neighboring points. Filters for orbit convolution are defined in the following way: For the orbit convolution demonstration we used the hexagon image, see Figure 2, and filtered it via convolution on R 2 and via C−orbit convolution on A 2 group to have a comparison for similar filters for both methods. The results are depicted on Figures 3, 4 and 5. The differences between the convolution on R 2 and orbit convolution via Corbit transform on A 2 group are very little. One of the reasons is the inequality of convolution kernels for both types of convolution. Concluding remarks (1) In the case of C 2 and G 2 orbit functions there are 7 more families of orbit function defined, the orbit convolution theorem can be formulated for each of them. This gives us bigger choice of the shape of the fundamental domain suitable for the image. (2) The method described here can be generalized to Weyl group of any rank. Therefore, it can be used for more general problems than the image processing. (3) The orbit convolution takes an advantage from the symmetry of the underlying Weyl group. On the other hand, as there is no fast algorithm yet, the computation takes more time than standard Fourier or cosine transform. One of our future projects is finding such a fast algorithm. The orders of the corresponding Weyl groups and even Weyl groups are: The determinants of the corresponding Cartan matrix are: The values of ε(x) and h ∨ λ are listed in Tables 1 and 2. Table 2. The coefficients h ∨ λ of A 2 , C 2 and G 2 . The variables t i , i = 0, 1, 2, are nonnegative integers and have the same meaning as in (15). Let x be in F e . For x ∈ r i (int F ) it holds that ε e (x) = \|W e \|. The values of ε e (x) for x ∈ F are listed in Table 3.	2014-02-17T01:42:37.000Z	2014-02-07T00:00:00.000	{ "year": 2014, "sha1": "3da7b6e97c53f7da04b06d87a181fd6f9fca9530", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "2ca331410dc620814ed2363b344a95aceddb33e3", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
250699156	pes2o/s2orc	v3-fos-license	Some definable types that cannot be amalgamated We exhibit a theory where definable types lack the amalgamation property. The theory Models M of the theory in which our counterexample lives are four-sorted, and are roughly obtained as follows. We start with a binary tree T(M ), with discrete level set L(M ). We then introduce two levelled sets A(M ) and B(M ), both with the same level set as the tree, namely L(M ), and cover each level x of T(M ) with a generic surjection from the cartesian product of the x-th levels of A(M ) and B(M ). In this section, we spell out this construction in detail. Definition 1.1. Let L be the following language. 1. L has four sorts A, B, T, L. 2. T has a binary relation ≤ T , a binary function , a unary function pred T , constants g T , r. 3. L has a binary relation ≤ L , a unary function pred L and constants g L , 0. (i) 0 = g L , and (L\{g L }, ≤ L ) is a discrete linear order with smallest element 0 and no largest element, with predecessor function pred L , with the convention that pred L (0) = 0. The "garbage" point g L is not ≤ L -related to anything, and pred L (g L ) = g L . (ii) (T \ {g T }, ≤ T , ) is a meet-tree with root r, binary ramification, 1 and, for every fixed element, its set of predecessors is a discrete linear order, with predecessor function pred T , with the convention that pred L (r) = r. The "garbage" point g T behaves similarly to the garbage point g L . every fixed element t ∈ T\{g T }, the restriction of T to the set of predecessors of t defines an order isomorphism onto an initial segment of Proof. It is easy to see that T is consistent. It is enough to prove quantifier elimination: since T admits a prime L-substructure M 0 , with underlying set the interpretations of the closed L-terms over ∅, completeness of T will follow, and (2) is a direct consequence of quantifier elimination. Let N 0 and N 1 be models of T with N 0 countable and N 1 ℵ 1 -saturated, and let M be a common L-substructure of N 0 and N 1 . It is an easy exercise to M -embed N 0 into N 1 , yielding quantifier elimination. Proof. This follows from quantifier elimination. Alternatively, one may use that no infinite branch is definable in the standard binary meet-tree (2 <ω , ω), e.g., since for any n ∈ ω and branches s, s ∈ 2 ω with s n = s n there is an automorphism σ over 2 <n with σ(s) = s . The types Let T be the theory defined in the previous section, and U T a monster model. Failure of amalgamation of definable types boils down to the following phenomenon. All elements y of A with level larger than L(U) have the same, definable, type, and similarly for z in B; nevertheless, if such y and z have the same infinite level, then f (y, z) can be used to produce an externally definable subset of T(U) which is not definable. More formally, we proceed as follows. Definition 2.1. Define the following sets of L(U)-formulas. 1. p(x) is the global type of an element x of sort L such that x > L(U). 2. q A (x, y) restricts to p on x, and says that y is an element of sort A with A (y) = x. 3. q B (x, z) restricts to p on x, and says that z is an element of sort B with B (y) = x. Lemma 2.2. All of p, q A , q B are complete types over U which are ∅-definable. Proof. Consistency and ∅-definability are clear. As for completeness, we argue as follows. 1. Completeness of p(x) follows from Proposition 1.3(2). 2. As for q A (x, y), note that, since y has a new level, it cannot be in A(U). Again because we must have f (y, b) = g T . By quantifier elimination, this is enough to determine a complete type. 3. The argument for q B (x, z) is symmetrical. Proposition 2.3. The types q A (x, y) and q B (x, z) cannot be amalgamated over p(x) into a definable type. In other words, no completion of q A (x, y) ∪ q B (x, z) is definable. Proof. Suppose r(x, y, z) is a completion of q A (x, y) ∪ q B (x, z). Then r(x, y, z) A (y) = x = B (z), thus r(x, y, z) T (f (y, z)) = x. Since p(x) is not realised, f (y, z), having a new level, cannot be in T(U). Consider the set {d ∈ T(U) \| r(x, y, z) f (y, z) > d}. If r(x, y, z) is definable, then this set is definable. As a set of predecessors, it must be linearly ordered, hence have a maximum by Lemma 1.4. But then f (y, z) / ∈ U contradicts binary ramification. Corollary 2.4. In T , global definable types do not have the amalgamation property. This partially answers [3, Question 9.3.1], asking whether there is such a theory which, additionally, has uniform definability of types; note that T does not. Moreover, T is easily shown to have IP.	2022-07-21T01:15:59.041Z	2022-07-20T00:00:00.000	{ "year": 2022, "sha1": "d72dfc8e00ce2f04b8848739c0b8beefc766eff7", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "d72dfc8e00ce2f04b8848739c0b8beefc766eff7", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
45559284	pes2o/s2orc	v3-fos-license	Effects of three surface conditioning techniques on repair bond strength of nanohybrid and nanofilled composites Background: Repair bond strength of different composite resins has been assessed in few studies. In addition, reports on the efficacy of surface treatments are debated. Therefore, this in vitro study was conducted to evaluate the effect of three surface treatments on two nanocomposites versus a microhybrid composite. Materials and Methods: In this experimental study, 135 composite blocks (45 specimens per composite) of microhybrid (Filtek Supreme Z250, 3M ESPE, USA), nanohybrid (Filtek Supreme XT, 3M ESPE), and nanofilled (Filtek Supreme Z350, 3M ESPE) were thermocycled (5000 rounds) and then surface roughened (except in a control group of 9 specimens of three composite types). Each composite type was divided into three subgroups of surface treatments: (1) Bur abrading and phosphoric acid (PA) etching, (2) sandblasting and PA etching, and (3) hydrofluoric etching and silane application (n = 15 × 9, complying with ISO TR11405). Composite blocks were repaired with the same composite type but of a different color. Microtensile bond strength and modes of failure were analyzed statistically using two-way analyses of variance, Tukey and Chi-square tests (α = 0.05). Results: There were significant differences between three composite resins (P < 0.0001) and treatment techniques (P < 0.0001). Their interaction was nonsignificant (P = 0.228). The difference between nanofilled and nanohybrid was not significant. However, the microhybrid composite showed a significantly higher bond strength (Tukey P < 0.05). Sandblasting was significantly superior to the other two methods, which were not different from each other. Conclusion: Within the limitations of this in vitro study, it seems that microhybrid composite might have higher repair strengths than two evaluated nanocomposites. Among the assessed preparation techniques, sandblasting followed by PA etching might produce the highest bond strength. INTRODUCTION Staining, fracture, or departures can clinically compromise composite restorations. [1,2] A questionable composite restoration can either be completely changed with a new restoration or be repaired. [2] A full replacement is the most frequent practice; however, it is over-treatment since it might deteriorate dental/ pulpal tissues, remove intact structures and etched enamel, and enlarge the cavities. [3] Hence, restoration repair seems preferable over total replacement. [2,3] Nonetheless, repair might weaken the restoration's retention potential. [2,4,5] The adhesion between fresh and old composite surfaces is achieved by a layer of oxygen-inhibited nonpolymerized resin. [2,6,7] Aging and water sorption might compromise the bond strength by removing this unpolymerized fi lm or reducing the unsaturated double carbon-carbon bonds. [2,6,8] The prognosis of this bond depends on multiple factors including old composite's surface properties as well as applied surface treatments. [3,[9][10][11][12] A variety of techniques are suggested to increase the composite-to-composite bond. These methods (including irrigating, disk/bur abrading, sandblasting, etching, or the application of silane/bonding agents) attempt to alter the composite surface topology. [2,7,10,12,13] Other important factors determining the surface characteristics of a composite resin are the composition and ratio of fi llers. [3] Having a high proportion of fi ller particles, nanocomposites are claimed to have promising physicomechanical properties. However, despite their broadening usage as esthetic materials, their repair bond strengths are not assessed except in a few studies. [3,5] Furthermore, the studied surface treatments for improving repair bond strength of composite-to-composite are highly controversial: [14] Some researchers have found promising effects using hydrofl uoric (HF) acid, roughening with a bur, or sandblasting. [4,5,11] On the other hand, some studies have failed to show a proper infl uence of these methods. [1,15] In view of the above-mentioned shortcomings and controversies and considering that results of a specifi c brand of a material cannot be necessarily generalized to other brands of the same material. [7] This study was conducted to assess the repair bond strength and mode of failure of three composites (two different nanocomposite types in comparison to a microhybrid composite), all under the effect of three different surface treatments. Since shear bond strength is not as reliable as microtensile bond strength (μTBS), we used the microtensile technique in this study. [3,16] MATERIALS AND METHODS This in vitro experimental study was performed on 144 composite specimens (9 experimental groups of 15 specimens each, and a control group of 9 specimens). The size of groups was in accordance with the ISO TR 11,405 standard. [17] All the experimental operations except the laboratory works were performed by a single operator. The laboratory works were performed by experienced laboratory experts. The materials used in this study are summarized in Table 1. Preparation of experimental composite blocks A total of 135 composite blocks were fabricated from the B1 color of three different composite materials (45 specimens per composite type) mentioned in Table 1. The composite blocks (4 mm high and 8 mm in diameter) were molded using stainless steel cylinders. The metal cylinders were incrementally fi lled with composite layers, from the bottom to the top [ Figure 1]. Each composite increment was 2 mm thick. After placing each layer, it was light-cured (40 s) vertically from a 1-mm distance, using a lightemitting diode unit (Demetron II, Kerr, Italy). After curing each composite block, the light-curing unit was calibrated at 600 mW/cm 2 . To create a smooth composite surface, after placing the fi nal layer of composite and before curing it, a transparent Mylar matrix strip was placed on the surface [ Figure 1], and the composite was light-cured. [18] After polymerization, the molds were gently removed. [2] Preparation of control group Similar to the above procedures, 9 composite blocks were fabricated incrementally from the above mentioned 3 composite types (each control subgroup contained 3 specimens). The only difference between the experimental and control composite blocks was that control stainless steel cylinders were of 8 mm high. The control composite blocks were built up in four increments of 2 mm each [ Figure 2]. Each layer was light-cured (40 s) vertically from a 1 mm distance. The light-curing unit was calibrated at 600 mW/cm 2 , after curing each composite block. In this group, no transparent matrix strip was used. The 8 mm high control stainless steel templates were later used for repairing the experimental composites as well. Surface treatments of aged experimental composites Each experimental composite group (n = 45) was randomly divided into three subgroups of 15 specimens each. Each experimental subgroup was subjected to one of the following three surface treatments. The control specimens were not surfaceabraded. Subgroup A (sandblast-phosphoric acid etching) The smooth and aged surface of each specimen was sandblasted for 10 s. This was done by spraying 50 μm aluminum oxide particles from a 5 mm distance under the air pressure of 60-100 PSI. The nozzle of the sandblaster device (Microsandblaster, Dento Prep, RØNVIG, Dental Mfg, Denmark) was held perpendicular to the composite surface. Subsequently, each composite block was etched by means of 35% phosphoric acid (PA) [ Table 1] for 30 s. Finally, they were rinsed (30 s) and dried (10 s) from a 5 mm distance using an oil-free air syringe. [16] Subgroup B (diamond bur abrading-phosphoric acid etching) Aged composite surfaces were abraded for 10 s with a cylindrical diamond bur with coarse particles (D&Z, Germany) attached to a high-speed water-spraying handpiece. After preparing every 5 specimens, used burs were replaced with new ones. Afterward, composite blocks were etched using 35% PA [ Table 1] for 30 s. Finally, they were rinsed (30 s) and air-dried (10 s) from a 5 mm distance. [16] Subgroup C (hydrofl uoric etching-silane bonding) Each aged composite surface was etched using 9.6% HF [ Table 1] for 60 s. It was then air-water-sprayed (20 s) and air-dried (10 s) from a 5 mm distance. Afterward, a two-part silane-coupling agent [ Table 1] was applied to the etched surface according to the manufacturer's instructions. Repairing the aged and conditioned experimental composites The 4 mm-high experimental blocks were mounted at the bottom of the 8 mm-high control template cylinder. An enamel-bonding agent [ Table 1] was applied to the abraded surface of the aged composite block. Afterward, 2 mm thick layers of fresh composite were placed incrementally, within the 8 mm high control mold. Each increment was then vertically photopolymerized (40 s) as stated above. [2] Aging the repaired experimental composites All experimental specimens (which had been thermocycled, surface-abraded, and repaired before), were stored in normal saline (24 h) at room temperature. [2] Preparing micron samples Merging in acrylic resin Since the composite specimens were small and light, they could not be conveniently cut by a high-speed disk cutter (Nemofanavaran Pars, Mashhad, Iran), as the disk would throw them out instead of preparing them. Therefore, they were fi rst modifi ed temporarily to a heavier and bulkier specimen with squared angles, so that the disk cutter would not throw them out. This was done by adding transparent acrylic resin [ Table 1] to the composite: First, a layer of red formaldehyde-free nail polish [ Table 1] was applied to all composite surfaces. When the nail polish was dried, the self-curable transparent acrylic resin was poured into a cubic plastic template of 10 mm height, 15 mm width, and 25 mm length. The composite cylinder was placed in the acrylic resin. Once the resin was cured, the block was removed from the plastic template and subjected to cutting machine [ Figure 3]. [19][20][21][22] Trimming A high-speed disk saw [2000-3500 rounds/min, Table 1] was used to trim the composite cylinders into rods of 1 mm 2 × 1 mm 2 cross-sections. The disk saw was 300-μm thick and of 110-mm diameter [ Figure 3]. [19][20][21][22] The device carved composite rods (8 mm length × 1 mm height × 1 mm width) out of the blocks of composite merged in transparent acrylic resin. [19][20][21][22] Microtensile assessment The 1 mm 2 × 1 mm 2 composite rods were mounted symmetrically from their both ends on the plates of a tensile testing machine (Micro Tensile Tester, Bisco, USA), using glue [ Table 1 and Figure 4]. The test machine applied the tensile force at 0.5 mm/min speed to the both ends of the composite blocks. The necessary force to debond each composite rod was recorded. The debonding force was divided by the surface area size of the rod base (1 mm 2 ) to calculate the μTBS in μTBS (MPa). Rods detached from any of their glued ends (instead of being debonded) would be excluded. This happened in the case of six composite rods, two of which were replaced with new rods. Mode of failure A stereomicroscope (Olympus SZX-12, Olympus, Japan) was used to assess the debonded surfaces at ×8 magnifi cation to determine the failure mode. The failure mode was defi ned as "adhesive" failure, if the detachment had been occurred through the junction of the composites. Cohesive failure meant a breakage through one of the composites (fresh or aged). Mixed failure meant a combination of both types. [2] Statistical analysis Descriptive statistics and frequency distributions were calculated for μTBS and mode of failure. The fi ndings regarding the μTBS values were analyzed using one-way and two-way analyses of variance (ANOVA), Tukey's post-hoc, Mann-Whitney U-test, and independent-samples t-test. The modes of failure were analyzed using Chi-square test. The level of signifi cance was set at 0.05. RESULTS Four of the specimens in one of the subgroups (subgroup C of nanofi lled composite [Z350 XT]) were failed (debonded before exerting tensile force) and excluded. The μTBS results are presented in Table 2. The two-way ANOVA showed signifi cant differences between the bond strengths of three composites (F = 55.320, P = 0.0000) and between three surface treatments (F = 17.609, P = 0.0000). However, the interaction of the variables treatment and composite was not signifi cant (F = 1.43, P = 0.228), meaning that the effect of treatments was similar in the case of all tested composites. The Tukey's post-hoc test showed that the difference between nanofi lled (Z350 XT) and nanohybrid (Z250 XT) composites was not signifi cant; however, microhybrid (Z250) composite showed signifi cantly better results compared to abovementioned composites [ Table 3]. The effi cacy of two treatments surface roughening with diamond burs and HF + silane application was similar. However, sandblasting was signifi cantly superior to the other two methods [ Table 3]. The one-way ANOVA showed a signifi cant difference between the subgroups (F = 18.53, P = 0.0000). The Tukey's post-hoc test showed signifi cant differences in 19 out of 36 pairwise comparisons with the highest difference seen between etched nanofi lled composite (Z350 XT) and sandblasted microhybrid (Z250). The lowest difference was observed between etched nanofi lled composite and etched nanohybrid composite. The Mann-Whitney U-test was used to compare the positive control groups of each composite with the three corresponding subgroups of the same composite. The independent-samples t-test was used to compare the whole control groups (all composites combined) with each type of composite in the control group. The independent-samples t-test was also used to compare surface treatments with the control [ Table 2]. The mixed mode of failure was the common type of failure in most of subgroups and groups. However, according to the Chi-square test, the comparisons were mostly nonsignifi cant [ Table 4]. Among the surface treatments, only surface roughening by diamond burs resulted in a signifi cantly higher rate of mixed failure type [ Table 4]. There was no signifi cant difference between the modes of failure of three composite types (P = 0.708). However, a signifi cant overall difference was observed between the modes of failure of three surface treatments (P = 0.035), indicating a shift in the modes of failure of diamond bur and acid etching/ silane application toward mixed failures [ Table 4]. DISCUSSION The results of this study showed that the tested nanocomposites were not superior, and the microhybrid composite had a higher bond strength in comparison with nanohybrid and nanofi lled composites, which had similar TBS rates. This pattern was visible both in the experimental and control groups. Microhybrid was the only composite in this study with an insignifi cant difference with control. Sandblasting was the most useful method, followed by two similarly ineffective methods of surface roughening by diamond burs and etching/ silane application. In addition, this order of treatment effi cacy was the same for all the tested composites. Compared to the cohesive strength of corresponding substrates, the interfacial repair bond strength had declined mostly in a signifi cant way, which confi rmed the previous results reporting bond strengths between 25% and 80% of control. [2,4,5] Aging can cause water infi ltration into the resin and into the junction of fi llers and matrix, deteriorate composite matrix by hydrolytic degradation of the silane fi lm over fi llers or matrix swelling and also remove its free radicals by water sorption and thermal stresses. [2,3,8,14] A substantial portion of the compositeto-composite bond is chemical and introduced by monomers in the oxygen-inhibited layer of the cured composite and monomers of the fresh composite. [6][7][8] Surface roughening is necessary or perhaps the most important factor for improving the repair bond strength because of creating micro-and macro-interlocking and broadening the surface. [2,[6][7][8]10,12,13,18,23] Moreover, shaving a layer of resin may expose a rough and fresh surface, which might improve the bond strength. [7,13] However, the bond strength did not increase up to the control levels in the present research. This might be due to the lack of oxygen-inhibited coating and the small amount of free monomers and photoinitiators in deeper layers of aged composite, which are now exposed. [7,23] Therefore, although this viscose coating consists of unpolymerized molecules that may produce covalent interfacial bonds, [8,12] the bonding ability of this layer never compares to fresh composites, as its free monomers and photoinitiators are reduced. [7,8,23] Moreover, water sorption might swell the matrix and/ or degrade the silane layer on fi llers. [3,8] Furthermore, thermal fl uctuations of thermocycling might produce microfractures in the resin or through its interface with fi llers. [3,25] This aging method was used in this study, since thermocycling for 5000 cycles might be more effective than acid citric storage or water boiling. [11,14] Repair bond strength necessary for an acceptable composite repair in vivo are not investigated. However, the strength of satisfactory resin-to-enamel bonds might be between 15 and 30 MPa. [2,26] Perhaps, repair bond strengths similar to composite-to-enamel bond strengths might be acceptable in clinical conditions. [2,27] According to certain authors, composite repair bond strengths need to be over 18 MPa to be clinically satisfactory. [2,26] Most of the assessed groups in this study produced repair bond strengths comparable or higher than the suggested minimum. Nanofi lled and nanohybrid composites did not show signifi cant differences. This was contrasting to another study which found nanofi lled composite of better repair retention. [28] A study compared two different nanohybrid composites with a microhybrid composite. [3] One of nanohybrid materials showed results similar to microhybrid, while the other one showed slightly better results. [3] It seems that besides numerous infl uencing factors, the type and brand of composites might be the case. [2,7] In the present study, three surface conditioning approaches were used. Two methods depended on an initial mechanical surface roughening followed by acid etching using PA. The other one relied on two chemical methods (etching using HF and then applying silane). The highest bond strength values in our study belonged to the group subjected to sandblasting followed by acid etching with PA. The effect might be mostly due to the sandblasting, since the other method (diamond bur application that had the acid etching step as well) did not provide similarly appropriate bond strengths. Moreover, PA etching alone has not been successful in increasing repair bond strength; it might only be a superfi cial cleanser. [1,2,18] Many studies have shown promising effects for silane application which might be related to new siloxane bonds created between resin and fi llers. [2,9,10,13,15,26] In this study, however, the silane application resulted in the weakest bond strengths and was the only group that failed to increase the bond strength to 20 MPa or higher. Few researches as well-reported that the addition of silane to the bonding system might not signifi cantly improve bond strength, or even might reduce it. [2,7,13] Methodological differences such as used brands and their qualities and compositions might contribute to the controversy. [7] The conclusive fi nding was the superiority of sandblasting followed by PA etching compared to the other methods, regardless of composite groups. In the nanofi lled group, sandblasting was the only subgroup among all subgroups that was not signifi cantly lower than control. This result was in line with previous studies showing aluminum oxide sandblasting as the best approach to regain proper bond strengths. [29] Nevertheless, from a clinical perspective, HF application and sandblasting are not as safe and convenient as surface roughening with diamond burs. [14] Contradictory results have been reported with the use of diamond burs for preparing composite surfaces prior to bonding. [9,12,14,15,18] The fi ndings of the present study should be evaluated further using negative control groups, in which thermocycling and composite repair would have been done however without any surface conditioning. Cohesive failures are fractures within the composite matrix and suggest a powerful adhesive bond between two composites, being greater than the cohesive bond within each of the composites. Therefore, they indicate acceptable clinical outcomes for a restoration repair. [1][2][3] On the other hand, samples with low bond strengths are likely to depart at the junction of two composites (adhesive failure) which imply lower success of repair bond or more robust structure of composite matrices. No pure cohesive failures were observed in this study. The composite types did not show a signifi cant effect on mode of failure. However, surface roughening with diamond burs improved the mode of failure to the cohesive end. It should be taken into consideration that adhesive failures are not easily detected under light microscopy, and the results should be carefully interpreted since a greater number of mixed failures might have happened. [2] CONCLUSION Microhybrid composite might have a better repair bond strength compared with nanocomposites, while nanofi lled and nanohybrid types are less likely different from one another. The assessed surface conditioning techniques might not recover bond strengths up to control levels. Sandblasting followed by PA etching might produce the highest repair bond strengths. Financial support and sponsorship Nil.	2018-04-03T06:05:57.262Z	2015-11-01T00:00:00.000	{ "year": 2015, "sha1": "4e8a1a296cd2553ff3a8ce47ea481e4ae51b70ce", "oa_license": "CCBYNCSA", "oa_url": "https://doi.org/10.4103/1735-3327.170575", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "32267d44cadbe6c4b282a8a5e2947aa69e97aa99", "s2fieldsofstudy": [ "Medicine", "Materials Science" ], "extfieldsofstudy": [ "Materials Science", "Medicine" ] }
9804045	pes2o/s2orc	v3-fos-license	Send Orders for Reprints to Reprints@benthamscience.ae the Open Civil Engineering Journal Deflection Behavior of Prestressed Concrete Beam Using Fiber Reinforced Polymer (frp) Tendon The application of prestressing steel is restricted in highly corrosive environment area. The behavior of structure changes due to corrosion of prestressing steel, which leads to reduction in strength and it may cause sudden failure. There are many research recommendations to resist corrosion of steel, however the durability of structure shall not be ensured during service life of structure. Fiber Reinforced Polymer (FRP) Tendon is considered as an alternate material due to its corrosive resistance property and high strength. An experimental and numerical analysis carried out to study the deflection behavior of FRP tendon prestressed beam and recommended design guidelines. There are four beam specimens casted and tested in laboratory and 51 experimental results collected from research article to carry out numerical study. The ACI, 2011 [1] recommended generalized deflection calculation for beam by softening the effective moment of inertia curve and also introduced the effect of shift of neutral axis once the member exceeds cracking stress of concrete. Based on experimental and numerical analysis study it is concluded that, the deflection behavior of FRP tendon beam depends on deformability of material, degree of prestressing and bond strength. Design chart proposed for calculation of effective moment of inertia and effective neutral axis distance with respect to deformability index. The error percentage of deflection values as per ACI 2011, is about 10 to 20% has reduced to less than 5% in the proposed method. INTRODUCTION The research on FRP as an alternate material for steel was started during 1950.Initially the usage of FRP was restricted due to its lack of ductile behavior, production of non-consistent property and risk of health and environment contamination.Due to the further continuous research during the past decade, different types of chemicals treated FRP bars with acceptable market standard and international code requirements have developed.Currently, FRP bars are widely used in corrosive environment areas due to their non-corrosive, high strength to weight proportion and fatigue properties.Carbon fiber reinforced polymer bars (CFRP), Glass fiber reinforced polymer bars (GFRP) and Aramid bars are widely used in Underground Structure, Marine Structure and Bridges.American Concrete Institute-ACI 440 recommended material manufacturing and testing methods and design guidelines for the FRP bars. Most of the long span structure need to control the deflection and member sizes.Prestressing of a member will facilitate to control the deflection and size of member.Generally, the deflection of member depends on stiffness of member and material properties.Prestressing force helps to balance the applied moment or alternatively increases the stiffness of member.The stiffness of member weakens due to cracks developed on the member, which leads to increase in deflection in a faster rate than un-cracked section.Hence, the detailed studies of deflection behavior of prestressing member were included below. DEFLECTION Many researchers carried out study on deflection behavior of structural member using prestressed bars.Branson, 1968 [2] derived a deflection equation by considering the moment curvature behavior of a beam.The effect of moment curvature is linear until concrete sustains tensile stresses (f st ).The moment of resistance of member upto the first crack is termed as cracking moment (M cr ).The linear nature of moment curvature shall extend Fig. (1), when the member is subjected to prestressing force.Alternatively concrete will start receiving tensile stress only after applied moment balances prestressing forces (P) on the member.When the applied moment M a is more than M cr (Pe+f st z-M selfwt ) then concrete attains permissible stresses and start cracking on the flexural tension side of member, hence the moment curvature behaves non-linearly.Further increase of applied moment will lead to move the curve linearly up to failure.The moment curvature ɸ of member behaves linearly up to point D. (1) The effect of curvature is related to the E c I g uncracked stiffness of member.The curvature is linear up to (M a -Pe)<=M cr =f st Z. where f st -Tensile strength of concrete, Z -Section modulus of member, P -Effective prestressing force, e -Eccentricity of prestressing with respect to neutral axis of member, E c -Young's modulus of concrete and I g -Gross moment of inertia of member. At point E, the member attains ultimate stage and moment curvature becomes linearly varying. (2) The effect of curvature is related to the E c I cr cracked stiffness of member at ultimate load.Where I cr is Cracked moment of inertia of member. At stage D to E, the moment curvature is non-linear, and the pattern of moment of inertia as shown in Fig. (2) changes from I g to I cr with respect to (M cr /M a ) ratio.Branson, 1968 assumed these variations as non -linear cubic power curve, and derived an equation for finding effective moment of inertia (I eff ).Hence constant k= (I g -I cr ) (3) Branson &Trost, 1982 andTadros, Ghali &Meyer, 1985 [3, 4] predicted that prestressing eccentricity is increasing due to shift of neutral axis during its cracking stage as shown in Fig. (3).The effect of neutral axis shift is non-linear due to the tension stiffening effect of concrete and prestressing.This leads to an increase in prestressing moment which reduces the deflection rate of increases.The pattern of neutral axis distance changes from Y g to Y cr with respect to (M cr /M a ) ratio as shown in Fig. (4).Where Y g is a neutral axis distance from extreme compression fiber of beam until first crack occurs and Y cr is neutral axis The effect of influence of above parameters leads to more shift of linear curve of deflection (shift of point D refer Fig. 1), shift of occurrence of cracked moment of inertia of member at ultimate stage (Shift of Point E refer Fig. 1) and the change of nonlinear curve of deflection between the un-cracked stage to cracked stage.Branson proposed simplified model which includes an influence of all the above parameters.The deflection errors by Branson, 1968 deflection model are converging to experimental results by adopting the transformation of value of effective moment of inertia as cubic power parabolic curve with respect to M cr /M a . A. Abdelrahman, G. Tadros, 1995 andS.H Rizkalla, 1999 [7 -9] studied the deflection behavior of prestressed concrete beam using CFRP Tendon.They predicted that, shift of Neutral axis distance (Yeff) with respect to M cr /M a are slower than steel tendon.It means the eccentricity (e) is increasing at a slower rate with respect to M cr /M a than steel tendon.So they considered the shift of neutral axis distance curve as square power curve with respect to M cr /M a ratio.The deflection values calculated using these concepts are 70% to 85% accuracy to the experimental results.ACI 440-4R -04, 2011 recommended to soften the moment of inertia values by introducing the additional factor βd considering the non-ductile behavior of FRP bar, because the application of Branson, 1968 equation is valid for calculation of effective moment of inertia of steel tendon. (5) (6) If E P = E S , then βd.= (0.5*(1+1) = 1 which is similar to steel tendon I eff equation.ACI recommended proportionate factor βd for FRP tendon with respect to steel tendon property by introducing the ratio of Young's modulus of FRP (E p ) to Young's modulus of steel (E s ) (refer equation 6).The deflection values are calculated using these concepts is 80% to 95% accuracy to the experimental results.The errors in deflection by ACI method are due to deformability behavior of particular type of FRP tendon to ductile behavior of steel tendon is not constant.Even the deformability behavior of particular type of FRP bar is varying with respect to the degree of prestressing and modulus of rupture of concrete.Hence this rational approach of introducing softening factor in moment of inertia does not carry any technical justification for calculating the deflection, because the failure pattern of FRP tendon is by deformability and rupture of bar, but not by the steel being ductile.This approach is completely irrelevant when partial prestressing with non prestressing steel or FRP bar is used. Branson, 1968 I eff & Y eff curve approach for calculating effective moment of inertia and shift of eccentricity is reasonable, because whatever factor impacts irrespective of tension stiffening effects on the member, the pattern of curve between un-cracked to cracked stage maintains a similar path.Hence Tadros, Ghali and Meyer, 1985 proposed a new deflection model for beam using FRP tendon by softening the curve to four power curves and generalized the equation. Toutanji and saafi, 1999 [10] proposed a new model for the GFRP reinforced member. Where ρ is a reinforcement ratio. Raed Al-Sunna1, Kypros Pilakoutas, Peter Waldron and Tareq Al-Hadee, 2006 [11] proposed a modified equation for the effective moment of inertia by considering the reinforcement ratio and bond strength.They introduced two additional rational parameters α & β to soften the moment of inertia.S. Khalfalla, 2009 [12] also proposed two additional rational parameters Ψ 1 & Ψ 2 to soften the moment of inertia. DEFORMABILITY P. Zou, 2003 and G.J. Mitolidis, T.N.Salonikios and A.J. Kappos, 2008 [13,14] stated that, FRP tendon is a nonductile material and the deflection behavior during its cracking stage has relying on deformability behavior of FRP material.The deflection of prestressed FRP beam follows the bilinear path.It consists of a linear curve upto prestressing strain and then bilinear curve for the balance strain available after prestressing strain, hence this bilinear effect influences the deflection curve pattern of the beam during cracking stage. A. Abdelrahman, 1995 compared the deflection of beams for the same percentage of CFRP tendon area by varying degree of prestressing.Test results as shown in Fig. (5) reveal that, the pattern of deflection curve between uncracked to cracked stage is changing with respect to degree of prestressing.Further, it shows that 50% degree of prestressed beams performs better deformation than 70% degree of prestressed beams.The initial moment of resistance of beam up to uncracked stage is higher for 70% prestresssed beam but further converging in a faster rate leads to an ultimate moment of resistance more or less same as 50% degree of prestressed beam.N. Grace, E. Jensen, V. Matsagar, and P. Penjendra, 2013 [15] observed that, the FRP tendon beam is better deformability behavior for the low degree of prestressed beams than high degree of prestressed beam.Abdelrahman, 1995 stated that FRP beam deformability shall be calculated by the ratio of deflection of beam considering as un-cracked stiffness at a load equal to ultimate load and to the deflection due to ultimate load considering the cracked stiffness.Dolan andBurke, 1996, andChad, 2001 [16, 17] proposed a simplified method for calculating deformability by strain approach.ACI 440-4R, 2011 amended their strain approach.The deformability of FRP prestressed beam shall be measured in terms of deformability index.It shall be defined as ratio of ultimate stain (ε pu ) to the stain at service stage (ε ps ) with the slight modification due to difference in neutral axis of elastic and inelastic behavior. (7) Where k is ratio of neutral axis depth to effective depth, β 1 is stress block factor for concrete, ρ is reinforcement ratio, d is effective depth to the FRP tendon, f pu is ultimate tensile strength of FRP tendon and f' c is compressive strength of concrete. MATHEMATICAL MODEL The literature study as narrated above by various researchers explicit that, FRP beam deflection model needs modification to match with experimental results.The following conclusion shall be made for the proposed new mathematical deflection model by considering their research recommendation for the deflection calculations. NUMERICAL ANALYSIS There are 51 numbers of experimental data as shown in Table 1, which contain an influence of various possible parameters considered for the numerical analysis.The deflection pattern of experimental results compared with assumed trial pattern as per proposed deflection model and then least square errors calculated.The optimum value of parameter m & n arrived by Lagrange Dual function convex optimization method.Design chart and equation are proposed to calculate the deflection of simply supported beam using FRP Tendon. Experimental works are carried out at research laboratories to validate the numerical analysis output.Four numbers of simply supported beam using Carbon fiber reinforced polymer (CFRP) Tendon casted and tested at Annamalai University Structural Engineering Research laboratory, India. Selection of Experimental Results for Numerical Study The influence of various possible parameters as stated in clause 3 considered for the selection of experimental results.The proposed numerical analysis models considered for prestressed tendon bar with no additional non prestressed reinforcements.The fully bonded prestressed FRP beam experimental results are only considered for the numerical analysis. Beam Configuration The beam span considered for the samples are varying from 2m to 10m and the beam shape includes rectangular, flanged T and I sections.Size of rectangular beams varies from 102mm to 200mm x 250mm to 550mm and the T & I beam flange width varies from 200 to 1220mm.Simply supported beam with two point or single point load test results are only considered for the sample selection. Configuration of FRP Tendon Arrangement Ttwelve types of beam configurations are considered based on the position & number of prestressing bars and shape of beam member as shown in Fig. (6).The Term R-2-H as stated in the Fig. (6) defines the shape of beam either Rectangular(R) or T beam (T) or I beam (I) and second term defines the number of prestressed bars and the third term defines the position of bar either Vertical (V) or Horizontal (H). Material The material properties considered for the samples include concrete compressive strength of 40MPa to 70 MPa, Young's modulus of 28 GPa to 41 GPa, tensile strength of 2.75 MPa to 6.3 MPa and FRP bar properties include of Young's modulus in the range of 50 GPa (GFRP Tendon) to 147 GPa (CFRP Tendon) and tensile strength of bar from 1200 MPa to 2700 MPa. Prestressing Tendon Degree of prestressing values of 23% to 70% are considered for selection of experimental results.The reinforcement ratio of section with balanced reinforcement is considered in the range of 0.09 to 1 and Tendon reinforcement percentage considered in the range of 0.04% to 0.52%.Straight tendon and harped tendon effects are also considered for numerical study. Lagrange Dual Function Convex Optimization There are 51 samples shown in Table 1 which are considered for arriving optimal values of m & n based on Lagrange dual function convex optimization method by varying the deformability index value.Deflection is calculated for every load increment of each sample by varying the m & n with all possible combinations.The lowest least square error corresponds to m & n is considered as proposed value for calculating deflections.Where, M Live is applied moment due to live load, M D is Moment due to self-weight, L is effective span of beam and d p is Effective depth of prestressing bar. Deflection for Sample 1 (T-4-V) The sample from Abdelrahman, 1995 experimental results of T-4-0.7-Vselected to demonstrate for calculation of optimum m & n value by numerical method.The power value of m & n varied at an interval of 0.1 and the deflections calculated for each possible combination of m & n values.There are 231 trials of deflection model calculated for each load increment of particular sample and the errors of each trial with respect to experimental results were tabulated.The deflection values calculated by numerical method are either higher or lesser than the experimental results for each increment of load, hence the errors are measured by least square error and calculated for each trial by squaring maximum and minimum errors. In order to study the convergence of lowest least square error solution, a graph is plotted for all the calculated least square error values with the possible combination of m & n value. Lower Bound Solutions Prediction of optimal solutions for m & n values will benefit to generalize the deflection equation considering all possible influence parameters.Hence, the lowest boundary of least square solutions profile as shown in Fig.The prestressed CFRP beam deflections of proposed method compared with experimental results and ACI method are shown in Fig. (9).It reveals that, the deflection of beam by ACI method is underestimating the actual experimental deflection value.Maximum Least square error by ACI method is 167 against proposed method error value of 2. Hence, the generalized ACI approach is not applicable for all conditions of FRP prestressed beams.Abdelrahman, 1995 study as shown in Fig. (5) demonstrates that, the deflection pattern is changing for the same percentage of reinforcement by varying degree of prestressing, hence the consideration of m=3 and n=2 as per ACI 2011 method will lead to erroneous deflection as compared to experimental results. Effective Moment of Inertia (I eff ) Effective Moment of Inertia of sample 1 as per proposed method is smoother curve than ACI method as shown in Fig. (10), because the shift of deflection is faster in low deformability index value as most of the strain is utilized for prestressing and the balance available strain only used to deform.The curve as per proposed method for the deformability index value of 1.54 is 4.55 power curve in place of 3 power curve is proposed in ACI 2011 method.There is no rational parameter like softening of equation (β d ) considered for calculation of moment of inertia. Shift of Neutral Axis Distance (Yeff) Shifts of neutral axis is slower phase as compared to ACI, 2011 method as shown in Fig. (11).The rate of shift of neutral axis is slower due to the high degree of prestressing leading to high tension stiffening of member at the tension zone.This effect is reverse when low degree of prestressing member is adopted.The selected sample 1 has less deformability behavior due to 70 % degree of prestressing, hence the shift of neutral axis fully relies on prestressed force and not on deformable behavior of FRP bars.The cracks of high prestressed beam penetrate faster than low degree of prestressed beam.The shift of neutral axis distance for the deformability index value of 1.54 and n = 1.4 power curve is given as: Proposed m & n Values The Lagrange dual function convex optimization as discussed in the previous clause is adopted for all 51 samples and predicted the optimum m & n for the various deformability index values.A graph plotted for all the optimal values of m & n is shown in Fig. (12) & n curve is plotted with respect to most coincidence dot points values to generalize the pattern. Proposed design chart as shown in Fig. (12) shall be used for selection of m & n for fully bonded with FRP tendon prestressed concrete beam with respect to deformability index value of member.The least square error for the proposed deflection model is less than the ACI, 2011 Deflection model.The best fit curve for 'm' is 3.15 power curve, so the value of m shall also be calculated by equation 12 for the value of deformability index within the range of 1.35 to 4.59.The value of m is higher at lower deformability index, because high degree of prestressing leads to less value of balance strain (ε m = ε pu -ε ps ) to deform.Due to the less balance strain the effective moment of inertia shift is faster which leads to increase in deflection faster and reaches the total deflection earlier than beams of high deformability index value.The value of m is varying from 3.43 to 4.79 with respect to the deformability index value of 1.35 to 4.59.The best fit curve for 'n' is 0.92 power curve, so the value of n shall also be calculated by equation 13 for the deformability index value within the range of 1.35 to 4.59.The value of n is high at high deformability index value which leads to shift of neutral axis in a faster rate due to lack of stiffening effect & low prestressing values.The deflection errors for the proposed method are within 5 % as compared to 20 % by ACI method.The errors by ACI methods are more when the deformability index is low, further the convergence of errors are less than 10% for the high deformability index values.The proposed deflection calculation method is based on the deformability index and it is calculated based on the ratio of strain at ultimate stage (ε pu ) to the strain at prestressing stage (ε ps ).The effect of balance strain (ε m = ε pu -ε ps ) decides the deformability nature of beam.Hence, the deflection calculation by the proposed method is realistic strain approach, wherein ACI method suggested rational approach.Proposed numerical methods for finding the deflection are near to true solutions for any loading stage as compared to ACI method, 2011. EXPERIMENTAL METHOD Experimental test carried out to validate the proposed design charts and equation for the deflection of FRP prestressed beams.Four beams were casted and tested by varying the deformability index values. Test Specimen and Setup The rectangular beam specimen of size 150mm x 250mm and length of 3100mm casted with PVC void former and then, after initial setting of concrete the PVC tube removed.The Carbon fiber reinforced polymer (CFRP) tendon used as a post tension FRP rod.Fig. (13) shows the various beam specimens configuration adopted for experimental works.The term CFB1-2-H-0.5 means "CFRP Tendon specimen number -Number of Tendon -tendon positioned Horizontally (H) or Vertically (V) and the last term is Degree of prestressing in fraction.There are four specimens casted in which two specimens are with one number of 9.5mm dia CFRP tendon and balance two with two numbers of 9.5mm dia CFRP tendon positioned horizontally and vertically.The degree of prestressing varied from 35% to 70%.The beam member was designed as tension controlled member.The top reinforcement provided as hanger bar to support shear links.Shear links were designed in such a way that shear failure should not occur during test.Simply supported beam with two points loading of an equal distance of one meter is applied on the beam member. Material Property High strength concrete of grade C40/50 is considered for mix design and the compressive & tensile strength of the same is confirmed by trial mix test results.Further, additional cube/cylinder is prepared during casting of beam specimen and tested during the experimental test of beam members.The average test results of concrete are shown in Table 2. Prestressing of CFRP Tendon Hydraulic jack of 10T capacity is used for stressing the 9.5mm dia CFRP tendon.Special measures are considered for both ends of beam specimen for the effective anchorage of tendon.Steel plates of size 100mm x 150mm and 24mm thickness are used at both ends of the beam.The CFRP Tendon connected with threaded studs on both sides is used to lock the stress by nuts as shown in Fig. (15).The effective prestressing force of tendon is achieved by controlling the elongation of tendon during stressing.Elongation δ =PL/(A P E p ) = δ 1 + δ 2 + δ 3 where δ 1 & δ 3 is elongation of high strength stud and δ 2 is elongation of CFRP Tendon.Cement grout is injected through grout tube immediately after the stressing of tendon.The specimen is allowed for 3 days of curing for the grout.Elongation due to prestressing loss measured before start of testing.Net elongation calculated after deducting the losses.Table 4 shows the effective prestressing force and corresponding elongation achieved during prestressing. Beam Specimen Testing A test frame capacity of 50T was used to test the beam.Load cell used to measure the applied load on beam.Three dial gauges installed bottom of beam at mid span and 500mm from left and right side as shown in Fig. (16) to measure the deflections during testing.A Demec gauge was used to measure the strain at surface of beam 50mm from top and bottom in the mid span zone of 1m.The beams were loaded at an increment of 5kN upto first crack and then reduced to 2.5kN during cracking stage.Deflections were recorded at each load increment.Crack meter were used to measure the crack width for each increment of load. Experimental Test Results and Discussion The flexural behavior of tested specimen is compared with ACI method as shown in Table 5.The Moment of resistance at first cracked stage is slightly higher than the theoretical calculations by ACI method.Hence, it shows that the effectiveness of prestressing of tendon & concrete strength of beam are achieved as per design.The moment of resistance at ultimate stage of beams by experimental values is slightly lower than the calculated value.However, these deviation shall be accounted by multiplying moment reduction factor of 0.85 in theoretical calculation as stated in ACI, 2011.This approach will aid to achieve the effective design of beam using prestressed CFRP tendon.The deflection test results of a beam in relation with deformability index were assessed.The CFRP tendon has lesser value of ultimate deflection for the high degree of prestressed beam than low degree of prestressed beam (refer Fig. 18).The crack patterns of tested CFRP specimen are illustrated in Fig. (17).Cracks are mostly developed in the pure bending zone due to the application of two point loadings.The definite crack pattern, width and spacing at bottom of beam show that these beams were failed by rupture of CFRP tendon.The mode of failure of beams leads to tension failure.The crack spacing approximately of 250mm shows that tension stiffening of beams is effective due to proper aggregate interlocking and effectiveness of prestressing of tendon.The first crack developed near to middle of support and then extended towards neutral axis.There was no sign of shear cracks on beam near support, because the beam is designed to resist shear failure.6 show that the deflection pattern of 70% degree of prestressed member fails in lesser deflection value than the 35% degree of prestressed member.The beam CFB4-1H-0.70deflection pattern reveals that, 70% of CFRP bar strength is utilized for prestressing and the remaining 30% is only utilized for deflection of beam during its cracking stage, so it leads to less deflection than the 35% degree of prestressed beam.High degree of prestressed beams provide better uncracked moment of resistence than low degree of prestressed beam, however the ultimate moment of resistance is almost the same for both cases.Similarly Fig. (20) and Table 6 show that 61% degree of prestressed member fails in lesser deflection than 48% degree of prestressed member.Hence, we can conclude that, the changes of deflection pattern are with respect to degree of prestressing and deformability index value. COMPARISON Experimental results of four beams were compared with the proposed Numerical model and ACI-Abdelrahman deflection model.Fig. (22) & Table 6 show that the deflection values of member by ACI method, 2011 is close to the experimental results for the high deformability index value due to the more quantum of strength of CFRP bar available to deform and it behaves more or less same as steel tendon in proportion with Young's modulus of materials.Fig. (21) & Table 6 show that, ACI method is deviating away from experimental results when low deformability index value of member due to less quantum of strength of CFRP bar is available to deform.ACI predicts 11.8mm deflection for the beam CFB1-1-H-0.70, but the actual deflection by experimental is more than the anticipated value which leads to under estimating of actual deflection value of member.Proposed numerical model coincides with experimental results due to its deriving deflection curve with respect to strain ratio of member at ultimate stage to first crack stage.The ACI method follows the similar approach irrespective to the degree of prestressing & strain of member at ultimate and first crack stage.The moment of resistance of member using CFRP tendon at ultimate stage is approximately same irrespective to the degree of prestressing of member which leads to change of deflection curve with respect to degree of prestressing.Hence the change of deflection curve is with respect to deformability nature of CFRP tendon.In this proposed method, the pattern of deflection curve changes during its cracking stage with respect to the degree of prestressing accounted. Effective moment of inertia curve with respect to M cr /M a for the beam member CFB4-1-H-0.70 as shown in Fig. (23) is lowering steeper than the ACI method due to the pattern of crack penetrates faster than low degree of prestressed member and leads to lowering the moment of inertia of member.ACI, 2011 also assumes the same however the rate of reduction of effective moment of inertia is faster for the low deformability index value of member. The shift of eccentricity is a slower phase than the ACI method, 2011 [1] as shown in Fig. (24), because the degree of prestressing force increases leads to more tension stiffening effect than the less degree of prestressing force at tension face of member.The rate of shift of neutral axis is slower than steel due to the effect of non-ductile behavior.The steel is ductile nature hence the tension stiffening effect of bar will loosen in a faster rate than FRP rod. CONCLUSION AND RECOMMENDATIONS Numerical and Experimental study reveals that, ACI -Abdelrahman method underestimate the deflection value leads to unsafe design of member.In this paper, it is on attempt to study the behavior of deflection of member using CFRP tendon.There are 51 numerical samples considered for the numerical analysis and four experimental specimens casted and tested for validation of proposed design charts and equations. The deflection of member is related to deformability property of FRP tendon and it shall be measured in terms of strain behavior of FRP bar up to rupture stage.The proposed deflection calculation method is based on the deformability index and it is calculated based on the ratio of strain at ultimate stage(ε pu ) to the strain at prestressing stage(ε ps ).The effect of balance strain (ε m = ε pu -ε ps ) decides the deformability nature of beam.Hence the deflection calculation by the proposed method is realistic strain approach.Wherein ACI method suggested rational approach of introducing softening factor which is calculated based on the comparison of FRP Young's modulus with respect to steel young's modulus property.Proposed numerical methods for finding the deflection are near to true solutions for any loading stage as compared to ACI method.The maximum error due to proposed method is less than 5% as compared with experimental results, where in the error by using ACI, 2011 method is up to 20%. Abdelrahman, 1995 proposed two power curves for the shift of neutral axis for the member using FRP prestressing, but the shift of curve is also changing with respect to deformability index.The moment of resistance of FRP bars are approximately same irrespective of degree of prestressing, hence the deflection curve ultimate changes with respect to degree of prestressing which is not accounted in ACI method, 2011 [1].Proposed method predicted that the shift of neutral axis is with respect to strain at ultimate stage to the strain at first crack stage.Hence the value of n power curve is less than 2 for the low deformability index and more than two for the high deformability index value.The concept of Branson and abdelrahman is valid for the high deformability index where in it is deviating away from experimental results for the low deformability index. Design charts as shown in figure 12 and equation 12 & 13 for m & n power coefficient have been proposed with respect to deformability index value.The value of Deformability index shall be calculated with respect to Dolan & burkes [20,21] strain approach.This proposed method is applicable for fully bond strength beam member.Hence, a detailed study is required for the deflection behavior of member considering the un-bonded and partially bonded FRP tendon.The deflection behavior of member shall be reviewed considering the beam as partial prestressing with non prestressing steel.The effect of partial prestressing with non prestressing steel will be differing with respect to reinforcement bar properties. 