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234421749	pes2o/s2orc	v3-fos-license	Outcomes with fracture-level transpedicular screws used for thoracolumbar junction fractures Original Article  Likhachev S.V., Zaretskov V.V., Arsenievich V.B., Ostrovskij V.V., Shulga A.E., Zaretskov A.V. Outcomes with fracturelevel transpedicular screws used for thoracolumbar junction fractures. Genij Ortopedii, 2020, vol. 26, no 4, pp. 548-554. DOI 10.18019/1028-4427-2020-26-4-548-554 © Likhachev S.V., Zaretskov V.V., Arsenievich V.B., Ostrovskij V.V., Shulga A.E., Zaretskov A.V., 2020 INTRODUCTION Fractures of the thoracolumbar junction (T11-L2), the transition from the less mobile thoracic spine to the more dynamic lumbar spine, are the most common fractures of the spinal column. Thoracolumbar burst fractures (AOSpine A3, A4) are normally stabilized with surgical techniques. Common surgical goals are to obtain the most stable fixation, correct the deformity and prevent the recurrence, produce spinal decompression to allow early ambulation. Shortsegment transpedicular screw fixation (SSTSF) is the preferred treatment option for thoracolumbar burst fractures for stabilizing the three-column spine. The technique is characterized by a relatively simple performance, less surgical trauma and decreased blood loss. Polysegmental fixation, circular stabilization of the spine and procedures performed using the anterior approach are alternatives to the conventional transpedicular screw fixation. Disadvantages reported with SSTSF include unstable metal construct, loss of correction at a long term, recurrence of spinal stenosis, neurologic deficit and pain. Short-segment instrumentation can be augmented with additional pedicle screws placed at the fracture level. The intermediate screws inside the fractured vertebra can improve stability of the anterior column due to additional reinforcement and reduce stress on each pedicle screw. Decision on intermediate screws to be placed at fracture level is made by the surgeon. Objective To compare clinical outcomes of transpedicular fixation with and without additional screws in the fractured vertebral body after isolated uncomplicated fractures at the thoracolumbar junction. MATERIAL AND METHODS A retrospective cohort study enrolled 78 patients with Th11-L2 thoracolumbar burst fractures who underwent surgical treatment at the institute between January 2016 and December 2018. Inclusion criteria were an isolated uncomplicated fracture at the thoracolumbar junction (AOSpine А3N0M1, А4N0M1) [14], SSTSF, follow-up period of at least one year. Exclusion criteria were circular stabilization Original Article of the spine, polysegmental transpedicular systems, neurologic deficit, postoperative infection, osteoporosis. A total of 62 patients met the inclusion and exclusion criteria and were allocated to two groups depending on the type of surgery performed. Patients of group I (n = 30) underwent bilateral transpedicular four-screw fixation. Patients of group II (n = 32) underwent similar procedure with two screws added in the fracture body. Clinical evaluation and imaging parameters were measured preoperatively, at one week, 1, 6 and 12 months postoperatively. General characteristics of the patients are presented in Table 1. Surgical technique Surgeries were performed with use of general anesthesia (ALV + TIVA). Patients were in supine position with cushions placed under the thoracic cage and pelvis. The standard median approach provided exposure to the posterior vertebral structures and the adjacent vertebrae. Bilateral transpedicular four-screw fixation was performed for the patients of both groups with two screws added at fracture level in group II. The screws were connected by rods, distraction was provided for remodeling of the broken vertebra and the assembly accomplished. 6.5 mm screws 40 or 45 mm long were employed for the cases. For the construct, 5.5 mm pins were used. Implants from one manufacturer were used for the cases, and surgeries were performed by the same surgical team. Clinical and imaging measurements Patients were requested to fill out questionnaires to evaluate pain intensity with 10-point Visual analogue scale (VAS-10) and with Oswestry Disability Index (ODI) for low back pain to quantify disability preoperatively, at one week, 1, 6 and 12 months postoperatively. Preoperative and postoperative radiographs were used to assess positioning of the intermediate pedicle screws and measure kyphotic angle by Cobb method. Kyphosis was evaluated by measuring anterior vertebral body height (AVBH, mm) at the fracture level. Linear and angulation measurements were produced using Surgimap (Spine Software, version 2.2.15.1). Axial CT scans were used to measure spinal canal stenosis (SCS, %) at the level of maximal retropulsion of bone fragments and assess decompression at 7 days postsurgery. Statistical analysis Statistical data analysis was performed using Microsoft Excel 13 and Statistica for Windows -6.0 computer program. Non-parametric statistical tests (Wilcoxon) was used when distribution was different from the norm. The Kolmogorov-Smirnov and Shapiro-Wilk tests were employed to determine normality of distribution in the groups. Most of the variables were not normally distributed, and the median, 25 th and 75 th percentiles were used for description. Non-parametric two-sample Mann-Whitney U test and the Newman-Keuls method were used to compare differences between two independent groups. Pearson's chi-squared and twotailed Fisher's exact test with observations less than 5 in a table cell were applied to identify differences in scores evaluating outcomes between the groups. The Wilcoxon signed-rank test was used to analyze kyphotic angles at different follow-up periods. For calculations, a significance level of < 0.05 was adopted. RESULTS Median age in group I was 31.5 (range, 25-43) years with male/female ratio of 21:9. Median age in group II was 29.0 (range, 25-46.7) years with male/female ratio of 22:10 (Table 1). No statistically significant differences in age and sex were observed in groups I and II (p = 0.782 and p = 0.915, respectively). Mean body weight was 71 (range, 62.7-80.7) kg and 72 (range, 64-79.7) kg in groups I and II, respectively. There were no statistically significant differences in body weight between the groups (p = 0.832). Vertebral fractures were distributed in groups I/II as follows, Th11: 3/3, Th12: 7/9, L1: 15/14, L2: 5/6. Fracture patterns in groups I/II were A3: 20/21, A4 10/11. The groups appeared to be comparable by fracture level and morphology (p = 0.311 and p = 0.931, respectively). Patients of both groups underwent surgery at 7 (range, 4-8) days on average. Injuries in groups I/II resulted from a fall from height 12/11 and RTA 18/21. There were no statistically significant differences in operating time in the groups with the mean of 60 (range, 50-70) minutes. Mean intraoperative blood loss was comparable in both groups measuring 150 (range, 100-170) mL. No early postoperative complcations were recorded. Mean length of hospitalization was 6 (range, 5-7) days in both groups. Mean preoperative pain scored 7 (range, 7-8) on VAS scale. Antalgic effect was recorded postoperatively at all stages of questionnaire completion. No changes in pain dynamics were observed at 7 days, 1 and 12 months postsurgery. There were statistically significant differences in pain intensity noted at 6 months in the groups with mean VAS score of 2 (range, 2-3) in group I and 1 (range, 1-2) in group II (p = 0.043). Quality of life improved progressively on ODI scale in both groups with considerable differences in support of group II recorded at 6 and 12 months postoperatively (p < 0.01). Dynamics in VAS and ODI scores is presented in Figures 1 and 2. Imaging parameters showed benefits of SSTST with intermediate pedicle screws added to fracture level. Preoperative segmental kyphosis demonstrated no significant differences between the groups (p = 0.881) and measured 14.50 (range, 120-190) in group I and 150 (range, 120-190) in group II and was successfully corrected in both groups with the mean 50 (range, 30-50) in groups I and II at 7 days postsurgery. The correction was shown to equally decrease in both groups (p = 0.698) during the first postoperative month and kyphotic angle measured 50 (range, 40-50) and 50 (range, 30-60) in groups I and II, respectively. Loss of correction was more evident in standard SSTSF group at 6 and 12 months. Mean segmental kyphosis measured 80 (range, 60-100) on average, with loss of correction of 3.50 (range, 10-60) in group I against mean kyphotic angle of 50 (range, 50-60) with loss of correction of 10 (range, 00-20) in group II (p < 0.01) at 12-month follow-up. Dynamics in measurements of kyphosis and loss of correction is presented in Figures 3 and 4. Although there were no statistically significant differences in mean AVBH measuring 19 (range, 14.7-21) mm in group I and 19 (range, 14.2-21) mm in group II preoperatively and showing similar values at 7 days of surgical correction in both groups: 27 (range, 24-29) mm in group I and 27 (range, 23-30) mm in group II, AVBH was shown to decrease progressively in group I with statistically significant differences between the groups (p = 0.034) measuring 24 (range, 20-26) mm in group I and 27 (range, 22-29.5) mm in group II. SSTSF with intermediate screws placed at fracture level appeared to maintain the AVBH better as compared to standard procedure (Fig. 5). Statistically significant advantages of indirect decompression of the spinal canal (Fig. 6) were seen with the use of SSTSF added The best surgical approach and treatment of thoracolumbar burst fractures are still controversial due to the limited number of published comparative studies. There is lack of clear evidence-based consensus about the modalities of surgical management for these thoracolumbar fractures. Short-segment transpedicular screw fixation is the preferred treatment option for thoracolumbar burst fractures due to relatively low complication rate and simple performance [15][16][17][18][19][20]. However, several authors report a high rate of fixation failure or instrumentation migration to be associated with SSTSF [19,21,22]. Radiological signs of hardware failure are not always accompanied by clinical symptoms. SSTSF can be reinforced with additional intermediate pedicle screws placed bilaterally into the involved vertebra [23][24][25] and is seen as a feasible treatment strategy for thoracolumbar fractures [26]. The technique is superior to conventional SSTSF in stability due to the greater number of supporting components and additional reinforcement provided for the anterior spinal column avoiding negative effect on the spinal biomechanics relative to polysegmental fixation. There are few reports on the use of the technology in the Russian literature [27,28]. There are few studies exploring radiological and clinical outcomes with application of intermediate pedicle screws in the available literature [29]. More long-term follow-up studies are needed. There were no statistically significant differences in demographic data of both groups in our series. There were also no statistically significant differences in blood loss, operating time, preoperative and postoperative VAS and ODI scores. Imaging parameters measured with preoperative radiographs and films taken at one week and one month postoperatively were comparable in both groups. There were statistically significant differences in segmental kyphosis, AVBH and SCS observed at 6 and 12 months postoperatively. Loss of correction was more evident in standard SSTSF group at 6 and 12 months. More improvement in SCS was seen in group II. Therefore, use of intermediate pedicle screws at fracture level allowed reduction in the loss of kyphotic deformity correction and AVBH at a long term and improvement in indirect decompression of the spinal canal. Outcomes of standard SSTFS are in line with available literature data and benefits from intermediate screws placed at fracture level detected with clinical and imaging assessment are confirmed by reports based on in vitro studies or computeraided modeling. The study has limitations related to retrospective design with a small cohort of patients and a short follow-up period of one year. Greater cohorts of patients and a longer follow-up period can facilitate more accurate assessment of intermediate pedicle screws for SSTSF. SSTSF added with intermediate pedicle screws has been shown to provide better maintenance of intraoperative kyphosis correction and vertebral height restoration of the affected vertebral body. Indirect decompression of the spinal canal appeared to be more efficient with use of fracture level screws. Further prospective clinical trials can be required for comprehensive evaluation of the above technique.	2020-12-24T09:12:18.420Z	2020-12-21T00:00:00.000	{ "year": 2020, "sha1": "a4e98e2b28eb4f5e42005776f88881beb3286f43", "oa_license": "CCBY", "oa_url": "https://doi.org/10.18019/1028-4427-2020-26-4-548-554", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "acb23ad5d845c9bdf77a4be1b926509e5a3bc8dc", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
221846384	pes2o/s2orc	v3-fos-license	Novel LAA Waiting and Transmission Time Configuration Methods for Improved LTE-LAA/Wi-Fi Coexistence Over Unlicensed Bands Long Term Evolution-Licensed Assisted Access (LTE-LAA) has been pointed out as a key solution to cope with the increasing amounts of data traffic and the scarcity of the licensed spectrum. The 3rd Generation Partnership Project (3GPP) has standardised LAA to operate over the 5 GHz unlicensed spectrum which is mainly occupied by Wi-Fi. It is a challenging problem to ensure a fair coexistence between these technologies. Several studies have been proposed in the literature to allow a fair LAA/Wi-Fi coexistence. In this work, various methods are proposed to adapt/select the waiting times for LAA based on the activity statistics of the existing Wi-Fi network. The main novelty is that the knowledge of the existing Wi-Fi activities is exploited to tune the boundaries of the Contention Window (CW) for LAA and to select fixed waiting times for LAA. Moreover, a dynamic method is proposed to adapt the Transmission Opportunity (TxOP) times for LAA based on the Hybrid Automatic Repeat Request (HARQ) feedbacks. The methods are evaluated using ns-3 network simulator based on the 3GPP fairness definition. We show that selecting fixed waiting times for LAA based on the existing Wi-Fi activities is more friendly to the existing Wi-Fi and provides better total aggregated throughputs for both coexisting networks compared to the 3GPP Category 4 Listen Before Talk (Cat 4 LBT) algorithm. Moreover, the proposed dynamic TxOP method is more friendly to the existing Wi-Fi and provides better total aggregated throughputs compared to the fixed TxOP period approach of the 3GPP Cat 4 LBT scheme. I. INTRODUCTION Unlicensed spectrum bands have inspired researchers as a promising solution for the licensed spectrum shortage given the current exponential increase in demand for wireless data and services. The licensed spectrum is scarce and costly, and no longer provides simple ways to increase the mobile networks capacities. A significant amount of spectrum of approximately 600 MHz is available for various purposes over the unlicensed 5 GHz band [1], [2]. This unlicensed available spectrum has recently attracted the industry and researchers to be utilised for Long Term Evolution (LTE) The associate editor coordinating the review of this manuscript and approving it for publication was Mamoun Alazab . deployments. Thus, LTE has been recently deployed to operate over unlicensed bands providing enhanced mobile networks capacities [3], [4]. This same concept is likely to be introduced as well in the future 3GPP specification for 5G New Radio Unlicensed (5G NR-U) and therefore still constitutes a research topic of recent interest [5], [6]. However, unlicensed spectrum bands are mainly occupied by Wi-Fi networks. Despite the fact that deploying LTE over unlicensed bands achieves higher throughput and more capacity, a few issues need to be considered while deploying these heterogeneous technologies (i.e., LTE and Wi-Fi) in a shared spectrum band [4], [7], since special attention needs to be paid to coordinate this coexistence over the same unlicensed spectrum band [8]. In particular, the difference in VOLUME 8, 2020 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ the Medium Access Control (MAC) layers between LTE and Wi-Fi creates a challenging problem given that Wi-Fi follows a Carrier Sense Multiple Access with Collision Avoidance (CSMA/CA) mechanism while LTE has no sensing scheme for transmission. Considering this heterogeneous coexistence between LTE and Wi-Fi, Wi-Fi may experience a lower opportunity to access the unlicensed channel since Wi-Fi nodes have to check the channel availability before transmitting their own data, thus potentially leading to a performance degradation for Wi-Fi [9], [10]. The key idea of coexistence between LTE and Wi-Fi networks over unlicensed bands is to increase LTE network capacity but not to degrade the performance of the existing (Wi-Fi) networks. As a result, different requirements need to be taken into account to design an unlicensed LTE such that it allows a fair coexistence with Wi-Fi over unlicensed bands. 3GPP TR 36.889 describes the ''fairness'' between the coexisting networks (i.e., LTE and Wi-Fi) over the unlicensed 5 GHz band as the ability of an LTE network not to impact the existing network (i.e., Wi-Fi) active on the same carrier more than an additional Wi-Fi network in terms of throughput and latency [4]. Two main approaches have been proposed in the literature to achieve a fair coexistence between these heterogeneous technologies [3], [4]. In particular, for some markets, such as Europe and Japan, a Listen Before Talk (LBT) protocol for Clear Channel Assessment (CCA) is required for accessing unlicensed bands, while in other markets, such as USA and China, there is no need for such protocol. LTE-Unlicensed (LTE-U), which does not need an LBT protocol, was the first version of LTE over unlicensed bands and was proposed by the industry consortium LTE-U Forum [11]. LTE-U was aimed at allowing a quick deployment of LTE networks in 5 GHz bands in those countries that do not require an LBT protocol by reusing mechanisms already available in the 3GPP standard. The three main mechanisms on which LTE-U relies are carrier selection, ON/OFF switching and Carrier Sense Adaptive Transmission (CSAT) to adapt the Duty Cycle (DC) of the transmissions [12], [13]. However, LTE-U is unable to fully meet the requirement of fair coexistence as defined in 3GPP TR 36.889. As a result, the 3rd Generation Partnership Project (3GPP) in Release 13 proposed LTE-Licensed Assisted Access (LTE-LAA) for Supplementary Down-Link (SDL) where an LBT protocol is required for transmission over unlicensed bands [4]. A. PREVIOUS RELATED WORK Due to the increasing interest in spectrum sharing between LTE and Wi-Fi networks over unlicensed bands, various studies have been recently devoted to implement different spectrum sharing mechanisms enabling a fair coexistence between these two heterogeneous technologies. A comparison between the coexistence of LTE-U/Wi-Fi and LTE-LAA/Wi-Fi scenarios is provided in [14]. The simulation results show that coexisting LAA with Wi-Fi achieves better performance than deploying LTE-U with Wi-Fi over the unlicensed 5 GHz band. A numerical analysis is performed for LTE and Wi-Fi networks in [15]. The numerical results show that coexisting both technologies over the same unlicensed band without any modification to the existing protocols can severely degrade the Wi-Fi performance. The impact of the DC parameter on LTE-U/Wi-Fi coexistence over unlicensed bands is studied in [16] where different blank subframes are deployed within the LTE-U frame allowing Wi-Fi transmissions. The simulation results show that by deploying more blank subframes over the LTE frame, a higher Wi-Fi throughput can be achieved. The performance of LTE-U/Wi-Fi coexistence is investigated in [17] by exploiting the knowledge of the activity statistics of the existing Wi-Fi network to select a fixed DC for LTE-U. The simulation results show that the total aggregated throughputs for the coexisting networks can be improved by selecting both the DC value and the location of the blank subframes of LTE-U based on the Wi-Fi activity statistics. In [18], an hyper access point (HAP) is proposed that allows LTE-U take advantage of the Wi-Fi point coordination function protocol by dedicating a contention-free period to LTE-U users and allowing a contention period for traditional Wi-Fi users. In [19], a listen-before-talk access mechanism featuring an adaptive distributed control function protocol is proposed, whereby the backoff window size is adaptively adjusted according to the available licensed spectrum bandwidth and the Wi-Fi traffic load to satisfy the quality-of-service requirements of small cell users and minimise the collision probability of Wi-Fi users. On the other hand, the fairness of LTE-LAA/Wi-Fi coexistence has been widely studied in the literature [20]. An LTE-LAA module has been developed for ns-3 network simulator in [21] to investigate the performance of LTE-LAA/Wi-Fi coexistence scenario. The simulation results show that the fairness depends on the design parameters of the LBT algorithm for LAA. The work reported in [7] indicated that the transmission times of LTE evolved Node B (eNB) should be fixed at the beginning of the Distributed coordination function Inter-frame Space (DIFS) of Wi-Fi Access Point (AP) and the CCA period of LTE eNB should be shorter than DIFS period leading to no collisions between LTE and Wi-Fi networks. In [16] and [22], an LTE muting scheme is considered where LTE eNBs follow a predetermined muting pattern allowing the transmission of LTE nodes. The impact of the LBT design parameters for LTE-LAA/Wi-Fi coexistence is investigated in [23]. In particular, an alternative approach to increase the LAA Contention Window (CW) is proposed based on the observed number of free slots during a specific time interval. The results show that the standard algorithm outperforms the proposed one. An adaptive LBT scheme for LAA based on Markov chain model is proposed in [24]. Specifically, a partially-randomised initial Clear Channel Assessment (iCCA) scheme and an adaptive CW size scheme are considered for the LBT algorithm of LAA based on the detection by the LAA system. The simulation results show that the proposed strategy is effective for LAA to achieve the fairness in the downlink scenario for LAA/Wi-Fi coexistence. A CW size adjusting method within an enhanced LBT algorithm of LAA is proposed in [25]. The CW size is adjusted based on the exchanged information from the neighbor nodes of the considered scenario. The simulation results show that the proposed scheme achieves better performance compared to the fixed scheme of LBT for the various coexistence scenarios. In [26], a fair downlink traffic management scheme is proposed for LAA/Wi-Fi networks to tune the minimum CW values and to assign feasible weights for LAA eNBs under different traffic loads. The simulation results show that the proposed scheme improves the aggregated utility of LAA/Wi-Fi networks but the Wi-Fi throughput decreases slightly compared to the static approach of minimum CW configuration. In [27], a CW adjustment method is proposed based on a simple gradient approach enabling a fair coexistence between LTE-LAA and Wi-Fi networks but the authors did not provide an analytical throughput model for the proposed method. An adaptive LBT algorithm is proposed in [28] where the different design parameters of LBT are adapted dynamically based on the varying traffic load and the CW size of the existing Wi-Fi system. In [29], a modified model to investigate the Energy Detection (ED) threshold of LAA is proposed. The numerical and experimental results show that the fairness between LTE-LAA and Wi-Fi networks depends on the channel access parameters such as ED threshold and Transmission Opportunity (TxOP) period of LAA. In [30], the fundamental trade-off between co-channel interference and collision probability is investigated and addressed by means of a power allocation rule with double water-filling lines, which achieves the complete set of Pareto optimal solution by means of the weighted Tchebycheff method. In [31], a joint licensed and unlicensed resource block allocation scheme is proposed to maximise the energy efficiency of LAA taking into account fair resource sharing between LTE and WiFi networks. In [32], an adaptive p-persistent channel access scheme for LAA (named p-LAA protocol) is proposed to balance the trade-off between throughput and fairness for the coexistence network. A Q-learning algorithm is proposed in [33] to adjust the TxOP periods for Wi-Fi and LAA based on the current traffic load or expected capacity. The simulation results show that the proposed algorithm can achieve fairness while maintaining high throughput when the algorithm is applied. The work presented in [34] provides a Markov chain model analysis for LAA/Wi-Fi coexistence scenario where the data are transmitted in a single transmission opportunity backoff. The numerical results show that the proposed model provides better performance for Wi-Fi network and for LAA networks in dense deployments. B. CONTRIBUTIONS In this work, we focus on LTE-LAA given that it represents the most promising unlicensed LTE approach to achieve fairness between LTE and Wi-Fi networks because it is generally more fair to Wi-Fi compared to LTE-U. Current studies mostly focus on the design of mechanisms that enable a fair coexistence between LTE-LAA and Wi-Fi over unlicensed bands. Considering the latest LBT algorithm of 3GPP, Category 4 (Cat 4) LBT algorithm, it can be noticed that the coexistence performance of LTE-LAA/Wi-Fi over the unlicensed 5 GHz band does not perfectly match the fairness definition as described by 3GPP TR 36.889 [4], as it was shown in the results reported in [14]. Specifically, a Wi-Fi performance degradation can be noticed due to this heterogeneous coexistence between LTE-LAA and Wi-Fi networks. This degradation is due to some potential drawbacks of the 3GPP Cat 4 LBT algorithm which are described in Section II and that are addressed and overcome by the methods proposed in this work. The main novelty of the methods proposed and analysed in this work is the exploitation of the activity statistics of the Wi-Fi network for an adequate configuration and operation of the LTE-LAA method. As opposed to previous related work, where the activity statistics of the Wi-Fi network are not taken into account, the methods proposed in this work exploit the availability of this information in order to optimise the performance not only of LTE-LAA but also of the Wi-Fi network itself. The main contributions of this work are as follows: where the activity statistics of the Wi-Fi network are used to set fixed waiting times for LAA before transmission instead of following a CW-based approach. 4) Various variants are proposed to select the lower bound of the CW of LAA based on the minimum and mode of the activity statistics of the existing Wi-Fi network. Moreover, a fixed waiting time method for LAA is proposed based on these variants as well. 5) A novel dynamic TxOP period approach is proposed where the observed Wi-Fi transmission pattern is exploited to configure the maximum TxOP length for LAA using a dynamic scheme. The remainder of this work is organised as follows. First, Section II describes the channel access mechanisms in Wi-Fi and LTE-LAA technologies. In addition, the 3GPP Category 4 (Cat 4) LBT algorithm is introduced. Various methods are presented in Section III to adapt/select the waiting times for LAA. Section IV presents a dynamic approach to configure the transmission times for LAA. The considered methodology, simulation environment and used model are described in Section V. Section VI presents and analyses the obtained simulation results. Finally, the conclusions are summarised in Section VII. II. COEXISTENCE OF Wi-Fi AND LTE-LAA: MAC PROTOCOL MECHANISMS This section introduces a review of Wi-Fi and LTE-LAA technologies to highlight the main features and the basic differences between both technologies. A. Wi-Fi TECHNOLOGY Wi-Fi technology employs a Distributed Coordination Function (DCF) protocol which uses carrier sensing to maximise the throughput while preventing packet collisions. DCF is mainly based on the CSMA/CA MAC protocol [12]. In particular, if there is a Wi-Fi node that has data to transmit, it needs to sense the channel firstly to be idle for a DCF Inter-frame Space (DIFS) duration. If the channel is clear, it will transmit a Request-To-Send (RTS) to the destination node. Then, the destination will send a Clear-To-Send (CTS) if it is ready to receive data. The Wi-Fi node will transmit its data when the sender node receives the CTS message. In addition, the destination will send an Acknowledgment (ACK) to the sender node for the successful data reception after a Short Inter-frame Space (SIFS) time. On the other hand, if the channel is not clear, the node keeps monitoring the medium until it becomes idle for a DIFS time, then it picks a random backoff time and counts down (in particular, a random number of time slots which is within a CW that has lower and upper bounds as shown in Table 1). When the backoff timer reaches zero, the Wi-Fi node can perform the transmission for a maximum time determined by the Transmission Opportunity (TxOP) parameter as shown in Table 1. The process is illustrated in Fig. 1. It is worth noting that, given the Wi-Fi MAC protocol, Wi-Fi nodes may be unable to access the channel if it is heavily and selfishly used by other technologies in the same channel. B. LTE-LAA TECHNOLOGY In some countries, such as Europe and Japan, the regulations require an LBT protocol to be used for transmission over unlicensed bands. As a result, 3GPP proposed in Release 13 a new version of LTE which supports an LBT protocol for the Down-Link (DL) transmission over unlicensed bands [4]. In Release 14, the Up-Link (UL) scenario was considered in the context of enhanced LAA (eLAA). LTE-LAA uses the Carrier Aggregation (CA) concept in the DL to combine the LTE spectrum in the licensed band with the spectrum in the unlicensed band, thus providing higher data rates, better user experience and enhanced capacity [4]. Aggregating licensed and unlicensed carriers is a key milestone towards 5G. The licensed LTE MAC protocol has no frame for collision detection and this is the key difference between LTE and Wi-Fi technologies. This requires a modification for the LTE air interface in order to include an LBT algorithm within the LTE MAC. Coexisting LTE with Wi-Fi over the same unlicensed band without any fair mechanism can degrade the Wi-Fi performance given the lack of an LBT mechanism in LTE (since it was designed assuming exclusive access to the spectrum) and the fact that Wi-Fi nodes would frequently sense the channel as busy before attempting any transmission, thus preventing their access to the channel. As a result, an LBT algorithm for LAA was introduced in 3GPP Release 13, which is referred to as Category 4 (Cat 4) LBT [4], [37]. The 3GPP Cat 4 LBT algorithm is similar to the Wi-Fi DCF protocol as it can be seen in Fig. 2. In this algorithm, a CCA period is considered to check the availability of the channel before transmission. In particular, an LAA eNB is allowed to transmit its own data after sensing the channel to be free for an initial CCA (iCCA) period (e.g., 34µs); otherwise, the extended CCA (eCCA) stage starts. During the eCCA stage, a backoff process starts by selecting a random number N ∈ [0, q − 1], where N indicates the number of idle slots that need to be observed before transmission, while q − 1 represents the upper bound of the CW, which varies 162376 VOLUME 8, 2020 according to an exponential backoff. In particular, the channel is observed by LAA eNB for a time equal to N multiplied by the CCA slot time period (e.g., 9µs). When the channel is free, another eCCA period (e.g., 9µs) begins and N is decreased by one if the channel is clear. When N decrements to zero, the LAA eNB starts the transmission for a fixed configurable Transmission Opportunity (TxOP) time, which can be up to 10 ms depending on the channel access priority class (see Table 2 and [37, Table 15.1.1-1] for details). If the LAA eNB needs another transmission, the eCCA stage is repeated again. However, the value of N is related to the channel access priority class which categorises the traffic type. In particular, the CW size q−1 is initialised with CW min and it is exponentially increased based on Hybrid Automatic Repeat Request (HARQ) feedbacks. Table 2 provides the values of CW min and CW max for each channel access priority class. For example, for the priority class 3, the initial value of the upper bound of the CW, q − 1, is 15 and it is updated to 31 by doubling q if 80% of HARQ feedbacks from the first subframe of the latest transmission are Negative Acknowledgments (NACKs). The upper bound of the CW q − 1 is again updated to 63 if another 80% of HARQ feedbacks are NACKs. Otherwise, the upper bound of the CW q − 1 is reset [37]. There are a few drawbacks of the 3GPP Cat 4 algorithm which considers the HARQ feedbacks to update the LAA CW [38], [39]. In particular, the LAA CW size will not be updated if less than 80% of the users suffer from the collision since the collision remains undetected below this threshold. Moreover, the algorithm only considers the detection of the first subframe of the transmission to update the CW size but the collisions from other subframes are neglected. Furthermore, the performance of LAA/Wi-Fi coexistence is affected by the configuration of the LBT parameters such as the CW and the TxOP length of LAA [28], [40], [41]. However, it can be noted that the adaptation approach of the LAA CW in the standard Cat 4 LBT algorithm does not take into account the activity statistics of the existing technology (i.e., Wi-Fi) and it configures the upper bound of the LAA CW to be 15, 31 or 63 (for class 3) regardless of the existing Wi-Fi activity patterns. Moreover, the Cat 4 LBT algorithm allows LAA eNBs to transmit, after the channel availability check, for a fixed TxOP period. It can be noted that this static TxOP approach is not the most efficient approach for a fair coexistence between LAA and Wi-Fi networks where the LAA TxOP length is kept fixed for all transmissions regardless of the HARQ feedbacks. Therefore, to enhance the performance of the current Cat 4 LBT algorithm, different methods are proposed in this work to update/select the LAA CW boundaries. The dashed shaded boxes in Fig. 2 highlight the procedure of the standard Cat 4 LBT that will be modified to include these proposed methods. In addition, a novel method is proposed to configure the TxOP length in a dynamic manner which will be included instead of the dashed shaded diamond in Fig. 2. All these methods are described below. III. METHODS TO ADAPT LTE WAITING TIMES In this section, various methods are presented to adapt/select the lower and upper bounds of the LAA CW, which determine the waiting times of LTE-LAA, based on the Wi-Fi activity statistics. In addition, various methods are described to select fixed waiting times for LAA eNB. A. DYNAMIC CW (DynCW) METHODS As stated before, the standard 3GPP Cat 4 LBT algorithm follows a similar contention mechanism to that of Wi-Fi technology aiming to achieve a fair coexistence between LTE-LAA and Wi-Fi networks. In specific, LAA updates the upper bound of the CW, q − 1, by doubling q from 15 to 31 and to 63 based on the HARQ feedbacks when the channel is sensed to be busy. It is worth noting that this increase in the upper bound of the LAA CW is heuristic and ignores the actual ON/OFF activities of the existing Wi-Fi network which may lead to an inefficient spectrum utilisation. In particular, if the channel is sensed to be busy by the LAA eNB, the upper bound of the CW is doubled, which in many cases may lead to longer waiting times than the actual occupancy times of the Wi-Fi transmissions, then LAA would wait a long time before re-accessing a channel that could actually be empty since a long time ago. This behavior would degrade the LAA performance by increasing latencies and reducing throughputs for LAA. As a result, considering the activity statistics of the existing technology (i.e., Wi-Fi) should provide a more efficient channel access mechanism since the LAA waiting times would be aligned with the actual occupancy times of the Wi-Fi network, thus reducing the latency and increasing the throughput for LAA. Two adaptation methods for the upper bound of the CW of LAA, q − 1, are proposed here based on the activity statistics of the existing Wi-Fi network [42]. It is worth mentioning that the existing Wi-Fi activity statistics can be estimated by the LTE system without any coordination between the coexisting networks and this can be performed based on the energy detection sensing decisions of the LAA algorithm [12], [43], [44]. In particular, LAA eNB can periodically sense the Wi-Fi channel state when LAA is not transmitting to estimate the Wi-Fi ON time periods. After observing the ON Wi-Fi channel state for a sufficient large number of ON periods, the LAA network can compute the Cumulative Distribution Function (CDF) for the ON time periods of the existing Wi-Fi network. This CDF, which describes the activity pattern of the existing Wi-Fi network, can be exploited to adapt the upper bound of the CW of LAA in an efficient manner instead of following the standard adaptation method as specified by the 3GPP Cat 4 LBT algorithm which doubles the q value regardless of the activity statistics of the existing Wi-Fi network. To illustrate the proposed method, Fig. 3 shows the CDFs of the Wi-Fi ON times estimated by the LTE network. In addition, Table 3 provides the corresponding values of the upper bound of the LAA CW, q − 1, for different percentile points of the CDFs under different traffic loads. Fig. 3 is used to compute the values in Table 3 by dividing the ON times of each percentile point by the LAA slot duration (9 µs) and rounding the result to the nearest integer toward infinity (i.e., ceil function). For example, for λ = 1.5 packets/second, the 50% percentile point corresponds to a Wi-Fi ON time of around 70 µs, which divided by 9 µs and ceiled results in the value q − 1 = 8 shown in Table 3 for the 50% percentile and λ = 1.5 packets/second. All the provided values in Table 3 are calculated following the same procedure. It is worth mentioning that a percentile point of 100% in a theoretical CDF model is not feasible generally since the corresponding ON time would tend to infinity. However, the CDF that is used by LAA for the CW adaptation is based on empirical observations of Wi-Fi ON times, which necessarily have a finite maximum and this value is selected as the 100% percentile point. This approach allows various adaptation methods. Two dynamic adaptation methods for the LAA CW are discussed below. Notice that the CDF of the Wi-Fi ON times will be affected not only by the packet inter-arrival times as illustrated in Fig. 3 but also by other network conditions such as the number of Wi-Fi APs, LTE-LAA eNBs and total number of users. If any of these network conditions change, the CDF of the Wi-Fi ON times will change as well. However, the LTE-LAA system will not be required to have any prior knowledge of these conditions. Note that the estimation of the required CDF and the resulting percentile points can be implemented as an online, real-time learning process. Any changes in the network conditions will automatically result in a change in the estimated CDF and the resulting percentile points. The proposed methods will thus adapt automatically to the new network conditions every time these change. 1) DYNAMIC CW WITH 3 ADAPTATION POINTS (DynCW-3) Three adaptation points are defined in this method for the CW of LAA. These points are at the 50% (median value), 95% and 100% (maximum value) percentiles of the CDF of the existing Wi-Fi ON time periods. This method is implemented by setting the first upper bound of the LAA CW, q − 1, to be the median (i.e., 50% value) Wi-Fi ON time. The reason behind choosing this value to be the starting point of the upper bound of the LAA CW is that in 50% of cases the Wi-Fi ON times will be shorter than this value and in the other 50% of cases they will be longer. Thus, the median value is considered a reasonable starting point. If the LAA transmission fails, this means that the 50% percentile time is not long enough to find a clear channel for LAA transmission and in such case the upper bound of the CW, q − 1, will be increased to the 95% percentile point. In most cases, LAA should find an idle Wi-Fi channel after the new waiting time and therefore can transmit. For those cases where Wi-Fi has very long transmissions, the upper bound of the LAA CW will finally be updated to the 100% percentile point (maximum value), thus hopefully leading to a successful transmission in the next attempt. For this method, it can be noticed that the actual LAA waiting times are adapted based on the existing Wi-Fi traffic statistics. 2) DYNAMIC CW WITH 2 ADAPTATION POINTS (DynCW-2) Two adaptation points are defined in this method for the CW of LAA. This method is implemented based on the Wi-Fi activity statistics as well. In particular, it defines the first maximum CW value at the 50% percentile (median value) point and finally at the 100% percentile (maximum value) point. The motivation of this method is to allow for a faster convergence to the optimum value of LAA CW, in case it needs to be increased, and therefore provide better performance for LAA by reducing latencies and increasing throughputs. provided by the proposed methods for the different traffic loads. Thus, the 3GPP method may lead to unnecessarily long waiting times for LAA and therefore a degraded performance. B. STATIC CW (StatCW) METHOD We propose here a new static method to select the upper bound of the LAA CW based on the activity statistics of the existing Wi-Fi network instead of updating the upper bound of the LAA CW dynamically [45]. In particular, the 50% (median value), 95% or 100% (maximum value) percentile point of the CDF of the ON Wi-Fi times are considered as fixed upper bounds of the LAA CW. In this proposed method, q − 1 is considered to be a static value that is selected as the corresponding value for the percentile point of the CDF of the ON Wi-Fi times divided by the CCA slot duration (9 µs). Table 5 shows corresponding values of the LAA CW, q − 1, for these percentile points of the CDF of the ON Wi-Fi times under different traffic loads. Therefore, the upper bound of the LAA CW is fixed and there are no different sizes for the CW size as specified in the 3GPP Cat 4 LBT algorithm where the upper bound of the CW could be 15, 31 or 63. The main motivation of this method is to allow a faster convergence to the prospective optimum LAA CW, thus further reducing LAA waiting times that should lead to lower latency and higher throughput for LAA. C. FIXED WAITING TIME (FWT) METHOD In the 3GPP Cat 4 LBT algorithm, the channel is observed by the LAA eNB for a time equal to N multiplied by the CCA slot time period (e.g., 9 µs) where N is a uniform random number within the interval N ∈ [0, q − 1] and q − 1 is the upper bound of the LAA CW, which is updated based on HARQ feedbacks to 15, 31 and 63. It can be noted that the number of idle slots that need to be observed by the eNB is random and constrained by the upper bound of the LAA CW. The random choice for the number of idle slots that need to be observed by the LAA eNB before transmission may not VOLUME 8, 2020 be the most appropriate approach since such random choice is somehow arbitrary and independent of the actual Wi-Fi activity statistics. This suggests that a fixed waiting time, if properly configured based on the Wi-Fi activity statistics, may lead to a more efficient operation, which motivates the idea considered in this subsection. This knowledge of the existing Wi-Fi network activity statistics can be exploited to allow the LAA eNB to wait a fixed (rather than random) amount of slots before attempting a new transmission. Setting a fixed waiting time, if properly configured, should allow a faster convergence to the optimum operating point than dynamic approaches. Therefore, in order to enhance the 3GPP Cat 4 LBT algorithm, we propose a new method with a fixed waiting time before transmission for LAA based on the activity statistics of the existing Wi-Fi network. The dashed shaded boxes in Fig. 2 highlight the procedure of the 3GPP Cat 4 LBT algorithm that need to be modified to implement the proposed Fixed Waiting Time (FWT) method for LAA. Notice that three key changes to the 3GPP standard approach are considered in this method. Firstly, there is no backoff process, which to some extent reduces the complexity of the algorithm since random number generators are not required in this case. Secondly, there is no adaptation of the LAA CW based on the received HARQ reports (in fact, there is no CW in this case), which also contributes to simplify the algorithm and reduce its complexity. Thirdly, the knowledge of the activity statistics of the existing Wi-Fi network is required to set a fixed waiting time before transmission for the LAA eNB. The LAA eNB waiting time is set as N multiplied by the CCA slot time (9 µs) where N can be set based on the percentile point of the CDF for the ON time periods of the existing Wi-Fi network divided by the CCA slot time (9 µs) as shown in Table 5. D. VARIANTS OF THE PROPOSED WAITING TIME ADAPTATION METHODS The previous proposed dynamic and static CW methods, including the standard 3GPP Cat 4 LBT algorithm, consider various implementations to adapt/select the upper bound of the LAA CW. All these methods propose different approaches to tune the upper bound of the LAA CW but they have not proposed any strategy to adapt/select the lower bound of the LAA CW. Exploiting the knowledge of the activity statistics of the existing Wi-Fi network to select the lower bound of the LAA CW may provide a more efficient channel access mechanism for LAA. Various methods are proposed here to select the lower bound of the CW of LAA based on the activity statistics of the existing Wi-Fi network. The ideas suggested in this section to select the lower bound of the CW can also be applied to the FWT method, thus leading to new variants of this method as well. 1) VARIANTS BASED ON SHORTEST (MINIMUM) Wi-Fi ON TIME It can be noted that the lower bound of the LAA CW in the methods discussed so far (including the Cat 4 LBT and proposed CW-based methods) is set to zero regardless of the HARQ feedbacks or even of the activity statistics of the existing Wi-Fi network. This value means that the LAA eNB may start the backoff process by selecting a random number N ∈ [0, q − 1], which could be zero or any small value within the interval [0, q − 1]. This selection process may not be efficient for LAA transmission since this small value of N may not be long enough to find a clear channel given that it may lead to shorter waiting times than the actual occupancy times of the Wi-Fi transmissions, and as a result LAA would attempt to access a channel which is not free, thus leading to a backoff process repetition and therefore degrading the performance of the coexisting networks. Consequently, new variants of the methods discussed so far are here proposed by selecting the lower bound of the LAA CW based on the minimum ON activity time of the existing Wi-Fi network. Thus, the LAA eNB starts the backoff process by selecting a random number N ∈ [N Min , q − 1], where N indicates the number of idle slots that need to be observed before transmission, N Min is the lower bound of the CW and q − 1 represents the upper bound of the CW. The value of N Min is obtained as the minimum ON activity time of the Wi-Fi Network divided by the LAA slot duration (e.g., 9 µs) and rounding the result to the nearest integer toward infinity (i.e., ceil function), while the upper bound of the CW, q − 1, is selected as discussed for each proposed method in Sections III-A and III-B. The motivation of these variants is that selecting any random number by the LAA eNB below N Min may not be a wise decision since the channel will likely still be busy (i.e., in use by a Wi-Fi transmission) within that time period. Therefore, to minimise the number of unfruitful backoffs, the minimum waiting time of the considered methods is adapted according to the minimum Wi-Fi ON activity time. IV. METHOD TO ADAPT LTE TRANSMISSION TIMES Spectrum regulators impose constraints on the maximum transmission duration for any wireless communications system operating over unlicensed channels. As a result, a predefined transmission period (TxOP) for LAA eNB is mandatory for transmissions over unlicensed bands. This transmission period determines for how long an LTE-LAA transmission may last, after which the transmission must finish (even if there are more data to transmit) in order to allow other users to access the unlicensed channel. The standard 3GPP Cat 4 LBT algorithm implements a configurable but fixed TxOP parameter that depends on the channel access priority class (see Table 2 and [37, This static scheme for the transmission of LAA over unlicensed bands may be unsuitable since a fixed TxOP may not lead to an efficient spectrum utilisation. In particular, the unlicensed channel may suffer from different traffic conditions and a dynamic TxOP scheme would be more efficient for spectrum utilisation, thus leading to better performance for the coexisting networks. In specific, when the Wi-Fi traffic load is low, the channel can be expected to be idle for longer time periods and these periods can be exploited for LAA transmissions for longer intervals, thus providing better performance (i.e., using longer TxOP period). On the other hand, a shorter TxOP period for LAA would be more suitable when the Wi-Fi traffic load is high since the use of a long TxOP period in such cases would lead to more collisions in the channel and degrade the performance for the coexisting networks. As a result, the static TxOP period scheme may not be the most suitable scheme for an efficient coexistence between LTE-LAA and Wi-Fi networks over unlicensed spectrum bands. Thus, a novel scheme is proposed here to adapt the TxOP period for LAA dynamically in order to improve the performance of the coexisting networks. The dashed shaded diamond in Fig. 2 highlights the procedure of the 3GPP Cat 4 LBT algorithm that will be modified to implement the proposed dynamic TxOP method for LAA. The new proposed method selects the TxOP for LAA based on the current size of the CW for LAA, which is a parameter readily available in any practical implementation of LAA [46]. The 3GPP Cat 4 LBT algorithm adapts the size of the CW for LAA based on the HARQ feedbacks, which reflect incorrect data transmission due to a congestion or a collision in the channel. Therefore, the current CW size can be seen as an indication of the current level of congestion in the unlicensed channel and used to adapt the TxOP accordingly. The proposed method considers two adaptation points for the maximum TxOP period of LAA as shown in Table 6, where the TxOP can dynamically range from 4 ms to 20 ms. This range of values for the TxOP has been selected to illustrate the full potential benefits of the method proposed in this section, but can be adjusted, where required, to specific local spectrum regulations, or optimised for specific ranges of traffic loads. According to the adaptation points shown in Table 6, when the LAA CW size is 15 (i.e., the minimum LAA CW size), the TxOP parameter is set to its maximum value of 20 ms. Otherwise, the TxOP period parameter is set to 4 ms, which is the minimum TxOP period considered. The reason behind choosing the maximum TxOP period (20 ms) for the lower value of the CW size is that the lower CW size is associated with low volume of Wi-Fi traffic, therefore a longer LAA transmission should be reasonable in this case since this low volume of Wi-Fi traffic means more idle times TABLE 6. Value of the selected TxOP period as a function of the LAA CW size for the dynamic TxOP method. VOLUME 8, 2020 in the channel, which can be exploited for LAA transmissions by setting TxOP period to its maximum value, thus improving the LAA performance without degrading the Wi-Fi performance. On the other hand, the LAA CW size is increased to 31 or 63 in the 3GPP Cat 4 LBT algorithm due to the channel congestion or transmission collisions, therefore the TxOP period is set here to its minimum value (4 ms) in order to reduce this congestion/collision, thus providing better performance for LAA and not degrading the performance of the existing network (i.e., Wi-Fi). It is worth mentioning that the proposed dynamic approach for the TxOP period of LAA is based on the traffic statistics of the coexisting networks through the received HARQ feedbacks. Thus, the key change between the 3GPP standard and the proposed approach is the use of a dynamic TxOP period instead of a static one. This dynamic adaptation for the TxOP of LAA can achieve better alignment between Wi-Fi idle times and LTE-LAA transmission times, thus reducing the number of collisions and achieving a better performance for the coexisting networks compared with the 3GPP Cat 4 LBT algorithm, which considers a fixed TxOP period for LTE-LAA transmissions regardless of the congestion/collision over the unlicensed channel. In addition, the proposed approach can be easily implemented in a real system without adding any significant modifications to current commercial products since the approach is mainly based on the LAA CW parameter which is a readily available in any practical implementation of LAA. V. SIMULATION METHODOLOGY AND SETUP The indoor scenario defined in [4] is considered in this work to verify the effectiveness of the proposed methods in providing a fair coexistence between LTE-LAA and Wi-Fi networks over unlicensed bands in terms of throughput and latency. In particular, the proposed methods are evaluated based on the 3GPP definition of fairness, where the LAA network should not impact the Wi-Fi network performance more than an additional Wi-Fi network operating on the same carrier in terms of throughput and latency. In order to estimate the activity statistics of the existing Wi-Fi network, two Wi-Fi networks are deployed together over the same unlicensed band. The CDF of the ON times of the existing Wi-Fi network can be estimated for this scenario and exploited to adapt/select the CW boundaries, the transmission waiting times and the transmission opportunity time for LAA. In particular, various statistical values can be evaluated from this CDF such as the percentile point at the 50%, 95% and 100% of the CDF. In addition, the