4 ) 3 . I' = (Ig-Icr) = + ( − ) ( stage.Branson, 1968 assumed the non-linear patten of curve as 2.5 power curves, and derived an equation for effective neutral axis distance (Y eff ) in cracking stage for the prestressing beam using steel tendon.i.e., Y cr = Neutral axis depth (DEFLECTION MODEL FOR THE FRP PRESTRESSING MEMBER Y. Tan, W. Xue, 2012 and Constantinos B. Demakos, Constantinos C. Repapis and Dimitrios Driva, 2008 [5, 6] stated that, the following parameters influence the deflection behavior of prestressed beam using FRP Tendon.Deformability behavior of prestressing bar.Degree of prestressing.Effect of shift of neutral axis during cracking stage.Bond strength shall be of fully bonded, partially bonded and un-bonded.Modulus of rupture of concrete.Modulus of elasticity of concrete.Modulus of elasticity of FRP bar.Influence of creep and shrinkage. Ieff & Yeff are not an irregular variations with respect to Mcr/Ma and it resembles the same pattern of curve even by considering the influence of many parameters.The pattern of curve resembling parabolic curve with power of m & n.The deflection values are influenced by deformability behavior of FRP tendon and Bond strength between concrete and FRP tendon.So in order to simplify this approach, a new mathematical deflection model is proposed by Numerical methods and it is validated with experimental results.Power of curves m & n values are related to the deformability and bond strength of FRP tendon.The deformability of FRP bars is related to the degree of prestressing and strain ratio at ultimate and service state of beam member. Fig. ( 7 ) shows the graph for sample 1.The values of least square error are converging between the m values of 4 to 5 for all the possible n values and then start increasing.There are many local optimal solutions with respect to n values.The convex convergence of curve for the value of n is in between 1.4 to 1.8 which provides optimal solutions. (7) is separated from the source trails and lower bound convergence curve is plotted for m+n vs corresponding least square error values.The Curve shown in Fig. (8) shows the convergence pattern of least square errors with respect to m + n value.Average least square error values are calculated in the convergence region as shown in Fig. (8) and interpolate the m & n value corresponds to the average least square error value. Behavior of Prestressed Concrete Beam The Open Civil Engineering Journal, 2016, Volume 10 47 and indicated as dotted points with respect to deformability index values.The best fit m Table 2 . Concrete test results. Age in Days Average Cube Compressive Strength MPa Average Tensile Strength MPa Youngs Modulus Mpa Remarks (14)l of grage Fe 500 MPa is used for shear links.CFRP bar shown in Fig.(14)used as post tensioning tendon supplied by Dextra PVT Ltd Honghong, China.The properties of the materials are tabulated in Table3.	2016-10-26T03:31:20.546Z	2016-02-29T00:00:00.000	{ "year": 2016, "sha1": "e5e0dadfd813c31bb62538a7970bfe7e9d356b9a", "oa_license": "CCBY", "oa_url": "https://opencivilengineeringjournal.com/VOLUME/10/PAGE/40/PDF/", "oa_status": "HYBRID", "pdf_src": "Grobid", "pdf_hash": "e5e0dadfd813c31bb62538a7970bfe7e9d356b9a", "s2fieldsofstudy": [ "Engineering", "Materials Science" ], "extfieldsofstudy": [ "Materials Science" ] }
240493808	pes2o/s2orc	v3-fos-license	Clinical aspect, pathogenesis and therapy options of alopecia induced by hormonal therapy for breast cancer Adjuvant hormonal therapy is one of the most important treatments of hormone-receptor-positive breast cancer and includes selective estrogen receptor modulators, aromatase inhibitors, and luteinizing hormone-releasing hormone analogs. In patients receiving these drugs, a progressive recession of frontal-temporal hairlines is often observed, such as a certain grade of hair miniaturization in the same areas and the central scalp area, producing a pseudo-female androgenic alopecia, which has to be considered oncotherapy-induced alopecia. The aim of this work, is to describe the clinical aspects and pathogenesis of this type of alopecia and to analyze the different drugs which have been proposed until now. The authors concude that topical hormones should not be considered as a therapeutic approach because of their direct or indirect oncogenic potential. A therapeutic approach that could be both safe and effective is proposed. Clinical aspect of alopecia induced by HT for breast cancer The frequency and the clinical features of alopecia associated with cytotoxic chemotherapy are well-known, while the incidence of all-grade alopecia in patients receiving endocrine therapies is less frequently described. It has currently been reported to range from 0% to 25.4% [1]. Clinically, a progressive recession of frontal-temporal hairline is present, associated with a certain grade of hair miniaturization in the same area and also in the central area of the scalp [6][7][8][9][10]. It develops within 12 months of HT in most cases. It is not severe alopecia, being reported as a grade 1 in the 93% of patients using common terminology criteria for adverse events, nevertheless, alopecia-related quality of life (QoL) is deeply affected. The mean score of the Hairdex questionnaire is reported to be 25.6, with a higher negative impact on emotions [11]. Pathogenesis of alopecia induced by HT for breast cancer The role of estrogens in modulating hair growth is well known [12], and aromatase has a protective role for anagen hair follicles of the frontal hairline. Aromatase p450 (p450arom) is responsible for the conversion of testosterone to estradiol, with the result of reducing testosterone and dehydrotestosterone (DHT) levels [13]. Its inhibition is useful in all estrogen-responsive cancers. Aromatase is also localized in the inner root sheath of hair follicles, and in particular, it is most highly expressed at the frontal and occipital scalp areas of women than at the same areas of men [14]. Blocking estrogen synthesis, AI cause a relative increase of 5α-reductase activity because of an upstream testosterone accumulation with the effect of higher production of DHT around hair follicles. This increase in DHT levels leads to a shorter anagen phase (which is the phase of hair growth), with a consequent male pattern of hair loss, mimicking female androgenetic alopecia (FAGA) which we call "pseudo-male pattern androgenetic alopecia (AGA)". Sebaceous secretion also increases, with changes in the hair texture and appearance. It is important to clarify that it is not a FAGA, but it has to be considered oncotherapy-induced alopecia. Moreover, the lack of involvement of the anterior scalp area and vertex underlines the difference between this type of alopecia and a real FAGA [15] (Figure 1). Concerning the other drugs employed in HT of breast cancer, they act at several levels and with several mechanisms (upstream blocking LHRH release, or downstream inhibiting estrogen receptors), but they all have the same final effect of inhibiting estrogenic activity. For this reason, their effects on hair follicles will be visible not only where aromatase is more represented, but also at the anterior scalp area and the vertex. Anyway, the clinical aspect is similar to the one caused by AI, so we finally observe a pseudo-FAGA. No data are currently available about the hair loss associated with SERD treatment. Herein we report and discuss the therapy options which have been proposed for the prevention and treatment of HT-induced alopecia and we give our therapeutic proposal. Therapy options of alopecia induced by HT for breast cancer Estrogens Standing to the pathogenesis of this type of alopecia the most effective solution would be the topical application of estrogens. Even if reports about the use of these molecules exist, we do not recommend them because of their possible oncogenic effects if they enter the cardiovascular system and/or because they could favor skin metastasis when topically administered. It is important to remember that HT is administered in patients with hormone-sensitive breast cancer, and higher blood levels of estrogens could enhance tumor growth. 17β-estradiol is a hormone to all effects, able to bind estrogen receptors, so it should not be used in this kind of patient. 17α-estradiol is an isoform of 17β-estradiol which is incapable of binding estrogen receptor, and for this reason, it was considered suitable for the treatment of alopecia induced by HT [10]. It has been demonstrated that 17α-estradiol is able to increase testosterone transformation in 17β-estradiol and androstenedione in estrone, probably by increasing aromatase activity [16]. So even this isoform, although unable to bind hormonal receptors, could be indirectly dangerous. Anti-androgen molecules Since it was clear that no estrogens could be employed, it was proposed to use anti-androgen molecules, such as spironolactone [10]. It competes for the binding site of the aldosterone receptor in the kidney, but it is also able to bind the DHT receptor so that it also has anti-androgen activity. Anyway, by binding the DHT receptor, spironolactone could activate a series of events that end with an increased estrogen production ( Figure 2). Theoretically, spironolactone would increase DHT concentration with a consequently delayed transformation of testosterone, which would accumulate. Moreover, it has been demonstrated that high levels of DHT lead to an increased synthesis of prostaglandin D2 (PGD2), which has a negative effect on hair follicle growth [17] (Figure 2). Figure 2. Spironolactone impedes DHT to bind its receptor ( ) leading to a DHT accumulation. Consequently, DHT transformation into testosterone is delayed (thinner arrow), so that testosterone concentration increases. Testosterone conversion into 17β-estradiol by aromatase could increase (thicker arrow). Moreover, high levels of DHT lead to an increased synthesis of PGD2 by PDG2 synthase (thicker arrow), which has a negative effect on hair follicle growth (thinner arrow) Finasteride has also been proposed [10], but even this molecule could provoke an increased estrogen production. The block of 5α-reductase would lead to a higher testosterone concentration (Figure 3). An increased quantity of testosterone could have different effects depending on the HT agent in use. In the case of treatment with SERMs aromatase is able to transform testosterone to 17β-estradiol, and if its concentration grows, it could compete with SERM's binding to the receptor. If the patient is taking LHRH analogs, the greater quantity of testosterone would be transformed to 17β-estradiol and it would bind its receptors if treatment with SERMs is not also given. In the case of the patient receiving AI, testosterone transformation into 17β-estradiol is theoretically blocked, but it cannot be excluded that testosterone could compete with binding to the enzyme and, having a better affinity for it, it could be transformed anyway. For these reasons all the molecules which increase testosterone concentration (spironolactone and finasteride) should be avoided in the case of HT with LHRH analogs. In the case of HT with AI or SERMs, they could be used in reserve, even if the authors discourage their use until their safety is demonstrated. Non-hormonal agents If it is accepted that hormonal agents are best not employed, other molecules can be considered. Topical minoxidil 2% and 5% is commonly utilized. It induces vasodilatation by opening potassium channels localized on smooth muscle cells of peripheral arteria, slowing down circulation. Minoxidil, in addition, induces angiogenesis and activates prostaglandin-endoperoxides synthase 1 which stimulates hair growth [18]. The final minoxidil effect is to extend the anagen phase. The association with topical hydrocortisone could help follicular growth. Freites-Martinez et al. [11] reported a moderate to significant improvement in alopecia in 80% of patients using topical minoxidil. Bimatoprost 0.03%, a prostaglandin analog, could also be utilized [19]. It protects follicles in the anagen phase and improves follicular growth in anagen I. For these reasons, it should be utilized with the same indications of minoxidil. In addition, the costs and the availability of the molecule should be considered. Minoxidil and prostaglandin analogs are not recommended in preventing alopecia induced by cytotoxic agents [20], but in the case of HT, they could be useful. Probably because, differently from classic chemotherapeutic drugs, HT does not have a direct toxic effect on hair follicles, but it creates an unfavorable condition for hair growth. Moreover, even if minoxidil and prostaglandin analog increases drug concentration around the hair follicle, it is possible that beyond a certain concentration, HT molecules are not able to bind their receptor sites over more, because receptor saturation would be reached. Lastly, another promising molecule is cetirizine. It is an antihistamine anti-H1 receptor, which is able to increase prostaglandin prostaglandine E (PGE) and prostaglandine F2alpha (PGFα) synthesis, with an action similar to bimatoprost, and to reduce inflammation and prostaglandin PGD2 synthesis. This action seems to be independent of its antihistamine one. Available studies evaluated cetirizine efficacy for the treatment of AGA and other types of alopecia with good results. Recently, it has also been proposed for the treatment of alopecia induced by palbociclib [21]. For this reason and the absence of hormonal influence, cetirizine could be a good option for alopecia induced by HT [22]. Our proposal In light of what is described above, we propose the use of topical formulations containing minoxidil 2%, cetirizine 0.5%, and hydrocortisone butyrate in alcohol, applied once a day alongside HT duration or minoxidil 5% + hydrocortisone butyrate 0.1% in the most severe cases and/or when a previous FAGA was present. Conclusions Pseudo-FAGA induced by HT for breast cancer is probably underreported if we consider the prevalence of this tumor and the common use of these therapies. Maybe because oncologists are not interested in the diagnosis of alopecia, and patients do not refer to dermatologists or they refer only if they had a previous FAGA that worsen after HT. Anyway, even if this pattern of alopecia is similar to FAGA, its pathogenetic mechanism and management are different, so it has to be considered an oncotherapy-induced alopecia. For this reason, and for its direct or indirect oncogenic potential, topical hormones should not be considered for the treatment of alopecia induced by HT. With this work, we propose a therapeutic approach that could be both safe and effective in order to reduce hair miniaturization and stimulate hair growth even when a previous FAGA was present.	2021-11-03T15:15:38.455Z	2021-10-31T00:00:00.000	{ "year": 2021, "sha1": "88cef9d84926776dac78b33dac1359316ad556c2", "oa_license": "CCBY", "oa_url": "https://www.explorationpub.com/uploads/Article/A100259/100259.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "08a8d7b5e9eb8625dcf4e1a64baba78324d94e59", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
52301499	pes2o/s2orc	v3-fos-license	Occurrence of Highly Conjugative IncX3 Epidemic Plasmid Carrying blaNDM in Enterobacteriaceae Isolates in Geographically Widespread Areas The emergence of New Delhi metallo-β-lactamase (NDM) in common enterobacterial species is a major concern for healthcare. Early reports have revealed that the spread of NDM involved diverse and heterogeneous plasmids. Recently, the involvement of a rare, IncX3 subtype plasmid has been increasingly recognized. Here, we studied the prevalence of IncX plasmid subtypes in 198 carbapenem-resistant Enterobacteriaceae, originating from a territory-wide active surveillance in Hong Kong in 2016. The complete sequences and biological features of the blaNDM-carrying plasmids were investigated. A total of 62 NDM-type, 21 OXA-48 type, 14 IMP-type, 8 KPC-type, 4 IMI-type producers, and 89 non-carbapenemase-producers were tested for presence of IncX subtypes. IncX3 (n = 60) was the most common subtype, followed by IncX4 (n = 6) and IncX1 (n = 2). The prevalence of IncX3 subtype in isolates producing NDM, other carbapenemase types and non-carbapenemase producers were 75.8, 21.3, and 3.4%, respectively (P < 0.001). An IncX3 plasmid (size ∼50 kb) was confirmed to carry blaNDM in 47 isolates of different enterobacterial species. Thirteen IncX3 plasmids originating from six healthcare regions in Hong Kong were completely sequenced. The results showed that the IncX3 plasmids carrying blaNDM share a high degree of sequence identity with a previously reported plasmid, pNDM-HN380 (GenBank accession JX104760), over the backbone and genetic load regions. A blast search further revealed the occurrence of identical or nearly identical IncX3 plasmids carrying blaNDM in other part of China, Korea, Myanmar, India, Oman, Kuwait, Italy, and Canada. Two IncX3 carrying blaNDM were investigated further. Conjugation experiments demonstrated that the IncX3 plasmids could be efficiently transferred to multiple enterobacterial species at frequencies that are comparable or higher than the epidemic IncFII plasmid carrying blaCTX-M (pHK01). In addition, efficient transfer of the NDM plasmids occurred over a range of temperatures. In conclusion, this study demonstrated the important role played by IncX3 in the dissemination of NDM and the occurrence of pNDM-HN380-like plasmids in geographically widespread areas. The high mobility of IncX3 plasmid across different enterobacterial species highlights the ability of this plasmid replicon to be an important vehicle in worldwide dissemination of NDM. The emergence of New Delhi metallo-β-lactamase (NDM) in common enterobacterial species is a major concern for healthcare. Early reports have revealed that the spread of NDM involved diverse and heterogeneous plasmids. Recently, the involvement of a rare, IncX3 subtype plasmid has been increasingly recognized. Here, we studied the prevalence of IncX plasmid subtypes in 198 carbapenem-resistant Enterobacteriaceae, originating from a territory-wide active surveillance in Hong Kong in 2016. The complete sequences and biological features of the bla NDM -carrying plasmids were investigated. A total of 62 NDM-type, 21 OXA-48 type, 14 IMP-type, 8 KPC-type, 4 IMI-type producers, and 89 non-carbapenemase-producers were tested for presence of IncX subtypes. IncX3 (n = 60) was the most common subtype, followed by IncX4 (n = 6) and IncX1 (n = 2). The prevalence of IncX3 subtype in isolates producing NDM, other carbapenemase types and non-carbapenemase producers were 75.8, 21.3, and 3.4%, respectively (P < 0.001). An IncX3 plasmid (size ∼50 kb) was confirmed to carry bla NDM in 47 isolates of different enterobacterial species. Thirteen IncX3 plasmids originating from six healthcare regions in Hong Kong were completely sequenced. The results showed that the IncX3 plasmids carrying bla NDM share a high degree of sequence identity with a previously reported plasmid, pNDM-HN380 (GenBank accession JX104760), over the backbone and genetic load regions. A blast search further revealed the occurrence of identical or nearly identical IncX3 plasmids carrying bla NDM in other part of China, Korea, Myanmar, India, Oman, Kuwait, Italy, and Canada. Two IncX3 carrying bla NDM were investigated further. Conjugation experiments demonstrated that the IncX3 plasmids could be efficiently transferred to multiple enterobacterial species at frequencies that are comparable or higher than the epidemic IncFII plasmid carrying bla CTX−M (pHK01). In addition, efficient transfer of the NDM INTRODUCTION The worldwide dissemination of bacteria producing New Delhi metallo-β-lactamase (NDM) is a major public health concern. NDM was first described for Klebsiella pneumoniae from a patient who was previously admitted to a hospital in India in 2009 (Yong et al., 2009). Since then, NDM producing bacteria have been identified in >40 countries (Khan et al., 2017). Many reports have described patients who have visited NDM endemic areas, such as the Indian subcontinent and Balkan states, and then returning home with NDM-producing bacteria causing colonization or infection, and subsequent local spread (Nordmann et al., 2011). Molecular investigations have revealed that the spread of NDM involved complex pathways and a high level of inter-genus, -species, and -lineages gene transfer (Johnson and Woodford, 2013;Khan et al., 2017). Early reports further highlighted the involvement of diverse and heterogeneous NDMcarrying plasmids (Nordmann et al., 2011). However, it is possible that the epidemiology of NDM may change, as the gene may be carried by high risk international bacterial clones and epidemic resistance plasmids (Johnson and Woodford, 2013). In China, reports of NDM-producing bacteria are on the rise, especially among Enterobacteriaceae from hospitalized patients (Yang et al., 2014;Zhang et al., 2016;Dong et al., 2017). In 2012, our group reported the involvement of IncX3 plasmids (represented by pNDM-HN380) in the dissemination of bla NDM−1 in multiple geographic areas in China (Ho et al., 2012b). Subsequently, pNDM-HN380-like plasmids carrying bla NDM−1 or variants have also been described in India, the Arabian Peninsula, Europe, and Australia (Wailan et al., 2015;Petrosillo et al., 2016;Riazzo et al., 2017;Choudhury et al., 2018;Ho et al., 2018a). In a recent survey of more than a thousand carbapenem-resistant Enterobacteriaceae originating from hospitals located in 25 provinces and municipalities in China, the prevalence of bla NDM was found to be 31% . Of note, bla NDM was found to be harbored on IncX3 plasmids in a large majority (92%) of the Escherichia coli and K. pneumoniae isolates . In South Korea, among 146 NDM-producing Enterobacteriaceae recovered from 33 general hospitals in 2010-2015, IncX3 was the predominant plasmid type (77%) harboring bla NDM (Yoon et al., 2018). The IncX plasmids are narrow host range plasmids of Enterobacteriaceae (Johnson et al., 2012). These plasmids have been identified mainly from Salmonella and E. coli from diverse sources at low frequencies of 8.7 to 10.6% (Lo et al., 2014;Dobiasova and Dolejska, 2016). In general, subtypes IncX1, IncX2, and IncX4 have been detected more commonly than IncX3 subtype (Dobiasova and Dolejska, 2016). In this investigation, we studied the prevalence of IncX3 and other subtypes in a collection of carbapenem-resistant Enterobacteriaceae (CRE) originating from an active surveillance in Hong Kong. The biological features of the bla NDM -carrying IncX3 plasmids were investigated. Bacterial Isolates and Identification A total of 198 CRE isolates, collected from 14 hospitals in Hong Kong in 2016 were investigated. The isolates were collected from a CRE surveillance program involving all the healthcare regions (Ho et al., 2011(Ho et al., , 2016. This comprised 109 carbapenemase-producing Enterobacteriaceae (CPE) and 89 carbapenemase-negative Enterobacteriaceae (CNE). The isolates (∼25%) were randomly selected from a total collection of 497 CPE and 331 CNE isolates in that year. Only one isolate from each patient was included. The species distribution for the final collection of CPE and CNE strains were, respectively, as follows: 49 and 30 E. coli, 32 and 38 K. pneumoniae, 11 and 8 Enterobacter cloacae, 6 and 3 Citrobacter freundii, 4 and 6 Enterobacter aerogenes, 4 and 1 Klebsiella oxytoca, and 3 and 3 other Enterobacteriaceae. The carbapenemase types in the isolates included NDM (n = 62), OXA-48 group (n = 21), IMP (n = 14), KPC (n = 8), and IMI (n = 4). The CPE isolates included 19 (17.4%) isolates from clinical specimens (including 3 blood cultures, 9 urines, 5 wounds, 1 bile fluid and 1 tracheal aspirate) and 90 (82.6%) isolates from rectal swabs. All except one CNE were recovered from rectal swabs. The isolates were identified to species level by MALDI-TOF MS (Chen et al., 2013) and were stored in MicroBank (Pro-Lab Diagnostics) at −80 • C. All the isolates were retrospectively retrieved for this work. Susceptibility Testing Disk diffusion method was used to determine the susceptibility of the isolates to ertapenem, imipenem, and meropenem (5). All the isolates were resistant to at least one of the carbapenem. Phenotypic expression of carbapenemase in the isolates was confirmed with a strip Carba NP test (Ho et al., 2018b). For the subset of NDM-producing isolates carrying IncX3 plasmids, MICs of the carbapenems and a panel of drugs (amikacin, colistin, gentamicin, levofloxacin, minocycline, and tigecycline) with potential activities were determined by a broth microdilution procedure using Sensititre plates (Thermo Fisher Scientific, West Sussex, United Kingdom). Cation-adjusted a No X2 and X5 replicons were detected. b X3 replicon was detected in isolates producing OXA-181 (n = 5), OXA-232 (n = 4), and KPC-2 (n = 1). One of the OXA-181 producers was also positive for the X4 subtypes. Mueller-Hinton broth was used according to the manufacturer's recommendation. The plates were inoculated and the endpoints read after incubation in ambient air at 35 • C for 18 h using a Thermo Fisher Scientific TM Sensitre TM ARIS TM 2X instrument. Quality control was performed with Pseudomonas aeruginosa ATCC 27853 and E. coli ATCC 25922. EUCAST breakpoints were used for interpretation of colistin and tigecycline MIC (EUCAST, 2018). MICs for the other drugs were interpreted according to the CLSI (CLSI, 2018). Molecular Studies PCR assays were used to detect carbapenemase genes (bla KPC , bla IMI , bla IMP , bla NDM , bla VIM , and bla OXA−48 ). The allele of the carbapenemase genes were determined by Sanger sequencing (Ho et al., 2016;Kong et al., 2018). The isolates were surveyed for presence of the IncX1 to IncX5 subtypes plasmid replicons using previously described PCR procedures (Lo et al., 2014). The co-transfer of bla NDM and the IncX3 plasmid was investigated by filter mating using J53 as the recipient. Transconjugants were selected on Luria-Bertani (LB) agar plates supplemented with sodium azide (100 µg ml −1 ) and meropenem (0.25 µg ml −1 ). Plasmid location of the bla NDM and IncX3 replicon were confirmed by hybridization using specific PCR products as probes . PCR and Sanger sequencing was used to determine the multilocus sequence types (MLST) of K. pneumoniae and E. coli using the Pasteur Institute and the Warwick scheme, respectively (Diancourt et al., 2005;Wirth et al., 2006). Plasmid Sequencing Thirteen isolates with the bla NDM genes carried on IncX3 plasmids were sequenced. The isolates (one from blood culture, one from bile fluid and 11 from rectal swabs) were chosen to provide representation from different hospitals in the collection. The IncX3 plasmids carrying bla NDM in the original isolates (n = 13) were sequenced by an Illumina HiSeq platform at ∼100fold coverage. The plasmids were assembled de novo using a CLC Genomics Workbench (Qiagen, Redwood City, United States) and gaps were closed by additional PCRs and Sanger sequencing Ho et al., 2018a). ISfinder 1 was used to identify and annotate insertion sequences. (Ho et al., 2012c;Jiang et al., 2017). The frequencies of conjugational transfer of the plasmids to a panel of 11 isolates comprising P. aeruginosa (n = 3), Acinetobacter baumannii (n = 2), E. coli (n = 3, including J53 and two clinical isolates of ST131 and ST405), Shigella flexneri (n = 1), Salmonella enteritidis (n = 1), and K. pneumoniae (n = 1) were investigated. The two clinical E. coli isolates were chosen to represent two widespread clones of multilocus sequence types (ST) 131 and 405. In addition, the frequencies of plasmid transfer to J53 recipient was assessed at three different temperatures (30, 37, and 42 • C). All conjugation, unless otherwise specified, were performed at 37 • C at donor to recipient ratio of 1 to 2. Transconjugants were selected on either MacConkey, XLD, or UriSelect TM 4 (Bio-Rad, CA, United States) agar plates containing supplemented sodium azide (100 µg ml −1 ), meropenem (0.25 µg ml −1 ) and/or cefotaxime (1 µg ml −1 ) as appropriate (Supplementary Table S1). The frequency of transfer was expressed as transconjugants per donor cell (T/D) as previously described (Zhong et al., 2012). Donor and recipient cultures placed separately on filters were included as controls in each run and no growth was observed on the transconjugants selection media. All experiments were carried out four times (biological duplicates and technical duplicates) and mean values presented with standard deviations. Statistical Analysis Proportions were compared using Chi-Square or Fisher exact tests. Conjugation frequencies were logarithmically transformed and compared using one way ANOVA. All analyses were performed using IBM SPSS Statistics, version 24 (Hong Kong). A two-tailed P-value of <0.05 was considered as significant. Characteristics of the NDM-Producing Isolates Carrying IncX3 Plasmids The 47 NDM-producing isolates carrying IncX3 plasmids were investigated further. The species of the isolates include 27 E. coli, 11 K. pneumoniae, 3 E. cloacae, 3 E. aerogenes, 2 C. freundii and 1 K. oxytoca. PCR and sequencing showed that the isolates had bla NDM−1 (n = 11), bla NDM−5 (n = 35), and bla NDM−7 (n = 1). In all the isolates, hybridization localized the bla NDM gene to the IncX3 plasmids. Table S2). This subset of isolates was randomly chosen from among the 47 NDM-producing isolates with IncX3 plasmids, including isolates from hospitals in six different healthcare regions. Twelve plasmids carried bla NDM−5 and one plasmid carried bla NDM−1 . They have a plasmid scaffold typical of IncX3 plasmids. The genetic load regions in the plasmids were compared with two reference IncX3 plasmids (pIncX-SHV and pNDM-HN380) ( Figure 1A). Features shared among the bla NDMcarrying plasmids include: (a) an ISL3 with 8-bp flanking direct repeats (ATATGCAT) downstream of the resolvase gene; and (b) the umuD gene was split into two fragments (umuD 1 and umuD 2) at the same position resulting in a pair of 3-bp direct repeats (TGT). In the plasmids, bla NDM was inserted as a putative IS26-ISAba125 transposon. The genetic load region in one plasmid carrying bla NDM−1 (pNDM-HK3694) was virtually identical to that in pNDM-HN380, except for a small deletion downstream of the IS3000. In ten bla NDM−5 -carrying plasmids originating from different species (5 E. coli, 4 K. pneumoniae, 1 E. cloacae) and six hospitals, the sequences inserted between the two umuD fragments were 100% identical (10117 bp in length). This inserted sequence differs from that in pNDM-HN380 by a deletion of 7874 bp (Figure 1A). The remaining two plasmids exhibited one or two minor variations including an IS1203 inserted within IS3000 (pNDM-HK2967) or an additional deletion (616 bp) at the junction between the IS5 and ISAba125 1 remnant (pNDM-HK3774). In all the 12 NDM-5 plasmids, IS5 was inserted at the same position leading to the flanking 4-bp direct repeats (CTAA). In the two NDM-1 plasmids (pNDM-HK3694 and pNDM-HN380), IS5 was inserted at a different position in the opposite orientation. Next, the complete sequence of pNDM-HK2998 (chosen as a representative) was used to query the GenBank database. FIGURE 2 \| Conjugation frequencies of IncX3 and IncFII plasmids to six recipients of different species. The donor E. coli strains for the IncX3 plasmids (pNDM-HK1493 and pNDM-HK396) were CRE1493 and CRE396, respectively, while for the IncFII (pHK01) plasmid was combat2D2. The histograms show means and standard deviations (error bars). Conjugation frequencies were logarithmically transformed prior to statistical analysis. This allowed identification of another 34 plasmids related to pNDM-HK2998 (Figure 1B), including 30 from Asia (China, Korea, Myanmar, India), two from Middle East countries (Oman, Kuwait), and one each from Italy and Canada. This comprised plasmids originating from human (n = 26) and food animals (n = 8). Multiple NDM variants (4,5,7,and 17) which differ from NDM-1 by one or more amino acids (V88L, D130N, M154L, and E170K) were carried by the plasmids. Alignment and phylogenetic analysis showed that the plasmids were highly similar in both the backbone and genetic load regions ( Figure 1B). Host Range and Conjugation Frequencies In conjugation experiments, the IncX3 and IncFII plasmids could be successfully transferred from the original clinical strains to the E. coli, S. flexneri, S. enteritidis, and K. pneumoniae recipients (Figure 2). The highest frequencies of conjugative transfer of the IncX3 plasmids were observed for the S. flexneri and S. enteritidis recipients (≥10 −1 per donor cell), following by the K. pneumoniae recipient (10 −2 to 10 −3 per donor cell). These conjugative frequencies were comparable or higher than those for the reference IncFII plasmid. Conjugation frequencies of the IncX3 plasmids in the E. coli recipients were variable; higher in the J53 recipient than in the two recipients of ST405 and ST131 lineages. In the latter two recipients, conjugation frequencies of the two IncX3 plasmids were lower than those for the reference IncFII plasmid. Despite repeated attempts, conjugative transfer of the IncX3 plasmids to P. aeruginosa and A. baumannii recipients was not successful. Incubation temperatures had different effects on the transfer frequencies of the IncX3 and IncFII plasmids (Figure 3). At 30 and 37 • C, conjugation frequencies of the IncX3 plasmids were comparable or higher (by 10 1 -10 3 -fold) than those for the reference IncFII plasmid. Incubation at 42 • C inhibited the conjugative transfer of the IncX3 plasmids but not the IncFII plasmid. DISCUSSION This study demonstrated the importance of pNDM-HN380-like, IncX3 plasmids in the dissemination of bla NDM among bacterial isolates of multiple Enterobacteriaceae species in Hong Kong. These plasmids can be considered epidemic because they have previously been detected in multiple countries and among bacteria of human and animal host sources (Ho et al., 2012b(Ho et al., , 2018aWailan et al., 2015;Petrosillo et al., 2016;Choudhury et al., 2018). Our isolates carrying IncX3 plasmids were mostly recovered from patients who have history of previous hospitalization in a healthcare institute either locally or in mainland China. In the collection, approximately three-quarters of them were rectal swab isolates which were detected through active surveillance culture upon hospital admission (Ho et al., 2011). It is likely that hospitals are the main reservoirs of these bla NDM carrying plasmids. Additional reservoirs or sources of this resistance mechanism include commensals carried by food FIGURE 3 \| Conjugation frequencies of IncX3 and IncFII plasmids at three temperatures. The donor E. coli strains for the IncX3 plasmids (pNDM-HK1493 and pNDM-HK396) were CRE1493 and CRE396, respectively, while for the IncFII plasmid (pHK01) was combat2D2. The histograms show means and standard deviations (error bars). Conjugation frequencies were logarithmically transformed prior to statistical analysis. animals or household contacts of the patients (Ho et al., 2012a(Ho et al., , 2018a. In China, similar IncX3 plasmids carrying bla NDM have been detected in E. coli from swine in Guangdong, Henan, and Hunan provinces and from a chicken in Shandong Province Ho et al., 2018a). A high degree of synteny was demonstrated among the complete plasmid sequences in the present analysis. With the exception of a few insertions and deletions, insertion sequence elements bearing identical direct repeats and other genes within the genetic load regions harboring bla NDM were shared between IncX3 plasmids from geographically widespread regions and host sources (Figure 1). The finding points to a single bla NDM insertion into a common IncX3 plasmid ancestor (pNDM-HN380-like), rather than multiple, independent bla NDM insertions in the IncX3 platform. Variants of bla NDM−1 then arise through single nucleotide substitution in the bla NDM coding region. The NDM-1 variants that were identified in IncX3 plasmids including NDM-4, NDM-5, NDM-7, and NDM-17 have enhanced carbapenemase activity, leading to increased isolate resistance to carbapenem (Makena et al., 2015;Liu et al., 2017). We showed that the IncX3 plasmids carrying bla NDM could be transferred to different bacterial species at frequencies that are comparable or higher than the epidemic IncFII plasmid carrying bla CTX−M . The efficient transfer of bla NDM plasmids to two clinical isolates of S. flexneri and S. enteritidis is concerning as this will render all the beta-lactam antibiotics ineffective. The conjugal transfer of the IncX3 plasmids carrying bla NDM to recipient E. coli over a range of temperature is also of note. The finding has implications for the dissemination of bla NDM in many environmental sources such as abiotic touch surface, sewage, waterway, and soil in tropical countries as well as in the guts of mammalian and avian species Warnes et al., 2012). In many Asian and Middle East countries, the ambient temperature in the summer is often above 30 • C. Hence, this plasmid type may be more effective than the other plasmid types in the environmental dissemination of bla NDM . Interestingly, the conjugal transfer of a related subtype, IncX4 at 30 • C to recipient E. coli is also higher than IncFII, suggesting that this may be a property shared by all the subtypes of IncX plasmid (Lo et al., 2014). CONCLUSION This study identified a highly conjugative, pNDM-HN380-like IncX3 plasmid to be a major vehicle for dissemination of bla NDM among multiple enterobacterial species in Hong Kong hospitals. Analysis of complete plasmid sequences confirms that this epidemic NDM plasmid is widespread in the ecosystem. AUTHOR CONTRIBUTIONS P-LH and YW conceived and designed the experiments and wrote the paper. P-LH, VC, CT, AW, RL, W-KL, and DT collected the bacteria and related the data. YW, M-KT, and K-HC performed the experiments. P-LH, K-HC, and YW analyzed the data. All authors provide critical input to the manuscript and endorsed the final version. FUNDING This work was supported by grants from the Health and Medical Research Fund (HKM-15-M10 and CHP-PH-13) of the Food and Health Bureau of the Hong Kong Special Administrative Region Government (HKSAR), and the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the HKSAR Department of Health. The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.	2018-09-20T13:03:42.314Z	2018-09-20T00:00:00.000	{ "year": 2018, "sha1": "8814f3cf180966843bd395912c4f0721e12f0f0d", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmicb.2018.02272/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "8814f3cf180966843bd395912c4f0721e12f0f0d", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
10487600	pes2o/s2orc	v3-fos-license	Intra-articular injection of photo-activated platelet-rich plasma in patients with knee osteoarthritis: a double-blind, randomized controlled pilot study Background Improvements in knee osteoarthritis (OA) symptoms with platelet-rich plasma (PRP) have been attributed to its ability to modify intra-articular inflammatory processes. Photo-activation of peripheral blood also improves inflammatory mediators associated with OA, however combined photo-activated PRP (PA-PRP) has not been investigated. This pilot study assessed the feasibility, safety and symptomatic and functional change following injections of PA-PRP compared to hyaluronic acid (HA) in people with knee osteoarthritis (OA). Methods Thirty seven people with knee OA were enrolled in this double-blind randomized controlled pilot study set in a sports medicine clinic. Participants were randomly allocated to receive three injections of either PA-PRP or HA. The patients and the administering doctor were blinded to group allocation. Outcomes included recruitment and safety data, 100 mm visual analogue pain score (VAS), the Knee Osteoarthritis Outcome Score (KOOS), Knee Quality of Life (KQoL) scale, maximum hopping distance and number of knee bends in 30 s at four and 12 weeks. Results Twenty three (62 %) participants met the inclusion criteria, of which 12 (32 %) were randomized to the PA-PRP group and 11 (30 %) to the HA group. Two participants did not complete the intervention and two withdrew following their first assessment. Minor pain and swelling during the injection period was reported by two participants from the PA-PRP group. The PA-PRP group demonstrated significant improvements in the VAS (p < 0.01, ETA = 0.686), KOOS Pain (p < 0.05, ETA = 0.624), KQoL Physical (p < 0.05, ETA = 0.706) and KQoL Emotional subscales (p < 0.05, ETA = 0.715) at four and 12 weeks. The PA-PRP group also significantly improved hoping (p < 0.05, ETA = 0.799) and knee bends (p < 0.01, ETA = 0.756) at four or 12 weeks. The HA group showed improvements on only the KOOS Function subscale at 12 weeks (p < 0.01, ETA = 0.602). After controlling for baseline values, there were no significant between-group differences at either time-point. Conclusions This study provides proof-of-concept evidence concerning the feasibility and safety of PA-PRP injections necessary to inform a larger clinical trial in people with knee OA. Our preliminary results also suggest PA-PRP improves self-reported pain, symptoms and lower extremity function, however no between-group differences were found. Photo-activated PRP may provide a safe and effective novel treatment for knee OA. Trial registration ACTRN12611000651987 Background Osteoarthritis is a leading cause of musculoskeletal pain worldwide and the knee is one of the most commonly affected joints. Prevalence of knee OA is expected to increase with an aging population and growing rates of obesity, and projections of total knee replacements are predicted to increase by approximately 600 % over the next 25 years [1]. As there is currently no cure for OA, treatment has focused on symptomatic relief with the aim of reducing pain and disability and maintaining or improving joint mobility [2]. Non-surgical treatments including exercise and weight loss are recommended due to poor symptomatic and functional outcomes with surgical management [3]. However compliance with non-surgical treatments is poor [4], whilst drug treatments such as simple analgesics and non-steroidal antiinflammatory drugs are associated with adverse events [2,5,6]. The addition of intra-articular injections with hyaluronic acid (HA) products (viscosupplementation) has also been recommended in patients unresponsive to nonpharmacological or analgesic regimes [6], although this treatment is also uncertain as efficacy is variable and ongoing treatment is required [7][8][9]. Given the progressive nature of knee OA, and the serious limitations associated with existing therapies, studies in to effective treatments with potential disease-modifying effects are needed. Recent research suggests that growth factors and other cytokines released by platelets in response to injury or pathology may modulate inflammatory processes and contribute to the maintenance or regeneration of tissue structures [10,11]. Consequently, platelet-rich plasma (PRP) injections have become an emerging treatment for soft tissue healing associated with tendon and ligament injury, bone mineralisation and cartilage regeneration [12][13][14]. Upon application to the affected site, activated platelets release growth factors and other bioactive molecules, and coagulation occurs to form a matrix that promotes migration of additional cells to the area. Combined, these factors may promote tissue healing and modulate the aberrant inflammatory processes implicated in the pathophysiology of OA [10][11][12]. Recent unblinded and non-randomized pilot and prospective studies investigating the clinical efficacy of intra-articular injections of PRP in patients with knee OA have demonstrated clinical improvement in selfreported pain and symptoms with no major adverse events [10,[15][16][17]. Furthermore, a recent systematic review found six randomized controlled trials reporting clinical benefits of PRP in patients with knee OA [18], however, only two were double-blinded with a matched control procedure [19,20], and neither of these evaluated the effects of PRP on objective measures of lower extremity function. This limits the ability to determine whether symptomatic benefits translate to improved mobility, which is critical given 80 % of people with OA have movement limitations and 25 % cannot perform daily activities [21]. The use of low-level light irradiation to activate peripheral blood (photo-activation) has also been shown to improve biological factors associated with osteoarthritis [22,23]. Studies have reported that photo-activation decreases proinflammatory cytokines (interleukin 2 and 6) and increases the concentration of leucocyte-derived antiinflammatory factors (interleukin 1 receptor antagonist) [22,23]. As such, this activation technique could be beneficial in PRP preparations higher in leukocyte concentration. To date, only two case studies have investigated combined photo-activation and PRP (PA-PRP) in degenerative conditions, reporting symptomatic improvements in one patient with a chondral defect [24] and another with knee OA [25]. There are currently no clinical trials of PA-PRP in knee osteoarthritis. Therefore, the aim of this double-blinded, randomized, controlled pilot study was to determine the feasibility, safety and changes in pain, symptoms and lower limb functional ability following intra-articular injections of PA-PRP compared to HA in patients with mild to moderate knee OA. Trial design This was a single centre, double-blind, randomized controlled pilot study comparing PA-PRP to HA. We chose HA as the active comparator as it is one of the most commonly used injective treatments for knee OA [26], and recent network meta-analyses have demonstrated clinical benefits above intra-articular placebo injections [27,28]. Participants were recruited from the community using online advertising and through existing databases between June and August, 2011. An information letter outlining potential risks and benefits was provided, and participants were fully informed about the testing protocol and procedures. The study was approved by the Australian Catholic University Human Research Ethics Committee, and all participants gave written informed consent prior to the commencement of testing. Participants Based on recommendations for determining sample sizes for pilot studies [29,30], we enrolled 37 people with knee OA to participate in the study (Fig. 1). To be eligible, participants were required to have a diagnosis of knee OA based upon the American College of Rheumatology knee OA clinical classification criteria [31], radiographic evidence of Kellgren-Lawrence grade 2 or 3 knee OA [32] and be willing to discontinue analgesics and anti-inflammatory medications (except Paracetamol) for at least two weeks prior to commencing the intervention and for the duration of the study. Only people with mild to moderate radiographic OA disease were chosen based on recommendations from a recent systematic review of PRP in people with degenerative knee pathology [33]. Exclusion criteria were systemic or inflammatory joint disease, history of crystalline or neuropathic arthropathy, cancer or other tumour-like lesions, immunosuppression or acute infective processes, pregnancy or lactation, other intra-articular lesions or treatments in the previous six months or allergy to any test substance. Procedures Group allocation and concealment was performed by an independent staff member not involved with the assessment of participants. Participants were randomly allocated following baseline data collection on a 1:1 basis using a computer generated randomization list. Both groups completed the PA-PRP collection and activation phase of the intervention as explained below, and the syringe was then provided to the independent staff member. The staff member either retained or discarded the syringe containing the PA-PRP depending on group allocation. The syringe containing the PA-PRP, or an identical looking syringe containing HA (Synvisc® Hylan G-F 20, Genzyme Biosurgery, Ridgefield, NJ, USA), was then occluded and returned to the treating doctor for administration. This process ensured the blinding of both the patient and treating doctor. All injections were performed at weekly intervals. To obtain the PRP, 48.5 ml of the patient's blood was collected using venipuncture, then centrifuged (Premiere XC-2000) at 2,000 rpm for five minutes. The plasma and buffy coat containing platelets was drawn from the top of the sample and placed in a sterile tube and again at 3,000 rpm for three minutes. This double-spin approach has been shown to produce PRP that is higher in leukocytes [34], which was preferred for this study as photo-activation is thought to act at least in part by influencing the pro-and anti-inflammatory properties of leukocytes [22,23]. Three quarters of the plasma was removed, 0.2 ml of sodium bicarbonate 8.4 % was added to the tube and the platelet pellet was reconstituted using the remaining plasma in a 3 ml syringe. To activate the PRP, the syringe containing the PRP then underwent low-level ultraviolet light irradiation (Adilight-1, Adistem Ltd.) for five minutes consistent with published protocols [23]. Participants were placed in a supine position and sterile drapes were placed around the surrounding area. Next, the participant's symptomatic knee was cleaned with chlorhexadine and iodine solution and the knee was anaesthetised using an intra-articular injection of 5 ml of 1 % Xylocaine. Following activation, 3 ml of PA-PRP or HA was injected under ultrasound guidance (Logic i, GE Healthcare) into the symptomatic region using an anteromedial approach. Following the injection, passive flexion and extension of the knee was performed 10 times, after which the participant remained resting in the supine position for approximately 10 min. Participants were advised to take paracetamol if they experienced any pain, and to limit their weight bearing activities for the subsequent 24 h, followed by gradual resumption of normal activities. Outcomes Prior to treatment, baseline demographic data was collected. Feasibility was recorded using recruitment and retention rates, and safety was assessed by recording the number and nature of adverse events. Adverse events were recorded weekly for the first month using participant phone calls, and at one and three month during the follow up assessment visits. To evaluate symptom severity, participants first completed a 100 mm Visual Analogue Scale (VAS) to rate their average knee pain over the previous week, with terminal descriptors of "no pain" and "worst pain possible" [35]. To document self-reported symptoms, participants also completed the Knee Injury and Osteoarthritis Outcome Score (KOOS) and the Knee Quality of Life 26-item questionnaire (KQoL-26). The KOOS is an OA disease-specific instrument for the assessment of patient-relevant treatment effects. The KOOS uses a five point Likert scale for scoring the three Western Ontario and McMaster Universities Osteoarthritis (WOMAC) sub-scales of Pain, Stiffness and Physical Function, with the additional inclusion of the sub-scales Quality of Life and Sport and Recreation [36]. The KQoL-26 is a self-reported quality of life instrument developed to assess the severity of knee symptoms and activity and work limitations in patients with knee injuries. It is comprised of three sub-scales, including Physical Functioning, Activity Limitations and Emotional Functioning, and uses a five point Likert scale for scoring [37]. Finally, recent research suggests that validated selfreport questionnaires and objective functional tests should be employed in combination to fully assess mobility-related outcomes in people with knee OA [38]. Consequently, two objective measures of lower extremity functional ability (maximum single leg hop and number of knee bends in 30 s) were also completed by participants as described previously [38,39]. The maximum single leg hop for distance required a hop to be performed from a starting position of balancing on one leg, and finishing position of landing and balancing on the same leg to a stationary stance position. The maximum number of knee bends performed in 30 s required the participant to balance on one leg and perform as many shallow knee bends (until they couldn't see their toes past the bent knee, or approximately 30 degrees) as they could in a 30 s period without touching the elevated foot on the ground. All surveys and functional tests were completed at baseline, four weeks and at a final follow-up at 12 weeks following the final treatment injection, as this timeframe has been shown to demonstrate symptomatic improvements in previous studies using other PRP preparation methods [19,20,40]. Statistical analysis The Statistical Package for Social Sciences (SPSS) software (Version 17) was used to analyse the data. Normality was assessed using skewness and kurtosis statistics and the Shapiro-Wilk test. To determine whether PA-PRP and HA had an effect on outcome variables, one way Repeated Measures Analysis of Variance (RM ANOVA) were conducted to compare baseline, four and 12 week follow-up scores for the VAS, KOOS, KQoL-26 and functional tests for each group. An analysis of covariance (ANCOVA) was used to assess between-group changes in the primary and secondary outcomes, with group allocation as the fixed factor and corresponding baseline outcome values as covariates. This technique has been reported to be the optimal method to analyse continuous data in clinical trials [41]. An alpha level of p < 0.05 was used to determine statistical significance. Results Twenty three (62 %) of the 37 enrolled people with knee OA (males = 16, females = 7, age = 51.20 ± 12.00 years, mass = 96.35 ± 18.14 kg, height = 178.00 ± 10.10 cm, BMI = 29.24 ± 9.52) met the inclusion criteria. Two participants did not complete the allocated intervention; one from the PA-PRP group who withdrew due to minor injection-related pain and swelling which resolved without further treatment, and one from the HA group who suffered an acute meniscal tear unrelated to the treatment. Two additional participants withdrew from the study due to other unrelated injuries after the four week follow up assessments. Consequently, data was available for 21 participants (PRP = 11 participants, HA = 10 participants) for the four week follow up and for 19 participants at 12 weeks (Fig. 1). Participant characteristics are displayed in Table 1. No differences were found between the two groups (p > 0.05). No treatment-related major adverse events were experienced by participants. Two participants from the PA-PRP group experienced minor pain and swelling during the injection period believed to be related to the injection technique. Both participants completed the injection course, and had resolution of symptoms by the following week. No further adverse events were experienced during the intervention or follow-up period. Average pain recorded using the 100 mm VAS reduced from baseline at the four and 12 week follow up time points for both the PA-PRP and HA groups ( Table 2). Although there was a slight increase in pain at the 12 weeks in the PA-PRP group, this still represented a 24 % improvement above baseline pain. Repeated Measures ANOVA revealed the reduction in pain was significant for the PA-PRP group only (p = 0.017). Post hoc tests showed that the reduction in pain at four weeks was significantly less than baseline (27.67 mm, 95 % CI 12.39 to 42.95, p = 0.003, ETA = 0.686), however the 11.4 mm reduction in VAS at 12 weeks was not statistically significant. No significant reductions in pain were found for the HA group. For the PA-PRP group, significant improvements were found at both follow up time points in the KOOS Pain subscale (4 weeks: 13 14.56, p = 0.002, ETA = 0.756) performance, whereas changes in physical function in the HA group were not found to be significant. To investigate betweengroup differences in mean change scores on the 100 mm VAS, KOOS, KQoL-26 and functional tests, ANCOVA was performed adjusting for baseline scores (Table 3). No significant between-group differences were found for any of the self-reported measures or for either of the functional tests (p > 0.05). Discussion This was a double-blind randomized controlled pilot study comparing intra-articular injections of a novel form of PRP activated with ultraviolet light compared to HA. The results demonstrate the feasibility of this technique in people with knee OA and show no serious adverse events were reported. There was a significant decrease in VAS pain for the PA-PRP group at four weeks that was greater than the minimal clinically important improvement level of 19.9 mm. The 24 % decrease below baseline pain levels at 12 weeks was not found to be statistically significant and no improvements in pain were found in the HA group. The PA-PRP group also demonstrated significant improvements in the KOOS Pain subscale at four and 12 weeks, and significant improvements in the KQoL-26 Physical and Emotional subscales. In contrast, the only significant improvement in the HA group was in the KOOS Functional subscale at 12 weeks. Finally, whilst the PA-PRP group significantly improved their performance on the functional measures of hopping and knee bend at 12 weeks, no significant improvements in lower extremity function were found in the HA group. Despite these improvements in the PA-PRP compared to the HA group, there were no significant between-group differences on any measures after adjusting for baseline variation. These preliminary results provide initial feasibility, safety and treatment data that may be used to inform a future large clinical trial to investigate whether this novel form of photo-activated PRP has symptomatic and functional benefits in people with knee OA. Of the 23 people who met the inclusion criteria, two participants did not complete the allocated intervention and two additional participants withdrew from the study following the four week follow-up appointment. These four patients were evenly spread between groups, and with the exception of one, were all due to new injuries unrelated to the treatment. One of the PA-PRP patients however did withdraw due to minor injection-related soreness, whilst another two also reported minor pain and swelling following an injection. In all instances, symptoms resolved within a week and no further adverse events were reported during the intervention or followup period. Previous RCTs have investigated the efficacy of PRP compared to HA or saline [19,20,34,40,42,43] however to our knowledge this is the first to objectively evaluate lower limb function and the only one to use photo-activation of PRP. Photo-activation of peripheral blood has been previously shown to improve the inflammatory cytokine profile of healthy adults [23] and people with psoriasis [22], and therefore has the potential to improve outcomes in OA where pro-inflammatory cytokines are critical mediators in the pathophysiology of the disease [44]. Combined with PRP, which releases growth factors and other proteins responsible for tissue repair and inflammatory modulation [45,46], this may offer a novel method for improving symptoms and function in people with knee OA. Our results provide mixed support for this new treatment approach. Although the PA-PRP group showed significantly greater improvement in Table 2 Mean (SD) for VAS, KOOS, KQoL and functional tests at baseline and four and 12 weeks following final injection PA-PRP HA Baseline (n = 11) 4 weeks (n = 11) 12 weeks (n = 10) Baseline (n = 10) 4 weeks (n = 10) 12 weeks (n = 9) self-reported symptoms and lower limb physical function compared to HA, between-group differences were not significant. It is possible that the small sample size of our pilot study may have reduced statistical power making definitive between-group conclusions difficult. The lack of between-group differences found in our pilot study may also be partly due to our PRP preparation method. We used a double spin technique which has previously been shown to produce PRP that is higher in leukocyte concentration when compared to PRP prepared using a single spin technique [34]. Indeed, our findings are consistent with two recent RCTs reporting comparable between-group improvements in selfreported pain and symptoms following injections of either PRP prepared using a leukocyte-rich technique or HA [19,26]. In contrast, two recent studies investigating the effects of PRP prepared using a leukocyte-poor technique, and a recent meta-analysis [47], have suggested this form of PRP produces improvements in self-reported symptoms above that of HA. Cerza and colleagues [40] showed significantly improved overall WOMAC score at 12 and 24 weeks in knee OA patients who had injections of PRP prepared using a leukocyte-poor technique compared to patients who received HA. Similarly, Sanchez and colleagues [43] reported that patients with knee OA who received leukocyte-poor PRP had significant benefits at 24 weeks compared to those who received HA. The findings of our study, and those from these previous clinical trials, suggests that leukocyte concentration of PRP may play a crucial role in clinical outcomes achieved for knee OA patients. However, future studies should explore the effects of photo-activation in both leukocyte-rich and leukocyte-poor PRP preparations given photo-activation is thought to act at least in part by modulating the pro-and anti-inflammatory properties of leukocytes [22,23]. There are some limitations that may have influenced the outcomes of our study. Firstly, as mentioned the small sample size of this pilot trial reduces statistical power which may make more definitive conclusions problematic, and the relatively short follow-up period of our study may have been too brief to determine betweengroup differences. A larger clinical trial is needed to confirm our results. Furthermore, we did not include a minimum VAS pain score as part of our screening criteria, resulting in greater variation in baseline pain and selfreported symptoms. This produced high baseline standard deviations which would have decreased the probability of finding differences between the PRP and HA groups, particularly with the small sample size. Finally, we did not examine how photo-activation modifies PRP. Given the benefits of photo-activation have been attributed to its influence upon the concentration of proinflammatory cytokines and leucocyte-derived antiinflammatory factors [22,23], and the potential role of leukocyte concentration in PRP, this warrant further investigation. Conclusions This randomized controlled pilot study provides feasibility and safety data for the use of PA-PRP in people with knee OA that may help to inform a larger clinical trial. Although under-powered to assess efficacy, our preliminary results also provide some evidence that PA-PRP improves self-reported pain, subscales of the KOOS and KQoL-26, and tests of lower extremity functional ability in knee OA patients. However these improvements were not found to be significantly greater than those of the HA group. Future clinical trials with larger sample sizes and longer follow up periods should investigate whether this novel photo-activated PRP method improves symptoms and function over that of HA in people with knee OA. Submit your next manuscript to BioMed Central and we will help you at every step:	2016-05-12T22:15:10.714Z	2016-02-09T00:00:00.000	{ "year": 2016, "sha1": "32d95194b60ac964f88c5de3fb283d2a441cf558", "oa_license": "CCBY", "oa_url": "https://bmcmusculoskeletdisord.biomedcentral.com/track/pdf/10.1186/s12891-016-0920-3", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "32d95194b60ac964f88c5de3fb283d2a441cf558", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
7880291	pes2o/s2orc	v3-fos-license	Analysis of Gene expression in soybean (Glycine max) roots in response to the root knot nematode Meloidogyne incognita using microarrays and KEGG pathways Background Root-knot nematodes are sedentary endoparasites that can infect more than 3000 plant species. Root-knot nematodes cause an estimated $100 billion annual loss worldwide. For successful establishment of the root-knot nematode in its host plant, it causes dramatic morphological and physiological changes in plant cells. The expression of some plant genes is altered by the nematode as it establishes its feeding site. Results We examined the expression of soybean (Glycine max) genes in galls formed in roots by the root-knot nematode, Meloidogyne incognita, 12 days and 10 weeks after infection to understand the effects of infection of roots by M. incognita. Gene expression was monitored using the Affymetrix Soybean GeneChip containing 37,500 G. max probe sets. Gene expression patterns were integrated with biochemical pathways from the Kyoto Encyclopedia of Genes and Genomes using PAICE software. Genes encoding enzymes involved in carbohydrate and cell wall metabolism, cell cycle control and plant defense were altered. Conclusions A number of different soybean genes were identified that were differentially expressed which provided insights into the interaction between M. incognita and soybean and into the formation and maintenance of giant cells. Some of these genes may be candidates for broadening plants resistance to root-knot nematode through over-expression or silencing and require further examination. Background Plant parasitic nematodes cause about US $100 billion in crop losses annually [1,2]. Root-knot nematodes (RKN; Meloidogyne spp.) are sedentary endoparasites. The most economically important species are Meloidogyne incognita and M. arenaria. Both are widespread and are considered as major crop pathogens worldwide. The RKN can be easily recognized by the "knots" or "galls" that form where they feed on roots [3,4]. These nematodes cause dramatic morphological and physiological changes in plant cells. Some plant genes are subverted by nematodes to establish feeding cells, and transcripts of several nematode genes were identified during infection [5]. Root-knot nematode damage to soybean (Glycine max) can be severe, especially when fields previously planted in cotton are rotated into soybean [6]. The RKN life cycle is complex [for review see: [3][4][5]7]]. The egg is laid in the soil or in plant tissues. The first stage juvenile develops inside the egg and molts one time to the second-stage juvenile (J2). When the J2 hatches from the egg, it infects the root close to the root tip in the elongation zone and migrates to the vascular tissue, where it establishes a feeding site by injecting esophageal proteins into several plant cells and it recruits host genes to alter the morphology of the host cells. Host cells become binucleate and then undergo multiple rounds of synchronous mitosis without cell division to form a giant cell [3][4][5]7]. These multinucleate cells can contain more than 100 polyploid nuclei. The cells surrounding the giant cell undergo hypertrophy and hyperplasia to form a root gall [3][4][5]7,8]. Thus, expression of numerous host genes is modified to produce these extensive changes in the root. The J2 males and females molt three more times to reach maturity. The mature female produces an egg mass in a gelatinous protective sac that is extruded from the female nematode onto the root surface. Within the nematode's esophageal gland cells, different proteins are produced to help the nematode establish a feeding site. Some of the proteins secreted by the nematode are injected into host cells and cause modification of the plant cells to form giant cells. Other proteins secreted by the nematode may interact with the host's extracellular receptors to influence signal transduction [8]. Similarly, gene expression is altered in the cells that are selected to be the feeding sites of the soybean cyst nematode. Klink et al. [9][10][11][12][13] demonstrated that numerous changes in gene expression occur in roots and in syncytial cells in soybean roots infected by either compatible or incompatible populations of soybean cyst nematodes. They used microarrays to study gene expression in laser capture microdissected (LCM) syncytium cells for susceptible and resistant reactions of soybean during infection with soybean cyst nematode (Heterodera glycines; SCN) [9,10,12]. Many genes were shown to be up-and down-regulated in both susceptible and resistant responses. Also, they identified many genes that are involved in plant-pathogen interactions, which provided new insights into the interaction between the cyst nematode and its host plant. In another microarray study by Klink et al. [12], distinct expression patterns at different developmental stages of the SCN feeding site were detected in gene expression studies of syncytial cells collected by LCM from SCN-susceptible and resistant soybean cultivars. Gene expression patterns at the first stage were found to be similar in both the susceptible and resistant reactions, when the nematode first attempts to establish itself in the host. This stage is called the parasitism phase. The second stage depends on the defense response of the host plant. If the soybean plant exhibits resistance to the parasite, the nematode will fail to establish and will develop very slowly or die. If the plant is not resistant to the nematode, the soybean host and SCN are compatible and the nematode will establish its permanent feeding site. Using microarray analysis Ithal et al. [14,15] studied transcript expression in syncytium cells induced by SCN in soybean roots after infection. They reported that several pathways are involved in the induction of syncytia. For example, genes involved in solidifying and lignifying the cell wall of the syncytium were shown to be up-regulated. Interestingly, they also reported down-regulation of the plant defense system, specifically the pathway leading to jasmonic acid. On the other hand, Klink et al. [16] examined the response of a resistant cultivar of soybean against SCN by studying gene expression using microarrays. The levels of transcripts of genes encoding enzymes involved in jasmonic acid biosynthesis, phenylpropanoid biosynthesis, suberin biosynthesis, adenosylmethionine biosynthesis, ethylene biosynthesis from methionine, flavonoid biosynthesis and the methionine salvage pathway were greatly altered during the defense response of soybean to H. glycines. Also, changes in gene expression have been monitored in Heterodera glycines-susceptible soybean cultivar (Kent) using microarray at 6, 12, and 24 hours after infection as well as 2, 4, 6, and 8 days after infection [17]. In that study, the level of genes encoding WRKY6 transcription factor and lipoxygenase were shown to be up-regulated at most time points tested (12 hours after infection (hai) -8 days after infection (dai)) after infection with Heterodera glycines. Analysis of microarray data can be complex, as datasets are very large and it is difficult to analyze and integrate changes in metabolic pathways. Tremblay et al. [18] used the PAICE program to analyze microarray data of soybean leaves infected with soybean rust (Phakopsora pachyrhizi). The PAICE program overlays gene expression results from microarrays onto biochemical pathways found in the Kyoto Encyclopedia of Genes and Genomes (KEGG). PAICE makes key changes in gene expression in biochemical pathways stand out and makes comparison of pathway changes among treatments and across time points easier. New targets for nematode control could be developed through the identification of genes that are involved in the establishment of the nematode in the host plant and which participate in the formation of the permanent feeding site for the nematode. Ibrahim et al. [19] were able to control M. incognita development in soybean plants after silencing four M. incognita genes using the RNA interference mechanism. In this study, portions of the genes encoding mitochondrial stress protein and tyrosine phosphatase were shown to have the greatest effect among four tested genes on nematode development and on the number of galls formed on the RNAi-expressing roots. Also, Dalzell et al. [20] were able to silence the gene encoding FMRF amidelike peptide (flp) with 21 bp siRNAs, specific to that gene in infective (J2) stage juveniles of potato cyst nematode, Globodera pallida, and Meloidogyne incognita. Charlton et al. [21] reduced the number of Meloidogyne incognita by 50% after simultaneous suppression of two genes, dual oxidase and a subunit of a signal peptidase required for the processing of nematode secreted proteins, respectively. In this paper we used the 37,500 probe set Affymetrix Soybean GeneChip to assay gene transcript abundance in galls formed in soybean by M. incognita at two stages, small galls at 12 dai and large galls at 10 wai. These time points were chosen to contrast active nematode feeding at 12 dai with plant gene expression in a mature infection at 10 wai. The latter time point is particularly interesting as gene expression in plant roots after prolonged infection has not been reported previously. We used PAICE [18] software to visualize the expression of genes related to major biochemical pathways and we identified a number of different pathway genes that were affected by nematode infection. Although we are using biochemical pathways as a format to visualize gene expression results, the changes in gene expression overlaid on these pathways do not imply similar changes in the levels of the encoded protein. This study provides insights into the interaction between M. incognita and soybean and into the formation and maintenance of giant cells. Our long-term objective is to identify possible gene targets for manipulation to develop broad resistance of plants to RKN by using gene silencing technology or to over express certain soybean genes. Plant and nematode procurement Glycine max cv Williams 82 and M. incognita population LESREC (Lower Eastern Shore Research and Education Center) were grown in a greenhouse at the United State Department of Agriculture Soybean Genomics and Improvement Laboratory, Beltsville, MD, USA. M. incognita eggs were harvested from roots of G. max cv Williams 82 2-4 months after inoculation using a method modified from those previously described in Meyer et al. [22] and Nitao [23]. Soybean seedlings were grown in Promix (Premier Horticulture INC., Quakertown, PA, USA) for one week in 20 × 20 × 10 cm flats, then moved to sand (The Stone Store, Hanover, MD). Three thousands eggs were used to inoculate roots of 7 day old soybean seedlings (cv. Williams 82). Soybean roots at 12 dai, 10 wai, and control uninfected plants were washed with sterile water, flash frozen in liquid nitrogen, ground to a fine powder and frozen at -80°C until use. The infected roots were collected at 12 days after infection. Nematodes were stained in infected roots using a modified protocol of Byrd et al. [24] and Mahalingam et al. [25]. Briefly, roots were washed in gently flowing tap water to remove soil and debris, cut to 2 cm segments, and placed in a small beaker, then soaked in 20-30 ml of 10% commercial Clorox (chlorine bleach, 5.25% NaOC1) for 3 min. The roots were rinsed in tap water and then transferred into a 50 ml glass bottle containing 20 ml of distilled water and left to boil in a microwave with loosened caps. A 500 μl of acid fuschin (0.15 g/10 ml H2O) and 500 μl of glacial acetic acid were added to the root samples and heated to boiling in a microwave twice. The roots were left to cool to room temperature before removing the excess stain with running tap water using Miracloth on the top of the bottle. A 20 ml of clearing reagent (1/3 lactic acid+ 1/3 glycerol + 1/3 distilled water) were added to roots and roots were left to destain for two hours to overnight. The nematodes were stained red as observed in the roots under a dissecting microscope. General chemical reagents were obtained from Sigma Chemical Co (St. Louis, MO). RNA extraction and microarray analyses RNA was extracted from 100 mg each of the three different root samples using the Ultra Clean Plant RNA Isolation Kit (MOBIO, Carlsbad, CA). Gene expression analysis was performed using the GeneChip ® Soybean Genome Array (Affymetrix ® , Santa Clara, CA, USA) containing more than 37,500 probe sets as described in Klink et al. [9,10]. In this GeneChip technology, each high density spot (gene) is represented by 11-probe pairs (11 μm feature size), which allows multiple independent measurement for each transcript. GeneChip ® Soybean Genome Array details are available at the Affymetrix ® website [26]. The microarrays were hybridized and scanned at the Laboratory of Molecular Technology, SAIC-Frederick, National Cancer Institute at Frederick, Fredrick, MD, USA. Affymetrix © soybean Genechip data was analyzed as described in Klink et al. [9,10,15] with additional analysis using PAICE [18]. Briefly, microarray gene expression data was imported into MATLAB (Natick, MA) Bioinformatics Toolbox. Normalization of the probe sets was performed using RMA (Robust Multiarray Analysis). The resultant calculated output was the log base 2 of the expression values, enabling scaling of the dataset. Volcano plots were produced, which graphically illustrate gene expression fold-change with respect to statistical significance. The plots were produced using foldchanges >= \|2.0\| and p-values < = 0.05 with respect to the control (untreated roots of cv. Williams 82). The ttest was used in calculating p-values. False Discovery Rate analysis was further utilized against significantly expressed genes [16]. The False Discovery Rate tool 'Significance Analysis of Microarrays' (SAM v3.0; [8] was used with a false discovery rate of 10%. RT-PCR for confirmation of differential gene expression in infected soybean roots at 12-dai and 10-wai To confirm the microarray results conducted obtained for M. incognita infected soybean roots at 12-dai and 10-wai, quantitative real time PCR (qRT-PCR) was performed on specific genes that were shown to be differentially expressed during the infection. Fourteen genes were chosen according to the changes in their expression at 12-dai and 10-wai (Table 1). The genes were classified and placed in three different groups according to their function [8,9]; Table 1). Soybean ubiquitin-3 (Accession No. D28123) was used to normalize the results. RNA samples also used for microarray analysis were used in qRT-PCR analysis. RNA from three different biological replicates of each time point (12 dai and 10 wai), and the control (noninfected roots) were used to synthesize first-strand cDNA using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlesbad, CA) following the manufacturer's instructions. Quantitative real time PCR was performed using the Stratagene Mx3000P RT-PCR system as described by the manufacturer (Stratagene, La Jolla, CA) with 10 ng/reaction of cDNA for all genes. Primer sequences specific to each gene are presented in Table 2. Other controls for qRT-PCR included reactions containing no template or no reverse transcriptase. These controls resulted in no amplification. qRT-PCR was performed in two biological replicates and each reaction was replicated three times. DNA accumulation was detected by SYBR Green and the Ct value (cycle at which there is the first clearly detectable increase in the fluorescence) was calculated using the software provided with the Stratagene Mx3000P RT-PCR system. Dissociation curves showed amplification for only one product for each primer set. Data analysis was performed according to the sigmoidal method described by Rutledge and Stewart [27] for absolute quantification of transcripts. Absolute quantification of fluorescence intensity per ng dsDNA was obtained using 100 fg lambda gDNA in quadruplicate to calculate the optical calibration factor. Absolute quantification of the transcript level of the RNAi targeted genes was calculated using specific equations according to Ibrahim et al. [19] and Tremblay et al. [28]. Pathway Analysis Biochemical pathway analysis was conducted using PAICE (Pathway Analysis and Integrated Coloring of Experiments) [18]. This software program maps expression levels of genes encoding enzymes found in the KEGG biochemical pathways database. Gene expression levels are denoted using color codes displayed at the pathway nodes depicted by enzyme EC numbers. Besides the pathway mapping feature, PAICE colors EC accessions using gradients of green and red to represent induced and suppressed gene expression respectively. The colors can be tailored to the analysis through the software package. PAICE has the unique ability to color in yellow the expression of genes having multiple family members that lack a consensus in gene expression, i.e. some members are over-expressed and others are underexpressed. Histological examination of RNK infection At 12 dai, galls can be identified as small swellings along the soybean root (Figure 1a). Within the gall the nematode has started feeding and can be visualized by staining with acid fuchsin [24,25] to monitor nematode invasion and development inside the roots (Figure 1b). Mature galls are present on soybean roots at 10 wai ( Figure 1c). Within the gall, mature female M. incognita can be identified easily by staining (Figure 1d). Transcript profiling of galls formed by M. incognita infection A comparison of gene expression at12 dai compared to control led to the identification of 1867 genes with greater than 1.5-fold change in expression ( Figure 2). Of these, 1278 genes increased and 589 genes decreased in Table 1 List of the 14 induced and suppressed annotated genes in soybean cv. Williams 82 at 12 dai and 10 wai by M. incognita (p-value ≤ 0.05) that were used in qRT-PCR for confirmation of microarray results expression. Transcripts encoding leghemaglobin C1 (AI973819) increased the most at 386-fold. The most down-regulated gene was BF070134 with homology to a putative senescence protein 12 and to ERD7; its transcripts were 77-fold lower than in the control. There were 2108 genes with altered expression in galls at 10 wai. Of these, 1460 genes increased in expression and 648 genes decreased in expression. The transcript of the gene encoding pathogenesis related protein PR1a (CK605838) increased the most at 258-fold. As in the 12 dai experiment, the most down-regulated gene was Table 2 Primers used in RT-PCR reaction to confirm microarray expression data. BF070134 with transcripts 172-fold lower than the control. When gene expression at 10 wai was compared directly to 12 dai, 827 genes were up-regulated, while 535 genes were down-regulated. In this case, transcripts of the gene encoding the cysteine-rich plant defense protein, defensin (PDF2.3; BI321308), increased the most at 63-fold, while the transcripts of the gene encoding xylene serine peptidase 1, subtilase (AW309540) decreased the most at 126-fold. Mitosis and cell division Our data reflect changes in expression of numerous genes involved in nuclear regulation and cell division in the gall at 12 dai and 10 wai (Table 3). For example two genes were increased in transcript abundance that are regulators of the cell cycle. These genes encode two NDR (nuclear Dbf2-related) family members of AGC kinase, and they are increased in expression 24.5-fold (BI968028) and 5-fold (CA802074)) at 12 dai. By 10 wai genes of several NDR family members (NHL1 and NHL3) are expressed less than at 12 dai, i.e. BI968028 at 5.5 fold, AW156706 at -2.6 fold, and CF806406 at -9.6 fold. Transcripts of numerous cyclin-dependent protein kinases are in greater abundance at 12 dai than in control tissues (Table 3). This correlates well with the increase in nuclear division that occurs in giant cell. In addition, the gene encoding RBR1 retinoblastomarelated protein, which modulates E2F-transcripton factors that inhibit cell proliferation, is also increased at 12 dai. Expression of a gene encoding the plant hormone, phytosulfokine 3 growth factor (BK000117) involved in positive regulation of plant cell proliferation, is increased by 44-fold at 10 wai. Cell wall modifications Numerous genes involved in cell wall remodeling were shown to be differentially expressed after infection with M. incognita (Table 4). Genes encoding four members of the expansin enzyme family (expansins A1, A5, A16, and 45) were up-regulated at both time points (Table 4) Figure 3). Also, in phenylpropanoid biosynthesis, genes encoding a family of 21 extensin peroxidases [EC 1.11.1.7] that participate in lignin biosynthesis were differentially regulated ( Figure 4). The extensin gene with the highest increase in expression (AW309606) increased by 95-fold while the extension gene with the largest decrease in expression (BI970840) decreased by -16-fold. Defense-related genes There are multiple changes in expression of genes encoding enzymes of the alpha-linolenic acid pathway leading to several important defense-related compounds, including jasmonic acid (Figure 6). At 12 dai, the change Genes encoding transcription factors and other proteins Changes in gene transcripts were accompanied by changes in expression of transcription factors, especially those in the WRKY family of transcription factors. Our microarray results indicated that genes encoding several family members of WRKY genes were down-regulated at 12 dai, including genes encoding WRKY6, 15, and 22 (-10.5, -14.3, and -8.5 FC, respectively). In contrast, at 10 wai, genes encoding WRKY 21 and 70 were up-regulated at 117 and 42 FC, respectively. Several pathogenesis-related proteins are induced in plants during infection with any pathogen or by wounding, including nematode infection, and induction of many of these is affected by salicylic acid, jasmonic acid or ethylene [29]. In our microarray data, genes encoding pathogen related proteins (PR) such as PR3 were down-regulated at 12 dai and genes encoding PR3 at 10 wai showed a mixed response; some were up-regulated while others were down-regulated. The three copies of the pathogen related protein PR1 gene were over-expressed by 78.23, 97.56, and 138.50 fold, respectively (Data not shown). Confirmation of differential gene expression by quantitative PCR Quantitative PCR was conducted to confirm gene expression patterns revealed by microarray analysis. We measured transcript abundance of 14 genes that showed increased or decreased transcript abundance by microarray analysis (Table 5; Figure 7). The trends in up-or down-regulation of gene transcripts were consistent between microarrays and quantitative PCR results except for expression of the gene encoding lipoxygenase family member LOX1 at 10 wai. However, we did observe differences in levels of expression between methods. Differences in fold-change in gene expression as measured by microarray and qRT-PCR have been reported in previous studies [15,18,28]. Discussion When M. incognita infects and feeds in a soybean root, numerous genes are altered in expression in the root. M. incognita not only triggers the defense response of the root, but also redesigns the morphology of the root to form a gall and converts a soybean cell into a giant cell for feeding. The timing of these changes coincides Table 4 Enzymes involved in cell wall modification and remodeling, for which their respective RNAs were found to be differentially regulated in our microarray dataset at both time points ( with changes in gene expression as seen in our microarray experiments. Regulators of the cell cycle and cell division The cell cycle is regulated by two types of cyclin-dependent kinases. CDKA is required for cells to enter the S and M phases. CDKB1 and CDKB2 are expressed during the G2 and M phases and are responsible for the G2-M transition [30]. Our microarray results indicate that genes encoding some members of the cyclin-dependent kinases family were differentially expressed at 12 dai and 10 wai. Over-expression of the gene encoding CKB2 at 12 dai (5.2 and 4.6 FC) correlates with the increase in plant nuclear division that occurs at the infection site due to M. incognita infection and feeding. Cells selected by M. incognita for feeding become multinucleate giant cells. This increase in nuclear division continues until 14 dai [3][4][5]. Cell cycle activation in giant cells has also been observed by Engler et al. [31]. In that study, the transition from S to G2 and G2 to M phase was reported after the over-expression of a GUS gene driven by the cycB2 or cycA2 promoters at one to nine days after infection with M. incognita. Expression of the CDKB2 gene at 12 dai was higher than at 10 wai, i.e., 5.2-versus 3.1-fold, respectively (Table 1). Ramsay et al. [32] found that cyc D3 is essential to stimulate the G1 phase of the cell cycle in root knot nematode infected giant cells. In this investigation, the two types of CycD3 (CycD3.2 and CycD3.3) were shown to be relatively more strongly expressed as compared to that of LeCycA1.1, LeCycB1.1 and LeCycD3.1 in giant cells induced by Meloidogyne spp. compared with other cyclin-dependent kinases. They observed PCR amplification of CyD3.2 and CycD3.3, while no amplification of cycA.1, CycB1.1 and CycD3.1 was observed. Our data showed a suppression of gene expression of the gene encoding cycD3 which is important for the regulation of the G1-S transition. In addition, at 10 wai we found an increase in gene expression of CKS1 (CDK-Subunit 1), a protein that prevents CDK from driving the cell cycle into S phase. This result suggests that at the earlier time point, the giant cells reach maturity and then the genes required for nuclear division are turned off. Cell wall modification and remodeling Due to multiple nuclear divisions of selected cells with no coincident cell division, the giant cells sometimes reach more than 400-times the size of a normal cell and may contain more than one hundred nuclei [4]. This expansion of the giant cell is accompanied by extensive cell wall modification. Our microarray data indicate altered expression of many genes involved in cell wall extension and remodeling (Table 4). We found that the genes encoding a cell wall-modifying xyloglucan endotransglycosylase/hydrolase [EC 2.4.1.207] and endoxyloglucan transferase A2 are differentially expressed in soybean roots after infection with M. incognita (12 dai and 10 wai). These enzymes play a role in softening and breaking down the cell wall [33]. Genes encoding many endo-1,4-glucanase family members were up-regulated at both time points (12 dai and 10 wai). Endo-1,4-glucanase is involved in cell wall remodeling and expansion. LCM was used to isolate giant cells formed in tomato by M. javanica to examine gene expression [34]. Numerous transcripts of genes involved in cell wall remodeling were also identified in the cDNA library of giant cells 4 dai, including transcripts of genes encoding pectin methylesterase and pectinesterase. Goellner et al. [35] identified genes encoding endo-1,4-glucanases that were upregulated in feeding cells formed by M. incognita and cyst nematode in tobacco plants. Also, Mitchum et al. [36] found that the promoter of an endo-1,4-β-glucanase gene was strongly activated in feeding cells formed by Meloidogyne incognita as indicated by strong promoter-driven GUS expression. The increase in expression of the gene encoding expansin A in our results is consistent with other investigations, wherein the expansin (LeEXPA5) genes in A. thaliana and tomato were shown to be up-regulated in developing giant cells after infection with Meloidogyne [37,38]. Moreover, down-regulation of genes encoding cellulose synthase and over-expression of genes encoding pectin esterase that degrades pectin to pectate coincide with a breakdown of the cell wall during the early time points of infection with Meloidogyne. Our results are consistent with those of Jammes et al. [38], who found genes encoding pectin esterases and pectate lyases were activated in Arabidopsis thaliana roots after infection with Meloidogyne incognita and the cell wall loosening process was activated during the development of the giant cell as well. In giant cells formed in tomato by M. javanica, there is an 8-fold and 7.3-fold increase in expression of the gene encoding pectinesterase U1 precursor [34]. Giant cells formed by M. javanica in roots of Arabidopsis were collected by LCM and analyzed by Barcala et al. [39]. Genes encoding cellulose synthase, expansin, pectate lyase, endoxyloglucan transferase also were all up-regulated in these cells coinciding with cytoskeleton rearrangements that occur during giant cell development. Nutrients supply for M. incognita The nematode uses a large amount of plant resources to develop and reproduce. This demand for energy and carbon is reflected in the numerous genes involved in glycolysis and gluconeogenesis that are up-regulated in the soybean root ( Figure 5). For example, we found many genes encoding enzymes in the glycolysis pathway and amino sugar synthesis to be up-regulated. Mostly, the changes in gene expression occurred early in infection (12 dai). In addition to their roles in pathways that provide energy and carbon for the nematode, some of these genes have an essential role in the soybean-M. incognita interaction. For example, at 12 dai, the gene encoding UDP-glucuronate 4-epimerase (EC 5.1.3.6) is highly down-regulated (-21 fold). In Arabidopsis, a mutation in this gene resulted in hyper-sensitivity to the cyst nematode, Heterodera schachtii [40]. In Azospirillum brasilense, this enzyme is important for lipopolysaccharide synthesis which is important in the bacteriumplant root interaction. A mutation in this gene resulted in the failure of the bacteria to respond to several stresses and antimicrobial compounds. It also affected the ability of the bacteria to form biofilms [41]. This enzyme may be important in allowing the host to respond to M. incognita invasion [42,43]. In the glycolysis and gluconeogenesis pathways, many genes were shown to be up-regulated, including the gene encoding glucose-6-phosphate isomerase [EC: 5.3.1.9]. The gene encoding this enzyme was also shown to be up-regulated in cucumber plants after treatment Note: Gray boxes refer to untested genes in a specific time point. with Trichoderma asperrellum T34 [44]. The enzyme is essential in salinity tolerance in the alga Dunaliella salina [45]. Not only do nematodes require large quantities of carbon and energy from its host, they also use starch during juvenile development. Starch is stored in syncytia formed by Heterodera schachtii in roots of Arabidopsis [46]. The authors postulate that the starch is also used to compensate for fluctuating levels of sugar during the course of nematode development and feeding. The high metabolic rate of cells was suggested by the increased expression of ribosomal genes in giant cells induced by M. javanica in tomato roots [47]. Degradation of the cell walls could result in release of sugar which in turn will be channeled to glycolysis as reflected in the activation of the genes encoding enzymes in the glycolytic pathway. Also, since some enzymes in glycolysis participate in the biosynthesis of pentose, purines and pyrimidines, there could be an increase in production of nucleotides required for DNA replication. Plant defense system When a nematode invades a plant root, it must repress or control the plant defense response so it can successfully establish its permanent feeding site [4][5][6]. Our microarray data showed significant changes in expression of genes related to the defense response against pathogens. The pathway leading to jasmonic acid biosynthesis is one of the pathways associated with pathogen resistance and genes in this pathway were significantly affected by Meloidogyne incognita infestation at both time points (12 dai and 10 wai; Figure 6). At 12 dai, six of seven members of the lipoxygenase gene family were up-regulated. Lipoxygenase (LOX) is essential to oxylipin biosynthesis and has an important function in the plant defense response against wounding and pathogen attack [48]. Reduction of LOX activity resulted in an increase in susceptibility of transgenic potato plants to insect attack [49]. Over-expression of the gene encoding lipoxygenase could mean that more 9-HPOTrE would be produced. This is one of the major products of lipoxygenase ( Figure 6). Interestingly, 9-HPOTrE is involved in the activation of the plant defense response directly or through its metabolites. In potato plants, 9-HPOTrE is produced in response to injury or infection. The role of 9-HPOTrE in the plant defense response suggests that there may be another pathway of LOX-mediated defense response [-50]. 9-HPOTrE could also be a substrate for allene oxide synthase ( Figure 6) to produce OPDA, the precursor for jasmonic acid. At 10 wai, the abundance of the lipoxygenase transcript was much lower than the 12 dai time point. Three of seven gene family members encoding lipoxygenase were down-regulated. Also, all of the allene oxide synthase family members were greatly down-regulated in addition to some other genes encoding enzymes in the jasmonic acid biosynthetic pathway ( Figure 6). This indicates that at 12 dai the plant defense system is still struggling to fight the infestation, but after prolonged infection (10 wai) most of the genes that are responsible for the production of one major defense hormone, jasmonic acid, were turned off. Genes in this pathway could be targets for testing whether resistance to nematode infestation can be increased in transformed plants by over-expression of these genes. We found a number of genes encoding PR proteins that were differentially expressed in soybean roots 12 dai with M. incognita. The genes encoding PR-1, PR-2 and PR-5 protein families increased in expression. Genes encoding PR-2 and PR-5 are expressed in sid mutants of Arabidopsis that do not accumulate SA. However, genes encoding PR1 are known to be induced by salicylic acid (SA) [50,51]. This suggests that salicylic acid or its derivatives may be synthesized at 12 dai and 10 wai by M. incognita infection. Interestingly, there are two different possible routes to salicylic acid production [52]. The pathway that has the most scientific support involves isochorismate synthase [53] and its genes are not represented on the microarray. The other pathway leading to SA production involves phenylalanine. In this pathway, we found a large increase in the expression of the gene encoding tyrosine aminotransferase [TAT; EC:2.6.1.5]. If the abundance of this enzyme is increased, then this could lead to increased phenylalanine production. Genes encoding phenylalanine ammonia-lyase [EC:4.3.1.24]; and salicylate 1-monooxygenase [1.14.13.-] were over-expressed 6.9 and 2.9 FC, respectively. SA induces the expression of PR-1 [54]. In giant cells of tomato formed by M. incognita 4dai, several genes involved in the phenylpropanoid pathway were detected, notably phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase (35). Transgenic tobacco overexpressing PR-1 was more resistant to blue mold and black shank caused by Peronospora tabacina and Phytophthora parasitica f. sp. nicotianae, respectively [29]. Although SA treatment of tomato plants that were inoculated with Meloidogyne incognita did not completely eliminate nematode infection, it enhanced the synthesis of PR-1, which resulted in a significant increase in resistance to the nematode [55]. In contrast, Portillo et al. [56] reported a decrease in transcripts of the PR-1 precursor in giant cells collected from tomato infected with M. javanica by LCM as measured using qRT-PCR. Genes encoding PR-3 and PR-4 family proteins are reported to be up-regulated by jasmonic acid and ethylene [29]. Also, PR-4 showed ribonuclease activity against fungal protein in wheat [29]. Furthermore, a gene encoding a pathogenesis-related protein was reported as up-regulated in the interaction of M. incognita with Arabidopsis at 21 dai [57]. They also reported the increased expression of genes encoding several proteinase inhibitor proteins and leucine-rich repeat family proteins. Conclusion There are major changes in host gene expression between 12 dai and 10 wai by M. incognita. In the pathway leading to jasmonic acid synthesis, several genes are down-regulated at 10 wai. We identified changes in important genes and pathways during parasitism. Some host genes encode proteins that participate in the establishment of the feeding site, i.e., giant cells, required by M. incognita and for gall formation. These results provide new insights into host-parasite interactions. In the future, some of these genes may be used to control the plant parasitic nematode infestation through plant genetic engineering to overexpress defense genes or silence genes that promote giant cell and gall formation.	2014-10-01T00:00:00.000Z	2011-05-10T00:00:00.000	{ "year": 2011, "sha1": "b1f59b989ce5a6acda99d63e25f5b3917e4cb384", "oa_license": "CCBY", "oa_url": "https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/1471-2164-12-220", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "d66d8d3bae10c723ed5699b7a3015974acc7b6d7", "s2fieldsofstudy": [ "Biology", "Environmental Science" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