minimum and mode can be evaluated from the CDF as well. These various statistical values are used in the implementations of the different proposed methods. Afterwards, one of these deployed Wi-Fi networks is replaced with an LTE-LAA network allowing an LTE-LAA/Wi-Fi coexistence scenario and assessing the validity of the various proposed methods. The LTE-LAA/Wi-Fi coexistence scenario is compared with the Wi-Fi/Wi-Fi scenario in order to determine how the introduction of an LTE-LAA network operating with the proposed methods affects and existing Wi-Fi network with respect to the introduction of an additional Wi-Fi network, therefore providing an accurate assessment of the coexistence fairness as defined by the 3GPP. The methodology for evaluating the coexistence performance of LTE-LAA and Wi-Fi follows the 3GPP TR 35.889 simulation conditions except the updating rule of the LAA CW, where the proposed CW methods are implemented. In addition, a dynamic TxOP approach is implemented instead of the static TxOP approach of the 3GPP to assess the validity of the proposed dynamic TxOP method. In this study, all methods are evaluated using the event driven simulator ns-3 with LAA extension [47]. This simulator is an open source simulator and it allows researchers to share their contributions [4], [48]. In this simulator, WifiNetDevice can coexist with other NetDevices and an LTE module was implemented and developed by the LENA project to evaluate the performance of issues in LTE systems such as radio resource management algorithms, cognitive LTE systems and DL/UL MAC schedulers [49]. In this work, an indoor scenario in a single floor building is adopted as specified by 3GPP by considering two operators; operator A (Wi-Fi) and operator B (LAA) using the same 20 MHz channel over the unlicensed 5 GHz band [4]. The LAA/Wi-Fi indoor scenario is shown in Fig. 5. Operator A (Wi-Fi) deploys four APs while operator B (LAA) deploys four eNBs. All the base stations (i.e., APs and eNBs) are equally spaced and centred along the shorter dimension of the building. Moreover, each operator deploys 20 stations (STAs)/User Equipments (UEs) randomly distributed in a one floor building with a rectangular area. All base stations (i.e., APs and eNBs) and users (i.e., STAs and UEs) are equipped with two antennas for 2×2 Multiple Input Multiple Output (MIMO) operation. The traffic is modelled as a File Transfer Protocol (FTP) Model 1 operating over User Datagram Protocol (UDP) considering DL scenario. This model simulates file transfers according to a Poisson process with an arrival rate of λ packets/second. The file size considered is 0.5 MB with different recommended arrival rates (λ = 0.5, 1.5, 2.5 packets/second), which are simulated to generate different load levels [4]. Notice that the packet size of 0.5 MB is the size of the Protocol Data Unit (PDU) at the application layer. The ns-3 simulator implements the whole set of layers of the protocol stack and these packets are split into smaller pieces of data for transmission according to the PDU size at each level of the protocol stack. The details of the employed simulation parameters are shown in Table 7 along with the 3GPP reference scenario. The coexisting Radio Access Technologies (RATs) detect each other based on an Energy Detection (ED) principle. Wi-Fi nodes can detect other Wi-Fi nodes at -82 dBm and LAA nodes at -62 dBm. On the other hand, LAA nodes can detect Wi-Fi nodes at -72 dBm. This means that Wi-Fi will defer to weaker Wi-Fi signals sensed on the channel compared to LTE signals, which are detected at -62 dBm. It is worth noting that the selected decision thresholds can have a significant impact on the performance of both coexisting networks in some scenarios. As extensively investigated and shown in [50], a careful selection of the thresholds can lead to an improved performance. This aspect, however, is out of the scope of this work and thus the default values presented above, which are defined in [4], are here considered. The parameter that describes the maximum length of transmission for LAA (i.e., the TxOP) is configurable and is set to be 8 ms in the Cat 4 method, CW methods and FWT methods. On the other hand, the TxOP parameter is configured dynamically within the dynamic TxOP method. The CW size is adapted as defined in Section II-B for the 3GPP Cat 4 LBT method and in Section III for the proposed CW selection methods. The performance of the considered LTE-LAA/Wi-Fi coexistence methods is evaluated based on the main performance metrics described in 3GPP TR 36.889 (i.e., throughput and latency). Throughput is measured as the amount of data correctly transmitted within a specified time period as observed at the Internet Protocol (IP) layer, while latency is measured as the time elapsed since the packet leaves the transmitter until it reaches the receiver. VI. SIMULATION RESULTS In this section, the performance of LTE-LAA and Wi-Fi networks is analysed using the proposed methods. The results are shown in terms of the individual throughputs and latencies for each network as well as the total aggregated throughput for both networks. To validate the performance of the proposed methods, the fairness definition as specified by 3GPP is considered based on the throughput and latency for 95% of the users. The results at various percentiles (90%, 95% and 100%) were evaluated in the context of this work and it was observed that the main trends and conclusions are similar in all cases. However, only the results for the 95% percentile case are provided here for the sake of brevity. A. DYNAMIC CW (DynCW) METHODS The coexistence of LTE-LAA and Wi-Fi networks is analysed here when LTE-LAA implements the proposed dynamic CW methods. The throughputs for the existing Wi-Fi network (i.e., operator A) under different traffic loads (i.e., different arrival rates) for various methods are presented in Fig. 6. The reference case represents a homogeneous scenario where the existing Wi-Fi network (operator A) coexists with another Wi-Fi network (operator B), while the other cases correspond to heterogeneous coexistence scenarios (i.e., Wi-Fi and LAA) where operator A is a Wi-Fi network and operator B is an LTE-LAA network. Based on the 3GPP fairness definition, an ideal LAA coexistence mechanism should allow the Wi-Fi network achieve at least the same performance as in the reference case without (ideally) experiencing any performance degradation. It can be seen that operating LAA using the Cat 4 LBT algorithm leads to a lower throughput performance for the Wi-Fi network than in the reference case for all traffic loads, which contradicts the 3GPP fairness definition. Compared to the 3GPP Cat 4 LBT method, the proposed dynamic CW methods (in particular the DynCW-2 method) achieve a comparable Wi-Fi throughput performance under low traffic loads (λ = 0.5 packets/second) and a slightly FIGURE 6. Wi-Fi throughput performance of the proposed dynamic CW methods. VOLUME 8, 2020 better performance under higher traffic loads (λ = 1.5 and 2.5 packets/second). Even though it can be seen from Fig. 6 that the fairness definition in terms of Wi-Fi throughput is not fully met with the proposed dynamic CW methods (DynCW-3 and DynCW-2 methods) for the different traffic loads, the proposed dynamic CW methods degrade the throughput performance of the existing Wi-Fi network to a lesser extent and therefore can be considered to be more friendly to Wi-Fi networks than the 3GPP Cat 4 LBT method. It is worth noting that the DynCW-2 method in general outperforms DynCW-3; this suggests that the two-point adaptation process of the CW performed by DynCW-2 allows a faster convergence to an appropriate CW size when this is required by the Wi-Fi traffic conditions. Fig. 7 shows the Wi-Fi latency performance under different traffic loads for the various methods. It can be noted that all methods lead to a similar latency performance as the reference case, which means that the latency experienced by the existing Wi-Fi network is not significantly affected by the presence of other (Wi-Fi or LTE-LAA) networks. The LAA throughput performance is illustrated in Fig. 8. It can be noticed that the proposed methods achieve better LAA throughputs compared to the standard Cat 4 LBT method as the traffic load increases. The LAA throughputs are improved using the proposed methods due to the smart selection of the upper bound of the LAA CW based on the Wi-Fi activity statistics. This approach in the proposed dynamic CW methods allows the LAA eNB to access the channel faster than the Cat 4 LBT method, thus removing unnecessary waiting times for the LTE-LAA network and providing better LAA throughputs. As high traffic demands are expected in the future, high performance at high values of λ is therefore more desirable. Finally, the total aggregated throughputs for both coexisting networks (i.e., Wi-Fi and LAA) are shown in Fig. 9. It can be seen that the proposed dynamic CW methods achieve better total aggregated throughputs compared to Cat 4 LBT at higher traffic loads. Specifically, the performance improvement in the total aggregated throughputs using the DynCW-3 method compared to the Cat 4 LBT method is 1.5% (1.3 Mbps) and 1.2% (1 Mbps) for λ = 1.5 and 2.5 packets/second, respectively. Moreover, the performance improvement in the total aggregated throughputs using the DynCW-2 method compared to the Cat 4 LBT method is 6.8% (6.1 Mbps) and 2% (1.6 Mbps) for λ = 1.5 and 2.5 packets/second, respectively. Overall, it can be noticed that the proposed dynamic CW methods can achieve a slightly better performance (for both Wi-Fi and LTE-LAA networks) compared to the standard Cat 4 LBT method under high traffic loads and therefore constitute more convenient coexistence approaches in such scenario (in particular the DynCW-2 method). B. STATIC CW (StatCW) METHOD The performance of LTE-LAA and Wi-Fi networks is analysed here using the proposed static CW method. The throughputs for the coexisting networks (i.e., Wi-Fi and LAA) for the different percentile points at 50%, 95% and 100% of the CDF of the ON times of the existing Wi-Fi network using the proposed static CW method are provided in Table 8. It can be seen that for the different traffic loads (i.e., different arrival rates) the 100% percentile point (maximum value) achieves the best performance in terms of Wi-Fi throughput with a rather constant performance in terms of LAA throughput. Table 9 provides the Wi-Fi latencies at the different percentile points of the CDF using the proposed static CW method. It can be seen that all percentile points (i.e., 50%, 95% and 100%) provide comparable performances in terms of Wi-Fi latency. Thus, the 100% percentile point of the ON Wi-Fi times will be considered to select the upper bound of the LAA CW in the proposed static CW method since this choice leads to the best Wi-Fi performance (LTE-LAA performance is unaffected by the selected percentile point). The Wi-Fi throughput performance of the proposed static CW method is presented and compared to the reference and 3GPP Cat 4 LBT cases in Fig. 10. It can be observed that the proposed static CW method achieves better throughput for the existing Wi-Fi network (i.e., operator A) for all traffic loads compared to the standard Cat 4 LBT method. In addition, it provides better throughput for the existing Wi-Fi network compared to the reference case at lower traffic loads (λ = 0.5 packets/second) and comparable throughput performance at medium and higher traffic loads (λ = 1.5 and 2.5 packets/second). Even though the fairness requirement in terms of throughput is not fully met for all traffic loads (concretely, for λ = 1.5 packets/second), the proposed static CW method provides a very close approximation, with a noticeably better performance than the standard Cat 4 LBT method. Comparing the results shown in Figs. 6 and 10, it can be noted that the static CW method provides in general a better Wi-Fi throughput performance than the dynamic CW method as well. The latencies of the existing Wi-Fi network are presented in Fig. 11 for the reference, Cat 4 LBT and static CW methods under different traffic loads. Comparable latencies for all traffic loads can be seen compared to the reference case. As a result, both Cat 4 LBT and static CW methods do not degrade the existing Wi-Fi performance in terms of latency. The throughputs for LAA (i.e., operator B) using the Cat 4 LBT and static CW methods under different traffic loads are presented in Fig. 12. Comparing this figure with the results shown in Fig. 8 for the dynamic CW methods, it can be noted that the LAA throughput performance is quite comparable in both cases, with the LAA throughput for the static CW method being slightly lower than that attained by the best dynamic CW method. This slight reduction of the LAA throughput with the static CW method compared to the dynamic CW method is the price to be paid in order to achieve a better Wi-Fi throughput performance that, as shown in Fig. 10 for the static CW method, meets more closely the 3GPP definition of fairness. The existence of a tradeoff VOLUME 8, 2020 between the Wi-Fi and LAA throughput performances seems reasonable. However, it is interesting to note that the slightly degraded LAA throughput of the static CW method with respect to the dynamic CW method leads to a comparatively larger improvement of the Wi-Fi throughput. This can be clearly seen by comparing the total aggregated throughput of the static CW method (shown in Fig. 13) with the total aggregated throughput of the dynamic CW methods (shown in Fig. 9). The static CW method outperforms the 3GPP Cat 4 LBT method in terms of aggregated throughput for all traffic loads (including λ = 0.5 packets/second, where the performance of the best dynamic CW method was still lower than that of the 3GPP Cat 4 LBT). Moreover, the performance improvements of the static CW method with respect to the Cat 4 LBT method are greater than those achieved by the best dynamic CW method, concretely 6.8% (7.1 Mbps), 7.8% (7 Mbps) and 1.6% (1.3 Mbps) for λ = 0.5, 1.5 and 2.5 packets/second, respectively. Based on the obtained results, it can be concluded that the static CW method not only is more convenient than the 3GPP Cat 4 LBT method but also than the best dynamic CW method in terms of fairness. In general, the static CW method allows the Wi-Fi network experience a higher throughput performance that is closer to the scenario of fair coexistence as defined by the 3GPP. On the other hand, the LTE-LAA performance remains quite stable and, as a result, the overall aggregated performance of both networks is significantly higher with the static CW method. C. FIXED WAITING TIME (FWT) METHOD The performance of LTE-LAA and Wi-Fi networks is investigated here using the proposed FWT method for LAA. Fig. 14 depicts the throughputs for the existing Wi-Fi network under different traffic loads for the reference, Cat 4 LBT and fixed waiting times methods. It can be seen that the proposed FWT method provides better throughput for the existing Wi-Fi network for all traffic loads compared not only to the standard Cat 4 LBT method but also to the reference case. This means that the existing Wi-Fi network (operator A) experiences a better throughput performance when the coexisting network (operator B) is an LTE-LAA network using the proposed FWT method than when it is another Wi-Fi network. This means that the proposed FWT method not only meets the 3GPP fairness requirement in terms of throughput but in fact leads to an improved throughput performance for the existing Wi-Fi network when an LTE-LAA network is introduced (compared to the introduction of another Wi-Fi network). This may be explained by the ability of the proposed FWT method to select a suitable amount of waiting time before attempting a transmission such that there is a high chance to find a free channel without waiting unnecessarily long times, which can in turn be ascribed to the selection of such waiting time based on the Wi-Fi activity statistics. comparable latencies for all traffic loads. As a result, both Cat 4 LBT and FWT methods do not degrade the performance of the existing Wi-Fi network in terms of latency. Fig. 16 depicts the throughputs for LAA (i.e., operator B) using the Cat 4 LBT and FWT methods under different traffic loads. It can be seen that the significant throughput performance improvements for the existing Wi-Fi network provided by the FWT method (as observed in Fig. 14) are not obtained at the expense of the LTE-LAA throughput performance, which is comparable under medium and higher traffic loads (λ = 1.5 and 2.5 packets/second) and even better at lower traffic loads (λ = 0.5 packets/second). As a result, the total aggregated throughput of both networks is significantly enhanced as it can be appreciated in Fig. 17, which shows very significant throughput performance improvements with respect to the 3GPP Cat 4 LBT algorithm of 39.2% (41.1 Mbps), 13% (11.6 Mbps) and 6.5% (5.2 Mbps) for λ = 0.5, 1.5 and 2.5 packets/second, respectively. These improvements are larger than those observed for the dynamic and static CW methods analysed in the previous sections and can be explained by the ability of FWT to select an adequate waiting time, which ultimately results in a reduced number of collisions between both coexisting networks and consequently in an improved performance for both networks, thus making the proposed FWT method a more suitable candidate for LTE-LAA. D. VARIANTS OF THE PROPOSED WAITING TIME ADAPTATION METHODS The performance of LTE-LAA and Wi-Fi networks is here analysed when the minimum and mode variants of the proposed waiting time adaptation methods are considered. Fig. 18 shows the throughput of the existing Wi-Fi network under different traffic loads for all the waiting time adaption methods considered in this work, including the 3GPP Cat 4 LBT and proposed methods, both in their standard versions and with the minimum and mode variants. It can be noticed that the minimum variants either provide a similar Wi-Fi throughput performance as the original versions of the respective method or, in some cases, lead to a slight throughput performance degradation, but in no case the minimum variants lead to a throughput performance improvement in the Wi-Fi network. These results indicate that the minimum variants are not suitable for a fair coexistence of the LTE-LAA network with the existing Wi-Fi network. The mode variants lead in most cases to higher Wi-Fi throughputs than their minimum counterparts, however they do not necessarily perform better than the standard versions of their respective methods (if fact, the mode variant leads to a higher throughput for all traffic loads only for the 3GPP Cat 4 LBT and DynCW-2 methods). As it can be observed in Fig. 18, the best Wi-Fi throughput performance for all traffic loads is attained with the FWT method in its standard version. Fig. 19 presents the latencies of the considered methods and their variants under different traffic loads. As it can be appreciated, and in line with the latency performance results presented in previous sections, comparable latencies are obtained with all methods and for all traffic loads. Fig. 18, noting the existence of a performance trade-off between the Wi-Fi and LTE-LAA networks such that an increase in the Wi-Fi throughput can usually be associated with a corresponding decrease in the LTE-LAA throughput and vice versa. The aggregated throughput performances of both Wi-Fi and LTE-LAA networks for the various methods considered in this work and their variants are shown in Fig. 21. As in previous figures, there are some specific cases where one of the variants provides a better performance than the original version of the corresponding method, however there is no clear trend that can guarantee an improved performance in all cases when a variant is employed. When jointly taking into account all the methods considered in this work to select/adapt the waiting times of LTE-LAA and their variants, it becomes apparent that the FWT method (in its standard version) is the most suitable candidate. On the one hand, the FWT method is the only candidate that in all cases (i.e., all traffic loads) ensures that the Wi-Fi throughput performance will not be degraded (with respect to the reference case) by the introduction of an LTE-LAA network, and therefore leads to a fair coexistence. As a matter of fact, and interestingly, the introduction of an LTE-LAA network using the proposed FWT method results indeed in a higher throughput performance for the existing Wi-Fi network than the introduction of another Wi-Fi network, as observed in Fig. 18. On the other hand, the FWT method yields the highest aggregated throughput between both networks. Therefore, the proposed FWT method is the only method out of all the methods considered in this work (including the 3GPP Cat 4 LBT method) that guarantees a fair coexistence with the existing Wi-Fi network (and in fact improves its performance) while at the same time providing the highest aggregated throughput between both networks. E. LTE TRANSMISSION TIMES ADAPTATION METHOD The performance of LTE-LAA and Wi-Fi networks is investigated here using the proposed dynamic TxOP period method. Fig. 22 depicts the throughputs for the existing Wi-Fi network under different traffic loads for the homogeneous coexistence (i.e., Wi-Fi and Wi-Fi) and the heterogeneous coexistence (i.e., Wi-Fi and LTE-LAA) scenarios. In particular, the 3GPP Cat 4 LBT method with various static TxOP periods is considered for Wi-Fi/LAA coexistence. Moreover, the proposed dynamic TxOP approach is investigated for the Wi-Fi/LAA coexistence scenario as well. It can be seen that the standard Cat 4 LBT method achieves lower Wi-Fi throughputs compared to the reference case for the different static TxOP periods for all traffic loads. The proposed dynamic TxOP method, when compared to the static TxOP method, provides a comparable Wi-Fi throughput performance for λ = 1.5 and 2.5 packets/second, and a slightly better throughput for λ = 0.5 packets/second, which in all cases is lower than the throughput experienced by the Wi-Fi network in the reference case. These results indicate that the proposed dynamic TxOP method does not provide in general an improved fairness for the existing Wi-Fi network. The corresponding latencies for the existing Wi-Fi network are depicted in Fig. 23. It can be noticed that all methods achieve very similar performance in terms of Wi-Fi latency. As a result, the Cat 4 LBT method using a static TxOP approach and the proposed dynamic TxOP method do not degrade the existing Wi-Fi latency. Fig. 24 presents the LAA throughput performance for the Wi-Fi/LAA coexistence scenario using the static approach of Cat 4 LBT and using the proposed dynamic TxOP approach. For low traffic loads (λ = 0.5 packets/second), it can be noticed that the dynamic TxOP method provides the same throughput as the static TxOP method with a 20 ms TxOP. This can be explained by the fact that, under low traffic loads, the channel is sparsely used, long idle times are frequent and collisions are unlikely to occur. As a result, the vast majority of transmissions are performed with the lowest value of the CW (i.e., 15) and therefore most of the time the dynamic TxOP method selects the highest TxOP available (i.e., 20 ms as illustrated in Table 6). In this scenario of low traffic load (λ = 0.5 packets/second), the dynamic TxOP method is equivalent to the static TxOP method with a 20 ms TxOP and, as a result, both methods achieve the same throughput. Notice that the achieved throughput is the highest attained for λ = 0.5 packets/second, which indicates that a constant selection of a 20 ms TxOP in such a case is the optimum choice when it comes to the LTE throughput. For medium traffic loads (λ = 1.5 packets/second), Fig. 24 shows that the static TxOP method is unable to achieve the same throughput as the proposed dynamic TxOP method. This is because under this higher traffic load, the channel usage increases and so does the number of collisions. As a result, a constant 20 ms TxOP is not the optimum choice anymore since this long transmission time will lead to more frequent collisions and therefore a lower throughput (this is also suggested by the fact that, under a static TxOP, the same throughput is obtained for 12 ms and 20 ms TxOP, showing that there is no benefit from performing longer transmissions). In this scenario, and as a result of the presence of some collisions in the channel, the CW will be increased sometimes from the lowest value (i.e., 15) to the next value (i.e., 31) and, when this occurs, the proposed dynamic TxOP method will accordingly reduce the TxOP from 20 ms to 4 ms in order to reduce the likelihood of more frequent channel collisions. As appreciated in Fig. 24, this dynamic adaption of the TxOP performs well and leads to a higher throughput for the LTE-LAA network than any of the static configurations. This is also confirmed for higher traffic loads (λ = 2.5 packets/second), where it can be clearly appreciated that the proposed dynamic TxOP yields a significantly improved throughput performance as a result of this smart adaption of the TxOP length to the instantaneous occupancy activity in the Wi-Fi channel. The total aggregated throughputs for the coexisting networks (i.e., Wi-Fi and LTE-LAA) for the various methods under different traffic loads are depicted in Fig. 25. The results presented in Fig. 25 show not only that the proposed dynamic TxOP method provides the highest aggregated throughput for all traffic loads compared to the standard Cat 4 LBT method based on a static TxOP configuration, but also obtains more significant performance improvements with respect to the static TxOP method as the traffic load increases. Specifically, the performance improvement in the total aggregated throughputs for both networks using the dynamic TxOP method compared to the static TxOP method for λ = 2.5 packets/second is 60.1% (42. F. DISCUSSION The methods proposed in this work fall into two categories, namely those focused on the LTE-LAA waiting times and those focused on the LTE-LAA transmission times. For the first category, the results presented in Section VI-D concluded that the FWT method is the most convenient approach within its category since it is the only method out of all the methods considered in this work (including the 3GPP Cat 4 LBT method) that guarantees a fair coexistence with the existing Wi-Fi network (i.e., it does not produce a degradation more significant than that caused by another Wi-Fi network) while at the same time provides the highest aggregated throughput between both networks when compared to the rest of methods in the same category. For the second category, a method has been proposed based on the dynamic adaption of the TxOP length, which is unable to improve the fairness offered by the 3GPP Cat 4 LBT method but yields a higher aggregated throughput between both networks, in particular as a result of a significantly enhanced throughput for the LTE-LAA network. Following the performance evaluation carried out individually for the methods in each category, a natural question is which of these methods would be more convenient in a practical coexistence scenario. This question can be answered based on the Wi-Fi throughput results shown in Fig. 26 and the total aggregated throughputs shown in Fig. 27, which compare together the main results shown in previous sections. As it can be appreciated, the FWT method is the only method that can fully meet the fairness requirement as defined by the 3GPP. Thus, this method may be more appealing to scenarios where the Wi-Fi and LTE-LAA networks are owned by different operators, where the LTE-LAA operator is strictly required to avoid causing unacceptable performance degradation to the Wi-Fi operator. On the other hand, the dynamic TxOP method provides the highest aggregated throughput at the expense of a slightly degraded Wi-Fi throughput and thus it may be more suitable to scenarios where both networks are owned by the same operator, for example where a mobile cellular operator offers Wi-Fi hotspots to its clients and therefore the ultimate interest is in maximising the overall aggregated throughput (i.e., the total capacity) of the owned network infrastructure. Finally, it is worth noting that, while in this work the traffic load has been varied by modifying the packet inter-arrival rates at the application layer (λ = 0.5, 1.5 and 2.5 packets/second), this can also vary based on the network scale (i.e., number of Wi-Fi APs, LTE-LAA eNBs, users). While the numerical results may change for different network conditions, the main conclusions of this study remain the same. In order to illustrate this, Figs. 28 and 29 show the counterparts of Figs. 26 and 27 when the total number of users in the system is doubled. As it can be appreciated, the main conclusions discussed above for the methods proposed in this work are also valid under larger network scales. VII. CONCLUSION Current studies aim to enable a fair coexistence between LTE-LAA and Wi-Fi networks over unlicensed spectrum bands. The current 3GPP Cat 4 LBT algorithm does not perfectly meet the fairness definition given by 3GPP. In particular, a Wi-Fi throughput degradation can be noticed due to deploying LTE-LAA with Wi-Fi over the same unlicensed band. Different design parameters of the LBT algorithm play a key role in this heterogeneous coexistence such as the waiting and transmission times for LAA. Therefore, novel methods have been proposed to tune the waiting and transmission times for LAA as an alternative to the traditional contention window-based approach and the fixed configuration of the TxOP period for LAA proposed by the 3GPP. The obtained simulation results have shown that, for LAA/Wi-Fi coexistence, selecting fixed waiting times for LAA based on the knowledge of the activity statistics of the existing Wi-Fi network achieves better performance compared to the contention window-based approach of the standard 3GPP Cat 4 LBT algorithm, and moreover it also results in less complex coexistence mechanisms. In addition, the dynamic TxOP period method achieves better performance compared to the fixed TxOP period approach of the standard Cat 4 LBT algorithm. The most convenient method to use depends on the particular business scenario and target of the network operator as discussed in this work but, in any case, the proposed methods can provide significant performance improvements compared to the standard 3GPP Cat 4 LBT method.	2020-09-10T10:21:34.400Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "3ce97ace27060f095b0d76aa0c0defa42d38b0d2", "oa_license": "CCBY", "oa_url": "https://ieeexplore.ieee.org/ielx7/6287639/8948470/09186015.pdf", "oa_status": "GOLD", "pdf_src": "IEEE", "pdf_hash": "cfec2bc477d0d274a4e4066726ac2b2235847e46", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
12770971	pes2o/s2orc	v3-fos-license	HIV-1 Tat protein perturbs diacylglycerol production at the plasma membrane of neurosecretory cells during exocytosis Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator (Tat) viral protein that is required for efficient HIV gene transcription. We recently reported that extracellular Tat is able to enter uninfected neurosecretory cells. Internalized Tat escapes endosomes to reach the cytosol and is then recruited to the plasma membrane by phosphatidylinositol 4,5-bisphophate (PtdIns(4,5)P2). Tat strongly impairs exocytosis from chromaffin and PC12 cells and perturbs synaptic vesicle exo-endocytosis cycle through its ability to interact with PtdIns(4,5)P2. Among PtdIns(4,5)P2-dependent processes required for neurosecretion, we found that Tat impairs annexin A2 recruitment involved in the organization of exocytotic sites at the plasma membrane. Moreover Tat perturbs the actin cytoskeleton reorganization necessary for the movement of secretory vesicles toward their plasma membrane fusion sites during the exocytotic process. Here, we investigated whether extracellular Tat affects PtdIns(4,5)P2 metabolism in PC12 cells. Using a diacylglycerol (DAG) sensor, we found that ATP stimulation of exocytosis triggers the production of DAG at the plasma membrane as seen by the relocation of the DAG probe from the cytosol to the plasma membrane. Exposure to Tat strongly delayed the recruitment of the DAG sensor, suggesting a reduced level of DAG production at the early phase of ATP stimulation. These observations indicate that Tat reduces the hydrolysis rate of PtdIns(4,5)P2 by phospholipase C during exocytosis. Thus, the neuronal disorders often associated with HIV-1 infection may be linked to the capacity of Tat to interact with PtdIns(4,5)P2, and alter both its metabolism and functions in neurosecretion. The human immunodeficiency virus type I (HIV-1) encodes three structural (gag, pol and env), four accessory (vif, vpr, vpu and nef ) and two regulatory (tat and rev) genes, the products of which are responsible for establishing sophisticated interactions between the virus and human host. HIV-1 transcriptional activator (Tat) is a small (~11 kDa) basic protein that binds to the transactivation-response element (TAR) on the nascent viral RNA and recruits molecular components for efficient transcriptional elongation. 1 Thus, Tat displays an array of functions that are essential for viral gene expression and replication, but also affects host gene expression. 2 Tat is an early regulatory protein that has a variable length of 86-103 aa (depending on viral isolates) and is encoded by two exons. The first exon encodes the first 72 amino acids and the second exon encodes for the remaining residues. Tat is actively released by infected T-cells into the extracellular medium by an unconventional mechanism 3,4 and the concentration of Tat in the serum of HIV-1 infected patients reaches nanomolar levels. 5 Tat can cross the blood brain barrier 6,7 and be produced in situ by HIV-1 infected cells such as perivascular macrophages, astrocytes and microglia. Tat is thus present within Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator (Tat) viral protein that is required for efficient HIV gene transcription. We recently reported that extracellular Tat is able to enter uninfected neurosecretory cells. Internalized Tat escapes endosomes to reach the cytosol and is then recruited to the plasma membrane by phosphatidylinositol 4,5-bisphophate (PtdIns(4,5)P 2 ). Tat strongly impairs exocytosis from chromaffin and PC12 cells and perturbs synaptic vesicle exo-endocytosis cycle through its ability to interact with PtdIns(4,5)P 2 . Among PtdIns(4,5)P 2 -dependent processes required for neurosecretion, we found that Tat impairs annexin A2 recruitment involved in the organization of exocytotic sites at the plasma membrane. Moreover Tat perturbs the actin cytoskeleton reorganization necessary for the movement of secretory vesicles toward their plasma membrane fusion sites during the exocytotic process. Here, we investigated whether extracellular Tat affects PtdIns(4,5)P 2 metabolism in PC12 cells. Using a diacylglycerol (DAG) sensor, we found that ATP stimulation of exocytosis triggers the production of DAG at the plasma membrane as seen by the relocation of the DAG probe from the cytosol to the plasma membrane. Exposure to Tat strongly delayed the recruitment of the DAG sensor, suggesting a reduced level of DAG production at the early phase of ATP stimulation. These observations indicate that Tat reduces the hydrolysis rate of PtdIns(4,5)P 2 by phospholipase C during exocytosis. Thus, the neuronal disorders often associated with HIV-1 infection may be linked to the capacity of Tat to interact with PtdIns(4,5)P 2, and alter both its metabolism and functions in neurosecretion. Keywords: HIV-1 Tat, neurosecretion, PI(4,5)P 2 the central nervous system and could potentially be involved in HIV-1 neuropathogenesis. HIV-1-associated neuronal symptoms include motor or cognitive dysfunctions and behavioral changes affecting nearly one third of patients undergoing intensive tritherapy. Moreover, one patient out of ten develops HIV-1 associated dementia at the late stage of AIDS. 8 Although some signs of neuronal death have been reported in these patients, they most likely do not explain the extent of cognitive alterations, suggesting alternative unknown mechanism altering neuronal functions. Extracellular Tat has been shown to penetrate several cell types through different pathways 9 and the capacity of Tat to reach the cytosol of uninfected cells likely enables circulating Tat to interfere with key cellular processes in these cells. Moreover, the recent breakthrough identifying the membrane inner leaflet phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] as a high affinity ligand for Tat, 10 suggests that key cellular activities relying on PtdIns(4,5)P 2 such as exocytosis might be affected in neurosecreting cells. HIV-1 Tat protein perturbs diacylglycerol production at the plasma membrane of neurosecretory cells during exocytosis In our efforts to unravel the potential effect of Tat on neurosecretion, we first tested the ability of extracellular Tat to enter A2 plays an essential role in calcium-regulated exocytosis by promoting the formation of exocytotic platforms in neuroendocrine cells. 15,16 Inhibition of annexin A2 translocation in cells exposed to Tat led to a reduction in the number of functional exocytotic sites and to a concomitant decrease in the number of exocytotic events. 14 Furthermore, we found that Tat hampers the actin cytoskeleton reorganization during neurosecretion, an effect that may also be a direct consequence of Tat binding to PtdIns(4,5)P 2 . Indeed, available PtdIns(4,5)P 2 can be a limiting factor for exocytosis efficacy since it orchestrates the actin-mediated movements of secretory granules to exocytotic sites. 17 In chromaffin cells, the cortical F-actin network constitutes a negative clamp preventing the movement of secretory vesicles to release sites, and it must be locally disassembled and reorganized in a PtdIns(4,5)P 2 -dependent manner to allow recruitment of secretory vesicles in preparation for exocytosis. 18 Thus, the inhibitory effect of Tat on subplasmalemmal actin may also explain its capacity to impair the exocytotic response. In our attempt to decipher the mechanism(s) by which Tat alters PtdIns(4,5)P 2 -dependent membrane trafficking processes in neurosecretory cells, we explored the ability of Tat to directly affect PtdIns(4,5)P 2 metabolism. Indeed, the quantity of available PtdIns(4,5)P 2 can influence the efficacy of exocytotic process. PtdIns(4,5)P 2 synthesis and hydrolysis is regulated by a complex set of inositol kinases, lipases and phosphatases, including PtdIns(4,5)P 2 -specific phospholipase C (PLC). PtdIns(4,5)P 2 hydrolysis by PLC generates two second messengers, namely diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, which both play a major role in neuronal plasticity and exocytosis. 19 To examine whether Tat interferes with the production of second messengers through hydrolysis of PtdIns(4,5)P 2 by PLC during exocytosis, we monitored DAG production in PC12 cells in situ upon neurosecretion stimulation. To this end, cells were transfected with a high affinity DAG sensor using the tandem C1 domains (C1ab) of protein kinase D tagged with GFP at the C terminus (pKDC1ab-GFP). 20 This probe enables to track the in situ production of DAG from PtdIns(4,5)P 2 upon ATP stimulation, used here as a secretagogue to trigger exocytosis. In resting PC12 cells, we found that pKDC1ab-GFP mostly localized in the cytoplasm and in perinuclear Golgi-like structures, but also weakly in the nucleus of some cells. Indeed, pKDC1ab-GFP possesses a nuclear export signal at the N terminus of the probe that may influence its nuclear accumulation. 20 Cell stimulation induced a dramatic and transient recruitment of pKDC1ab-GFP to the cell periphery (Fig. 1, upper panel), revealing the rapid and robust production of DAG at the plasma membrane of stimulated PC12 cells. Image quantification indicated that recruitment of the DAG probe to the plasma membrane peaked between 20 sec and 40 sec after stimulation and thereafter decreased to reach basal level 3 min after stimulation (Fig. 2). In cells exposed to exogenous Tat, the translocation of the DAG sensor to the plasma membrane in response to secretagogue stimulation was significantly delayed reaching a plateau only 200 sec after stimulation (Fig. 1, middle panel and Fig. 2). Interestingly, the highest fluorescence intensity reached at the plasma membrane in Tat-treated cells was similar to that conventional neurosecretory cell models i.e., chromaffin and PC12 cells. 11 Consistent with the Tat uptake pathway observed in other cell types, 12,13 our results indicate that internalized Tat is first found in early endosomal structures, as revealed by co-localization with EEA1 and then in late endo-lysosomal Lamp-1 positive structures. 14 After 4 h, Tat-containing vesicular structures were barely detectable and, concomitantly, Tat signal became visible on the inner leaflet of the plasma membrane. 14 In addition, when overexpressed in PC12 cells, Tat localized essentially at the plasma membrane. 14 The binding of Tat to PtdIns(4,5)P 2 is largely responsible for this recruitment of Tat to the plasma membrane because both neomycin, a PtdIns(4,5)P 2 masking drug, and PtdIns(4,5)P 2 5-phosphatases, which reduce cellular PtdIns(4,5)P 2 levels when overexpressed, efficiently displaced Tat from the plasma membrane to the cytosol. 14 Most strikingly, once internalized by chromaffin and PC12 cells, Tat strongly inhibited the number of exocytotic events as revealed by a reduction in amperometric spike frequency and growth hormone release, respectively. 14 Inhibition of secretion was observed at nanomolar concentrations of Tat, comparable with the concentrations reported for circulating Tat, 5 thereby arguing for the physiological significance of our findings. Expression of Tat mutants unable to bind PtdIns(4,5)P 2 did not inhibit secretion, demonstrating that the effect of Tat on secretion is directly linked to its interaction with PtdIns(4,5)P 2 at the plasma membrane and does not result from a transcriptional effect of Tat. 14 Overexpression of phosphatidylinositol 4 phosphate 5-kinase was able to partially rescue secretion from cells exposed to Tat, in line with the idea that Tat blocks secretion through its capacity to specifically bind PtdIns(4,5)P 2 . Interaction of Tat with PtdIns(4,5)P 2 occurs with such a high affinity that Tat sequesters this phosphoinositide and thereby strongly impairs the translocation of the PtdIns(4,5)P 2 -binding protein annexin A2 to the plasma membrane following cell stimulation. Annexin DAG biosensor (pKDC1ab-GFP) recruitment to the plasma membrane of PC12 cells was recorded using a SP5II Leica confocal microscope in xyt mode using a 63X oil-immersion objective and a heated stage (37°C). Recordings were started 45 sec before cell stimulation, and a total of 601 frames were taken at every 500 ms during 5 min. Data and image analysis. The recruitment of GFP tagged DAG biosensor was analyzed using the SP5II Leica quantification software (LAS AF Lite). Three regions of interest (ROI) were determined to delimitate cell periphery including plasma membrane, cytoplasm and nucleus of each cell. Average fluorescence intensities of different ROIs were analyzed through the 601 frames of recorded videos for each condition. Time dependent variation of fluorescence intensities at plasma membrane level and cytoplasm is expressed as mean ± SEM. Values are normalized to starting point fluorescence intensities that are fixed as 100%. Data were generated from 8 control, 8 Tat-treated and 5 neomycin-treated cells respectively issued from two independent cell cultures. Statistical significance was estimated using Student's t-test and data were considered significantly different when p < 0.01. Gaussian distribution of the data was verified. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. in control cells. Thus, the total level of DAG production was similar in Tat-treated and control cells, but with strikingly different kinetics. DAG generation was rapid and transient in control cells whereas it increased slowly and progressively in cells exposed to Tat (Fig. 2). In Tat-treated cells the DAG probe remained recruited at the plasma membrane for up to 8 min and subsequently slowly translocated back to the cytoplasm (not shown). To test the idea that Tat may affect PLC activity by reducing substrate [PtdIns(4,5)P 2 ] availability, we tested the effect of neomycin, a non-specific PLC inhibitor known to prevent DAG production by sequestering PtdIns(4,5)P 2 . As illustrated in Figure 1 (lower panel), neomycin treatment (4 mM for 2 h) completely blocked the recruitment of the DAG probe to the plasma membrane in response to exocytosis stimulation. This result suggests that neomycin and Tat affect DAG production through distinct mechanisms in ATP stimulated PC12 cells that undergo exocytosis. Hence, Tat may be progressively displaced from PtdIns(4,5)P 2 allowing its slow hydrolysis by PLC or Tat might be able to directly affect the enzymatic activity of PLC. The latest possibility is however unlikely because we recently reported that Tat has no impact on the fast and transient calcium increase in PC12 cells undergoing exocytosis, 14 which reflects normal PLC activity in these cells under these conditions. An alternative possibility is that in the presence of Tat, the cell differently handles DAG. It is noteworthy to mention that DAG can be transformed into phosphatidic acid (PA) by DAG-kinase. On the other hand PA itself, which is produced at exocytotic sites in neurosecreting cells, 21 can also be converted into DAG by PA phosphohydrolase. This PA phosphohydrolase activity, as an alternative pathway in these cells, might thus be responsible for some of the lately produced DAG explaining the delay in the pKDC1ab-GFP probe recruitment to the plasma membrane. It is likely that precise lipidomic analyzes will be required to fully understand the profound effect of Tat on lipid turnover in neurosecretory cells. In conclusion, we found that Tat dramatically inhibited neurosecretion by a mechanism that most likely reflects a general alteration of the availability and/or the metabolism of PtdIns(4,5)P 2 . These effects of Tat might contribute to the neuronal disorders associated with HIV-1 infection. Materials and Methods Cell culture, transfection and imaging. PC12 cells were cultured as described previously 22 and transfected with pKDC1ab-GFP expression vector (0.8 to 1 μg total DNA, 15 × 10 4 cells, 24-well plate) using Lipofectamine 2000 as previously reported. 23 Under these conditions, transfection efficiency was around 50%. Twenty-four h after transfection, PC12 cells were treated 2 h with 4 mM neomycin or incubated for 30 min on ice with 50 nM Tat or citrate buffer (vehicle, 100 mM NaCl, 50 mM Na-citrate, pH 7) and chased for 4 h at 37°C as described. 14 Cells were then washed three times with Locke's buffer (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgSO 4 , 11 mM glucose and 15 mM HEPES, pH 7.4) and stimulated with 300 μM ATP in Locke's buffer 24 to trigger exocytosis. GFP-tagged grants of Agence National de Rercherche, ANR-09-BLAN-0264 to N.V. and from The Agence Nationale de la Recherche sur le SIDA, (ANRS) and Sidaction to B.B.	2017-10-01T01:10:06.881Z	2013-05-31T00:00:00.000	{ "year": 2013, "sha1": "180901052127f1d758ab6546c9ba5e68103b3846", "oa_license": "CCBYNC", "oa_url": "https://www.tandfonline.com/doi/pdf/10.4161/cib.25145?needAccess=true", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "180901052127f1d758ab6546c9ba5e68103b3846", "s2fieldsofstudy": [ "Biology", "Chemistry", "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
104468798	pes2o/s2orc	v3-fos-license	R-loops: formation, function, and relevance to cell stress Exposure of genomic, single-stranded DNA (ssDNA) during transcription and replication creates opportunities for the formation of inappropriate secondary structures. Cells manage this exposure by using topoisomerases and helicases to reduce the inherent topological stress that arises from unwinding the double helix and by coating ssDNA with protective protein complexes. Interestingly, specific DNA-RNA hybrids, known as R-loops, form during transcription and exist in homeostasis throughout the genomes of prokaryotes and eukaryotes. These hybrids nucleate from guanine rich clusters in the template strand and extend across GC rich spans of transcribed genes. In vivo regulatory functions have evolved from R-loops, including regulation of gene expression and telomere lengthening. However, they also exist as a form of stress, particularly when replication forks collide with the transcription machinery. New methodologies and models are being developed to delineate the biology of R-loops, including those related to cell stress-based diseases like cancer. As accumulation of R-loops is associated with disease, targeting molecular pathways that regulate their formation or removal could provide new avenues for therapeutic intervention. This review covers recent understandings of the molecular basis for R-loop formation, removal, and biological outcomes in the context of cellular stress. INTRODUCTION A variety of topological, structural and hybridization events occur during DNA replication and gene transcription. Unwinding of the DNA double helix provides access for polymerase to a template strand, and creates torsional stress that can manifest anomalous formation of "non-traditional" moieties. One such structure, known as an R-loop [1,2], occurs during transcription (Figure 1). As RNA polymerase progresses along the DNA double helix, newly transcribed RNA threads back to hybridize with the transiently accessible template strand and displace the non-template strand. Structurally, the hybrid adopts an intermediate conformation between B-form double-stranded (ds)DNA and Aform dsRNA. This form carries more stability than dsDNA and must be enzymatically resolved in order to restore the native double helix. A homeostasis emerges from the constant formation and removal of R-loops throughout the genome. Moreover, their presence has biological relevance in regulating gene expression and specialized rearrangement events. Misregulation of R-loop homeostasis promotes genomic instability is associated with progressive neurodegenerative disorders or cancer. The purpose of this review is to detail the current understanding of R-loop contribution to cell stress and disease. search demonstrates that RNA containing four or more consecutive guanine residues near the 5' end possesses significantly higher rates of R-loop formation due to this nucleation requirement. After initiation of the R-loop, RNA-DNA hybridization is elongated and stabilized by a subsequent stretch of guanine rich (G-Rich) sequence. Elongation becomes less favorable when G-rich content decreases and the structure terminates. Other factors also promote R-loop formation and reduce the requirement for G-Rich sequence. For example, increased negative supercoiling on the trailing fork of the transcription bubble extends the opportunity for potential interactions between newly transcribed RNA and the template strand (reviewed in [5]). Additionally, nicks in the non-template strand can promote DNA-RNA hybridization of nascent RNA to the template strand, even when the G-Rich sequence is distal to the initiating G-cluster [3,4]. The build-up of R-loops is counteracted through a variety of mechanisms that maintain transcription bubble integrity, including nuclease activity and resolution of topological stress (Figure 1). These mechanisms are detailed in recent review articles [5][6][7][8][9]. Briefly, distinct enzymes carry out complementary actions to prevent accumulation of R-loops. RNase H1 and RNase H2 utilize 5'-3' exonuclease activity to remove RNA from the loop. These enzymes are evolutionarily conserved in prokaryotes and eukaryotes and are the only RNA-specific ribonucleases known to digest hybridized RNA. Complementary to RNase H1/2, cells also possess helicases that "untangle" DNA-RNA lesions like mammalian DHX9 and aquarius (AQR) [10,11]. Other helicases known to resolve R-loop structures include the yeast Sen1p orthologue, senataxin (SETX) [12] and an ATPdependent DNA helicase PIF1 [13]. Preventative mechanisms exist as an additional measure of protection against R-loop accumulation (Figure 1). Topoisomerases work to decrease negative supercoiling that increase access of nascent mRNA to G-clusters on the template DNA. Topoisomerase 1 (TOP1) and Topoisomerase 2 (TOP2) have both been shown to relieve torsional stress and prevent R-loop accumulation at the rDNA locus [14]. Moreover, topoisomerase 3B (TOP3B) reduces R-loop formation at highly expressed genes like the protooncogene MYC [15]. Biogenesis of messenger ribonucleoprotein particles (mRNP) also prevents R-loop accumulation. During mRNP biogenesis, RNA-binding proteins (RBP) interact with nascent mRNA to promote mRNA processing FIGURE 1: Model of R-loop homeostasis. A homeostasis exists between formation and removal of R-loops across the genome. Normally, mRNP biogenesis machinery targets nascent RNA and processes and prepares the strand for nuclear export (top). Topoisomerases and Helicases aid in preventing R-loop formation by reducing exposure times of ssDNA during transciption. RNase H removes R-loops by specifically digesting RNA in helical formation. Together these processes reduce R-loop accumulation during transcription. Factors that lead to pausing of RNA polymerase render conditions favorable for R-loop formation (replication stress, DNA damage, fork collisions, etc). R-loops nucleate from G-clusters (dashed green line). The loop then extends along GC rich sequences within the gene during elongation (bottom). The balance between removal and formation creates an equilibrium at nucleation sites across the genome. and export from the nucleus. In eukaryotic cells, the THO/TREX complex, as well as Serine/Arginine Rich Splicing Factor (SRSF1), binds ssRNA to initiate mRNP maturation, and these interactions prohibit R-loop formation [16][17][18]. Additional mRNA splicing and surveillance machinery such as the exon junction complex (EJC) and the exosome also reduce R-loop accumulation by sterically prohibiting interactions between the nascent mRNA and exposed ssDNA [18,19]. Epigenetic mechansims act upstream of topoisomerase and helicase function in R-loop homeostasis. For example, the MYST family acetyltransferase MOF aids in preventing R-loop accumulation that arises from replicative stress. Depletion of MOF decreases replication fork (RF) speed and potentiates fork stalling, factors that contribute to Rloop nucleation. Furthermore, recruitment of the repair machinery to sites of DNA damage occurs through MOFmediated ubiquitination [20], while an interaction between THO and SIN3A has been described where histone deacetylation prevents R-loop formation [21]. How acetylation regulates R-loop formation is likely context dependent and further studies should delineate these differences. Collectively, the cooperative activities of transcription bubble maintenance and mRNA processing machinery resolve and prevent R-loop accumulation. Each individual component contributes to the homeostasis of R-loops, and their respective regulation determines the local prevalence of these hybrid structures throughout the genome. TECHNIQUES DEVELOPTED TO STUDY R-LOOPS Since being first described in 1976 [2], several techniques have been developed to study eukaryotic R-loop biology in vitro and in vivo. Various plasmid-based systems have been created to study the regulation of R-loop formation and removal. Known R-loop sequences are inserted into plasmids in order to examine the effect of these structures on replication or transcription in vitro. Classically, a series of electrophoresis techniques have been developed to identify sequences that favorably form R-loops in vitro. The R-loop structure migrates more slowly in agarose gel electrophoresis and can be visualized with ethidium bromide (EtBr) after in vitro transcription [22,23]. Moreover, the shifted band shows resistance to digestion by RNase A and sensitivity to digestion by RNase H1. These exonucleases target ssRNA and helical RNA, respectively. Using labeled uridine during the in vitro transcription step increases sensitivity of the assay. Moreover, it eliminates signal from dsDNA that appears when using a general nucleic acid stain like EtBr. As a method to identify perturbations in the dsDNA helix, bisulfite modification can be used to detect R-loops [22]. Briefly, unpaired cytosines on the displaced anti-sense ssDNA are converted to uracil through deamination by addition of bisulfite. The uracil subsequently converts CG basepairs to AT basepairs during PCR amplification and the change can be observed by sequencing. Because this technique targets accessible strands of ssDNA, R-loop formation should be verified through additional methodologies. A powerful, genome-wide technique, known as DNA-RNA immunoprecipitation followed by sequencing (DRIPseq), uses the S9.6 antibody to specifically pull down DNA-RNA hybrids that are subsequently identified by high throughput sequencing methods to map these structures to the genome [24][25][26][27]. While the use of DRIP-seq and its derivatives give an overall picture of R-loop distribution across the genome, called peaks should be validated as some inherent bias has been reported [28]. An alternative high throughput technique known as DNA-RNA in vitro enhancement followed by sequencing (DRIVE-seq) utilizes an epitope-tagged, catalytically dead RNase H1 and affinity pulldown to recover DNA-RNA hybrids for sequencing [29,30]. Depending on experimental conditions, high throughput strategies identify between 1,000 and 20,000 R-loops across the genome. Interestingly, R-loops are largely mapped to gene promoters and terminator regions in human cell lines, showing their potential involvement in regulating RNA pol II-mediated transcription. However, several specialized regions also show enrichment for R-loops where they may act as a more structural component to the genome, including telomeres, ribosomal DNA (rDNA), and transposable elements. Recently, IP-mass spectrometry techniques were used to identify R-loop interacting proteins in a high throughput format. The study identified the RNA helicase DHX9 and characterized its role in suppressing R-loop accumulation, reducing DNA damage, and promoting transcriptional termination [31]. R-loops can also be detected with microscopy to visually confirm their formation using in vitro transcription. For example, the S9.6 antibody, as well as a GFPtagged DNA-RNA hybrid binding domain of RNase H1, allow for visualization of R-loop foci with fluorescence microscopy [26,32] or fluorescence in situ hybridization (FISH) based techniques [33][34][35]. Electron microscopy provides another mechanism to study and visualize R-loop structures [36]. The continued development of techniques and methodologies to study R-loop dynamics and the downstream consequences of their accumulation will further detail the contribution that these structures have in gene regulation and genomic instability. IN VIVO ROLES FOR R-LOOPS IN EUKARYOTIC CELLS Predictably, formation of a DNA-RNA hybrid creates structural lesions in the genome that can affect replication, transcription, and recombination. Ongoing research continues to detail the in vivo role of R-loops as regulatory elements of specialized events in the eukaryotic cell. Previously, mitochondrial origins of replication contain R-loops that are used to facilitate replication of the mitochondrial DNA (mtDNA), a process likely conserved from prokaryotic ancestors [37]. Studies in activated B-lymphocytes also show that R-loops are used at the switch (S) region during immunoglobulin class switching recombination (CSR) to promote deaminization of cytosine and generate a doublestrand break (DSB) [22,38]. In this case, activation-induced deaminase (AID) gains access to the WRC motifs (W=A/T, R=A/G) within the repetitive S regions in the displaced, non-template strand downstream of the IgG promoter [39]. Recently, researchers describe a mechanism where the RNA helicase DDX1 resolves G-quadruplex structures into R-loops at the S region of immunoglobulin heavy chain locus (IgH) in order to recruit AID for CSR [40]. While R-loops contribute to specialized events or structures in the human genome, they also have distinct roles in regulating transcription. In one instance, R-loops regulate epigenetic mechanisms of gene repression. When R-loops form in downstream CpG island promoter regions, they inhibit the activity of DNA methyltransferases to promote expression [26]. In a separate study, R-loop formation can facilitate binding of transcription factors to regulatory elements on DNA. Invasion of anti-sense non-coding RNA (ncRNA) into the promoter region of the vimentin (VIM) gene reduces local nucleosome density. The formed R-loop subsequently increases gene expression by providing access of nuclear factor kappa B (NF-κB) to its binding element [41]. Interestingly, this mechanism runs counter to previously described repressive activities of non-coding (nc)RNA, and provides new insight into how these moeities can positively regulate gene expression. R-loop formation in G-Rich sequences downstream of the 3' polyadenylation (polyA) signal can promote RNA Pol II pausing [42]. The R-loop structure promotes antisense transcription and formation of dsRNA. As a result, RNAi machinery and associated histone modification machinery, including G9a, localize at the pause site. Subsequent deposition of the repressive H3K9me2 mark on chromatin by G9a leads to recruitment of heterochromatin protein 1γ (HP1γ), which reinforces the pausing. Transcriptional termination subsequently occurs and prevents run-through at regions of high gene density to maintain gene integrity. MECHANISMS OF STRESS INDUCTION VIA R-LOOP ACCUMULATION Accumulation of R-loops often results in increased cellular stress that leads to genomic instability. The current hypothesis states that stabilized, co-transcriptional R-loops disrupt RF progression during S-phase. This disruption causes stalling that can lead to fork collapse, DSBs, or incomplete replication before entry into mitosis. Recently, perturbation of the mRNA splicing, processing, and export factors have been shown to increase R-loop mediated genome instability. For example, disruption of the THO and Transcription Export (TREX) complex (discussed above) leads to a hyper-recombination phenotype in yeast and human cells [43,44]. Interestingly, these mutants show genome-wide increases in histone H3 serine 10 phosphorylation (H3S10P) to promote chromatin condensation during mitosis. Evidence suggests that this modification, along with R-loop associated H3K9me2 deposition, impairs replication and leads to stress [45]. Independent studies demonstrate that dysfunction of the SRSF1 splicing complex increases R-loop formation detrimental to genomic stability [16]. In these cases, the observed phenotypes can be rescued through overexpression of RNase H, indicating the importance of resolving DNA-RNA hybrids to prevent DSBs or recombination. Resolving topological stress remains an important aspect of genome maintenance where accumulation of R-loops after TOP1 inhibition increases genomic rearrangements [46]. These events result from replication fork collisions with a transcription bubble that was likely paused by the formation of an R-loop. The deposition of R-loops at promoter and terminator regions has been shown to regulate initiation, pausing, and termination of transcription. Perturbation of these regulatory events could lead to inappropriate changes in gene expression or genetic stress. Studies indicate that mutation of the THO complex impairs elongation, possibly through the formation of R-loop intermediates that prevent RNA Pol II processivity [47]. Moreover, these impairments facilitate the hyper-recombination effects described above. A second form of genetic stress occurs from the exposed ssDNA segment in the R-loop. This feature is susceptible to alterations via DNA-modifying or repair enzymes, as well as chemical mutagens [48]. AID activity or other factors may inappropriately target the ssDNA strand of the R-loop and create lesions in the genome that lead to point mutations or other forms of deleterious repair. Unsurprisingly, stabilization of co-transcriptional R-loops can localize mutagenesis to gene regions and exacerbate their effect by targeting coding sequence [44]. DNA damage sensing and repair proteins have recently emerged as inhibitors of R-loop accumulation in eukaryotic cells (Figure 2). For example, the Breast Cancer Type 1 and 2 susceptibility genes (BRCA1/2) prevent potential harmful effects of R-loops through two distinct manners. BRCA1 recruits the helicase SETX to R-loops that form at transcription termination pause sites or sites of Negative Elongation Factor (NELF) mediated Pol-II pausing [49,50]. BRCA1dependent recruitment of SETX resolves R-loop structures at these sites and suppresses DNA damage. Interestingly, studies of breast cancer models with a BRCA1 mutation show increased insertion-deletion (in-del) mutations at BRCA1-bound termination sites known to inhabit R-loops. Depletion of BRCA2 from cells also increases R-loop accumulation, suggesting an important role for this protein in R-loop homeostasis. However, dissecting the specific role (or roles) of BRCA2 in resolving R-loops has been difficult due to its contributions in DNA repair, RF stability, and other mechanisms associated with mitosis. Its inherent involvement in these processes may alone prevent R-loop formation by preventing RF collapse and recruiting the ssDNA binding protein, Rad51, to DSB sites [51]. However, recent evidence shows that BRCA2 associates with the TREX complex subunit, PCI-domain containing protein 2 (PCID2), which suggests a protective role for BRCA2 against the co-transcriptional formation of R-loops [33]. Separately, research shows that BRCA2 recruits RNA polymerase II associated factor-1 (PAF1) to promoter-bound Pol-II, potentiates pause release, and decreases R-loop formation [52]. Both BRCA1 (FANCS) and BRCA2 (FANCD1) are part of the Fanconi Anemia (FA) mediated DSB repair pathway. Therefore investigators elucidated that this complex may regulate genome stability through R-loop resolution. In addition to the protective functions of BRCA1 and BRCA2, the disruption of critical FA complex members FANCD2, FANCA, and FANCM lead to genomic instability and DNA damage from R-loop mediated RF collapse [53,54]. The activity of the FA complex during replication allows RFs to traverse intrastrand crosslinks (ICL), and promotes postreplicative repair of this lesion [55]. Briefly, the FA complex is recruited by ATR and subsequently activates FANCD2 through a mono-ubiquitination step. The complex then utilizes the DNA translocase activity of FANCM to traverse the lesion and prevent RF collapse. Emerging evidence demonstrates that the same FA complex mediated mechanisms facilitate RF progression through transcriptionmediated conflicts, including R-loops. The FA complex is recruited to the stalled RFs due to collisions with the transcription machinery. It subsequently removes R-loops and restarts replication to prevent RF collapse. RNase H1 overexpression rescues the phenotypes observed during disruption of FA function and validates the role of R-loops in these events. Conversely, defects in the FA complex lead to increased R-loop formation upon treatment of ICL-inducing agents. This suggests that R-loops may be an outcome of genotoxic stress in FA patients. Interestingly, depletion of BRCA1 or BRCA2 individually shows that each gene regulates R-loop formation in distinct regions of the genome. Sequence or epigenetic differences that determine recruitment of BRCA1 or BRCA2 to R-loops remain an area of keen interest in the field. The induction of stress due to R-loop accumulation largely results from collisions that occur between RFs and the transcriptional machinery, particularly in a head-on orientation [56]. Genomic instability and genome rearrangements occur if these structures cannot be resolved through the replication stress responses of the FA complex, DNA repair, and R-loop homeostasis. This creates a positive feedback model where genomic instability can potentiate additional R-loop accumulation. Buildup of R-loops during stress may identify new mechanisms that contribute to the pathology of these structures. BIOLOGICAL OUTCOMES OF INAPPROPRIATE R-LOOP HOMEOSTASIS R-loops can arise from a variety of cellular stresses, and lead to complications including replication defects, transcriptional irregularities, and genomic instability. The biological consequences of disrupted R-loop homeostasis has been described in multiple neurodegenerative diseases that largely associate with ataxia (Reviewed in [57]). GCrich trinucleotide expansions of gene regions provide an extended landscape for R-loop formation that disrupt transcription and proper gene expression (Figure 2A). For example, Huntingtin (HTT; Huntington's Disease), Frataxin (FXN; Friedreich Ataxia), and Ataxin 1/2 (ATXN1/ATXN2; Spinocerebellar Ataxias) all have GC-rich trinucleotide expansion tracks that form R-loops in vitro and associate with disease progression [58,59]. R-loop induced disruption of gene expression occurs through distinct mechanisms in trinucleotide repeat disorders. Expansion of the repeat region in the first intron of FXN leads to R-loop formation, increased histone H3K9 dimethyl (H3K9me2) chromatin deposition, and impeded Pol II mediated transcription [60]. Chemical inhibition of TOP1 in Friedreich Ataxia (FRDA) model cell lines with FXN expansions potentiates R-loop accumulation and H3K9me2 enrichment, providing further evidence that R-loop formation increases deposition of this repressive mark. Fragile X syndrome harbors a mechanism where trinucleotide expansion in the 5' UTR of the FMR1 gene leads to DNA methylation mediated silencing of this locus [60,61]. However, removal of DNA methylation from this locus reactivates expression to only twenty-five percent of wildtype cells. The reactivation of FMR1 expression is partial because of R-loop formation after depletion of DNA methylation. Examination of the relationship between DNA methylation and R-loop formation at this locus could reveal a distinct mechanism of gene silencing in this model. Separately, trinucleotide repeat expansions can form R-loops that induce RNA Pol II pausing and aborted transcripts, which subsequently sequester nucleolin (NCL) and cause nucleolar stress [62,63]. Furthermore, mutations in the machinery that maintain R-loop homeostasis also associate with neurodegenerative disease. Dysfunctional TREX or RNaseH2 are linked to Aicardi-Goutières Syndrome (AGS) while SETX mutations contribute to ataxia with oculomotor apraxia (AOA2) [64,65]. Mutations of SETX also occur in the motor neuron disease amyotrophic lateral sclerosis 4 (ALS4). In this case, the SETX mutation decreases R-loops in the promoter region of BMP and activin membrane bound inhibitor (BAM-BI). Local R-loop loss increases DNMT-1 dependent methylation at the promoter, and downregulation of BAMBI leads to the activation of the TGF-β pathway, a process implicated in ALS [66]. As expected, the increased genome instability, chromosomal rearrangements, and DNA damage that occur during aberrant accumulation of R-loops is linked to oncogenesis [53,67,68] (Figure 2B). Loss of BRCA1 and BRCA2 function has already been well established in breast cancer. Cells show an increase in DSBs when lacking the protective effects that these genes carry against RF collapse [33,49,50,69]. Moreover, the increased frequencies of in-dels in BRCA mutant cell lines localize these mutations to gene regions and can have deleterious effects on gene expression. The cancer-associated genotoxic stress that arises from mutations in BRCA1/2 or members of the FA complex can all be partially rescued through overexpression of RNase H1 in cancer cell lines, suggesting that the aberrant R-loop formation contributes to malignant progression. The Tudor containing protein 3 (TDRD3) plays a critical role in maintaining proper R-loop homeostasis at the c-MYC locus [15]. TDRD3 recruits TOP3B to relieve negative super-coiling at c-MYC and prevents R-loop accumulation. Loss of TDRD3 function in B-lymphocytes leads genomic rearrangements between c-MYC and the Ig heavy chain (IGH) locus due to AID activity on R-loop associated ssDNA. This translocation commonly occurs in Burkitt's lymphoma and multiple myeloma and causes oncogenic levels of c-MYC expression. Loss-of-function in mRNP biogenesis machinery also associates with known cancers. For example, sequestration of the THO/TREX complex through Kaposi's sarcoma herpesvirus expression of ORF57 leads to increased genomic instability and subsequent malignant progression in immunodeficiency-associated models [70]. In Saccharomyces cerevisiae, the formation of R-loops at the noncoding telomere repeat-containing RNA (TERRA) locus facilitates type II survival in telomerase mutants [71]. The survival mechanism depends on radiation sensitive (RAD52) dependent recombination events that result in long heterogenous tracts of telomeric repeats. Extended tracts of recombination dependent repeats appear in mammalian cells utilizing the alternative lengthening of telomere (ALT) pathway during bypass of senescence. Therefore, accumulation of R-loops at TERRA may also play a role in promoting ALT activation in some types of cancer. Together, these models provide a wide variety of mechanisms where stress associates with increased R-loop formation and can lead to progressive neurodegenerative disease or cancer. The diversity of these mechanisms lends credence to the importance of proper R-loop homeostasis. Redundancy in the machinery that resolves R-loops suggests these structures are dynamic and widespread (studies indicate that R-loops occupy up to 5% of the genome [25]). As we increase our understanding of the formation and resolution of these lesions in vivo, we may discover new disease associations of R-loops. CONCLUDING REMARKS Emerging evidence demonstrates the importance of proper R-loop homeostasis. These structures provide critical regulatory functions for gene expression and, in some cases, gene rearrangements. However, their formation and resolution must be tightly regulated to prevent topological events that disrupt replication through impaired RF progression. Such impairment provides an avenue for genome instability and contributes to the development of progressive diseases like cancer. Moreover, prolonged R-loop occupancy exposes ssDNA to genotoxic stress, which produces deleterious effects on gene expression. Future examination of R-loops in cancer models should determine whether the seemingly independent functions of BRCA1 and BRCA2 are coordinated to prevent RF collapse through FANC-complex-mediated mechanisms. Specifically, research should elucidate whether BRCA1 recruits SETX to unwind R-loops while BRCA2 acts to traverse the lesion in a manner to complete replication and allow DNA repair to occur at a later phase. Moreover, delineating the coordination between RNase H, helicases, and topoisomerases to prevent R-loop accumulation could provide insight into why specific cell types are susceptible to diseases associated with improper R-loop homeostasis. Finally, the link between the epigenetics and sites of Rloop formation has been established, but further examination of the machinery that recruits chromatin-modifying enzymes is warranted. Discovery of mechanisms that link R-loop homeostasis to chromatin modification would lend further clues into the development of specific cancers or neurodegenerative diseases. Specifically, the field would benefit from identification of R-loop "readers" that allow cells to detect these structures and modify chromatin to repress gene expression or promote transcriptional termination. R-loops provide an interesting new source of replicative and transcriptional stress that clearly have disease-causing consequences. As new methodologies and models are developed to study their regulation and homeostasis, re-searches will continue to integrate this under-studied structure into cell biology and pathogenesis.	2019-04-10T13:13:14.523Z	2019-01-21T00:00:00.000	{ "year": 2019, "sha1": "1f3ad3acc13d76e430d1ce03a06c8de1d5d884a1", "oa_license": "CCBY", "oa_url": "http://www.cell-stress.com/wp-content/uploads/2019/01/2019A-Allison-Cell-Stress.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "1f3ad3acc13d76e430d1ce03a06c8de1d5d884a1", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
214732668	pes2o/s2orc	v3-fos-license	Effect of T-shape Shoulder Fillet on the Plastic Deformation Properties of SS400 and LYS160 Steel. Shoulder fillets are widely used in the structural optimization design of metal dampers. However, the plastic deformation property of dampers affected by stress concentration, owing to different fillets, has not been explored in-depth. In this study, two typical metal damper materials with different plastic deformation, i.e., ordinary steel SS400 and low-yield-strength steel LYS160, were investigated. The strengthening effect of fillets under different loading is evaluated by comparing the mechanical properties of different fillet heights. Furthermore, the effect of the stress concentration caused by different fillet shapes, based on the failure mode of materials, is discussed. Subsequently, the fatigue degradation effect under the reciprocating shear loading is studied. Based on a series of studies on the deformation properties of fillets in different ductile materials, the basis for the structural optimization design under plastic deformation is provided. Introduction As an important energy-dissipating component, metal dampers are widely used in seismic engineering. It is crucial to optimize the design of metal dampers, to improve their energy-dissipation capacity, by fully utilizing the plastic deformation of metal materials. Different types of dampers can be designed, using different deformation mechanisms of metal, such as shear [1], tension [2], bending, or their combination [3]. To utilize these dampers more effectively in energy dissipation, the core energy-dissipation structure is typically optimized. In the optimization design of a shear panel, a uniform stress distribution in the damper is desired. Hence, local weakening [4] or local strengthening [5] is generally performed. Local weakening is typically realized in shoulders with local penetration [6] or by holes with overall penetration [7]. Meanwhile, local strengthening is realized by adding stiffeners at weak positions [8]. In the case of dampers with tension and compression energy dissipation, the strength of the fixed part of the damper is generally significantly greater than that of the energy-dissipation core, to ensure that the latter can perform reliably. Accordingly, the thickness of the fixed part is greater than that of the energy-dissipation core [9,10], and a shoulder is set between them. From the optimization design of these dampers, it is clear that, regardless of the damper type, a reasonable design of shoulders is indispensable for an optimized design. The fatigue performance of shoulders is generally evaluated by the stress concentration. Some studies focused on the stress concentration of shoulders under different loads, such as tension [11,12], bending [13], and shear [14,15]. At the same time, fillet height [16], fillet length [17], and fillet shape [18,19] of the shoulder are other research concerns. The results show that, when the fillet length is long enough, the stress concentration of the shoulder under different loads can be effectively improved. Accordingly, in order to reduce the influence of stress concentration, the length, height, and radius of the shoulder fillet are clearly specified in the national standard of material performance test [20]. However, a large arc cannot be adopted in the thickness direction of a shear panel damper, owing to its thickness limitation. Therefore, a small arc fillet or a T-shape fillet [21] cannot be avoided. Through the rational design, the stress can be evenly distributed on the shear panel [22], as shown in Figure 1. The stress concentration under the traditional elastic deformation is usually represented as a narrow band of the maximum stress distributed at the fillet. The stress concentration of shear panel damper under plastic deformation is quite different with that of traditional shoulder under elastic deformation. Energy is typically dissipated by the plastic deformation of a damper instead of the elastic deformation. With the increase of loading cycle, the stress at the edge of shear panel decays rapidly when the plastic deformation reaches the critical value of fatigue [23]. Studies regarding the stress concentration of shoulder fillets under plastic deformation and its effect on the deformation properties of dampers are scarce. In view of the complexity of shoulder fillet damage under plastic fatigue loading, it is more convincing to analyze the deformation properties of shoulder fillet by experiment. Compared with traditional ordinary steel, low-yield-strength steel is becoming more widely used in metal-damper design owing to its large plastic deformation. However, the effect of shoulder fillets in large plastic deformation on the structure's performance has not been studied in-depth. Hence, the performance difference caused by shoulder fillets under different plastic deformation has not been compared and analyzed. In this study, ordinary carbon steel and low-yield-strength steel were selected as test materials. The mechanical properties of ordinary carbon steel and low-yield-strength steel with different shoulder fillet shapes and heights under different loads were analyzed. The effect of T-shape shoulder fillet on the plastic deformation ability of structures fabricated by using these two different ductile materials is discussed in detail. Test Plan Low-yield-strength steel (yield strength 160 MPa, LYS160) and ordinary carbon steel SS400 were adopted to design the specimens, as shown in Figure 2. The effective diameter of the specimen was 10 mm, and the length was 50 mm. To investigate the effect of fillet height under tension and shear, three different diameters (12/14/16 mm) of the clamped end were adopted. The diameter ratio of the clamped end to the specimen were 1.2, 1.4, and 1.6, respectively (Figure 2a,b). Based on the three different fillet heights, the effect of fillet shape on structural deformation performance is further discussed. The properties of the standard specimen with a large arc transition can be obtained easily, according to previous studies [24]. Hence, they were not tested in this study. The fillet shape was designed as two types, i.e., T-shape with right angle and small arc, denoted as T and ARC, respectively. Flanges ( Figure 1) are typically fixed on the left and right sides of the shear panel, to improve the stress. In the actual operating condition, the angle of these T-shape shoulders is no longer a constant value; it will change with the shear amplitude. Accordingly, three angles (R = 30 • , 45 • , and 60 • ) were designed in OBLIQUE T-shape shoulders (Figure 2c), to investigate the change in mechanical properties to clarify its effect on the plastic deformation capacity. Test Setup Tensile and shear tests were performed, using a universal material testing machine and a torsional fatigue testing machine with loading speeds of 1 mm/min and 180 • /min, respectively ( Figure 3). The force, displacement, torque, and twist angle of the specimens were measured directly by a computer. The experimental process was captured with a camera, and micro-section characteristics were observed through SEM (Scanning Electron Microscope). Tension The failure modes of specimens with different fillet heights under tensile loading are shown in Figure 4. No plastic deformation was observed in the clamped end of the specimens, even when the fillet height was the smallest (1 mm, Ten-1, Ten-4, and Ten-10). For LYS160 or SS400, a small fillet height can cause plastic deformation to be concentrated in the middle area. Under tensile loading, a smaller fillet height can yield a better step-strengthening effect. All the specimens were necked and fractured in the middle parts. This implies that no stress concentration existed, or the stress concentration at the fillet was negligible under tensile loading. This phenomenon is primarily determined by the characteristics of tensile deformation, based on the following formula under tensile loading: where F is the tensile force; A is the section area, i.e., A = πd 2 /4; and d is the diameter of the specimen. As the diameter of the clamped ends is larger than that of the middle part, the corresponding stiffness of the clamping part is greater than that of the middle part. Once the tensile loading is applied, the middle part will deform first. With the increase in loading, the middle diameter of the specimen decreases and the stiffness becomes smaller. The diameter and rigidity of the clamped part at both ends remained unchanged. Hence, the reduction in stiffness of the middle part caused the deformation to be concentrated in the middle part. The reinforcement effect of the clamped end can be realized when the diameter of the middle deformation area is slightly larger than that of the middle deformation area. Moreover, the stress concentration of the structure under tensile loading has little correlation with the fillet shape and angle of the shoulder. Torsion Red and blue lines were drawn on the opposite sides of the specimen, along the axial direction, to observe the torsion deformation of the specimen. The failure modes of the specimens under shear loading are shown in Figure 5. When the fillet height was small, plastic deformation was observed at two clamped ends. This indicates that, when the fillet height was 1 mm (D/d = 1.2), the clamping end could not produce a strengthening effect. When the fillet height was 2 mm (D/d = 1.4), the strengthening effect was demonstrated in specimen SS400. When the fillet height was 3 mm (D/d = 1.6), the strengthening effect was achieved in specimen LYS160. Compared with the tensile test, the maximum shear stress of the torsion test is expressed as follows: where W t = πd 3 /16; d is the diameter of the specimen; and T is the torque. Without necking occurring in torsion, the shear area in the middle area of the specimen and the corresponding W t remained unchanged. The torque increased with the deformation, owing to its stress-hardening effect. When the antitorque of the clamping end was lower than the loading torque, torsional deformation was produced in the clamping ends (Figure 5a,b,d,e). Therefore, the strengthening effect of the clamping end under torsional loading should be designed with consideration of the material's hardening characteristics. It was discovered that the fractures typically appeared at the fillet of the specimens under shear loading ( Figure 5). High stress concentrations tend to be produced in the sharp transition part. When the clamping end shoulder was beveled, the stress concentration distributed at the shortest point of the middle specimen at the beveled edge. In fact, the effective length of the intermediate specimen was affected by the bevel angle. The stress concentration of the structure under shear loading was affected by the fillet height, fillet shape, and shoulder angle, simultaneously. Fracture Surface The fracture transversal surfaces of specimens SS400 and LYS160 under different unidirectional loads are shown in Figure 6. Under tensile loading, necking was observed in both specimens SS400 and LYS160. The diameter of the necking section of specimen LYS160 was much smaller than that of specimen SS400 (Figure 6a,b), demonstrating a better plastic deformation capacity. Under shear loading, several peaks and troughs distributed at the fracture transversal surface of specimen SS400 (Figure 6c). The fracture transversal surface of LYS160 under shear loading exhibited a smooth plane (Figure 6d). The microstructures of specimen LYS160 under tension and torsion are shown in Figure 7. Under tensile loading, many dimples appeared in the fracture transversal surface of specimen LYS160 (Figure 7a). Meanwhile, the surrounding surface of the specimen was distributed with grooves similar to those after plowing. Under shear loading, concentric circular slip lines were observed in the fracture transversal surface of specimen LYS160 (Figure 7b). Many parallel slip lines were also observed on the cylindrical surfaces of specimen LYS160 (Figure 7c). This demonstrated the good slip performance of LYS160 in both the axial and radial directions. Seen from the fracture surface, the essence of failure mode of LYS160 and SS400 under tension was necking. The sole difference between them is the necking degree. The failure mode of LYS160 is totally different than that of SS400 under shear loading. LYS160 presents uniform interlayer slip deformation, with a story height of about 500 µm. A smooth slip surface can be formed between the layers, which is helpful for the stable energy dissipation of large plastic deformation. Cyclical Shear The failure mode of the T-shape shoulder specimen under the reciprocating torsion is shown in Figure 8. The former five figures are the failure modes of specimen LYS160, and the latter five figures are those of specimen SS400. When the fillet height was 3 mm (D/d = 1.6), plastic deformation was not observed at the clamping end. The strengthening effect of the clamp can be ensured under cyclical shear loading. The failure mode of specimen LYS160 under cyclical shear loading (Figure 8a-e) was the same as that under unidirectional shear loading, all of them fractured from the T-shape shoulder fillet. The failure mode of CT6 (Figure 8f) was the same as that of the specimen under monotonic shear loading. Except for CT6, the failure mode of specimen SS400 under cyclic shear loading was consistent with that of the standard specimen under unidirectional loading. They almost cracked at the middle of the specimen (Figure 8g-j). The initial crack direction was consistent with the direction of the lines drawn on specimen SS400. The fatigue failure mode of specimen SS400 with a T-shape shoulder under cyclic shear loading depended on the loading amplitude. When the amplitude was large, the specimen with an abrupt shoulder fractured. When the amplitude was small, the failure mode of the specimen with a T-shape shoulder was the same as that of the standard specimen [24]. Similar to the monotonic shear loading, there were many parallel microcracks in the middle of the LYS160 specimen under the reciprocating shear loading, demonstrating a uniform interlayer slip. It suggests that the stress concentration is negligible in the middle part of the LYS160 specimens. Meanwhile, there was a sharp change of stress in the shoulder fillets, and the stress concentration was severe. Therefore, the corresponding failure mode is fillet crack. Under the reciprocating shear loading, the uniform deformation in the middle part of SS400 specimens cannot be obtained. The corresponding stress concentration is serious, and the failure mode of SS400 specimens was the fatigue fracture of middle part. Monotonic Loading The stress-strain curves of specimens with different shoulders are shown in Figure 9, and they did not differ vastly when tensile loading was applied (Figure 9a,b). The stress-strain curves of SS400 and LYS160 were nonlinear in the plastic range. The ductility of SS400 was poor, and it would break soon after necking, resulting in the sudden disappearance of the force. The ductility of LYS160 was pretty good. The force of LYS160 decreased gradually, from the maximum value to 0, after necking. The stress-strain curves of SS400 and LYS160 under torsion were approximate with linear in the plastic range. No matter the fillet form and material, the stress value dropped sharply once the fillet broke. Cyclical Torsional Loading The hysteresis curves of specimen LYS160 under the reciprocating torsional loading are shown in Figure 10a; and they exhibit a regular rectangular that can be simplified as a perfect elastic-plastic model. The hardening effect appeared in the second cycle, and there, no stress-hardening or stress-softening effect was observed from the second cycle. A significant decrease in force was observed once the specimen cracked. The hysteretic curves of specimen SS400 under the reciprocating torsional load are shown in Figure 10b; these curves were also approximately rectangular and could be simplified as a bilinear model. The hardening effect was observed in the second cycle. Subsequently, the continuous softening effect was observed from the third cycle. As shown in Figure 11, Specimen LYS160 exhibited a stable energy-dissipation capacity, whereas specimen SS400 exhibited stress degradation with the increase in cycle number. Deformation Capacity under Monotonic Loading When the shoulder was perpendicular to the loading direction, the effect on the plastic deformation capacity could be ignored. The maximum deformation capacity of LYS160 and SS400 was approximately 47% and 30%, respectively. Their ultimate strain values are approximate with that of the standard tensile specimen. The deformation capacity of specimen LYS160 changed with the shoulder, under shear loading ( Figure 11a). To compare with the standard torsional specimen with a large transition arc, the stress concentration can also be alleviated when the fillet radius is small. When the fillet radius was small (Tor-4, Tor-5, and Tor-6), the maximum shear strain was approximately 850%, which is similar to that of the standard torsional specimen [24]. When the shoulder fillet was T-shape, the stress concentration was distributed around the shoulder fillets of the specimen (Tor-1, Tor-2, and Tor-3). The deformation capacity of the specimen would be decreased by 21% if the T-shape shoulder fillet was adopted. When the T-shape shoulder was inclined, the stress concentration was concentrated at one point (Tor-7, Tor-8, and Tor-9). Compared with the T-shape shoulder specimens, the deformation capacity of the OBLIQUE T-shape shoulder specimen would be decreased by 17%. Compared with standard torsional specimen, the deformation capacity of SS400 decreased from 440% to 135% shear stain when the T-shape shoulder fillet was adopted. The reduction of deformation capacity was around 69% (Figure 11b). In the case of unidirectional shear loading, the material with poor plastic deformation capacity was more sensitive to fillet shape. Deformation Capacity under Cyclical Loading The fatigue performances of the T-shape shoulder fillet and traditional standard specimens are shown in Figure 12. The red and blue lines are fatigue curves of the traditional standard and T-shape shoulder fillet specimens, respectively. As shown in Figure 12, the fatigue life of specimen LYS160 with a T-shape fillet was lower than that with an arc transition. When the number of cycles was 10, the strain amplitude of the traditional standard specimen was 82%, while that of the T-shape shoulder fillet specimen was 43.4%. The fatigue performance deteriorated by 47% in the large plastic deformation range when the T-shape fillet was adopted. The red and green circles in the figure are the fatigue cycles when the fillets of specimen SS400 are arc and T-shape, respectively. The green curve is the fatigue life curve. Although the maximum shear strain of sharp transition specimen SS400 was 69% lower than that of the standard specimen under unidirectional shear, the fatigue performance did not differ, owing to the small loading amplitude. The transition form of the fillet imposed little effect on the fatigue behavior of specimen SS400 under a small deformation. Under reciprocating shear, the deformation in the middle of the LYS160 specimens was uniform, and the stress concentration was not significant. The stress concentration at the fillet caused the sharp decline of strain amplitude, and the advantage of large plastic deformation capacity of LYS160 could not be utilized. The fatigue performance of the specimens depends on the fatigue performance of the fillet, while it is less affected by the fatigue performance of the material. On the opposite, when the loading amplitude of SS400 is not more than 61% shear strain, the fatigue fracture occurs in the middle of the SS400 specimens. The fatigue performance of the specimens depends on the fatigue performance of the material, while it is not affected by the fillet. The difference of fatigue performance between different plastic materials is narrowed when the T-shape shoulder is adopted. Conclusions In this study, the deformation properties affected by different shoulder fillets were investigated by tensile and shear loading. The main conclusions are as follows: (1) For LYS160 or SS400, its deformation capacity was not significantly affected by the transition form of fillets under tension loading. (2) The shear deformation capacity was affected by the fillet shape, while it was insensitive to the fillet radius. (3) Under reciprocating shear, the deformation capacity of large plastic material (LYS160) was affected by the T-shape fillet slightly, while the deformation capacity of small plastic material (SS400) was affected by the T-shape fillet greatly. (4) Under repeating shear, stable interlayer deformation could be obtained in the middle part, and the deformation capacity of LYS160 specimens depended on the T-shape fillet. There was no stable deformation in the middle part, the deformation capacity of SS400 specimens was not affected by the T-shape fillet. (5) When the fatigue cycle was 30, the corresponding amplitude of the LYS160 specimens with a T-shape fillet was 25% shear strain, which can meet most engineering applications.	2020-04-01T13:05:13.817Z	2020-03-26T00:00:00.000	{ "year": 2020, "sha1": "6fad47e33e98f5c7835b1fd7b0efa4b648e2ee14", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1996-1944/13/7/1528/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "ae3e0ef5d9865871cae55d3cdf6f280da626136f", "s2fieldsofstudy": [ "Engineering", "Materials Science" ], "extfieldsofstudy": [ "Materials Science", "Medicine" ] }
118480940	pes2o/s2orc	v3-fos-license	Reconstruction of Markovian Master Equation parameters through symplectic tomography In open quantum systems, phenomenological master equations with unknown parameters are often introduced. Here we propose a time-independent procedure based on quantum tomography to reconstruct the potentially unknown parameters of a wide class of Markovian master equations. According to our scheme, the system under investigation is initially prepared in a Gaussian state. At an arbitrary time t, in order to retrieve the unknown coefficients one needs to measure only a finite number (ten at maximum) of points along three time-independent tomograms. Due to the limited amount of measurements required, we expect our proposal to be especially suitable for experimental implementations. I. INTRODUCTION Tomographic maps [1] can be considered a very useful tool for reconstructing the physical state or some other properties of many physical systems, both in a classical (e.g. medical physics, archaeology, biology, geophysics) and in a quantum perspective (e.g. photonic states [2], photon number distributions [3,4,5], longitudinal motion of neutron wave packets [6]). The tomographic analysis is based on a probabilistic approach towards physical system investigation. In particular, its key ingredient is the Radon transform [7]. Given the phase-space of the system, this invertible integral transform allows to retrieve the marginal probability densities of the system, i.e. the probability density along straight lines. However, while in the classical regime the state of the system can be fully described by means of a probability distribution on its phase space, this is no longer the case of quantum systems. Indeed, due to the Heisenberg uncertainty relation, it is not possible to write a probability distribution as a function of both momentum and position. In this case, the Wigner function [8,9] can be employed as a quantum generalization of a classical probability distribution. This function is a map between phase-space functions and density matrices. Even if the Wigner function can take on negative values, by integrating out either the position or the momentum degrees of freedom, one obtains a bona fide probability distribution for the conjugated variables. From this point of view, the Wigner function corresponding to a quantum state can be regarded as a quasi-probability distribution and interpreted as a joint probability density in the phase space [10]. In this paper we apply quantum symplectic tomography to the investigation of open quantum systems [11,12] which, due to the coupling to an environment (bath), undergo a non-unitary dynamical evolution. A complete microscopic description of system-plus-bath dynamics is a complex many-body problem. Hence, as in general one aims at describing the dynamics of the system, only basic information about the bath is retained, according to the so-called open system approach. The state of the system is then expressed by means of a reduced density matrix, obtained from the total density matrix by tracing out the environmental degrees of freedom. The system dynamics is then governed by the so-called quantum master equation. The master equation approach can be seen as the generalization of the Schrödinger equation to the possibly incoherent evolution of a density matrix. In this case, the generator of the time evolution is the Liouville dissipative operator. The integration of a time-dependent Liouvillian being a highly involved task, e. g. see [13,14], it is highly preferrable to deal with a time-independent Liouvillian, i.e. to assume a Markovian dynamics. Several approximations allow a Markovian description, such as the weak coupling limit, the singular limit and the low density limit [11,12,15]. Nevertheless, a proper derivation of the master equation still requires complete information about the bath. The lack of this knowledge leads to the derivation of phenomenological master equations with unknown coefficients. Indeed, recent investigations [16,17,18] provide a more accurate approximation than the weak coupling limit, due to a more refined coarse grained dynamics. Even in this case, the obtained master equation has unknown coefficients, as it depends phenomenologically on the system investigated. In this paper, we will focus on a class of Markovian master equations with unknown coefficients modeling a one-dimensional damped harmonic oscillator. In particu-lar, we choose Lindblad operators [19] linear in both momentum and position degrees of freedom, such that the dynamical evolution of the system preserves the Gaussian form of the states. Our goal is to show how, by means of a tomographic approach, it is possible to measure indirectly the unknown coefficients by using Gaussian wave packets as a probe. This paper is organized as follows. In section II we introduce the class of master equations we want to investigate. In section III we derive the expressions for the coefficients of the master equation as a function of the first and second evolved momenta (cumulants) of a Gaussian state. In section IV we introduce the Wigner function and the Radon transform for an arbitrary Gaussian wave packet at a generic time t . We show that in order to measure the cumulants of a Gaussian state, and then indirectly the unknown parameters of the master equation, we need only a finite number (eight or ten) of time-independent tomograms. In section V we summarize and discuss our results and outline some feasible applications. Finally, in appendix A, we propose an alternative procedure to obtain the cumulants of a Gaussian state by means of time-dependent tomograms. This approach however appears to be less convenient for practical implementations. II. DESCRIPTION OF THE SYSTEM We want to investigate a class of master equations describing a Gaussian-shape-preserving (GSP) evolution of a quantum state. In the Markovian approximation, the non-unitary time evolution of a quantum system is described by the following general master equation [19,20]: whereρ(t) is the reduced density operator of the system. Eq. (1) is exactly solvable if the Lindblad operatorsV j and the system HamiltonianĤ are, respectively, at most first and second degree polynomials in position (q) and momentum (p) coordinates [21,22]. For systems like a harmonic oscillator or a field mode in an environment of harmonic oscillators (i.e. collective modes or a squeezed bath),Ĥ can be chosen of the general quadratic form where δ is the strength of the bilinear term inq andp, m is oscillator mass, and ω its frequency. The operatorsV j , which model the environment, are linear polynomials inq andp:V with a j and b j complex numbers. The sum goes from 1 to 2 as there exist only two c-linear independent operatorŝ V 1 ,V 2 , in the linear space of first degree polynomials inp andq. We can safely omit generic constant contributions inV j as they do not influence the dyamics of the system. Given this choice of operators, the Markovian master equation (1) can be rewritten as: where λ = −Im j=1,2 a * j b j is the unknown friction constant and are the unknown diffusion coefficients, satisfying the following constraints which ensure the complete positivity of the time evolution [21,22]: Markovian GSP Master equations of the form Eq. (4) are used in quantum optics and nuclear physics [23,24,25], and in the limit of vanishing ω can be employed for a phenomenological description of quantum Brownian motion [26,27,28]. Also, in the case of a high-temperature Ohmic environment the time-dependent master equation derived in [13,14] can be recast in this time-independent shape. It must be noted however that in the hightemperature limit the third constrain in (6) seems to be violated. Nevertheless, even if D qq = 0, D qp = 0 and λ = 0, D pp diverges only linearly with temperature. Therefore, we can recover the complete positivity by means of a suitable renormalization. This renormalization consists in adding a suitable subleading term D qq (e.g. D qq ∝ T −1 ). Otherwise, we can consider an high frequency cut-off for the environment [13,14]. In this way the master equation is not Markovian anymore. Anyway, since it involves only regular functions, it should give a completely positive dynamics (as the microscopic unitary group does). III. GAUSSIAN STATES EVOLUTION In this section we investigate the evolution of an initial Gaussian state according to Eq. (4). In particular we derive invertible expressions for the cumulants of the state at a time t in terms of the parameters of the master equation. Due to the Gaussian shape preservation, the evolved state at time t is completely determined by its first and second order momenta: Due to the linearity of theV j 's in phase-space, the timeevolution of the first and second order cumulants can be decoupled. We then obtain the following two sets of solvable equations [21,22]: The above equations allow to obtain the time-dependent momenta as a function of the Master equation coefficients λ, D qq , D pp , D qp . We now show how to invert these relations in order to express the parameters λ, D qq , D pp , D qp as a function of the evolved cumulants at an arbitrary time. The solution of Eqs. (8) is given by [21,22] where η 2 = δ 2 − ω 2 . If η 2 < 0 we can set η = iΩ and the previous equations hold again with trigonometric instead of hyperbolic functions. The coefficient λ can then be obained by inverting Eqs. (10). The elements of the diffusion matrix can be retrieved from the second set of equations (9), whose solutions can be expressed in a compact form as where ¿From the invertibility of matrices T (T 2 = 1) andK = K −1 e Kt − 1 (invertible for bounded K also if some of its eigenvalues are 0), we can derive the expression of D qq , D pp and D qp using Eq. (11): We emphasize that the time t at which we are considering the cumulants is completely arbitrary. For instance, the expression of the coefficients D qq , D pp , D qp in terms of the asymptotic second cumulants and the parameter λ reads: IV. CUMULANTS RECONSTRUCTION THROUGH TOMOGRAPHY In this section we introduce a procedure based on symplectic tomography in order to measure the first and second cumulants of a Gaussian wave packet at an arbitrary time t. This will allow us to indirectly measure the parameters λ, D qq , D pp , D qp , them being functions of the evolved cumulants at an arbitrary time (see previous section). The tomographic approach is very useful when dealing with a phenomenological master equation of the form of Eq. (4), as the dependence of the coefficients of the master equation from the physical parameters is in principle unknown. A. Symplectic tomography Given a quantum stateρ(t) its Wigner function reads: If the system dynamics is described by the master equation (4), and the initial state is Gaussian, the Wigner function preserves the Gaussian form of the state. Indeed, it can be expressed as a function of its first and second order momenta: Let us now consider the line in phase-space The tomographic map of a generic state along this line, i.e. its Radon transform, is given by: ¿From equation (17) it follows that for a Gaussian wave packet the Radon transform can be explicitly written as: , (20) with the following constraint on the second cumulants: This constrain is obeyed for each value of the parameters µ and ν iff ∆q 2 t ∆p 2 t − σ(q, p) 2 t > 0. This inequality is always satisfied as a consequence of the Robertson-Schrödinger relation. Eq. (19) also implies a homogeneity condition on the tomographic map: \|c\| (cX, cµ, cν) = (X, µ, ν). This condition can be used in the choice of parameters µ, ν. In fact, if one uses polar coordinates (r, θ), i.e. µ = r cos θ, ν = r sin θ, the homogeneity condition can be used to eliminate the parameter r. From Eq. (18) it emerges that the coordinates of the phase space need to be properly rescaled in order to have the same dimensions. For instance, we can set q → mω q and p → 1 mω p. In particular if ω = 0, i.e. for a free particle interacting with the environment, we can choose the same rescaling with a fictitious frequency defined by ω = ∆p 2 0 /2m, imposing q → ∆p0 √ 2 q and p → 1 √ 2∆p0 p. In general, every rescaling assigning the same dimensions to q and p is suitable for our purpose. B. From tomograms to cumulants Let us now consider the tomograms corresponding to two different directions in phase space, i.e. to two different couples of parameters (µ, ν), e. g. X = q and X = p. These lines in phase space are associated respectively to the position and momentum probability distribution functions: ¿From Eqs. (22)- (23) we see that the tomographic map depends only on a single parameter X. This reduces the dimensionality of the problem with respect to the Wigner function, that is a function of both p and q. The lines individuated by the choices (µ, ν) = (1, 0) and (µ, ν) = (0, 1) correspond to tomograms depending on the time average and variance respectively of position and momentum. In order to determine the latter quantities we have to invert Eq. (22) and (23) for different values of X, i.e. for a given number of points to measure along a tomogram. Thus, our first goal is to determine the number of tomograms required to measure the cumulants of our Gaussian state. To answer this question, we first focus on the direction µ = 1, ν = 0. In Fig. 1 we plot the Wigner function of our system at a generic time t and some straight lines along the considered direction. In Fig. 2 we plot the GSP tomogram defined by Eq. (22). Inverting Eq. (22), we obtain: Using the value of the tomogram (0, 1, 0) we can get q t as a function of ∆q t : If we know the sign of q t then we need only the value of the tomogram (0, 1, 0) to get q t , otherwise we need another point. Using Eq. (25), Eq. (22) becomes an equation for ∆q t only, and it can be rewritten as This equation is trascendental, therefore we will solve it numerically. We can graphically note in Fig. 3 that for each X and corresponding (X, 1, 0) there may be two values of ∆q t satisfying the previous equation. In order to identify one of the two solutions, it is enough to consider two points, {(X 1 , (X 1 , 1, 0))} and {(X 2 , (X 2 , 1, 0))}, and to choose the common solution for the variance. This is made clear by Fig. 3, where the ratio between right and left side of Eq. (26) for two different values of X is plotted. The common solution (i.e. when both ratios are equal to 1) is labeled ∆q t . As a consequence, whether we know or not the sign of the average q t , we need three or four points to determine q t and ∆q t in Eq. (22). Analogously, we need other three or four points for p t and ∆p t in Eq. (23). Let us now compute the covariance σ(q, p) t . To this purpose, we consider the tomogram: This is a Gaussian whose average value is already determined. Indeed, according to the previous steps, we need two more points of this tomograms to determine the spread (∆q 2 t + ∆p 2 t )/2 + σ(q, p) t , from which we can retrieve σ(q, p) t . Hence, we have shown that by means of eight or at most ten points belonging to three tomograms, the first and second order momenta of a Gaussian state can be measured at an arbitrary time t. One can then use these measured cumulants in order to infer the master equation parameters describing the system under investigation. We note also that we can reasonably infer that the number of tomograms needed to reconstruct the system density operator is minimized by employing Gaussian wave packets as a probe. Indeed these states have minimum uncertainty, and are the only states having positive Wigner function [29]. V. CONCLUSIONS In this paper we have proposed an approach to the study of open quantum systems based on quantum symplectic tomography. In many contexts the reduced dynamics of a system coupled with its environment is modeled by phenomenological master equations with some general features, but with unknown parameters. Hence, it would be highly appealing to find a way to assign some values to these parameters. We have tackled this problem for a wide class of Markovian master equations, which are the Gaussianshape-preserving ones. We have proved that it is possible to retrieve their unknown parameters by performing a limited number (ten at maximum) of time-independent measurements using Gaussian wave packets as a probe. This result leads to some interesting applications. Once retrieved the unknown master equation coefficients, it is possible to compute the dynamical evolution of any physical quantity whose analytical expression is known. The indirect-measurement scheme we propose could be then employed to make predictions on system loss of coherence due to the external environment. In order to perform this kind of analysis one can consider some quantities such as the spread and the coherence length in both position and momentum [30], provided their analytical expressions are available for an arbitrary time t (e.g. see Ref. [28]). Working in the coherent state representation, the evolution of the system of interest from an arbitrary initial state can be in principle predicted. Therefore, it is possible to perform the proposed indirect analysis of the decoherence processes. For example, if we consider an initial Schrödinger-cat state, highly interesting due to its potentially long-range coherence properties and its extreme sensitivity to environmental decoherence [31], we can re-write it as a combination of four Gaussian functions. Therefore, due to the linearity of the master equation, it can be possible to derive analytically the state evolution and to analyze its loss of coherence by means of the procedure we propose. VI. ACKNOWLEDGEMENTS We warmly thank Dr. P. Facchi, Prof. G. Marmo and Prof. S. Pascazio for many interesting and useful discussions. In particular we thank Prof. G. Marmo for his invitation at the University of Naples "Federico II" which gave us the chance of starting this work. APPENDIX A: ALTERNATIVE PROCEDURE Here we propose an alternative time-dependent procedure to compute the second cumulants of a Gaussian state, by means of tomograms, given the knowledge of the first cumulants time evolution. To this purpose we need to consider the following three tomograms: 2π ∆q 2 t /2 + ∆p 2 t /2 + σ(q, p) t . (A1) Inverting the previous equations one can infer ∆q t , ∆p t and σ(q, p) t from the knowledge of 1 , 2 and 3 . However, this procedure presents two drawbacks. In fact, the evolved averaged values q t and p t are required and we need tomograms evaluated on time-dependent variables. These problems do not arise in the time-independent procedure, based only on tomograms for which no a priori knowledge on the Gaussian state is required. Nevertheless, in this alternative time-dependent scheme only three tomograms are required.	2009-09-13T17:18:01.000Z	2009-09-13T00:00:00.000	{ "year": 2009, "sha1": "b181fddbb927f9b0231f556e509524e6a1a4f02a", "oa_license": null, "oa_url": "http://arxiv.org/pdf/0909.2250", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "b181fddbb927f9b0231f556e509524e6a1a4f02a", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Physics" ] }