54951382	pes2o/s2orc	v3-fos-license	Microbiological Quality of Organic and Conventional Leafy Vegetables The aim of this study was to assess the microbiological profile of leafy vegetables from organic and conventional farming (n = 70). Themicrobiological parameters analyzed consisted of aerobic mesophilic bacteria, yeasts and molds, coliforms at 30 and 45C, and Salmonella ssp. A biochemical identification of the Enterobacteriaceae species was carried out. Some Enterobacteriaceae species were identified in the produce from both farming systems and Hafnia alvei was the most widespread specie observed. Salmonella spp. was not detected in the samples analyzed. The microbial counts for indicator microorganisms were, in general, higher for conventional leaves when compared to those produced by organic farming. Introduction The organic food and beverage market sold about 81.6 billion US dollars' worth of produce worldwide in 2015.Nowadays, the highest per capita consumption of organic food is found in European countries, such as Switzerland, Luxembourg, and Denmark [1].The Institute of Development Promotion (IPD-Instituto de Promoc ¸ão do Desenvolvimento) estimated there were more than 10,000 organic production units in Brazil in 2014.However, the lack of accurate data about the Brazilian market limits a precise number, since the majority of sales occur directly between small farmers and consumers [2]. The reasons for the consistent growth in the consumption of organic vegetables go from ethical and political reasons to life quality, safety, and environmental concerns [3,4].Organic vegetables, generally, presents lower amount residues of pesticides when compared with conventional ones [5].This fact increases consumer trust in organic food due to a lower perceived risk and increased perceived quality [6].Perceived quality is one of the most important predictors of organic willingness to buy and consume [4].The French Agency for Food Safety (ANSES) points out that the use of safe chemicals, such as alcohols and chlorine compounds, and the banishment of Genetically Modified Organisms (GMOs) constitute the most significant characteristics of organic production [7]. The relationship between microbiological risk and organic production is still uncertain since many factors may contribute to the development of microorganisms, including differences between the cropping system.The use of animal manure in organic farming naturally raises concern about the microbiological safety.Since there is a restriction concerning the chemicals allowed in organic production [8] the potential for microbial contamination exists when this type of fertilization is used, representing an increased risk to public health [9][10][11].In Brazil the use of manure in organic agriculture is allowed; however, the composting of manure into soils is mandatory for this cropping system [12]. In some scientific studies greater risk with the consumption of organic food was not identified [8,13] but controversially, several foodborne disease outbreaks are related to organic food [14].One example is the outbreak of Shiga toxin-producing Escherichia coli O157:H7 linked with organic Applies the water through small emitter to the soil surface; drip irrigation allows the water to drip slowly to the roots of plants minimizing the water evaporation; microspray applies water to the soil surface by a small spray or mist Farm of conventional cultivation Manual with manure River water (surface water) Sprinklers Sprinkler irrigation is a method similar to natural rainfall; water is delivered through a pressurized pipe network to sprinklers which spray the water into the air * Source: Solomon et al. [23]. spinach consumption reported in five States of United States affecting 33 persons in 2012 [15].No deaths were reported; however two persons developed hemolytic uremic syndrome (HUS).Leafy vegetables do not generally represent the main type of food reported in outbreaks.From 1998 to 2008 only 5.2% of bacterial foodborne illnesses in the USA were related to leafy vegetables [16].The data from the Brazilian Health Ministry shows that vegetables represented about 0.8% of this epidemiology in the last 10 years; but this proportion could be higher because most outbreaks are subnotified in the USA [17] and Brazil.In addition, raw vegetables must be sanitized before consumption.However, food handlers, both professional and domestic, fail to follow hygiene procedures, increasing the risk of consuming raw vegetables [18,19]. In this context, the present work proposes to microbiologically evaluate certified organic leafy vegetables and conventional ones from the same cultivars.Well water is used for irrigation which is performed by microirrigation (drip and microspray irrigation).The fertilization is manual and made with manure composting into soils.The vegetables are collected by a work team composed of 8 persons, and each employee is responsible for the collection of a particular plant in order to observe possible changes in the crop.The organic vegetables presented national certification by Ecocert (a system which adopts external audits) and the leafy vegetables that have been evaluated are produced organically there since 2014.The production area for conventional agriculture has 1.5 hectares, in which 19 different species of vegetables are grown throughout the year.The irrigation process is only by sprinkling and the irrigation water comes from a river from to the PCJ watersheds (belonging to Piracicaba, Corumbataí and Jaguari cities).Manure is also used for fertilization using a manual process.The work team is composed by 3 employees.Both producers used the same plant seedlings from the same provider, so the production only differed with respect to the type of cultivation.Five units of each leafy vegetable, organically and conventionally grown, were collected.The following species were analyzed: green leaf lettuce varieties "Milena" (LM) and "Vanda" (LV) and iceberg lettuce (IL) (Lactuca sativa L.), common chicory (CC) (Cichorium intybus), escarole (ES) (Cichorium endivia), collard greens (CG) (Brassica oleracea L.), and parsley (PA) (Petroselinum crispum).Table 1 summarizes the characteristics of the two farms. Material and Methods The analyses were performed from February to November 2016.The analysis routine was the following: each type of leafy vegetable obtained by conventional and organic cultivation was harvested at the same day and taken to the Microbiology Laboratory.All analyses were performed on the day the vegetables were harvested.They were placed in plastic bags without modifying the atmosphere and stored at 4 ± 3 ∘ C until analyzed.All samples presented a fresh appearance at the time of analysis.The vegetables were washed in running water before analysis, leaf by leaf, immersed in a sodium hypochlorite solution (200 mg/L) for 15 minutes, and then rinsed in running water [20].This step aimed to compare the microbiological quality of the leave vegetables under the consumption conditions.Both the conventional and organic leafy vegetables of the same type/variety were analyzed at the same time and under the same conditions.Since the production areas are nearby (approximately 40 Km distant), the conditions of climate and rain during cultivation were equivalent for each type of leafy vegetable growth at the same time. Microbiological Analyses. The samples were analyzed for aerobic mesophilic bacteria, yeasts and molds, coliform (at 30 ∘ C and 45 ∘ C) counts according to Downes and Ito [21], and the presence of Salmonella according to Andrews and Hammack [22]. Plate Count Agar (PCA) (Difco/BD, USA) was used for the aerobic mesophilic bacterial count with the addition of 1% triphenyl tetrazolium chloride (TTC) (Merck, USA), using the pour plate technique and incubation at 35 ± 1 ∘ C/48 h.To determine the yeast and mold counts, the spread plate The method described by Andrews and Hammack [22] was used to evaluate the presence of Salmonella.Briefly, the method consists of the following steps: (A) preenrichment (Lactose Broth, Difco/BD, USA); (B) selective enrichment (Tetrathionate broth (TT) and Selenite Cysteine broth (SC), Oxoid, England); (C) differential plating (Hektoen Enteric agar (HE), Salmonella Shigella agar (SS) and Xylose-Lysine-Desoxycholate agar (XLD), Oxoid, England); and (D) biochemical tests (Lysine Iron Agar (LIA) and Triple Sugar Iron agar (TSI), Difco/BD, USA).After application, each culture medium was incubated at 35 ∘ C/24 h, and when a positive reaction was obtained in the biochemical tests, a serological test was carried out using polyvalent antisera (Probac, Brazil). Biochemical Identification of the Enterobacteriaceae. To identify the species of Enterobacteriaceae present in the samples, the commercial systems Bactray5 I and II (Laborclin, Brazil) were used.These consist of a miniature kit indicated for gram-negative oxidase negative bacilli, glucosefermenting and glucose nonfermenting bacteria.Initially, MacConkey media for the growth of typical colonies was used, with incubation at 35 ∘ C/48 h, and then transferred to Brain Heart Infusion broth (BHI) and incubated at 35 ∘ C/24 h.The BHI broth was adjusted to a 0.5 McFarland turbidity reading in a spectrophotometer at 600 nm. An aliquot of 1.0 mL of the suspension was then transferred to the Bactray I and II systems and incubated at 35 ∘ C/18 h-24 h.An electronic program provided by Laborclin revealed the species found. Reference Standards. The data obtained were compared with the Brazilian legislation (RDC no.12) (Resoluc ¸ão da Diretoria Colegiada = Resolution of the Board of Directors) which indicates a maximum count for coliform bacteria at 45 ∘ C of 2 log CFU/g and the absence of Salmonella in 25 g [24].The Compendium of Methods for the Microbiological Examination of Foods published by the American Public Health Association (APHA) was also consulted in order to establish the parameters not contemplated by the Brazilian legislation [21]. Statistical Analysis. The variables were expressed as the means followed by the standard variation.The distribution histograms, mean, standard deviation, and minimal and maximum values were verified.The Kolmogorov Smirnov test was used to verify the adherence of distributions with the normal curve. The means obtained for organic and conventional cultivation were compared using Student's -test (variables with normal distribution and homoscedasticity) or Mann-Whitney's (variables without normal distribution and/or homoscedasticity).All tests were carried out using the SPSS software version 15.0.1.,considering a significance level of < 0.05. Results and Discussion The comparison between aerobic mesophilic bacteria, coliforms at 30 ∘ C and 45 ∘ C, and yeasts and molds can be observed in Table 2 (mean counts for each group of microorganism) and Figure 1 (mean counts for each leafy vegetable).Leafy vegetables from conventional cultivation presented higher mean counts of coliforms at 30 ∘ C, coliforms at 45 ∘ C, and yeasts and molds than the organically grown ones. Of all the samples analyzed, only one conventional vegetable (LV) and two organic ones (LM and CC) presented aerobic mesophilic bacterial counts higher than 6 log CFU/g, but they showed no signs of deterioration.When the aerobic mesophilic bacterial counts reach 6 log CFU/g, the vegetables may show signs of deterioration and become unsuitable for consumption [25]. A range from 4 to >7 log CFU/g for yeasts and molds in organic and conventional vegetables was reported by the literature [26,27].In the present work mean counts for yeasts and molds for each leafy vegetable were lower than 4 log CFU/g; however the samples were sanitized before analysis. The contamination with coliforms at 30 ∘ C found presently reached 1.08 log MPN/g for organic vegetables and 1.96 log MPN/g for conventional vegetables.Even though high levels of coliforms at 30 ∘ C may not represent an eminent health risk, they suggest poor hygiene conditions.A study with organic lettuces cultivated in Brazil [28] showed a range from 2.7 to ≥3.38 log MPN/g for coliforms at 30 ∘ C. The occurrence of coliforms at 45 ∘ C can indicate fecal contamination.Lotto and Valarini [29] showed that conventional lettuces produced in Brazil presented higher counts for coliforms at 45 ∘ C than organic lettuces, in accordance with the results of the present study, where the three types of lettuce evaluated presented statistically higher counts for coliforms at 45 ∘ C for conventional leaves (Figure 1(d)).For fresh, unpacked, unprocessed, and sanitized vegetables, the maximum count accepted for coliforms at 45 ∘ C is 2 log MPN/g [24]. There was a statistical difference between the microbial load (i.e., coliforms at 30 ∘ C and at 45 ∘ C and yeasts and molds) of the leafy vegetables obtained by the two cultivation systems; however, the absolute numbers of the counts were similar, less than 1 log different.Although the averages were close, in the individual analyses of two conventionally cultivated leafy vegetables (LV (Vanda lettuce) and IL (iceberg lettuce)) higher counts of coliforms were observed at 45 ∘ C than the established by the Brazilian legislation [12] indicating risk to the consumers. The presence of Salmonella spp. was not detected in any of the samples analyzed.Some studies refer to the absence of Salmonella spp. in fresh vegetables from the Brazilian market [26,30]; however other researchers found the presence of Salmonella in a range from 0.7 to 20% of the samples [7,28,31]. In some Brazilian studies, higher microbial counts were observed for organic vegetables [7,26,28,32] than for conventional ones.However, the microbial comparison of organic and conventional vegetables is controversial and depends on several factors including inadequately composted or raw animal manure and irrigation with bad quality water [7,8,33]. The irrigation system is another important factor in the microbiological safety of a crop, since contaminated water can reach the leaves during irrigation [3].The control of this source of contamination has been indicated as one of the most promising targets to reduce microbial contamination [34].During this study, technical visits were made to both production areas (conventional and organic cultivation).The agricultural production unit responsible for the organic vegetables uses good quality water and microirrigation systems.On the other hand, the conventional unit uses river water with just a sprinkler system.According to ICMSF [35] good quality water is less susceptible to contamination by pathogens than surface water taken from rivers, for example.Groundwater commonly contains less organic matter and microorganisms than surface water because the rock tends to act as a filter to remove some contaminants.Considering these aspects, the source and type of irrigation used may have affected the results. The indiscriminate use of herbicides and pesticides in conventional agriculture systems has been indicated as one of the causes of water and soil contamination and an imbalance in the environment, with direct impacts on the microbial loads of the soil and on several other species [36].On the other hand, some studies have shown that organic fertilizers can benefit the soil microbiota, increasing the availability and uptake of nutrients and also contributing to plant growth [37]. Although animal manure benefits the soil microbiota and can be used as a fertilizer in both organic and conventional farming, the risks in the case of organic production can be higher.To minimize this problem, a composting process is recommended, which involves mineralization, moisturizing, and decomposition of the organic matter, resulting in a stable, pathogen free product with an improved nutrient content [38].During the thermophilic phase of composting, the temperature can rise to 60 ∘ C and this is enough to reduce the microbiological counts, including those of pathogens [39].Costa et al. [40] demonstrated that the coliform population suffered a progressive reduction during composting, and Enterobacteriaceae such as Salmonella were destroyed.The use of composting is mandatory for organic farmers in Brazil, as described in Normative Instruction number 46 [12].However, for conventional agriculture, although the use of manure is allowed, composting is not required.It is suggested that composting must be required by Brazilian government in both the organic and conventional systems given the importance of this process in product safety.Table 3 shows the counts for the Enterobacteriaceae species found on the samples of leafy vegetables.The specie present in most samples was Hafnia alvei, which was found in both organic and conventional produce.Other genera/species identified in the samples were Pseudomonas luteola, Acinetobacter baumannii/calcoaceticus, Klebsiella oxytoca, Enterobacter asburiae, Pantoea dispersa, and Yokenella regensburgei.Although Enterobacteriaceae are present in the soil, water, plants, and the human microbiota, they can be a source of foodborne diseases.Some species observed in the present study in both organic and conventional leaves are epiphytic bacteria, which commonly colonize on the surface of plants [41].Their occurrence was also documented by Al-Kharousi et al. [42] in about 91% of vegetable samples. Although not necessarily harmful to the vegetables themselves, all the species found here have been somehow related to hospital-acquired infections.Hafnia alvei appeared 5 times in this study and can be found in human and animal feces, as well as in water, soil, and diverse products of animal origin [43] playing a role in the spoilage of refrigerated meat and vegetables [44].Moreover, H. alvei can be resistant to penicillin, ampicillin, cephalothin, tetracycline, and amoxicillin/clavulanic acid [45,46] which can represent a threat from the clinical perspective. It is also important to point out that, with respect to the species reported in the current study, the reports found in the literature concerning infections caused by them occurred mostly in hospital environments and possibly with immunocompromised subjects.It is also important to highlight that the increase in outbreaks has been strongly associated with the consumption of vegetables by immunocompromised individuals [47]. Organic agriculture production can be associated with other benefits, especially regarding sustainability and public health.Brazil, for example, is the leading country in the use of pesticides (5.2 L per capita/year), causing a strong impact on the environment in terms of the depletion of natural resources, the persistence of certain residues, and a decrease in biodiversity.The effects on public health due to the consumption of contaminated food, as well as on farm workers due to pesticide intoxication, can be considered as a violation of human rights: violating the right to health and to adequate and healthy food [48]. Conclusion The present study showed that the same cultivars of leafy vegetables obtained by organic cultivation had lower microbial counts than those obtained by conventional cultivation. The methodological approach of the present study does not allow to conclude which cropping system is safer; however, the observed results indicate that leafy vegetables cultivated in a conventional way can present a higher count of microorganisms even after the use of chemosynthetic pesticides and fertilizers.In this sense, good farming practices, independent of the cropping system, tend to be more effective in controlling the microbiological load.The quality of the water and the type of irrigation used in production can affect the quality of the leafy vegetables, so they should be carefully evaluated and implemented. Anyway, the detection of some Enterobacteriaceae species (but no Salmonella specie) in various types of leafy vegetable from both farming systems raises concern about the consumption of raw vegetables by susceptible individuals, such as infants, the elderly, and immunocompromised persons. Additional Points Practical Applications.The organic market is increasing worldwide.Agriculture for local consumption and exportation is the main economic activity in Brazil.As a result of that, the country is leading pesticide use (5.2 L per capita/year) and the study of organic production is important to give the opportunity to the population to reduce chemical exposure.The microbiological quality of leafy vegetable from organic farming was compared to conventional farming and showed no greater microbial load to them. Figure 1 : Figure 1: Counting of aerobic mesophilic bacteria (a), yeasts and molds (b), and coliforms at 30 ∘ C (c) and 45 ∘ C (d) among the 7 species of organic and conventional vegetables.Equal letters for the same vegetable indicate no statistical difference in Student's -test ( < 0.05). Table 1 : Summary of the characteristics of the two evaluated farms. Table 2 : Mean counts of aerobic mesophilic bacteria, yeasts and molds, and coliforms at 30 ∘ C and 45 ∘ C from 70 vegetable samples from organic and conventional cultivation.wasadopted using Sabouraud Dextrose Agar and incubation at 25 ± 1 ∘ C for 3 to 5 days.All results were expressed in log CFU/g.The coliform counts at 30 ∘ C and 45 ∘ C were obtained using the Conventional Multiple Tube Technique with Lauryl Sulfate Tryptose Broth (Oxoid, England) and Brilliant Green Bile Broth (Oxoid, England), with incubation at 35 ± 1 ∘ C/48 h.Positive samples were transferred to EC broth (Difco/BD, USA), with incubation at 45±1 ∘ C/24 h.The results were expressed as the log most probable number per gram (log MPN/g). Table 3 : Enterobacteriaceae species identified by miniature kits Bactray I and II from samples of organic and conventional cultivation of leafy vegetables.	2018-12-14T05:54:20.665Z	2018-02-08T00:00:00.000	{ "year": 2018, "sha1": "c3cece054a641c4f34dde918e824b39029771468", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/jfq/2018/4908316.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "c3cece054a641c4f34dde918e824b39029771468", "s2fieldsofstudy": [ "Agricultural And Food Sciences", "Biology" ], "extfieldsofstudy": [ "Biology" ] }
263795612	pes2o/s2orc	v3-fos-license	Concept of spectrometer for resonant inelastic X-ray scattering with parallel detection in incoming and outgoing photon energies A spectrometer for resonant inelastic X-ray scattering (RIXS) is proposed where imaging and dispersion actions in two orthogonal planes are combined to deliver full two-dimensional map of RIXS intensity in one shot with parallel detection in incoming hvin and outgoing hvout photon energies. Preliminary ray-tracing simulations with a typical undulator beamline demonstrate a resolving power well above 11000 in both hvin and hvout near a photon energy of 930 eV, with a vast potential for improvement. Combining such a spectrometer - nicknamed hv2 - with an XFEL source allows efficient time-resolved RIXS experiments. Introduction High-resolution RIXS (Resonant Inelastic X-ray Scattering) is a synchrotron radiation based photon-in / photon-out experimental technique, which gives information about charge-neutral lowenergy excitations in correlated electron systems (e.g.crystal field, charge transfer or spin excitations) in solids, liquids and gases (Kotani & Shin 2001). Detalization of the physical information available from RIXS is directly connected to the resolution and detection efficiency of the RIXS instrumentation (for some entries see Nordgren et al. 1989;Hague et al. 2005;Tokushima et al. 2006;Ghiringhelli et al. 2006).Soft-X-ray RIXS spectrometers of the last generation (Ghiringhelli et al. 2006) based on variable line spacing (VLS) gratings and high-resolution CCD detectors allow routine operation with a resolving power E/ΔE better than 10000 at 1 keV photon energy, which takes the RIXS experiment from the energy scale of chargetransfer and crystal-field excitations to that of orbital and magnetic excitations (Schlappa et al. 2009).The position-sensitive CCD detector allows parallel detection in outgoing photon energies hv out .Modern beamlines, delivering the spot size on the sample in a few μm range, enable slitless operation of the RIXS spectrometers, dramatically increasing their detection efficiency.Interesting is a non-conventional concept of active grating monochromator -active grating spectrometer (Fung et al. 2004) which promises an increase of the detection efficiency by 2 orders of magnitude. The full set of RIXS data is a two-dimensional map of X-ray scattered intensity I(hv in ,hv out ) depending on the incoming hv in and outgoing hv out photon energies.Presently, one acquires I(hv in ,hv out ) in a sequental fashion by measuring one-dimensional I(hv out ) spectra over a series of separate hv in selected by the beamline monochromator.Here, a concept is presented of the RIXS spectrometer to enable acquisition of the whole I(hv in ,hv out ) map in one shot with parallel detection in hv in and hv out . Concept Optical scheme of such a RIXS spectrometer -nicknamed hv 2 for simultaneous detection in hv in and hv out -is shown in Fig. 1.The monochromator produces in its (stigmatic) focal plane a line image of light with vertical dispersion in energy hv in .A refocusing KB optics consisting of the vertically and horizontally refocusing (plane-elliptical) mirrors V-RM and H-RM brings this image into a line focus on the sample.Extreme vertical demagnification of the refocusing stage is necessary to squeeze the vertical extension of the image, important for inhomogeneous samples, and extreme horizontal demagnification to deliver small source size and thus high energy resolution for the spectrometer operated slitless.The light scattered from the sample is intercepted by a vertically focusing (plane-elliptical) mirror FM of the spectrometer stage.It operates in the vertical plane to bring the scattered light image into a magnified image on the two-dimensional positionsensitive detector (PSD) with vertical dispersion in hv in .A (spherical) VLS grating operates in the horizontal plane to disperse the scattered light in hv out and focus it onto the PSD with horizontal dispersion in hv out .In this way the full two-dimensional image of RIXS intensity I(hv in ,hv out ) is formed on the PSD in one shot with parallel detection in hv in and hv out . A few comments: • If one inserts a slit in the monochromator focal plane to select one single hv in , the hv 2 spectrometer becomes fully equivalent to standard RIXS spectrometers with VLS gratings (Ghiringhelli et al. 2006) where the dispersive plane is horizontal and the H-FM acts to focus the beam on the CCD in the non-dispersive vertical plane, increasing the spectrometer acceptance by a factor of ~3 compared to the single-grating design (Hague et al. 2006).Therefore, the hv 2 concept adds simultaneous detection in hv in as a 'free lunch' without compromising the detection efficiency in hv out compared to the standard RIXS spectrometers.There is no compromise on resolution either provided the horizontal demagnification is sufficient; • The hv 2 spectrometer inherently includes an option for XAS data acquisition in the total fluorescence yield (TFY) in one shot of parallel detection in hv in .This measurement mode is realized simply by setting the spherical grating to the zero diffraction order.The vertical line formed in this case on the PSD is the XAS spectrum as a function of hv in .Note that aberrations from the grating affect in this case only the horizontal image profile and, by integration in the horizontal direction, have no influence on the XAS spectrum.In this connection one should mention the Energy-Dispersive XAS (see a review in Pascarelli et al. 2006) which is an efficient technique for one-shot XAS data acquisition with hard X-rays.This techniques uses however transmission of X-rays through the sample and is not applicable in the soft-X-ray range; • Response of the hv 2 spectrometer in hv in critically depends on homogeneity of the sample along the line focus.However, the vertical refocusing can reduce its extension below 100 μm within an hv in bandwidth of a few eV (see the ray tracing analysis below) which is acceptable for most of the samples.The sample homogeneity is not an issue for studies on liquids and gases; • An important aspect of the hv 2 concept compared to the usual sequential data acquisition is that the full RIXS snapshot I(hv in ,hv out ) of the electronic structure is acquired in the same instant of time.This is a crucial advantage for studies of time evolution processes, including kinetics of chemical reactions; • The total optical length of the spectrometer stage has to be constant under variation of photon energy in order to stay focused in hv in .Maintaining high resolution in hv out requires then two degrees of freedom of the optical system to cancel both the defocus and coma aberrations (Strocov et al. 2008).They can be delivered by (coordinated) variation of the grating pitch angle and the grating translation. The hv 2 concept promises adequate performance already with common 3 rd generation synchrotron sources (see below).However, its full potential unfolds with X-ray free-electron laser (XFEL) sources.Their inherently pulsed operation ideally combines with the hv 2 spectrometer ability to produce full I(hv in ,hv out ) snapshots in the same instant of time, allowing efficient time-resolved measurements (Patterson et al. 2009).Furtermore, the second data dimension radically alleviates the problem of low average intensity of the XFEL radiation.On the technical side, an important advantage of XFELs is a round spot profile.Much smaller horizontal source size compared to the synchrotron sources allows further reduction of the horizontal spot size on the sample and thus increase of resolution in hv out .Furthermore, small angular divergence of XFEL radiation helps reduction of the size of the optical elements. Ray-tracing simulations Expected performance of the hv 2 spectrometer installed at a typical undulator beamline of a 3 rd generation synchrotron source was estimated with preliminary ray tracing simulations.No attempt ws made to optimize exact parameters of the optical scheme.The simulations were peformed with the code PHASE (Bahrdt et al. 1995).In order to reduce influence of the polynomial approximation of the ellipsoidal shape (Peatman 1997) in this code, the ray tracing was restricted around the central ray within an angular divergence of 0.01 mrad for the refocusing stage and 0.1 mrad for the spectrometer.The refocusing and spectrometer stages each had a length of 5500 mm.The light incidence angle was 88 o for all optical elements.The slope errors of all optical elements were taken as 0.5 μrad, which is a conservative state-of-art value for the (bendable) plane optics.The energy was chosen as 930 eV (near the 2p 3/2 core level of Cu). Monochromator adopts the classical collimated-light PGM scheme (Follath & Senf 1997) with stigmatic focus.The parameters of the source and positions of the optical elements are chosen close to the ADRESS beamline of SLS (Strocov et al. 2009).In order to increase the horizontal demagnification, the collimating mirror is chosen cylindric, with the horizontal focusing performed by the focusing mirror having the toroidal shape.With a grating of 1400 lines/mm and C ff = 2.75, the monochromator delivers E/ΔE ~ 15000.The image in its focal plane formed by 5 monochromatic rays separated by 1 eV is shown on Fig. 2 (left). Refocusing stage has a total length of 5500 mm.The V-RM is installed in 500 mm from the sample and H-RM in 200 mm, delivering extreme horizontal demagnification of the spot (combining the two KB mirrors in one ellipsoidal mirror results in a 'smiley' distortion of the image on the sample). With the light incident on the sample at a grazing angle of 30 o and scattering angle 90 o , the image seen by the spectrometer is shown in Fig. 2 (center).Its vertical extension is less than 100 μm.Note certain distortion of the vertical scale due to spatial extension of our polychromatic source.Each spot has a vert.× hor.FWHM size of 1.7 × 9.2 μm 2 . Spectrometer stage has the V-FM in 200 mm from the sample, delivering extreme vertical magnification of the image on the PSD to reduce influence of its pixel size.The optical element dispersing the light in hv out is a spherical VLS grating with a central groove density a 0 of 3500 lines/mm installed in ~1500 mm from the sample (optimization of the grating position and VLS parameters followed the procedure from Ghiringhelli et al. 2006).The PSD is turned to a grazing incidence angle of 20 o in the horizontal plane to reduce the effective pixel size for hv out .Certainly the spectrometer stage can use other optical schemes such as the Hettrick-Underwood one (Hague et al. 2006) bringing larger acceptance in the dispersive plane. The image formed on the PSD is shown in Fig. 2 (center).The monochromatic lines are now dispersed in hv out (= hv in in our case) in the horizontal direction.This image is therefore the full map of scattered intensity resolved in hv in and hv out .In convolution with an effective PSD spatial resolution of 24 μm (typical of the nowadays CCD detectors) the image demonstrates E/ΔE in hv in and hv out better than 11100 and 13700, respectively, allowing state-of-art RIXS experiments. Certain distortion of the hv in scale can easily be corrected by post-processing of the data. The above preliminary optical scheme has a vast room for improvements.The resolution in hv in can be increased by optimization of the refocusing stage vertical demagnification, which defines the vertical image size on the sample and thus on the PSD.Most important, one can implement the monochromator without any horizontal focusing to deliver divergent light directly to the H-RM. The concomitant increase in the nominal horizontal demagnification by a factor ~3 will dramatically reduce the horizontal spot size on the sample and thus improve the spectrometer resolution in hv out . Summary A concept of spectrometer for resonant inelastic X-ray scattering (RIXS) -nicknamed hv 2 -has been presented where imaging and dispersion actions in the vertical and horizontal planes, respectively, are combined to deliver full two-dimensional map of RIXS intensity in one shot with parallel detection in hv in and hv out .This scheme is free of any compromise on energy resolution or detection efficiency compared to the conventional RIXS spectrometers.Preliminary ray-tracing simulations with a 3 rd generation synchrotron source demonstrate a resolving power above 11000 in hv in and above 13700 in hv out at 930 eV photon energy, with a vast room for further improvements such as a monochromator without intermediate horizontal focusing.Combining the hv 2 spectrometer with an XFEL source allows efficient time-resolved RIXS experiments with further increase of resolution. Fig. 1 .Fig. 2 . Fig. 1.Operational principle of the hv 2 spectrometer.The image formed on the PSD is the full two-dimensional image of RIXS intensity I(hv in ,hv out ) acquired with parallel detection in hv in and hv out .	2019-04-12T19:56:29.963Z	2009-09-26T00:00:00.000	{ "year": 2009, "sha1": "74b77d93b9ec7a4cc7b713d628b6b24b529a6c62", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "74b77d93b9ec7a4cc7b713d628b6b24b529a6c62", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
236706936	pes2o/s2orc	v3-fos-license	Performance analysis of AS-SOFC fuel cell combining single and sinusoidal flow field: numerical study The performance of a solid oxide fuel cell (SOFC) was examined using 3D computational fluid dynamics to model mass and heat flows inside the channels. In the present investigation, a SOFC fuel cell with a new flow field based on a sinusoidal flow has been studied. The latter was tested and compared with a single flow using ANSYS FLUENT. The obtained results showed that at a given operating voltage, the maximum power for the sinusoidal and the single flow fields were 1.43 and 1.35W/cm, respectively. By taking in addition, into account the concentration, activation and Ohmic losses; it was noticed that the distribution of velocity and temperature for the sinusoidal flow led to bettered results. Furthermore, it was observed that the maximum use of H2 mass fraction consumed in sinusoidal and single flow field designs were 60% and 55% respectively. Similarly, the highest H2Omass fraction values produced for the sinusoidal and single flow designs were 42% and 34% respectively. This model was validated and confronted to previous data. The present results agree well with reported studies in literature. Introduction Nowadays, fuel cell technology is a fast growing scientific and technical field. It is in the process of constituting the core of the next industrial revolution. Recently, solid oxide fuel cells studying has attracted the attention of many researchers worldwide for clean energy production [1]. They are regarded as the most advantageous types appropriating renewable energy sources to produce electricity and provide great power density, which can be employed to versatile power generation systems [2]. SOFCs provide several advantages such as: high efficiency [3,4], low emission of pollutants, working in silence [1], fuel flexibility [5], long-term stability [6], use of low-cost catalyst material [7] and the ability of utilization for cogeneration [8,9]. Flow channel design has a vital importance on the performance of SOFC fuel cells. Consequently, various investigations have been carried out to explore the influence of flow channels design. Amongst these, the most often mentioned model given by Achenbach [10] studied with time-dependent the effects of flow manifolding with utilizing differential and finite equations that allow determining heat and mass transfer in SOFCs. The researchers found that the counter-flow design has an impact in improving the performance compared to cross and co-flow. Xu et al. [11] conducted a numerical model to study the planar solid oxide electrolyzer cell (SOEC) with diverse flow forms (co-flow, counter-flow and cross-flow fields). The authors arrived at that the cross-flow design presented the best performance as it produced hydrogen the most compared to the co-flow and counter-flow under similar boundary conditions. Hawkes et al. [12,13] presented 3D simulations on a SOEC stack under cross-flow configuration. They have discussed profiles of activation overpotential, temperature, operating potential, current density, Nernst potential, the gas composition of anode-side and cathode-side and hydrogen production at several deferent operating conditions of the stack using ANSYS FLUENT. Wuxi et al. [14] examined the impact of geometric parameters such as the dimension of the channel, the repeating cell height and the width of the manifold on the flow improvement. The simulation results showed that the ratio of the outlet to the inlet manifold width was affected significantly by the flow uniformity. Valery et al. [15] proposed a modified planar SOFC fuel cell with internal reforming. The study combined parallel flow and a new flow field design. The simulation analysis confirmed that the new modified anode configuration enhanced the performance cell and realized more uniform distribution of species concentration, velocity and current density. Saied et al. [16] compared diverse flow fields including helical, modified parallel design, traditional parallel, single-entry serpentine, double-entry serpentine and triple-entry serpentine. Their results noted that the triple-entry serpentine configuration achieved regular distributions for fuel and oxygen and presented better performance compared to other designs. Huang et al. [17] considered a new design using short guide vanes equally spaced around the feed header of the doubleinlet/single-outlet flows of rib-channel interconnects to examine the flow uniformity in several interconnectors and its impact on cell performance. They established that the new design enhanced the degree of flow uniformity in interconnects and achieved a greater value of peak power density. Lin et al. [18] developed analytically the influence of rectangular ribs on the concentration polarization of planar SOFCs. The study showed that the best design was achieved by reducing the overall concentration and Ohmic polarization of the ribs. Qiuwan et al. [19] performed numerical studies examining the performance of SOFC with and without rectangular obstacles. This attempt indicated that the fuel cell with obstacles enhanced the cell performance as it attained better current density, species concentration and temperature distribution. Clearly, the flow field configuration has a vital effect on the performance of the fuel cell and is the key factor in SOFC design optimization. Therefore, further studies on new field designs are recommended to obtain more acute and reliable results on cell performance. The main objective of the present investigation is to enhance the performance of a solid oxide fuel cell (SOFC). This study contributed and help to choose the optimum configuration for the SOFC fuel cell system manufacturers and their applications and commercialization. Thus, a new flow field based on a sinusoidal flow has been proposed and studied. The SOFC is an electrochemical device as other fuel cells; it is a combination of a reactant (oxygen) and a fuel to produce electricity and heat. The electrochemical reaction occurring in a planar SOFC involves the following individual reactions, at the anode and cathode [20], which could be expressed as follows: At cathode Overall Cell reaction H 2 þ The cell voltage [21][22][23][24] could be calculated from: where h Ohm , h act and h con are the Ohmic, activation and concentration losses over-potential respectively [25]. The Nernst equation is also an open-circuit voltage (OCV); its value is related to the gas composition, the operating pressure, the operating temperature… etc. It is defined by equation (5) [26][27][28][29]: where T (K) is the operating temperature, P (Pa) the partial pressures of reacting species, R (J mol À1 K À1 ) is the gas constant, F (C mol À1 ) is Faraday number, and DG (J mol À1 K À1 ) is the Gibbs free energy. Theoretical analysis Mathematical models of a planar SOFC involve the governing equations of mass, momentum, energy and transport of charges. Moreover, knowledge of the Butler-Volmer equation was necessary for analyzing a model. In this study, a SOFC model was implemented in ANSYS FLUENT 18.1 software, for solving 3D and steady-state governing equations by using the finite volume approach. The applicable equations are: -Mass conservation equation [30]: The steady-state mass conservation equation is given as: The source terms, which are applicable at the interface electrode-electrolyte, are obtained from equations (8)−(10), calculated as [31][32][33][34]: where r (kg m À3 ) is the gas density, v (m s À1 ) the velocity, e is the porosity and S m (kg m À3 s À1 ) the mass source. -Momentum equation [30]: Due to low Reynolds and the steady-state momentum conservation equation can be written as: where k g is the gas phase permeability (m 2 ) and m (kg m À1 s À1 ) the gas viscosity. where y i is the mass fraction of species i, D eff (m 2 s À1 ) the effective diffusivity coefficient between species i and j, S i (kg m À3 s À1 ) the source term for species i and x j the mole fraction for species j. -Energy equation [30]: The energy conservation equation can be formulated as: where k eff (W m À1 K À1 ) is the coefficient of thermal conductivity, c p (J kg À1 K À1 ) is the specific heat capacity, and S T (W m À3 ) the heat source. -Charge transport [16,[31][32][33][34][35]: In order to solve the charge conservation equation, electrons and ions transport should be considered. The electronic charge occurs in the electrodes and interconnectors; while, the ionic charge only exists in the electrodes and electrolyte. Thus, the governing equations for charge by Ohm's law are as follows: Electronic charge balance: Cathode electrode layer ∇: Ionic charge balance: Anode electrode layer ∇: Cathode electrode layer ∇: where s a and s c (s m À1 ) denote the electrical conductivity for anode and cathode, respectively. While J a and J c designating the volumetric current densities of anode and cathode; based on the Butler−Volmer equation [30] could be computed from equations (19) and (20) as: where J 0,a and J 0,c (Am À2 ) are the reference exchange current densities of anode and cathode and A V (m 2 m À3 ) is the reactive surface area per unit volume. The reference exchange current density for anode and cathode could be calculated as [36]: k a and k c refer to the pre-exponential coefficients. E act,a and E act,c are the activation energies of the anode and cathode exchange current densities, respectively. P is the partial pressure of chemical species [36]. Methodology The geometrical SOFC single cell was executed in the commercial software GAMBIT (Version 2.4.6). Table 1 [37]. The subsequent step after producing cell geometry was the creation of high-quality mesh. This was done by an ANSYS WORKBENCH MESH. In order to achieve the best quality, the mesh independency was studied by creating three different meshes with 76,800, 194,560 and 304,000 hexahedral cells. The results of mesh independency are presented in Table 2, from which it was noticed that mesh of 304,000 cells led to a reasonable agreement when compared to the numerical results of [37]. For more details about the mesh independency, refer to [34,38,39]. Once the computational mesh was created, it must be imported into the solver ANSYS FLUENT 18.1. After examining the mesh file, setting the parameters, materials and defining the boundary conditions for the two geometries; as listed in Tables 3 and 4, respectively. Model validation The predicted current density versus voltage (J−V) results for our numerical model showed an acceptable accuracy with the data obtained from references [37,46] as shown in Figure 2. Nonetheless, the simulation manifested different values of current density at lower values of voltage. This is most probably due to errors and hypotheses associated with the SOFC model. Conversely, when using default parameters, the results were somewhat far from the other curves. This is why all conditions must be scrutinized and carefully readjusted according to the physical properties, to obtain results comparable to the experimental results. Figure 3a and b. It could be deduced that the distribution near GDL had improved, due to deflection design which increased the anode velocity and augmented the distribution mechanism. As a result, the velocity in the anode channel for the sinusoidal design was higher than the simple single-channel which led to cell performance enhancement. Distribution of hydrogen and water mass fraction The hydrogen (H 2 ) mass fraction contours on the anode channel for both designs are represented in Figure 4 at 0.6 V. The mass fraction reduction along the z-channel is due to consumption of hydrogen by the electrochemical [44,45] reaction. It is also found that the mass fraction diminished from 0.97 to 0.38 and 0.43 for the sinusoidal and single flow design respectively. These results signify that the reaction rates at the sinusoidal flow design are greater than those detected in the single flow. The maximum use of hydrogen mass fraction consumed in the sinusoidal and single flow field designs are 60% and 55% respectively for the operating voltage of 0.6 V, as given in Table 5. According to Figure 4 along with Table 5, the sinusoidal form gave a greater level of hydrogen mass fraction. Consequently, this configuration is considered more uniform in diffusing of hydrogen mass fraction because of the best use of the active area in the CL (catalyst layer). On the other hand, increasing hydrogen mass fraction consumption increases the water (H 2 O) mass fraction produced as displayed in Figure 5. The sinusoidal flow design has a better distribution compared to the single flow field as demonstrated in Figure 5. The highest water mass fraction values produced for the sinusoidal and single flow designs were 42% and 34% respectively for the operating voltage 0.6 V, as shown in Table 6. This uniform distribution leads to regularly generate heat and decrease thermal stresses, suggesting that the sinusoidal cell aids in reducing losses. This could be explicated by the higher rate of reaction. Specifically, the highest temperatures for the sinusoidal and single flow were 1128.30 K and 1126.95 K respectively for the operating voltage 0.6 V. For all the geometrical configurations, the highest temperature value was always located in the center of the cell which represents the electrolyte due to the counter-flow direction as marked in the literature [47,48]. Concentration, activation and ohmic overpotentials The mathematical model was able to predict the overpotentials inside the SOFC for both sinusoidal and single cells, which is necessary for testing their performance. As presented in the literature, there are three overpotentials, caused by various mechanisms. The concentration overpotential is based on the resistance to transport the fraction of reactants and oxidant. As shown in Figure 7a the sinusoidal flow field led to a reduction in the concentration over potential, whereas, the concentration over potential increased with the current density for both designs. The activation overpotential was slightly higher for the single cell than the sinusoidal as shown in Figure 7b. This is due to increasing the temperature in the sinusoidal cell which contributed in reducing activation polarization. In addition, the sinusoidal cell took part in minimizing the Ohmic overpotential as displayed in Figure 7c. This is due to the improvement of the operation temperature which raises the conductivity of the electrolyte. From the above mentioned arguments, it could be deduced that the sinusoidal cell significantly contributed to the performance enhancement and to minimization of the concentration, activation and Ohmic overpotentials compared to the single cell. Figure 8 displays the polarization (J−V) and the power density curves for the two designs; from which it could be readily noticed that the sinusoidal flow design was more performable than the single flow. Additionally, the maximum power densities of the sinusoidal and single flow designs were 1.43 and 1.35 W/cm 2 , respectively. This result indicates that the sinusoidal flow design offers the most use of the hydrogen as considered in the previous section. Moreover, the difference in performance between the two designs is well remarked at low voltages due to decreasing in the concentration losses. As a consequence, the sinusoidal flow design has contributed significantly in ameliorating the performance. Conclusions In this paper, two different flow designs of SOFC have been numerically developed in the software ANSYS FLUENT 18.1. The main motivation behind this research was to ameliorate the performance of SOFC fuel cells. The results obtained from this model were confronted to available research data in the literature. Simulation results including velocity distributions, gas species, temperature, concentration overpotantial, activation overpotantial, Ohmic overpotantial and cell performance for both flow designs have been introduced and discussed. The sinusoidal design gave a better distribution of velocities and temperatures than the single flow, which improved the transport of hydrogen of the cell. As a result, more water was produced at the anode channel and accordingly, the cell performance rose. On the other hand, it was established that the channel design performed an important role in enhancing the performance and decreasing the overpotantials losses of SOFC fuel cells. The CFD results indicated that, when the cell was run at high voltage, the influence of channel design on the cell performance was negligible; whereas, when treated at the low voltage the channel design influence became significant. Lastly, it could be concluded that the	2021-08-03T00:06:30.888Z	2021-01-01T00:00:00.000	{ "year": 2021, "sha1": "0f1da9ff6c082335765b258cdf5f42229fd8df82", "oa_license": "CCBY", "oa_url": "https://www.rees-journal.org/articles/rees/pdf/2021/01/rees210009.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "666b5e23e6ba61aa92a5b0deb6c149f8b0ad98d4", "s2fieldsofstudy": [ "Engineering", "Materials Science", "Physics" ], "extfieldsofstudy": [ "Materials Science" ] }
257520306	pes2o/s2orc	v3-fos-license	Spherical Attapulgite/Silica Aerogels Fabricated via Different Drying Methods with Excellent Adsorption Performance Dye wastewater has caused great harm to the environment, which is an urgent problem to be solved. As typical three-dimensional porous materials, aerogels have attracted great interest in dye wastewater treatment. In this work, spherical attapulgite/silica (ATP/SiO2) gels were initially prepared by easily scalable sol-gel dripping methods and then dried to aerogels with three drying techniques, namely, supercritical CO2 drying (SCD), freeze-drying (FD), and ambient pressure drying (APD). The effect of the drying techniques and heat-treated temperature on the physical characteristic, morphological properties, microstructure, and chemical structure of the spherical ATP/SiO2 aerogels were investigated. The macroscopic morphology of the spherical ATP/SiO2 aerogels was homogeneous and integrated without local cracking. The average pore diameter and specific surface area of the spherical ATP/SiO2 aerogels prepared by the three drying techniques were in the range of 6.8–8.6 nm and 218.5–267.4 m2/g, respectively. The heat treatment temperature had a significant effect on the pore structure and the wetting properties of the aerogels. The 600 °C heat-treated aerogels were subjected to adsorption tests in methylene blue (MB) solution (60 mg/g, 100 mL), which exhibited a great adsorption capacity of 102.50 mg/g. Therefore, the resulting spherical ATP/SiO2 aerogels possessed multipath preparation and exhibited an efficient adsorption performance, with the potential to be applied as an adsorbent for dye wastewater. Introduction Highly toxic, carcinogenic, and teratogenic dye wastewater has caused serious damage to the ecological environment [1,2]. Dyes in wastewater can destroy the self-purifying function of water bodies, since they can reduce water clarity, consume oxygen in the water, and hinder photosynthesis in plants [3]. Remarkably, most organic dyes are difficult to degrade naturally and can persist in the natural environment [4]. Methyl blue (MB) is a hydrophilic organic compound with free solubility in water, representing typical cationic dyes [5,6]. Adsorption has been recognized as one of the most efficient methods for removing dyes from wastewater due to the fact of its widespread use, effectiveness, and reusability. As typical three-dimensional porous materials, SiO 2 aerogels have attracted great interest in adsorption fields [7]. SiO 2 aerogels can be employed as dye adsorbents owing to the following two advantages: a high specific surface area that enables numerous adsorption sites and properties that can be easily tuned by surface modification to adapt to different application environments [8]. Yao et al. [8] used water as a solvent for preparing MTMS/tetraethoxysilane silica aerogels with a high porosity of 96.36%. Jiang et al. [9] synthesized amine-grafted silica aerogels by sodium silicate to adsorb CO 2 . tact structure and low density. The main disadvantages of the SCD method are the required energy, maintenance cost, and high-pressure conditions [30]. FD is the process of removing water or other solvents from frozen samples through sublimation in a vacuum environment. The freezing process can evade the shrinkage of the aerogel skeleton, but the crystallization of the solvent will enlarge the pore size and destroy the original pore structure [31]. The principle of APD is to elevate the temperature of the solvent above the boiling point and transform it into gas, which is the most convenient technique for removing solvents from porous materials. The convenient evaporation of the APD method with its low cost is very suitable for mass production and wide application [19,32]. However, the capillary pressure during the drying process exceeds the elastic limit of the solid structure, which may result in the shrinkage and collapse of the porous aerogel materials [33]. To reduce the effect of capillary forces and control the irreversible shrinkage of SiO 2 aerogels during the APD process, surface hydrophobic modification by silylation is of great importance. Selay Sert et al. [34,35] investigated the effects of different silylation agents (including MTMS, trimethylchlorosilane, and methyltriethoxysilane) on the microstructure and chemical properties of SiO 2 aerogels under APD. The results show that the MTMS-modified SiO 2 aerogel had a highly developed three-dimensional network structure and high specific surface area. Therefore, MTMS was used as a silane modifier in this work. To synthesize an excellent dye adsorbent, ecofriendly spherical ATP/SiO 2 aerogels were successfully prepared via three drying methods (SCD, FD, and APD). Hydrophobization of the spherical ATP/SiO 2 aerogels was achieved by MTMS modification. The structure of the as-prepared spherical ATP/SiO 2 aerogels with three drying techniques was analyzed and compared. The effects of the heat treatment temperatures on the physicochemical properties of the resulting aerogels were studied. Furthermore, the adsorption performance of the spherical ATP/SiO 2 aerogels dried by APD and heat treated at 600 • C was also investigated. The spherical ATP/SiO 2 aerogels have excellent adsorption properties and can be prepared in multiple ways. This suggests that the prepared aerogels have promise as an adsorbent for dye wastewater. Synthesis of Spherical ATP/SiO 2 Gels The ATP/SiO 2 solution was prepared according to the following steps. Alkaline silica sols, H 2 O, and ATP were directly separated in a pot with a mass ratio of 1:1:0.5. Acetic acid was used as a catalyst to adjust the pH value of the solution to 4.5~5.5. Then, the mixture was stirred for approximately 30 min at room temperature. Subsequently, the ATP/SiO 2 solution was obtained by ultrasonic treatment for 15 min. Furthermore, the spherical ATP/SiO 2 gels were prepared by the dripping method. The ATP/SiO 2 solution was dripped into dimethicone oil at 80 • C to form spherical ATP/SiO 2 gels. Finally, the spherical ATP/SiO 2 gels were washed five times with deionized water at 60 • C to remove impurities and then aged in water for 12 h. Preparation of Spherical ATP/SiO 2 Aerogels Using Different Treatment Techniques The hydrophobic spherical ATP/SiO 2 gels were obtained by the surface modification process. Trimethoxymethylsilane (MTMS) was employed as a hydrophobic modification agent. The spherical ATP/SiO 2 gels were firstly placed in a mixed MTMS/ethanol solution with a volume ratio of 1:4 for 12 h and then soaked in ethanol solution for 24 h to wash off the excess MTMS and reaction byproducts. Subsequently, the primed samples were dried to hydrophobic spherical ATP/SiO 2 aerogels using the SCD method in an autoclave for 4 h (50 • C, 10 MPa, HELIX 1.1 system, Applied Separations, Inc., Allentown, PA, USA), denoted as HAS-S. The samples without hydrophobic modification were denoted as AS-S. In addition, the spherical ATP/SiO 2 gels were initially placed in a mixed MTMS/tertbutanol solution with a volume ratio of 1:4 for 12 h and then washed with tert-butanol solution for 24 h to exchange the excess MTMS and reaction byproducts. Finally, the hydrophobic spherical ATP/SiO 2 aerogels were prepared using the FD method (frozen at −15 • C in a fridge for 5 h and then dried at −80 • C for 12 h), denoted as HAS-F. Moreover, the previous hydrophobic spherical ATP/SiO 2 gels were adequately washed with N-hexane for 24 h to displace the water and byproducts from the samples. The volume of N-hexane was 5 times larger than the volume of the gel. After aging and solvent exchange, the hydrophobic spherical ATP/SiO 2 aerogels were obtained by APD in an air oven (50 • C for 6 h, and 80 • C for 4 h), denoted as HAS-A. To study the variation in the physicochemical properties of the spherical aerogels, the HAS-A was heat treated at different temperatures (200 • C, 400 • C, 600 • C, and 800 • C) for 3 h in a muffle furnace with a heating rate of 5 • C/min. The 600 • C heat-treated HAS-A is denoted as AS-A. Characterization The apparent densities (ρ) of the samples were calculated by ρ = m/v, where m and v are the apparent mass and volume. Scanning electron microscope (SEM) images of the samples were carried out using an Ultra-55 (Zeiss, Oberkochen, Germany) at an operating voltage of 15.0 kV. Transmission electron microscope (TEM) images were recorded on a JEOL JEM 2100F microscope at 200 kV. X-ray photoelectron spectroscopy (XPS) was carried out using an Axis Ultra DLD equipped with Al Ka (1486.6 EV). Fourier transform infrared spectroscopy (FT-IR) measurements were recorded on an FT-IR spectrometer (Spectrum 100, Perkin Elmer, Waltham, MA, USA) for KBr pellets. The contact angles were carried out using a JC2000D1 by grinding the aerogels into powder and then pressing them into pieces. The thermogravimetric (TG) analysis was conducted on a Netzsch STA449F5 thermal analyzer (Selb, Germany) in a temperature range of 30 • C to 800 • C with a heating rant of 10 • C/min under flowing air. The BET specific surface area (S BET ), pore volume, and pore distribution were measured using a V-sorb 2800P after pretreatment for 6 h at 120 • C. The zeta potential was analyzed and measured using a Malvern Zetasizer Nano ZS90 (Malvern, UK). Adsorption Experiment The AS-A was used as an adsorbent for studying the adsorption performance. The MB solution was used to simulate the wastewater, and all adsorption tests were carried out at room temperature. A total of 50 mg AS-A and 100 mL MB solution were mixed in a centrifuge tube and shaken for 100 min (200 rpm/min) at room temperature. After a sufficient adsorption process, the solution was centrifuged for 5 min (5000 r/min), and the concentration of residual dye in the supernatant was analyzed using a UV-Vis spectrometer at 663 nm. The influence of the pH, contact time, and MB concentration were systematically studied to determine the adsorption performance of the AS-A for MB. The removal rate of MB was calculated by a comparison of the initial concentration and the residual concentra-tion after adsorption. The equations for the adsorption efficiency (R) and the adsorption capacity (q e ) of AS-A are shown as follows: where C 0 and C e represent the initial and residual concentration of MB (mg/L), respectively; V is the volume of the MB solution (L); and m is the mass of AS-A (g). Formation Mechanism of the Spherical ATP/SiO 2 Aerogels The schematic diagram of the synthesis process for hydrophobic ATP/SiO 2 aerogels is shown in Figure 1a. Because of the incompatibility between water and oily solvent, the hydrophilic ATP/SiO 2 mixed sols can disperse in the oily solvent. When the surface tension of the sol droplet is much greater than the gravity, the isotropic spherical ATP/SiO 2 sol forms in the oil phase [16]. Considering the influence of the temperature on sol-gel kinetics, the oil bath temperature was set at 80 • C for forming the spherical ATP/SiO 2 gel [9]. The final spherical ATP/SiO 2 aerogels were obtained by three drying methods. Figure 1b presents the synthesis mechanism of the spherical hydrophobic ATP/SiO 2 aerogels. Silica sols exist as nanoscale silica particles, which are distributed uniformly in the water. Both silica sol particles and ATP contain many active -OH groups, which can favorably translate into the ATP/SiO 2 gels by a hydrolysis polycondensation reaction under the appropriate conditions of the silica concentration, pH value, and temperature. In addition, the silica sol is bound with ATP via hydrogen bonding, and then the MTMS used as hydrophobic modifier are grafted onto the ATP/SiO 2 gels by the immersion modification method. Effect of Drying Techniques and MTMS Modification on Physical Characteristic and Morphological Properties of Spherical ATP/SiO 2 Aerogels A comparison of the different drying methods for preparing the spherical ATP/SiO 2 aerogels is shown in Figure 2. All spherical ATP/SiO 2 aerogels exhibited an integrated macroscopic morphology with a diameter of approximately 2.5 ± 0.15 mm (2.65 mm, 2.52 mm, and 2.35 mm for HAS-S, HAS-F, and HAS-A, respectively). By contrast, the shrinkage rate of the HAS-A was larger than the other samples resulting from the drying capillary pressure of the solvent evaporation during the APD process. Table 1 shows the physical parameters of the ATP and spherical ATP/SiO 2 aerogels prepared by different drying methods. The densities of the ATP/SiO 2 aerogel composites (0.46-0.65 g/cm 3 ) decreased greatly due to the loose three-dimensional skeleton structure instead of the relatively dense structures of the ATP (2.41 g/cm 3 ). Meanwhile, the densities of the ATP/SiO 2 aerogels modified by MTMS were larger than the samples without hydrophobic modification, which is attributable to the grafting of CH 3 -(SiOH) 3 groups. The FT-IR spectra of the as-prepared aerogels are presented in Figure 3a. A broad transmittance appeared at approximately 3200-3700 cm −1 , which is attributed to the stretching vibrations of -OH groups [36]. The symmetric stretching and tetrahedra bending vibration of Si-O-Si and Si (or Al)-O appeared at 1035 cm −1 and 1100 cm −1 [37]. The peaks at approximately 1450 cm −1 and 1640 cm −1 are assigned to the deformation vibration of the adsorbed water and bound water [32,38]. By contrast, there was a new peak at 780 cm −1 , which is normally observed in the hydrophobic group and corresponds to the stretching vibration of -CH3 in MTMS [39]. Moreover, all of the modified samples showed the symmetric deformation vibration of Si-C bonds and symmetric stretching vibrations of the terminal -CH3 groups at 2975 cm −1 and 1275 cm −1 ,which is proof of the The FT-IR spectra of the as-prepared aerogels are presented in Figure 3a. A broad transmittance appeared at approximately 3200-3700 cm −1 , which is attributed to the stretching vibrations of -OH groups [36]. The symmetric stretching and tetrahedra bending vibration of Si-O-Si and Si (or Al)-O appeared at 1035 cm −1 and 1100 cm −1 [37]. The peaks at approximately 1450 cm −1 and 1640 cm −1 are assigned to the deformation vibration of the adsorbed water and bound water [32,38]. By contrast, there was a new peak at 780 cm −1 , which is normally observed in the hydrophobic group and corresponds to the stretching vibration of -CH 3 in MTMS [39]. Moreover, all of the modified samples showed the symmetric deformation vibration of Si-C bonds and symmetric stretching vibrations of the terminal -CH 3 groups at 2975 cm −1 and 1275 cm −1 ,which is proof of the presence of Si-CH 3 [40]. Thus, the FT-IR spectrums have proved that the hydrophobic -CH 3 groups were successfully coupled to the HAS-S, HAS-F, and HAS-A. The XPS measurement (Figure 3b) was subsequently used to quantify the elemental composition of AS-S and HAS-S. The XPS spectra of AS-S and HAS-S show four peaks at 100, 150, 285, and 531 ev, which are assigned to Si 2s, Si 2p, C 1s, and O 1s, respectively [39]. Compared with AS-S, the relative strength of the C 1s peak of the HAS-S increased significantly, while that of the O 1s peak became weak. It can be inferred that with the addition of MTMS, the dehydration condensation reaction between the -OH and CH 3 -(SiOH) 3 groups resulted in the loss of some O elements and the grafting of -CH 3 groups. The surface wettability of the spherical ATP/SiO 2 aerogels was evaluated by contact angle measurements, which are presented in Figure 4 shows the SEM and TEM images of the prepared spherical ATP/SiO2 aerogels and pure ATP. As shown in Figure 4e, the pure ATP displays the features of a club-shaped structure with a diameter of approximately 30 nm and partial agglomeration. As shown in Figure 4a-d, all of the ATP/SiO2 aerogels exhibited inherent threedimensional porous network structures, which consisted of club-shaped ATP, SiO2 nanoparticles, and nanopores. As seen from the magnified SEM images, the unordered nano-scaled ATP did not influence the formation of the porous structure of the ATP/SiO2 aerogels. Additionally, there were some large pores in the HAS-F and densification phenomenon in the HAS-A, which is detrimental to the specific surface areas. By contrast, the AS-S and HAS-S samples exhibited significant homogeneous pore structures. Furthermore, the TEM images ( Figure 4f) show that the as-prepared ATP/SiO2 aerogels using different drying methods displayed random interconnected networks of nanometer-sized SiO2 aerogel particles and club-shaped ATP, with interconnected amorphous silica particles surrounding the ATP. Meanwhile, the ATP could form Figure 4 shows the SEM and TEM images of the prepared spherical ATP/SiO 2 aerogels and pure ATP. As shown in Figure 4e, the pure ATP displays the features of a club-shaped structure with a diameter of approximately 30 nm and partial agglomeration. As shown in Figure 4a-d, all of the ATP/SiO 2 aerogels exhibited inherent three-dimensional porous network structures, which consisted of club-shaped ATP, SiO 2 nanoparticles, and nanopores. As seen from the magnified SEM images, the unordered nano-scaled ATP did not influence the formation of the porous structure of the ATP/SiO 2 aerogels. Additionally, there were some large pores in the HAS-F and densification phenomenon in the HAS-A, which is detrimental to the specific surface areas. By contrast, the AS-S and HAS-S samples exhibited significant homogeneous pore structures. Furthermore, the TEM images ( Figure 4f) show that the as-prepared ATP/SiO 2 aerogels using different drying methods displayed random interconnected networks of nanometer-sized SiO 2 aerogel particles and club-shaped ATP, with interconnected amorphous silica particles surrounding the ATP. Meanwhile, the ATP could form hydrogen bonds with silica particles during the reaction, which could strengthen the crosslinked structures of the ATP/SiO 2 aerogels and resist the greater capillary pressure during the drying process. Satisfactorily, compared to other aerogel composites strengthened by coarse fibers, the nanoscale club-shaped ATP was conducive to improving the mechanical performance of the prepared spherical ATP/SiO 2 aerogels without destroying the internal pore structures [19]. The EDS spectrums of AS-S and HAS-S are presented in Figure 5, and the concentration of C, O, and Si elements are presented in Table 2. The apparent concentration of the C element increased from 23.8 wt% (AS-S) to 40.1 wt% (HAS-S). In addition, the homogenous distribution of the C element of HAS-S further proves the successful modification of MTMS. The N2 adsorption-desorption isotherms and BJH pore size distribution of the spherical ATP/SiO2 aerogels are described in Figure 6. All isotherms are Type IV based on the IUPAC classification, reflecting the characteristic of mesoporous materials. The adsorption-desorption curves form closed hysteresis loop, which is generally caused by the capillary condensation in the mesopores. The rapid adsorption process in the lowpressure region (0-0.1) is caused by the micropores inside the aerogel matrix. It is shown in Table 1 that the specific surface area of the as-prepared ATP/SiO2 aerogels (with the content of ATP over 60 wt%) was much higher than that of pure ATP, resulting from the improvement in the network skeleton structures of the ATP/SiO2 aerogel composites. Additionally, the specific surface areas of the aerogels obtained by different treatment techniques showed a decreasing trend (SSCD > SFD > SAPD), corresponding to the values of 248.7-267.4 m 2 /g, 241.7 m 2 /g, and 218.5 m 2 /g, respectively. The HAS-A showed the minimum specific surface area because of the particle agglomeration and closed pores appearing during the APD process (shown in Figure 4d). The HAS-S had higher specific surface areas than AS-S, which can be explained by the fact that the grafting of MTMS improves the hydrophobic property of ATP/SiO2 aerogel without damaging the nanoporous microstructure. Comparing AS-S to HAS-S, the average pore size and pore volume decreased from 8.3 nm and 0.41 cm 3 /g to 7.9 nm and 0.37 cm 3 /g, respectively. This is because some organic groups enter the larger pores of spherical ATP/SiO2 aerogels with the grafting of the CH3-(SiOH)3 group, which is favorable to increasing the porosity of HAS-S [41]. Furthermore, owing to the high capillary pressure of the solvent evaporation and shrinkage of the pore structure in the APD process, the pore volume of the HAS-A decreased significantly in comparison with the other hydrophobic samples. Compared with HAS-S, HAS-F exhibited lower specific surface area and higher pore volume due to the destruction of the original pore structure during the crystallization and sublimation of the solvent. As revealed in Figure 6b, all spherical ATP/SiO2 aerogels showed a broad pore size distribution in the range of 2-50 nm. After the hydrophobic modification, the peak of the micropores slightly shifted to higher values (Figure 6c), which can be explained by the fact that the pores in the aerogel are filled with the hydrolysis products of MTMS [41]. This indicates that the hydrophobic modification was beneficial to the microporous structure of the aerogels. Therefore, all spherical ATP/SiO2 aerogels had a good specific surface area and porous network structure, revealing the feasibility of the multipath preparation via the SCD, FD, and APD techniques. The N 2 adsorption-desorption isotherms and BJH pore size distribution of the spherical ATP/SiO 2 aerogels are described in Figure 6. All isotherms are Type IV based on the IUPAC classification, reflecting the characteristic of mesoporous materials. The adsorptiondesorption curves form closed hysteresis loop, which is generally caused by the capillary condensation in the mesopores. The rapid adsorption process in the low-pressure region (0-0.1) is caused by the micropores inside the aerogel matrix. It is shown in Table 1 that the specific surface area of the as-prepared ATP/SiO 2 aerogels (with the content of ATP over 60 wt%) was much higher than that of pure ATP, resulting from the improvement in the network skeleton structures of the ATP/SiO 2 aerogel composites. Additionally, the specific surface areas of the aerogels obtained by different treatment techniques showed a decreasing trend (S SCD > S FD > S APD ), corresponding to the values of 248.7-267.4 m 2 /g, 241.7 m 2 /g, and 218.5 m 2 /g, respectively. The HAS-A showed the minimum specific surface area because of the particle agglomeration and closed pores appearing during the APD process (shown in Figure 4d). The HAS-S had higher specific surface areas than AS-S, which can be explained by the fact that the grafting of MTMS improves the hydrophobic property of ATP/SiO 2 aerogel without damaging the nano-porous microstructure. Comparing AS-S to HAS-S, the average pore size and pore volume decreased from 8.3 nm and 0.41 cm 3 /g to 7.9 nm and 0.37 cm 3 /g, respectively. This is because some organic groups enter the larger pores of spherical ATP/SiO 2 aerogels with the grafting of the CH 3 -(SiOH) 3 group, which is favorable to increasing the porosity of HAS-S [41]. Furthermore, owing to the high capillary pressure of the solvent evaporation and shrinkage of the pore structure in the APD process, the pore volume of the HAS-A decreased significantly in comparison with the other hydrophobic samples. Compared with HAS-S, HAS-F exhibited lower specific surface area and higher pore volume due to the destruction of the original pore structure during the crystallization and sublimation of the solvent. As revealed in Figure 6b, all spherical ATP/SiO 2 aerogels showed a broad pore size distribution in the range of 2-50 nm. After the hydrophobic modification, the peak of the micropores slightly shifted to higher values (Figure 6c), which can be explained by the fact that the pores in the aerogel are filled with the hydrolysis products of MTMS [41]. This indicates that the hydrophobic modification was beneficial to the microporous structure of the aerogels. Therefore, all spherical ATP/SiO 2 aerogels had a good specific surface area and porous network structure, revealing the feasibility of the multipath preparation via the SCD, FD, and APD techniques. The TG curves of the prepared spherical ATP/SiO2 aerogels are displayed in Figure 7, under flowing air. The weight loss rates of the AS-S, HAS-S, HAS-F, and HAS-A were 14.62%, 15.72%, 12.85%, and 13.67%, respectively. The weight loss stage was mainly divided into three parts. The first stage, consisting of the volatilization of the residual solvent and the adsorbed water, appeared at 50-200°C. In this stage, the weight loss rate of the hydrophobic samples was lower than AS-S (4.51%), which reflects the hydrophobicity of the MTMS modification. Considering the residual solvent, water molecules, and CO2 during the SCD process, the weight loss rates of AS-S (4.51%) and HAS-S (3.87%) were significantly higher than those of HAS-F (2.59%) and HAS-A (2.17%). In the second stage between 200 °C and 500 °C, the weight loss was assigned to the elimination of the crystal water in the ATP and the condensation of Si-OH. The weight loss in the third stage (over 500 °C) was mainly attributed to the oxidative decomposition of Si-CH3 and the removal of structural water in the ATP [27]. It is noted that a characteristic temperature point appeared in the hydrophobic sample at approximately 630 °C, which implies the complete decomposition of the Si-CH3 group [42]. Effect of Heat Treatment on Physical Characteristic and Morphological Properties of HAS-A The influence of the heat treatment temperatures on the morphology, microstructure, and pore structure is shown in Figures 8 and 9. Heat treatment is a simple and efficient way to modify the crosslinking structure and chemistry of aerogels [43]. The 600 °C heat- The TG curves of the prepared spherical ATP/SiO 2 aerogels are displayed in Figure 7, under flowing air. The weight loss rates of the AS-S, HAS-S, HAS-F, and HAS-A were 14.62%, 15.72%, 12.85%, and 13.67%, respectively. The weight loss stage was mainly divided into three parts. The first stage, consisting of the volatilization of the residual solvent and the adsorbed water, appeared at 50-200 • C. In this stage, the weight loss rate of the hydrophobic samples was lower than AS-S (4.51%), which reflects the hydrophobicity of the MTMS modification. Considering the residual solvent, water molecules, and CO 2 during the SCD process, the weight loss rates of AS-S (4.51%) and HAS-S (3.87%) were significantly higher than those of HAS-F (2.59%) and HAS-A (2.17%). In the second stage between 200 • C and 500 • C, the weight loss was assigned to the elimination of the crystal water in the ATP and the condensation of Si-OH. The weight loss in the third stage (over 500 • C) was mainly attributed to the oxidative decomposition of Si-CH 3 and the removal of structural water in the ATP [27]. It is noted that a characteristic temperature point appeared in the hydrophobic sample at approximately 630 • C, which implies the complete decomposition of the Si-CH 3 group [42]. The TG curves of the prepared spherical ATP/SiO2 aerogels are displayed in Figure 7, under flowing air. The weight loss rates of the AS-S, HAS-S, HAS-F, and HAS-A were 14.62%, 15.72%, 12.85%, and 13.67%, respectively. The weight loss stage was mainly divided into three parts. The first stage, consisting of the volatilization of the residual solvent and the adsorbed water, appeared at 50-200°C. In this stage, the weight loss rate of the hydrophobic samples was lower than AS-S (4.51%), which reflects the hydrophobicity of the MTMS modification. Considering the residual solvent, water molecules, and CO2 during the SCD process, the weight loss rates of AS-S (4.51%) and HAS-S (3.87%) were significantly higher than those of HAS-F (2.59%) and HAS-A (2.17%). In the second stage between 200 °C and 500 °C, the weight loss was assigned to the elimination of the crystal water in the ATP and the condensation of Si-OH. The weight loss in the third stage (over 500 °C) was mainly attributed to the oxidative decomposition of Si-CH3 and the removal of structural water in the ATP [27]. It is noted that a characteristic temperature point appeared in the hydrophobic sample at approximately 630 °C, which implies the complete decomposition of the Si-CH3 group [42]. Effect of Heat Treatment on Physical Characteristic and Morphological Properties of HAS-A The influence of the heat treatment temperatures on the morphology, microstructure, and pore structure is shown in Figures 8 and 9. Heat treatment is a simple and efficient way to modify the crosslinking structure and chemistry of aerogels [43]. The 600 °C heat- Effect of Heat Treatment on Physical Characteristic and Morphological Properties of HAS-A The influence of the heat treatment temperatures on the morphology, microstructure, and pore structure is shown in Figures 8 and 9. Heat treatment is a simple and efficient way to modify the crosslinking structure and chemistry of aerogels [43]. The 600 • C heat-treated HAS-A is denoted as AS-A. As the calcination temperature increased, the appearance of the color of the HAS-A gradually changed from off-white to brick red (Figure 8a), which is mainly caused by the component of ATP. It is found from Figure 8b-e that the HAS-A heat treated at 400 • C showed a highly homogeneous porous structure. Due to the pyrolysis of -CH 3 and condensation among -OH, there was a large amount of silica particles aggregated inside the aerogel, and the uniform pore structure was damaged after calcination at 600 • C. A denser silica structure of the as-prepared HAS-A heat treated at 800 • C was clearly observed. All the curves in Figure 9a are type IV isotherms, suggesting that mesopores are still present in the aerogel despite the high temperature heat treatment. Consequentially, the pore size distributions of the heat-treated samples are shown in Figure 9b,c. From 200 • C to 400 • C, the peak of the micropores increased and then decreased after 600 • C. This is because at 200-400 • C, the excess organic matter inside the pores of the HAS-A decomposed, resulting in extra pores. At 600-800 • C, the aerogel particles inside the HAS-A will be aggregated and block the pore channels [44]. The pore structure of the HAS-A at different calcination temperatures is shown in Table 3. The specific surface area increased dramatically from 231.5 m 2 /g to 337.7 m 2 /g, with the treatment temperature increasing from 200 • C to 400 • C. Simultaneously, it was observed that the total pore volume changed from 0.40 cm 3 /g to 0.43 cm 3 /g, while the average pore size changed from 11.3 nm to 10.6 nm. However, owing to the oxidation of -CH 3 groups and condensation between -OH, a further increase in the calcination temperature was not favorable for the specific surface area and total pore volume. treated HAS-A is denoted as AS-A. As the calcination temperature increased, the appearance of the color of the HAS-A gradually changed from off-white to brick red (Figure 8a), which is mainly caused by the component of ATP. It is found from Figure 8be that the HAS-A heat treated at 400 °C showed a highly homogeneous porous structure. Due to the pyrolysis of -CH3 and condensation among -OH, there was a large amount of silica particles aggregated inside the aerogel, and the uniform pore structure was damaged after calcination at 600 °C. A denser silica structure of the as-prepared HAS-A heat treated at 800 °C was clearly observed. All the curves in Figure 9a are type IV isotherms, suggesting that mesopores are still present in the aerogel despite the high temperature heat treatment. Consequentially, the pore size distributions of the heattreated samples are shown in Figure 9b,c. From 200 °C to 400 °C, the peak of the micropores increased and then decreased after 600 °C. This is because at 200-400 °C, the excess organic matter inside the pores of the HAS-A decomposed, resulting in extra pores. At 600-800 °C, the aerogel particles inside the HAS-A will be aggregated and block the pore channels [44]. The pore structure of the HAS-A at different calcination temperatures is shown in Table 3. The specific surface area increased dramatically from 231.5 m 2 /g to 337.7 m 2 /g, with the treatment temperature increasing from 200 °C to 400 °C. Simultaneously, it was observed that the total pore volume changed from 0.40 cm 3 /g to 0.43 cm 3 /g, while the average pore size changed from 11.3 nm to 10.6 nm. However, owing to the oxidation of -CH3 groups and condensation between -OH, a further increase in the calcination temperature was not favorable for the specific surface area and total pore volume. Figure 10a shows the XRD pattern of the heat-treated HAS-A. The characteristic peaks at 2θ = 27.6° and 30.9° were assigned to the (240) and (400) crystal planes of the ATP [27]. After a 600 °C calcination, the characteristic peak at 30.9° decreased significantly, indicating that the crystal structure of the ATP had been disrupted. After a 800 °C calcination, the characteristic peak at 30.9° gradually disappeared, indicating the complete destruction of the ATP crystal structure. A broad peak at 2θ = 21.5° was clearly identified in all samples, indicating that the silica aerogels preserve their original amorphous structure despite high temperature calcination [44]. The FT-IR spectra of the HAS-A heat treated at different temperatures are depicted in Figure 10b. The -CH3 groups are observed at the absorption peaks of 780 cm −1 , 1275 cm −1 , and 2970 cm −1 . Those bands of the −CH3 groups disappeared after a 600 °C calcination, indicating the pyrolysis of -CH3 [42]. Moreover, Figure 10c shows the water contact angles of the HAS-A treated at different temperatures. The hydrophobicity of the HAS-A remained stable at 500 °C (200 °C, 400 °C, and 500 °C, corresponding to the values of 140.4°, 136.7°, and 131.2°, respectively), while the HAS-A converted to hydrophilic AS-A completely at 600 °C, with a water contact angle value of 0°, which is in accordance with the representation results in the TG (Figure 7) and FT-IR (Figure 10b). The transition from hydrophobic to hydrophilic in the spherical ATP/SiO2 aerogels is conducive to the adsorption of soluble dyes in aqueous solutions. Therefore, the AS-A were used as adsorbents for studying the adsorption performance. Figure 10a shows the XRD pattern of the heat-treated HAS-A. The characteristic peaks at 2θ = 27.6 • and 30.9 • were assigned to the (240) and (400) crystal planes of the ATP [27]. After a 600 • C calcination, the characteristic peak at 30.9 • decreased significantly, indicating that the crystal structure of the ATP had been disrupted. After a 800 • C calcination, the characteristic peak at 30.9 • gradually disappeared, indicating the complete destruction of the ATP crystal structure. A broad peak at 2θ = 21.5 • was clearly identified in all samples, indicating that the silica aerogels preserve their original amorphous structure despite high temperature calcination [44]. The FT-IR spectra of the HAS-A heat treated at different temperatures are depicted in Figure 10b. The -CH 3 groups are observed at the absorption peaks of 780 cm −1 , 1275 cm −1 , and 2970 cm −1 . Those bands of the −CH 3 groups disappeared after a 600 • C calcination, indicating the pyrolysis of -CH 3 [42]. Moreover, Figure 10c shows the water contact angles of the HAS-A treated at different temperatures. The hydrophobicity of the HAS-A remained stable at 500 • C (200 • C, 400 • C, and 500 • C, corresponding to the values of 140.4 • , 136.7 • , and 131.2 • , respectively), while the HAS-A converted to hydrophilic AS-A completely at 600 • C, with a water contact angle value of 0 • , which is in accordance with the representation results in the TG (Figure 7) and FT-IR (Figure 10b). The transition from hydrophobic to hydrophilic in the spherical ATP/SiO 2 aerogels is conducive to the adsorption of soluble dyes in aqueous solutions. Therefore, the AS-A were used as adsorbents for studying the adsorption performance. Adsorption Studies To observe a visible change in the MB [7], Figure 11a exhibits the absorbance curves of MB at different concentrations. The adsorption capacity of the AS-A at different pH values, times, and MB concentrations is discussed as follows. Effect of the Solution pH on MB Removal by AS-A In this experiment, the pH was adapted from 2 to 11, and 50 mg of AS-A was used to adsorb MB at 50 mg/L for 100 min. The result in Figure 11b shows that the removal efficiency improved with the increase in the value of pH. The MB preferred to adsorb on AS-A under alkaline conditions, and the adsorption efficiency could reach 93.2% at a pH value of nine. Under acidic conditions, the adsorption capacity of AS-A is limited. This is due to the presence of excess hydrogen ions under acidic conditions, which leads to the protonation of functional groups on the AS-A surface during the adsorption process. Under alkaline conditions, AS-A has more adsorption sites on its surface, and the adsorption efficiency of MB will increase. Consequently, the pH value for the subsequent experiments was set to nine. Adsorption Studies To observe a visible change in the MB [7], Figure 11a exhibits the absorbance curves of MB at different concentrations. The adsorption capacity of the AS-A at different pH values, times, and MB concentrations is discussed as follows. Adsorption Studies To observe a visible change in the MB [7], Figure 11a exhibits the absorbance curves of MB at different concentrations. The adsorption capacity of the AS-A at different pH values, times, and MB concentrations is discussed as follows. Effect of Adsorption Time on MB Removal In this part, the adsorption quantities from 0 to 100 min were studied to explore the adsorption equilibrium time. The pH value and MB concentration were set to 9 and 40 mg/L, respectively. As shown in Figure 11c, the adsorption capacity increased rapidly to 69.2% within 10 min. Figure 11e presents the spectra of the absorbance of MB with time. After 90 min, the adsorption equilibrium was achieved. The corresponding adsorption efficiency and capacity of the MB were 92.45% and 73.96 mg/g, respectively. After another 10 min, there was no noticeable change in the adsorption rate. Therefore, in subsequent experiments, the adsorption time was set to 100 min. Effect of Initial Concentration on MB Removal The effect of the initial MB concentration (10-100 mg/L) on the adsorption efficiency is shown in Figure 11d. Figure 11f shows the variation in the absorption spectra for different initial concentrations of MB. The adsorption capacity increased and then gradually stabilized with the increasing MB concentration. When the MB concentration reached 60 mg/L, the adsorption capacity reached 102.50 mg/g. As the concentration of MB was further increased, there were no significant changes in the adsorption capacity. This indicates that 50 mg of AS-A could not provide enough adsorption sites to remove the excess MB from the solution. Adsorption Kinetics The adsorption kinetics is closely related to the contact time, which can reflect the adsorption mechanism of the absorbent. Kinetic models were used to evaluate the experimental data: Pseudo-first-order equation: ln(q e − q t ) = ln q e − k 1 t Pseudo-second-order equation: where k 1 and k 2 (g/(mg·min)) represent the pseudo-first-order and pseudo-second-order rate constants, respectively; q e (mg/g) is the equilibrium adsorption quantity; q t (mg/g) is the quantity adsorbed at different times ("t"). The model fitting results and the calculated kinetic parameters are shown in Figure 12 and Table 4. Compared with the pseudo-first-order, the experimental data confirm that the adsorption process was consistent with the pseudo-second-order model with high R 2 values of 0.99955. This suggests that the adsorption efficiency is highly dependent on the quantity of unoccupied active sites [45]. The q e values of the pseudo-second-order model (76.90 mg/g) were found to be essentially the same as the experimental results (73.96 mg/g), indicating that the pseudo-second-order kinetic model can be employed for the adsorption of MB on AS-A. Adsorption Isotherms The equilibrium adsorption isotherms play an important role in the interaction between the adsorbent and the adsorbate, which can explore the maximum adsorption capacity of the adsorbent. The Langmuir equation and the Freundlich equation are shown as follows: Langmuir equation: Freundlich equation: where qe (mg/g) is the adsorption amount at adsorption equilibrium; qmax (mg/g) stands for the maximum adsorption capacity; Ce (mg/L) represents the equilibrium concentration; kL and kF are the constants of the Langmuir isotherm equation and Freundlich isotherm equation; and 1/n is the adsorption intensity parameter. The fitting results of the Langmuir and Freundlich equations are shown in Figure 13 and Table 5. Adsorption Isotherms The equilibrium adsorption isotherms play an important role in the interaction between the adsorbent and the adsorbate, which can explore the maximum adsorption capacity of the adsorbent. The Langmuir equation and the Freundlich equation are shown as follows: Langmuir equation: where q e (mg/g) is the adsorption amount at adsorption equilibrium; q max (mg/g) stands for the maximum adsorption capacity; C e (mg/L) represents the equilibrium concentration; k L and k F are the constants of the Langmuir isotherm equation and Freundlich isotherm equation; and 1/n is the adsorption intensity parameter. The fitting results of the Langmuir and Freundlich equations are shown in Figure 13 and Table 5. The equilibrium adsorption isotherms play an important role in the interaction between the adsorbent and the adsorbate, which can explore the maximum adsorption capacity of the adsorbent. The Langmuir equation and the Freundlich equation are shown as follows: Langmuir equation: Freundlich equation: where qe (mg/g) is the adsorption amount at adsorption equilibrium; qmax (mg/g) stands for the maximum adsorption capacity; Ce (mg/L) represents the equilibrium concentration; kL and kF are the constants of the Langmuir isotherm equation and Freundlich isotherm equation; and 1/n is the adsorption intensity parameter. The fitting results of the Langmuir and Freundlich equations are shown in Figure 13 and Table 5. The Langmuir model fits the experimental data best, with R 2 values of 0.9986, which proves that the sorption process of MB on AS-A belongs to monolayer adsorption [45]. The maximum adsorption capacity derived from the Langmuir adsorption model was 109.05 mg/g, which closely corresponds to the experimental data (102.50 mg/g). The adsorption performance of the AS-A was compared with the other reported adsorbents ( Table 6). The AS-A obtained by heat treatment at 600 • C had excellent adsorption performance on MB in comparison with the other adsorbents. Although it has no advantage in terms of specific surface area, the combination of attapulgite and silica aerogel greatly improved the adsorption of the composite. This reveals that AS-A is a potential adsorbent for dye wastewater. The regenerative adsorption of the adsorbent is a vital factor in measuring its suitability in practical applications. In the recycling test, AS-A was heat treated at 600 • C for 3 h in a muffle furnace to eliminate the adsorbed dye. As shown in Figure 14a, the adsorption capacity of AS-A remained at 82.32% after five adsorption cycles, indicating that the adsorption of MB on AS-A was stable for multiple cycles. After the regeneration, the disappearance of MB aromatic group at 1603 cm −1 illustrates that MB can be completely removed by calcination at 600 • C (Figure 14b) [52]. The Langmuir model fits the experimental data best, with R 2 values of 0.9986, w proves that the sorption process of MB on AS-A belongs to monolayer adsorption [ The maximum adsorption capacity derived from the Langmuir adsorption model 109.05 mg/g, which closely corresponds to the experimental data (102.50 mg/g). adsorption performance of the AS-A was compared with the other reported adsorb ( Table 6). The AS-A obtained by heat treatment at 600 °C had excellent adsorp performance on MB in comparison with the other adsorbents. Although it has advantage in terms of specific surface area, the combination of attapulgite and s aerogel greatly improved the adsorption of the composite. This reveals that AS-A potential adsorbent for dye wastewater. The regenerative adsorption of the adsorbent is a vital factor in measuring suitability in practical applications. In the recycling test, AS-A was heat treated at 60 for 3 h in a muffle furnace to eliminate the adsorbed dye. As shown in Figure14a, adsorption capacity of AS-A remained at 82.32% after five adsorption cycles, indica that the adsorption of MB on AS-A was stable for multiple cycles. After the regenerat the disappearance of MB aromatic group at 1603 cm −1 illustrates that MB can be comple removed by calcination at 600 °C (Figure 14b) [52]. Adsorption Mechanism The zeta potential of the AS-A was negative in the pH range of 5-9 (Figure 15a), indicating that the surface of the AS-A exhibited negative charges. In addition, the amount of negative charge on the AS-A surface tended to increase with the increase in the pH. As shown in Figure 15b, MB is a typical cationic dye with a positive charge [53]. The negatively charged AS-A had a significant adsorption effect on the positively charged MB, resulting in electrostatic attraction between MB and AS-A. Therefore, the adsorption mechanism of AS-A on MB is mainly an electrostatic effect. The surface negative charge of AS-A has great attraction to MB with positive charge, which leads to an excellent adsorption performance on MB. 9, and t = 100 min); (b) FT-IR spectra of AS-A before and after regeneration. Adsorption Mechanism The zeta potential of the AS-A was negative in the pH range of 5-9 (Figure 15a), indicating that the surface of the AS-A exhibited negative charges. In addition, the amount of negative charge on the AS-A surface tended to increase with the increase in the pH. As shown in Figure 15b, MB is a typical cationic dye with a positive charge [53]. The negatively charged AS-A had a significant adsorption effect on the positively charged MB, resulting in electrostatic attraction between MB and AS-A. Therefore, the adsorption mechanism of AS-A on MB is mainly an electrostatic effect. The surface negative charge of AS-A has great attraction to MB with positive charge, which leads to an excellent adsorption performance on MB. Conclusions In this work, the spherical ATP/SiO2 aerogels were successfully synthesized by three different drying techniques (SCD, FD, and APD). The spherical ATP/SiO2 aerogels dried with three techniques possessed a complete macromorphology and homogeneous porous network structures. The control of the size of the obtained spherical particles will be included in the next step of the study. The club-shaped ATP was well dispersed in the aerogel matrix without agglomeration, and the silica particles were closely attached to the surface of the ATP. The average pore diameter and specific surface area of the spherical ATP/SiO2 aerogels prepared by three drying techniques were in the range of 6.8-8.6 nm and 218.5-267.4 m 2 /g, respectively. The BET specific surface area of the HAS-A after the 400 °C heat treatment sharply increased to 333.7 m 2 /g. Compared to the other adsorbents, AS-A showed a higher adsorption capacity of 102.50 mg/g for MB. After five cycles of regeneration, the adsorption efficiency of AS-A could still maintain 82.32%, exhibiting a good reusability. Therefore, these porous spherical ATP/SiO2 aerogels with multipath preparation and excellent adsorption performance are expected to have applications in dye wastewater treatment. Conclusions In this work, the spherical ATP/SiO 2 aerogels were successfully synthesized by three different drying techniques (SCD, FD, and APD). The spherical ATP/SiO 2 aerogels dried with three techniques possessed a complete macromorphology and homogeneous porous network structures. The control of the size of the obtained spherical particles will be included in the next step of the study. The club-shaped ATP was well dispersed in the aerogel matrix without agglomeration, and the silica particles were closely attached to the surface of the ATP. The average pore diameter and specific surface area of the spherical ATP/SiO 2 aerogels prepared by three drying techniques were in the range of 6.8-8.6 nm and 218.5-267.4 m 2 /g, respectively. The BET specific surface area of the HAS-A after the 400 • C heat treatment sharply increased to 333.7 m 2 /g. Compared to the other adsorbents, AS-A showed a higher adsorption capacity of 102.50 mg/g for MB. After five cycles of regeneration, the adsorption efficiency of AS-A could still maintain 82.32%, exhibiting a good reusability. Therefore, these porous spherical ATP/SiO 2 aerogels with multipath preparation and excellent adsorption performance are expected to have applications in dye wastewater treatment.	2023-03-15T15:10:25.494Z	2023-03-01T00:00:00.000	{ "year": 2023, "sha1": "5841809eaefe257848e3ea2cf6283b6f6933a3d6", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1996-1944/16/6/2292/pdf?version=1678683192", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "1fffc7c6272391738cde7635002fc5b6a7bcd9cc", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Medicine" ] }
215768834	pes2o/s2orc	v3-fos-license	A cryogenic magneto-optical device for long wavelength radiation We present here a small-scale liquid Helium (LHe) immersion cryostat with an innovative optical setup suitable to work in long wavelength radiation ranges and under applied magnetic field. The cryostat is a multi stage device with several shielding in addition to several optical stages. The system has been designed with an external liquid Nitrogen boiler to reduce the liquid bubbling. The optical and mechanical properties of the optical elements were calculated and optimized for the designed configuration while the optical layout has been simulated and optimized among different configurations based on the geometry of the device. The final design has been optimized for low noise radiation measurements of proximity junction arrays under applied magnetic field in the wavelength range $\lambda$=250-2500 $\mu$m. We present here a small-scale liquid Helium (LHe) immersion cryostat with an innovative optical setup suitable to work in long wavelength radiation ranges and under applied magnetic field. The cryostat is a multi stage device with several shielding in addition to several optical stages. The system has been designed with an external liquid Nitrogen boiler to reduce the liquid bubbling. The optical and mechanical properties of the optical elements were calculated and optimized for the designed configuration while the optical layout has been simulated and optimized among different configurations based on the geometry of the device. The final design has been optimized for low noise radiation measurements of proximity junction arrays under applied magnetic field in the wavelength range λ=250-2500 µm. I. INTRODUCTION Cryogenic temperatures are required in many researches such as superconductivity and superfluidity, surface and interface, advanced spectroscopies and in the R&D of low-noise detectors. As an example, superconducting radiation detectors integrated into surface Paul traps for scalable quantum information processing require operation at cryogenic temperatures [1][2][3]. Optical spectroscopies at low temperature usually allow to resolve narrow features and trap unstable intermediates [4]. Recently, long wavelength radiations have come to attention being a particular region of the electromagnetic spectrum interesting for multi disciplinary applications for both basic researches and technologies [5][6][7]. This e.m. domain λ=250-2500 µm (energy range 0.5-4 meV) allows the investigation of several fundamental physical phenomena, e.g., phonon and plasmon dynamics, elementary particle physics and possibly also cold dark matter [8][9][10][11][12]. Furthermore, with its high transmission through a wide range of non-conducting materials, long wavelength radiation and, in particular terahertz (THz) radiation, holds a significant potential in several applications. In the recent years, the investigation of semiconducting and superconducting behavior of many materials and the modulation of the electronic properties of systems with low dimensionality have been investigated [13][14][15][16]. It has been demonstrated that superconducting proximity junction arrays can be designed to behave as long wavelength radiation detectors [17,18]. The superconducting dynamics of such devices, based on non-localized vortex dynamics, can be modulated via external magnetic and electric fields [19]. The response of these devices in the radio frequency domain, well below the expected plasma frequency of the proximity junction, can be tuned via non-equilibrium phenomena induced by external perturbations. It has been also speculated that such devices may represent a new pathway towards robust, low noise, broadband and with high sensitivity low energy detectors. However, the investigation of the dynamics in such devices requires a low noise cryostat with a highly stable temperature control that can be operated under external magnetic and electric fields. These cryogenic systems should also be designed with suitable optical arrangements compatible with this energy range. In a conventional cold finger optical system, the sample is cooled through its mechanical link to the cooling element, either by boiling liquid Helium or a mechanical refrigerator. This approach presents several problems. The first is that the sample is cooled relatively slowly and often we observe the formation of crystallites on the sample surface. In addition, reaching the required temperatures and its fine control under applied electric and magnetic fields is not an easy task. Finally, the optimization of the optical arrangement to operate in this extreme photon energy range is complex. Within the framework of the project TERA [20], we have developed a small scale liquid Helium immersion cryostat with an embedded optical setup, aimed at the detection of the photo-response of novel proximity junction arrays under an applied magnetic field. In the following we will present and discuss the different cooling stages, the shields and the optical layout. The latter has been simulated and optimized based on the different in-operando configurations. The optical transmission and the mechanical resistance of the optical components have been calculated, optimized and characterized. II. CRYOSTAT AND OPTICAL SET UP DESCRIPTION A schematic view of the immersion cryostat is shown in Fig.1. The cryostat is designed to be a table top small scale cryostat for facile optical alignments. The section views of the cryostat (Fig. 1b) shows the vacuum chamber (2) surrounding the liquid Nitrogen tank (3) in the upper half, and the polished copper radiation shield in the lower one (11). The shield has a hole in correspondence of the optical path. At the center, the liquid Helium container (4), inside which the experimental apparatus (1) is hosted. The inner container has a 10 cm diameter, a value that sets the maximum size of both magnetic coils and sample holder. The inner stainless steel tube is provided at the bottom with a window assembly mounted on a CF flange that holds a 29 mm diameter optical window set in front of the sample. The copper cooling jacket extends down the sides as well as below the window assembly to improve cooling and to speed up the initial cooling process. The external container is equipped with four vacuum-tight optical windows. All inner metal-metal connections, except the copper shield and the cryostat cover part that is connected by bolts, have been connected either by soldering or brazing. Inside upper part of the liquid Helim container are housed two concentric copper disks acting as thermal shields and connected to the sample holder support with two Rexilon rods (10 mm diameter), useful to minimize the thermal conduction between the immersed components and the thermal shields. The sample holder support consists of a PEEK holder support housing two superconducting NbN coils attached with concentric tubes with 21 mm apertures pointing to the sample set in the center. Considering the aperture dimension of the magnetic coils the maximum sample size can be 20× 20 mm and can be replace by simply opening the cryosts top flange (top dark part of figure1b), since the sample holder and the magnetic coils are connected to the copper disks shields and connected to this flange. The coils generate a magnetic field normal to the sample surface 3) the liquid Nitrogen reservoir; 4) the liquid Helium reservoir 5) connector for signal, thermometer and gauss-meter wiring; 6) liquid Nitrogen insert tube ; 7) liquid Helium pressurizing and pumping valve; 8) vacuum jacket pumping valve; 9) liquid Helium insert tube; 10) LHe level sensor feedthrough; 11) copper radiation shield. Inside the inner tube there are two copper disks as radiation shields. The sample holder is connected to the lower one via two Rexilon rods to minimize thermal losses. plane (see Fig. 2) up to 150 mT at 4.2 K calibrated with a calibrated gaussmeter. Under operation the vacuum jacket chamber reaches up to 1 × 10 −4 mbar rapidly while the vacuum level lowers to 5 × 10 −6 mbar, introducing the liquid Nitrogen inside the LN bath. The latter should be refilled periodically prior to introduction of the liquid Helium. The thermal conduction from the bottom and the contact thermal conduction lead to a temperature of ∼ 100 K in the main cylinder upon insertion of the liquid Nitrogen. The LHe insertion takes place in over pressurized He inner cryotube to prevent the crystallization on the sample surface. The temperature is read by a calibrated Cernox thermometer connected to the PCB sample support and the applied magnetic field is measured via a previously calibrated