119467930	pes2o/s2orc	v3-fos-license	Kramers equation algorithm for simulations of QCD with two flavors of Wilson fermions and gauge group SU(2) We compare the Hybrid Monte Carlo (HMC) and the Kramers equation algorithms for simulations of QCD with two flavors of dynamical Wilson fermions and gauge group $SU(2)$. The results for the performance of both algorithms are obtained on $6^312$, $12^4$ and $16^4$ lattices at a pion to $\rho$ meson mass ratio of $m_\pi/m_\rho \approx 0.9$. We find that the Kramers equation algorithm gives an equally good performance as the HMC algorithm. We demonstrate that the classical equations of motion used in these algorithms lack reversibility in practical simulations and behave like those of a chaotic dynamical system with a Liapunov exponent $\nu \approx 0.75$. Introduction Numerical simulations have been proven to be an important tool in obtaining information about non-perturbative properties of a physical system. Finding e cient algorithms is therefore one of the major research subjects. In particular, improved algorithms for models containing fermions, notably QCD, are needed. Up to now, the Hybrid Monte Carlo (HMC) algorithm 1] has been the most often used update scheme for fermionic systems. It is an exact Monte Carlo method, easily implementable on vector and parallel machines and it has been proven to work 1 well in practice. Still, simulations of fermionic systems turn out to be very di cult and time consuming. Improvements on this situation are therefore clearly welcome and alternatives to the HMC algorithm should be tried out. Recently, a new idea has been put forward by M. L uscher 2] that establishes another exact method for QCD simulations. It consists of a \bosonization" of the fermionic system. One arrives at a completely local bosonic action. The price to pay is the introduction of a number of scalar eld copies, where the number of copies typically is O(100) on a 16 4 lattice. Unfortunately, unexpected long autocorrelation times have been encountered in practical simulations within a QCD like theory 3,4], where the gauge group was chosen to be SU(2) instead of SU (3). So far, it remains to be seen whether the problem of these large autocorrelation times can be overcome. In this paper, we want to continue the search for fermion algorithms by studying the Kramers equation algorithm. This algorithm was proposed by Horowitz 5] already some time ago and is a generalization of the HMC algorithm. The main modi cation is that, in the refreshment of the momenta, a term proportional to the momenta themselves is added. It can be made exact by introducing a global accept/reject step. At least for a free eld theory, it can be shown to have a dynamical critical exponent of z = 1. However, in contrast to the HMC algorithm, this result can be obtained without going to the large trajectory length limit. The hope is therefore that, as far as the critical slowing down is concerned, it works equally well as the HMC algorithm. However, due to the shorter trajectory lengths, it might consume less computer time. The Kramers equation algorithm was introduced and discussed in 5]. A particular implementation and further discussion can be found in 6], where a test of the algorithm was performed on the 2-dimensional Gross-Neveu model. The results in 6] indicate that the Kramers equation performs as well as the HMC algorithm. A test of the Kramers equation algorithm for QCD has not been performed so far. Here we want to ll this gap and study the Kramers equation algorithm in QCD with SU(2) gauge group. The reason why we have chosen SU(2) instead of SU(3) is that we want to continue the algorithm tests as initiated in 3,4] with L uscher's fermion algorithm. Our results for Wilson QCD show that, in the actual computer time, the Kramers equation algorithm performs at least as well as the conventional Hybrid Monte Carlo algorithm. Another aspect which is in favor of the Kramers equation algorithm, is the lack of reversibility of the discretized classical equations of motion in the HMC algorithm. Since for lattices used in todays simulations, the number of steps within a trajectory can reach O(100) 7], one may fear that due to accumulations of rounding errors, the HMC algorithm lacks its reversibility property, necessary for the detailed balance condition. Indeed, in numerical experiments violations of reversibility have been observed 8]. In the Kramers equation algorithm, this e ect is drastically reduced since only one step in the leapfrog integration is used. This paper is organized as follows. After de ning our model in the next paragraph, we recapitulate shortly the HMC algorithm in section 2. In section 3, we introduce the Kramers equation algorithm. Two improvements we have used, namely preconditioning and a better leapfrog integration scheme as proposed in 9] are explained in section 4. The performance tests of the Kramers equation against the HMC algorithm are presented in section 5. Section 6 is devoted to the investigation of reversibility in the HMC algorithm and we will conclude in section 7. The theory that we would like to study is the standard lattice Wilson QCD with gauge group SU(2). We will work on a 4-dimensional euclidean space-time lattice with volume = L 3 s L t . We introduce gauge elds U (x) 2 SU(2) where = 0; 1; 2; 3 designates the 4 forward directions in space-time and quark elds Aa (x) where A,a and are avor, color and Dirac indices, respectively. The full partition function for our model is given by, Z = Z DUD D exp ( S g S w ) ; (1) where the gauge action S g and the Wilson fermion action S w are given by: The Wilson di erence operator D which appears in the above expression is given by (setting the Wilson parameter to one): and U P is the usual plaquette term on the lattice. In the following, we will consider two degenerate avors of Wilson fermions. As usual, for the simulations the fermion determinant is written in terms of Gaussian scalar elds such that the path integral reads Z = Z DUD y D e S eff ; S eff = S g + y (M y M) 1 ; (4) with M = 2 (D + m) the fermion matrix. The Wilson hopping parameter is related to the bare quark mass via = (8 + 2m) 1 . Hybrid Monte Carlo Algorithm Let q represent some stochastic variable, i.e. the gauge link elds and the scalar elds of eq. (4) in our case. Let us furthermore introduce a ctitious Monte Carlo time t and a set of momenta p which are conjugate to q. In the HMC algorithm, the system de ned by the 3 euclidean action S(q) evolves then according to (stochastic) Hamilton's equations of motion which read, considering time to be continuous for the moment, _ p = H q ; _ q = H p : (5) Here the Hamiltonian H = 1 2 p 2 + S(q) and the initial momenta are obtained from random numbers with Gaussian measure of unit variance. A leapfrog integrator 1] with N md molecular dynamics steps and a discrete step size is used to integrate eqs. (5) numerically. The product N md is called the trajectory length and a trajectory consists of N md molecular dynamics steps. In the simulations N md = O(1). In the HMC algorithm, due to the nite step size errors of the leapfrog integration, a global accept/reject step at the end of a trajectory is needed to ensure exactness of the algorithm. In a free theory, one can show that the acceptance rate of the Hybrid Monte Carlo algorithm is given by 10, 11]: P acc erfc(cN md 3 p ) ; (6) where erfc is the error function and c a constant. If the total trajectory length N md is xed, an increasing will drive the acceptance rate to zero exponentially. Therefore, one should take as large a step size as possible, while keeping the acceptance rate at a reasonably high level. To maintain a constant acceptance rate, one also has to scale the step size according to 1=4 . In our simulations, we have tried to maintain the acceptance rate at 80 to 90 percent level. Kramers equation algorithm The Kramers equation algorithm for simulating quantum eld theories was introduced and described in 5]. It amounts to add a second order time derivative term to the usual Langevin equation and originates from the theory of Brownian motion (see 12] for a discussion of the Kramers equation in this context). The equations describing the time evolution of the system are very similar to eqs. (5) and read _ p = H q p + (t) ; _ q = H p ; (7) where the stochastic variables (t) are the so-called \white noise". Of course, eqs. (7) are to be understood only on a formal level. We see that the main di erence between eqs. (5) and eqs. (7) is the white noise term and an addition of a friction term which introduces a new tunable parameter . In this paper, we adopt a particular variant of the Kramers equation algorithm 5, 6] which in its discretized version consists of the following steps. 1.) Generate momenta p 1 according to a Gaussian distribution with zero mean and unit variance P(p 1 ) / e p 2 1 2 : (8) Given the initial con guration (q 1 ; p 1 ), 2.) update the momenta, using a discretized time step p = e p 1 + p 1 e 2 ; (9) where is again selected from a Gaussian distribution with zero mean and unit variance. 3.) Leapfrog integrationp = p + 2 F(q 1 ) ; q 2 = q 1 + p ; p 2 =p + 2 F(q 2 ) ; (10) where F = S= q is the force. 4.) Perform a Metropolis test, by accepting the candidate con guration with probability P(q 1 ; p ! q 2 ; p 2 ) = minf1; e H(q 1 ;p) H(q 2 ;p 2 ) g : (11) On rejection, set q 2 = q 1 ; p 2 = p ; (12) where the negation of the momenta is necessary to guarantee exactness of the algorithm. In the appendix, we show that the above scheme ful lls the stability criterion, which {in combination with ergodicity{ ensures the convergence to the ground state probability distribution 13]. In the simulations, steps 2.)-4.) are repeated k-times before the momenta are refreshed again in step 1.). In our simulations, a value of k = 4 was chosen. Obviously, in the limit = 1 and k = 1, the above scheme reduces to a one step Hybrid Monte Carlo algorithm. A continuum free eld analysis of eqs. (7) 5, 6] reveals that the exponential autocorrelation time behaves as 1=! min , where ! min is the slowest mode of the system, i.e. the inverse correlation length . This behavior, corresponding to a dynamical critical exponent of z = 1 as in the HMC algorithm, is assumed at = 2! min . The remarkable property of the Kramers equation algorithm is that this result can be obtained already for short trajectory lengths, whereas for the HMC algorithm, a value of z = 1 can only be reached in the large trajectory length limit 11,14]. This property of the Kramers equation algorithm has important consequences. First, one obviously saves computer time per trajectory. Second, since we have only one step in the trajectory, e ects of the (non)-reversibility 5 in the HMC algorithm (see Section 6) are drastically reduced. Third, according to the free eld analysis of the tuning of the step size (6), the step size is expected to scale like / 1=6 . This softer volume dependence of the step size is indeed seen in our practical simulations. The drawback, of course, is that the trajectory lengths become shorter, resulting in larger autocorrelation times. Whether the advantages mentioned above will merit the increase of the autocorrelation times, will be investigated in section 5, where we give results for the performance of the HMC and the Kramers equation algorithms in simulations of QCD with gauge group SU(2). In the next section, we will rst explain two improvements that we have implemented for both the HMC and the Kramers equation algorithms. Improvements Even-odd Preconditioning Preconditioning 15] is by now standard for simulations in QCD. We write the fermion matrix as M 0 @ 1 D eo D oe 1 1 A : (13) The nondiagonal part of the fermion matrix D eo only connects the odd lattice points with even lattice points and similarly the matrix D oe only connects the even lattice points to the odd lattice points. The preconditioned matrixM is now and the path integral (4) can be written equally in terms of the preconditioned matrixM. The preconditioned matrix has two advantages over the original fermion matrix. The rst is a reduction of the memory requirement. SinceM only connects odd with odd (or even with even) sites, we save a factor of two in the memory requirement. The second advantage is that the matrixM yM , which is used in the simulations, is better conditioned than the original matrix M y M. Let us denote max and 0 to be the largest and the lowest eigenvalue of the matrix M y M and~ max ,~ 0 will correspond to the largest and the lowest eigenvalue of the matrixM yM . One can show that q max = jjMjj 1 + jjDjj 1 + 8 ; (15) and similarly q~ max = jjMjj 1 + 64 2 , where the notation jj:jj stands for the l 2 -norm of the matrix 16]. The ratio (1 + 64 2 )=(1 + 8 ) is close to one for all practical values of in numerical simulations. Therefore, when comparing the condition number max = 0 of the matrix M y M with~ max =~ 0 of the preconditioned matrixM yM , the main di erence comes from the ratio of the lowest eigenvalue in the two cases, assuming that the largest eigenvalue is close 6 (4) 54 to the bound given above. It is expected that the lowest eigenvalue~ 0 of the preconditioned matrixM yM is about a factor of 4 larger than the lowest eigenvalue of the original matrix M y M. This is motivated by noticing that for any eigenvalue of the matrix M, ( 2 2 ) is an eigenvalue of the matrixM. Therefore, when j j is small, the absolute value of the corresponding eigenvalue ofM is about a factor of two larger than that of M. However, since the eigenvalues of M are not directly related to the eigenvalues of M y M, we have to test this expectation numerically. Indeed, the e ect is con rmed by our numerical simulation results. Since the number of Conjugate Gradient iterations is proportional to the square root of the condition number, we expect to save about a factor of two in computer time. We have tested the even-odd preconditioned version of the HMC algorithm on a small lattice (4 4 ) for various values at = 1:75 and compared with the version using the original matrix M y M. The results are summarized in table 1. The lowest eigenvalue 0 (~ 0 ) of the matrix M y M (M yM ) was measured by minimizing the Ritz functional, ( ) = jjM jj 2 =jj jj 2 , using a Conjugate Gradient technique 17,18]. One sees that indeed the average value of the lowest eigenvalue <~ 0 > ofM yM is always about a factor of 3:5 larger than < 0 > of the original matrix M y M. Correspondingly, the number of Conjugate gradient iterations N CG for each inversion of the matrix is decreased. The average plaquette values < U P > in both cases are also listed for comparison. A similar improvement was also found in the larger volume simulations. For the lattice size of 6 3 12 and = 2:12, = 0:15, we found the average value <~ 0 > to be larger by about a factor of 3:6 as compared to < 0 >. Sexton-Weingarten Integration Another improvement can be made to the leapfrog integration scheme used in the conventional Hybrid Monte Carlo algorithm. This has been suggested by Sexton and Weingarten in 9]. The basic idea is the following. The force F needed for the update of the momenta consists of two parts. One originates from the gauge staples and the other from the fermionic force. While the pure gauge part needs only moderate computer time, the fermionic force part includes a matrix inversion and consumes considerably more CPU time. One may, however, introduce di erent time scales for the leapfrog integration corresponding to the two force terms. For the pure gauge part, the leapfrog can be done with ner time steps. Although we then have to do more arithmetic operations for this part, we nally gain in the increase of the acceptance rate due to partial cancellations of the nite step size errors coming from the force. Consider the Hamiltonian H = 1 2 p 2 + S 1 (q) + S 2 (q) ; (16) for which the number of arithmetic operations required to evaluate the force due to S 1 is far less than that due to S 2 . Then, the partial time evolution operators are given by T 1 ( ) = exp( 1 2 L(S 1 )) exp( L( 1 2 p 2 )) exp( 1 2 L(S 1 )) ; T 2 ( ) = exp( L(S 2 )) : (17) In eq. (17) L is a linear operator, representing a symplectic integrator 9, 6]. Sexton and Weingarten suggest that one can de ne a full time evolution operator T( ) by: T( ) = T 2 ( 2 ) T 1 ( n ) n T 2 ( 2 ) : (18) As has been shown in 9], errors induced by nite time step sizes will be reduced by the above method due to ner step sizes, leading to a better integration scheme. A more complicated scheme can be constructed by adding one more T 2 insertion in each step. The scheme we used is given by the following formal expression: T( ) = T 2 ( 6 ) (T 1 ( 2 )T 2 ( 2 3 )T 1 ( 2 )T 2 ( 3 ) N md 1T 1 ( 2 )T 2 ( 2 3 )T 1 ( 2 )T 2 ( 6 ) ; T 1 ( 2 ) = T g ( 12n ) T k ( 4n )T g ( 3n )T k ( 4n )T g ( 6n ) n 1 T k ( 4n )T g ( 3n )T k ( 4n )T g ( 12n ) : (19) In the above formula, a factor of T 2 ( ) stands for an update of the momenta by a step , taking into account the force coming from the fermionic part only. Similarly, T g ( ) stands for an update of the momenta by a step due to the gauge part alone and T k ( ) stands for an update of the gauge elds. In the practical simulation, the integer n is chosen to be around 4. Increasing this number will not result in a further improvement of the acceptance rate 9]. The total trajectory length in such a sequence of updates is t = N md and the number of matrix inversions is equal to (2N md + 1). More complicated schemes than the above will require more T 2 insertions in each step and will not lead to further improvements. The tests of the above scheme as given in eq. (19) on the small lattice (4 4 ) runs turned out to be quite promising. In table 2, we have listed the results of the tests for the conventional 8 (2) integration scheme and the Sexton-Weingarten scheme for the HMC algorithm. These simulations were done on a 4 4 lattice with = 1:75 and = 0:15. We xed the trajectory length to be one in all these runs, which is a reasonable value for the practical runs as well. Then, we systematically changed the number of steps of the trajectory hence the step size. The number of conjugate gradient iterations needed for each trajectory (N iter ) and the acceptance rate (P acc ) of the runs are listed in table 2. The ratio N iter =P acc should be minimized for optimal performance. From table 2 we see that, for the conventional leapfrog integration scheme, the best performance is reached at about 80 percent acceptance rate. The corresponding value of N iter =P acc is about 420. The Sexton-Weingarten integration scheme, however, can achieve a N iter =P acc ratio of about 270, which is about 50 percent better than the conventional scheme. Using this new integration scheme, we can increase the step size quite substantially. The e ect of these improvements on larger lattices is expected to be more pronounced 9]. We also tried the biconjugate gradient method. However, similar to the conclusions in 6], we did not nd an improvement on the APE computer. As mentioned in the introduction, we want to continue the search for an alternative to and hopefully better algorithm than the HMC algorithm. We therefore performed our tests at the same parameter values as chosen in 3, 4], i.e. = 2:12, = 0:15 and lattice sizes of 6 3 12, 12 4 and 16 4 . For the lattice size 16 4 , we only ran the Kramers equation algorithm. We have measured the average plaquette value < U p >, the average lowest eigenvalue <~ 0 > of the preconditioned matrixM yM , the pion correlation functions and the meson correlation functions in our runs. When we give values of the pion mass m and the meson mass m , we use as a de nition cosh m = C(L t =2 + 1) C(L t =2) ; (20) where C is the correlation function for the pion or meson. At the chosen values of and , we obtain a ratio of m =m 0:9. In order to get an estimate for the performance of the Kramers equation and the HMC algorithms, we have studied the integrated autocorrelation times of the plaquette U p , the lowest eigenvalue~ 0 of the preconditioned matrixM yM , the pion C and the meson C correlation functions at distance L t =2 + 1. We list the values of < U p >, <~ 0 >, m as well as m in table 3 for both algorithms, demonstrating that they give consistent results. In an ideal situation, the integrated autocorrelation time int of some observable O is ob- where O (t) is the connected autocorrelation function of the observable O. In reality, the summation can, of course, only be taken over a nite data set. Summing over the whole data set, assuming that the statistics is much larger than the autocorrelation time, may also be misleading, since the noise in the tail of the autocorrelation functions gives an unwanted bias. Therefore, we have used the \window" method suggested by Sokal 13] to extract the integrated autocorrelation times. This method basically suggests that one should only sum the autocorrelation function up to a certain distance T cut , thereby introducing the integrated autocorrelation function In practice, the integrated autocorrelation time of some observable O, int (O), is determined by searching for a plateau behavior of the corresponding integrated autocorrelation function in the range of T cut = int (O) 4 10. The cut parameter T cut should also satisfy T total =T cut 1, where T total is the total number of measurements. In Fig. 1, we plot the integrated autocorrelation function for~ 0 to give an example. One indeed observes a plateau behavior where the integrated autocorrelation function does not depend on the value of T cut . In table 4, we give the results for the integrated ( int ) and exponential ( exp ) autocorrelation times for the plaquette value U p and the pion correlation function C at distance L t =2 + 1 measured per trajectory. For the runs on the 6 3 12 lattice, we ran eight copies of the program on single nodes of the APE Q1 machine. This allowed us to determine the integrated autocorrelation times for each copy independently, from which we obtained the error quoted in table 4. For the 12 4 and 16 4 lattices, the error was obtained by splitting the total run into smaller parts and analyze these parts independently. We checked the values of the integrated autocorrelation times as given in table 4 by a blocking analysis of the error of the observables. Our statistics is su ciently large that we can reach an error plateau. The integrated autocorrelation times determined in this way are in complete agreement with the ones obtained from the integrated autocorrelation functions. In addition, we determined the exponential autocorrelation times exp by tting the time behavior of the autocorrelation functions to an exponential. Again, exp int (see table 4) in all cases. In the last column of table 4, we give the product of the integrated autocorrelation time int (U p ) for the plaquette value and the average number of Conjugate Gradient iterations per trajectory. This quantity gives a direct estimate for the computer time consumption of both algorithms and hence should be used for comparison. Two comments are in order concerning table 4. First, comparing the results for the autocorrelation times on the 6 3 12 and the 12 4 lattices, one notices int (U p ) to be smaller on the larger lattice. This demonstrates that, by a more careful tuning of the parameters, substantial improvements can be achieved. As we started our investigation with the 6 3 12 lattice, we did not choose the optimal values for the parameters. Second, on the 12 4 lattice, we ran the Kramers equation algorithm at two values of : (a) = 0:5 and (b) = 2:0. The behavior of the integrated autocorrelation function is given in Figure 2. It shows that tuning of this free parameter in the Kramers equation algorithm can change the autocorrelation times by almost a factor of two. It is interesting to note that, whereas the autocorrelation times of the fermion correlators are somewhat increased, the ones for U p and~ 0 are decreased. We found a similar e ect of the tuning of in runs on 8 3 12 lattices. Again, while increasing , the autocorrelation times for the fermionic correlators increased whereas the ones for U p and~ 0 dropped. The detailed balance proof of the HMC algorithm requires exact reversibility 1] of the discretized equations of motion. Of course, on a computer, the algorithm runs with only a nite precision and one may wonder, whether accumulations of rounding errors can lead to violations of the exact reversibility. This is particularly true for todays simulations where O(100) molecular dynamics steps are used to obtain one trajectory 7]. One may investigate this question by taking an initial con guration C ini , integrating it along a trajectory and then integrating it back to reach the end con guration C end . Indeed, measuring several observables on C ini and C end discrepancies were encountered 8]. We decided to study this problem in numerical simulations using the HMC algorithm. The nonlinear nature of Hamilton's equations of motion that are used in the HMC algorithm suggests that the dynamics is chaotic. To test this proposal, let us de ne a quantity, jjdUjj, to measure the di erence between two gauge eld con gurations kdUk 2 = 1 4 X x; ;a (U a (x) V a (x)) 2 : Here U a (x); V a (x) are two SU(2) gauge link variables with lattice point index x, direction and group index a. To show that the suggested chaotic behavior is really a property of Hamilton's equations of motion, we proceeded in the following way. Given a gauge eld con guration obtained in the course of some run, we added a small noise U (x) to the gauge eld variable U (x), such that V (x) = U (x)+ U (x). Then, we took both con gurations and iterated them according to the leapfrog integration scheme used in the HMC algorithm. We measured kdUk after some number of steps N md in the leapfrog integration. If the system is chaotic, we expect that asymptotically ( N md 1) kdUk = Ae N md : (24) In eq. (24) is the {to be determined{ Liapunov exponent, characterizing a chaotic system and is the step size used in the program. In Fig. 3a, we show lg(kdUk) as a function of N md , where lg stands for the 10-based logarithm. Clearly, an asymptotic linear behavior is seen, giving a Liapunov exponent of = 0:78. The step size was chosen to be = 0:01 and the lattice is = 4 4 . The data are obtained by averaging over 10 independent gauge eld con gurations. Errors are smaller than the symbol size. Obviously, the noise that has been added is faking some rounding errors that occur in the actual simulations. If such rounding errors appear, we predict then that the errors blow up 14 Figure 3: We plot lg(kdUk) as de ned in eq. (23). In (a) jjdUjj is obtained by adding a small noise U to the initial con guration and then iterate the leapfrog integration. In (b) jjdUjj is obtained by reversing the trajectory. exponentially according to eq. (24) with the Liapunov exponent as found above. To see whether this e ect really happens, we performed a Monte Carlo run with our dynamical Wilson fermion program as described above on 4 4 lattices. Every third trajectory, we reversed the time and integrated back, measuring kdUk using the initial con guration before starting the leapfrog integration and the nal con guration at the end of the reversed trajectory. By xing the step size = 0:03, we obtained kdUk as a function of the trajectory length N md . Indeed, we nd in Fig. 3b again a linear behavior of lg(kdUk) with a Liapunov exponent that is compatible with the previous one. These gures were obtained from runs on workstations with 32 bit arithmetic. We repeated the runs with 64 bit arithmetic. We found the same asymtotic exponential behavior with the only di erence that the amplitude A in eq. (24) gets smaller and the asymptotic behavior 15 sets in much later. We also measured the Liapunov exponent on the APE machine which has 32 bit arithmetic but where we used double precision or Kahan summations 20, 21] to avoid rounding errors in scalar products. Again, we nd a Liapunov exponent around = 0:75. Notice that, for smaller trajectory lengths, the value of jjdUjj is less than what the asymptotic formula (24) predicts. For example, for a 4 4 lattice, when comparing the value of jjdUjj at trajectory length one (typical for HMC algorithm) and at trajectory length 0:1 (typical for the Kramers equation algorithm), we found a factor of 2:5 di erence. This factor will increase further for larger lattices. From this point of view, the Kramers equation algorithm appears to be a safer algorithm than the HMC algorithm. To see whether the Liapunov exponent is really representing the chaotic behavior in the continuum time, we tried to extract it for di erent values of the integration step size . We nd that is independent of the step size, provided the step size is small enough. Therefore, we expect the Liapunov exponent to represent the property of the continuum time integration. We have also investigated the dependence of the Liapunov exponent on bare parameters. On the 4 4 lattice, we have performed the study of the quantity kdUk at various values for = 1:75. We found that the Liapunov exponent has very weak dependence on . Runs on a larger 6 3 12 lattice revealed again a value of 0:75, strengthening our conclusion that it is representing the continuum time chaotic behavior of the classical equations of motion. Conclusion In this paper, we compared the performance of the HMC and the Kramers equation algorithms in simulations of QCD with two avors of dynamical Wilson fermions and gauge group SU (2). Adopting the same improvements for both algorithms, preconditioning and the Sexton-Weingarten 9] leapfrog integration scheme, we found that the Kramers equation algorithm performs as well as the HMC algorithm, as can be read o from table 4. Although the integrated autocorrelation time is larger for the Kramers equation algorithm (see table 4), the average number of Conjugate Gradient iterations per trajectory is much less, resulting in a comparable performance for the Kramers equation algorithm. Therefore, we think that the Kramers equation algorithm should be used more often in QCD simulations so as to obtain more experience with it. In particular, the tuning of the parameter in the Kramers equation algorithm seems to be important to obtain optimal performance for larger lattices. As the description of the Kramers equation algorithm in section 3 shows, it is easy to change an existing HMC code to a Kramers equation one. We demonstrated that Hamilton's equations of motion used in the HMC algorithm, due to their nonlinear nature, behave like those of a chaotic dynamical system. Therefore, if rounding errors occur during a simulation, they accumulate and blow up exponentially / expf N md g with N md the trajectory length and the Liapunov exponent which we determined to be 0:75. Therefore, the HMC algorithm may lack the reversibility property. Since in the Kramers equation algorithm, a trajectory consists of only one step, this e ect is substantially reduced.	2019-04-14T02:30:11.159Z	1995-06-13T00:00:00.000	{ "year": 1995, "sha1": "38639daea6c3f0c5f101ad93b47493f38f38ddfb", "oa_license": null, "oa_url": "http://arxiv.org/pdf/hep-lat/9506020", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "38639daea6c3f0c5f101ad93b47493f38f38ddfb", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
259521448	pes2o/s2orc	v3-fos-license	Changes in physical properties of some proprietary safflower ( Carthamus tinctorius L.) cultivars after film coating application Safflower is one of the economically important oil plants. There are some studies on safflower, but none on its seed properties. This study was carried out in the Biosystem Engineering laboratories of the Faculty of Agriculture of Bingöl University in 2021. The seeds of four different proprietary safflower cultivars were randomly sampled, and some of their physical properties were examined under two separate groups, that is, control and film-coated. The film coating material was applied in a single layer, and its effects on the seed size were compared between the study groups. The coating dose should be at a level that will not adversely affect the germination and emergence performance of the seed. As a result of the study, it was found that the safflower seeds were long and oval in shape. This study is important in that its results will help the researchers carry out mechanization and breeding studies on safflower. Introduction Safflower (Carthamus tinctorius L.), a valuable oil plant with an annual herbaceous structure, has been cultivated for about 3000 years (Koç et al. 2017;Culpan and Arslan, 2018;Baydar and Erbaş, 2020). It has a high tolerance to conditions such as drought and low temperature, thanks to its behaviour in the consumption of water and nutrients in the soil depending on its root structure. Due to these features, safflower is important in making use of fallow lands, controlling erosion control, and expanding cultivation areas (Baydar and Erbaş, 2020;Esendal, 1981;Bayraktar, 1991;Geçit et al. 2018;Köse et al. 2021). Safflower is sown in two seasons, summer and winter. Depending on the irrigation conditions, it can be thorny or thornless and has yellow, white, red, and orange flowers with a height of 50-150 cm. Its seeds can be white, brown, striped white, and rarely black (Culpan and Arslan, 2018;Nacar et al. 2016;Toprak and Tunçtürk, 2018). The flowers and seeds of the safflower plant are used in many sectors. Safflower oil is used in biodiesel production as well as in the food, medicine, textile, and paint industries (Kırıcı, 1998;Yılmaz and Tunçtürk, 2018). It is also used as animal feed and ornamental, medicinal, and aromatic plant (Koç and Güneş, 2021). The unsaturated fatty acids (oleic acid and linoleic acid) account for about 90% of the total fatty acid content of the safflower seed. It contains 32-34% carbohydrate, 14-15% protein, 5-8% moisture, and 2-7% ash (Weiss, 2000; Çoşge et al. 2007;Kalafat et al. 2009). Since safflower is economically valuable and durable, mechanization trials and breeding studies have been carried out to expand its cultivation areas. In these studies, it was aimed to place the seeds in the soil with the least product loss by choosing the appropriate tools, machines, and systems for the physical properties of the seeds. In addition, in breeding studies, the characteristics of seeds are important in the development of new varieties. Seed technologies are used to increase the resistance of seeds to ecological conditions and increasing planting opportunities. Researches are being carried out to increase seed quality through methods such as the use of plant nutrients, film coating, and pelleting. In this study, the seeds of different safflower cultivars were examined separately under two groups, control and film-coated, and some physical properties of the seeds were determined. Material and method This study was carried out in the laboratories of the departments of Biosystem Engineering and Field Crops, Faculty of Agriculture, Bingöl University in 2021. In this study, the seeds of four different Safflower cultivars (Balcı, Dinçer, Yektay, and Yenice) were used as plant material. The safflower seeds were obtained from the Transitional Zone Agricultural Research Institute, Ministry of Agriculture and Forestry, Republic of Türkiye. The seeds were examined in two groups, control group and the film-coated group. SPSS v.22 statistical packages was used to analyze the data, and the TUKEY test was used to determine the differences. The statistical significance was set at p<0.05. In the study, a water-based polymeric material from a commercially sold brand in the market was used in the film coating application. The safflower seeds were film coated (single layer) by spraying and then dried at room temperature (approximately 24°C) in a moisture-free, dry, and dark environment for 24 hours (ISTA, 2007). The length (mm), width (mm), and surface area (mm 2 ) of the seeds were measured using a stereo microscope (Nikon SMZ 745T) (Dumanoğlu and Geren, 2020;. The obtained data were evaluated according to the seed classification criteria specified in Table I. The mean arithmetic diameter of the seeds (mm) (), their mean geometric diameter (mm) () and sphericity () [L: Seed length (mm) W: Seed width (mm)] were calculated using the data (Mohsenin, 1970;Alayunt, 2000;Kara, 2012). In addition, 1000-grain weight was measured in triplicate randomly for both groups (Dumanoğlu and Öztürk, 2021). Results and discussion We examined some physical properties of four different safflower cultivars under two separate groups (control and film-coated) and found that, in the control group, the seeds of the variety Balcı had the highest length and surface area (7.599 mm; 22.027 mm 2 ) and those of the variety Yenice had the lowest values (6.502 mm; 18.792 mm 2 ); the seeds of the variety Dinçer had the highest width (3.721 mm) and those of the variety Yektay had the lowest width (3.508 mm) (Table II) In the control group, the seeds of the variety Balcı had the highest mean of both arithmetic (5.572 mm) and geometric (79.738 mm) diameter and sphericity (10.700) and those of the variety Yenice had the lowest (5.062 mm; 56.458 mm; 8.589). The variety Balcı was found to have the highest 1000-grain weight (40.434 g) and the variety Yenice had the lowest (34.843 g) ( Table II). As for the film-coated group of four different safflower varieties, it was found that the seeds of the variety Balcı had the highest length (7.427 mm) and those of the variety Yektay had the lowest length (6.452 mm); the seeds of the variety Yektay had the highest width (3.661 mm) and the seeds of the variety Yenice had the lowest width (3.503 mm); and the seeds of the variety Yenice had the highest surface area (21.248 mm 2 ) and those of the variety Yektay had the lowest surface area (18.709 mm 2 ). It was found that, in the film coated safflower cultivars, the seeds of the variety Yenice had the highest mean arithmetic diameter (5.480mm) and those of the variety Yektay had the lowest (5.056 mm); the seeds of the variety Yenice had the highest mean geometric diameter (76.184 mm) and those of the variety Yenice had the lowest (55.729 mm); and the seeds of the variety Yenice had the highest sphericity (10.082) and those of the variety Yektay had the lowest (8.561). In the film-coated group, the variety Balcı was found to have the highest 1000-grain weight (40.562 g) and the variety Yenice had the lowest (35.645 g) (Table I). Based on these results, it can be asserted that all the safflower seeds in the control group and the film-coated group had a long and oval seed structure. As a result of the measurement and calculation processes, it was found that the safflower varieties in the control and film coated groups had similar values in all parameters. This is primarily due to the application of a single layer film coating. There was not much difference between the coated and uncoated seeds in terms of size, and the film material did not cover the seed like a barrier. This is extremely important for germination and emergence performances. In addition, it should be noted that the seeds randomly selected for film coating were slightly smaller than those in the control group. The thousand grain weights found in this study were in agreement with those reported in previous studies. Koc et al. (2017) examined the yield components of five safflower lines (106-2, 11-1, 77-1-d, 89-1-c, BDYAS-9) developed by selection between 2015 and 2016 in Konya and some standard safflower varieties (Göktürk, Balcı, Linas, Olas, Dinçer) and reported that the 1000-grain weights of the varieties ranged from 36.5 g to 43 g. On the other hand, in their study, Geçit et al. (2018) reported that the safflower varieties had a 1000-grain weight of 30-45 g. The 1000 grain weights of the safflower cultivars examined in the present study were similar to those reported in these studies.. The safflower seeds were also coated using a film coating material in order to improve their properties and quality, and the film coated seeds were compared with those in the control group. Measurement and calculation processes, which form the basis for mechanization and breeding research, were carried out in this study. Based on the results of the study, it can be asserted that the variety Balcı in the control group and the variety Yenice in the film coated group came to the fore. This study is important in that it will form a basis for future research on seeds of oil plants.	2023-07-11T18:32:06.620Z	2023-06-22T00:00:00.000	{ "year": 2023, "sha1": "7aec57fe3f260c8c265db92633b79a031585a324", "oa_license": "CCBYNC", "oa_url": "https://www.banglajol.info/index.php/BJSIR/article/download/60570/45007", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "c2e1a0b91ef24f8ef63861fdd0792700b977c156", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [] }