Hall probe inserted perpendicularly inside the coil at the back of sample. The cryostat with the capacity of the ∼ 5 Lt, is stable and maintains the temperature to 4.2 K for about six hours without incident radiations. The LN evaporator jacket reduces bubbling inside the cryotube a condition that significantly reduces the light scattering along the optical path [4]. The temperature can be further reduced down to ∼2 K with a continuous pumping of the pressurized LHe bath. The detailed cryogenic optical arrangement is described in figure 3. The layout consists of a 2 mm Zeonex window mounted with a Vyton O-ring to the vacuum chamber jacket and connected to the ambient temperature. Along the optical path the second window is in contact with the LHe under a heavy tensile pressure (due to the differential pressure between the two volumes). The window is made by 2.2 mm thick crystal quartz as will be discussed later. The window is connected to the LHe cryotube by a modified Conflat flange and the vacuum is sealed by a teflon ring on the vacuum side and an Indium wire on the liquid side (see figure 3-inset 1). The window was tightened in gradual cross contraction using a torque driver up to 2 N/m. A 2 mm diameter Zeonex focusing lens with a 20 mm focal distance is set in from of the sample, screwed within the coil aperture inside the PEEK cylinder support (see figure 3-inset 2). The cylinder itself is fixed by a PEEK nut tightening the cylinder in place. This design further facilitates the setting along the axis of the lens focal point with regard to the sample positioning. The optimal thickness of all windows, being the minimum thickness capable of withstanding the pressure difference insuring its high transmittance, was calculated considering the materials tensile strengths as where, d is the slab thickness, R is the unsupported area, P is the pressure difference, S the safety factor and η is the modulus of the rupture. The relative thickness behavior vs. differential pressure of the crystal quartz with η = 9427 psi with a maximum 3 mm unsupported area is shown in figure 4. Considering the differential pressure to sustain, a minimum thickness in the range d = 1.8 − 2.2 mm was considered. While for the outer Zeonex window with η = 6090 FIG. 4 : The curve of the Zeonex window slab thickness as a function of the pressure difference between the cryostat jackets. psi withstanding the atmospheric pressure a relatively smaller thickness of the windows has been considered (2 mm). Both Zeonex and crystal quartz windows were tested under these conditions and passed the tests. The Zeonex window was tested also under vacuum down to 1 × 10 −6 mbar. In order to evaluate the effective transmission and the efficiency of the optical layout, the optical response of the Zeonex windows were measured in the spectral region of interest. The optical response of the polymeric Zeonex material, was investigated with terahertz time-domain spectroscopy (THz-TDS), described in detail in Refs [21][22][23]. The in house THz-TDS spectrometer in the transmission configuration, used for this purpose, is based on two photo-conductive switches, one as emitter of the broadband THZ radiation (0.1-2.5 THz) and one as receiver [24], respectively (see figure 5a). Two twin G10620-11 Hamamatsu photo-conductive antennas (PCAs) were operated by a mode-locked ultrafast laser (FemtoFiber NIRpro, Toptica), at 780 nm, temporal pulse width 100 fs, repetition rate of 80 MHz and output power of 150 mW. After the laser power reduction, a beam splitter (BS) 50:50 splits the main beam into two separated pump and probe beams. Dielectric mirrors propagate the beams towards emitter and receiver PCAs. The THz produced by the emitter, is then collected and collimated by TPX lenses with focal length of 50 mm. The THz beam is focused on the sample and the transmitted radiation is recollected, re-collimated and refocused on the receiver PCA. Simultaneously, the probe beam is used to gate the THz-detector PCA. Two motorized stages are used in the system configuration (see figure 5). The fist one, a delay line (DDSM100/M, Thorlabs) allows the collection of temporal THz electric field varying the optical path between pump and probe beams [21]. The second one is a 3-axes motor stage that ensures an accurate positioning and alignment of the sample in the THz beam focal spot. The output signal from the THz-detector, filtered by a lock-in amplifier, was acquired and digitized and then transferred to the PC. The optical system provides a spectral bandwidth ranging from 0.1 to 2.5 THz, with a spectral resolution of 50 GHz. This spectral resolution is sufficient to characterize the response of the non-resonant and transparent window set. We recorded 5 THz electric field profiles with 5 scans. The THz electric fields for the free space (without sample) and for the Zeonex window with thickness 2 mm are shown in figure 5b. The temporal delay between the air and the Zeonex signal was 3.75 ± 0.01 ps. The power spectra of free space and Zeonex slab are also shown in figure 5c along with its total transmittance (see Fig. 5d). The transmittances was calculated as The results indicate a transmission of about 80% in the range below 1 THz, which is slightly higher than that of crystal quartz [25]. The total transmission of the optical arrangement was then calculated using the Beer-Lambert law [26] and considering the transmission of the Zeonex derived from experimental results (see figure 6). In order to prevent any contamination of visible and IR radiation inside the cryotube a 0.2 mm flexible HDPE filter (with T∼ 85% at 1 THz [27]) was attached to the entrance window. Based on the thickness of the Zeonex windows, crystal quartz and the filter transmission a total transmission of about 45-50% onto the sample was calculated. Based on the geometry of the cryogenic system, three different layouts of the optical paths were considered as described in Fig.7. All of them are possible configurations of this optical system. Key parameters, considered mainly in the lens design, were the resolution, the focal length and the lens diameter. Both focal length and lens diameter are determined by geometrical constraints. Furthermore, we planned to have a focal spot diameter less than 1 mm in order to guarantee a sufficient number of photons on the sample. In the layout (1), a THz plane wave illuminates the focusing lens (L1) with a focal length of 130 mm. A polymeric/quartz window (W1) is located between the lens (L1) and its focal plane. The maximum diameter for the lens in this location is 50 mm. In the layout (2) the THz radiation pass the polymeric window (W1) and it is focused by the lens (L1) with a focal length of about 50 mm that acts also as the second window. In the layout (3), the plane wave is incident on two polymeric/quartz windows, (W1) and (W2), and it is finally focused by the lens (L1) with a focal length of about 20 mm (length of the magnetic coil aperture L=32 mm). Considering the constraints of the coil, the maximum lens diameter is 20 mm since the diameter of the magnetic coil aperture is D=21 mm. All layouts have been numerically simulated by means of a commercial software based on the finite element method in the frequency domain [28]. The material selected for both windows and lenses is the Zeonex by Zeon Corporation, a cyclo-olefin with a refractive index of = 1.518 − j0.001 at 1 THz, successfully employed in other devices operating at THz frequencies [29,30]. The windows have been designed with a thickness of 2 mm. The lens (L1) in the layout 1 is a plano-convex lens with a radius of curvature R = 67.6 mm and a maximum thickness of 1 cm. The lens (L1) in the layout (3) is a plano-convex lens with a radius of curvature R = 10.7 mm and a maximum thickness of 1 cm. The lens (L1) in the layout (2) is a plano-convex lens with a radius of curvature R = 26.2 mm and a maximum thickness of 1 cm. As a proof of principle, a linearly polarized plane wave has been selected as a radiation source at 1 THz. The results of the numerical simulations are described in Fig.7. They confirm the occurrence of a focal spot with a diameter comparable with the theory [26]. The full width half maximum (FWHM) of the absolute value of the electric field in the focal area corresponds to a measure of the focal spot diameter of the selected lenses. The FWHM of the lens (L 1 ) focal spot is equal to about 1 mm for the layout (1), 0.48 mm for the layout (3), and 0.65 mm for the layout (2). As expected, the presence of the windows slightly affects the focusing properties in terms of maximum values of the electric field modulus at the focal point, due to the mismatch of the refractive index between vacuum and Zeonor and to the moderate absorption of the polymer at 1 THz. The optical configuration presented in layout (3) shows a higher resolution of the lens (L 1 ) at 1 THz, compared with the other configurations. Hence, we selected the latter optical arrangement for the cryogenic system that was designed and manufactured [27] according to this layout (3). Furthermore, to implement the tuning of the lens depth of field (DoF), defined as the distance along the optical axis in which the power does not decrease below the 80% of the maximum, a second lens with a focal length of 30 mm alternative to the lens (L1) of the Layout (3) was numerically simulated. The modulation of the DoF facilitates the compensation of the photon intensity attenuation due to sample positioning misalignments. The plano-convex lens with a focal length of 30 mm has a diameter of 20 mm, a radius of curvature R = 15.9 mm, and a maximum thickness of 6.5 mm. Simulations of this lens in layout (3) are reported in Fig.7. The FWHM of the resulting focal spot is equal to about 0.56 mm. Simulations confirm that a longer focal length allows a longer DoF. We also evaluated the lens orientation in term of DoF by numerical simulations. As reported in 7, when the lens is oriented as in Fig.7 c and e, DoF is 2.3 mm and 3.5 mm for the lens with a focal length of 20 mm and 30 mm, respectively. On the other hand, when the lens is oriented as in Fig.7 d and f, DoF is 3.5 mm and 4.2 mm for the lens with a focal length of 20 mm and 30 mm, respectively. The simulations confirm that the selected lenses guarantee the necessary flexibility with respect to the sensor position and with acceptable losses. Finally, the total transmission and efficiency of the optical configuration was measured at the optical exit using a continuous diode THz emitter at 270 GHz (Virginiadiode). The total transmission of the optical line was measured The lens was shifted in a 5 mm range to optimize the focal distance and the transmission. The measurement was performed looking at the peak to peak amplified voltage response of the pyroelectric detector triggered by the output frequency of the chopper (see figure 8a,b). The lens distance was varied via the perforated support designed for an accurate translation inside the magnetic coil aperture. The lens with the focal distance of 20 mm was used in these tests and the distance changed from d=19 to d=23 mm using the mounted screw. As can be seen in the figure 8c, the lens shows a relatively asymmetric response while the highest intensity was measured at f =21 mm for the value of V p−p =210 mV. The deviation from the maximum in the 4-5 mm distance range was measured to be of the order of the ∆I d =± 24%, ranging from V p−p =175 mV to V p−p =210mV. The result is in agreement with the simulation of the optical response of the lens. The depth of focus determined allows measurements with minor displacements of the sample positioning. The overall transmission of the optical line in the cryotube without LHe was also calculated to be 42% in a relatively good agreement with the estimated value of ∼ 50% actually, slightly lower due to the transmission difference at these wavelengths. The cryogenic optical set up shows an acceptable response and hence could be successfully used for the investigation of new proximity array devices that require a sufficient number of photon and the control over external magnetic and electric field [17,31]. III. CONCLUSIONS We presented here the characteristics of a compact liquid Helium immersion cryostat designed and built with an optical setup optimized for experiments using radiation at long wavelengths. The system has been specifically designed to characterize novel proximity junction array detectors and other 2D imaging detectors working at LHe temperature. The cryostat allows to maintain the sample at the temperature of 4.2 K for six hours, while it may reach the lowest temperature of 2 K upon LHe bath pumping. A liquid Nitrogen boiling chamber is used to reduce bubbling inside the cryotube. The optical configuration of the device is made by three stages of optical elements: a Zeonex window, a crystal quartz window and a Zeonex focusing lens, out of which the latter two are immersed in liquid Helium. All materials of the optical windows were selected on the base of accurate simulations and have a thickness of about 2 mm, i.e., 2 mm for Zeonex and 2.2 mm for crystal quartz, considering both materials tensile stress and the differential pressure they are withstanding inside the cryostat as well as their optical parameters. The transmission of the Zeonex windows were measured using a two stage photo-diode configurations and for a thickness of 2 mm are characterized by a transmission of 80% at 1 THz. Based on the optical layout, different configurations were simulated with a spatial resolution ranging from 0.48 to 1 mm and the configuration with the highest estimated resolution was realized. Furthermore, several other configurations were simulated and compared to verify the tunability of the DoF in the range 2.3 mm to 8 mm in order to compensate sample positioning misalignments. The total transmission of the optical configuration exhibits a maximum value of 42% while a DoF deviation of 24% was measured by changing the lens focal distance. These results measured in cryogenic conditions are in good agreement with theoretical estimations. Summarizing, this original cryogenic systems matches the demanding requests of the investigation of samples and devices to be probed at low temperature, long wavelengths and when external electric and magnetic fields have to be applied. IV. ACKNOWLEDGMENTS This project has been supported within the framework of the TERA project of the V th Committee of the INFN. We like to thank S. Cibella and P. Carelli for their support in the assembly of the apparatus for pyroelectric measurements and A. Grilli and A. Raco for their valuable support in the construction of the apparatus. We acknowledge the financial support of the Bilateral Cooperation Agreement between Italy and Japan of the Italian Ministry of Foreign Affairs and of the International Cooperation (MAECI) in the framework of the project of major relevance N. PGR0072. V. DATA AVAILABILITY The data that supports the findings of this study are available within the article.	2020-04-28T01:00:57.875Z	2020-04-15T00:00:00.000	{ "year": 2020, "sha1": "1858808347fd4e83ce318a426e852f391f1e5b0c", "oa_license": null, "oa_url": "https://iris.uniroma1.it/bitstream/11573/1553572/1/Rezvani_A-cryogenic_2020.pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "1858808347fd4e83ce318a426e852f391f1e5b0c", "s2fieldsofstudy": [ "Physics", "Engineering" ], "extfieldsofstudy": [ "Physics", "Materials Science", "Medicine" ] }
37697515	pes2o/s2orc	v3-fos-license	Long-range potential and the fine structure of the diffraction peak The possibility of oscillations in the differential elastic cross section of hadron scattering at small momentum transfer is studied. It is shown that string-like quark potentials at large distances can lead to such small oscillations, and an analysis of the experimental data at small $\|t\|$ allows the determination of the parameters of the potential. The AKM theorem [1] predicts that the differential elastic cross section will contain a structure periodic in the scale q = \|t\| for t → 0 [2]. We study this effect through models for the hadron-hadron interaction at large distances. This will lead to a new parametrisation of experimental data with a high confidence level in a wide energy region. We consider many experimental data on nucleon-nucleon elastic scattering at small momentum transfer in a very large energy interval from √ s = 2 GeV to 1800 GeV. We find that the oscillations are present in most of the datasets. The differential cross sections are given by the formula where F C is the Coulomb scattering amplitude, F N the hadronic amplitude, ρ(s, t) the cotangent of its phase, and ϕ(s, t) the Coulomb-hadron interference phase [3]. Here we neglect the hadron spin-flip amplitudes and take into account all parts of the electromagnetic amplitudes. The spin-non-flip amplitudes are written in the standard form for small momentum transfer: where α is the fine-structure constant and G(t) the proton electromagnetic form factor squared. This formula is used by experimentalists to extract ρ from their data to obtain the value of ρ(s, t). If an additional periodic amplitude has a sizeable real part ReF osc (s, t), the oscillation in the differential cross sections will be proportional to The determination of ρ(s, t) and ReF osc (s, t) then clearly depend on each other. We considered several forms for F osc (Bessel functions J 0 or J 1 , sines, functions of q or q 2 ...), and the best results are obtained for We considered experimental data forpp scattering at low energies ( √ s = 3.1 − 6.2 GeV) and high energies ( √ s = 52.6, 541, 546, 1800 GeV) [4]. The We again obtain an improvement in the χ 2 between 15% and 25%. More remarkably, the half period of the oscillation for all these experiments lies near 12 MeV (see Fig. 2a). The normalization constant of the additional term fluctuates around 0.15 GeV −2 , and it may grow with energy as ln s, as shown in Fig. 2b. In all cases we obtain a substantial decrease in the value of the χ 2 . It is clear that for such a high frequency, the improvement is unlikely to be accidental, or to correspond to fluctuations of the data. We believe that it is evidence for the existence of such oscillations in the real part of the scattering amplitude. Note that to obtain the above fits, we also resorted to an unusual method. Indeed, the direct minimization of the χ 2 works poorly, as one should first fix the model producing oscillations for the small-\|t\| part of the scattering amplitude, and as the effect is small, so that outside of the exact fit, it will give an insignificant change to the χ 2 . Therefore, we also used a method comparing two statistically independent sets of data, for example [5]. The whole interval of q is divided into small intervals ∆q i , i = 1, ...n, equal to dq. The deviations of the experimental data from an exponential, weighted by the inverse the experimental errors, are added in two separate sums for i even or odd. If the intervals correspond to the period of the cross section, then these two sums will be significantly different in sign. If the starting point of the first interval is moved by one half period, the two sums will become identical, and close to zero (see the middle lines of Fig. 1b and [6]). We can calculate the significance level by comparing the two fits, with the interval moved or not. Most remarkably this method leads to a similar value for the half period, dq ≃ 12 MeV. Note that the effect comes mostly from the lowest q, where it gets enhanced by the Coulomb-hadron interference term. The convergence of the two methods to equivalent fits, and the significant improvement in the quality of the fit for many independent experiments, suggest that the oscillations are not due to statistical fluctuations, but rather reflect a physical phenomenon. The hadron potential at large distance Such a small period of oscillation may be related with the properties of the hadron interaction at large distances. In some calculations of the scattering amplitude, determined by the gravitational potential in an n + 4 dimensional world in the framework of the ADD-scenario [7], one obtains a periodical structure (our calculations and [8]). In general, we can assume that an additional potential has a small constant value at large distances and is sharply screened at a given distance, which provides a cut-off for the integrals. In the q-representation, the corresponding amplitude will oscillate with a period that depends on the distance at which the potential is cut, as shown in Fig. 3a. Such a scenario could also be realized during fireball processes via the screening of the electromagnetic interactions at large distances. Indeed, let us take the additional potential in our case in b-space in the following form: where b scr is the distance at which the additional potential has a screening effect, and K 1 (ix) is the MacDonald function of imaginary arguments. If b scr is sufficiently large we obtain an oscillating amplitude with a small period in q (see Fig. 3b). Note that in this case the integration has no specific cut. However, the oscillations can be characterized by an amplitude and a period that do not depend on energy, or depend on it very weakly. This, together with the small size of the coupling and the long range of the interaction, may point to an electromagnetic origin of this effect. Conclusion We have shown that oscillations, periodic in √ −t, exist in many experimental data sets at a significant level. The confirmation of the existence of such a periodic structure in the elastic-scattering amplitude at the LHC would give us important information about the behavior of the hadron interaction potential at large distances which may be connected with the problem of confinement. acknowledgments O.S. would like to thank the organizers R. Fiore and A. Papa for the invitation and support of his participation in conference. Work supported in part by a grant from FNRS.	2010-11-18T12:09:38.000Z	2010-11-18T00:00:00.000	{ "year": 2010, "sha1": "869fc56e3e690b10bb5ec214941d5f640dbeac66", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1011.4177", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "869fc56e3e690b10bb5ec214941d5f640dbeac66", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
269222682	pes2o/s2orc	v3-fos-license	Treatment of atopic dermatitis with upadacitinib: adcare single center experience Introduction The role of upadacitinib in the management of moderate to severe atopic dermatitis seems promising, but more data on its efficacy and safety are needed. This study endeavors to assess the practical impact and safety of upadacitinib in patients with moderate to severe atopic dermatitis. The study aims to evaluate the efficacy and safety of upadacitinib in the treatment of moderate to severe atopic dermatitis, focusing on analyzing patient responses to the treatment. Methods In this study, adult patients diagnosed with moderate to severe atopic dermatitis received upadacitinib at daily doses of 15 mg or 30 mg, as prescribed by their attending physicians. The therapeutic efficacy of upadacitinib was meticulously assessed using established clinical metrics. Simultaneously, a comprehensive safety assessment was conducted through monthly monitoring, including the evaluation of potential effects of upadacitinib intake on hepatic function, lipid profile, and hematopoiesis using the pertinent laboratory tests. Results Sixteen participants were enrolled in the study. At 1month follow-up, there was a significant reduction in the mean Eczema Area and Severity Index (EASI) score to 18.8 points, which further increased to 24 points at the 4-month mark. Additionally, 9 participants (56%) demonstrated an EASI-50 response after 1 month of treatment, with this response increasing to 9 participants (90%) after 4 months. Furthermore, enhanced therapeutic responses were observed at 4 months, with 6 patients (38%) achieving an EASI-75 response at 1month and 8 patients (80%) achieving this milestone at the 4-month follow-up. This study highlights the potential of upadacitinib as an effective treatment option for moderate to severe atopic dermatitis. While it demonstrates improved symptom management, close monitoring for potential adverse events, particularly infections and the known risks of Janus kinase inhibitors, is essential. Further research is essential to determine the long-term safety and efficacy of upadacitinib. Introduction: The role of upadacitinib in the management of moderate to severe atopic dermatitis seems promising, but more data on its efficacy and safety are needed.This study endeavors to assess the practical impact and safety of upadacitinib in patients with moderate to severe atopic dermatitis.The study aims to evaluate the efficacy and safety of upadacitinib in the treatment of moderate to severe atopic dermatitis, focusing on analyzing patient responses to the treatment. Methods: In this study, adult patients diagnosed with moderate to severe atopic dermatitis received upadacitinib at daily doses of 15 mg or 30 mg, as prescribed by their attending physicians.The therapeutic efficacy of upadacitinib was meticulously assessed using established clinical metrics.Simultaneously, a comprehensive safety assessment was conducted through monthly monitoring, including the evaluation of potential effects of upadacitinib intake on hepatic function, lipid profile, and hematopoiesis using the pertinent laboratory tests. Results: Sixteen participants were enrolled in the study.At 1month follow-up, there was a significant reduction in the mean Eczema Area and Severity Index (EASI) score to 18.8 points, which further increased to 24 points at the 4-month mark.Additionally, 9 participants (56%) demonstrated an EASI-50 response after 1 month of treatment, with this response increasing to 9 participants (90%) after 4 months.Furthermore, enhanced therapeutic responses were observed at 4 months, with 6 patients (38%) achieving an EASI-75 response at 1month and 8 patients (80%) achieving this milestone at the 4-month follow-up.This study highlights the potential of upadacitinib as an effective treatment option for moderate to severe atopic dermatitis.While it demonstrates improved symptom management, close monitoring for potential adverse events, Introduction Atopic dermatitis (AD) is a common, heterogeneous, chronic, flaring, and systemic inflammatory disease characterized by eczematous skin lesions and intense pruritus and a negative impact on patients' quality of life (QoL).It is the most common in the pediatric age group with an estimated prevalence of 15-30% (1).Nevertheless, studies have shown that up to 14% of adolescents and adults can also experience AD symptoms (2)(3)(4)(5).However, the proportion of severe disease forms in the adult cohort is higher compared to children (6). Adherence is essential for successful treatment.However, studies have shown that up to 33% of patients do not comply with prescribed topical therapy, and adherence drops sharply immediately following the first doctor's visit (7).This can lead to a more severe course of the disease and an increase in skin process progression.At the same time, even with a high level of patient compliance to treatment with topical medications and a responsible attitude toward skincare, it is often not possible to achieve significant results without systemic therapy. The therapeutic management of AD is mostly based on topical and/or systemic immunosuppressive/immunomodulant therapies and can be challenging in the long-term period, particularly for moderate-to-severe AD.Beside conventional systemic agents, such as cyclosporine, targeted therapies approved for the treatment of AD are currently available (8). The progress of novel therapy concepts and an increased understanding of AD pathophysiology has provided the basis for new drug development for systemic therapy of moderate to severe forms of AD (3). In the past, nonspecific immunosuppressive drugs were used, which had lack of efficacy and caused significant side effects (2,3).Contemporary options of systemic therapy for severe atopic dermatitis have now become routine clinical practice.The pioneer in this field was dupilumab, a biological anti-IL4R monoclonal antibody.Dupilumab has demonstrated a good safety profile, although the most undesirable side effect is conjunctivitis, occurring in 4.7-28% of patients with AD.It also has good efficacy profile, effectively controlling the disease in up to 40% of patients, with time to response from 16 to 24 weeks of treatment.However, it doesn't address all the unmet needs, i.e., it cannot be applied in patients with a tendency to inflammatory eye diseases or hypereosinophilia.In addition, the injectable form of drug administration may present additional difficulties when using dupilumab (9,10). Recently, the variety of targeted systemic treatment options has been further enriched with a new class of Janus Kinase inhibitors (JAKi) molecules -abrocitinib, baricitinib, and upadacitinib.The inhibition of the JAK signaling pathway is a promising therapeutic technique for reducing the activation of many pro-inflammatory mediators involved in the pathogenesis of AD.The efficacy and safety of these molecules [biologics and/or Janus kinase (JAK) inhibitors] are being evaluated in clinical trials, and several of them have already received marketing authorization.However, clinical trials are conducted in controlled situations and with selected populations, which do not necessarily reflect prescribing conditions in daily practice.Therefore, the evaluation of these molecules in real-life settings is essential for clinical practice because they assess treatment outcomes within patient populations displaying a heterogeneity of AD phenotypes (11).All molecules have received approval in clinical trials with findings indicating that upadacitinib, a selective JAK-1 inhibitor, had a slightly better safety profile at a dosage of 15 mg per day compared to Upadacitinib 30 mg per day and other classes of JAK-inhibitors in standard doses (12,13). Despite the fact that the results of randomized cohort studies are extremely important for evaluating the efficacy and safety of drugs, they nevertheless do not allow elucidating many issues related to the individual characteristics of patients.In these cases, the results of real clinical practice are of paramount importance. However, the difficulty in conducting this routine clinical studylies in the current lack of consensus on what would constitute a successful treatment outcome in cases of atopic dermatitis, as the criteria used to assess response to therapy can vary significantly.These criteria include endpoints and duration of treatment. Objective: To perform a step-by-step assessment of the efficacy and safety of upadacitinib for the treatment of moderate to severe atopic dermatitis in a real-world setting, analyzing patient responses to treatment. Materials and methods A cohort study was conducted in two ADCARE (Atopic Dermatitis Center of Reference and Excellence) centers.The study was approved by the Ethics Committee and all participating patients provided informed consent. A total of 19 patients (11 female) with moderate to severe AD (4 moderate AD, 15 severe AD) were candidates for targeted therapy with upadacitinib (Supplementary Diagram 1).The clinical and demographic characteristics of the cohort are presented in Table 1.Following the full examination, 3 patients withdrew their informed consent and refused to receive systemic targeted therapy.As a We sourced data from individual outpatient electronic medical records and an electronic register system of a reference center.The analysis included initial patient characteristics and observations on their dynamics, in addition to results from laboratory (complete blood count, blood chemistry test, coagulation tests, total IgE levels, specific IgE levels, Hepatitis serologic tests) and instrumental (chest X-ray) tests. Inclusion criteria were as follows: 1. Adults (at least 18 y.o.) patients irrespective of gender. 2. The presence of AD symptoms for ≥ 3 years. 3. Moderate to severe forms of atopic dermatitis (EASI ≥ 7, SCORAD ≥ 25, IGA ≥ 3; transcripts and descriptions of the questionnaires are listed below).4. Inadequate response, such as no reduction in inflammatory foci or itching, to topical treatment with glucocorticoids and/or calcineurin inhibitors. 5.The ability to understand and fill out questionnaires related to participation in the study.6.The ability to visit the hospital, according to the proposed plan. Exclusion criteria were as follows: 1. Any existing contraindications to the use of upadacitinib as outlined in the product label.2. Any conditions which would inhibit participant's ability to engage in the study, as determined by a healthcare professional (e.g., failure to adhere to clinic visits, complete questionnaires, contact with physician due to alcohol abuse, use of illicit drugs, mental health issues, cognitive impairment etc.).3. Refusal to consent and participate in the study at the start of treatment. The SCORAD questionnaire was used to assess disease severity only at the first visit to evaluate if the patient was suitable for the study.Scores between 0 and 20 on this questionnaire correspond to mild AD, between 21 and 50 − to moderate AD, and above 51 − to severe AD. The following tools were used to assess disease severity, response to treatment and quality of life throughout the study: -BSA (body surface area) calculated the percentage of the affected body surface. -EASI (Eczema Area and Severity Index): a moderate response to therapy was considered to be a decrease in the initial index value by 50% to 75%, while an optimal response was defined as a decrease of the initial EASI by 75% to 100%.The minimum clinically significant difference or mean difference in change scores on the index was 6.6 points, with an EASI-50 defined as a minimal criterion for an early response to therapy according to EASI was evaluated at baseline, 1-month and 4-month follow-up. -The POEM and DQLI questionnaires were used for subjective patient assessment. The optimal response according to POEM (Patient-Oriented Eczema Measure) was considered 0-2 points, suboptimal (moderate) − 3-7 points, 8 or more points − non-response to therapy regarding patient's feelings.The DLQI (Dermatology Life Quality Index) had a score range from 0 to 30, with score bands indicating the following effects on patients' lives: 0-1 indicating no effect; 6-10 indicating moderate effect; 11-20 indicating strong impact; 21-30 showing an extremely strong impact.A decrease in the DLQI index by 4 points or more after therapy was defined as a moderate response, while an optimal response was defined as a decrease to 0 or 1 score after treatment (14).The minimal difference in change scores is 4 points, according to the criteria for inflammatory skin diseases (15,16).DLQI was evaluated at initiation, 1-month and 4-month follow-up. -the numerical rating scale (NRS) of pruritus was assessed according to a 0 to 10-point scale. -the disease impact on sleep (Insomnia NRS) was also observed on a 0-10-point scale (17).POEM, NRS pruritis and insomnia were evaluated at baseline and at 1 month.Before the treatment initiation, a hepatitis screening and QuantiFERON test were performed in order to prevent exacerbation of a possible persistent infection due to drug immunosuppression. Data were visualized using Statistics 13 software.The nonparametric Wilcoxon test for dependent samples was used to identify any significant differences; significance levels p < 0.05 ( * ), p < 0.01 ( * * ) and p < 0.001 ( * * * ) were identified.The Spearman correlation coefficient was used to determine the relationship between the indicators. History of previous atopic dermatitis treatment strategies in the studies patients Prior to upadacitinib initiation, one patient switched from therapy with dupilumab due to insufficient disease control.Additionally, two patients have been taking a standard dose of cyclosporine for 3 and 12 months but discontinued due to lack of efficacy.In four cases of the baseline cohort, PUVA-therapy (photochemotherapy with psoralen and A-wave ultraviolet radiation) was performed, but had an incomplete effect.Thirteen patients used systemic corticosteroids for relief during exacerbations and all patients used both topical glucocorticoids and concomitant moisturizers.The sensitization spectrum of patients treated with upadacitinib (n = 16). Patient characteristics at baseline The baseline characteristics of the primary cohort (19 patients) are presented in Table 1.According to the World Health Organization (WHO) gradation, all patients of the cohort belonged to the young age group with a median age of 33 years (ranging from 19 to 46).Early onset of atopic dermatitis (AD; within the first year of life) was reported in most patients, and the average duration of AD matched the age range and was 33 years (ranging from 19 to 46).Initially, peripheral blood eosinophilia (more than 300 cl/µl) was detected in 11 patients.High total immunoglobulin E levels (> 100UI) were observed in 16 patients.Atopy was observed in most patients before treatment with Upadacitinib (n = 18), with sensitization to at least one allergen as follows: dust mites (67%), epidermal (72%), pollen (14%), molds (10%) and food (17%).Polysensitization dominated with 15 out of 19 total cases, whereas monosensitization was determined in only three cases.One patient was not sensitized to any of the allergens tested. A total of 19 patients (11 female, 8 male) with moderate to severe AD (4 moderate AD, 15 severe AD) were candidates for targeted therapy with upadacitinib (Supplementary Diagram 1).The clinical and demographic characteristics of the cohort are presented in the Table 1.After the full examination, 3 patients withdrew their informed consent and refused to receive systemic targeted therapy, thus 16 patients continued the trial and were initiated with upadacitinib 15 mg/day from January to July 2022 inclusive.The planned dynamic monitoring period was 4 months, with 5 control visits, including the initial one.Six patients had not reached 4 months of follow-up at the time of statistical data processing; therefore, the analysis at the 4-month follow-up was carried out for 10 patients who had entered the study earlier than the other participants. The sensitization spectrum of the 16 patients who have started taking upadacitinib is shown in Figure 1.Polysensitization (sensitivity to two or more groups of allergens) was observed in 12 of the 16 patients.In most cases, the patient exhibited sensitivity to multiple allergens at the same time.Monosensitization (sensitivity to only one allergen) was found in 3 patients (2 were sensitive to epidermal allergens, while one was sensitive to dust mites).Proportion of patients achieving improvement on EASI response at 1-month and 4-month follow-up. Figure 2 shows the Type 2 inflammation-related comorbidities of patients treated with upadacitinib.12 patients had rhinoconjunctivitis, 6 of whom also had bronchial asthma, and 6 patients had food allergies.Three patients had no Type 2 comorbidities. The dynamics of the skin process under upadacitinib therapy was assessed through the EASI scores, which are represented in Figures 3, 4. At baseline, the mean EASI score was 29.5 points; a mean reduction to 18.8 points was seen at 1-month follow-up, with further gain to 24 points at 4-month follow-up (Table 2).At 1-month follow-up, 9 (56%) participants had achieved a 50% reduction in their EASI score from baseline (EASI-50), increasing to 9 (90%) at 4-month follow-up (Figure 3).Further improvement was seen toward 4 months: 6 patients (38%) achieved an EASI score decrease of 75% (EASI-75) at 1 month and 8 patients (80%) met this goal after 4 months.In addition, 50% reached the highest responses level evaluated in this study, the EASI 90 response rate, after 4 months (Figure 3).All patients were initially administered 15 mg/day of upadacitinib and 4 patients had their dose increased to 30 mg/day after 1 month due to suboptimal response. In six cases, the optimal response to therapy was demonstrated 4 weeks after initiation.One patient (No. 12) only achieved a response at 3 months of therapy, but the dose wasn't escalated due to the patient's concerns (Table 3). Three patients (No. 6, 7, 15) exhibited a wave-like pattern in their responses throughout the course of treatment, which could have been triggered by external stressors such as intense physical activity, exposure to cold air and/or allergens.Notably, patient No. 9 achieved satisfactory results with improved EASI skin scores after the dose of upadacitinib was escalated to 30 mg/day during the second month of therapy.Moreover, similar benefits were observed in patients No. 6 and 7 when their daily dose of upadacitinib was escalated to 30 mg/day. The results showed a statistically significant decrease in both NRS-itch and NRS-insomnia scores from the baseline point to both 1-month and 4-months follow-up (Figures 5, 6).At the 1-month follow up, the mean percentage reduction of pruritus score from baseline was 63.5%, while the NRS insomnia score had a mean percentage reduction of 64.6% at the same time (Figure 5). A strong positive correlation was revealed between an increase in the quality of life and a decrease in the absolute values of EASI score (correlation coefficient = 0.966) at the 4-month follow-up. Treatment safety Among 16 patients continuing treatment, 3 (18.8%)experienced acne, and 3 (18.8%)reported more frequent acute respiratory infections compared with the same period in the previous year before systemic treatment had been initiated.One patient (6.25%) noted an exacerbation of the labial form of herpetic infection; this was resolved without requiring antiviral therapy.An isolated transient elevation of ALT (100.2 u/l) was observed in one case; no other clinically significant deviations in analyses were observed. During the study, two non-severe adverse events that led to the discontinuation of therapy were registered.A 46-year-old female reported experiencing dyspeptic phenomena (flatulence and moderate epigastric pain) during the second week of therapy.The patient chose to interrupt the treatment, and subsequently the symptoms abated.After reintroducing upadacitinib one week later, the symptoms resurfaced; thus, it was decided jointly to end the treatment. The second case was a 28-year-old male patient who experienced acne and pyoderma periauricular skin infection after 3 weeks of treatment initiation.The symptoms resolved upon treatment discontinuation.In addition, the patient had been suffering from frequent upper respiratory viral infections up to once a month; additionally, an increase in the frequency of labial herpes infections was observed.Upon administration of oral acyclovir optimal response was seen; however, the patient decided to discontinue the treatment due to safety reasons. Discussion Upadacitinib is an oral, selective and reversible small-molecule Janus kinase (JAK) inhibitor, taken once-daily.It has been specifically engineered to have greater inhibitory power against JAK1 than against JAK2, JAK3, and tyrosine kinase 2 (TYK2).JAK1 is an intracellular molecule involved in the signaling of Box plots showing significant reduction in mean Eczema Area and Severity Index (EASI) score at baseline and at 1-and 4-month follow-ups. several important cytokines that play a role in AD pathology.This medication has been approved for the treatment of moderate to severe AD in adults and adolescents over 12 years old (18).The current study cohort showed improved signs and symptoms of AD, with a statistically significant reduction in EASI, DLQI, pruritus intensity, and sleep disorder scores from baseline to 1-month and 4-month follow-ups on upadacitinib therapy. Of the 10 patients who reached 4 months of treatment, 8 achieved an optimal response according to the EASI 75-100 criteria, 1 achieved a moderate response ( EASI 50-75), and 1 did not respond to therapy ( EASI ≤ 50). Despite the predictable short response time to upadacitinib therapy, described in other real-world studies (19, 20), 44% of patients did not reach a EASI 50 by the end of 1 month of follow-up.The scientific community has to determine an effective strategy for these cases.Options include escalating the dose of upadacitinib immediately or continuing with a standard dose for longer treatment and then assessing effectiveness criteria at week 16.However, it is certain that therapy should be continued, in the absence of any clinically significant side effects, for at least 4 months. The timing of therapeutic response in the study by Chiricozzi et al. (8) eczema severity index (EASI) 75, EASI 90 and EASI 100 were achieved by 78.2, 47.6, and 28.2% of patients at week 16 and 87.6, 69.1, and 44.3% at week 48, respectively.The percentage of patients achieving these therapeutic goals increased until week 32, followed by a plateau. If objective criteria of effectiveness are not met after 4 months of therapy (i.e., 1 out of 10 patients did not reach a EASI-50, 2 patients did not reach a EASI-75), a comprehensive assessment of the therapy's effectiveness should be conducted by considering subjective indicators (such as POEM, DLQI, severity of itching, and sleep disorders) along with the patient.The decision to either continue or discontinue the current therapy should be jointly made N/A, not accessed.Yellow filling: achievement of moderate response; green filling: achievement of optimal response; white filling: no response, up arrow: escalation of upadacitinib dosage from 15 to 30 mg/day.The percentage difference between the EASI score at baseline and that at follow-up month is referred to as EASI ( EASI, %).Moderate response is considered to have been achieved if the EASI is 50-75%, while an optimal response is defined as a EASI of 75-100%. by the patient and clinician; however, according to the National Institute for Health and Care Excellence (NICE) recommendations (21), failing to reach EASI-50 by 4 months should most likely suggest switching/stopping the current treatment regimen. It is interesting to note that the efficacy of upadacitinib in clinical trials was evaluated at both 16 and 52 weeks, with the primary endpoint being a EASI-75, as we previously described. The results of our study show greater effectiveness of upadacitinib therapy compared to those reported in phase III clinical trials (MEASURE-UP1, MEASURE-UP2, and AD-UP) (22-24) (Table 3).The effectiveness of the treatment was evaluated through a percentage reduction in EASI scores at 4 months, including EASI-75 and EASI-90. A deep analysis on upadacitinib refractory patients shows that several possible factors may have contributed to this lack of response to therapy (Table 4).In addition to atopic dermatitis, female patient 3was diagnosed with a mild form of bronchial asthma and allergic rhinoconjunctivitis.Furthermore, the patient tested positive for sensitization to several groups of allergens, including dust mites and pet hairs.Upon contact with the allergens, the patient experienced shortness of breath, difficulty breathing, skin rashes, skin itching, and rhinoconjunctivitis.Additionally, patient 3was found to be sensitive to pollen (weed and tree) and various fungal allergens.Despite attempting to advise patient 3 to eliminate these factors from her environment in order to reduce inflammation and restore the epidermal barrier's integrity, the patient refused to make any change.Furthermore, a 15-year inhalation of cigarette smoke (at 10 cigarettes per day) was noted; smoking has been shown to influence the integrity of the epidermal barrier (25) and increase proinflammatory immune response functions (26,27).In addition to these factors, it is worth noting that the patient had an elevated Body Mass Index of 28.7 which may have contributed to inflammatory activity (28-30).The patient had several chronic diseases, including calculous cholecystitis, hypertension, chronic tonsillitis, dyslipidemia, and osteochondrosis with associated ischial pain syndrome.No medications to treat those comorbidities were used.It is our belief that the constant non-cutting pain syndrome has had a significant negative impact on the patient's mental health, leading to a failure to achieve relief of subjective symptoms such as reduced itching and normalization of sleep, thus contributing to a cycle in the pathogenesis of atopic dermatitis. There is no clear indication in the initial dose of the drug.We suggest that for adults with a long history of the disease, upadacitinib therapy should be initiated at 30 mg per day.It may take longer for a clinical effect to become apparent.Further studies are needed to confirm this hypothesis. The results of upadacitinib clinical trials showed that most of the cohort (9), were adults aged 18-75 who had moderate to severe atopic dermatitis, with 40% of them being women (REF). We also reviewed several case reports which highlighted the remarkable efficacy and safety of upadacitinib in patients with severe atopic dermatitis who had previously received systemic immunosuppressants or dupilumab.The study by Dal Bello et al. (31) examined a cohort of predominantly male (80%) patients with mean age of 35 who presented with severe atopic dermatitis (median EASI score of 34) and had a history of T2 comorbidity (50% had bronchial asthma and/or allergic rhinoconjunctivitis).They had all previously been treated with various systemic therapy options (azathioprine, cyclosporine, methotrexate, UVB therapy), before being subsequently treated with dupilumab.The study showed adequate disease control on upadacitinib treatment in ten patients that failed dupilumab treatment.The meta-analyses by Drucker et al. (32) and Sedeh et al. (33), which examined systemic therapy drugs for severe atopic dermatitis, concluded that Abrocitinib 200 mg and Upadacitinib 30 mg/day were the most acceptable treatment options.Upadacitinib 15 mg/day was also found to be equally as effective compared with Dupilumab 300 mg/2 weeks.Outcomes included change in Eczema Area and Severity Index (EASI), Patient Oriented Eczema Measure (POEM), Dermatology Life Quality Index (DLQI), and Peak Pruritus Numeric Rating Scales (PP-NRS).The drugs evaluated in these studies included Abrocitinib 100 and 200 mg, Baricitinib 2 and 4 mg, Upadacitinib 15 and 30 mg, Dupilumab 300 mg/2 weeks, and Traclokinumab 300 mg/2 weeks.In terms of safety, all treatments were generally well tolerated. Janus Kinase (JAK) inhibitors versus Dupilumab were compared in a meta-analysis conducted by Nusbaum et al. (34), which demonstrated that the JAK inhibitor class was superior to Dupilumab regarding efficacy, without sacrificing safety.Of all JAK inhibitors evaluated, upadatinib at a dose of 30 mg showed the highest efficacy.However, the data demonstrated by Napolitano et al. (35) in a real-life dual-center experience demonstrated a statistically significant superiority of upadacitinib over dupilumab for both skin clearance and relief of pruritus, although the incidence of serious adverse events (SAEs) and adverse events (AEs) leading to discontinuation was higher in the upadacitinib-treated cohort (35). Within the Janus Kinase Inhibitor class, an interesting metaanalysis by Zhang et al. (36) showed that upadacitinib 30 mg was superior to other oral JAK inhibitors with regard to its lack of serious adverse events.However, the cohort of patients consisted of individuals whose disease severity ranged from mild to severe. Regarding the safety of upadacitinib, Qiu et al. (37) showed that the group receiving the escalated dose had a higher risk of acne and elevated levels of creatine phosphokinase (CPK), but no serious adverse events were detected. According to the results of our study, as well as studies published by other authors (11, 38), severe infections and serious adverse cardiovascular events were not recorded. As for conventional immunosuppressants, there are three main options: cyclosporine A, azathioprine, and methotrexate.According to Schram et al. (39) (41,42).However, no comparison has been made with those using Janus kinase inhibitors.The most frequent adverse events associated with methotrexate (MTX) are elevation of liver enzymes and gastrointestinal issues.Neurotoxicity and atrial hypertension have been reported in clinical trials involving cyclosporine.Azathioprine is associated to myelosuppression and hepatotoxicity (43). A direct comparison was conducted in a randomized cohort trial by Blauvelt et al. (10) between upadacitinib and dupilumab (9), to ascertain if there is an equal safety profile.The results showed that, with an equal safety profile, upadacitinib was superior; at 4 month post-treatment, 71% of patients in the upadacitinib group reached EASI reference point, while only 61% of patients in the dupilumab group achieved this benchmark. Over the past decades, our understanding of the pathophysiology of AD has grown, leading to a revolution in effective targeted therapy, such as the JAK-inhibitor upadacitinib.This is the first study to examine upadacitinib in real clinical practice with relevant issues raised concerning its dosing and response.It is important to consider both the doctor's opinion regarding the dosing and response to therapy, as well as the patient's point of view.Successful treatment outcomes depend on forming effective partnerships between doctors and patients that are based on communication and involve assessing both objective measures of disease activity, and subjective feelings reported by the patient. The limitations of our study are inherent in the design of real-world registry studies.There were missing data, including clinical questionnaires and laboratory markers, for some patients.As with all registry data, there may be a bias and erroneous entery of data, limiting data quality and representativeness.Additionally, the single center data were analyzed which may be a subject to bias related to clinical practice.However, in line with the objectives of our study, at present, this represents real-life usage of this medication for AD in clinical practice. Due to the small cohort size, the study results should be interpreted with caution, and further studies with a larger sample and longer follow-up are needed to make better recommendations for clinical use. In conclusion, the initial real-life results with upadacitinib show promising effectiveness, even in patients with comorbidities or ones who received prior systemic treatments. FIGURE 5 FIGURE 5Box plots showing significant reduction in mean NRS-itch score at baseline and at 1-month follow-up. FIGURE 7 FIGURE 7Box plots showing significant reduction in mean DLQI score at baseline and at 1-and 4-month follow-ups. TABLE 2 AD severity assessment at baseline and follow-up after 1 and 4 months on upadacitinib therapy. TABLE 3 Dynamics of skin cleansing according to the EASI index (n = 16). TABLE 4 Proportion of patients who had achieved coprimary endpoints at 4 month in phase III clinical trials and current study.	2024-04-19T15:19:24.229Z	2024-04-17T00:00:00.000	{ "year": 2024, "sha1": "8080cdab4cfc662818ca728946a82037cc32473f", "oa_license": "CCBY", "oa_url": null, "oa_status": "CLOSED", "pdf_src": "PubMedCentral", "pdf_hash": "513e74d2bb60b277cdf2bfc248ce66a32036fdb4", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