6544463	pes2o/s2orc	v3-fos-license	Determination of Elemental Composition of Malabar spinach, Lettuce, Spinach, Hyacinth Bean, and Cauliflower Vegetables Using Proton Induced X-Ray Emission Technique at Savar Subdistrict in Bangladesh The concentrations of 18 different elements (K, Ca, Fe, Cl, P, Zn, S, Mn, Ti, Cr, Rb, Co, Br, Sr, Ru, Si, Ni, and Cu) were analyzed in five selected vegetables through Proton Induced X-ray Emission (PIXE) technique. The objective of this study was to provide updated information on concentrations of elements in vegetables available in the local markets at Savar subdistrict in Bangladesh. These elements were found in varying concentrations in the studied vegetables. The results also indicated that P, Cl, K, Ca, Mn, Fe, and Zn were found in all vegetables. Overall, K and Ca exhibited the highest concentrations. Cu and Ni exhibited the lowest concentrations in vegetables. The necessity of these elements was also evaluated, based on the established limits of regulatory standards. The findings of this study suggest that the consumption of these vegetables is not completely free of health risks. Introduction Minerals containing food plants are of prime concern as these plants contain the prime resource of essential elements (without hydrogen and oxygen) in the human nutrition [1]. A little quantity of these minerals originates from soil, water, air, and rocks. The elements which are confined in the plants are obtained directly through fruits and vegetables, or indirectly through milk and meat from those animals that consume the plants. Trace elements do not provide any calorie to the human body but play an important role in the adjustment of the body pH, osmotic regularity and are used as coenzyme which regulates the elemental metabolic processes in the human cell. In recent years trace elements have become essential to the biological cell cycling and homeostasis process [2,3]. In the human body excessive accumulation of potentially toxic elements or lack of essential trace elements for a long period is responsible for various diseases [4]. Improvement of the mineral regulation, reduction of cardiovascular diseases, and certain cancer risks can be achieved through the increase in the consumption of various fruits and vegetables. The levels of trace elements in fruits and vegetables may be regulated by various factors like genetic, soil chemistry and the pattern of agricultural system [5]. The edible components of the plants like leaves and stems and the concentrations and the types of elements available in these parts are greatly influenced by atmospheric contamination. If tubers and roots are consumed as food, the elements that are found in the soil may reveal the maximum effect on the levels of minerals of fruits and vegetables, whereas elements confined in fruits are prone to be significantly influenced by the variations in the geochemical settings. Minerals can be divided into three groups: major physiological elements, trace elements, and nonessential elements [6]. Major physiological elements are phosphate, bicarbonate, Na, K, Ca, Mg, Cl, and so forth. Essential and trace elements are Co, Cr, Cu, Fe, I, Mn, Mo, Ni, S, Se, Zn, and so forth. Nonessential elements like As, Al, Br, Ba, Bi, Cs, Cd, Ge, Hg, Li, Pb, Rb, Sr, Si, Sm, Sb, Sn, Ti, Tl, and W are not currently considered essential, even though some of them have a beneficial pharmacological action in appropriate dosage [6]. The nonnutritive toxic minerals which are commonly known to have toxic properties even in small quantities (<100 ppm) are Pb, Sb, Cd, As, Se, and Hg [7]. For this reason, the estimation of both physiological and trace levels of elements obtained in food are significant for safety, efficacy, and nutritional values of food [4]. The aim of this study was the precise determination of the contents of minerals and heavy elements from the most known and available vegetables present in Bangladeshi market. Before selecting these vegetable samples, at first short discourse was performed with the vegetable vendors in some local markets at Savar. Vegetable vendors were asked to suggest some vegetables which are sold most. They provided some popular names of vegetables. Most of the people in Savar area eat these vegetables to receive nourishment and for the availability of these vegetables at affordable cost. This information proves that most important vegetables, which are mostly consumed by the people living in Savar area, were selected to perform this study. Consumers living in Savar area usually welcome these types of foodstuffs into their regimens. These foods are cheap and contain low fat. Moreover, they are very tasty, easy to find in the grocery store, and also suitable to cook. Both cooked and raw vegetables are very rich sources of different types of minerals and vitamins. After that, high nutrition values of these selected vegetables were ensured by studying different literatures. From the preliminary selected vegetables, finally the top 5 vegetables were selected for our research. Collection of the samples was performed several times a week from nearby markets in Savar area. Sample Preparation. Five different vegetables Basella alba (Malabar spinach), Lactuca sativa (lettuce), Spinacia oleracea (spinach), Lablab niger (hyacinth bean), and Brassica oleracea (cauliflower) were collected from different places of Savar area, Bangladesh. For each category of sample, we collected 10 vegetable samples. All determinations of essential and trace elements in collected vegetables were performed by the technique Proton Induced X-ray Emission (PIXE). The technique PIXE is being used in Accelerator Facilities (AF) Division of the Atomic Energy Centre, Dhaka (AECD), Bangladesh. The fundamental benefit of the PIXE method is to determine the concentrations (absolute or relative) of elemental constituents in the sample under investigation. The collected samples were washed thoroughly with distilled water and then dried in an oven at 80 ∘ C and consequently grinded into fine powder through using mortar and pestle. Then pellets having a diameter of 12 mm were made from powder of samples by using a 15 ton Perkin Elmer pellet maker. PIXE Technique. Proton Induced X-ray Emission (PIXE) is one of the important and widely used analytical techniques at MeV energy accelerators. Sven Johansson at the department of Physics, Lund University, first introduced this technique [8]. When charged particles with sufficient energy hit a sample, a vacancy in the inner shells of an atom may be created. For MeV ions the probability (cross section) for ejecting inner shell electrons is very high [8]. Such a vacancy can be occupied in a number of techniques and one of the processes may discharge X-rays with energy characteristic of that specific atomic number. In the PIXE technique Xrays are detected using semiconductors. An energy dispersive analysis of the detector signals can disclose the distinctiveness of different elements existing in the samples analyzed and more notably by quantifying the charge; that is, the number of incoming elements and the concentrations of the elements can be precisely quantified. PIXE technique has excellent detection limits, so the concentrations of the elements can be accurately quantified [9]. This technique was chosen because of its advantages. It is a nondestructive, very sensitive, and multielemental technique which permits rapid and simultaneous detection of elements between Na and U [10,11]. PIXE technique is the reliable measurement of toxic elements in the bioenvironmental samples in parts per million (ppm) levels and also supports detecting the presence of any essential elements. It also buttresses in making decision about conservation of the public health and improving environmental quality [8]. Instrumentation 2.3.1. Experimental Setup. The elemental analysis was carried out at Accelerator Facilities (AF) Division of the Atomic Energy Centre, Dhaka (AECD), Bangladesh. The prepared pellet samples were irradiated at the PIXE station for elemental analysis with 2.5 MeV collimated proton beam (size approximately of 2 mm) at a current of about 10 nA. The experimental samples were irradiated with approximately 10 C charge and it was obtained from the 3 MeV Van de Graff Accelerator. All experiments were done in the vacuum chamber. The vacuum in the range of the order of 2.0 × 10 −5 mbar is regularly achieved in the chamber. The turbo pumping system is used to evacuate the accelerator tube and rest of the beam line. A small turbo pump is being used with the IBA scattering chamber for vacuum purpose. Characteristic X-rays from targets were measured with a Si(Li) detector with respect to the beam line. Detector was connected to a multichannel analyser (MCA) in order to convert analogue data into digital. 170 m thick Mylar absorber was used in front of the X-ray detector for two reasons. Firstly, to save the X-ray detector from radiation damage from the scattered high energy particles from the sample and secondly to decrease the count rate in the X-ray detector which is very important in order to decrease pulse pile up and the dead time in the data-acquisition system [8]. Accurate charge measurement is very essential for the complete quantification of elemental concentrations of a sample [8]. The most imperative factors that were considered during charge measurements were loss of secondary electrons, loss of scattered particles, and charge build up in insulating samples. An electron flood gun was used in all experiments in order to prevent charge build up in the samples [12]. The charge measurement was actually done in the Faraday cup which was positioned at the end of the beam line after the chamber. The low elements present in the sample that produce most of the counts were compared to the tracer level of high elements in the sample. PIXE Calibration. GUPIX was chosen to analyse complex PIXE spectra of sample from a number of software packages available in this field. It was developed in the Guelph University, Canada. GUPIX uses all the modernized and best databases available like cross sections, fluorescence, stopping powers, Coster-Kronig probabilities and attenuation coefficients. It has many convenient options such as: the possibility to add invisible elements to the fit, matrix iteration, layer thickness iteration, analysis of any batch spectra, and so forth. It also possesses the possibility to include an energydependent calibration factor and low energy tailing descriptions of parameter of the X-ray detector in the database [13]. All of these measurements greatly improve the accuracy of the quantification of elemental concentrations of samples through using PIXE technique. Mainly the value method was followed during the PIXE calibration process [14,15] that is based upon the equation given below: where ( , ) is the measured X-ray yield computed by the fitting program. ( , ) is the theoretical X-ray yield per unit beam charge, per unit solid angle, and per unit concentration computed from the GUPIX database which includes the matrix correction and secondary fluorescence for the thick targets. is the measured beam charge or some value proportional to the charge. is the concentration of the element quoted by the manufacturer or measured by some other method. ( ) is the fractional transmission of X-rays through absorbers. is the intrinsic detector efficiency. is the product of the detector solid angle and any correction factor for the charge measurement. So in this case, also includes the effects of possible inaccuracy in the description of the detector solid angle, the thickness of the detector crystal, the thickness of the absorber layer, and so forth. It also includes any imperfection in the various databases used in GUPIX [16,17]. The energydependent -values and low energy tailing parameters are detector specific [13]. To evaluate the -values a whole set of thin Micromatter standard was irradiated. First, by using the actual solid angle as the value in the GUPIX the concentration values were calculated and then the thickness of the absorber was adjusted to get the closest concentration value, the manufacture quoted value, especially for those elements whose X-ray was sensitive to absorb that particular thickness. Then values were calculated by comparing the calculated concentration values to the manufacturer quoted concentration values multiplied by the detector solid angle [13]. The values were then plotted as function of X-ray energy, which was a scattered curve, and fitted by a suitable trend line to obtain an equation. Then this equation was used to generate a smooth set of values as function of Xray, which was stored inside the GUPIX library for further PIXE analysis of unknown samples. These values were used as function of energy and this was suggested by GUPIX. Depending on the element, the precision was well below 1% and the accuracy was within 5-10%. An excellent procedure has been established by Kim et al. [18] for determining the elemental composition of any biological sample. The limit of detection (LOD) meant the sensitivity of any measurement system. Limit of detection is clearly proportional to the solid angle. In PIXE analysis, detection limits are usually calculated by the following equation: PIXE Standardization Procedure. Calibration and analytical methodologies of the PIXE technique were performed by analyzing appropriate reference material called IAEA-V-10 (Hay powder). The concentrations of the standard were also prepared in a similar manner with the studied vegetable samples. In order to validate our results, the elemental concentrations of standard samples were measured several times in the same experimental conditions of the analyzed vegetable samples. Relative standard deviations are mostly within ±5-10%, which may recommend good precision. Data-Acquisition System. The data-acquisition system is one of the important parts of an ion beam experimental setup like PIXE technique [19]. MAESTRO-32 (Ortec, USA, version 6.05) interface software was used for spectrum data acquisition. X-ray spectra of vegetable samples were analyzed using GUPIX. GUPIX software with the help of DAN32 interfacing software has become convenient to analyze any complex PIXE spectra [20]. Results and Discussion In case of reference material IAEA-V-10 (Hay powder), the deviations of the measured values were mostly within ±5-10% which may represent a good agreement between the measured and certified values (Table 1). Table 2 shows a wide variation in the elemental concentrations of the different vegetable samples studied. Several trace elements are important micronutrient to the human body and are required for wellbeing of body immune system. The elements that were found in this study are silicon, phosphorus, chlorine, potassium, calcium, titanium, manganese, iron, cobalt, zinc, strontium, bromine, rubidium, chromium, ruthenium, nickel, sulfur, and copper ( Table 2). The concentration level of each element in the selected vegetables was found in the decreasing order as K > Ca > Fe Table 2). The distributions (in log scale) of bromine, rubidium, phosphorus, sulfur, strontium, ruthenium, chlorine, potassium, chromium, manganese, iron, cobalt, copper, calcium, titanium, zinc, nickel, and silicon within five different vegetables are shown using a side-by-side box plot ( Figure 1). Calcium contents in five vegetables varied in wide range of 98652-80743 ppm. The highest concentration of Ca was found in Basella alba (98652 ppm) and the lowest concentration was found in Brassica oleracea (80743 ppm). Ca is important due to the dietary significance of such cations and is the 5th most common element found in the human body. It is an obvious structural material, being present in cell walls, bones, and teeth. Calcium intakes protect against the risk of hypertension and decrease bone loss and the risk of kidney stones [21]. Calcium initiates changes in protein shape through binding to thousands of cell proteins. Thus it governs cellular functions like muscle contraction and cell movement to nerve transmission, glandular secretion, and cell division [22]. Adequate intakes (AI) levels of Ca for infant from birth to 6 months and from 6 months to 1 year are 210 mg/day and 270 mg/day, respectively. The RDAs of Ca for these same age groups are 200 and 260 mg/day, respectively. AI levels of Ca for children of 1-5 years and 4-8 years are 500 and 800 mg/day, respectively, and RDAs of Ca for these same age groups are 700 and 1000 mg/day, respectively. AI and RDA levels of Ca for adolescents of 8-18 years, adults of 19-50 years, and >50 years are 1300, 1000, and 1200 mg/day, respectively. AI and RDA levels of Ca for pregnant and lactating women of 14-18 years and >19 years are 1300 and 1000 mg/day [23,24]. Mean calcium intakes will be higher than the recommended intake for calcium in persons dependent on these types of vegetables. Thus people of these areas are at high risks of excess dietary calcium. Pregnant women and elderly persons are vulnerable to the development of some diseases like hypercalcemia, metabolic alkalosis, and possibility of renal insufficiency. It has revealed that calcium may protect against the development of colon and breast cancer [25,26]. Cancer patients may be relieved from chemotherapy and radiation therapy induced osteopenia and osteoporosis due to proper calcium intake. Some of these vegetables also provide protection against cancer and osteoporosis that is correlated well with significant concentrations of calcium present in them. The elemental concentration of potassium in Lactuca sativa vegetable was 97723 ppm, which was the highest amount of potassium among various samples studied. The lowest amount of potassium was exhibited by Brassica oleracea (83412 ppm). The elemental concentrations of potassium of Basella alba, Spinacia oleracea, and Lablab niger were 91632 ppm, 88021 ppm, and 92381 ppm, respectively. An increased level of potassium seems to augment the necessity of sodium and vice versa. The high level of potassium in these types of vegetables analyzed supports the idea that these plants may have high absorption rate of trace heavy metals from the soil where it grows and there may be plenty of potassium available in the soil of those vegetable plots. Potassium plays a notable role in maintaining the human's body fluid balance. Potassium is the primary cation inside cells; notably all of these vegetables are rich sources for potassium and calcium. So these vegetables can be eaten as DASH (Dietary Approaches to Stop Hypertension) diet, which also helps to reduce blood pressure. Potassium insufficiency directs to an Values are presented as mean ± SD ( = 10) for 18 elements in each vegetable. = number of samples analyzed for each vegetable. enhancement of the basic amino acid amount of the tissue fluids and some raise of cellular sodium levels as a means of sustaining anion-cation balance. Potassium deficiency affects the collecting tubules of the kidney, ultimately causing the incapability of concentrate urine. It is also responsible for the alterations of intestinal motility and gastric secretions [27]. 1875-5625 mg/day is the Recommended Dietary Allowances (RDAs) of potassium for adults and children above the age of 4 years [6]. Elemental concentrations of K in five different vegetables are fortunately above the RDA value and can serve as rich sources of K. Only one vegetable sample, namely, Lactuca sativa, had concentrations of Ru at a level of approximately 6301 ppm. Ruthenium has no recognized therapeutic importance to the human body [28]. Ru has the propensity to be mobile in the soils and thus can be easily taken up and gathered in various plant species. The concentration of Rb was detected in both Spinacia oleracea (1595 ppm) and Lactuca sativa (4426 ppm). Rubidium is normally observed in animal tissue and it is similar to the potassium during its delivery and excretory pattern [29]. The inclusion of rubidium to potassium-deficient diets helps to avert the lesions characteristic of potassium reduction in rats [30]. It was observed that the aqueous extract of Spinacia oleracea eaves has protective effect, for example, attenuating the spread of lesions [31]. The presence of Rb in Spinacia oleracea substantiates the therapeutic usage of it against the development of lesions. From reviewing of different literatures on Lactuca sativa, it can be postulated that the presence of Rb was not correlated with its potential healing effects. So, further investigation is needed to understand the correlation. Nickel was detected in only one sample named Brassica oleracea and the amount was 1214 ppm. Most of the nickel in food remains unabsorbed by the gastrointestinal tract and usually less than 10% of nickel ingested with food is absorbed [32]. The maximum permissible concentration of Ni in vegetables and fruits is 0.5 mg/kg [33]. In the present study the value of Ni observed in Brassica oleracea actually violated the permissible limit of Ni consumption as set by International Programme on Chemical Safety (IPCS) in its Health and Safety Guide number 62. Ni is well known as an activator of a number of enzymes, for example, alkaline phosphatase and oxaloacetate decarboxylase. So taking of Brassica oleracea (cauliflower) along with diet may be advantageous for the activation of these enzymes. The concentration of Mn differed markedly among vegetable samples. The highest Mn content (59861 ppm) was found in Lactuca sativa and the lowest value (1852 ppm) was determined in Basella alba. The concentrations of Mn found in Spinacia oleracea, Lablab niger, and Brassica oleracea were 13222, 39402, and 9018 ppm, respectively. Manganese functions as a cofactor of decarboxylase, transferase, and hydrolase enzymes in human body. In this sense, the determination of trace elements like Mn in food is of great importance since the deficiency or excess of Mn could promote several clinical disorders resulting in serious health problems. Deficiency of Mn in human body is the prime cause of myocardial infarction and other cardiovascular diseases. Mn deficiency is also characterized by CNS malfunctions, bone abnormalities, defective growth, reproductive dysfunction and disturbances in lipid and fat metabolism, and so forth [34]. The safe and sufficient level of daily manganese recommended intake range from 1.2 to 1.5 mg/day for children up to 8 years, 1.9-2.3 mg/day for male up to 70 years, and 1.6-1.8 mg/day for female up to 70 years [35]. However, it is essential to note that the levels of Mn in vegetable samples were very high for these aged groups. Manganese appears to be the ideal metal for oxidative stress protection. Protection of Mn against radiation, oxidative stress, and backup for SOD (Super Oxide Dismutase) enzymes were collectively referred to as manganese antioxidant [36]. The presence of Mn in all five vegetable samples analyzed also substantiates that these vegetables may give protection against radiation and oxidative damage. Phosphorus is a ubiquitous mineral and acts as an integral component of the body's vital energy source, ATP. Phosphorus-containing compounds such as cAMP, cGMP, and IP3 regulate many intracellular signaling processes in the body [37]. Brassica oleracea had the highest phosphorus concentration (87856 ppm) followed by Lactuca sativa (78342 ppm). The lowest phosphorous concentration (14314 ppm) was showed by Lablab niger. Both Basella alba and Spinacia oleracea showed phosphorus concentration of about 22827 and 65105 ppm, respectively. The recommended dietary allowances of phosphorous for children of 1-3 years, for children of 4-8 years, for males/females of 9-18 years, for males/females of 19-70 years, and also for males/females of >70 years are 460 mg/day, 500 mg/day, 1250 mg/day, 700 mg/day, and 700 mg/day, respectively [23]. The values observed in five different vegetables were above these dietary allowances proposed by Food and Nutrition Board. These vegetables can be used as a staple food source for phosphorous. These are also useful for the primary prevention of osteoporosis. The biological role of titanium is not fully understandable and has been studied for more than 90 years [38]. There is no strong evidence of a biochemical role of titanium. Some plants depend on Ti to perform photooxidation of nitrogen compounds and some processes of photosynthesis [39]. The presence of Ti in two of vegetable samples may be due to commencement of these biochemical pathways. The concentration of titanium was high in Spinacia oleracea (33682 ppm) whereas low in Basella alba (24026 ppm). High concentrations of titanium can possibly be found in foods or vegetables available in local market as a result of soil contamination by fly ash fallout [40][41][42], use of industrial sewage, and household waste. Titanium has no well-known biological use in human body, although it is known to act as a stimulating agent. No adequate intakes (AI) or recommended dietary allowances (RDAs) have been determined for Ni. A study was conducted in 1969 by Asmaeva and Il'vickij revealed that levels of titanium in foods were affected by climatic conditions and the region of growth [43]. Micromineral copper content was found to be nil in four vegetables except Brassica oleracea that showed an amount of 1264 ppm. Copper deficiency in human may lead to hypochromic, microcytic anemia, neutropenia, cardiovascular system abnormalities, bone and cartilage instability, hypopigmentation, and abnormal keratinization of hair [37,44]. Two copper containing superoxide dismutase enzymes protect human body against free radicals. Cu also helps to heal any type of wound. Adequate intake (AI) levels for copper have been recognized for infants of 0 to 6 months of age and for those between 7 and 12 months as 200 and 220 g/day, respectively. The RDAs for 1-3 years, 4-8 years, 9-13 years, 14-18 years, and 19-50+ years of age are 340, 440, 700, 890, 900, and 900 mg/day, respectively. The RDAs during pregnancy (14 through 18 years and 19 through 50 years) and lactation (14 through 18 years and 19 through 50 years) are 1000 and 1300 g/day, respectively [45]. Unfortunately Cu concentration observed in Brassica oleracea was higher than allowance. Although copper is indispensable to the good health but excessive consumption may result in serious health problems like kidney and liver damage [46]. Because of these possible adverse consequences from high copper ingestion, an endurable upper intake level (UL) of 10 mg/day has been established for adults who are older than 19 years of age. According to the results of this study conducted in five vegetable samples, Brassica oleracea is a highly rich vegetable for its Cu content. Thus this vegetable may be associated with some potential healing effects on free radicals and also may protect humans from developing cardiovascular diseases. Zinc is an indispensable or essential element for animals [47] and almost 300 mammalian enzymes are related to Zn [37]. The results indicated that all vegetable samples contained Zn. The amount of Zn was in the range of 14022-82467 ppm. Zn concentration was considerably higher in Lactuca sativa (82467 ppm) than in rest of the four different vegetable samples, for example, Basella alba, Spinacia oleracea, Lablab niger, and Brassica oleracea. Previous studies revealed that optimal level of Zn is required for the human body to reduce morbidity resulting from diarrheal and respiratory diseases [48]. Additionally, Zn deficiency can also cause retardation of growth and development, immune system deficiencies (i.e., lymphopenia, thymic defects, reduced phagocytosis, depressed T-cell function, and impaired cytokine production), behavioral disturbances (including impaired hedonic tone), impaired taste (hypogeusia), delayed healing of wounds and burns, decubitus ulcers, and cognitive function impairment [37]. The RDAs of Zn per day are 4 mg for children of about 4-8 years, 8 mg for children of about 9-13 years, 11 mg for male children of about 14-18 years, 11 mg for male adults of about 19-71+ years, 9 mg for female children of about 14-18 years, and 8 mg for female adults of about 19-71+ years of age [35]. The observed concentration of Zn in vegetables strengthens its abundance. It was observed that there is a direct association between zinc deficiency and cancer [49][50][51]. Zinc has played protective role against carcinogenesis and the presence of considerable amounts of zinc in all these vegetable samples just buttresses their usage for the treatment of cancer. An interesting observation made in this work is the absence of chromium in Basella alba, Spinacia oleracea, Lablab niger, and so forth. The amounts of chromium in Lactuca sativa and Brassica oleracea were approximately 9335 and 9112 ppm, respectively. These considerable levels were attributed to external sources such as processing, preparation, and storage. Chromium is another essential trace element and is necessary for optimal growth. Chromium has been shown to normalize the impaired glucose tolerance of some diabetes, old patients, and malnourished children [6]. 50-200 g/day is the RDA of Cr for adults and children above 4 years [52]. This highest level of Cr in Brassica oleracea and Lactuca sativa may be influenced by the local soil and water conditions of the vegetable plots. Vegetable plants do not need Cr and normally contain <0.2 mg/kg but plants could bioaccumulate Cr from soil and water. When different types of vegetable plants are grown in soil polluted from Cr-emitting industries or when sewage sludge is used as a fertilizer, these plants can accumulate high concentrations of chromium [53]. An upper tolerable limit or UL value for Cr has not yet been established. Cr has played a vital role in the metabolism of cholesterol and in-heart diseases [54]. The presence of considerable amounts of Cr in Lactuca sativa and Brassica oleracea may be correlated with their therapeutic properties against diabetes mellitus and cardiovascular diseases. Within the selected vegetables, the highest concentrations of Fe were noticed in Lactuca sativa (90525 ppm) followed by Spinacia oleracea (83411 ppm), Lablab niger (80761 ppm), Basella alba (72325 ppm), and Brassica oleracea (72123 ppm) in decreasing concentrations. The U.S. Food and Nutrition Board (FNB) has already assessed that the iron bioavailability from the diet of a vegetarian is only 10%, while mixed diet has iron bioavailability of 18%. Therefore, the RDAs for iron are of 10 mg/day for children of 4-8 years, 11 mg/day for males of 14-18 years, 15 mg/day for females of 14-18 years, 8 mg/day for 19-50 years of male adults, 18 mg/day for 19-50 years of female adults, and 8 mg/day for >50 years of male/female adults [35]. In the least-developed countries like Bangladesh the frequency, severity, and nature of the adverse effects of nutritional deficiency of iron are perhaps more problematic. Preterm or low birth weight infants, young children, pregnant women, and women in their reproductive years are more likely to benefit from dietary recommendations to Lactuca sativa and Spinacia oleracea, Lablab niger, Brassica oleracea, and Basella alba that have high levels of iron to satisfy their greater needs. According to World Health Organization (WHO), 39% of children less than 5 years of age, 48% of 5to 14-year-old children, and 42% of women in developing countries suffer from anemia and half of these individuals are in iron deficiency. Thus the beneficiary effects of iron can be correlated with significant concentrations present in all vegetable samples. A very narrow range of concentration (11253-15152 ppm) of cobalt was found between Basella alba and Lablab niger. Cobalt acts as a cofactor of enzymes that is involved in DNA biosynthesis and metabolism of amino acid [55]. Cobalt is a unique key mineral in Cobalamin or vitamin B12. Cobalt deficiency disease or symptoms are noticeable in vitamin B12 deficiency and pernicious anemia. In humans, cobalt deficiency symptoms include hypothyroidism, goiter, and heart failure. Very little information is available on its concentrations in different kinds of foodstuffs. Basella alba and Lablab niger are incredibly high in mineral Co. So these kinds of vegetables may prevent diseases like pernicious anemia. RDA per day of Co is 1-2 mg for a normal adult male of 22 years of age established by the Food and Nutrition Board (FNB), National Research Council, 1968 [6]. Cobalt showed quite lower toxicity than many other elements present in the soil of vegetable plots [56]. Cobalt in dietary sources like vegetables and fruits comes from soil contaminated by phosphate fertilizers, sewage sludge, burned fossil fuels, and industries that use or process cobalt containing compounds [57]. Children can be easily affected by coming in contact with high concentration of cobalt found in these vegetable samples because of their smaller body weights. The amounts of Sr present in both Basella alba and Lactuca sativa were 1043 and 3015 ppm, respectively. The concentration of Sr in the Lactuca sativa grows in polluted areas and may be due to the absorption of the Sr from the polluted soils and water. These types of plants can also be applied to control soil pollution, make the soil safe, and facilitate further cultivation of vegetables in these plots. Strontium has little or no beneficial action. Sr can replace calcium in bone formation and has been used to hasten bone remineralization in diseases like osteoporosis [6]. The presence of Sr in these two vegetable samples might boost the development of collagen and cartilage in joints of human body. Silicon helps human body to shield against cancers and other severe diseases by neutralizing some heavy metals and scavenging of free radical as well as highly reactive singlet oxygen ( 1 O 2 ) [28]. Powell et al. reported that fruit and vegetables were the richest sources of Si with substantial quantities present in Kenyan spinach [58]. This study has revealed that Malabar spinach (Basella alba) and spinach (Spinacia oleracea) contain the silicon and may play a vital role in bone formation and also may give protection against cancers. The existence of Si in these vegetables is therefore also helpful for cleaning human teeth, as the plants make the teeth strong and healthy. Only Basella alba and Spinacia oleracea contained Si in the concentrations of 3271 and 3124 ppm, respectively. Total Diet Study of FDA revealed that only 14% grain products and 8% vegetables were the contributors of Si to the U.S. diet [59]. Si was not detected in Lactuca sativa, Lablab niger, and Brassica oleracea. The Food and Nutrition Board (FNB) has not established a DRI/RDA for silicon since it is not considered essential [37]. Cl is another very indispensable element to human body. For example, Cl joins with Na and K to bring electrical charge when it is dissolved in body fluids that allows working of nerve cells. Its ion is used as an electrolyte, as well as making the hydrochloric acid. It is used by the stomach for digestion of food properly [28]. It also maintains the electrolyte balance in human body. It also helps in absorbing of crucial elements that are required for man's survival. All five vegetables had concentrations of Cl showing difference between 35188-75013 ppm. It also contributes to the maintenance of the electrolyte balance. RDA per day of Cl is 1700-5100 mg for adults and children over 4 years of age [52]. These vegetables may be capable of exhibiting some unique beneficial effects particularly in regulation of the body fluid balance and circulation of ions in the bloodstream. Both Spinacia oleracea and Lablab niger analyzed in this work had concentrations of S observed through PIXE were 74442 and 72168 ppm, respectively. But S was not detected in the remaining vegetables. Sulfur is considered to be therapeutically important. Sulfur is widely distributed throughout the body as sulfhydryl groups of cysteine and disulfide linkages in protein from cysteine, for example, insulin [6]. According to National Research Council, 1968, RDA per day of S is 2-3 gm for a normal adult male of 22 years of age [6]. Spinacia oleracea and Lablab niger with their abundant quantities of S may lower blood cholesterol and speed production of carcinogen-destroying enzymes and thus protect against cancer development. Bromine contents showed the lowest value of approximately 6395 ppm in Spinacia oleracea as compared to those in the Lablab niger (7485 ppm) and Brassica oleracea (6643 ppm). Bromine remained undetected in Basella alba and Lactuca sativa. Bromine has no known benefit in life processes in plants and animals. So there is no Dietary Reference Intake (DRI)/Recommended Dietary Allowance (RDA) for pregnancy, children, adults, and nursing. It was revealed from the results that all three vegetable samples have higher Br concentration than what these should normally be in vegetables. The uptake of this high concentration of Br by vegetables is influenced not only by plant species and physicochemical characteristics of soil but also by temperature and rain fall which may exert considerable effects. Conclusion PIXE technique was used for the elemental analysis of five different vegetables available in the local market of Savar, Dhaka 1342, Bangladesh. Significant differences were observed during analysis of the concentrations of 18 elements of five different vegetable samples. Considering the order of decreasing concentrations of elements in five different vegetables, it can be concluded that all of them were observed as rich sources of macro elements, that is, K, Ca, Cl, P, and S as well as trace essential elements, that is, Fe, Mn, Cr, and Zn. These vegetables are also free from toxic elements including Cd and Pb. Some elements like Ti, Sr, Ru, and so forth which have no known biological action in human body were found in very low concentrations. This study also revealed that overall distributions of K, Ca, Fe, Cl, P, and Zn in all vegetable samples analyzed are high. The use of fertilizers and metal based pesticides in agriculture may be responsible for these excessive levels of elements which may lead to serious health related complications. The results of this study supply valuable pieces of information about the metal contents in vegetables available in the local market of Bangladesh. Moreover, these results can also be used to investigate the chemical quality of other vegetables available here in Bangladesh and to avoid all the possible health risks associated with their consumption by humans. Conflict of Interests In this research work the authors declare that they have no interests in competing with other researchers.	2016-05-04T20:20:58.661Z	2015-07-02T00:00:00.000	{ "year": 2015, "sha1": "756acde99f605d1986da67a9d749120d879e73d2", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/bmri/2015/128256.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "33315a3e555786f3900d837fd9a303dfaa5feb95", "s2fieldsofstudy": [ "Agricultural and Food Sciences", "Environmental Science" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
52409196	pes2o/s2orc	v3-fos-license	POPULATION AND LAND COVER DYNAMICS OF SUNDARBANS IMPACT ZONE IN BANGLADESH Sundarbans is the largest mangrove forest of the world that lies in Bangladesh and India. This paper examined the correlation among population dynamics of Sundarbans impact zone (SIZ) from 1974 to 2011; impact of climate change on the forest; and changes in land cover of the forest from 1973 to 2010. Population size of SIZ was increased by 1.6 times between 1974 and 2011, whereas decreased by 2% during 2001 to 2011. During 1973 to 2010, water bodies, barren land and vegetated land reduced by 7.35%, 49.56% and 15.92% respectively; while grass land increased by 228.14% during the same period. But both population size and vegetated land declined during 2001 and 2011. This was due to the landfall of two severe cyclones in 2007 and 2009 through Sundarbans which resulted thousands of human casualties and out migration, and destruction of the forest. In addition, anthropogenic interventions like low flow from Ganges River and policy constraints also contributed to the demolition of Sundarbans. Thus, population growth, climate change and anthropogenic interventions are playing a decisive role to the depletion of forest resources from the Sundarbans mangrove forest. Introduction The densely populated and climate fragile Bangladesh has shared the world's largest mangrove forest with India.Around 38% of the world's mangroves occur in Asia of which Indonesia alone accounts for 19% (FAO 2007).Sundarbans mangroves forest (SMF) is the largest contiguous coastal wetland system in the world.This forest is also one of the productive mangrove wetland ecosystems in the Ganges-Brahmaputra delta (Islam -Gnauck 2008).The area of the Sundarbans forest cover is about 10,000 km² in southwest Bangladesh (known as Sundarbans reserve forest, SRF) and West Bengal (known as Sundarbans National Park) of India (UNESCO WHC).Around 62% of the forest covers lies in Bangladesh, while the remaining 38% is in India (Siddiqi 2001;Lacerda 2001).The SRF is located at the southern edge of Gangetic delta bordering Bay of Bengal and is bounded by Baleswar River on the east and Harinbanga River (international boundary with India) on the west (UNESCO WHC).The SRF covers an area of 6,017 km² which accounts for 4.07% of total area of Bangladesh and 40% of total area managed by Forest Department of Bangladesh (BBS 2014).The Sundarbans Reserved Forest is both a Ramsar site, since 1992, and a World Heritage site of the UNESCO since 1997(FAO 2007).This forest provides natural protection to the lives and properties of coastal communities from cyclones and storm surges. Around 2% of the total labor of Bangladesh was engaged in the forestry sector in 2013 and contributed about 2% of total Gross Domestic Product (GDP) of Bangladesh (BBS 2014).The SRF contributes about 41% of the total forest revenue (Islam 2010).The SRF is surrounded by a very densely populated area who are mostly depends on Sundarbans for their livelihoods.Approximately 2.5 million people live in the small villages surrounding the Sundarbans, while the number of people within 20 km of the Sundarbans boundary is 3.14 million (MARC 1995).Numerous people engage in commercial exploitation of Sundari (Heritiera fomes), Gewa (Excoecaria agallocha), Goran (Ceriops decandra), and other tree species, while local people depend on forest for firewood, timber for boats, poles for house-posts and rafters, golpata (Nipa fruticans) leaf for roofing, grass for matting and fodder, reeds for fencing, and fish for their own consumption.There is no permanent settlement in SRF (Chaffey et al. 1985).But the forest is under threats due anthropogenic interventions.The SMF was about 16,700 km 2 around 200 years ago (Banglapedia 2015) which has now dwindled down to half of its original size, the rest of the land being deforested and converted to agricultural landscape (Hussain -Archarya 1994). Mangrove ecosystems of the world are threatened through various forms of human pressure, in particular extraction, pollution and reclamation (Akhter 2006).High population pressure in coastal areas has led to the conversion of many mangrove areas to other uses (FAO 2007).According to 2011 census, the total population of Bangladesh was 149.77 million with a density of 1015 per km 2 (BBS 2011).The heavy population pressure is placing growing demand on natural resources, especially forest sector.At approximately 0.0002 km 2 per person of forest, Bangladesh currently has one of the lowest per capita forest ratios in the world (Zaman 2011). There is a reciprocal relationship between population factors (size, distribution and composition) and environmental factors (air, water and surface forms).This relationship (population-environment relationship) is affected by some intervening factors which include social, political, technological and cultural factors.The objective of this paper was to explore the correlation among population dynamics of Sundarbans impact zone (SIZ), impact of climate change on Sundarbans and changes of land cover in SRF.To describe the correlation, this research used population and environment framework which was proposed by MacKellar et al.Islam (2014) discovered that Sundarbans mangrove forest (both in Bangladesh and India) decreased by 19.3% due severe tropical cyclone in 1977and 1988. Islam (2014) ) argued that the fate of Sundarbans depends to climatic issues e.g., salinity, frequency of severe tropical cyclone and tidal effect those directly depend to global warming.Rahman et al (2013) studied on the effectiveness of different classification methods for extraction of Mangrove forest in Sundarbans using Landsat ETM+ data and found that the band ratio/supervised classification approach produces the best accuracy in detecting the mangrove class.Giri et al. (2007) studied on Sundarbans forest dynamics (both India and Bangladesh) using multi-temporal satellite data from 1970s, 1990s, and 2000s through supervised classification approach.Their study found that areal extent of Sundarbans mangrove forest has not changed significantly (approximately 1.2%) in the last 25 years and the forest is constantly changing due to erosion, aggradation, deforestation and mangrove rehabilitation programs.The study of BIDS (2010) described the sociodemographic dynamics of SIZ. Therefore, this present research work Sundarbans has a highly fragile ecosystem and its delicate balance may be adversely affected if these (demographic and climate change) pressures are not dealt with. Population data of SIZ were collected from census reports for 1974, 1981, 1991, 2001 and 2011 of Bangladesh Bureau of Statistics (BBS).Policies and research works related to climate change impact on Sundarbans mangrove forest were systematically reviewed to understand the impact of climate change on Sundarbans.To detect changes in vegetation cover of SIZ between 1973and 2010, this study used Landsat images of January-1973, January -1978, January -1989, January -2001 and January -2010 obtained from United States Geological Survey (USGS), Global Visualization Viewer (Glovis).Multi Spectral Scanner (MSS), Thematic Mapper (TM) and Enhanced Thematic Plus (ETM+) sensor used to generate land cover maps in satellite image.For digital image processing, ERDAS Imagine 2013 was used.Layers stacking method was applied because of using different bands (or layers) of information.In order to make clear visualization of the objects, histogram equalization method applied and haze reduction tool used for improving the quality of the images.Then radiance was calculated from the digital number.After visual interpretation, a land cover class scheme was developed (Tab.1).Normalized Difference Vegetation Index (NDVI) was calculated from the visible and near-infrared light reflected by vegetation using the following formula (NASA-EO, 2015): NDVI= (RNIR -RRED)/ (RNIR+RRED) where, RNIR: near-infrared reflectance RRED: visible reflectance RNIR: near-infrared reflectance RRED: visible reflectance NDVI of Landsat images were generated using the conditional logic.Then the area covered by each category was derived.NDVI value of raster layer was classified using the supervised classification scheme as barren land (0-0.2 μm), grass land (0.2-0.3 μm) and vegetated land (0.3-1.0 μm).NDVI value of less than and equal to 0 (zero) was kept out as water body.A detail classification scheme was shown in Tab. 1. Finally, vector maps of 1973, 1978, 1989, 2001 and 2010 were prepared using ArcGIS 10.1 software. Results of the study Population factors Sundarbans impact zone covers an area of around 6% land of Bangladesh.More than half of total impact zone is shared by Shyamnagar, Koyra and Dacope sub-district.According to 2011 census, SIZ districts had a population of 7.8 million which constituted about 5.4% of total population of Bangladesh and around 2.2 million people inhabited in SIZ sub-district (Fig. 3) which was almost 1.5% of country's total population (BBS 2011b;BBS 2011c;BBS 2012;BBS 2013). In 2011, population density in SIZ districts (556 per km 2 ) and SIZ sub-districts (426 per km 2 ) were below the national average (976 per km 2 ).In fact, in all SIZ localities population density increased since 1981.The highest absolute change in population density observed for Bagerhat Sadar (from 763 per km 2 in 1981 to 977 per km 2 in 2011).On the other hand, during 2001 to 2011, the density of population decreased remarkably in some SIZ sub-districts like Mongla (8.8%), Morrelganj (15.7%) and Dacope (3.1%).This tendency was also recorded at some SIZ districts like Bagerhat (4.6%) and Khulna (2.4%). Environmental factors The coast of Bangladesh is a global hotspot for tropical cyclones.Coastal zone represents around 32% of Bangladesh's total land area and about 10% of the country's total land lies within one meter above mean sea level.Over the last few decades, the numbers of cyclone formations and landfalls has been increasing in Bay of Bengal.In the last east to west.Height and growth of different mangrove species in Sundarbans are correlated with salinity level.For example, Sundari trees grows in low saline zone (5~10 ppt), Gewa in moderate saline zone (10~25 ppt) and Goran in high saline zone (over 25 ppt).The degree of salinity has strong influenced on regional distribution of species and stand-height of tress in Sundarbans.Sundari trees are being destroyed by outbreak of top-dying disease.Sea level rise also poses another threat to Sundarbans. Land cover change of Sundarbans reserve forest The Landsat images of SIZ were shown as Landsat images of Sundarbans (Fig. 7.).NDVI of different land cover classes (Fig. 8.) were calculated from the subsets of Landsat images of 1973, 1978, 1989, 2001 and 2010.These values were converted into areas and presented in Table 3. Areas occupied by water bodies in 1973, 1978, 1989, 2001 and 2010 were 5488.04 km 2 5456.85km 2 , 5341.20 km 2 , 5218.19 km 2 and 5084.92km 2 , respectively.Percentage occupied by water bodies to total area decreased from 58.79% in 1973 to 54.52% in 2010.Water bodies decreased about 7.35% over the last 37 years.On the other hand, area covered by barren land in 1973 was 8.78%, which was decreased in 2010 and made up 4.43% to total study area. The most significant changed of land cover found for grass land (Fig. 9.).Grass land made up around 5.54% of total area in 1973 and increased to reach 18.51% by 2010.Dense vegetated land (both mangrove and nonmangrove) decreased notably during 1973 to 2010 (Fig. 9.).Vegetated area occupied The highest rate of decline of vegetated land observed during 2001 to 2010 period which was 6.04%, followed by 5.00% between 1978 and 1989.Regional variations of vegetation coverage were also prominent.Dense vegetation coverage was apparently distributed uniformly in forest except 2010.During this period vegetated land was more agglomerated in western part than eastern part (Fig. 8.). Intervening factors National and international policies and legislations play a significant role to conserve as well as depletion of forest resources.The legal forest cover of Sundarbans has not changed in the last four decades and theoretically, it is now well conserved.But it is difficult to conserve Sundarbans from the least flow of Gorai River that adversely affected SMF.India constructed a barrage (Farakka barrage) to divert Ganges River water, and as a result, southern part of Sundarbans is facing serious salinity problems.Reduction in freshwater flow through Sundarbans is one of the reasons of decreasing the abundance of Sundari tree form Sundarbans. Institutional response represents a significant mechanism through which humans react to environmental change.In particular, policy plays a key role in determining ultimate effect of humans on environment.Hypothetically, if government of Bangladesh takes effective and efficient legal measures to conserve Sundarbans from illegal harvesting and if there is no climate change impact, even Sundarbans will further be degraded because of lack of policy integrations.The oil spills (9 December, 2014) at Shela River threatened trees, plankton, and vast populations of small fish and dolphins of Sundarbans.Even after such a disaster government did not show any attempt to restrict vessels movement through Shela River and other linked channels of Sundarbans.In addition, government has also planned to install 1320 megawatt coal-fired power station at Rampal sub-district of Bagerhat district which is situated 14 kilometers north of Sundarbans.This project will not only worsen ecosystems of Sundarbans, but may also degrade ecologically sensitive SIZ locality through changing micro-climatic variability. Population and poverty processes are intimately linked to forest cover change.The percentage of extreme poor (lower poverty line) people in 2010 in SIZ sub-districts (for example, Sarankhola, 28.2%; Shyamnagar, 33.8%; Koyra, 29.1%; Dacope, 24.9%) were higher than the national average (17.6%).These extreme poor people generally engaged themselves in extracting natural resources either from Sundarbans or from common property.The impact of climate change in south-western coast of Bangladesh is now visible.Population growth and climate change may speed up poverty trap in SIZ locality.Demand for timber from Sundarbans is also another cause for forest degradation.Unfortunately, this demand leads to illegal logging and cutting down of major species from Sundarbans (e.g.Sundari, Gewa, Goran trees). Discussions The linkage between environment and population trends is a less documented phenomenon for SIZ.The high rates of population growth in SIZ locality during 1980s and 1990s had significant environmental implications on Sundarbans.Increase in population in SIZ localities also caused increased demand for food and arable land.Moreover, agricultural expansion in the form of shrimp farming encouraged deforestation and encroachment of coastal land, which in turn contributes to microclimatic change.Population growth also associates with increased demand for energy, especially fuel wood from forest, which provides energy to virtually all rural frontier population of SIZ. According to FAO (2007), Sundarbans Reserved Forest is well protected, and no major changes have been found during 1980 to 2005.This study was also found similar results.However, this study found significant changes within the land cover classes of Sundarbans throughout the study period. In general, population size and composition, and urbanization are interrelated with consumption and depletion of resources.During 1981 to 1991, population size of SIZ increased by 20% whereas number of trees per square kilometer in SRF decreased by 25% (Forest Inventory of 1983 and 1996) (Chaffey et al. 1985;Choudhury -Hossain, 2011).Population size is associated with harvesting of wood for fuel.Around 57% (BBS 2011) population of SIZ districts used wood for cooking.This figure was higher for rural settings of SIZ.Level of urbanization in SIZ sub-districts were very slow and below national level.Therefore, most of the frontier people basically depend on primary level economic activities especially collecting and gathering of natural resources from Sundarbans.At the same time, commercial shrimp cultivation increased in many folds that played a significant role to alter climatic variability of SIZ localities. During 1973 to 2010, water bodies, barren land and vegetated land decreased by 7.35%, 49.56% and 15.92% respectively; whereas grass land increased by 228.14% during same period.This implied density of evergreen vegetation and its canopy closure decreased.This study found significant relationship between changes in population size and vegetated land.and some intervening factors contributed to change land cover of Sundarbans over the last four decades.This research thus proposed a modified version of population and environment interaction in the context of Sundarbans reserved forest. Sundarbans mangrove ecosystems hold remarkable value for south-west coastal communities and for country as a whole.But forest resources are being destroyed at alarming rates, although total forest cover has not changed significantly.To stop and reverse current trend of resource depletion from SRF, effective, time fitting and efficient measure need to take by forest department of Bangladesh.It is also urgent to improve institutional and management capacity of forest department.Geographical information system (GIS) and remote sensing base regular monitoring system could be a sophisticated tool to identify temporal and spatial change of forest cover.To stop illicit resource extraction by influential people and frontier communities, patrolling systems need to be increased and modernized.Thousands of people of SIZ actively involves in SRF resource collection.Thus, creation of alternative livelihoods for Sundarbans dependent communities can reduce overload from Sundarbans.Outcome oriented planning for alternative livelihood generation is also indispensible.In addition, temporal ban of Sundarbans resource extraction (such as leaves collection, timber collection, fish collection and honey collection) may foster conservation efforts.Political commitment and willingness on trans-boundary river management also crucial for conserving Sundarbans from human induced climate change impacts.The role of frontier communities to conserve forest is necessarily important and their representation and active involvement could functionalize forest management committees more energetically.And, finally, it is required to ensure transparency, accountability and awareness of communities and forest managers to conserve Sundarbans from human interventions. Fig. 4 . Fig. 4. Comparison of population growth rate of 2001 and 2011 in SIZ sub-district decade (2001s), several cyclones landfall through Sundarbans, the most devastating were cyclone Sidr (landfall on 15 November 2007) and cyclone Ayla (landfall on 25 May 2009).These two cyclones endangered lives and livelihoods of coastal communities and impact of climate change in southern coastal zone has become more pronounced after the cyclones.The Sundarbans is already affected by increasing salinity and extreme weather events.The freshwater flow in Sundarbans depends on upstream river flow (Transboundary Rivers).Gorai River (a distributary of Ganges) carries freshwater for Sundarbans.But the natural flow of Gorai River has been disrupted after the construction of Farakka barrage on Ganges River by India in 1975 about 190 km upstream of the Gorai River mouth to divert Ganges River water through Hoogly River to Kolkata port.Since then dry season flow of Ganges River or Padma River dropped dramatically.Since 1988, Gorai River has completely disconnected from Ganges flow during dry season (November to April).Freshwater flow through creeks and rivulets of Sundarbans helps to flush salinity off from the forest floor of Sundarbans.This chronic disturbance of freshwater flow altered salinity regime of Sundarbans.Salinity of Sundarbans increases from east to west and, hence, density of vegetation growth and canopy closure decreases from Fig. 6 . Fig. 6.Age group wise population composition of SIZ districts During 2001 to 2011, population growth rate of SIZ sub-district was negative, yet vegetated land decreased.This finding could be explained by climate change only.Coastal zone was affected by two major consecutive cyclones (Sidr and Ayla) in 2007 and 2009.As a result, thousands of people were displaced on embankments, roads or other collective centers.Many people migrated from SIZ for searching livelihoods.Therefore, it can be summarized that population growth, climate change Fig. 10 . Fig. 10.Population and environment relation of Sundarbans (as proposed by the author) Table 1 . Land cover classification scheme of SIZ Table 3 . Areas of different land cover classes of Sundarbans (areas in km2) land 2501.05km 2 in 1973 which further dropped to 2102.94 km 2 in 2010. Fig. 9. Areas occupies by water bodies, barren land, grass land and vegetated	2018-09-18T17:28:52.497Z	2017-04-14T00:00:00.000	{ "year": 2017, "sha1": "73b60900185308f2b1998d966753c962843607fc", "oa_license": "CCBYSA", "oa_url": "https://doi.org/10.21120/le/11/1/1", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "73b60900185308f2b1998d966753c962843607fc", "s2fieldsofstudy": [ "Environmental Science", "Geography" ], "extfieldsofstudy": [ "Geography" ] }