264405849	pes2o/s2orc	v3-fos-license	Large zeta sums In this article, we investigate the behaviour of values of zeta sums $\sum_{n\le x}n^{it}$ when $t$ is large. We show some asymptotic behaviour and Omega results of zeta sums, which are analogous to previous results of large character sums $\sum_{n\le x}\chi(n)$. Introduction Let q be a large integer and χ (mod q) be any non-principal Dirichlet character.The study of character sums n≤x χ(n) has a long history.In 1918, Pólya and Vinogradov proved independently the nontrivial upper bound n≤x χ(n) ≪ √ q log q. This uniform upper bound remains the best possible up to the implied constant till now and is called the Pólya-Vinogradov inequality.Assume the generalized Riemann hypothesis is true, in 1977 Montgomery and Vaughan [10] showed that n≤x χ(n) ≪ √ q log 2 q.This is the best possible conditional upper bound up to the implied constant.Also conditionally, Granville and Soundararajan [8] showed that log x/ log 2 q → ∞ as q → ∞ implies n≤x χ(n) = o(x). Like character sums, zeta sums n≤x n it have many similar properties.It is not hard to show the Pólya-Vinogradov type inequality (Hao Zhang) School of Mathematics, Hunan University, Changsha 410082, P. R.China E-mail addresses: zikangdong@gmail.com,weijiawang@tsinghua.edu.cn,zhanghaomath@hnu.edu.cn. assuming x < t are both large.Unconditionally, the Vinogradov-Korobov method yields that log x/(log t) 2/3 → ∞ as t → ∞ implies n≤x n it = o(x).Though the analogous results are less celebrated than those of character sums, the study of zeta sums is important as well.The bounds of zeta sums on a wide range of x is related to those of the Riemann zeta function close to the 1-line, while obtaining a larger saving on a more limited range of x is related to the values of the Riemann zeta function on the critical line.These two kinds of sums can be modelled by the same sums of random multiplicative functions n≤x X n , where X n is the Steinhaus random multiplicative functions.Recently, based on his celebrated work on moments of random multiplicative functions, Harper [6] unconditionally showed the low moments of zeta sums (and also character sums) have "better than squareroot cancellation": for 1 ≤ x ≤ T and 0 where L T = min{x, T /x}.This draws much new attention to zeta sums.The best known lower bounds are due to La Bretèche, Munsch and Tenenbaum [5].Yang [14] showed the following conditional asymptotic formula, which is an analogue of a similar result of character sums by Granville and Soundararajan [8].In 2019, Lamzouri [9] Theorem.[14, Theorem 5] Assume the Riemann hypothesis and let T be large.If 2 ≤ x ≤ T , T + y + 3 ≤ t ≤ T 1000 and y ≥ (log T ) 2 (log x) 2 (log 2 T ) 12 , then and Yang conjectured (see [14,Conjecture 1]), the conditions of the above Theorem can be extended to t ≍ T and y = (log T + (log x) 2 )(log 2 T ) A for some positive A. This conjecture is very strong.Assuming Yang's conjecture, one can deduce very sharp upper bounds for the derivatives of the Riemann zeta function (See [14,Theorem 7]).Thus the asymptotic behaviour of values of zeta sums is very important. Without the Riemann hypothesis, we can show that, except a small set of t, zeta sums can be approximated by the sums over friable numbers. Theorem 1.1.For all 1 ≤ t ≤ T but a set of measure at most T 1−1/ log x , whenever 2 ≤ x ≤ T 1 3 , y ≥ log x log T (log 2 T ) 5 we have For all 1 ≤ t ≤ T but a set of cardinal at most T 1−1/(log 2 x) 2 , we have Yang's conditional result is sharp, since we can show that x = (log T ) A for some In fact, zeta sums can gain large values in any direction.Denote by ρ(•) the Dickman function. Now we will introduce the resonance method for large zeta sums.This method can date back to Voronin's work in 1988, and developed significantly by Soundararajan [12].Firstly, when x is not very large compared with exp((log T ) 1 2 ), we can use the so-called "long resonance method" to detect large values of zeta sums.This method for character sums is due to Munsch [11], which improves previous work of Hough [7]. When log x is a small power of log T , we can write the lower bound in a more compact way. When x is even smaller (power of log T ), we can write the lower bound more precisely. Corollary 2. Let x = (log T ) A for some A > 1.Then we have When x is very close to exp((log T ) 2 ), we would use the method of Hough [7], which is essentially Soundararajan's resonnance method in [12]. 2 ) but smaller than √ T , we can combine the resonance method with GCD sums (also called Gál-type sums).This kind of method was originated from Aistleitner [1], and subsequently developed by Bondarenko and Seip [2,3], and La Bretèche and Tenenbaum [4]. Theorem 1.5.Let exp((log T ) When x is larger than √ T , we may need to look for the relation between the quantities 1 In comparison, the Poisson summation formula for character sums suggests the "symmetry": In fact, our Theorems 1.1, 1.2 and 1.4 can be generalized to the case of n≤x f (n)n it in shorter interval [T, 2T ], where f (n) is any completely multiplicative function satisfying \|f (n)\| = 1 for any n ∈ N * .Xu and Yang have the same treatment in [13] and get a similar result of our Theorem 1.4. This article is organized as follows.We will present some preliminary lemmas in §2.We will prove Theorems 1.1-1.5 separately in §3-7. Preliminary lemmas In this section, we give some lemmas that we will use later. Here and throughout this paper, we will put X p as a sequence of i.i.d random variables equidistributed on the unit circle for all prime p.If n = i p a i i , let X n = i X a i p i be multiplicative random variables. Lemma 2.1.Let r(n) be any bounded arithmetic function and E(•) be the expectation.Then Proof.Rearranging the sum, we get (2.1) ) and y = log T log x(log 2 T ) 8 .Then for t in at least a set of measure 3 ). We pick only those X n for which \| arg X p \| ≤ π log T for all p ≤ y.For these choice n≤x n∈S(y) With the same argument as the proof of Lemma 7.1 in [8], we get the desired result. According to [8, Theorem 6.1], if y ≥ C log 2 x then there are a certain constant c > 0 so that For A > 1, we deduce that there are t of measure at most T A −2k not satisfying exp O log 2 x log 2 x y . Take y = log x log T (log 2 T ) 5 and A = 2.We get the first part of Theorem 1.1. For the second part, let y = (log T + log 2 x)(log 2 T ) 4 and A = exp log x (log 2 T ) 2 .Then there are at most t of measure Proof of Theorem 1.2 Put y = log T log x(log 2 T ) 8 and y 1 = log T (log log T ) 7 .With the same argument as the proof of [8, Theorem 3], we get that except for a set of measure at most We see that with an exceptional set of measure at most Proof of Theorem 1.3 Let ε be a very small positive number.For let a k be completely multiplicative with a 1 = 1, a p = 1 − log y log x(log 2 T ) 1+δ for p ≤ y and a p = 0 for p > y.Here δ is a positive number smaller than ε.We have the following result for a k , which follows directly from [11, P. 35-36]. Lemma 5.1.Let a k and y be defined above.We have k≤x k∈S(y) Define the resonators where a k is defined above.We have and where S t (x) = n≤x n it .Trivially we have by the upper bound of R(t), and by the rapid decreasing of φ(•), we have For I 1 (R, T ), we have For I 2 (R, T ), we have . By using the formula (2.1), we have Now by combining Eq. (6.1) and Eq. ( 6.2) we get max Finally, the result follows from the proof of Theorem 3.2 in [7, P.103] 7. Proof of Theorem 1.5 Before we prove Theorem 1.5, we present the following result on GCD sums.For J := {j ≥ 0 : M j = ∅}, let M ′ = {m j = min M j : j ∈ J }. Then we define the resonator We define For M 1 (R, T ), we have and we have Now we focus on I 2 (R, T ).We have For the inner sum, we have Inserting into (7.2),we have where the last inequality follows from Lemma 7.1.Thus we complete the proof of Theorem 1.5. n≤x n it ≪ √ t log t , ( Zikang Dong) School of Mathematical Sciences, Tongji University, Shanghai 200092, P. R.China (Weijia Wang) Yanqi Lake Beijing Institute of Mathematical Sciences and Applications & Yau Mathematical Sciences Center, Tsinghua University, Beijing 101408, P. R.China also generalized their work to the sums of Hecke eigenvalues of holomorphic cusp forms.Denote by S(y) the y-friable integers and define Ψ(x, y) := n≤x n∈S(y) 1, Ψ(x, y; t) := n≤x n∈S(y) n it . So we deduce that I 2 ( 6 . 1 √ T − 1 R, T ) I 1 (R, T ) ≥ k≤x k∈S(y) a k ≥ Ψ(x, (1 + o(1))y), by Lemma 5.1, which completes the proof.Proof of Theorem 1.4 Proof.Let y = T /x and a = log x log 2 x, we define a completely multiplicative function r(n) by r(p) = a √ p log p where a 2 ≤ p ≤ e (log a) 2 is prime and r(p) = 0 for other primes.We define the resonator R(t) = n≤y r(n)n it , then we have max t∈[1,T ] n≤x log m i k m j l m∈M i ,n∈M j mk=nl 1 =≫ 1 min{r(m i ) 2 , r(m j ) i,j∈J m∈M i ,n∈M j mk=nl φ T log T log m i n m j m .Since m ∈ M i , n ∈ M j implies log m i n m j m ≪ log T T and thus φ T log T log m i n m j m I 2 ( 1 . R, T ) ≫ T log T k,l≤x m,n∈M mk=nl 1 = T log T m,n∈M k,l≤x mk=nlFor fixed m, n, mk = nl implies k = nL/(m, n) and l = mL/(m, n) for some integer L. Since max M ≤ 2 min M, we have for the inner sumk,l≤x mk=nl 1 ≥ x max{ m (m,n) , n (m,n) log(T /x) log 3 (T /x) log 2 (T /x) ,	2023-10-23T01:01:21.920Z	2023-10-20T00:00:00.000	{ "year": 2023, "sha1": "0e47c5d861c9e3bad6a3da5f30079070fca8abd0", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "c33bb1a1797495c4f46750bd299317b808d40a46", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
247902218	pes2o/s2orc	v3-fos-license	Experimental Study on Ultrasonic-Assisted End Milling Forces in 2195 Aluminum-Lithium Alloy To achieve high-quality machining of the 2195 aluminum-lithium alloy, this paper presents an experimental study on the effect of milling processing parameters on milling forces and surface topography, during which conventional milling and longitudinal-torsional ultrasonic vibration milling of the 2195 Al-Li alloy were performed. The characterization of the tool tip trajectory illustrates some of the advantages of ultrasonic machining, which include variable depth of cut and tool chip pulling. The differences in milling forces between conventional milling and longitudinal-torsional ultrasonic vibration machining were compared using orthogonal tests, and the effect of ultrasonic vibration on milling forces was investigated in detail. The maximum reduction of milling force Fy in the feed direction under the influence of torsional vibration is 62% and 54% for larger feed per tooth and cutting depth, respectively. The high-frequency impact generated by the longitudinal vibration not only reduces the chip accumulation on the surface, but also smooths out the tool-tooth scratches and creates a regular surface profile. In addition, the characteristics of the milling force signals of the two machining methods were analyzed, and the analysis of the spectrum of the collected milling forces revealed that the ultrasonic vibration caused the high-frequency components of the milling forces Fy and Fz. The orthogonal result analysis and single-factor result analysis verified the superiority of ultrasonic machining, provided parameter selection for subsequent aluminum-lithium alloy machining, and bridged the gap of longitudinal torsional ultrasonic vibration machining of 2195 aluminum-lithium alloy in the study of milling force. Introduction Aluminum-lithium alloys are lightweight aerospace structural materials with excellent comprehensive properties such as low density, low weight, high elastic modulus, high specific strength and specific stiffness [1][2][3]. Among them, 2195 aluminum alloy is one of the most complex grades in the wrought aluminum-copper family (2000 or 2xxx series), with at least 91.9% aluminum by weight. The high strength-to-weight ratio of this alloy made it lucrative for aerospace applications. Thus, it successfully replaced the 2219 alloy in the Space Shuttle fuel outer storage tank, reducing its mass by 5% and increasing the storage capacity by 3.4 t [4]. More recently, 2195 alloy was used for the propellant tanks of the Falcon 9 Full Thrust orbital launcher and the new ULA Vulcan first stage. Like most other aluminum-copper alloys, 2195 is a high-strength alloy, with bad workability and poor corrosion resistance. As a wrought alloy, it can be welded, particularly by friction stir welding, and is fracture resistant at cryogenic temperatures. Alternatively, the processing of aluminum-lithium alloys has been achieved by the chemical etching method [5][6][7][8][9], which was not environmentally-friendly due a series of pollution problems. Nowadays, mechanical milling has made great progress, which improves the milling efficiency and avoids environmental pollution. The ease of combustion and oxidation of Mg and Li elements in 2195 Al-Li alloy leads to increased difficulty in thermomechanical treatment and poor machinability of Al-Li alloy under high-speed milling [10]. The study of processing methods and process parameters [11] of aluminum-lithium alloy products is a good guide for engineering applications. Mou et al. [12] studied the surface integrity of aluminum-lithium alloys obtained by milling under different coolant conditions, and the results showed that the milling direction and the angle of contact between the milling tool and the test piece have a large effect on the surface roughness. Rong et al. [13] studied the surface roughness of aluminum-lithium alloys using high-speed milling and investigated the variation law of process parameters on surface roughness. So far, the difficulty of machining aluminumlithium alloys has diminished, but the biggest challenge remaining is how to achieve good surface integrity and ideal chip control by controlling the machining parameters [14]. Most researchers have focused more on the surface morphology of Al-Li alloys, while machining parameters often affect the variation of milling forces and change the surface morphology. The magnitude of the milling force determines the power consumed during the milling process and the deformation of the machining system [15,16], while the machining parameters have a direct influence on the milling force and surface topography. Tej Pratap et al. [17] investigated the high-speed milling process by the finite element modeling (FEM) of the titanium alloy milling process, which showed that strain rate and temperature significantly influenced the cutting force. Sela et al. [18] established a model of milling force under high-speed milling of 7455-T7351 aluminum alloys and studied the predicted value of cutting force under orthogonal test. Girish et al. [19] designed an end mill tool for ultrasonic milling, based on the orthogonal test method for ultrasonic milling of the Al6063 aluminum alloy, and established regression equations to assess the cutting forces during ultrasonic milling. The results showed that the feed rate had the strongest effect on the cutting forces among other machining parameters. Ultrasonic vibration machining techniques can reduce milling forces [20,21] and improve surface quality. In recent years, ultrasonic vibration machining has been gradually applied to alloys with poor machinability and to processes such as grinding and drilling of conformal materials [22,23], which provides a new idea for the machining of aluminumlithium alloys. Compared with conventional machining, machining with longitudinal vibration coupled with torsional vibration can change the tool tip variation from linear to intra-dimensional space motion, thus further reducing the milling force and improving the machining quality [24,25]. So far, ultrasonic vibration processing has mostly focused on titanium alloys and other composite materials, while domestic and foreign scholars have not conducted longitudinal-torsional ultrasonic vibration milling of aluminum-lithium alloy materials. Therefore, it is necessary to study the longitudinal-torsional ultrasonic vibration on the milling force of the 2195 aluminum-lithium alloy. This paper presents the results of an experimental study on the effects of machining parameters on milling forces and surface topography for conventional and longitudinal torsional ultrasonic vibration milling of the 2195 aluminum-lithium alloy. This study can fill the gap in the field of longitudinal ultrasonic vibration milling for the 2195 Al-Li alloy, thus laying the foundation for high-quality machining of the 2195 Al-Li alloy. Materials Tests were performed on the 2195 Al-Li alloy (Ocean Power Rare Metal Products Co, Suzhou, China), which chemical composition and mechanical properties at room temperature are listed in Tables 1 and 2, respectively. Workpieces used in all tests were made from the same sheet to prevent additional effects caused by different batches of various specifications. The sheet was cut into 15 mm × 7 mm × 8 mm blocks of specimens by wire cutting (Cyang, Guangdong, China). Test Setup The test setup for milling force testing is shown in Figure 1. A CNC machining center (Henfux-HFM 700L, Taiwan, China) with conventional milling and ultrasonic milling capabilities and a maximum rotational speed of 30,000 r/min was used in the tests. Its spindle includes a piezoelectric transducer, an ultrasonic transducer and a tool holder. The induction is generated by a transmitting coil fixed to the machine and a receiving coil rotating coaxially on the spindle to power the tool. Ultrasonic vibrations are applied to a piezoelectric transducer, which is to be amplified by a variable amplitude horn, resulting in longitudinal vibrations and torsional vibrations applied to the tool. To study the milling force of ultrasonic-assisted milling of the 2195 aluminum-lithium alloy, a Kistler 5080 piezoelectric crystal force meter (Kistler, Winterthur, Switzerland) was set to 150 kHz acquisition frequency and 150 N acquisition range. The milling force signal of longitudinaltorsional ultrasonic assisted milling (L-UVAM) was collected online in real-time scale, and the average milling forces in three directions (x, y, and z) during smooth cutting were recorded via the signal amplifier. Materials 2022, 15, x FOR PEER REVIEW 3 specifications. The sheet was cut into 15 mm × 7 mm × 8 mm blocks of specimens by cutting (Cyang, Guangdong, China). Test Setup The test setup for milling force testing is shown in Figure 1.A CNC machining ce (Henfux-HFM 700L, Taiwan, China) with conventional milling and ultrasonic millin pabilities and a maximum rotational speed of 30,000 r/min was used in the tests. Its s dle includes a piezoelectric transducer, an ultrasonic transducer and a tool holder. induction is generated by a transmitting coil fixed to the machine and a receiving rotating coaxially on the spindle to power the tool. Ultrasonic vibrations are applied piezoelectric transducer, which is to be amplified by a variable amplitude horn, resu in longitudinal vibrations and torsional vibrations applied to the tool. To study the mi force of ultrasonic-assisted milling of the 2195 aluminum-lithium alloy, a Kistler 508 ezoelectric crystal force meter (Kistler, Winterthur, Switzerland) was set to 150 kHz ac sition frequency and 150 N acquisition range. The milling force signal of longitud torsional ultrasonic assisted milling (L-UVAM) was collected online in real-time scale the average milling forces in three directions (x, y, and z) during smooth cutting recorded via the signal amplifier. Tool-Workpiece Kinematics Analysis To improve the material removal rate, a four-flute integral general-purpose car end mill (model: GM-4E-D8.0 R1.0, Zhuzhou cemented carbide Cutting Tools Co., zhou, China) was used. The milling cutter specifications were as follows: diameter mm, edge length of 20 mm, total length of 60 mm and coated with AlCrXN. Tool-Workpiece Kinematics Analysis To improve the material removal rate, a four-flute integral general-purpose carbide end mill (model: GM-4E-D8.0 R1.0, Zhuzhou cemented carbide Cutting Tools Co., Zhuzhou, China) was used. The milling cutter specifications were as follows: diameter of 8 mm, edge length of 20 mm, total length of 60 mm and coated with AlCrXN. Figure 2a shows the schematic diagram of longitudinal torsional ultrasonic vibration milling process. The analysis of the cutting edge trajectory during ultrasonic milling is essential for the analysis of milling forces in milling tests. The radius of the tool is R, the Materials 2022, 15, 2508 4 of 16 tool tooth number is N, and the coordinate system O-xyz is established between the tool and the center point of the surface contacted by the 2195 Al-Li specimen, where the point P on the edge of the end mill is used as the reference point. The x-direction is the radial direction, the y-direction is the tool feed direction, and the z-direction as the direction of applied ultrasonic vibration. We define A as the amplitude of ultrasonic vibration, A l as the amplitude of longitudinal vibration and f l as the frequency of longitudinal vibration. Accordingly, A t is the amplitude in the torsion direction and f t is the frequency in the torsion direction. The rotation speed of the spindle is n s and the speed in the feed direction is v w . The feed per tooth is f z and the depth of cut is a p . Materials 2022, 15, x FOR PEER REVIEW Figure 2a shows the schematic diagram of longitudinal torsional ultrasonic vibr milling process. The analysis of the cutting edge trajectory during ultrasonic mill essential for the analysis of milling forces in milling tests. The radius of the tool is R tool tooth number is N, and the coordinate system O-xyz is established between th and the center point of the surface contacted by the 2195 Al-Li specimen, where the P on the edge of the end mill is used as the reference point. The x-direction is the r direction, the y-direction is the tool feed direction, and the z-direction as the directi applied ultrasonic vibration. We define A as the amplitude of ultrasonic vibration, the amplitude of longitudinal vibration and fl as the frequency of longitudinal vibr Accordingly, At is the amplitude in the torsion direction and ft is the frequency in th sion direction. The rotation speed of the spindle is ns and the speed in the feed dire is vw. The feed per tooth is fz and the depth of cut is ap. Figure 2c. The trajectory of the reference point P the application of torsional torque is shown in Figure 2d. In longitudinal torsional sonic vibration, the frequency and initial phase of both longitudinal and torsional v tion is identical, because the vibrations in both directions come together from the s amplitude. Thus, fl = ft, the ratio of amplitude in both torsional and longitudinal vibr is 0.6:1, and the initial phase j is the same. As a result, the equations of motion for con tional milling and longitudinal vibration are expressed by Equations (1) and (2), re tively. The trajectory of the point P in torsional vibration is shown in Equation (3) Figure 2c. The trajectory of the reference point P after the application of torsional torque is shown in Figure 2d. In longitudinal torsional ultrasonic vibration, the frequency and initial phase of both longitudinal and torsional vibration is identical, because the vibrations in both directions come together from the single amplitude. Thus, f l = f t , the ratio of amplitude in both torsional and longitudinal vibration is 0.6:1, and the initial phase j is the same. As a result, the equations of motion for conventional milling and longitudinal vibration are expressed by Equations (1) and (2), respectively. The trajectory of the point P in torsional vibration is shown in Equation (3). The trajectory of the longitudinal torsional vibration motion of the point P is shown in Equation (4). According to the milling trajectories presented in Figure 2b,d, the path where the reference point P is located is a three-dimensional curve with "sinusoidal" fluctuations, while conventional milling is a simple two-dimensional curve. In actual machining, the longitudinal and torsional vibrations of the tool can form a series of vibration traces on the surface. Due to the variable tool acceleration, the cutting depth of the tool will also change. This machining feature reduces milling forces and creates a regular texture on the workpiece surface, optimizing surface integrity. Test Program The force gauge was turned on before the milling test, and the cutting fluid was not added during the test. The dynamometer was closed to terminate data collection when milling was complete. To ensure the accuracy of the milling force, the milling force signal was finally processed to acquire the mean value of the milling force. Firstly, the test values of milling force under each machining parameter were analyzed by the method of an orthogonal test to acquire the results of machining parameter interaction. The polarization difference and variance of the machining parameters were then analyzed to finally determine the order of the effect of each machining parameter on the test results. The machining parameters that had the least effect on the results were used quantitatively, the machining parameters that had the greatest effect on the results were used as variables, and then single factor analysis of the milling force values was undertaken. The effect of milling force on the machined surface was obtained by observing the surface morphology after machining. The test program is summarized in Table 3. During the test, there were many factors affecting the cutting force. Of these, four factors, namely milling speed n s , feed per tooth f z , cutting depth a p and amplitude A, were used in the test program. Results and Discussion The results of the orthogonal test on the milling force of the 2195 Al-Li alloy are presented in Table 4. A total of 27 sets of tests were conducted considering the interactive effects of milling speed, feed per tooth and ultrasonic amplitude. Since the diameter of the milling tool is larger than the end face of the workpiece, the entire surface can be milled in one tool feed. In the coordinate system O-xyz, the tool rotated counterclockwise, and the milling force Fx direction was along the opposite direction of the x-axis. The tool feeding in the opposite direction of the y-axis, the milling force Fy, is a positive number. Influenced by the longitudinal ultrasonic vibration, the "sinusoidal" motion of the tip of the tool and the workpiece had the characteristics of contact and separation, so there were positive and negative alternating phenomena of Fz. Fx and Fy did not alternate between positive and negative intervals because the workpiece width was much larger than the cutting depth. The interaction (coupling) effects of milling speed (n s ), ultrasonic amplitude (A) and feed per tooth (f z ) on the milling forces Fx, Fy and Fz are depicted in Figure 3. As seen in Figure 3a,d,g, a simultaneous increase in feed per tooth and rotational speed leads to a significant increase in the cutting forces Fx, Fy and Fz. At a high milling speed (n s = 8000 r/min) and using a smaller feed per tooth f z = 10µm/r can effectively reduce the milling force. At large feed per tooth (f z = 50 µm/r), the milling force can be reduced by decreasing the milling speed to n s = 6000 r/min. Analysis of the Coupled Effect of Machining Parameters on the Milling Forces The interaction (coupling) effects of milling speed (ns), ultrasonic amplitude (A) and feed per tooth (fz) on the milling forces Fx, Fy and Fz are depicted in Figure 3. As seen in Figure 3a,d and g, a simultaneous increase in feed per tooth and rotational speed leads to a significant increase in the cutting forces Fx, Fy and Fz. At a high milling speed (ns = 8000 r/min) and using a smaller feed per tooth fz = 10μm/r can effectively reduce the milling force. At large feed per tooth (fz = 50 μm/r), the milling force can be reduced by decreasing the milling speed to ns = 6000 r/min. The analysis of coupled effects in Figure 3b,e and h revealed that higher milling speeds weakened the effect of ultrasonic vibration, while large ultrasonic amplitudes re sulted in a significant reduction of cutting forces at low rotational/milling speeds (A = 5 μm, ns = 6000 r/min). At small feed per tooth value fz = 10 μm/r, the superimposed ultra sonic amplitude A = 5 μm was more likely to result in lower milling forces as shown in Figure 3c,f and i. The analysis of coupled effects in Figure 3b,e,h revealed that higher milling speeds weakened the effect of ultrasonic vibration, while large ultrasonic amplitudes resulted in a significant reduction of cutting forces at low rotational/milling speeds (A = 5 µm, n s = 6000 r/min). At small feed per tooth value f z = 10 µm/r, the superimposed ultrasonic amplitude A = 5 µm was more likely to result in lower milling forces as shown in Figure 3c,f,i. Analysis of Main Effect Trends The trend of the influence of the interaction between the machining parameters is assessed through the experiment of milling force. Data processing of the test values is required to ensure the results are not due to chance. The order of the influence of each machining parameter on the milling force was obtained by the Polarization Difference analysis of the test data. The value of polarization difference (PD) is denoted by K. This orthogonal test is a four-factor three-level test with three levels of polarization difference as K 1 , K 2 and K 3 . The results are shown in Table 5. The decreasing order of the influence of each milling parameter on the milling force components was as follows: on Fx:f z > a p > n s > A, on Fy:f z > a p > A > n s , on Fz:f z > A > a p > n s . To clarify the effect of machining parameters on milling forces, main effect plots were obtained by Polarization Difference analysis, as shown in Figure 4. These plots were also categorized and discussed to assess the specific effect of each parameter on the cutting force. It can be seen in Figure 4 that plots of Fx, Fy and Fz exhibit similar patterns with s quantitative differences. This is because the longitudinal ultrasonic vibration directl fects the longitudinal milling force (Fz), and the superimposed torsional ultrasonic vi tion indirectly affects the tangential milling force (Fy). The main patterns observed in ure 4g,k imply that the milling force tended to decrease significantly with the ultras amplitude due to the increased ultrasonic amplitude, promoting the contact and sep tion between the specimen and the tool. It also prolonged the duration of this intermi milling, reducing the milling force. In general, aluminum material is softer. When the milling speed is increased, the generated by the material in the shear range increases, which reduces the elastic de mation capacity of the aluminum alloy and reduces the shear strength of the mate thus the milling force has a tendency to decrease. An increase in spindle speed will red the shear angle and shear area, and the change in shear surface area leads to a reduc in shearing force. In the main effect diagram, due to the high specific strength and spe stiffness of the 2195 Al-Li alloy, increasing the friction between the tool and the mat during high-speed milling increases, resulting in a slight increase in milling force It can be seen in Figure 4 that plots of Fx, Fy and Fz exhibit similar patterns with slight quantitative differences. This is because the longitudinal ultrasonic vibration directly affects Materials 2022, 15, 2508 9 of 16 the longitudinal milling force (Fz), and the superimposed torsional ultrasonic vibration indirectly affects the tangential milling force (Fy). The main patterns observed in Figure 4g,k imply that the milling force tended to decrease significantly with the ultrasonic amplitude due to the increased ultrasonic amplitude, promoting the contact and separation between the specimen and the tool. It also prolonged the duration of this intermittent milling, reducing the milling force. In general, aluminum material is softer. When the milling speed is increased, the heat generated by the material in the shear range increases, which reduces the elastic deformation capacity of the aluminum alloy and reduces the shear strength of the material, thus the milling force has a tendency to decrease. An increase in spindle speed will reduce the shear angle and shear area, and the change in shear surface area leads to a reduction in shearing force. In the main effect diagram, due to the high specific strength and specific stiffness of the 2195 Al-Li alloy, increasing the friction between the tool and the material during high-speed milling increases, resulting in a slight increase in milling force. As shown in Figure 4a,e,i. the effect of rotational speed on the milling force of the 2195 aluminumlithium alloy is not significant because the shearing force decreases and the friction force increases at the same time by increasing the spindle speed. As shown in Figure 4b,d,f-h,l, the increase in shear stress exceeded the strength of tool in contact with the material, so that the milling force increased slightly. Meanwhile, increased milling force was linked to increased feed per tooth and depth of cut. The latter resulted in higher cutting thickness and closer tool contact with the specimen, thus increasing the milling force. Single-Factor Analysis The effect of rotational speed on milling forces is superimposed on other machining parameters when the interaction effect is considered. However, the polarization difference analysis of the experimental values indicates that the effect of rotational speed on milling forces is negligible, therefore a single factor analysis of the variation of feed per tooth and cutting depth and ultrasonic amplitude can be applied with the available experimental values, as shown in Figures 5-7. Figure 5 shows a single factor analysis of the milling force Fx. Increase in feed per tooth and cutting depth causes an increase in milling force. The tool is in closer contact with the workpiece in the x-direction because of the torsional vibration, which results in higher milling force at larger feed and cutting depths. Figure 6 shows the single factor analysis of milling force Fy. Torsional vibration significantly reduces the milling force in the tool feed direction, and the higher amplitude reduces the milling force more effectively. Such a phenomenon is the result of the chip pulling effect and tool separation characteristics caused by torsional vibration. In Figure 6a, an amplitude of 5 mm reduces the milling force by 62% at feed per tooth of 50mm. In Figure 6b, an amplitude of 5 mm reduces the milling force by 54%. Figure 7 shows the single factor analysis of the milling force Fz. As shown in Figure 7a,b, the milling force Fz for conventional milling (CM) has a tendency to increase and then decrease followed by an increase due to the increase in feed per tooth and cutting depth; the milling force is minimal at f z = 30 mm and a p = 1 mm. The contact-separation characteristic of the longitudinal vibration causes a change in the depth of cut in the vertical direction, which causes the milling force to change to the opposite direction. Higher amplitude leads to higher longitudinal high-frequency shocks and more pronounced contact-separation characteristics. then decrease followed by an increase due to the increase in feed per tooth and cu depth; the milling force is minimal at fz = 30 mm and ap = 1 mm. The contact-separ characteristic of the longitudinal vibration causes a change in the depth of cut in the tical direction, which causes the milling force to change to the opposite direction. H amplitude leads to higher longitudinal high-frequency shocks and more pronounced tact-separation characteristics. Figure 8 shows the test values of milling forces (Fx, Fy and Fz) for different amplitudes with the same other milling parameters (ns = 8000 r/min, fz = 10 μm/r and ap = 0.5 mm). Milling Force Signal Spectrum Characteristics As shown in Figure 8a,b, the ultrasonic vibration with amplitude A = 3 μm caused a slight increase in the peak milling force, but its mean value decreased. This was due to the superimposed effect of the overall system vibration frequency and ultrasonic vibration resulting in an increase in the peak cutting force. However, the ultrasonic vibration caused a change in contact and separation between the tool and the test piece, resulting in a decrease in the mean value of the peak cutting force. At an amplitude A = 5 μm, the peak Figure 8 shows the test values of milling forces (Fx, Fy and Fz) for different amplitudes with the same other milling parameters (ns = 8000 r/min, fz = 10 μm/r and ap = 0.5 mm). Milling Force Signal Spectrum Characteristics As shown in Figure 8a,b, the ultrasonic vibration with amplitude A = 3 μm caused a slight increase in the peak milling force, but its mean value decreased. This was due to the superimposed effect of the overall system vibration frequency and ultrasonic vibration resulting in an increase in the peak cutting force. However, the ultrasonic vibration caused a change in contact and separation between the tool and the test piece, resulting in a decrease in the mean value of the peak cutting force. At an amplitude A = 5 μm, the peak and mean values of the transverse milling forces (Fx and Fy) were reduced. That is, the To investigate the effect of ultrasonic vibration with different amplitudes on the spec tral characteristics of milling forces, the fast Fourier transform (FFT) was performed fo conventional milling, as shown in Figure 9a As shown in Figure 8a,b, the ultrasonic vibration with amplitude A = 3 µm caused a slight increase in the peak milling force, but its mean value decreased. This was due to the superimposed effect of the overall system vibration frequency and ultrasonic vibration resulting in an increase in the peak cutting force. However, the ultrasonic vibration caused a change in contact and separation between the tool and the test piece, resulting in a decrease in the mean value of the peak cutting force. At an amplitude A = 5 µm, the peak and mean values of the transverse milling forces (Fx and Fy) were reduced. That is, the effect of ultrasonic vibration exceeded that produced by the equipment resonance. A longer separation window between the tool and the specimen reduced both the peak and mean values of the milling forces. Thus, the longitudinal ultrasonic vibration is one of the main factors in the change of tool trajectory, as shown in Figure 8c. Compared with conventional milling, the peak milling force (Fz) of the test at A = 5 µm was smoother and less volatile, which can be considered as the result of the "sinusoidal" variation of the tool motion. When the amplitude was increased, as shown in Figure 8d-f, the increase in the amplitude led to a backward shift in the peak test milling force. Milling Force Signal Spectrum Characteristics To investigate the effect of ultrasonic vibration with different amplitudes on the spectral characteristics of milling forces, the fast Fourier transform (FFT) was performed for conventional milling, as shown in Figure 9a The spindle rotation frequency in conventional (CM) and ultrasonic-assisted milling (L-UVAM) did not change with increasing amplitude. The tool cutting frequencies of Fx and Fy under normal milling were 1027 Hz and 821 Hz, respectively. By applying ultra sonic vibration with a 3 mm amplitude, the tool cutting frequencies of Fx and Fy were increased to 1232 and 2053 Hz, respectively. At an ultrasound amplitude of 5 μm, the too cutting frequency of Fx continued to increase to 2258 Hz, while Fy was reduced by the effect of torsional vibration, resulting in a stable tool cutting frequency of 1027 Hz. The tool plunge frequency of Fz was reduced by applying ultrasonic vibration. In addition, the ultrasonic vibration with applied longitudinal torsion induced the high-frequency com ponents of Fy and Fz. The spindle rotation frequency in conventional (CM) and ultrasonic-assisted milling (L-UVAM) did not change with increasing amplitude. The tool cutting frequencies of Fx and Fy under normal milling were 1027 Hz and 821 Hz, respectively. By applying ultrasonic vibration with a 3 mm amplitude, the tool cutting frequencies of Fx and Fy were increased to 1232 and 2053 Hz, respectively. At an ultrasound amplitude of 5 µm, the tool cutting frequency of Fx continued to increase to 2258 Hz, while Fy was reduced by the effect of torsional vibration, resulting in a stable tool cutting frequency of 1027 Hz. The tool plunge frequency of Fz was reduced by applying ultrasonic vibration. In addition, the ultrasonic vibration with applied longitudinal torsion induced the high-frequency components of Fy and Fz. Surface Morphology Analysis The magnitude of the milling force has a direct correlation to the surface topography. Figure 10 shows the microscopic morphology images of CM and L-UVAM machined surfaces observed by scanning electron microscopy. As the images of the surface morphology in Figure 10a-c show, there are obvious tool marks on the CM machined surface. The increase in feed per tooth increases the milling force, the chip residue on the surface increases, and the lateral plastic flow of the machined surface material becomes more severe, resulting in significant scraping. As shown in Figure 10d-f, the surface topography is changed due to the reduction of milling force at f z = 10 µm and A = 3 mm. The torsional vibration smooths out the tool marks and creates a "sinusoidal" texture on the surface in the direction of the feed. As shown in Figure 10g-i, increasing amplitude causes higher density of traces left by the tool trajectory, surface smearing effect is more obvious, in addition, the impact load increases to increase the material removal rate and reduce the surface chips. Ultrasonic vibration creates a unique texture on the workpiece, which efficiently removes chips and also reduces surface roughness, ultimately improving the surface quality of the workpiece and frictional wear and other characteristics. Materials 2022, 15, x FOR PEER REVIEW 14 resulting in significant scraping. As shown in Figure 10d-f, the surface topograph changed due to the reduction of milling force at fz = 10 μm and A = 3 mm. The tors vibration smooths out the tool marks and creates a "sinusoidal" texture on the surfa the direction of the feed. As shown in Figure 10g-i, increasing amplitude causes hi density of traces left by the tool trajectory, surface smearing effect is more obviou addition, the impact load increases to increase the material removal rate and reduc surface chips. Ultrasonic vibration creates a unique texture on the workpiece, which ciently removes chips and also reduces surface roughness, ultimately improving the face quality of the workpiece and frictional wear and other characteristics. Conclusions This paper presents the results of a study of the effect of two machining proce CM and L-UVAM, on the milling forces of 2195 Al-Li alloys with varying machining rameters. The interaction effect analysis was combined with the single factor analys analyze the variation of milling force. The effects of feed per tooth and depth of cu milling forces were analyzed, as well as changes in force signals and spectra in ultras machining, and changes in surface topography due to changes in milling forces. The s lays a theoretical foundation for further research and optimization of aluminum-lith alloys in the machining milling process, and provides some experimental basis for machining of aluminum-lithium alloys. The following main conclusions were obta from the investigation: (1) In general, cutting depth directly affects the amount of milling force. Explo the effects of milling speed, feed per tooth and ultrasonic amplitude on the interactio Conclusions This paper presents the results of a study of the effect of two machining processes, CM and L-UVAM, on the milling forces of 2195 Al-Li alloys with varying machining parameters. The interaction effect analysis was combined with the single factor analysis to analyze the variation of milling force. The effects of feed per tooth and depth of cut on milling forces were analyzed, as well as changes in force signals and spectra in ultrasonic machining, and changes in surface topography due to changes in milling forces. The study lays a theoretical foundation for further research and optimization of aluminum-lithium alloys in the machining milling process, and provides some experimental basis for cold machining of aluminum-lithium alloys. The following main conclusions were obtained from the investigation: (1) In general, cutting depth directly affects the amount of milling force. Exploring the effects of milling speed, feed per tooth and ultrasonic amplitude on the interaction of milling forces revealed that higher speeds with f z = 50 µm and lower speeds with f z = 10 µm both have a significant effect on milling forces Fx and Fy. Both L-UVAM milling with A = 5 µm at f z = 10 µm and CM machining with f z = 50 µm decreased Fz. (2) Longitudinal torsional vibration significantly reduces the milling force, and the reduction increases with increasing feed per tooth and cutting depth. Compared to CM milling, the milling force Fy can be reduced by up to 40% for f z = 50 mm and up to 50% for a p = 1.5 mm at A = 3 mm. The maximum reduction of milling force Fy is 62% for f z = 50 mm and 55% for a p = 1.5 mm at A = 5 mm. (3) The L-UVAM milling process through the tool motion trajectory analysis can be known longitudinal torsion of ultrasonic vibration to the tool tip from the original twodimensional motion to three-dimensional motion, longitudinal vibration and torsional rotation on the tool superposition to make the collected milling force signal to change. L-UVAM caused the cutting force signal to exhibit oscillation characteristics; with an increase in the ultrasonic amplitude, the periodic contact separation of the tool became more pronounced, delaying the peak time of the milling force. L-UVAM caused significant changes in the low-frequency components of the milling force signal spectrum (spindle frequency and tool plunge frequency, as well as other multipliers), as well as high-frequency components in the spectrum of milling forces Fy and Fz. (4) The reduction of milling force has an important effect on surface topography, and longitudinal torsional ultrasonic vibration can not only reduce the milling force but also change the surface topography characteristics. In CM machining, the milling force increases with the increase of feed per tooth, which leads to serious lateral plastic flow of material on the machined surface of the Al-Li alloy, and the surface scratches are obvious, the chip accumulation is serious, and the surface morphology is not regular. L-UVAM machining reduces the milling force, so it improves the surface topography. The high frequency impact of longitudinal vibration increases material removal rates, reduces chip build-up on the surface, and the variable cutting depth created by the oscillating feature creates texture on the surface. Torsional vibration reduces milling forces in the feed direction and has a smoothing and "ironing" effect, reducing surface scratches. In conclusion, CM and L-UVAM machining of the 2195 Al-Li alloy has collected a large number of results on milling forces and some regular conclusions have been obtained. This paper fills some gaps in the field of longitudinal torsional ultrasonic vibration milling processing for 2195 Al-Li alloys, but more research is worthy of deeper investigation, for example, whether the test parameters could be applied when milling 2195 Al-Li alloys at low speed. As the surface integrity of different materials under different processing methods is greatly affected, the surface morphology is associated with the surface integrity, and further research is needed to demonstrate whether the milling force has an effect on the residual stress, surface roughness and other factors. In the future, research on 2195 aluminum-lithium alloys should consider such content. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ma15072508/s1, Figure S1: Effect of milling force on surface topography No. 1; Figure S2: Effect of milling force on surface topography No. 2; Figure S3: Effect of milling force on surface topography No. 3; Figure S4: Effect of milling force on surface topography No. 4; Figure S5: Effect of milling force on surface topography No. 5; Figure S6: Effect of milling force on surface topography No. 6; Figure S7: Effect of milling force on surface topography No. 7; Figure S8: Effect of milling force on surface topography No. 8; Figure S9: Effect of milling force on surface topography No. 9.	2022-04-03T16:31:05.512Z	2022-03-29T00:00:00.000	{ "year": 2022, "sha1": "61082066bf4ba0305e9f6037777f8b576f27bc25", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1996-1944/15/7/2508/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "306286ad2461e7401b9110b1f897e1bafb8fdc22", "s2fieldsofstudy": [ "Materials Science" ], "extfieldsofstudy": [ "Medicine" ] }