253119915	pes2o/s2orc	v3-fos-license	Nonparametric estimation of trend for SDEs with delay driven by fractional Brownian motion with small noise We investigate the problem of nonparametric estimation of the trend for stochastic differential equations with delay and driven by a fractional Brownian motion through the method of kernel-type estimation for the estimation of a probability density function. 1 Introduction Guschin and Kuchler (1999) investigated asymptotic inference for linear stochastic differential equations (SDEs) with a time delay of the type dX(t) = (aX(t) + bX(t − 1))dt + dW t , t ≥ 0 driven by the standard Brownian motion W = {W t , t ≥ 0} with the initial condition X(t) = X 0 (t), −1 ≤ t ≤ 0 where {X 0 (t), t ∈ R} is a continuous process independent of the process W. They investigated the asymptotic properties of the maximum likelihood estimator (MLE) of the parameter θ = (a, b) based on the observation of the process {X(t), 0 ≤ t ≤ T }. They showed that the asymptotic behaviour of the MLE depends on the ranges of the values of a and b. Prakasa Rao (2008) considered the problem of estimation of the parameter (a,b) for linear SDE of the form dX(t) = (aX(t) + bX(t − 1))dt + dW H t , t ≥ 0 (1. 1) driven by the fractional Brownian motion W H = {W H t , t ≥ 0} with known Hurst index H and with the initial condition X(t) = X 0 (t), −1 ≤ t ≤ 0 where {X 0 (t), t ∈ R} is a continuous process independent of the process W H . Asymptotic properties of the MLE for the parameter θ = (a, b) is studied in Prakasa Rao (2008). For some details, see Prakasa Rao (2010), pp.156-163. Applying the results in Mohammed and Scheutzow (1990), it can be shown that there exists a unique solution X = {X(t), t ≥ −1} of the equation (1.1) and it can be represented in the form This process has continuous sample paths for t ≥ 0 almost surely and conditionally on X 0 , the process X is a Gaussian process. Furthermore the function x 0 (t) defined for t ≥ −1 is the fundamental solution of the differential equation Guschin and Kuchler (1999) gives a complete discussion on the existence and representation of the fundamental solution of the equation (1.3). Kutoyants (2019) studied nonparametric estimation for SDE with delay driven by a standard Wiener process with small noise. We wlll now investigate a similar problem for SDE with delay driven by a fractional Brownian motion with small noise. Preliminaries Consider the SDE with delay given by where W H is a fractional Brownian motion with known Hurst index H and the function S(t, x) is unknown. We assume that the delay τ ≥ 0 is known. Consider the ordinary differential equation For any given value x, let t = t x be defined by the equation x tx−τ = x. The problem is to estimate the function S(t x , x) based on observations X T = {X t , 0 ≤ t ≤ T }. Observe that the equation (2.1) can be represented in the form Parametric inference for SDEs of delay type and driven by Wiener process was investigated by several authors. A survey of these results is given in Kutoyants (2005). Nonparametric inference for such processes with small noise was surveyed in Kutoyants (1994Kutoyants ( , 2004. Parametric inference for SDEs of delay type and driven by fractional Brownian motion was investigated by Prakasa Rao (2008) and a discussion was presented in Prakasa Rao (2010). We now initiate the study of nonparametric inference for processes with time delay but driven by a fractional Brownian motion. Main Results We assume that the the following conditions hold. The function S(t, x) has two continuous bounded derivatives with respect to t and x. (C 3 ) The delay parameter τ and the Hurst index H are known. Note that the condition (C 2 ) implies that there exists constants M > 0 and L > 0 such that x) denotes the partial derivative of S(t, x) with respect to t and S ′ x (t, x) denotes the partial derivative of S(t, x) with respect to x. We now consider the problem of estimation of the function S(t, x) for t > τ. For t ≤ τ, the function S(t, x t ) = S(t, x 0 ) and hence a function of one variable t ∈ [0, τ ]. This problem was investigated in Mishra and Prakasa Rao (2011) and in Section 5.3 in Prakasa Rao (2010). Here after, we assume that t > τ. Let G(.) be a real-valued function which is bounded and satisfies the conditions: Let φ ǫ > 0 be a bandwidth function tending to zero as ǫ → 0. We now construct a kernel type estimator for the function S(t, x) for t > τ based on the kernel G(.). For a study of kernel-type estimators and their properties for the estimation of a probability density function, see Prakasa Rao (1983). Let as an estimator for the function S(t, x) for t > τ. We will prove a lemma which will be used in the sequel. In the following discussion, the constant C may be different from one inequality to another. Lemma 3.1: Suppose that the function S(t, x), 0 ≤ t ≤ T, x ∈ R has a continuous bounded derivative with respect to x and the the process almost surely. Applying the Grownwall lemma, it follows that there exists a constant C > 0 such that by the estimate for the supremum of a fractional Brownian motion over an interval [0, T ](cf. Novikov and Valkeila (1999); Prakasa Rao (2010), Prop. 1.9). for some positive constant C. Applying the transformation s = t x,ǫ + φ ǫ u and using the Taylor expansion, we get that where R (3.9) and rearranging, we obtain that Applying the condition (C 1 ), it follows that where the random variable H(t x,ǫ ) has bounded moments by Lemma 2.1 and the maximal inequalities for the fBm. Note that by the conditions on the function S(s, x). Applying the inequality derived above, it follows that \|t x,ǫ − t x \| ≤ ǫα −1 H(t x,ǫ ) (3. 13) and the last term tends to zero as ǫ → 0. Observe that and hence, on the set Γ ǫ , it follows that from the inequality (3.7). Let χ Γǫ denote the indicator of the set Γ ǫ . Observe that ≤ Cǫ 2 by the conditions (C 1 ) and by the inequality given in (3.13). Furthermore, there exists an absolute constant c(2, H) depending on H such that (by Memin et al. (2001), Prakasa Rao (2010), Theorem 1.7)) for some constant C depending on H. Let us denote t x,ǫ + φ ǫ u by t x,ǫ,u in the following computations. Note that for some positive constant C by an application of Lemma 2.1. Combining the bounds obtained above, we get that We now estimate the P (Γ c ǫ ). Observe that and hence, for φ ǫ < 1 4 τ, it follows that by Theorem 1.4 in Prakasa Rao (2014) for the fractional Brownian motion. A similar estimate can be obtained for the term P (T − t x,ǫ ≤ 2φ ǫ ). We can now estimate the mean squared error on the set Γ c ǫ . Observe that The last inequality follows by observing that and noting that the random variable S ǫ (t, x) has polynomial moments of all orders from the conditions on the kernel G(.) and from the bounds on Wiener integrals with respect to the fractional Brownian motion due to Memin et al. (2001). Furthermore the function S(t.x) is bounded by assumption. Combining the inequalities obtained above, we get that for some positive constants C and c. The optimal rate can be obtained by choosing φ ǫ such that φ 4 ǫ = ǫ 2 φ 2−2H ǫ or equivalently φ ǫ = ǫ 1/(3−H) . Hence we obtain the bound (3. 22) 4 Comments : Remarks 4.1: (C 4 ) Suppose that the function S(t, x) is Lipschitz and the function g(t) = S(t, x t−τ ) has k continuous derivatives and the k-th derivative g (k) (t) satisfies for some positive constant C 0 and for some 0 ≤ β ≤ 1. Let F k,β be the class of functions g(.) satisfying the conditions stated above. Following the arguments stated in Kutoyants (2019), it can be shown that for some positive constant C. Choosing φ ǫ such that we obtain the inequality By a similar analysis, it can be shown that, for any p > 0, sup S(.,.)∈F ( k,β) for some positive constant C. It can be shown that , for t = t x such that x tx−τ = x, following the arguments given earlier, implying that the estimator S ǫ (t, x) is asymptotically efficient in the sense of Hajek-Lecam (cf. Ibragimov and Khasminskii(1981)). Remarks 4.2: Suppose the delay τ is not known and we want to estimate the function f (t) = S(t, x t − τ ), 0 ≤ t ≤ T.Suppose the function f (t) is k times continuously differentiable and the k-th derivative satisfies the condition (4.1). We can estimate the function f (t) by the kernel estimator Note that S(s, X s−τ ) = S(s, x s−τ ) + O p (ǫ) by Lemma 2.1 and for some positive constant C from the earlier computations. It is sufficient to study the limit behaviour of the term The normalizing function φ ǫ = ǫ 1/(k−H+β+1) will give the bound for the mean square error as given in (4.3).	2021-04-09T01:15:36.235Z	2021-04-08T00:00:00.000	{ "year": 2021, "sha1": "fc608e0728e19000a8afec3692111ef1b0fbcb0a", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "fc608e0728e19000a8afec3692111ef1b0fbcb0a", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
211009307	pes2o/s2orc	v3-fos-license	Discriminative Feature Learning Constrained Unsupervised Network for Cloud Detection in Remote Sensing Imagery Cloud detection is a significant preprocessing step for increasing the exploitability of remote sensing imagery that faces various levels of difficulty due to the complexity of underlying surfaces, insufficient training data, and redundant information in high-dimensional data. To solve these problems, we propose an unsupervised network for cloud detection (UNCD) on multispectral (MS) and hyperspectral (HS) remote sensing images. The UNCD method enforces discriminative feature learning to obtain the residual error between the original input and the background in deep latent space, which is based on the observation that clouds are sparse and modeled as sparse outliers in remote sensing imagery. The UNCD enforces discriminative feature learning to obtain the residual error between the original input and the background in deep latent space, which is based on the observation that clouds are sparse and modeled as sparse outliers in remote sensing imagery. First, a compact representation of the original imagery is obtained by a latent adversarial learning constrained encoder. Meanwhile, the majority class with sufficient samples (i.e., background pixels) is more accurately reconstructed than the clouds with limited samples by the decoder. An image discriminator is used to prevent the generalization of out-of-class features caused by latent adversarial learning. To further highlight the background information in the deep latent space, a multivariate Gaussian distribution is introduced. In particular, the residual error with clouds highlighted and background samples suppressed is applied in the cloud detection in deep latent space. To evaluate the performance of the proposed UNCD method, experiments were conducted on both MS and HS datasets that were captured by various sensors over various scenes, and the results demonstrate its state-of-the-art performance. The sensors that captured the datasets include Landsat 8, GaoFen-1 (GF-1), and GaoFen-5 (GF-5). Landsat 8 was launched at Vandenberg Air Force Base in California on 11 February 2013, in a mission that was initially known as the Landsat Data Continuity Mission (LDCM). China launched the GF-1 satellite. The GF-5 satellite captures hyperspectral observations in the Chinese Key Projects of High-Resolution Earth Observation System. The overall accuracy (OA) values for Images I and II from the Landsat 8 dataset were 0.9526 and 0.9536, respectively, and the OA values for Images III and IV from the GF-1 wide field of view (WFV) dataset were 0.9957 and 0.9934, respectively. Hence, the proposed method outperformed the other considered methods. Introduction Remote sensing imaging technology such as multispectral (MS) imaging and hyperspectral (HS) imaging can perceive targets or natural phenomena remotely [1][2][3]. Depending on the wide-scale In view of this, we propose a discriminative feature learning constrained unsupervised network for cloud detection (UNCD) in MS and HS images. The proposed UNCD method depends on an important observation that clouds are sparse and modeled as sparse outliers [28][29][30]. Adversarial feature learning is conducted on an unsupervised neural network, namely, an autoencoder (AE) [31,32], to extract a compact representation of the original input image in the deep latent space. An image discriminator is introduced to correct image features in order to avoid the generalization of out-of-class features that is caused by the adversarial feature learning. With sufficient training samples, the background can be more distinctively reconstructed than the clouds. Besides, a multivariate Gaussian distribution is adopted to extract a discriminative feature matrix of the background in the latent space; hence, if the dataset contains clouds, the encoder will encourage the learning of the background distribution of the dataset. As a consequence, the residual error between these two lower-dimensional representations is beneficial to cloud detection. We conducted experiments on both MS and HS images and evaluated the performance of the proposed UNCD framework in terms of detection accuracy and generalization. The contributions of this paper are fourfold: 1. A novel UNCD method is proposed to address the issue of insufficient training data in remote sensing images, especially hyperspectral data, in the field of cloud detection. To the best of our knowledge, in this paper, such an unsupervised adversarial feature learning model is utilized for the first time for MS and HS cloud detection. 2. Latent adversarial learning is introduced such that the AE focuses on extracting a compact representation of the input image in the latent space. 3. An image discriminator is used to prevent the generalization of out-of-class features. 4. A multivariate Gaussian distribution is adopted to extract a discriminative feature matrix of the background in the latent space, and the residual error between the low-dimensional representations of the original and background pixels is beneficial to cloud detection. The remainder of this paper is organized as follows. In Section 2, AE and adversarial learning models are briefly described. Section 3 introduces the proposed UNCD framework. The experimental results and discussion are presented in Section 4. The discussion of the experimental results is introduced in Section 5. Finally, Section 6 presents the conclusions of this study. Generative Adversarial Network A generative adversarial network (GAN) is a highly efficient generation model proposed by Goodfellow et al. [33] in 2014. GANs consist of a generator G and a discriminator D, which are trained against each other. The generator G inputs a random data z whose probability distribution is p z (z) and outputs a data x f ake = G(z). The discriminator D receives two inputs, real data x with a probability distribution p data (x) and generated data G(z). The discriminator is trained to identify the real data. Via successive adversarial learning, G generates increasingly many real data. The objective of this adversarial learning process can be expressed as [33]: Due to its advantages in terms of generation and adversarial learning performance, we constructed a GAN model that is based on an encoder and a decoder for the improved production of discriminative features. Variational Autoencoders An AE updates the parameters by minimizing the distance between the input data and the reconstructed data, which is an unsupervised neural network. Many extensions that are based on AEs have been proposed and broadly used, one of which is the variational AE (VAE) [32]. The VAE conducts variational inference to match the distribution of the hidden code vector of the AE with a predefined prior distribution. From the perspective of the probabilistic graph model, the encoder and decoder of the VAE can be interpreted as a probabilistic encoder and a probabilistic decoder, which are denoted as p θ (x\|z) and q φ (z\|x), respectively [32]. To match the distribution of the hidden code vector z with a predefined prior distribution, variational inference is performed to optimize the variational lower bound on the marginal log-likelihood of each observation. Thus, the objective function of VAE can be expressed as: where x i refers to a sample from the training dataset. The first term is the KL-divergence term, which represents the difference between the distribution of the extracted latent feature samples and the Gaussian distribution. The smaller the difference, the closer the distribution of the extracted latent features is to the Gaussian distribution. The second term is the AE's reconstruction error, which represents the expectation of the reconstruction error, namely, the gap between the input and the output. The smaller the expected value of the reconstruction error, the closer the output can be to the input. The hidden code vector z is sampled from Both the encoder and the decoder originally leverage the sigmoid function as the nonlinear activation function. Recently, the rectified linear unit (ReLU) [34] has been widely used as the nonlinear activation function. Figure 1 illustrates the overall framework of our proposed UNCD method. Let Y= y i M×N i=1 , y i ∈ R L×1 denote an input remote sensing image with M × N spectral vectors, and each spectral vector contains L dimensions. Firstly, the underlying characteristics are extracted by an encoder E and a decoder De trained via an adversarial approach, thereby yielding a compact representation Proposed Method , l < L of the redundant remote sensing images. Based on the observation that clouds are sparse and modeled as sparse outliers, some powerful constraints are imposed on the unsupervised neural network for the extraction of a discriminative feature matrix E (De (Z)) of the background, which has the same dimension as Z. Due to the reconstruction ability of AEs and the generation capability of GANs, it is possible to obtain features of the full image and the background in deep latent space, respectively. As a consequence, Z and E (De (Z)) are compact representations of the original image and background, respectively. Specifically, the residual error between Z and E (De (Z)) is beneficial to cloud detection. More details of each step are described as follows. Constructing the Residual Error in the Latent Space The proposed UNCD method relies on two assumptions for cloud detection. One is that the background can be distinguished from the clouds in the latent feature space and most of a remote sensing scene is background, while clouds are sparse. When a remote sensing image Y that contains clouds is input into the network, the network is expected to generate a compact representation of Y and to reconstruct a corresponding background via an unsupervised approach. Consequently, the residual error refers to the deep latent feature space in which the clouds are enhanced, and the background is suppressed: The encoder learns a mapping from input Y into the deep latent space, which produces a feature matrix Z that preserves the essential information including the clouds and background. Then, Z is input into the decoder De to reconstruct Y. Since sufficient training samples are available for most of the background and clouds are sparse with limited training samples in a remote sensing scene, the decoder produces small reconstruction error for the background region but relatively large reconstruction error for the clouds. The adversarial leaning terms and physical constraints regarding the characteristics of the cloud-contaminated images are imposed on the network to enhance the capability of discriminative feature learning performance. Adversarial Feature Learning Term Adversarial feature learning is used in the latent space to extract distinctive features. As shown in Figure 1, the encoder0 acts as a generator to produce latent feature variables z i ∼ q E 0 y i , which strives to fool the latent discriminator D z with z i ∼ p z i sampled from the prior distribution. The latent discriminator D z aims to distinguish between input that comes from the encoder z i ∼ q E 0 y i versus that from prior distribution z i ∼ p z i . As a consequence, the generator (encoder) E 0 attempts to minimize this objective against the latent discriminator D z that attempts to solve min where Loss adv_z is denoted as: Here, we set the prior distribution z i ∼ p z i as multivariate Gaussian distribution to extract feature variables that are more beneficial to cloud detection considering that clouds are sparse and the majority is background. Adversarial Image Learning Term The adversarial feature learning in the previous part may generate out-of-class features due to the capability of GAN to generate new variants. To generate features of the original remote sensing images, we constrain the network by the image discriminator D I to avoid the generation of out-of-class features, as illustrated in Figure 1. Via this approach, the decoder De and the image discriminator D I are combined into a GAN again, in the training phase of which the decoder De is changed to a generator to ensure that the generated image can fool the image discriminator D I . The image discriminator D I aims to distinguish whether its input comes from the generator De or the real input. The optimization problem is to achieve min De max D I Loss adv_I , where Loss adv_I can be expressed as: The first term indicates that the discriminator attempts to maximize the output of the real sample to make it closer to 1. The second term indicates that by optimizing the discriminator, the output to the generated sample becomes closer to zero. At the same time, the generator is optimized, thereby making the output of the discriminator on the generated samples closer to 1. Latent Representation of the Background The Gaussian distribution that is imposed on the network focuses on reconstructing the majority class, namely, the background, with relatively sufficient samples. Consequently, the generated image De (Z) with the same size as the input is closer to the background than to the clouds. Due to the strong representational capability of AE, it can still reconstruct the sparse pixels (i.e., the clouds). To further reduce the proportion of clouds in the reconstructed image De (Z), we impose the representation consistency constraint in the deep latent feature space, that is, we enforce the satisfaction of a multivariate Gaussian distribution by E (De (Z)) via an unsupervised approach that better accords with the background characteristics: where E De z i,l = µ i + σ i ε l and ε l ∼ N (0, I). The encoded output of the second encoder is represented by E De z i , and the output of the first decoder is denoted by De z i . During the training of encoder1, only the parameters of the encoder are optimized, while the parameters of the decoder De are kept fixed as, illustrated in Figure 1. E De z i,l is the projection of the reconstructed spectral vector De z i in the low-dimensional latent space, which is the enhanced background representation obtained by imposing the representation consistency constraint. Thus, the distinctive residual error for distinguishing between the background and the clouds in the low-dimensional latent space can be calculated via Equation (3), and an example is represented in Figure 2. Reconstruction Loss In a traditional AE network, the input data are encoded by the hidden layers to be decoded correctly in the output. The parameters are selected such that the objective of minimizing the following cost function is realized: where y i denotes an input spectral vector with L dimensions andŷ i denotes the corresponding reconstructed spectral vector. Finally, the combination of the aforementioned losses yields Loss = Loss adv_z +Loss adv_I +Loss Gauss +Loss r . The model is trained and updated via the stochastic gradient descent (SGD) algorithm. When the model loss converges, the parameters, including the weight matrix and the bias, are obtained. According to Figure 2, the compact representation of the original image, Z, contains features of both clouds and background regions, whereas E (De (Z)) can well represent the background information with the clouds restricted. As a consequence, the residual error ∆Z obtained from the pixel-wise difference enhances the clouds while suppressing the background in the lower-dimensional feature space, which facilitates the detection of clouds as shown in Figure 3. Subsequently, an adaptive weighting method that was proposed in our previous work [35,36] is applied on each dimension of the residual error ∆Z to discard redundant information and to construct a comprehensive map, where the structure tensor (ST) is utilized. The ST of the ith dimension in the residual error ∆Z can be defined as [35,36]: where ∆Z i x = ∂∆Z i ∂x and ∆Z i y = ∂∆Z i ∂y represent the derivatives of the ith dimension of the residual error ∆Z along the x and y directions, respectively. Since this structure tensor S i of the ith dimension is a semi-determined matrix, it can be decomposed into [35,36]: where λ i 1 and λ i 2 are the non-negative eigenvalues of the ith dimension and η i 1 , η i 2 are the corresponding eigenvectors. The larger eigenvalue is represented by λ i 1 . As discussed in our previous work [35,36], the relative larger eigenvalue can represent the response intensity of each pixel in the corresponding dimension of ∆Z. Therefore, the weight vector for each dimension of ∆Z is calculated from the relative larger eigenvalue. The larger the edge intensity of the ith dimension, the more structural information the ith dimension of ∆Z contains. Consequently, the ith dimension should occupy a larger proportion. The (i − 1)th dimension should also occupy a larger proportion. If the larger eigenvalues of all the dimensions are same, all the dimensions will have the same proportion, which is the same as the result obtained with the averaging function. In summary, the weighted residual error ∆Ẑ is calculated as [35,36]: where l is the dimension of ∆Z. To further consider that the adjacent pixels have high correlations with each other, we utilize a guided filter [37] to move the neighboring pixels to the same object for both the background and clouds. Finally, we use an iterative optimization step to further increase the detection accuracy, namely, we multiply the initial detection map by the residual error ∆Z, and repeat the detection procedure until the following stopping rule is satisfied: where C f −1 D and C f D represent the ( f − 1)th and f th detection maps, respectively. According to this stopping rule, if the ( f − 1)th and f th detection maps are highly similar, the iterative optimization will terminate. Landsat 8 Dataset The Landsat 8 dataset was made available online https://www.usgs.gov/land-resources/nli/ landsat/spatial-procedures-automated-removal-cloud-and-shadow-sparcs-validation, and is widely used to evaluate the cloud detection methods due to its wide coverage and high resolution. This dataset consists of 80 MS remote sensing images, with details described in [38]. The Landsat 8 dataset was collected globally using two instruments: OLI (Operational Land Imager) and TIRS (Thermal Infrared Sensor). We use the data from both of these. All the channels are used for cloud detection. The spatial resolution of each Landsat image is 30 m-that is, each pixel represents information on an area of 900 m 2 . The location of the dataset is global. Each image contains 1000 × 1000 pixels in the spatial domain and ten bands in the spectral domain. The corresponding reference maps are shown in Figure 4. GF-1 WFV Dataset The GaoFen-1 (GF-1) satellite was launched by China. Gaofen means "high resolution" in Chinese. Detailed information on the GF-1 satellite and wide field of view (WFV) images are available online http://sendimage.whu.edu.cn/en/resources/mfc-validation-data/. The GF-1 WFV image considered here is a Class 2A product produced via relative radiation correction and system geometry correction. The Class 1A data are the original digital product for regular radiation calibration, while the Class 2A data were generated after system geometry correction, where all pixels are re-sampled to 16-m resolution with 10-bit data. The false-color images and the corresponding reference maps are shown in Figure 5. GF-5 Hyperspectral Dataset The GaoFen-5 (GF-5) satellite can capture hyperspectral observations in the Chinese Key Projects of the High-Resolution Earth Observation System. Six payloads were carried on it: an Advanced Hyperspectral Imager (AHSI), a Visual and Infrared Multispectral Imager (VIMI), an Atmospheric Infrared Ultra-Spectral Sounder (AIUS), a Greenhouse Gases Monitoring Instrument (GMI), an Environmental Monitoring Instrument (EMI), and a Directional Polarization Camera (DPC). The spectral coverage of these sensors ranges from ultraviolet to long-wave infrared bands. Two HS images captured by the GF-5 AHSI sensor were used to evaluate the performance of the proposed method. Each image has a size of 430 × 430 × 180 pixels. The false-color images are shown in Figure 6. The reference maps for this dataset have not been published yet. Experimental Results We comprehensively evaluated the proposed method on three real datasets, which included MS and HS images captured by various imaging sensors over various scenes. First, we describe the dataset. Then, we introduce the compared methods and evaluation criterion. Third, we further investigate the performance of the proposed UNCD method, both qualitatively and quantitatively. Finally, we analyze the impact of the network structure on the detection performance. Experimental Setting Due to insufficient samples in the remote sensing imagery, we fixed the depth of our UNCD to 2 to avoid overfitting. Inspired by [39], the number of hidden nodes was fixed to √ L + 1, where L is the number of bands of the input remote sensing image. The leaky ReLU (LReLU) was used as the nonlinear activation function with a slope of 0.2 to compress the negative input and retain the negative part of the information to a certain extent. As the number of epochs increases, both the detection performance and the computational complexity increase. Thus, we set the number of epochs to 1000 as a trade-off. The batch size was fixed to the number of pixels in the spatial domain, namely, M × N for each remote sensing image. The learning rate was set to 0.01. The parameters introduced above were set to the default values in our experiments, and they can be tuned by the user for the optimal results. We conducted experiments on 8 NVIDIA Tesla k80 graphics cards based on a system running Python 3.6.0 and TensorFlow 1.10.0. All compared methods were implemented in MATLAB R2017a. Compared Methods and Evaluation Criterion To evaluate the performance of the proposed UNCD model, several representative cloud detection methods that are frequently cited in the literature, namely, K-means, PRS, SVM, PCANet, and SL, were employed for comparison in terms of both visual effects and quantitative evaluations. The K-means method is a typical unsupervised method. The PRS method yields satisfactory results on several remote sensing images, and is also an unsupervised method. Since the PRS method is only applicable to RGB images, we combined band 4-red, band 3-green, and band 2-blue into RGB images. The SVM, PCANet, and SL methods are supervised learning methods for cloud detection. To comprehensively evaluate the cloud detection results, the three commonly used evaluation criteria, namely, the area under the curve (AUC) [40] of the receiver operating characteristic (ROC), the overall accuracy (OA), and the kappa coefficient (Kappa), were employed. The area under the curve (AUC) [40] is widely used in objective evaluation indices. The AUC can identify general trends in the detection performance. The larger the AUC value of the ROC curve, the better the performance. The OA is defined as [41]: where true positives (TPs), true negatives (TNs), false positives (FPs) and false negatives (FNs) represent the number of correctly detected cloud pixels, the number of correctly detected non-cloud pixels, the number of false-alarm pixels, and the number of missed cloud pixels, respectively. The kappa coefficient reflects the agreement between a final cloud detection image and the ground-truth map. Compared with the overall accuracy (OA), the kappa coefficients can more objectively reflect the accuracy of the results. The larger the value of the kappa coefficient, the higher the accuracy of the result will be. The kappa coefficient is calculated as [41]: Landsat 8 Dataset Results The reference maps and the visual cloud detection results that were obtained by the competing methods for two images from the Landsat 8 dataset are shown in Figures 4 and 5. The reference map is published at the same time as the dataset and used together with the data. Moreover, the provider of the dataset marks the annotation. Image I is an example of a case with thin clouds, and Image II is an example of a case with thick clouds. With these two images, we compared our method with K-means, PRS, SVM, PCANet, and SL. We implemented these compared methods via the publicly released codes. According to Figures 4 and 5, the proposed UNCD method caused the smallest visual difference between the reference map and the detection map compared to K-means, PRS, SVM, PCANet, and SL. As illustrated in Figures 4 and 5, the K-means method yielded many noise-like detection results. The PRS method realized minimal improvement for Image I with respect to the K-means method, and thin clouds were undetected in large areas in Image I. The SL method generated some detection mistakes in the image due to the thinness and thickness of the clouds. The PCANet and SVM methods outperformed the K-means, PRS, and SL methods. In particular, the proposed UNCD method could accurately distinguish clouds and complex background scenes. The reason is that the proposed detection method extracts the spectral features of the image while utilizing the spatial background information between adjacent pixels. In comparison to these methods, the advantage of the proposed UNCD method is its robust detection performances for different clouds with different scales in different scenes. The objective evaluations, including AUC, OA, and Kappa obtained by all the considered algorithms, are reported in Table 1, which complies with the visual observation. Concretely, the AUCs obtained by the proposed UNCD method were 0.9543 and 0.9637 for Images I and II, respectively, which are much higher than those obtained by the second-best approach in each case, 0.8485 (PRS method) and 0.8848 (K-means method). The OA and Kappa obtained by the proposed UNCD method were also the highest, and much higher than those obtained by the second-best method. GF-1 WFV Dataset The reference maps and detection maps obtained by the compared methods are shown in Figures 6 and 7. The detection results obtained by the proposed method were similar to the reference maps, indicating that the proposed method could obtain a satisfactory detection performance. It is apparent that the UNCD method achieved better detection results than the K-means, PRS, PCANet, SVM, and SL methods. For the GF-1 WFV dataset, Table 2 lists the corresponding quantitative metrics. In addition, the competing methods are also reported in Table 2. From Table 2, the AUC values, OA, and Kappa obtained by the proposed UNCD method were the best among the compared methods. The reported OA scores on Images III and IV (0.9957 and 0.9934) obtained by the proposed UNCD method were higher than that obtained by the second-best methods (0.9835 for SVM on Image III and 0.9690 for SL on Image IV). The GF-5 dataset was used to evaluate the feasibility of our proposed UNCD method on real hyperspectral data. The cloud detection results are displayed in Figures 8 and 9. Image V in Figure 8 contains large clouds, while Image VI in Figure 9 contains many small clouds. It can be observed that our method was capable of discriminating clouds of different sizes from the background pixels. The K-means method introduced many false positives. PRS and PCANet failed to detect all the clouds. By contrast, the SVM and SL methods had better performance while also generating some detection mistakes. For example, the buildings in Image V were still detected as clouds by the SVM and SL methods. Component Analysis This section analyzes the effects of the significant processing components on the detection performance on each dataset. Since the reference maps of the GF-5 hyperspectral dataset are not publicly available, the objective evaluations could not be obtained for this dataset. Therefore, four remote sensing images coming from the Landsat 8 dataset and the GF-1 WFV dataset were used to evaluate the effect of each component objectively. Three comparison experiments were conducted. In the first experiment, only the AE was considered, which is a basic model for joint encoder-decoder training. The second considered AE with additional adversarial training (a latent feature discriminator). The third utilized the proposed method. The AUC, OA, and Kappa values were calculated as reported in Table 3. The better the detection performance, the higher the AUC, OA, and Kappa values. The AUC values were 0.9254, 0.9025, 0.9436, and 0.9462 for four images when only using AE. When applying the adversarial feature learning, the AUC values were improved to 0.9477, 0.9379, 0.9506, and 0.9689, respectively. When the multivariate Gaussian distribution was introduced, the method yielded the best AUC values, which reached 0.9543, 0.9637, 0.9676, and 0.9860, respectively. Similarly, the other two indicators (i.e., OA and Kappa) also increased. These results demonstrate that each component of the proposed UNCD method has a positive influence on the cloud detection performance. Discussion According to the values of AUC, OA, and Kappa in Tables 1 and 2 Figures 4-9 for various types of datasets, compared to several state-of-the-art methods, the proposed UNCD method performed the best in detection according to both the objective evaluation results and the visual observations. The superior performance of the proposed method is due to the latent adversarial learning constrained encoder, the image discriminator, and the multivariate Gaussian distribution in the network architecture. It can be concluded from the component analysis experiments that each part had a positive impact on the detection results. While the proposed UNCD method yielded promising results in cloud detection, several areas were identified during experiments for improvement in future work. It is worthwhile to further utilize the data characteristics of remote sensing images in order to optimize unsupervised networks and to improve the cloud detection performance. In addition, the network architecture can be enhanced by adding some loss functions and constraints. and the visual observations in The OA values of Images I-IV that were obtained by the proposed UNCD method were 0.9526, 0.9536, 0.9957, and 0.9934, respectively. Our method outperformed the second-best method by 1.44%, 5.23%, 1.24%, and 1.96%, respectively. While the performance of our method was the best among the considered methods, there is still room for improvement. Moreover, the accuracy of cloud detection was not high, and there were still many missed detections. However, other comparison methods also have this problem. Meanwhile, the proposed UNCD method is devoted to cloud detection. In the future, we will further realize cloud shadow detection. At the same time, to make the method universal, we will conduct additional experiments on datasets acquired by additional sensors. Conclusions In this paper, we proposed a discriminative feature learning constrained unsupervised network for cloud detection (UNCD) for remote sensing imagery. The induced latent discriminator, image discriminator, and multivariate Gaussian distribution consider the fact that clouds are sparse and modeled as outliers and realize a discriminative residual map between the original input and the background. Based on the analysis of the strong correlation between adjacent pixels, a guided filter is employed on the residual map to obtain an initial detection map. To further improve the detection performance, an iterative optimization algorithm is introduced which terminates automatically if the stopping condition is satisfied. Extensive experimental results on several datasets demonstrate that the proposed UNCD not only realizes a more favorable detection performance but also generalizes better to different datasets compared with other state-of-the-art methods. Moreover, the OA values for Images III and IV from the GF-1WFV dataset were 0.9957 and 0.9934, respectively. This signifies that our algorithm performwe better than other known algorithms. In future work, we will expand the datasets used for the experiments to improve the performance of the algorithm. Conflicts of Interest: The authors declare no conflicts of interest. Abbreviations The following abbreviations are used in this manuscript:	2020-02-03T02:10:27.917Z	2020-02-01T00:00:00.000	{ "year": 2020, "sha1": "194bf2e99d1fab93da0e4e77562ba4bb0e6951fd", "oa_license": "CCBY", "oa_url": "https://doi.org/10.3390/rs12030456", "oa_status": "GOLD", "pdf_src": "Unpaywall", "pdf_hash": "194bf2e99d1fab93da0e4e77562ba4bb0e6951fd", "s2fieldsofstudy": [ "Environmental Science", "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
269500498	pes2o/s2orc	v3-fos-license	Is a portable pressure plate an alternative to force plates for measuring postural stability and interlimb coordination of quiet standing balance control? Introduction Center-of-pressure (COP) synchronization and symmetry can inform adaptations in balance control following one-sided sensorimotor impairments (e.g., stroke). As established force plates are impossible to transport, we aimed to criterion validate a portable pressure plate for obtaining reliable COP synchronization and symmetry measures, next to conventional postural stability measures. Methods Twenty healthy adults participated. In a single session, three 40-s eyes-open and eyes-closed quiet stance trials were performed per plate-type, randomly ordered. Individual-limb COPs were measured to calculate between-limb synchronization (BLS) and dynamic control asymmetry (DCA). Net COP (i.e., limbs combined) area, amplitude, and velocity were used to describe anteroposterior (AP) and mediolateral (ML) postural stability. Criterion validity was evaluated using Spearman correlations (r) and Bland-Altman plots. Test-retest reliability was tested using intraclass correlation coefficients (ICC). Results Strong correlations (r > 0.75) and acceptable reliability (ICC > 0.80) were found regarding individual-limb COP velocity and DCA, net COP ML amplitude and AP and ML velocities. Bland-Altman plots yielded possible proportional bias; the pressure plate systematically underestimated COP scores by force plates and a larger error associated with a larger measurement. Conclusions Despite correlations between instruments and sufficient reliability for measuring postural stability and DCA, this technical note strongly suggests, due to a systematic deviation, using the same plate-type to accurately assess performance change within subjects longitudinally over time. Background Pathologies causing one-sided sensorimotor impairments (e.g., stroke) compromise balance control and increase the risk of falls. 1 However, balance recovery studies in this field remain scarce and often fail to distinguish behavioral restitution from compensations by relying on clinical scales, such as the Berg Balance Scale (BBS), 2 that have considerable ceiling effects 3 and do not show qualitative changes on the rated tasks. 4,5Likewise, most posturographic studies measuring, for example, the center-ofpressure (COP) area, velocity, or amplitude as a more precise postural stability measure are confounded by using only one force plate.This is insufficient to separate the limbs and inform adaptations in standing balance as reflected by, for example, the between-limb synchronization (BLS) 6,7 and dynamic control asymmetry (DCA). 8,9These metrics are currently utilized in the stroke recovery literature to inform, respectively, how well both limbs work together to control balance and how much each limb contributes.Hence, these measures are complementary in providing greater insight into compensatory changes in balance performance following one-sided loss of sensorimotor function. Measuring BLS and DCA requires posturographic systems with two floor-mounted force plates by established brands, which are currently considered the "gold standard" method. 10,11However, these force plates are expensive and impossible to transport, which makes clinical studies difficult to conduct over the first 3 to 6 months poststrokethe period of significant balance recovery 2 -while patients are being discharged from stroke services to their own homes or care facilities in the region.3][14] Although not yet investigated, these instruments can record individual-limb COP movements using a single plate due to a larger number of embedded sensors.This advantage may further mitigate the need for extensive infrastructure to measure BLS and DCA, thus improving clinical feasibility of conducting serial measurements as part of trials. As a "first step" toward its criterion validation, the current study involving healthy adults compared a portable pressure plate with gold standard force plates for measuring COP synchronization (i.e., BLS) and symmetry (i.e., DCA) while standing, next to conventional descriptors of postural stability.Acknowledging that repeatable measures are conditional for showing agreement between assessment methods, 15 we investigated the test-retest reliability of each plate type before comparing them.Our research questions were as follows: (1) Are three immediate test-retest repetitions using a pressure plate or gold standard force plates sufficient to achieve reliable measures of postural stability, BLS, and DCA in healthy adults standing quietly?(2) Are averaged pressure plate measures of postural stability, BLS, and DCA in agreement with those obtained using gold standard force plates in healthy adults standing quietly? We expected to confirm the literature by showing a high test-retest reliability (i.e., intraclass correlation coefficients (ICC) ≥ 0.80) of AMTI (Advanced Mechanical Technology Inc., MA, USA) force plates for measuring postural stability, 10,11 and hypothesized to find similar results with respect to BLS and DCA.Furthermore, we expected that a pressure plate's reliability for measuring postural stability, BLS, and DCA would be comparable to force plates.][14] Because correlations alone are insufficient to detect systematic biases, we additionally aimed to explore the level of agreement with a Bland-Altman analysis. 15,16Measurement bias with lower COP values by a pressure plate was priorly suggested, 12,14 warranting further investigation. Design Ten female and 10 male adults with a perfect BBS score volunteered to participate.No back or lower limb injury, use of medication, or neurological condition were reported.All volunteers provided written informed consent according to the policy of the local ethics committee (Antwerp University Hospital, BE; protocol no.19/18/233; date 24/ 06/2019). In a single session, a 0.5 m Footscan (RS Scan, Materialize, BE) pressure plate (578 x 418 x 12 mm, sampling frequency 500 Hz) and two AMTI Type OR 6-7 force plates (500 x 400 mm, sampling frequency 1000 Hz) were used to quantify quiet standing balance during separate, randomly ordered measurements to avoid interference of the differently sized, rigid plates.As such, per instrument six 40-s trials were performed, alternatively with the eyes open or closed.The bare feet were positioned in a standardized way (8.4 cm heel-to-heel distance, 9°toe-out angle), and participants were instructed to stand as still as possible while keeping the eyes fixated at a 3-m distant target placed in front of them or with the eyes closed.This protocol aligns with typical clinical posturographic testing. 8,9o assess test-retest reliability per instrument, variation in three immediate test repetitions in the same participant and under the same condition (i.e., eyes-open or eyesclosed) was analyzed using ICCs. 17Regarding criterion validity, defined following COSMIN 18 as the extent to which outcomes reflect results by the gold standard, averaged outcomes per condition and participant were compared between instruments. Data processing For each eligible trial, we calculated the net (i.e., both limbs combined) and individual-limb COP with anteroposterior (AP) and mediolateral (ML) coordinates from the last 30 s to avoid starting effects.The reference axes were rotated by 9°t o coincide with the foot axis.We followed these steps, as described per instrument: • Force plates: Raw tri-axial force data from four load sensors -one in each corner -were collected with Nexus (Vicon Motion Systems Ltd, UK).Then, a custom-made MATLAB (version R2018a) algorithm was used to compute the COP for each side using these equations: Here, x, y, and z are the AP, ML, and vertical directions, respectively; F are the forces; M are the moments; and C is the offset from the geometric plate center.Subsequently, the net COP was calculated as a weighted average using this equation: • Pressure plate: Many embedded sensors (2.6 sensors/ cm 2 ) record the plantar distribution of vertical forces, or Fz.COP was then computed using the system's own software (Footscan 9, RS Scan, Materialize, BE) as the point of application of the summed forces using all sensors bearing weight at the entire plate (i.e., net COP), or at either geometrical side to extract individual-limb COPs. Finally, the COP signals from both instruments were low-pass filtered (2 nd order Butterworth, 10 Hz) using the same MATLAB algorithm, and the same scripts were used to calculate outcome metrics. Outcome metrics Peak-to-peak sway amplitude in mm (nCOP amp-ap , nCOP amp-ml ) and root mean square velocity in mm/s (nCOP vel-ap , nCOP vel-ml ) were determined in the AP and ML directions as traditional descriptors of postural stability. 19In addition, the net COP area (nCOP area ) was calculated in mm 2 as an ellipse that covered 85% of the entire signal using principal component analyses. 20mplitudes (iCOP amp-ap , iCOP amp-ml ) and velocities (iCOP vel-ap , iCOP vel-ml ) were also determined for each individual limb, next to BLS as a cross-correlation coefficient between COP movements at a zero time-lag 6,7 and DCA as a symmetry index 8 following this equation: The loaded and unloaded sides were determined by dividing the mean vertical force under each limb.We focused on the AP direction, and BLS and DCA were not calculated with ML COPs because frontal plane sway is mainly controlled by a loading-unloading mechanism that is not reflected by COP changes. 6 Statistical analysis Regarding question 1, we computed the ICCs using a twoway mixed-effects model (ICC 3,3 ) as a measure of agreement between the three test-retest measurements. 17A multiple-measurement type was chosen because the actual application is based on averaging trials. 10,11Acceptable reliability was defined as an ICC 3,3 > 0.80.Specifically, ICC 3,3 ≥ 0.90 was interpreted as excellent and 0.80-0.90as good reliability. 21or question 2, averaged COP scores were plotted per participant to observe general trends between instruments.Spearman's rank correlation coefficients (r) were calculated for each outcome and r ≥ 0.75 were interpreted as strong, 0.50-0.74as moderate, and <0.50 as low or no relationship. 21Mean differences were statistically analyzed using Wilcoxon singed rank tests because the averaged outcomes per instrument were not normally distributed.In conjunction, Bland-Altman plots (i.e., subject-specific mean scores by difference scores) were created, including the mean difference line with its standard error and the limits of agreement (LOA). 15Narrower LOA encompassing zero reflect better agreement, whereas the distribution of difference scores was visually analyzed for bias.According to Ludbrook, 16 a significant difference by a constant amount is interpreted as fixed bias and a slope pattern as proportional bias.The significance level of all analyses was set two-tailed at 0.05. A sample of 20 subjects was defined a priori, by offering 80% power to detect a correlation coefficient of a least r = 0.60 between both devices at a significance level of 0.05. Results One participant had a corrupted dataset, and 19 participants (10 female) with a mean (±SD) age of 35.4 ± 15.9 years were included in the analyzes.Their body mass and length were 77.3 ± 13.7 kg and 171.2 ± 7.0 cm, respectively. Table 1 shows the reliability outcomes with ICC 3,3 pointestimates and their confidence intervals.Regarding the force plates, ICC 3,3 ≥ 0.80 were found for eyes-open and eyesclosed conditions regarding nCOP area , nCOP amp-ml , nCOP vel-ap , nCOP vel-ml , iCOP vel-ap , and DCA.A pressure plate yielded ICC 3,3 ≥ 0.80 regarding the nCOP vel-ml , iCOP vel-ap , and DCA under both visual conditions.In addition, the pressure plate reached sufficient reliability for measuring nCOP amp-ml and nCOP vel-ap under the eyesclosed condition.In general, ICCs < 0.80 were found for measuring amplitudes (i.e., nCOP amp-ap , iCOP amp-ap ) and BLS, irrespective of the instrument and visual condition. Figure 1 illustrates that a relatively higher COP score by the pressure plate corresponds to a higher force plate score, and vice versa.In line with this observation, Table 2 shows strong correlation coefficients of r ≥ 0.75 for both visual conditions regarding nCOP vel-ml , iCOP vel-ap , and DCA.In addition, nCOP area , nCOP amp-ml , nCOP vel-ap , and iCOP amp-ap were strongly correlated between instruments with r ≥ 0.75 with respect to the eyes-closed measurements. A second observation from Figure 1 is that the pressure plate COP scores are consistently lower than those of the force plates.In the same vein, mean differences between instruments were highly significant with respect to each COP metric investigated (p < .001,Table 2).Bland-Altman plots further show that most difference scores were positioned within the LOA with broad ranges that were situated below zero, except for nCOP area (Figure 2).In addition, an inclined distribution slope is shown for nCOParea, nCOPamp-ml, nCOPvel-ap, nCOPvel-ml, iCOPvel-ap, and iCOPvel-ml, such that an increasingly larger difference was observed relative to the mean score, indicating proportional bias.Mean differences in BLS and DCA were, however, nonsignificant (p > .05,Table 2) and Bland-Altman plots show difference scores that are evenly distributed within the LOA, ranging above and below zero. Discussion In the current study, a reliability analysis (question 1) and head-to-head comparison were performed between a pressure plate and gold standard force plates to criterion validate the former (question 2) for measuring COP while quiet standing.We advanced prior validation experiments [12][13][14] by addressing the capability of a single pressure plate to capture individual-limb COP movements to address postural stability, as reflected by the total amount of COP sway, and interlimb coordination in terms of BLS and DCA.We found acceptable test-retest reliability for measuring velocity-based COP metrics with the pressure plate, including DCA, with strong correlations relative to force plates.COP amplitudes and BLS were found less reliable.Despite strong associations between instruments, we identified a systematic deviation.The pressure plate systematically underestimated force plate outcomes of COP with proportional bias.Seemingly, both instruments acted in a similar, but not identical way.These findings are discussed with implications for clinical use. Similar to prior validation studies, [12][13][14] we found strong associations between a pressure plate and established force plates for measuring postural stability.In our analyzes, this association was strongest for velocity-based measures of COP (Table 2), and we have shown for the first time a similar correlation strength with respect to DCA, i.e., a symmetry index of individual-limb COP velocities. COP velocities are known to be particularly consistent, requiring two to three trials to reach reliable outcomes. 10,11his reliability is thought to result from a sensitivity to highfrequency changes in the COP signal that are more consistent and reflect stabilizing responses to body sway. 19In agreement, we found AMTI force plates to yield good-toexcellent reliability for measuring nCOP vel-ml , nCOP vel-ap , iCOP vel-ap , and DCA, whereas displacement-based measures including COP amplitudes and BLS were less reliable, despite averaging three 30-s trials (Table 1).A low reliability of force plates for measuring BLS agrees with a The mean values and their standard deviations (SDs) are shown per instrument.Mean differences are presented between devices with a standard error (SE).p values are the probability statistics that the mean values are the same (null hypothesis) assessed with Wilcoxon signed rank tests.Results highlighted in bold are statistically significant (p < .05).Lastly, r estimates show the strength of the Spearman correlation coefficients between instruments with , reflecting a statistically significant correlation of moderate strength (r = 0.5-0.74);and , reflecting a significant strong (r ≥ 0.75) correlation.previous study in stroke patients. 22In comparison, the pressure plate exhibited a similar reliability in the same measures during the eyes-closed condition, whereas eyesopen measurements often yielded ICCs < 0.8, or insufficient reliability (Table 2).This may suggest that a pressure plate's measurement precision is lower relative to a force platform, but improves when sway is provoked by closing the eyes, which typically causes larger and more regular COP movements. 23otwithstanding strong correlations and acceptable testretest reliability, we identified a systematic deviation.COP scores by the pressure plate were significantly smaller than those by force plates (Table 2), and this deviation shows increment proportionally to the magnitude of the measure (Figure 2).To gain more insight into this deviation, we have added a limited power spectral analysis in a single subject for each instrument (see attachment A).The energy content of the COP signal measured with the pressure plate is lower compared the force platform.Furthermore, the attached figures suggest that the pressure plate particularly misses out on the higher-frequency COP components. Proportional bias in summary outcomes of COP was earlier suggested, 12,14 pointing toward an underlying cause inherent to the use of different soft-and hardware.Although speculative, this may include the sensor type because, relative to force plates, horizontal forces are not recorded by a pressure plate.Furthermore, averaging forces over many embedded sensors could result in a smoothing effect, causing a lower COP velocity and amplitude.Alternatively, the different sampling frequencies (pressure plate 500 Hz vs force plates 1000 Hz) may have caused bias, acknowledging that COP measures are sensitive to sampling frequencies, 24 although both instruments exceed the recommended 100 Hz. 11 Interestingly, Bland-Altman plots show agreement between instruments for measuring DCA, yet with fairly large LOA (eyes-open [52.5;À66.2], eyes-closed [35.2;À51.3]; Figure 2).The systematic deviation appears to be controlled for by using two instrument-dependent COP measures to calculate this symmetry index.This underpins our finding of systematic rather than random deviation from force plates when measuring COP with a pressure plate.However, whether both plate-types exhibit agreement in the case of asymmetric balance due to, for example, post-stroke hemiplegia 7,8 cannot be addressed here. Implications First, strong correlations between instruments indicate that the pressure plate can serve as an alternative; however, because of systematic bias, it cannot replace force plates.Therefore, we strongly recommend that researchers and clinicians use the same instrumentation between testing sessions to evaluate performance changes in standing postural control within subjects.Second, the pressure plate yielded similar reliability to gold standard force plates when visual input was suppressed.This finding may suggest that a pressure plate's utility to measure COP is limited to challenging test conditions, or when assessing quiet standing balance in clinical populations who exhibit greater spontaneous sway.However, this requires confirmation of our findings in these populations.Third, velocity-based measures are particularly reliable and therefore advised for describing (change in) balance performance, irrespective of the choice of plate-type.Finally, general similarities between instruments suggest that existing recommendations for standardizing force plate measurements are applicable to pressure plates.This includes that at least 90 s of COP data should be collected over several test repetitions within a session. 10,11In line with prior reliability studies, 10,11,19 averaging three 30-s trials was found sufficient to achieve reliable outcomes in our study.This is important for clinical use because pathological populations are often unable to adhere to longer assessment durations.Our own protocol, as part of a recently completed stroke recovery study, 25 is attached in supplement (see attachment B). Limitations We included a small sample of mostly younger adults.Replication in larger samples, including healthy agematched subjects, is needed to confirm our findings and provide normative values in higher-age categories as a reference for clinical trials.Moreover, generalization of our findings to people with asymmetric balance remains unknown.Second, we did not record COP data simultaneously by "stacking" plates to avoid interference.However, simultaneous recordings are recommended for future studies to allow more-accurate comparisons by eliminating withinsubject variability.Third, our analyses should be viewed within the context of specific devices, and outcome metrics and balance conditions that were chosen to align with established clinical testing protocols.This includes, for example, cropping the first 10 s of each recording and calculating summary measures of the resultant 30 s of data.Alternatively, future studies may investigate epoch-based metrics, as recently argued. 26Finally, we tested intrasession test-retest reliability.However, assessing test-retest reliability between sessions is required to further develop standardized testing protocols for tracking changes in balance performance due to ongoing sensorimotor recovery or an intervention, as outlined above (see implications). Conclusion The current results suggest that the pressure plate is a reliable assessment tool, yielding strong correlations with gold standard force plates for measuring postural stability and individual limb contributions to balance control, as reflected by DCA.However, there are some concerns with its criterion validity as we found a systematic deviation causing lower COP scores compared with outcomes by force plates.This strongly suggests using of the same instrumentation to accurately assess performance changes in a consistent way within a specific subject.If this limitation is considered when designing data collection protocols, a pressure plate may hold promise as a clinical tool to make serial measurements as part of longitudinal studies in populations with impaired balance and an increased risk of falls feasible.Therefore, further validation experiments investigating measurement properties of pressure plates for assessing balance, next to pressure insoles to measure COP movements during various upright activities 27 and portable force plates that are emerging, 28 are encouraged in pathological populations. Figure 1 . Figure 1.Mean COP scores per participant measured with either a pressure plate or gold standard force plates.Peak-to-peak amplitude (amp, shown in purple) and root mean square velocity (vel, shown in green) of the COP in anteroposterior and mediolateral directions, recorded at the limbs separately (ie, left-sided and right-sided) and combined (ie, net), are plotted per individual subject (N = 19).Thicker bars reflect pressure plate scores and thinner bars reflect force plates as the gold standard.These bars are overlapped per participant to visually compare outcomes obtained from both instrument types for measuring COP when quiet standing. Figure 2 . Figure2.Bland-Altman plots with subject-specific differences and mean scores between a pressure plate and two force plates as the gold standard.The Y-axis shows the subject-specific difference scores between the two measurement instruments, and the X-axis represents the mean scores per subject.Solid red lines represent the mean difference score of the entire group of healthy participants (N = 19), dotted red lines represent the standard error of the mean score, and blue dotted lines and the blue-colored area in-between represent the upper and lower ends of the limits of agreement.On the left, the net COP metrics reflecting postural stability are displayed.On the right, individual-limb COP and interlimb coordination metrics are displayed.A downwards shift of the mean scores deviating from the zero score, as can be observed in most of the shown figures, reflect a systematic difference with lower pressure plate value compared with force plates that increases relative to the magnitude of the measure (ie, proportional bias).PP, pressure plate; FP, force plates. Table 1 . Test-retest reliability of a pressure plate and laboratory-grade force plates for measuring postural stability and interlimb coordination in quiet standing balance control.ICC 3,3 point-estimates with 95% confidence intervals are shown, reflecting variation between three immediate 30-s trial repetitions within a single measurement session. Results are shown per instrument and visual condition.ICC 3,3 estimates highlighted in bold reflect a statistical significance finding with , reflecting good reliability (ICC 3,3 0.90-0.80),and **, reflecting excellent reliability (ICC 3,3 > 0.90).a discrepancy between instruments of ICC 3,3 > 0.20 favoring the marked value.b ICC 3,3 ≥ 0.80 reflecting good reliability across instruments for the eyes open condition.c ICC 3,3 ≥ 0.90 reflecting excellent reliability across instruments for the eyes closed condition. Table 2 . Criterion validity of a pressure plate for measuring postural stability and interlimb coordination in quiet standing control using two laboratory-grade force plates as the gold standard.	2024-05-03T06:17:28.925Z	2024-03-04T00:00:00.000	{ "year": 2024, "sha1": "e0787de63b6553f6729388ff64848882689d8a37", "oa_license": "CCBYNC", "oa_url": "https://journals.sagepub.com/doi/pdf/10.1177/20556683241234858", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "849e3f2cb10eab1987c6f4e6d4e652058204128a", "s2fieldsofstudy": [ "Medicine", "Engineering" ], "extfieldsofstudy": [ "Medicine" ] }
267112883	pes2o/s2orc	v3-fos-license	Improvement of Recalcitrant Dissecting Cellulitis of the Scalp After a Trial of Upadacitinib Dissecting cellulitis of the scalp (DCS) is a rare condition characterized by painful inflammatory nodules and abscesses on the scalp, often leading to sinus tracts and scarring alopecia. We present a case of DCS in a 26-year-old male who experienced significant clinical improvement following a short course of upadacitinib, a Janus kinase (JAK) inhibitor. The patient received multiple standard treatments such as topical antimicrobials, oral antibiotics, corticosteroids, and intralesional triamcinolone injections, with limited success. However, following the initiation of upadacitinib, the patient reported reduced pain, pustular draining, and bleeding, with significantly improved quality of life. To our knowledge, there is currently a paucity of literature documenting the use of JAK inhibitors for DCS. This case aims to highlight the potential of JAK inhibitors as a therapy for refractory DCS, a condition with limited treatment options. Introduction Dissecting cellulitis of the scalp (DCS) is a rare condition that is a part of the follicular occlusion tetrad of hidradenitis suppurativa (HS), acne conglobata, and pilonidal disease [1].It commonly occurs in 20-40-yearold African American males and presents as painful, inflammatory nodules or abscesses that can precipitate the development of sinus tracts, keloids, and scarring alopecia [2].Although the precise etiology and inflammatory cytokines involved in DCS are unclear, it is suspected that an error in follicular keratinization leads to the obstruction and rupture of hair follicles, resulting in a chronic inflammatory response [1].HS is a condition closely linked with DCS and thus, one of the pathways that could possibly be involved in the chronic inflammatory response is the Janus kinase (JAK) signaling pathway [3].JAK inhibitors bind to JAK, an intracellular tyrosine-kinase protein, which downregulates pro-inflammatory signaling molecules such as IL-10, IL-12, and IL-23 [4].Upadacitinib is highly selective for the JAK1 isoform and inhibits IL-6 and interferon gamma-related signaling [5].We present a case of recalcitrant DCS that displayed an impressive clinical response after a course of upadacitinib. Case Presentation The patient is a 26-year-old male, non-smoker with a history of obesity and atopic dermatitis.On his initial visit, he reported an 11-month history of enlarging, painful cysts on the posterior scalp that drained purulent exudate and blood.He had not experienced similar cutaneous symptoms at other sites, including the axillae or groin, and never had prior treatment for his scalp lesions.On physical examination, the posterior scalp exhibited large, tender, draining pustules, nodules, and interconnected, firm sinus tracts with absent hair growth (Figure 1A, 1B).A complete blood count and basic metabolic panel were within normal limits.Inflammatory markers, including C-reactive protein (1.4 mg/dL, ref <0.8), erythrocyte sedimentation rate (60 mm/h, ref 0-15), and IL-6 (5.73 pg/mL, ref <5.0), were elevated. FIGURE 1: Prior to the trial of upadacitinib Posterior scalp with large pustules, nodules, and interconnected, firm, edematous sinus tracts that drain pus and blood, with an absence of hair growth. Prior to therapy with oral JAK inhibitor upadacitinib, the patient was given topical antimicrobials (benzoyl peroxide 10%), oral antibiotics (sulfamethoxazole-trimethoprim 800-160 mg twice daily), and oral prednisone (20 mg three times a day).Intralesional triamcinolone injections (40 mg/ml) were also administered to the affected areas over multiple follow-up visits.Adalimumab could not be ordered due to insurance and financial issues.Undergoing surgery or taking isotretinoin was also not preferred by the patient.Four months following his initial visit, the patient stated that only short-term relief was felt with the corticosteroid injections and there was little to no improvement in his overall condition since beginning treatment.A physical examination revealed continued tenderness, inflammation, and drainage from his scalp lesions.Due to limited therapies available for the treatment of DCS and the failure of standard therapies, a trial of upadacitinib 15 mg twice daily was initiated and added to his regimen of topical antimicrobials, oral antibiotics, and corticosteroid injections.At a one-month follow-up visit, the patient reported substantial improvement in pain, pustular draining, and bleeding.According to the patient, this was the longest period where he experienced minimal to no flares and reported a dramatic improvement in his quality of life.At around a two-month follow-up visit, a physical exam revealed significantly fewer pustules, smaller sinus tracts, and overall decreased inflammation with no visible drainage (Figure 2A, 2B).No major side effects were reported and the continuation of upadacitinib was planned. FIGURE 2: After two months of upadacitinib Posterior scalp with fewer pustules and smaller, less inflamed sinus tracts without pustular drainage or bleeding.Areas of prior inflammation now exhibit scarring. Discussion Features of DCS overlap with many core qualities of HS, such as the type of lesions present, recurrence of lesions, and its suppurative nature, with the main difference being the location of the disease.These two diseases often coexist [1].Oral JAK1 inhibitors have shown preliminary efficacy in treating moderate-tosevere HS in two phase II studies, have demonstrated high clinical responses in cohort studies, and are currently undergoing a phase III trial for HS [3,6].However, it is currently only FDA-approved for varying inflammatory and rheumatologic conditions such as rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, and recently for moderate-to-severe Crohn's disease [7,8]. Current standard pharmacological treatments for DCS remain limited and include oral antibiotics, isotretinoin, and corticosteroids, which all have variable efficacy [9].In recent case studies, tumor necrosis factor-alpha inhibitors have shown some promise in DCS refractory to conventional treatment, a therapy also indicated for moderate-to-severe HS [10,11].One recent case of an adolescent with DCS demonstrated a successful response to treatment with adalimumab and baricitinib [1].However, there is no literature detailing the use of JAK inhibitors as a potential therapy for DCS, and to our current knowledge, this is the first case of recalcitrant DCS successfully responding to a trial of upadacitinib.Although our patient was not taking upadacitinib as a monotherapy for his condition and was on other concomitant medications, it was only after the addition of upadacitinib to his regimen that a significant clinical response was observed after months of failing to respond to standard therapies. Conclusions Our case adds to the increasing number of literature on the use of anti-inflammatory agents like JAK inhibitors for the treatment of inflammatory and suppurative dermatoses.This single-patient observation introduces the need for further investigation and consideration of including JAK inhibitors in the management of refractory DCS, a chronic, difficult-to-eradicate disease for which current treatment options are limited.	2024-01-24T17:58:08.413Z	2024-01-01T00:00:00.000	{ "year": 2024, "sha1": "4baf2f9094eb6caa4247b410ff3465c2efff7caf", "oa_license": "CCBY", "oa_url": "https://assets.cureus.com/uploads/case_report/pdf/222394/20240116-8370-z9hw9m.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "952cc1f87a8c7ebe25a4f473a83136556edef8ac", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
246241490	pes2o/s2orc	v3-fos-license	Technical procedures and REDCap tools for internet-based clinical trials In March of 2020 our team of researchers developed and opened three clinical trials to investigate hydroxychloroquine as prophylaxis or treatment for the coronavirus disease 2019 (COVID-19). We simultaneously built corresponding Research Electronic Data Capture (REDCap) projects for these low-touch, remote trials that relied on participant-reported data. REDCap has built-in features that support pragmatic, internet-based studies, and REDCap is flexible enough to allow creative solutions for specific trials. We describe challenges, choices, and suggestions based on our experience with REDCap for our COVID-19 trials. Background The coronavirus disease 2019 (COVID-19) was declared a global pandemic by the WHO on March 11, 2020. The novelty of this disease and its level of impact required immediate and rapid research to test options for mitigation, including clinical trials of potential prophylaxis and treatment [1]. Our team of researchers quickly prioritized our efforts and resources to respond to the public health crisis. Within 8 days we had drafted and received IRB approval for a protocol to investigate hydroxychloroquine as a possible prophylaxis for those who had been recently exposed to the virus [2]. Protocols to investigate the drug as an early outpatient treatment for COVID-19 [3] and as a pre-exposure prophylaxis (PrEP) [4] followed shortly thereafter. At the same time, we designed and built databases for electronic data capture for these three outpatient trials. From the outset we knew that these trials needed to be pragmatic since the virus was completely novel and healthcare experts were continuously learning about its behavior and effects. At the onset of the pandemic, all interpersonal interaction was dramatically restricted, including within our research team. We were all suddenly working remotely, so we had limited face-to-face interaction with each other, much less with potential participants. We therefore planned for trials that would be entirely internet-based, with self-screening and a data-collection system that was flexible as the situation developed. We selected the online electronic data capture software REDCap (Research Electronic Data Capture) [5], primarily because we had immediate access through our institution and our team had experience using this platform. Additionally, REDCap is extremely user-friendly and flexible, allowing the entire data-collection system including surveys and reporting to be developed rapidly. REDCap also has all of the requirements for security of health data already in place. Here we describe the technical procedures and tools our team implemented to conduct three rapid, internet-based, pragmatic randomized clinical trials via REDCap. (Summarized pearls in Table 1) Some concerns we present affected us because of the speed and urgency of the early COVID-19 pandemic, but most issues are generalizable to any remote low-touch, automated clinical study in REDCap. Screening and enrollment All three trials were designed with a similar survey structure: Screening survey, Enrollment survey, and Follow-up surveys. We achieved this in REDCap by creating each of these as a separate instrument, activated as a survey. Screening These trials needed to recruit as many participants as possible from around the continent, so we activated REDCap's Custom Public Survey URL for the Screening surveys and publicized these links widely. We initially created and utilized a study-specific email address to provide an "out of office" automated reply providing potential participants with a participant information sheet in the text of the message along with the To send an informational email to a batch of participants, schedule an Automated Invitation for a survey without a link. Survey Settings Allows modifications for survey appearance and access The splash screens that accompany a REDCap survey are useful for including instructions to participants. These can be edited throughout the trial as FAQs come in from participants. Allow or limit a participant's ability to return to and change a survey once submitted. Ensure logic is true before sending Automated Invitation An option on the Automated Invitation Especially useful for a project that gets updated often. "REDCap will re-evaluate the logic against the record's data values whenever the record values are changed AFTER the invitation has been scheduled but BEFORE it has been sent to the respondent". Survey Distribution Tools Public Survey Link Participant List Survey Invitation Log A Custom Public Survey URL is a personalized link to the first survey in a project and is useful for marketing a trial.Participant List is useful for viewing lists of participants by contact information and for sending surveys in a batch.Survey Invitation Log is useful for confirming when surveys were sent and for editing scheduling of future survey invitations. Reports Creating a report by filtering on certain fields is useful for tracking data. For proactive tracking it can be useful to use the datediff function in the filter logic to remove participants who have passed a time threshold. Alerts Can be set to send an email to a particular recipient if the value of a field meets specified conditions. Useful for immediate notification of an adverse event. Rule H Updates or corrects the values of Calculated fields. Useful if any Calculated field needs to be updated regularly. Piping Values of any field can be piped into the text of any other field and into the text of a survey invitation. Among other things, this can be useful for including study medication shipping number at study start, reminding a participant of an answer on a previous survey during followup, and for informing the participant of their randomization assignment at study end. History All values ever entered for a field are automatically saved within that field's history. These can be retrieved if a value ever gets accidentally deleted (e.g. if a participant manages to return to a survey, changes something, and re-saves the survey). URL for the Screening survey. Eventually we created a website for each trial [6]. Regardless of how participants accessed the URL, once potential participants accessed the Screening survey they submitted their email address and answered questions to determine their eligibility. Eligibility criteria were listed on clinicaltrials.gov, but not otherwise publicly stated. The eligibility questions were a series of REDCap fields for all of the trials' inclusion and exclusion criteria. A set of Calculated fields following these questions then processed the responses and determined eligibility (Supplemental Material 1). These Calculated fields were hidden from the participants using the @HIDDEN-SURVEY Action Tag. Once eligibility was determined from a submitted Screening survey, the Automated Invitation for the Enrollment survey was immediately triggered, and potential participants received an email with a link to the Enrollment survey. This two-part screening and then enrollment verified participants had working email addresses. Participants were warned that an immediate email would be sent and if not received to look in their spam email folder. Another option would have been to put the Screening and Enrollment surveys into a Survey Queue so that the Enrollment survey appeared immediately if qualifications were met. However, by using our method with the Automated Invitations, eligible participants were forced to find emails from our trials in their inboxes. This way we were only enrolling participants who successfully interacted with our emailed links, which was critical since all of our followup data were collected via emailed surveys. Participants who were ineligible received an email notifying them of their status. To achieve this we created a "Not enrolled" instrument, which was activated as a survey. If the conditions to not be enrolled were met in the Screening survey, then the Automated Invitation to this survey was sent out, but with a link to complete a survey removed from the body of the emailed invite. Instead, we included links to the CDC website for further information about the COVID-19 disease. Eligibility requirements changed multiple times and quickly as our understanding of the behavior of the COVID-19 disease evolved and from FDA communications. For the post-exposure prophylaxis (PEP) and early treatment trials, which started enrolling in March 2020, this meant that our calculated eligibility fields needed to be updated even as enrollment was underway. So as to not disturb the value of the eligibility field of already enrolled participants, we created a new eligible Calculated field with each new update and edited the logic of the Automated Invitation for Enrollment. We found it useful to include the date change in the new field name to easily reference when the updated eligibility field was implemented. REDCap has a feature in the Data Quality Application called "Rule H", which updates "incorrect values for Calculated fields." In order to ensure that the Automated Invitations for Enrollment survey only triggered for new participants (and did not re-send to previous participants), we made sure we never executed Rule H in the project for the PEP and treatment trials. Additionally, we also updated the Conditions on the Automated Invitation to include appropriate logic. The preferable logic to do this would have been logic such as "[scr_date] > (date of update);" however, REDCap does not have the ability to read dates into logic. In addition, as we were screening hundreds of participants a day for these trials, the exact time of update was important. We therefore used logic based on the last participant to screen prior to the moment of update implementation (e.g. "[study_id] > 13098"). Additionally, REDCap has a very useful option on the Automated Invitation that can verify logic. When the "ensure logic is true before sending automated invitation" option is selected, "REDCap will re-evaluate the logic against the record's data values whenever the record values are changed AFTER the invitation has been scheduled but BEFORE it has been sent to the respondent." We tracked changes with a diagram and detailed notes. This aided in intra-team communication to make sure we all agreed. Tracking these changes both in notes and with dated fields names also aided in data management to determine who was eligible based on the time they were screened and guided our development of the CONSORT diagram in the presentation of final results. Fig. 1 depicts the final flow diagram of inclusion criteria for the PEP and treatment trials [2,3]. Notes on updates were also important for communication with Canadian database developers as they had to make the exact same changes to their REDCap project run in parallel. These projects were required to remain separate to abide by privacy guidelines specific to each country. Maintaining two separate but identical REDCap projects added another layer of challenge to managing the data for these pragmatic trials. Consistent and detailed communication was vital to identical data collection. Thus, in addition to comprehensive notes, we downloaded the full Data Dictionary after every update, filed it by date in our records, and delivered it to our Canadian co-investigators. Enrollment These were high-volume studies and the immediate nature of the pandemic necessitated expeditious enrollment. Our studies were designed entirely to allow potential participants to self-screen. This allowed us to reach as many people as possible, from all over North America, very quickly, and allowed us to enroll participants even as the pandemic was limiting face-to-face interactions. Given the emergency situation created by the pandemic, the IRB and FDA approved this design and the use of e-consent. Our website, surveys, and emails were our primary interactions with participants. Therefore, wording and study definitions had to be very clear. We listed study-specific definitions in a REDCap Descriptive field at the top of the screening form (Supplemental Material 2). We embedded our informed consent documentation into a Descriptive field in the Enrollment survey then followed it with a series of questions to test comprehension. Hidden Calculated fields assessed comprehension and, if achieved, indicated the participant as enrolled. Participants signed their informed consent directly into a Signature field. REDCap does have an eConsent feature in the Survey Settings. Although REDCap has randomization applications, we conducted ours manually. As an internet-based trial, persons could potentially try to screen themselves multiple times, so as to meet eligibility criteria. There was no mechanism within REDCap to stop the same email address or name from being entered multiple times or nonsensical data entered, thus accuracy of information was manually verified by study personnel. Our statistician created the randomization schedule using a permuted block design with random blocks of sizes 2, 4, and 8. This generated list of randomization codes and assignments was sent to the pharmacy, while a blinded list of randomization codes was sent to the study team. Further details of the logistics of this process are summarized in Pullen et al [6]. Once randomization was confirmed, our team tracked this result and study medication shipping in a separate Enrollment Processing instrument. Follow-up The fields confirming randomization and shipment of study medication from the Enrollment Processing instrument triggered the automated invitations to deliver surveys for follow-up data collection. The PEP and treatment trials had follow-up surveys sent on days 1, 3, 5, 10, and 14 while the PrEP trial had surveys sent weekly for 12 weeks. Any participant who reported hospitalization during follow-up was sent surveys post-trial end to continue tracking their vital status. Survey structure The PEP trial and the treatment trial involved the exact same intervention, nearly the same follow-up schedule, and included participants of the same out-patient population -with the major difference being a positive test or the onset of symptoms [7]. Given this, we developed one REDCap project to work for both trials at once. With the PEP and treatment trial housed in the same REDCap project, when it was determined that participants in the PEP trial who became symptomatic within 1 day of enrollment prior to receiving the study medication would be considered in the treatment trial, we were able to easily transition these participants seamlessly. We collected the same data on all participants. Structuring fields and wording to guide self-reported data and cover possible scenarios is important. We wrote the text for our follow-up surveys so that if someone skipped a day or week the phrasing still made sense to the participant and for our analysis. For example, in the PrEP study, every weekly follow-up survey began with the following text in a Descriptive field: "The following questions pertain only to the past week, not since enrollment." This was reinforced in the wording of each question on the survey (e.g. "In the past week, how many tablets have you taken?"). Since the PrEP trial followed participants weekly for up to 12 weeks, we structured the fields regarding hospitalizations in a way that allowed for the possibility of multiple hospitalizations and possible missed visits. The reality of having a participant complete a survey while hospitalized was likely low, but by asking on every survey, we were more likely to capture the data if available. We also considered having separate hospitalization surveys, but we decided that streamlined surveys were more acceptable for participants and simpler for data management. On any given week a participant could either be not hospitalized, be currently hospitalized, or have been discharged from a hospitalization since the previous survey. We decided to ask a specific hospitalization question on every weekly survey: "Have you spent any days hospitalized (including a discharge day) since the last survey?" By specifying that a discharge day would count as a day in the hospital we were able to cover the various scenarios. An example of one week (8) of fields and branching logic is displayed in Supplemental Material 3. Automated invitation scheduling There are four steps REDCap outlines to schedule an automated invitation to a survey. In "STEP 1" one can select if the invitation will be sent my email, SMS message, or a combination. "STEP 2" involves composing the invitation message and specifying the "sender". There are two options for scheduling the timing of a Follow-up survey using the Automated Invitations: by indicating the number of days after a specific status is achieved (e.g. enrollment) in "STEP 4" or by programming the "STEP 3: Conditions" logic using the datediff function. Both can achieve the same timing: The benefit of the latter is that it puts any delay condition in STEP 3, freeing up STEP 4 so that one can choose the exact time of day a survey is sent. Additionally, the invites do not get into the queue until the day they are scheduled, which can be useful for a pragmatic trial in which changes might need to be made. The Automated Survey Invitation scheduling is an extremely useful feature of REDCap, though it is not without its challenges. For example, when the PEP and treatment trials opened, the Day 3 survey was only meant for treatment participants, so we wrote the logic in the Automated Invitation so that the Day 3 survey was delivered if enrolled into treatment or symptomatic by Day 1: It is important to note that updating the Automated Invitations for any reason can affect previous participants. We only wanted this change to apply to participants who had not yet reached Day 3, but in making the change in logic we inadvertently set the Automated Invitation to send to previous participants as well. We noticed this before it was actually sent, but because these invitations had been saved and scheduled, we had to manually delete all unnecessary Day 3 survey emails using the Survey Invitation Log. Scheduling Automated Invitations should take into account realworld issues. For example, scheduling Follow-up surveys based on completion of the previous survey can cause problems because (a) if a participant missed a survey then the next survey would never be sent, or (b) if a participant was delayed in completing a survey then the next survey would be delayed in being sent. For the PEP and treatment trials especially, we wanted data from short visit windows, as close to our scheduled days as possible. We therefore found it most useful to time all Follow-up Automated Invitations based on completion of the Enrollment survey. Another option that we considered and could be useful for other trials is the Survey Queue, which "displays a list of your surveys to a participant all on a single page, in which the queue comprises all surveys that are to be completed (like a 'to-do' list) as well as the surveys that the participant has already completed." Because our Follow-up surveys needed to be completed over several days to weeks, we decided this would not be useful for our participants. We did include language in our emails to inform our participants about what surveys to expect. If we had more planning time available, it would have been better to include the entire schedule in the initial email with the Enrollment survey, including time of day to expect survey emails. Complete data Assuring complete data was a priority, and we established settings within the Automated Invitations such that reminder emails were sent repeatedly up to three times or until that survey was completed. Even with automated email reminders, some participants missed surveys. The reporting system in REDCap allowed us to identify any participants in the PrEP trial who were consistently missing surveys and continually check in with them throughout follow-up with manual phone calls and mobile phone text messages. To be as proactive as possible with assuring follow-up, we established running reports of participants who had missed each weekly survey as well as reports of participants who had missed multiple surveys in a row (Weeks 1-2, Weeks 1-4, Weeks 5-8, Weeks 9-12). We used the datediff function in the Filter logic to keep these reports updating continuously. For example, the Week 2 survey was automatically sent on day 16 post-enrollment. One email reminder was sent 32 hours later, but if by day 19 a participant had still not completed the Week 2 survey, they would appear on the "Missed Week 2 Report" for 3 days or until their survey was complete: Our team reviewed these Reports and acted upon them on a coordinated schedule; the logistical aspects have been summarized in Pullen, et al. [6]. We encouraged participants to complete their surveys, but we also implemented a vital status instrument to record basic information from any brief phone call or text message exchange. The REDCap programming for Automated Invitations and Reports for the PrEP trial are presented in Table 2. We also created a field called enr2today = datediff([enr_date],"today","d","mdy") which was useful for reporting how far into follow-up a participant was. In order to update the "today" element, we ran Rule H every day for the PrEP trial. We found in all of our trials that participants who decided ultimately to not take the study drug often decided to not complete the surveys. Common misconception that if people stopped taking study medication that we were not interested in their outcome. Standard templated wording of informed consent stating that one can stop participating in research at any time is unhelpful for intention-to-treat analysis. Clarity is needed to separate stopping the study intervention and continuing follow up from stopping all research participation. We amended wording to emails and on the screening forms to remind them of the importance of collecting their follow-up data regardless of their adherence or side effects. We used the example that if only the people who did well on the study medication completed follow up, that the study medication would look as if it performed better than it actually did. We cared about everyone's experience with the study medication, even if they stopped taking the medicine due to side effects or other reasons. Particularly for remote trials, this distinction should be made clear upfront of giving participants options if they want to stop the study medication versus stopping all research participation. Project Set-up Testing the REDCap project as much as possible is critical. Creating a duplicate, practice REDCap project was a useful tool for testing issues both during the development of the data collection system, and throughout enrollment as changes were made. REDCap makes it easy to copy an entire research project over to a duplicate project. When a project is in production, one can easily download an updated Data Dictionary, and upload it to the duplicate project to ensure that they remain identical. Collaboration is indispensable on any clinical trial, even a completely internet-based study. Prior to starting the trial, our team of investigators, colleagues, friends, and family played the roles of mock participants in the duplicate practice project in order to test out different scenarios. What if a participant misses a survey? What if a participant needs to make changes later? Additional beta-testing by non-medical personnel is recommended to assure the language is appropriate. Physicians routinely under-estimate the complexity of medical language for the general public. Although we had set up our fields and wording to be as specific as possible, we did have some issues arise once we were ready for analysis. For example, we had to use the survey date completed as the date of an event because we did not ask specifically for the date of onset of illness. This is important to consider depending on whether it is a time-to-event analysis or not. In the PrEP trial with weekly follow up surveys, for example, we collected the date of PCR testing but not the date of symptom onset. This created a variable time lag of up to 6 days in a 12 week trial. While such time lag would be equally distributed across treatment arms, it was less precise than we would have liked in retrospect. Conversely, the PEP and treatment trials did not use time-to-event Accessed link a n/a n/a n/a n/a analyses, thus this detail was unnecessary. Once our trials were underway our team developed some sub-studies including COVID-19 antibody testing and hydroxychloroquine drug level testing [4,8]. We were able to easily implement consenting and follow-up surveys for these sub-studies as further surveys nested within the parent trial REDCap projects. Communication We established email addresses for the trials that would interact with participants. For the PrEP trial, for example, we wrote text at the end of the Screening survey (using the Survey Settings) that potential participants should expect all surveys to be sent from this email: covid19prepfa q@umn.edu. By using a University of Minnesota email address, we were less likely to be delivered to potential participants' spam folders. This University-designated account was blocked from automatic forwarding, so we also set up faq.covid19@gmail.com to allow our team to receive participant questions forwarded to their personal emails as alerts to the questions. They could then log into this account to respond. We created an on-call schedule of investigators to field questions from this email address in a timely manner approximately 18 hours per day. We could have further customized our email messaging to include pictures of the investigators to make it more personalized. REDCAP allows pictures via the addition of html to messages, via: <img src="URL" alt="Alternative text"> where the URL is an internet-accessible image. We also utilized REDCap for each of the trials to send out a final email to all trial participants on the day of publication. Many participants appreciated receiving prompt notification of the trial results as well as knowing their randomization assignment. To send these messages en masse, we piped the participants' randomization assignment from a variable added into REDCap after trial completion into the email message for a new Final Result survey. We specified the STEP 3: Conditions based on logic that the participant was randomized: ending an email to many participants that is not tied to a survey instrument is not straightforward within REDCap. To accomplish this when necessary we made a Report of emails of participants we wanted to email, downloaded the Excel file for this Report, and used an external program to send the batch email. For internet-based trials, consider verifying important endpointrelated information by email or telephone. FDA recommended at least 10% of endpoint information be verified, which we did. We also verified information for people who were outliers, such as PCR-positive persons who remained asymptomatic, to verify that this was indeed correct. The REDCap Alerts feature can be useful for immediate follow-up of events. Logic can be set such that an email is sent to a specified recipient with the relevant participant ID and fields. We set Alerts to notify our PIs of hospitalizations and other adverse events if a participant reported one on a Follow-up survey. We also used Alerts in the treatment trial for Screening surveys that required expert review to determine eligibility (see Fig. 1). REDCap quirks When designing the data collection system, fields can be marked as "required", forcing a pop-up to alert the participant if they skip the field and try to submit the survey. However, this does not prevent the participant from submitting the survey with the skipped required field. Such an incomplete survey, once submitted, cannot be updated. There is an option in the Survey Settings to allow the participants to "return and modify completed responses." We allowed this initially but discovered that this situation led to one participant deleting all of their data. Fortunately, the value History is recorded on each field and can be manually re-entered. However, on the Enrollment survey the signature field was lost from the survey and the value History. Thankfully the REDCap administrator for our institution was able to retrieve these. One survey on which we did allow participants the option to save and return was the Drug start survey in PrEP. We wanted to make sure participants completed this survey after they had physically received the study medication. Since we could not ensure that the survey arrived after the study medication, we started with a basic question of "Did you receive the study medication?" If the answer was "No", branching logic was set so that a Descriptive field appeared with instructions to contact the team and select "Save & Return Later." We based the Follow-up surveys on the field "[shipped] = 1" which our team entered manually into the Enrollment Processing instrument once randomization was completed and the study medication was shipped. To assist with tracking study medication delivery, we later included the FedEx shipping number as a piped-through field on the 13 July, 2020 n/a n/a n/a n/a n/a n/a n/a n/a a Anyone could access the link for screening. Eligibility was determined through survey questions. email with the Drug Start survey. As this field was manually completed, it was important to make sure that the FedEx number was entered into Enrollment Processing before or at the same time that [shipped] was set to 1 (yes) and the instrument was saved, otherwise a blank line would have appeared in the email where the number should have been. On numeric fields there is an option to set minimums and maximums; however, on date fields one cannot easily stop future dates from being entered, especially when there is not a set timeframe when the survey could be completed. Relatedly, REDCap Calculated fields cannot read dates written within a function. In order to help prevent a future date from being entered, we created Hidden Calculated fields that compared the date entered to the date the form was completed using the datediff function. As soon as a future date was entered, the negative value of the Calculated field would trigger the branching logic to display a Descriptive field stating "Error! The date ... cannot be a future date. Please correct." For those with COVID-19 symptoms, we collected data on symptom severity using a 0-10 visual analog scale. We discovered that the default appearance of the visual analog scale field in REDCap displays the mark in the middle of the scale at a 5 on a scale of 0 to 10. It was possible for a participant to feel a level 5 and therefore not move the marker; however, if the marker was unmoved then REDCap records the score as 0. Also, since the field was required, these surveys were registered as Incomplete in REDCap. Once we discovered this issue we changed the instructions and followed up with any participant who this might have affected. Conclusion We designed and ran three large-scale, internet-based, double-blind randomized clinical trials for outpatients during the first 3 months of the U.S. COVID-19 pandemic [2][3][4]. We knew from the start that these trials would be pragmatic and our data collection system would have to be able to respond to such needs. With REDCap we were able to update automatic eligibility criteria assessment fields and Automated Invitation logic while maintaining data integrity. For trials that rely on self-screening and self-report, structuring and writing text for survey fields that guide participants through the processes is critical. Automated Invitations allow for scheduling surveys strategically, prompting participants to record their own data. Proactively tracking and reaching out to participants who miss surveys allows for more complete data. Testing the REDCap projects is essential. We tested our REDCap projects as much as possible with limited time prior to enrollment and continued to test features using identical practice REDCap projects throughout follow-up. Pragmatic, internet-based clinical trials are possible and are a useful tool for low-cost studies in the modern world. REDCap has excellent features and flexibility to support such trials. Funding Our Covid-19 clinical trials were supported by Jan and David Baszucki, Steve Kirsch, the Rainwater Charitable Foundation, the Alliance of Minnesota Chinese Organizations, the Minnesota Chinese Chamber of Commerce, and the University of Minnesota Foundation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.	2022-01-25T14:09:52.811Z	2022-01-01T00:00:00.000	{ "year": 2022, "sha1": "a50f16af6d62bf05923bf4fcd7f3a6ba15ef3f0d", "oa_license": null, "oa_url": "https://doi.org/10.1016/j.cct.2021.106660", "oa_status": "BRONZE", "pdf_src": "PubMedCentral", "pdf_hash": "7dc843d4bf0aa673d42af009591e6a9d902aac02", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
40943936	pes2o/s2orc	v3-fos-license	Abstract: Effect of Local Flaps Used for the Reconstruction of Nasal Tip Tumors on the Function of Nasal Valves METHODS: 60 patients who had non-melanocytic skin cancer on the only nasal tip were included in this study. There was no previous history of nasal surgery, allergic rhinitis, concha hypertrophy and other breathing problems in any patients. Six patients were treated with forehead flap, ten patients nasolabial island flap, twenty patients bilobe flap that based on inferior, 24 patients bilobe flap that based on superior. Function of the internal and external nasal valves were evaluated by preoperative and postoperative comparison of Cottles test, nasal endoscopy and digital phography of nares during forced inspiration and expiration and were followed up 1-years. INTRODUCTION: Nasal valves are the most important regulator of nasal airway. 1,2 Nasal valve dysfunctions are observed after removal of skin cancer at the nasal ala, crease and lateral side wall. 3 We studied that effect of forehead flap, nasolabial island flap and bilobe flap on function of the internal and external nasal valve in the reconstruction of nasal tip tumors. METHODS: 60 patients who had non-melanocytic skin cancer on the only nasal tip were included in this study. There was no previous history of nasal surgery, allergic rhinitis, concha hypertrophy and other breathing problems in any patients. Six patients were treated with forehead flap, ten patients nasolabial island flap, twenty patients bilobe flap that based on inferior, 24 patients bilobe flap that based on superior. Function of the internal and external nasal valves were evaluated by preoperative and postoperative comparison of Cottles test, nasal endoscopy and digital phography of nares during forced inspiration and expiration and were followed up 1-years. RESULTS: There was no breathing problem in patients treated with forehead and nasolabial flap. Chronic stuffy nose, external valve collapse and positive Cottles test was observed in three, two and one of twenty patients who treated with bilobe flap based on inferior and in four, four and two of 24 patients who treated bilobe flap based on superior, respectively. CONCLUSIONS: Nasal reconstruction has not only aesthetic, but also functional consequences. The nasal valve dysfunction can be substantially parallel with the amount of dissection during surgery on the nasal skin. 3 According to our study, rejyonel flaps don't effect nasal valves functions. Affiliation: Selcuk University, Konya INTRODUCTION: Nasal tip reconstruction is a very challenging surgical procedure in aesthetic and reconstructive surgery. 1,2 The main purpose of nasal tip reconstruction is to repair with similar texture tissues without disturbing facial aesthetics and function. In this study, reconstruction of the nasal tip defects with superior-based tunnellized pedicle nasolabial island flap is presented. METHODS: 10 patients who were operated for non-melanoma skin cancer including nasal tip area were included in the study. Patient's medical records were reviewed, digital photography was taken before surgery. All of patients were assessed by detailed physical, ultra-sonographic, endoscopic nasal-airway examination. Each lesion was excised with at least 5mm margin. An elliptical-shaped superior pedicle nasolabial flap was designed based on angular artery. Pedicle of flap was dissected cephalic direction and subcutaneously to the superior of the defect area. The flap was moved to the defect by a tunnel. At the 12th month postoperatively, symmetry of nasal shape and face were evaluated with patient satisfaction scale (-1: not satisfied, 0: neutral, 1: moderate, 2: good, 3: very satisfied). RESULTS : 4 patients were male, 6 female and ages ranged from 61 to 83 years (mean 72.1). In all patients, there was no pathological lymphnode and no internal and external nasal valve dysfunction. Defects were between 4 cm2 and 7.56 cm2 after surgical excision. There were no infection, hematoma and total flap failure; but a partial (20%) flap necrosis due to venous congestion was seen in one diabetic patient and treated	2018-05-31T06:24:15.407Z	2017-09-01T00:00:00.000	{ "year": 2017, "sha1": "b3ec476838b3e5cd766e69b667eebdacd54c62dd", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1097/01.gox.0000526170.37929.51", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "b3ec476838b3e5cd766e69b667eebdacd54c62dd", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