264511357	pes2o/s2orc	v3-fos-license	Enhancement of Thermal and Gas Barrier Properties of Graphene-Based Nanocomposite Films Poly(vinyl alcohol) (PVA), a naturally occurring and rapidly decomposing polymer, has gained significant attention in recent studies for its potential use in pollution preventive materials. Its cost-effectiveness and ease of availability as well as simple processing make it a suitable material for various applications. However, the only concern about PVA’s applicability to various applications is its hydrophilic nature. To address this limitation, PVA-based nanocomposites can be created by incorporating inorganic fillers such as graphene (G). Graphene is a two-dimensional carbon crystal with a single atom-layer structure and has become a popular choice as a nanomaterial due to its outstanding properties. In this study, we present a simple and environmentally friendly solution processing technique to fabricate PVA and graphene-based nanocomposite films. The resulting composite films showed noticeable improvement in barrier properties against moisture, oxygen, heat, and mechanical failures. The improvement of the characteristic properties is attributed to the uniform dispersion of graphene in the PVA matrix as shown in the SEM image. The addition of graphene leads to a decrease in water vapor transmission rate (WVTR) by 79% and around 90% for the oxygen transmission rate (OTR) as compared to pristine PVA films. Notably, incorporating just 0.5 vol % of graphene results in an OTR value of as low as 0.7 cm m–2 day–1 bar–1, making it highly suitable packaging applications. The films also exhibit remarkable flexibility and retained almost the same WVTR values even after going through tough bending cycles of more than 2000 at a bending radius of 2.5 cm. Overall, PVA/G nanocomposite films offer promising potential for PVA/G composite films for various attractive pollution prevention (such as corrosion resistant coatings) and packaging applications. INTRODUCTION Polymers offer a variety of excellent environmental and physical features such as density, linearity, humidity, molecular weight, crystallinity, and more, making them highly competitive in the food packaging industry. 1The industry frequently requires materials with specific degrees of polymerization, chemical inertness, water and oxygen permeability, density, thermal properties, mechanical properties, and morphological properties 2 all of which polymers can provide.Unfortunately, nonbiodegradable polymers are commonly used in food packaging, contributing to environmental problems. 3,4t is crucial to use polymers that decompose easily and quickly to reduce environmental pollution.Biopolymers are the ideal solution because they are biodegradable, environmentally friendly, and renewable 5−8 The remarkable properties of biopolymers make them suitable for a wide range of applications.Biopolymers can be divided into two types: natural or synthetic.Natural biopolymers include proteins, polysaccharides such as cellulose and starch, and others, while synthetic biopolymers include polylactic acid (PLA), 9 poly-(vinyl alcohol) (PVA), 10 polycaprolactone (PCL), 11 polyhydroxy butyrate (PHB), 12 and polyglycolic acid (PGA). 13These materials have applications in the medical, textile, and packaging industries.The only limitation to using biopolymers may be their unfavorable mechanical and barrier properties due to their hydrophilic nature.However, incorporating nanoparticles into these polymers and using them as polymer nanocomposites 5,14 can solve this problem. Poly(vinyl alcohol), also known as PVA, is a synthetic and relatively inert polymer that is water-soluble.It is a biocompatible and biodegradable polymer with many applications in different industries, such as packaging and adhesives. 15PVA is a semicrystalline and nontoxic material with excellent thermal stability, high transparency, good mechanical properties, flexibility, and high gas barrier properties.Poly(vinyl alcohol) (PVA)'s solubility in water and its tendency to absorb water can pose challenges in applications requiring gas barrier qualities.To address this, researchers use cross-linking PVA molecules, adding hydrophobic polymers, and controlling hydrolysis during production.They also insert hydrophobic agents or surface modifiers to prevent water penetration while maintaining gas barrier properties.Production process variables like temperature, pressure, and humidity also improve PVA's water solubility and gas barrier performance.Barrier coatings or laminates protect PVA from direct moisture exposure, ensuring that its gas barrier properties remain intact.The final PVA product is tested and controlled to meet the required standards for reduced water absorption and gas barrier qualities.These scientific techniques effectively address the challenges posed by PVA's water solubility and water absorption while maintaining its essential gas barrier properties in various applications.−18 To enhance the thermal and barrier properties of PVA, nanoparticles are often employed.Nanotechnology, which involves engineering materials with at least one dimension in the nano range (10−100 nm), 19 offers a way to gain new characteristics attributed to nanometer dimensions.One of the best and fastest routes to apply nanotechnology is by creating nanocomposites.Polymer nanocomposites have been used as barrier films to protect OLEDs, photovoltaics, food, medicines, drinks, and in many other applications because of their low cost, environmentfriendly processing and lightweight. 20Barrier films are designed to hinder gas and moisture permeation through them, which can be detrimental to packaged food quality. 21ifferent methods can be used to decrease the permeability of polymers, but the best alternative material is adding nanosized inorganic fillers, which possess a great boundary volume and fine grain size, such as clay, 22 silica particles, 23 ZnO, 24 carbon nanotubes, 25 graphene, 26 graphene oxide, 27 etc. 28A polymer nanocomposite coating consists of polymer matrices and nanofillers (usually inorganic nanoparticle fillers), such as metal oxides, SiO 2 , ZnO, Al 3 O 2 , TiO 2 , clay, graphene, graphene derivatives, carbon nanotubes, etc. Graphene is an extremely unique substance with a honeycomb lattice and a single sheet of sp 2 carbon atoms.It is the stiffest, strongest, but thinnest material known. 29It has been used in numerous sectors such as electronic, optical, light processing, mechanical, energy-related, thermal, and distinct biological, making it a low-cost material that can be utilized to improve the performance of a polymer. 26Graphene enhances the mechanical properties of composite materials by increasing their aspect ratios and covalent bonding.This increases tensile strength and Young's modulus, making it a more durable material.Graphene also distributes loads and transfers stress, increasing its mechanical resilience.However, variable water contents can affect mechanical strength in gas barrier applications as some polymers can be plasticized and softened by water, lowering the overall strength.To maintain mechanical robustness and gas barrier performance under various aqueous conditions, careful selection of polymer matrix and graphene concentrations is crucial.Singh et al. 30 studied graphene-based materials for gas and chemical sensors in wearable electronics, focusing on sensitivity and selectivity in hazardous gases, heavy metals, and environmental contaminants.They investigated potential applications in wearable electronic devices and the IoT, addressing challenges like flexibility, stability, and wireless integration.Other researchers like Tambe et al. 31 synthesized a PVA/graphene solution to spin coat graphene over a steel substrate.As the concentration of graphene in the PVA matrix nanocomposite coating on the steel substrate increased, the coefficient of friction also increased.A nanocomposite with improved mechanical strength, thermal stability, and electrical conductivity compared with pure PVA is produced by incorporating graphene into the PVA matrix.High aspect-ratio graphene and powerful covalent bonds enhance mechanical qualities like tensile strength and Young's modulus, making these nanocomposites appealing for structural applications.Additionally, because of the high thermal conductivity of graphene, the PVA matrix has better thermal stability, increasing its usefulness under hot conditions.By addition of graphene, it is possible to dramatically increase the electrical conductivity of PVA, which opens up opportunities for use in flexible electronics, sensors, and conductive coatings.Graphene can be added to PVA to enhance its gas barrier qualities, which makes these nanocomposites potentially useful as barrier coatings and packaging materials. 31Krishnan et al. 32 studied the graphenebased nanocomposite.In this research, biosensors are crucial in medical sciences for precise physiological evaluation.Graphene-based nanocomposites offer efficient, cost-effective solutions for biosensor development.These platforms detect various biomolecules with low detection limits, enabling biofunctionalization, enzyme immobilization, and various reactions on nanocomposite surfaces. To produce polymer matrix nanocomposites, researchers have explored several methods, including melt mixing, in situ polymerization, and mixing filler ingredients while electrospinning.However, these techniques can be expensive. 33,34he simplest and most effective method for fabricating polymer-based nanocomposites is solution mixing.This involves mixing colloidal suspensions of nanoparticles with polymer matrix solutions through simple stirring or shear mixing.The nanocomposite can then be recovered using solvent evaporation, distillation, or no solvent coagulation.Blade casting is another environmentally friendly method for producing nanocomposites, where a hot melt or solution is cast on substrates such as polyester or PET depending on the polymer and composite nature.The thickness of the film and the content of the casting resin can be controlled by a doctor's blade or film applicator.This method allows for the easy dispersion of nanoparticles without agglomeration.Vollenberg and Heikens 35 produced nanocomposite samples by thoroughly mixing filler particles with a polymer matrix.They used filler materials such as alumina beads with 35 and 400 nm dimensions and mixed them with polystyrene (PS), styreneacrylonitrile copolymer (SAN), polycarbonate (PC), and polypropylene (PP).The average volume fraction of filler particles was maintained at 25%. The main objective of this study is to create coatable thin films of PVA-based nanocomposites.Produced thin films will have PVA as a matrix and graphene nanosheets as nanofillers.The films will be produced by the cost-effective method, i.e., blade coating, as to mimic the high throughput roll-to-roll coating method.The cost-effective thin films will have improved barrier characteristics against heat and environ-mental attacks such as the diffusion of oxygen and moisture while maintaining the transparency.This coatable PVA-based solution can be applied to a variety of applications wherein thermal stability and barrier against diffusion of gases is prime requirement such as packaging of food or electronics and corrosion resistant applications for textile and polymeric items. Materials. The PVA used in this study was purchased from Aldrich Chemistry (Darmstadt, Germany) and had an average M w of 85,000−124,000 and an 87−89% hydrolyzed content.Graphene nanoparticles with an α value of 200 were supplied by Aldrich Chemistry (Darmstadt, Germany). Sample Preparation. The following method was used to prepare PVA/graphene nanocomposite films.First, 1 g of PVA powder was added slowly to 100 mL of distilled water at 90 °C and stirred for 1 h using a magnetic stirrer at 700 rpm.Graphene was dispersed in distilled water using ultra sonication.Next, the desired amount of graphene dispersion was gradually added using a glass dropper into the PVA solution and stirred for 1 h.To investigate the effect of the filler content on the characteristics of the nanocomposite, the weight percentage of graphene was varied from 0.1 to 0.5 vol %.Lower filler content was chosen to prevent agglomeration, which would weaken the thermal properties of the nanocomposite.High filler content would likely cause the filler to clump together in the nanocomposite.The PVA/graphene solution was then ultra sonicated again for 20 min to prevent agglomeration. The next step involved blade casting of the solution with the thickness of the applicator set at 50 μm.The resulting thin films were left to dry at room temperature for 24 h.The films were kept in a vacuum desiccator until further characterization was performed. Characterization. 2.3.1. UV−Vis Spectrophotometry. The UV/vis absorption spectra of the single photoactive layers (P3HT) were measured using a Shimadzu UV-1800 spectrophotometer.To ensure that the sample is hit at the same spot during the degradation test at various time intervals, a customized sample holder was utilized. 2.3.2.Fourier Transform Infrared (FTIR) Spectrophotometry.The FTIR spectra were recorded in ATR mode using a Bruker ALPHA-P FTIR spectrophotometer (operated with OPUS 7.2 software) in 64 scan summations at a resolution of 4 cm −1 . Scanning Electron Microscopy. The JSM-7610F type of JEOL scanning electron microscope (SEM) was used for the morphological investigation.A secondary electron image detector was used to produce high-resolution topographical imaging.The 20 kV accelerating voltage and a low probe current mode set to 65 nA were found to be the ideal operational conditions.Notably, the polisher played a critical role in the sample preparation procedure. 2.3.4.Tensile Strength.Tensile strength (TS) and elongation at break (EAB) of each film were evaluated using Z005 Zwick/Roell universal testing equipment from Germany.The samples were prepared in accordance with ASTM D 882-10 guidelines, using a crosshead speed of 5 mm/min and a starting grab separation of 50 mm.The films were divided into strips that were 130 mm long and 30 mm wide.To evaluate the samples' mechanical qualities, a 5N load cell was employed. 2.3.5.Water Vapor Transmission Rate (WVTR).A Thwing−Albert Instrument Company (West Berlin, NJ, USA) aluminum cup with a diameter of 6.35 cm, complying with the ASTM E-96 standard, was used.The experiment was conducted according to the methodology outlined by Channa et al. 36 2.3.6.Bending.A cyclic bend tester was used where one end was stationary and the other end moved forward and backward to create a radius with a predetermined value.The film's resistance to bending was determined after each set of bending cycles, and the sample size for this test was 3 × 10 cm 2 .A sample measuring 3 × 3 cm 2 was taken from the center of the bent sample for the permeation test. 2.3.7.Oxygen Transmission Rate Measurement of Films.The oxygen penetration rate was measured using a permeation chamber with an optical oxygen measuring spot, Pst9, made by PreSens Precision Sensing GmbH, Regensburg, Germany.The detection limit for oxygen permeation was 0.1 cm 3 m 2 day −1 bar −1 .The oxygen transmission rate (OTR) and permeability were calculated after the samples were positioned between the two chambers of the device and after 15 min of nitrogen gas flow in each permeation cell.Oxygen was then pumped for 30 s in the bottom chamber, and the increase in oxygen fraction in the top chamber was continually examined for a few days. Thermogravimetric Analysis (TGA). The thermal stability of the films was investigated using a thermogravimetric analyzer (SDT Q-600 TA Instruments, Artisan Technology Group, Champaign, IL, USA).The initial sample weight for each operation was set at 5−8 mg, and the sample was heated from 24 to 550 °C at a rate of 10 °C/min in a nitrogen environment using a low flow rate of 200 mL/min. RESULT AND DISCUSSION 3.1.Transparency.The main challenge for food packaging is to achieve a transparent barrier coatings.Figure 1 illustrates the UV−vis spectra of PVA and its nanocomposites with graphene.The pure PVA film exhibited high transparency across the visible light range (380−800 nm) with a total transmittance of 98% at 550 nm, making it suitable for optical applications such as polarizers or long-pass filters. 31,37owever, for the nanocomposites, the total transmittance remained almost unchanged, even after the addition of graphene content.A negligible decrease in transmittance was observed, which could be attributed to either the scattering of a small portion of light due to graphene particles or reflectance.Therefore, the maintenance of the transparency of the barrier nanocomposite is solely dependent on the graphene content within the matrix. 38Figure 2 represents the optical trans- parency of PVA and the PVA/G nanocomposite, indicating no difference in transparency.Similarly, Figure 3 shows transmittance measurements at 550 nm, indicating a decrease in total transmittance with increasing graphene content. 39 Compound Analysis via FTIR. The FTIR spectra of graphene, pure PVA film, and PVA/graphene nanocomposite films are presented in Figure 4. Graphene shows two distinct peaks at 1350 and 1620 cm −1 , which are also observed in the polymer and polymer nanocomposite films.The bands at 1088 cm −1 are attributed to the vibrational movement of functional groups such as C−O (bending) present in PVA.The peaks at 1322 and 1399 cm −1 are due to C�O bending.The two weak bands at 2907 and 2941 cm −1 can be attributed to C−H stretching and the existence of acetate groups in the PVA matrix. 40The peaks at 3262 cm −1 are assigned to normal polymeric O−H stretching, which is a result of intermolecular as well as intramolecular hydrogen bonds present in the PVA matrix. 41The addition of graphene did not lead to any change in the molecular interactions.Graphene and PVA (poly(vinyl alcohol)) with graphene may have similar FTIR spectra because the FTIR technique primarily measures the vibrational modes of chemical bonds in a material, and the presence of graphene in the PVA matrix may not significantly alter the fundamental chemical structure of PVA.Graphene is made up of carbon atoms organized in a 2D honeycomb lattice and lacks major distinctive absorption bands in the FTIR region.As a result, its presence in a composite material such as PVA with graphene may not result in distinguishable peaks in the FTIR spectrum.PVA, on the other hand, is a polymer composed of repeated −CH 2 −CH(OH)− units, and its FTIR spectrum often exhibits peaks associated with these functional groups.The chemical structure of PVA is not greatly altered when graphene is integrated into the PVA matrix, so the FTIR spectrum of the composite material may closely mirror that of pure PVA. 42.3.Microstructure.The SEM image of the complicated structure of the graphene nanoflakes is shown in Figure 5a.The graphene structures presented by Mutyala and Mathiyarasu 43 are also comparable.Due to their distinct presence within the substrate, these tiny graphene flakes appear as discrete entities.The SEM image provides a high-resolution perspective that enables the in-depth analysis of these graphene nanoflakes because it was created at an accelerating voltage of 20 kV, was magnified by a factor of 4000×, and covered a 5 μm area.Figure 5b,c, which exhibits PVA composite material with a 0.5% graphene content, then reveals a unique characteristic.At this particular condition, the SEM pictures clearly show a remarkable degree of homogeneous incorporation of graphene throughout the whole PVA matrix.Notably, there are no discernible fluctuations or clusters of graphene, demonstrating that the material has achieved a remarkably uniform dispersion of graphene within the polymer.This consistency is particularly important because it ensures that graphene's excellent features, such as great electrical conductivity and increased mechanical strength, are consistently integrated throughout the entire composite material.44 3.4. Beding.Bendability and flexibility at a smaller radius are two of the most desirable characteristics of films.18 To demonstrate the produced films' flexibility, a bending test was conducted and the outcomes are presented in terms of normalized WVTR (as shown in Figure 6).The films were bent with a radius of 6.35 cm, and identical films were then analyzed for WVTR.If the bent film maintains its initial WVTR even after multiple bending cycles, it would indicate that the films are flexible and bending does not impair their functionality (WVTR).Therefore, 20,000 bending cycles were applied to all created films, and they were frequently checked for WVTR.The results of the first and periodic WVTR studies were the same, indicating that perfect PVA films displayed exceptional bendability.This suggests that PVA films were unaffected even after 20,000 bending cycles.Additionally, the films showed no obvious signs of degradation after being bent.However, PVA films containing graphene also showed a negligible change in the composite coatings.PVA films containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 vol % graphene, respectively, showed losses of 0.1−0.3%.This could be explained by the strong link or wetting between PVA and graphene 2D structures, which implies that the graphene always returns to its original position after bending, maintaining its adhesion to the PVA matrix and causing no harm to it.45 Based on these findings, it is determined that basic PVA films bond to graphene better and that the films remain flexible overall. Tensile Strength. The mechanical properties of the composite material show a striking transition when compared between PVA (poly(vinyl alcohol)) in its purest form and PVA with 0.5% graphene reinforcement in the tensile strength tests.Pure PVA exhibits a remarkable tensile strength of 165 MPa (shown in Figure 7) in its original state, demonstrating that it can endure large tensile forces before failing.It is moderately stiff as evidenced by the Young's Modulus of 0.054 MPa.In contrast, a remarkable improvement is seen when just 0.5% graphene is added to the PVA matrix.The composite material is significantly reinforced, as seen by the composite material's explosively high tensile strength of 341 MPa.By making the material more resilient and able to endure significantly higher tensile stresses, graphene plays a crucial role in enhancing the mechanical integrity of the substance as demonstrated by this significant increase.Although the precise Young's modulus value for the PVA-graphene composite is 0.14 MPa, one may anticipate that it will rise as well, indicating the material's increased stiffness as a result of the addition of graphene.Despite being reduced to 129.1%, the elongation at the break still indicates a material with outstanding flexibility, especially in light of the significant increase in tensile strength.Cheng-an et al. 46 show that the composite film's tensile strength grew quickly as the GO content increased.Overall, these findings show graphene's enormous potential as a reinforcing material, providing novel perspectives for the creation of high-performance composite materials with outstanding mechanical properties. 47.6.Water Vapor Transmission Rate (WVTR).PVA is a hydrophilic material with poor moisture barrier properties.To evaluate the WVTR of PVA, a test was conducted using a film test area of 0.0031 cm 2 and a testing time of 10 days, while maintaining the test conditions at 23 °C and 50% RH.The WVTR of pure PVA was found to be 19.3 ± 2.23 g/m 2 day, but the addition of 0.1 vol % graphene reduced it to 6.09 ± 1.65 g/m 2 day.Further addition of 0.2, 0.3, 0.4, and 0.5 vol % graphene resulted in a reduction of 68, 79, 86, 92, and 93%, respectively, with corresponding WVTR values of 4.03 ± 1.29, 2.61 ± 0.93, 1.41 ± 0.68, and 1.22 ± 0.55 g/m 2 day.The WVTR and 1/WVTR values are listed in Table 1.Graphene's Figure 6.Normalized WVTR values of different concentrations of graphene film with 0−0.5 vol % films plotted against the number of bending cycles at a bending radius of 6 cm.The black curve represents the pristine PVA film, the red curve represents the PVA + 0.1G, the blue curve represents the PVA + 0.2G, the green curve represents the PVA + 0.3G, the purple curve represents the PVA + 0.4G, and the yellow curve represents the PVA + 0.5G vs the number of bending cycles.All the rested films had a thickness of 100 μm.impermeable nature creates a more tortuous path for gas molecules to pass through the barrier film, leading to the improved barrier quality. 40Table 1 also shows that increasing the graphene content reduces the weight loss of distilled water through the nanocomposites, indicating a decrease in moisture permeability.Graphene is a single layer of carbon atoms organized in a 2D honeycomb lattice with extraordinary features such as significant mechanical strength, thermal conductivity, and gas and liquid impermeability.When graphene is mixed with PVA, it produces a distributed network within the polymer.Water vapor molecules attempting to diffuse through the PVA composite may encounter a complicated path due to the presence of graphene in the material.The impermeability of graphene works as a barrier, preventing water vapor from passing through the PVA matrix. When compared to pure PVA, which contains the extra barrier supplied by graphene, this behavior reduces the WVTR of the composite.Graphene's sheet-like structure, good aspect ratio, and exfoliation properties also help it to disperse in the PVA matrix and create a hindrance for diffusing molecules by decreasing the free volume of the polymer.SEM also helps examine the film's stability and potential water vapor permeation channels by revealing the distribution of graphene layers and imperfections.Understanding the microstructure of graphene-PVA sheets for low WVTR applications can be guided by SEM investigation, as shown in Figure 5. Controlling the orientation and distribution of graphene sheets within the film can improve its barrier characteristics, lowering the rate of water vapor transmission.The prepared films have excellent moisture barrier properties and can be used in the packaging industry, electronics, fuel cell industry, etc.The improved moisture barrier performance is further illustrated in Figure 8, which shows the inverse of WVTR versus polymer nanocomposite films.Introducing nanoparticles or graphene can enhance the blocking effect of the film, and increasing the graphene content from 0.1 to 0.5 vol % decreases 79% of water vapor permeation rate.This indicates a direct relation between graphene content and the inverse of WVTR.The inclusion of nanoparticles has a significant impact on the PVA matrix, as demonstrated by Channa et al. 48The moisture permeability decreases with increasing glass-flake concentration, which produces an extended route and makes it harder for moisture molecules to disperse.The moisture permeability in the PVA layers with glass-flake concentrations of 5−25 vol % ranged from 6.2 to 1.2 g m 2 day −1 . Bharadwaj proposed a new model for relative permeability that included the aspect ratio of nanoparticles, order parameter S. 49 P P S (composite) (polymer) The experimental values of this work were compared to the theoretical permeation model proposed by Bharadwaj.The comparison of the theoretical Bharadwaj permeation model based on aspect ratio; α = 200 and order parameter; S = 0 (suggesting random orientation by supplier) showed similarity with current experimental results as shown in Figure 9.A graph was drawn using eq 1 as a guide.This graph demonstrates that the information provided by the supplier regarding graphene material is accurate and that graphene's interactions with polymers are beneficial to moisture permeability. Reduction in water vapor permeability through nanocomposite thin film concerning the permeability of pristine PVA is termed the barrier improvement factor (BIF). 50,51 It can be calculated by BIF permeability(polymer) where α is the aspect ratio of graphene, S is the order parameter, and Ø is the volume fraction of graphene.Figure 10 shows the increase in BIF with increasing volume fraction of graphene content, confirming the enhancement of barrier performance of graphene and PVA nanocomposites. Oxygen Transmission Rate (OTR). Coating and packaging materials need to have oxygen barrier properties to protect objects from internal damage and enhance long-term performance. 52PVA is known for its exceptional oxygen barrier properties due to its high degree of crystallinity and strong intermolecular force caused by the hydroxyl groups in repeating units. 53As shown in Figure 11, the addition of graphene to PVA decreased its OTR values (at 0% RH and 25 °C), possibly due to the graphene nanocomposite and PVA matrix being designed with an incredibly tortuous path to prevent oxygen molecules from penetrating through it.The development of highly oriented 2D filler-based coatings is responsible for the superior oxygen barrier property.Gaume et al. 54 also examined the permeability of nanocomposites and demonstrated that 5 wt % of MMt-Na+ enhanced helium permeability (by 70%), as well as oxygen and water permeability.The OTRs of PVA/G composite films are significantly lower compared to pure PVA films, indicating improved barrier properties of graphene by the polymerization of PVA.These findings imply that a PVA matrix with uniformly dispersed graphene can be produced due to the enhanced compatibility between PVA/G and the PVA matrix.Furthermore, the increased quantity of strong hydrogen bonds results in a longer diffusion pathway and more contact between PVA-G and the PVA matrix, which decreases oxygen transmission.Although the optical transmittance drops when the PVA-G concentration rises (Figure 1), the barrier characteristics are still suitable for gas barrier films due to the improved compatibility of PVA-G and the PVA matrix. 3.8.Thermal Stability.The thermogravimetric analysis (TGA) and corresponding differential thermogravimetric analysis (DTG) in an inert atmosphere were used to evaluate the nanocomposite materials' thermal stability. 55As shown in Figure 12, the weight loss of the PVA film steadily increases with temperature, with inflection points at 85.7, 210.9, and 390.5 °C.According to ref 56, this weight loss can be attributed to the dehydration of the PVA chain and the formation of a polyacetylene-like structure at temperatures between 200 and 300 °C, the evaporation of adsorbed water in the PVA chain at temperatures between 60 and 150 °C, and the main chain disintegration of PVA at temperatures between 380 and 470 °C (as shown in Table 2).The weight loss of the PVA-G film with 5 vol % exhibits a trend similar to that of the PVA film with increasing temperature, as shown in Figure 12.This suggests that adding nanomaterial can increase the thermal stability of the polymer matrix.At this stage, the PVA nanocomposite film and the pure PVA film showed similar levels of stability.The stages of deterioration observed in the PVA film also coincided with those of the PVA film containing graphene.At the end of the thermal deterioration process, the weight loss concentration of the PVA film with graphene was 7.65%. CONCLUSIONS Graphene-based nanocomposites are a novel type of material with potential applications in numerous fields.In this study, we present a general procedure for synthesizing poly(vinyl alcohol) (PVA)/graphene (G) nanocomposites using a simple solution-mixing approach.Both materials are cost-effective, widely available, and durable.To evaluate the properties of the produced materials, we conducted tests such as thermogravimetric, water vapor transmission rate, oxygen transmission rate, tensile strength, SEM, UV−vis spectroscopy, and FTIR spectroscopy.By using ultra sonication, we achieved an even distribution of graphene nanoparticles throughout the PVA matrix as confirmed by a SEM image, effectively preventing nanoparticle restacking.The strong interfacial connection between PVA and graphene, which was primarily due to hydrogen bonding and enhancement of the PVA crystallinity, improved the mechanical and thermal stability of the nanocomposites.Our tests showed that the addition of just 0.5 vol % graphene concentrations to the PVA film reduced the water vapor transmission rate from 19 to 1.22 g m −2 day −1 and the oxygen transmission rate from 10 to 0.5 cm m −2 day −1 Figure 2 . Figure 2. Optical transparency of (a) pure PVA film and (b) PVA with 0.5 vol % graphene film with logo on the back side.A black circle has been drawn around the sample. Figure 7 . Figure 7. Stress−strain relationships of PVA and PVA/GO nanocomposite films. Figure 8 . Figure 8. Barrier performance of the polymer nanocomposite film with 0−0.5% variation of graphene in PVA. Figure 9 . Figure 9.Comparison of experimental relative permeability of PVA films with increasing content of graphene with the Bharadwaj model for an aspect ratio of α = 200 and order parameters of S = 0. Figure 10 . Figure 10.Experimental barrier improvement factor based on graphene content compared with the Bharadwaj model. Figure 12 . Figure 12.Thermogravimetric analysis (TGA) of the simple PVA film is represented by a black dotted line and the simple black line represents the PVA + 0.5G film.Differential thermogravimetric analysis (DTG) of the simple PVA film is represented by a red simple line and a red dotted red line representing the PVA + 0.5G film. Table 1 . Moisture Barrier Property of Polymer and Polymer Nanocomposite Films Table 2 . Film Thermal Gravimetric Analysis Data °C bar −1 .Furthermore, graphene increased the thermal stability and tensile strength of the polymer nanocomposite film.We were able to produce the films at a lab scale for less than 0.6 USD/m 2 .These findings suggest that PVA/G nanocomposites have the potential for use in various coating and packaging films to protect against moisture and corrosion.IftikharAhmed Channa − Thin Film Lab as Part of Materials and Surface Engineering Group, Department of Metallurgical Engineering, NED University of Engineering and Technology, Karachi 75270, Pakistan; Email: iftikharc@neduet.edu.pk	2023-10-27T15:11:00.862Z	2023-10-25T00:00:00.000	{ "year": 2023, "sha1": "ad47360617e82d079ae658afb6a097bd3f2f9421", "oa_license": "CCBYNCND", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "0dde37dedfc66947c974aba8eef7d01beda33d24", "s2fieldsofstudy": [ "Chemistry", "Engineering", "Materials Science", "Physics" ], "extfieldsofstudy": [] }
54797350	pes2o/s2orc	v3-fos-license	Evaluating the Performance of Libyan Banks Using Return on Investment Return on Investment (ROI) is a financial ratio which commonly used to evaluate the overall performance of a company. Despite of the importance of ROI unfortunately not many Libyan banks use it to evaluate their performance. Most of Libyan banks use the result of income statements to evaluate their performance instead of other tools that use to evaluate performance of banks. This study will be focusing on the ROI. The objective of this research is to evaluate the performance of Libyan banks using ROI as a financial tool. The Libyan banks which involved in this research are: Alwahda bank, National Commercial bank, Commercial and Development bank, Mediterranean bank and Alejmaa Alarabi bank. In this research financial statements of the Libyan banks for period from 2005-2009 will be used to calculate the ROI. In general, the results of the ROI of the banks used in this research showed increase and then begin to decrease and then increase again. The results clearly indicate that the performance of Libyan banks is not stable depending on components of ROI. INTRODUCTION Financial ratios are the oldest and important method for analyzing company performance. Mainly it is used to study the financial and credit position of organizations, in this method the performance is based on the examination of the financial statements. However, the number included in a financial statement does not indicate if it is important and it does not always provide useful information. The importance of the number is only when compared with other numbers (Tofeeq, 1997). Brigham and Ehrhardt (2005) state that "financial ratios are designed to help evaluate financial statements". Financial ratios are useful as a planning and control tool. Financial ratios analysis is used to evaluate the performance of an organization. It aims to determine the strong and weak points and it offers solutions by providing appropriate plans (Zayod et al., 2005). A large number of standards and various financial ratios can be used when analyzing the credit and financial position of organizations. The choice of ratios used for analysis depends largely on the type of activity in an organization and the purpose of analysis (Tofeeq, 1997). Most researchers divide the financial ratios into four groups (Ross et al., 2007). Liquidity ratios this indicates an organization's ability to pay debt in the short run (Lasther, 2005) and Activity ratios Gitman et al. (2002) stated that "These are used to measure the speed with various accounts are converted into sales or cash". They are also used to measure how efficiently a company manages its assets (DUFR, 2011). After that Debt ratio these show how effectively the organization uses other people's money and whether it is using a lot of borrowed money (Lasher, 2005). The last one is Profitability ratios consist of several measures which assesses an organization's success in making money (Lasher, 2005). Profitability ratios are used to evaluate the company's ability to generate profits from its operational activities and include some ratios such as Net Profit Margin, Gross Profit, Return on Assets (ROI), Return on Equity (ROE), The nature of the organization influences the ratios employed. For example, in the case of a bank, the liquidity ratio is used to determine the amount of liquidity that a bank needs in order to meet its liabilities; a bank also uses profitability ratios (Tarawneh, 2006). Industrial organizations focus on profitability and activity ratios. On the other hand, non-profit organizations attempt to get enough financial resources to continue their activities, so they tend to focus on debt ratios. There are limitations to use financial ratios including; financial ratios analysis measure performance in the past and cannot predict what the firm will be in the future, also financial ratios analysis ignores many factors that effect on the performance of the company such as customer satisfaction and financial ratios are affected by all the problems that appear in the financial statements because it measures the relationship between the elements of financial statements in a certain moment (DUFR, 2011). Unfortunately not many Libyan banks use the information in the annual reports to evaluate their performance despite of the importance of ROI. So, in this study we focus on the Return on Investment (ROI) and we aim to evaluate the performance of Libyan banks as a financial ratio tool. This study is organized as follows: First some background information about ROI and then the methodology section that discusses the sample selection and variables measured. Following that are the results and conclusion. Return on Investment Organizations may have centers which control processes done such as cost centers. These centers have control over costs which are evaluated by measuring the range of achievements of decided standard costs (Gareson and Norain, 2002). Organizations may also have profit centers that have control over all costs and revenues that are evaluated by performance reports of income statements which explain the differences between actual and estimated statements. Organizations may also add investment centers which have control over costs, revenues and use of investment money, that can be evaluated by income statements too but one usually uses the ROI which determines the comprehensive performance of an organization. When a manager has all the responsibility and authority for his or her center's working capital and physical assets, the manager is running an investment center. The performance of such a unit is best measured with a metric that relates profits earned to the level of physical and financial assets employed in the center. Investment center managers are evaluated with metrics such as the ROI and economic value added (Kaplan, 2006). For a short term, it seemed as though nearly every firm that went public was only interested in 'share holder value' in an effort to drive up the value of the stock and of course, employee held stock options. The ROI may the most important ratio of all. It is the percentage of return on funds invested in the business by its owners (FRA, 2011). The ROI can be defined as the investment's ability to achieve a return due to its use and if we want to measure the investment's ability effectively we have to compare the achievement return with returns for previous periods of the same organization or with returns for other organizations which operate in the same sector. Therefore it is necessary to express this return as a percentage of the investment used (Almaghbob, 1991). ROI reveals area where capital is being effectively employed and this information is helpful in obtaining a desired balance in the use of facilities (Terry, 1985). There are three dominant reasons for using the ROI: First, most people can understand ROI easily. Second, it combines three critical performance measures variables scale, earnings and investment. Third, it is popular with financial analysts, investors, creditors and other external information users (ROI Vs ROS, 2011). There are some advantages for using ROI including that the ROI focus on profits, objective to cost and profits, also readily available data, different sized divisions, it is fair to different sizes and managers accept project with higher ROI's (MADPE, 2011). Also there are some advantages for using ROI including that the ROI is a historic measure, accounting policies, ignore risk, there are some methods better decision criteria more than ROI such as Net Present Value (NPV) and projects which have a slow payoff maybe rejected (MADPE, 2011). Methodology In this study five Libyan banks that headquarters located in Benghazi and Albida city were chosen as the sample. These banks are Alwahda was established under the law (153) For the purpose of this study ROI, is defined as follows. Revenues This is money which is obtained by sale goods or services. In the banking industry, this revenue is money which is obtained for the provision of mortgages, social loans, credit facilities and commercial loans. It also includes the following items: Interest earned, bank commissions, currency exchange and other miscellaneous revenues for example fines collected as a result of delays in payment of instalments for loans. Net Operating Income A net income before interests and taxes. Operating Assets Assets include cash, debtors, building, machinery and equipments and other assets. Assets that are not included as operating assets are land that is retained for use in future expansions or buildings rented to others. In this study we calculated using the following formula Equation (1-3): ROI Profit Margin x Turnover operating assets = RESULTS In this study data was collected from Alwahda Bank, National Commercial Bank and Commercial and Development Bank from 2005-2009 and Table 1 below shows the data were collected. Table 2 shows results that were calculated using Equation (1). The purpose of the study was to evaluate the performance of Libyan banks compared to the standard performance. As there were no standards available to compare with ROI in Libyan banks, in this study we compared the performance of Libyan banks historically. For Alwahda Bank The ROI increased by 13.25% from 2005-2006 because Profit Margin and Turnover of Assets were increased by 0.79%, 12.12% respectively. Then It decreased in 2007 depending on decreasing of Turnover of assets, because of increasing rate of assets was greater than increasing rate of revenues and it continued to decrease to 1.15% from 2008-2009. In 2008 the ROI decreased due to increasing of operating income, which resulted decreasing of Profit Margin and in 2009 the ROI decreased also because the Turnover of Assets was decreased due to decreasing in Revenues with increasing in Operating Assets. For National Commercial Bank The ROI increased by 15.38% from 2005-2006 because Profit Margin and Turnover of Assets were increased by 2.16%, 13.04% respectively. Then it decreased in 2008 because both of Profit Margin and Turnover of Assets were decreased to 0.84%. It later increased again to reach 1.21% in 2009 because the Profit Margin was increased by 44.47% and Turnover of Assets was not changed. For Commercial and Development Bank The ROI was not stable as it decreased from 2005-2006 by 23.12%, because of increasing in operating income lower than increasing in revenues. It then started to increase to reach its beak in 2008 and then it started to decrease again to 1.53% in 2009 due to the profit margin was decreased, because of the Operating Income decreased with increasing in revenues. Also, due to the turnover of assets decreased because each of revenues and operating assets were increased but the rate of increase of operating assets was greater than the rate of increase of revenues. For Mediterranean Bank The ROI decreased from 9.86% in 2005-to 3.94% in 2006 by 60% because Profit Margin and Turnover of Assets were decreased by 13.5%, 53.8% respectively. Then it increased in 2007 depending on increasing of Profit Margin. Then increased again to 4.38% in 2008 because the Profit Margin decreased by 5.86% but the Turnover of Assets increased by 10.71%. After that ROI decreased again to 3.97% in 2009 because the Profit Margin was decreased. For Alejmaa Alarabi Bank The ROI decreased from 3.24% in 2005 to reach to the lowest rate of ROI in 2007 because of decline in Profit Margin rate was greater than the rate of increase in the Turnover of Assets. DISCUSSION In this study, we use only the hestorical standards because there is no any standards available in banking system of Libya to compare with ROI that we are obtained from this study. Wu (2012) found that the ROI is more influncing factor to evaluate performance and has high value for chinese banks. There are many studies use the profitability ratios (ROI, Return on Assets (ROA) and Return on Equity (ROE)) as a deteminants to measure the efficiency and evaluate performance of banks such as Sufian (2007;2009;; Casu and Molyneux (2003); Isik and Hassan (2002); Ariff and Can (2008) and Miller and Noulas (1996) they found the profitability ratios have positive significant related with efficiencies of banks. We can apply the results that we are obtained from this study for all Libyan banks, because all Libyan banks work under the same environment conditions and all Libyan banks are totally or heavelly ownered by Central Bank of Libya (CBL). CONCLUSION ROI is a financial ratio which is commonly used to evaluate the performance of a company. In this study ROI was used to evaluate the performance of five Libyan banks namely Alwahda Bank, National Commercial Bank, Commercial Development Bank, Mediterranean bank and Alejmaa Alarabi bank. The data was collected from financial statements of these banks from 2005-Science Publications AJEBA 2009. The results clearly indicate that the performance of Libyan banks is not stable. In this study, we have faced standards limitition, because of that we have used only the historical standard because there is no any standards are available to use it to compare with results such as industrial standard, also this study include only five Libyan banks because we cann't collect the data from all banks because the conditions in Libya during this study. We can sugget that future studies can use more modern approches for evaluate the performance of Libyan banks such as Balanced Scorecard (BSC), Parametric approach such as Stochastic Frontier Approach (SFA) and non-parametric approach such as Data Envelopment Analysis (DEA).	2019-05-29T13:11:12.581Z	2013-02-01T00:00:00.000	{ "year": 2013, "sha1": "45b8ec8821d467a0abc0e057a2569721c9ae843b", "oa_license": "CCBY", "oa_url": "http://thescipub.com/pdf/10.3844/ajebasp.2013.84.88", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "56901d2b5985ba3cc754544dacf5ac5641308595", "s2fieldsofstudy": [ "Business", "Economics" ], "extfieldsofstudy": [ "Business" ] }
250010531	pes2o/s2orc	v3-fos-license	Kounis Syndrome Secondary to Laxative Administration Kounis syndrome (KS) is defined as an acute coronary syndrome triggered by the release of inflammatory mediators after an allergic attack. It usually occurs secondary to allergic injuries from foods, medications, and insect bites. However, there are no known reports of KS secondary to the intake of laxatives. This article reports the case of a 43-year-old woman who, after ingesting a dose of sodium phosphate monobasic/sodium phosphate dibasic, presented a maculopapular rash on the trunk and extremities. The electrocardiogram showed ST depression in V4-V5-V6 and signs of prolonged QTc; troponin I uptake was positive. Due to presumed myocardial injury and high suspicion of coronary disease, coronary angiography was requested, which showed epicardial coronary arteries, without angiographically significant stenosis, thus confirming the presence of KS secondary to the ingestion of a laxative. Introduction Kounis syndrome (KS) is defined as an acute coronary syndrome that presents as unstable angina that may be secondary to vasospastic or nonvasospastic etiology, and even as a manifestation of acute myocardial infarction, triggered by the release of inflammatory mediators after allergic aggression [1]. It has been reported that there are three types of KS associated with the same number of different pathophysiological processes derived from the activation and degranulation of mast cells, which lead to the release of powerful inflammatory mediators. us, type I is a coronary spasm in patients with normal or nearnormal coronary arteries [1,2]; established or pre-existing coronary artery disease in which inflammation leads to coronary vasospasm or plaque rupture (type II); or instent thrombosis and restenosis (type III) [2]. is syndrome generally occurs secondary to allergic injuries from foods, medications, and insect bites [2,3]. Anaphylactic reactions to polyethylene glycol are very rare [4], and there are no known reports of KS secondary to this type of medication. Case Report A 43-year-old female patient was referred to a high-complexity hospital for the suspected coronary syndrome. e patient reported a history of chronic low back pain and cholelithiasis, waiting for scheduling and management by general surgery on an outpatient basis. In the initial care hospital and due to her low back pain, a simple X-ray of the total spine was sent and a laxative preparation was ordered for this procedure. One dose of sodium phosphate monobasic/sodium phosphate dibasic (Travad oral ® ) was administered. Fifteen minutes after ingestion, the patient presented a maculopapular rash on the trunk and extremities, an event that later became generalized; this situation was associated with dyspnea and use of accessory muscles, in addition to diarrheal stools on multiple occasions. Subsequently, the patient consults again and was assessed in the emergency room, where the following vital signs were found: blood pressure 88/64 mm Hg, heart rate 122 bpm, respiratory rate 32, and oxygen saturation 88-89%. It was then interpreted as anaphylaxis, for which management was started with IM adrenaline until a total dose of 3 mg was reached. With this, improvement in blood pressure figures and control of skin and gastrointestinal symptoms were obtained. During her observation in the emergency department, the patient reported a sensation of chest discomfort, epigastric pain, and abdominal pain (after the administration of adrenaline); therefore, this was considered as a diagnostic challenge/differential. It was decided to perform an electrocardiogram in which ST depression was interpreted in V4-V5-V6 (Figures 1 and 2). Troponin I (ultrasensitive) was also taken, which was positive for the cutoff point (4.8 ng/ml; cutoff up to 0.3 ng/ml). Management was then started with aspirin 300 mg, atorvastatin 80 mg, furosemide 40 mg every 8 hours intravenously (IV), isosorbide dinitrate in case of angina, methylprednisolone 50 mg every 8 hours IV, and enoxaparin 60 mg subcutaneously every 24 hours. Subsequently, a referral was made to a hospital with a higher level of complexity due to a diagnosis of non-ST elevation myocardial infarction, with Killip III classification, due to crepitus and radiological signs of acute pulmonary edema. Upon her admission, there was no clear clinical evidence of angina or chest pain, she denied paroxysmal dyspnea, denied bendopnea, denied extremities edema, denied other symptoms, and reported improvement of the rash. She presented the following vital signs at that time: blood pressure 122/74 mm Hg, heart rate 78 beats per minute (bpm), respiratory rate 18 per minute, and oxygen saturation 98%. On physical examination, no use of accessory muscles, no respiratory effort, rhythmic heart sounds, and no presence of heart murmurs were observed. An electrocardiogram was taken, which reported sinus rhythm, heart rate of 79 bpm, QTc 521, QRS 70, PR 154, and prominent precordial T waves ( Figure 3). With the above, and due to signs of prolonged QTc, magnesium sulfate was administered, 1 gram every 8 hours IV. Moreover, due to suspicion of a coronary syndrome, high-sensitivity troponin (hs-cTN) was requested again, presenting a value of 10854, that is, positive. Furthermore, leukocytes 16,200/ul, neutrophils 78.9%, hemoglobin 12.1 g/dL, platelets 259,000/ml, INR (international normalized ratio) 1.0, potassium 3.8 mmol/L, magnesium 1.8 mg/dL, sodium 140 mmol/L, nitrogen urea 13.4, creatinine 0.7, alanine aminotransferase 16 IU/L, and aspartate aminotransferase 55 IU/L. Due to electrocardiographic findings and positive troponin (biomarker), a transthoracic echocardiogram was requested (Figure 4), reporting a normal-sized left ventricle, moderate hypokinesia of the middle segment of the anterior and lateral wall, normal contractility of the rest of the segments, LVEF (left ventricular ejection fraction) 55%, no diastolic dysfunction, the right ventricle of normal size and systolic function, TAPSE (tricuspid annular plane systolic excursion): 21 mm, nonquantifiable pulmonary systolic pressure, valvular apparatus without relevant alterations, size atria normal, pericardium and great vessels without relevant alterations, IS/IVS without defects or shunts, no intracavitary thrombi, and normal in the rest of the study. Due to presumed myocardial injury and high suspicion of coronary artery disease, coronary angiography was requested, which showed epicardial coronary arteries, without angiographically significant stenosis ( Figure 5), thus confirming the presence of Kounis syndrome. e patient was discharged 3 days after admission, asymptomatic, without complications, with outpatient cardiology follow-up, and secondary cardiovascular prevention started (acetylsalicylic acid 100 mg daily, atorvastatin 40 mg nightly). Discussion e cardiovascular system can be highly compromised in the presence of allergic reactions. is is how a dangerous and serious cardiac manifestation such as KS can occur, which can include potential symptoms such as hypotension, arrhythmias, and ventricular dysfunction [5][6][7]. Inflammatory cells, including eosinophils, macrophages, and mast cells, play a relevant role in hypersensitivity reactions. ey start an inflammatory cascade through the release of leukotrienes, thromboxane, IgE, tryptase, histamine, and cytokines, which act on different organic systems [8]. At the coronary level, histamine can cause vasoconstriction, which seems to be the main pathophysiological mechanism in KS [9]. In the present case, a KS secondary to the administration of a laxative is described. Although the literature reports KS secondary to drugs [2,3], especially antibiotics [2], there is no known report related to this type of medication. However, the clinical manifestations related to KS were similar to those presented in the patient reported in this case. Chest pain has been reported to be the most common sign, followed by allergy-related symptoms (pruritus and edema) [2]. Despite this, patients admitted to the emergency department without clear cardiac symptoms have been documented, but that the search for etiology and phenomena of cardiac ischemia are documented either due to changes in the electrocardiogram or due to elevation of biomarkers [10]; the presentation of patients with the acute coronary syndrome as the only manifestation of a hypersensitivity reaction is less frequent [11]. Due to this wide range of symptomatologic variants and the absence of cardiac risk factors, KS remains underdiagnosed [2]. Different electrocardiographic patterns associated with KS are presented in the literature, including STsegment elevation in the anterior and inferior face of the electrocardiogram, followed by repolarization disorders, heart block, atrial fibrillation, and ventricular ectopy rhythms [12]. Allergy studies include the measurement of serum levels of tryptase, histamine, immunoglobulin E, complement levels, and eosinophil count. However, these studies are not as widely available. erefore, among all patients with angina and a suspected allergy component, and the coronary angiography is normal, or indicates that there are no coronary arteries with lesions, and the KS picture is more suggestive. Troponin I usually presents elevation in 7-10% of patients with anaphylaxis, without having such a high range, as in patients with coronary syndrome due to atherothrombosis [13,14]. Regarding the management of KS, a great versatility between cases has been described, possibly due to the absence of predetermined algorithms. However, the recommended therapy consists of concomitant management of myocardial ischemia and allergic reaction [2]. e use of nitrates should be considered as long as the patient does not present some degree of hypotension, improving the signs and symptoms of vasospasm. It is not clear the use of acetylsalicylic acid as well as inhibitors of the glycoprotein IIb-IIIa complex in KS; however, until the active coronary disease has been ruled out, it should be recommended if the initial clinical picture is more in favor of an acute coronary syndrome. e use of other medications such as adrenaline, steroids, and antihistamines is recommended under the pretext of the distributive shock that these patients present, as long as they are indicated [15,16]. Finally, it is important to keep in mind that hypersensitivity reactions, regardless of the primary trigger, can be complicated by the acute coronary syndrome. Data Availability e clinical data utilized in this report are described in this article. Ethical Approval e Bioethics Committee of the Hospital San Vicente Fundación approved the publication of this report. Consent e patient signed the informed consent related to hospital care and for academic purposes. Case Reports in Medicine	2022-06-25T15:02:41.021Z	2022-06-22T00:00:00.000	{ "year": 2022, "sha1": "9d6f6c061e09477630059f6bd4c6fade804fb092", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/crim/2022/6087176.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "2d7627950e7ae956871284ebd3037149ad6eacc7", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }

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