246519106	pes2o/s2orc	v3-fos-license	Foliar spraying of MnO2-NPs and its effect on vegetative growth, production, genomic stability, and chemical quality of the common dry bean Abstract A large part of the income of most developing countries is coming from the agricultural field, where semi of their population relies on farming in their life. The world population is growing rapidly; consequently, there is a requirement to increase agricultural output through the implementation of newfangled technology such as nanotechnology. Herein, the manganese oxide nanoparticles (MnO2-NPs) were prepared as a foliar nano-fertilizer. The effect of different concentrations (0, 10, 20, 30, and 40 ppm) of MnO2-NPs on the common dry bean plant criteria, yield, chemical quality of leaves and seeds, genomic DNA, and some genes encoding proteins were investigated, which was planted throughout two sequent seasons 2020 and 2021 in clay soil. The results showed that MnO2-NPs with a concentration of 30 ppm enhanced the growth criteria and the yield percentage by (45.2 and 48.9%) during two seasons, respectively, of a common dry bean. The chemical quality for leaves and seeds varied in their response to MnO2-NPs. In addition, the genomic DNA and some genes encoding proteins of the plants were significantly affected by MnO2-NPs at concentrations of 30 and 40 ppm in comparison to other concentrations. Introduction The common bean is a fundamental legume in worldwide for straight human consumption. The crop is used fundamentally for its dry seeds, and green pods. In addition, the leaf of the common bean is sometimes utilized as a leaf vegetable and the straw is utilized for animal feed (Shaban et al., 2019;Zewail, 2014). The nutritional value of the common bean is comparatively rich with cheap sources of proteins, carbohydrates, fiber, folic acid, vitamins, and minerals for people in developed and developing countries (Cort es et al., 2013). Moreover, the common bean is described as the only best nonmeat source of iron which supplies 23% to 30% of the recommended levels from one serving each day (Shimelis & Rakshit, 2005). Also, it appears an inexpensive source of calcium, and several amino acids as lysine that using in the dietary regimen (El-Mohamedy et al., 2017). The highest productivity and the best nutritional value of common bean could be achieved via using nutrients, which could be applied in the form of nano-fertilizer. The application of manganese as a fertilizer leads to boosts the productivity and quality of the crop, due to enhancing the plant nourishment and rising photosynthesis in the plants, thus the productivity and goodness increase by improving photosynthetic efficiency (Mousavi et al., 2007). The results of using manganese nanoparticles treatment in mung beans are increased plant growth by about 52% and 38%, for root and shoot, respectively (Pradhan et al., 2013), while the eggplant improved the yield by about 22% (Elmer & White, 2016). This result is due to the small size ranging from 1 to 100 nm, higher particular surface area, and interaction of nano fertilizers that may improve the solubility, expansion, and availability of plants, that being so promoting the yield of plants compared with conventional fertilizers. Where, the essence of nanotechnology is size and control (Abobatta, 2018;Salama et al., 2021a;Shebl et al., 2020). Manganese is an essential micronutrient, with important functions in the plant as an ingredient of enzymes participatory in photosynthesis and other processes, and plays an important function in photosynthesis, as a structural part of the water-splitting CONTACT Dina M. Salama dinasalama@ymail.com Vegetable Research Department, National Research Centre, Dokki, Giza, Egypt. protein of photosystem II. It acts as an electron store and delivers it to the chlorophyll reaction centers (Millaleo et al., 2010). Moreover, manganese is the section of a major antioxidant frame such as superoxide dismutase that preserves the cells of the plant through inactivating the free radicals that cause spoilage to plants tissues. It is from the above that manganese participates in photosynthesis, respiration, and nitrogen (N) metabolism (Mousavi et al., 2011). Manganese is required necessary in little quantities, which is usually less than 1 kg/ha in cereals and about 2 kg/ha in sugar beets (Draycott & Christenson, 2003). In the same trend, the application of Mn-NPs as a foliar spray to plants is more active practice on plants responses than soil application (Dimkpa et al., 2018). Since the micronutrient is used in a small amount and doesn't straight contact the soil, which prevents losses during fixation (Abd El-Aziz et al., 2021;Teixeira et al., 2004). The plants' biological functions count on the activities that are happening at the molecular scale. Although, the insignificant advance has been made at the molecular scale affected through nanoparticles, which is a fundamental step in estimating probable mechanisms and plants' effects. Wherefore, it is important to understand plants' underlying mechanism and its response to nanomaterials, and the drift in gene expression through molecular approaches (Ali et al., 2021). So, this research aims to investigate the effectiveness of manganese oxide as nanofertilizers on the growth, yield, and quality of common dry bean plants. Additionally, the effect of manganese nanofertilizer on genomic stability of DNA. Supplies Seeds of common bean (Phaseolus vulgaris L., cv Nebraska) were purchased from the Agricultural Research Centre, Egypt. The agriculture fertilizer such as potassium sulphate, calcium superphosphate, agriculture sulfur, and ammonium sulfate were bought from Abu Qir Company, Egypt. Manganese nitrate was obtained from Sigma-Aldrich Company. Preparation of nanofertilizers Manganese oxide nanoparticles (MnO 2 -NPs) were prepared thermally by dissolving 100 g of manganese nitrate (Mn(NO 3 ) 2 4H 2 O) in 50 ml hot water then stirring for 10 min, after that the solution was heated to 100 C for 24 h in an oven till black viscous liquid was obtained. The deionized water was adding to viscous liquid and centrifugation at 7000 rpm for 15 min. The black precipitate was washed twice with deionized water and then dried at 60 C (Najafpour et al., 2012). Characterization of nanofertilizers The surface morphology of MnO 2 -NPs was analyzed using scanning electron microscope (SEM; JEOL/ Noran, JSM 6360LV). While transmission electron microscope (TEM; JEM-1230, Japan, magnification 6 Â 10 5, and a resolution of 0.2 nm) was used to illustrate the particle size and shape of MnO 2 -NPs. Where the dispersed solution of the MnO 2 -NPs was dropped on a carbon-coated copper grid, then was placed in the TEM device after to drying. The X-ray diffraction (XRD) pattern of MnO 2 -NPs was obtained by using a Diano X-ray diffractometer with radiation source CoKa under operating voltage 45 kV, and a Philips X-ray diffractometer with radiation source CuK (PW 1930 generator, PW 1820 goniometer, and). Experiment planning The experiment was conducted to study the effect of Foliar spraying of MnO 2 -NPs and its effect on vegetative growth, production, genomic stability, and chemical quality of the common dry bean. In half of February, about 120 kg/ha of common bean seeds were cultivated throughout two seasons, 2020 and 2021 on the farm with clay texture in El-Menofia governorate, Egypt. The seeds of the common bean were cleaned from dust and odd substances manually, after that it was immersed in water for one hour before planting to encourage the germination of common bean seeds. The seeds were cultivated with rate 2 seeds/hills, and the space between hills on one side of the ridge was 30 cm while between the ridges was 60 cm. The physical and chemical characteristics of the cultivated soil, mean of two seasons, have been analyzed as described by Cottenie et al. (1982) (Table 1). Experimentation treatments After 20 days of sowing common bean plants, they were sprayed with MnO 2 -NPs at various concentrations (0, 10, 20, 30, and 40 ppm), using hand-pump garden sprayer. Each treatment was sprayed with one liter of suspended nanomaterials in water. The trial layout was a complete randomized block design with three replicates. Vegetative growth characteristics Various vegetative growth criteria such as the length of plant and root, number of leaves, and flowers per plant, as well as fresh and dry weight for plants were studied on five plants that were taken randomly from each treatments in the vegetative stage (45 days of planting). Yield and its attributes The number and weight of pods and seeds per plant, seed index (weight of 100 seeds), weight of seeds (t/ha), shelling percentage, as well as shoot residues (kg/ha) were assessed from five plants that were randomly taken from each plot in harvest stage (90 days after planting). Chemical analyzes 2.8.1. Photosynthetic pigments After 45 days from cultivation, fresh leaves were collected to determine its content of photosynthetic pigments as (mg/g) according to the method mentioned by Salama et al. (2021b). The chlorophyll (a and b) and carotenoid contents were determined by spectrophotometer (SHIMADZU 240 UV/VIS spectrophotometer) at 663 and 644 nm and 452, respectively, after extraction in acetone (85%).The pigment content were expressed as mg/g fresh weight of plant material and was calculated by the equations suggested by Jiang et al. (2018) as follow: Chl a ¼ 10:3 E663 À 0:918 E644 lg=ml ð Þ 2.8.2. Proximate analysis Fresh leaves and seeds were dehydrated at 70 C in the oven to a constant weight and then grinded in an electric mill for a proximate analysis (Jayant et al., 2018). The content of moisture was obtained by desiccation the leaves and seeds at 105 C till constant weight. The ash content was specified from combustion at 550 C for 6 hours in a muffle furnace. The protein content was measured by the Kjeldahl method (N Â 6.25). The fiber was determined by acid digestion (1.25%), followed by alkali digestion (1.25%), then ether extraction. The total energy was qualified using equation described by Nwabueze (2007). Minerals determination The milled common bean plant samples (leaves and seeds) were digested with H 2 SO 4 -H 2 O 2 . The contents of potassium and phosphorous were estimated by spectrophotometer, while zinc, manganese, iron, and copper were determined in digested solutions by atomic absorption (Hao et al., 2019). Statistical analysis The data were statistically analyzed according to Snedecor and Cochran (1989) and the LSD was test at a 5% level. DNA extraction and RAPD-PCR analysis The mutagenicity or genotoxicity was studied by using the RAPD-PCR technique to examine and estimate the genomic variation. This technique is low expensive, rapid, reproducible, and does not require specific knowledge of the DNA sequence (Atienzar & Jha, 2006). The genomic DNA was isolated from a common dry bean by using the GeneJET Plant Genomic DNA Purification Mini Kit Kits (Thermo scientific K0791), and then was quantified by using a NanoDrop 1000 spectrophotometer (Thermo Scientific) according to the method mentioned by Osman et al. (2020). Eight random primers were used to perform RAPD-PCR as mention in Table 2. PCR amplification for isolated DNA was performed in 0.2 ml PCR Eppendorf having 12.5 ml Dream Taq Green PCR Master Mix 2X (Thermo scientific K1081), 1 ml primer 10 pmol (Metabion, German) and 1 ml Template DNA (50 ng/lL) then completed to 25 ml by water (nuclease-free). Thermocycler (Bio-Rad) was programmed as follows: 94 C for 5 min (one cycle) then 94 C for 1min, 37 C for 45 sec and 72 C for 45 sec (35 cycles) then 72 C for 5 min (one cycle) then held at 4 C. Then 100 bp DNA Ladder H3 RTU (GeneDirex, Cat No. DM003-R500) and 5 ml of DNA amplified PCR product was loaded in each well of agarose and then was placed in 1X TAE buffer (1%) and run at 100 V for about 2 hours. The gel was photographed by gel documentation (Bio-Rad) and was analyzed by the Total Lab program to find out the molecular size of each band. Protein banding patterns SDS-PAGE is widely used to describe the genetic structure of crop and to provide information about genes structure . It was performed according to the method of Laemmli (1970). The water-soluble proteins (W.S.P.) were extracted from the leaves samples of randomly selected shoots at seedling and flowering stages. The marker of used protein was BLUltra Prestained Protein Ladder (GeneDirex, Cat No. PM001-0500). The results were analyzed by the Total Lab program to obtain the molecular weight of each band. Toxicity test The seeds of common beans were dried in an oven at 105 C till constant weight after that they were grinded to a fine powder. An extraction was obtained from the powder of common bean in water at two concentrations 0.1% and 1.0%. Then Microtox analyzer 500 (USA) was used to measure the toxicity of the samples (Johnson, 2005). Characteristics of MnO 2 -NPs The morphological structure of the prepared nanoparticles was illustrated in Figure 1(a) and 1(b). The TEM diagram illustrated a regular diamond shape of MnO 2 -NPs as presented in Figure 1(a). In addition, it can be demonstrated that the prepared nanomaterials have particles size less than 100 nm. The SEM image (Figure 1a and 1b) supported this observation, since the particles are gathering to form forming a clear parallelepiped rectangles structure. The crystal structure of the MnO 2 -NPs was examined using X-ray diffraction measurements ( Figure 1c). The main peaks at 2h equal 28.6 , 37.3 , 40.9 , 42.8 , 56.6 , 59.3 , and 72.3 are corresponding to plane 110, 011, 020, 111, 121, 220, and 031, respectively. These diffraction peaks resembled the reported values of Reference code 98-005-6006 of MnO 2 , where the crystal system is Tetragonal, a space group is P 42/m n m, and the mineral name is Pyrolusite (Devaraj & Munichandraiah, 2008;Dewi & Yulizar, 2020). Pradhan et al. (2013) recorded that the mung beans plant treated with manganese nanoparticles led to increasing the growth of roots by about 52%, shoots by about 38%, and the number of rootlets by about 71% at a concentration of 0.05 mg/l. In another study, Liu et al. (2016) found that MnO x -NPs slightly minimized the germination percentage of lettuce seeds by about 63% as compared to control even at the higher concentration of 50 mg/l. Over and above, MnO x -NPs ameliorated the growth seedlings of lettuce by reinforcing root elongation. Shebl et al. (2019) represented that 20 ppm of manganese oxide nanoparticles was enough to enhance the growth criteria of the squash plant. These results might be related to the efficiency of the photosynthesis process with spraying MnO 2 -NPs on the plant with concentration 30 ppm (Table 5). In addition, this result can be imputed to the variation of proteins wished for metabolic processes in every stage. Where, the seedling and the flowering stage had a special protein at Mw 37 and 33 kDa as shown in Table 10. Common bean yield Yield and its components of common bean plants are shown in Table 4 during the two seasons of agriculture 2020 and 2021. The data showed that MnO 2 -NPs appeared a significant effect on the common bean yield and its components. The common bean plants treated with 30 ppm MnO 2 -NPs had a higher number and weight of seeds/plant, also weight of pods/plant, as well as the yield (t/ha). Where the yield percentage increased by (45.2 and 48.9%) during two seasons, respectively. Contrariwise, the highest value of the number of pods/plant, seed index, and weight of residues per plant and per hectare appeared with plants sprayed by 40 ppm MnO 2 -NPs, while the best shelling percentage was recorded by using the lowest concentration of MnO 2 -NPs (10 ppm). Where the shelling percentage increased by (18.0 and 15.3%) during two seasons, respectively. The increase in yield was due to the improvement of vegetative growth characteristics when spraying plants with MnO 2 -NPs concentration (30 ppm), as shown in Table 3. This effect shows that the MnO 2 -NPs concentration (30 ppm) is enough to enhancement the translocation of the metabolites from vegetative growth to the seeds, which reflected in the improvement of the seed yield of the common bean plant. Additionally, manganese has a significant function in stimulating the enzymes for the metabolism of carbohydrates and phosphorylation. Consequently, it leads to the enhancement of the efficient quantitative properties of the common bean yield by the systematization of the plant's growth. Therefore, the implementation of Mn is major for gaining the maximum profitable yield Nadergoli et al. (2011). Likewise, Ruttkay-Nedecky et al. (2017) illustrated that manganese is an fundamental micronutrient for growth regulation and plants development. Elmer and White (2016) found that treating eggplant plants with manganese nanoparticles improved productivity by 22% compared to control. Also, the application of MnO-NPs as foliar with a concentration 20 ppm gave the highest yield and the best characteristics of the fruits of the squash plant compared with other treatments (Shebl et al., 2019). These results can be expounded on the basis of the effect of MnO 2 -NPs on the genomic DNA of plants, where the most effective concentrations of MnO 2 -NPs on the plant genome were 30 and 40 ppm in contrast with other concentrations. Because some bands have vanished and others were appeared with these concentrations compared with the control, as shown in Table 8. Also, the improvement in the vegetative growth criteria such as the number of leaves, branches, and flowers per plant, Table 3, may be the reason for the enhancement of the yield and its components of common bean plants. Furthermore, many studies illustrate the effect of Mn in microscale on productivity. Teixeira et al. (2004) found that the application of Mn on the leaves of the common bean by 315 g/ha increases the yield by 34%, while its combination with Zn (280 g/ha) increases the yield by 60%. Herein, it can be found that 30 ppm of MnO2-NPs (%14 g/ha) enhanced the yield percentage by about 47%. This indicates the possibility of reducing the quantity of traditional fertilizer used and at the same time reaching a higher productivity. Proximate components It is evident from ( Table 6) that the leaves of the common bean plant treated with various concentrations of manganese nanoparticles resulted in significant increment on proximate components, during two sequential seasons 2020 and 2021. The content of moisture in leaves showed the highest with plants sprayed with a concentration of 40 ppm MnO 2 -NPs. A concentration of 30 ppm MnO 2 -NPs recorded the best percentage of protein and ash. In contrast, energy content significantly increased by application MnO 2 -NPs with a concentration of 20 ppm. In contrast, the percentage of carbohydrates, lipids, and ash was increased with traditionally grown common bean plants. This result might be due to that the proximate components were varied in their response to MnO 2 -NPs concentration. Where Schmidt et al. (2016) showed that manganese has an important role in the photosystem, respiration, and nitrogen assimilation as a catalyst. Also, Shebl et al. (2019) found that the application of 20 ppm Mn-NPs as foliar fertilizer recorded the best organic matter and protein percentage, as well as the highest total energy in the leaves of the squash plant. From the previous results, the application of nanomaterials boosted the nutritional values of plants as a result of ameliorating crop yields (Adisa et al., 2019;Salama et al., 2019). The data in Table 6 revealed that the proximate components of seeds common bean were affected with several concentrations of manganese nanoparticles, during two consecutive seasons 2020 and 2021. The marked increase of moisture percentage in seeds was observed with a 30 ppm MnO 2 -NPs. While the highest carbohydrates percentage showed in plants spraying with a 20 ppm MnO 2 -NPs. On the contrary, a concentration of 10 ppm MnO 2 -NPs gave the maximum energy percentage. Also, Fat, fiber, and ash are increased when common bean plants are grown conventionally. This result might be due to the proximate components varied in their response to MnO 2 -NPs concentration. Verma et al. (2021) found that the impact of plant species relies on the concentrations of nanoparticles. Furthermore, nano-fertilizers have a substantial part in the physiological and biochemical processes of crops through raising the availability of nutrients, that support to reinforce metabolic processes and photosynthesis (Adisa et al., 2019). Minerals content The results in Table 7 illustrated that the minerals content of leaves and seeds were significantly affected by spraying MnO 2 -NPs as a foliar application on the common bean plant, during two sequent seasons 2020 and 2021. The best value of nitrogen, phosphorus, potassium, and zinc was recorded with leaves of common bean treated with MnO 2 -NPs concentration (30 ppm). Reciprocally, 40 ppm MnO 2 -NPs appeared the highest content of manganese, iron, and copper as compared to concentrations and control. This effect shows that the content of minerals was significantly increased with raising the MnO 2 -NPs concentration. Alike results were recorded by Shebl et al. (2019), who indicated that the nitrogen percentage was increased in squash leaves when plants were sprayed with Mn-NPs at a concentration of 20 ppm. As for seeds of common bean in Table 7, the percentage of nitrogen showed the best content with a 10 ppm concentration of MnO 2 -NPs. On the other hand, the phosphorus and potassium percentage gave the highest with a concentration of 30 ppm MnO 2 -NPs. Instead, the seeds obtained from plants treated with 40 ppm MnO 2 -NPs showed the better content of zinc, manganese, iron and copper compared to other concentrations in both seasons of cultivation. Regarding the copper content of bean seeds, the concentration of 20 ppm showed the best copper content. The effect of MnO 2 -NPs concentrations on the content of minerals in the common bean leaves and seeds can be attributed to the function of the manganese in the metabolic processes and breakthrough the plant cell (Dimkpa et al., 2018;Schmidt et al., 2016). Also, manganese being applied to plants has a great effect on the multifariousness biological systems inclusively photosynthesis, respiration, and nitrogen absorption (Mousavi et al., 2011). It can be concluded that the increment in nutrients can be referred to as the increased production associated with an enhancement in nutrients at a higher nutritional supplements concentration in seed and other parts of the plant (Nagamani et al., 2020). Because, the nanoparticles interactions are by the absorption, transfer, and accumulation in the plant species (Rico et al., 2011). Effect of MnO 2 -NPs on genomic DNA using RAPD-PCR marker The deviation in the DNA fingerprints of common dry bean plants, which were sprayed with different concentrations of MnO 2 -NPs (0, 10, 20, 30, and 40 ppm), were estimated by using eight RAPD primers ( Figure 2 and Table 8). The primers OP-B18, OP-B20, and OP-P17 showed seven, ten, and eight bands ranged from 2780 to 330, 1770 to 100, and 1790 to 385 bp, respectively. These primers hadn't any polymorphic band. While the primer OP-P9 displayed nine bands with eight monomorphic bands and one polymorphic band at 1750 bp (Rf 0.409), which were present only in the treatment 20, 30, and 40. Primer OP-A18 showed seventeen bands ranged from 1400 to 155 bp, with twelve monomorphic and five polymorphic. Where one þ ve unique band and one À ve unique were obtained at molecular size 1955 bp (Rf 0.449) and 1550 bp (Rf 0.497), respectively, in 40 ppm MnO 2 -NP treatment. Otherwise the bands at 1260 bp (Rf 0.532) and 500 bp (Rf 0.732) were disappeared only in control. The band at 1665 bp (Rf 0.477) appeared only in 30 and 40 ppm treatments. Primer OP-P5 showed one monomorphic band and six polymorphic bands ranged from 1485 to 530 bp. Two þ ve unique bands (660 bp and 530 bp) and two À ve unique bands (1485 bp and 1300 bp) were shown in control only. In addition, the band at 1125 bp (Rf 0.526) was disappeared in 40 ppm treatment while the band at 1020 bp (Rf 0.538) disappeared in 30 and 40 ppm treatment. Primer OP-P14 showed nine bands with three monomorphic bands and six polymorphic bands ranged from 1900 to 440 bp. Only the control had one þ ve unique bands at 440 bp (Rf 0.720). While Table 6. Effect of MnO 2 -NPs on proximate components of the common bean during two successive seasons 2020 and 2021. the band at 1000 bp (Rf 0.542) appeared in 20 and 40 ppm. Finally, the primer OP-P16, had fourteen bands ranged from 1845 to 100 bp with ten monomorphic bands and four polymorphic bands. Since two À ve unique bands at 1320 bp (Rf 0.519) and 120 bp (Rf 0.934) disappeared only in 40 ppm treatment, but it had one þ ve unique at 100 bp (Rf 0.945). while the band at 1500 bp (Rf 0.493) was absent in treatments 30 and 40 ppm. It data illustrated that the concentrations 30 and 40 ppm of MnO2-were more effective on the plant genome compared to the control, where some bands were disappeared and others were appeared in those treatments. This illustrated that this concentration could acting as the genotoxic agent or DNA mutation in the priming sites. Atienzar and Jha (2006) stated that the variation in the priming sites performs a change in the annealing conditions and homologous recombination that causes the disappearance of certain bands and the appearance of new bands. Also, Ghosh et al. (2016) found that ZnO-NPs at a high concentration causes losing of membrane integrity for root meristem of Allium cepa cells, and increasing of chromosome aberrations, as well as breaking of DNA strand. The mutagenic effect of MnO 2 -NP could be qualitatively measured by genomic template stability percentage (GTS %), which measures the grade of DNA alteration and the efficiency of DNA repair and replication. The date in Table 9 showed that GTS% value was decreased with increasing the concentration of MnO 2 -NPs. These results pointed out that all MnO 2 -NPs concentrations had an influence on the genomic DNA of the common bean plants. Former studies showed that the GTS% was decreased with rising the foliar spraying concentration of Ag-NPs up to 80 ppm on tomato plant (C¸ekiç et al., 2017), ZnO-NPs up to 50 ppm on bean plant (Mahmoud, 2019), and MoO 3 -NPs up to 30 ppm on Phaseolus vulgaris plants (Osman et al., 2020), which was in agreement with our results. Figure 3 illustrates the effect of MnO 2 -NPs on the genome of the common dry bean, where the expression of some genes encoding proteins was switch on or off. Twenty-three bands wear appeared in the seedling stage, where fifteen bands were monomorphic and eight bands were polymorphic. The number of proteins bands in the control was 22 but in treatment 10 and 20 ppm MnO 2 -NP was 21 bands while in treatment 30 and 40 ppm were 17 and 16 bands, respectively. The protein with Mw 110 kDa displayed in all treatments except 40 ppm MnO 2 -NP, but the proteins with Mw 85, 72, 63, and 32 kDa were absent in the case of treatments 30 and 40 ppm. Whereas the protein with Mw 28 kDa was truant in the control but formed under all MnO 2 -NP treatments. Toxicity test The seeds of common beans obtained from the treatment of plants by the various concentrations of MnO 2 -NPs (0, 10, 20, 30, and 40 ppm) showed nontoxicity by using a Microtox analyzer. Where the effective concentration (EC 50 ) for all samples was !100 that indicates they are nontoxic and ecofriendly. Jiang et al. (2012) found that EC 50 values for chlorophyll a and phosphate content showed that silver nanoparticles (Ag-NPs) was less toxic than AgNO 3 . Conclusion The prerogative of the utilization of manganese in the form of nano is to increment its efficiency to spread through the pores of the plant cell. Moreover, the changes take place in the plant via the nanomaterials enhancing its growth and yield. We can recommend that the manganese nanoparticles can be sprayed on common bean plants at a concentration of 30 ppm to improve the growth, yield, and genetic characteristics, as well as enhanced the quality characteristics of the common bean in terms of its content of proximate analysis, minerals. We propose that more studies will be preferable around the foliar enforcement of manganese nanoparticles with other concentrations of various microelements in the future.	2022-02-04T16:03:05.386Z	2022-02-02T00:00:00.000	{ "year": 2022, "sha1": "d43a474c89c8b3866110d06bb873a7f2888affdb", "oa_license": "CCBY", "oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/25765299.2022.2032921?needAccess=true", "oa_status": "GOLD", "pdf_src": "TaylorAndFrancis", "pdf_hash": "8ac66170890bc18cb9aef023e86f49e60845b26a", "s2fieldsofstudy": [ "Agricultural and Food Sciences", "Environmental Science", "Chemistry" ], "extfieldsofstudy": [] }
14514993	pes2o/s2orc	v3-fos-license	High-Strength Bolt Corrosion Fatigue Life Model and Application The corrosion fatigue performance of high-strength bolt was studied. Based on the fracture mechanics theory and the Gerberich-Chen formula, the high-strength bolt corrosion fracture crack model and the fatigue life model were established. The high-strength bolt crack depth and the fatigue life under corrosion environment were quantitatively analyzed. The factors affecting high-strength bolt corrosion fatigue life were discussed. The result showed that the high-strength bolt corrosion fracture biggest crack depth reduces along with the material yield strength and the applied stress increases. The material yield strength was the major factor. And the high-strength bolt corrosion fatigue life reduced along with the increase of material strength, the applied stress or stress amplitude. The stress amplitude influenced the most, and the material yield strength influenced the least. Low bolt strength and a low stress amplitude level could extend high-strength bolt corrosion fatigue life. Introduction High-strength bolts were widely used in engineering to hold two or more parts together. Bolts and screws played an important role in the performance of machinery. The highstrength bolt connection was common in the steel structure erection joint. 1,500,000 sets of high-strength bolts were used on the Jiu Jiang Bridge [1], and more than 100 ten thousand sets of high-strength bolts were used on Chongqing Chao Tian Men Bridge [2]. The fatigue performance of highstrength bolt joint affected the bridge structure security. A review of the work done in 1943 was performed by Arnold [3]. He considered many aspects and identified heat treatment, physical dimensions, and shape of the thread form as important factors. Patterson [4] evaluated the different methods for bolt fatigue-life prediction. Burguete and Patterson [5] investigated the effect of mean stress on the fatigue limit of bolts. They compared the fatigue limits predicted using the Goodman line, the Soderberg line, and the Gerber parabola and models by Cook [6] which deal more specifically with notched specimens, with their own experimental data. Liao et al. [7] adopted Gurson-Tvergaard-Needleman (GTN) model to forecast high-strength bolt's breaking load. Zhang [8] used many kinds of analysis methods, carrying on analysis to the bolt's fracture and the substrate. Based on two-stage loose theory, Yamamoto and Kasei [9] proposed bolt and nut relative sliding quantitative model. In order to study the detention break reason during the use of high-strength bolt, Yang et al. [10] established high-strength bolt stress corrosion break mathematical model. Most studies focused on the ultimate bearing strength of the bolted joints. Xiao [11] investigated the bearing strength and failure process for double lap bolted joints under a static tensile load. Girard et al. [12] showed that the bearing strength of the bolted joint was largely depending on the clamping force. Improved numerical analysis methods were needed to reduce the expensive and time-consuming experiments. Many steel structures were in corrosion environment. The atmosphere revealed the presence of NaCl in coastal area and revealed the presence of SO 2 in industrial area. These factors accelerated structure corrosion. Excessive corrosion could eventually lead to a reduction in preload as parts weaken or to the total loss of clamping force through corrosion wastage or stress corrosion cracking (SCC) [13]. Many studies on corrosion fatigue had been done. However, there was no authoritative theory about it. The "wedge theory" was widely accepted [14]. The theory was that corrosion products played role as wedge in the cyclic loading process. It induced crack expansion. The fatigue life of high-strength bolt joint reduced under the coupling function of the corrosion and the fatigue load [15]. Hence, high-strength bolt's corrosion fatigue performance was worth paying attention to. The aim of the present paper was to evaluate high-strength bolt corrosion fatigue life. Based on the fracture mechanics, high-strength bolt corrosion fatigue question was analyzed. The model of the high-strength bolt corrosion fracture crack and fatigue life was established. Stress Intensity Factor of High-Strength Bolt The corrosion fatigue life of structure without the initial cracking was divided into two stages, namely, the actual life of corrosion fatigue crack and the propagation life of corrosion fatigue fracture, as shown in Figure 1. The failure process of high-strength bolt was described by cycle number: was the total cycle number, was initial life, and was the crack growth life. could be estimated with the partial strain method, and could be estimated with the fracture mechanics method. During the production process of bolt, the micro crack occurred at the bottom of the bolt or the head rod connection. Fracture of high-strength bolt generally occurred in thread root [16], as shown in Figure 2. The fracture mechanics holding that micro crack on the surface only started to expand if the alternating load achieves some threshold value. The stress intensity factor was calculated according to [17] where = ( ) ( / ). In the formula ( ) was influence coefficient of crack gap angle : ( / ) was the influence coefficient of the mutual interferes between threads: and were great diameter and minor diameter of the thread, was bolt applied stress, was the height of thread, and was the crack depth, as shown in Figure 3. Generally, was fit for typical plane model with penetrative crack. It belongs to two-dimensional fracture. However, the crack inside bolt belongs to three-dimensional fracture. The buried crack could be analyzed as flaky crack in infinite space. Hence, was appropriate for bolt with pessimistic results. The inflect factor of included applied stress and crack size. increased with increase of applied stress. If was big enough to expand the crack, this was critical state. in critical state was called as fracture toughness . was a material property, like the modulus of elasticity or the coefficient of thermal expansion: where was critical stress and was critical crack size. The Threshold Value of the High-Strength Bolt's Corrosion Cracking Growth If > , the crack would expand. This was fit for general environment. However, in corrosion environment, the fracture toughness should be Iscc . Iscc was the threshold stress intensity factor for stress corrosion cracking. Generally, ISCC was much less than because the corrosion environment was rich in hydrogen. Hence, the bolt was easy to fracture the corrosion environment. The high-strength bolt was often outside, corrupted by damp air and rain water. If the micro crack had existed in the thread root during the process, they would seriously reduce the crack growth life as the stress corrosion crack. At present there was not precise expression for ISCC . It could be estimated by Gerberich-Chen formula [18]. Gerberich-Chen believed that stress corrosion of steel was hydrogen induced cracking. They deduced Gerberich-Chen formula based on fracture mechanics: In the formula, was gas constant; = 8.314 × 10 −6 MN ⋅ m/ ∘ C Mol. was absolute temperature: = 273 + 20 = 293 K. The Wellman formula showed [19] that ∝ √ . However, according to the Gerberich-Chen formula, the threshold value ISCC decreased as the material yield strength increases. It was opposite to the Wellman formula. The -ISCC relational curve was shown in Figure 4. The Gerberich-Chen formula was fit for corrosion fatigue. This had already been verified by experiments. The Model of High-Strength Bolt Corrosion Fracture Crack According to formula (2) and formula (4), we could get Then the depth of allowed crack was Substitute formula (5) into formula (7), the crack depth of the high-strength bolt corrosion fracture may be gotten: The Model of the High-Strength Bolt Corrosion Fatigue Life Paris formula [20] was commonly used to calculate the fatigue life in the fracture mechanics; namely, where was crack size. was the load cycle number. , was material constant. Δ was stress intensity factor range. According to the fracture mechanics theory, Substituting formula (9) into (8), the result was After integration Substituting formula (8) into formula (12), the fatigue life of the high-strength bolt corrosion fracture can be estimated. Discussion If ≈ 1, according to formula (2) and (6), the relations among the material yield strength, applied stress, and the allowed corrosion crack can be obtained, as shown in Figure 5. From Figure 5, it was shown that the crack depth of high-strength bolt was influenced by material yield strength , applied stress . decreased with the increase of the material yield strength and applied stress . The material yield strength had significant effect on the crack depth . Hence, the bolt strength level should be low. Moreover, suppose = 3, = 6.9 × 10 −12 . If 0 = 0.001 mm and Δ = 100 Mpa, according to the formula (6) and (9), the relations of the material yield strength, the applied stress, and the corrosion fracture fatigue life was obtained, as shown in Figure 6. In addition, with assumption 0 = 0.001 mm, = 100 Mpa, the relations of these factors were obtained, as shown in Figure 7. According to Figures 6 and 7, it was found that the corrosion fatigue life of high-strength bolt was influenced by material strength , applied stress , and stress amplitude Δ . The fatigue life reduced with the increase of material strength , applied stress , and stress amplitude Δ . Stress amplitude Δ had significant effect on fatigue life, and the influence of material strength was the smallest. Hence, the stress amplitude should be controlled to a low level. Conclusion Based on the fracture mechanics, the corrosion fracture crack estimation model and the fatigue life estimation model of high-strength bolt were established. Then the crack depth of the high-strength bolt breaks in the corrosion environment, and fatigue life could be estimated. The factors affecting highstrength bolt corrosion fatigue life were discussed. The biggest crack depth of the corrosion fracture high-strength bolt decreases with the increase of material yield strength and applied stress , in which material yield strength had significant effect on crack depth . Hence, the bolt strength level should be low. The fatigue life of the corrosion fracture high-strength bolt decreases with the increase of material yield strength , applied stress , and the stress amplitude Δ . Stress amplitude Δ had significant effect on fatigue life, and the influence of material strength was the smallest. Hence, the stress amplitude should be controlled to a low level.	2016-05-04T20:20:58.661Z	2014-07-24T00:00:00.000	{ "year": 2014, "sha1": "4f6a2cbe822e61106c78b09156d4b9082201b7c2", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1155/2014/567318", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "4f6a2cbe822e61106c78b09156d4b9082201b7c2", "s2fieldsofstudy": [ "Engineering", "Materials Science" ], "extfieldsofstudy": [ "Materials Science", "Medicine" ] }
119202845	pes2o/s2orc	v3-fos-license	Field dependence of magnetic correlations through the polarization flop transition in multiferroic TbMnO3 : evidence for a magnetic memory effect The field-induced multiferroic transition in TbMnO3 has been studied by neutron scattering. Apart strong hysteresis, the magnetic transition associated with the flop of electronic polarization exhibits a memory effect: after a field sweep, TbMnO3 does not exhibit the same phase as that obtained by zero-field cooling. The strong changes in the magnetic excitations across the transition perfectly agree with a rotation of the cycloidal spiral plane indicating that the inverse Dzyaloshinski-Moriya coupling causes the giant magnetoelectric effect at the field-induced transition. The analysis of the zone-center magnetic excitations identifies the electromagnon of the multiferroic high-field phase. The magnetoelectric eect couples the electric polarization P to an applied magnetic eld H and the magnetization M to an external electric eld E [1,2]. Very recently, giant magnetoelectric coupling has been reported for several transition-metal oxides, such as e. g. perovskite manganites RMnO 3 with R=Gd, Dy, and Tb [3,4], spinel chromate CoCr 2 O 4 [5], spin-chain cuprate LiCu 2 O 2 [6], and huebnerite MnWO 4 [7,8]. Common to all these systems is a non-collinear magnetic ordering, the key element to understand the multiferroic order [9,10,11]. Two non-collinear spins S i , S j at a distance r ij induce a spontaneous electric polarization via an inverse Dzyaloshinski-Moriya coupling P ∝ r ij × (S i × S j ). (1) A spiral structure with a nite angle between the spinrotation axis S i ×S j and the propagation vector k , therefore, induces a nite electric polarization, and a large magnetoelectric eect may arise from the response of the spiral to an applied magnetic eld [10,12]. TbMnO 3 is particularly well suited for the investigation of the oxide multiferroics, as it exhibits a rather large electric polarization and as its crystal and magnetic structure is simpler than that of other multiferroics. TbMnO 3 crystallizes in the orthorhombic symmetry, space group P bnm [13], and the Mn magnetic moments order below T N =42 K in a collinear spin-density wave (SDW) with incommensurate (IC) propagation vector k Mn ≈ (0 0.28 0) [14,15]. In a second magnetic transition at T FE =28 K, the Mn magnetic order continuously transforms into an elliptical cycloid with the spiral basal plane being parallel to the bc plane (i. e. the spins rotate around the third crystallographic axis a) [16]. According to the general concept of eq. 1, the spiral order implies a spontaneous electric polarization P c along c, which is observed experimentally below T FE [3], rendering TbMnO 3 multiferroic, see Fig. 1. Applying a magnetic eld, giant magnetoelectric eects are observed. A critical eld H c =7 T along c completely suppresses the FIG. 1: (color online) Sketch of the magnetic spiral structure in zero eld inducing an electric polarization Pc (above). The rotation from Pc to Pa in the high-eld phase is supposed to originate in a concomitant op of the spiral rotation axis from a to c (below). electric polarization [17,18], while a eld of 10.5 T (6 T) parallel to a (b) ops the electric polarization by 90 • from P c to P a [3]. Since the magnetic modulation in the FE high-eld phases remains along [0 1 0], the 90 • -rotation of the electric polarization suggests the op of the spiral rotation axis from a to c, see Fig. 1 [10,12]. However, a proof that inverse Dzyaloshinski-Moriya coupling according to equation (1) accounts for the giant magnetoelectric eects at the eld-induced transition is still missing. Measuring selected superstructure reections by neutron or x-ray diraction techniques [18,19,20] it has been demonstrated, that a magnetic eld H parallel to c melts the IC ordering and stabilizes a paraelectric phase with simple AFM ordering [18]. The op of the electric polarization for a eld along a or b coincides with a rst order transition into a high-eld commensurate (HF-C) ordered phase with propagation vector q HF Mn = (0 0.25 1) [19,20]. The mechanism in eq. (1) provides for a close coupling between the dielectric properties and magnetism which leads to hybridized phonon-magnon excitations arXiv:0711.2930v1 [cond-mat.str-el] 19 Nov 2007 [9,10,11] as they were proposed long ago [21]. While such electromagnon was proposed on the basis of Infrared (IR) optical-spectroscopy on TbMnO 3 [22], only the correspondence of the peaks in the IR spectrum with neutron scattering measurements of the IC zone-center magnetic excitations [23] documents the mixed phononmagnon character of the excitation. This rather soft electromagnon mode possesses the correct symmetry to rotate the spiral plane from the bc to the ab plane and appears to be activated when eld is applied along a or b. In this work we follow the antiferromagnetic zonecenter excitations through the polarization op transition with H applied parallel to a using inelastic neutron scattering. Our measurements show pronounced changes in the magnetic excitations at the critical eld, while analyses of the spectra are consistent with a magnetic spiral where the magnetization rotates within the ab plane as predicted by the inverse Dzyaloshinski-Moriya coupling. Detailed measurements of the propagation vector k as a function of magnetic eld reveal a magnetic memory eect that is driven by domain wall locking. Neutron scattering experiments were performed at the cold triple-axis spectrometer PANDA (FRM-II, Garching) using the same single crystal as in our previous studies [23]. The sample was mounted with the scattering plane dened by [0 1 0] and [0 0 1] in a 15 T-cryomagnet and the eld applied along the vertical a-axis. The P bnm-lattice constants are a = 5.302 Å, b = 5.857 Å, and c = 7.402 Å, which in the following will be used to index all vectors of reciprocal space. Monochromatic neutrons were selected and analyzed using the (0 0 2)-Bragg reection of pyrolytic Graphite (PG). In the standard set-up the energy of the analyzed neutrons was xed to E f =4.66 meV (k f = 1.50 Å −1 ), but in order to enhance the experimental resolution selected scans were repeated In the vicinity of H a ↑ , a region of coexistence of low-eld incommensurate (LF-IC) and HF-C phases exists, and the transition is clearly of rst order, in good agreement with recent scattering experiments [19,20] and thermodynamic studies [24]. On the downward run a pronounced hysteresis is observed, as the system switches back into the LF-IC phase at H a ↓ 8 T [24]. Again, around H a ↓ a region of coexistence of both phases is found, which is broader than that in the upward stroke. Furthermore, 6 8 10 b determined before the eld sweep. We can only recover the original zero-eld incommensurability by heating the system up into the paraelectric SDW-phase and subsequent re-cooling it to 17 K. In the FE spiral phase, TbMnO 3 memorizes its magnetic history as the size of the incommensurability ε 0 b at zero eld depends on the magnetic diary of the sample. Measurements of magnetization from a single crystal of TbMnO 3 with H a show that there is no large eect that tracks the hysteresis of the magnetic wave vector as we found in our neutron measurements. The magnetization hysteresis that is associate with the transition to the HF-C phase is very small, see Fig. 2c. This indicates that the ferromagnetic alignment of Tb-spins that occurs with applied magnetic eld is of little consequence to the memory eect we nd here. A more plausible explanation is that the transition into the HF-C phase induces domain walls which at least partially remain when switching back to the LF-IC phase. These domain walls are only suppressed when fully leaving the spiral phase upon heating [25]. Memory eects at combined spin and polarization op transitions should be of general importance in view of future applications in magnetoelectric data storage. We now turn to the eld dependence of the magnetic excitation spectrum. Inelastic scans at the magnetic zone-center Q = (0 ε b 1) for T = 17 K are presented in Fig. 3. At zero eld, several magnetic signals are detected. The feature around 4.5 meV corresponds to a crystal eld (CF) excitation in the Tb-subsystem [23]. In the following discussion we will ignore this CF-excitation, as up to the maximum eld investigated, H = 12 T, no signicant changes can be observed in the spectra above ≈ 4 meV, see Fig. 3a. In the regime of the Mn spin-wave excitations below 4 meV the impact of the magnetic eld is, in contrast, remarkable, see Fig. 3c and d. At zero eld, the magnon spectrum consists of three dierent low-energy branches: One mode with very low energy, and two modes at nite energies, 1.0 meV and 2.5 meV, respectively, are observed at the magnetic zone center [23]. With increasing eld, this characteristic structure of the spectrum remains unchanged up to H a = 8 T, and the data for 4 T and 8 T can well be described using similar parameter sets as those for the zero-eld data. The observed spectral weight of all modes increases following the elastic signal with increasing eld, but for H a ≤ 8 T we do not observe signicant changes in the spin-wave frequencies. Note, that the broad response close to ω = 0 meV, which has to be ascribed to the lowlying spin-wave mode, can not be fully separated from the elastic signal. With the transition into the HF-C phase the spectrum exhibits prominent changes, see Fig. 3d. The signal around 1.0 meV is completely suppressed, and for H a = 12 T the spectrum can be decomposed into two intense excitations centered around 0.5 meV and 2.25 meV, and a weaker feature around 3.0 meV. A fourth, rather sharp signal around ≈ 1.5 meV, marked by small dia-monds in Fig. 3 can be ascribed to a spurious contribution [26]. The low-energy spin-wave spectrum of the HF-C phase consists of three branches, similar to the spectrum in the LF-IC phase [23]. The data measured at 10 T-data can be described by a weighted summation of the 8 T and 12 T spectra, see Fig. 3c, consistent with the co-existence regime of the C and IC wave vectors shown in Fig. 2b. In the zero-eld phase with P \|\|c longitudinal polarization analysis allowed for the unambiguous identication of the phason mode and of the Goldstone Boson of the FE spiral structure [23]. The applied magnetic eld prohibits a similar polarization analysis for the P a HF-C phase. However, the systematic survey of the Q-dependence of the magnon signal can determine the character of the different magnon branches in the HF-C phase, as only magnetic polarization perpendicular to the scattering vector, S ⊥ , contributes to the neutron scattering intensity. In Fig. 4 we compare the excitation spectra recorded at the three dierent A-type zone centers Q 1 = (0 0.25 1), Q 2 = (0 1.75 1) and Q 3 = (0 0.25 3). At these three equivalent q-points, the observed magnon frequencies are identical, ω 1 = 0.44(1) meV, ω 2 = 2.20(2) meV and ω 3 = 3.06(2) meV. However, signicant changes can be observed in the distribution of spectral weight among the three modes. The mode ω 2 is strongest for Q = Q 1 , whereas the branches ω 1 and ω 3 are most intense at Q 2 and are remarkably suppressed at Q 3 . With f (Q) denoting the magnetic form factor and α denoting the angle between Q and the magnetic polarization, the observed intensity is given by With the three chosen Q points the geometrical conditions signicantly vary as the angle with c, α c , changes : α c1 = 17.5 • at Q 1 , α c2 = 65.5 • at Q 2 , and Q 3 is almost parallel to c, α c3 = 6 • . The strong suppression of spectral weight for ω 1 upon rotating the scattering vector towards c thus implies a magnetic uctuation polarized predominately along c. Indeed, from the observed intensities we nd I obs to the expected value for an entirely c-polarized mode, I c Q1 : I c Q2 = 6.2. The second mode ω 2 exhibits opposite behavior. The loss of scattering intensity from Q 1 to Q 3 is fully explained by the magnetic form factor f (Q), implying that ω 2 is polarized perpendicular to c. The third mode ω 3 , although by far the weakest of the three magnon excitations, exhibits similar behavior as ω 1 with the most intense signal for Q = Q 2 , see Fig. 4b, and we may thus conclude that ω 3 also possesses a dominant component along c. The magnetic structure in zero eld is described by a magnetic cycloid in the bc-plane [16], and it has been shown both theoretically and experimentally that the low-energy spin-wave spectrum consists of three branches with dierent character [23,27]. One of the modes is the sliding mode of the spiral and is polarized within the spiral plane. The two other modes correspond to magnetic uctuations perpendicular to the spiral plane and are polarized along a. These modes rotate the spiral basal plane around c and around b, respectively. The latter one is identied as the electromagnon observed in IR spectroscopy [22]. This mode thus possesses the symmetry to rotate both the magnetic spiral plane and the ferroelectric polarization. Applying the arguments of the zero-eld analysis to the spectrum of the HF-C phase, our observations are fully consistent with the eld-induced op of the spiral to the ab-plane, as the polarization patterns of the spinwave excitations are rotated by 90 • . One of the three modes in the HF-C phase, ω 2 , is polarized within ab, and this mode should correspond to the sliding mode of the commensurate spiral. Compared to the LF-IC phase the phason energy is strongly enhanced, increasing from ≈ 0.2 meV for H = 0 T to 2.20 meV in the HF-phase. The large phason energy indicates a strong pinning in the HF-C phase resulting in a very anharmonic magnetic modulation associated with domain boundaries. This further corroborates our interpretation that the magnetic memory eect arises from domain boundaries introduced in the HF-C phase. Furthermore, the magnetic eld will additionally trap the phase of the spiral. The two other HFmodes are both polarized predominantly along c, i. e. perpendicular to the opped spiral plane. These branches, thus, correspond to the two a-modes of the zero-eld spiral, and following the above argumentation, at least one of these modes is expected to couple strongly to an alternating electric eld along c and should be visible in optical spectroscopy. This mode is the multiferroic electromagnon, but our neutron scattering experiment cannot determine which of the two c-modes at high eld corresponds to the electromagnon. We are,unfortunately, not aware of an optical study to observe the electromagnetic response of TbMnO 3 in the HF-C phase. In conclusion, we have studied the eld dependence of elastic and inelastic neutron scattering in multiferroic TbMnO 3 . The magnetic superstructure reections exhibit strong hysteresis across the magnetic transition accompanying the op of the electronic polarization and, most interestingly, there is a clear magnetic memory ef-fect. After the eld sweep at low-temperature, the system is not found in the same magnetic phase as the one obtained by zero-eld cooling. Such memory eect should be a generic feature at incommensurate to commensurate spin-op transitions in spiral magnets. The magnetic excitation spectrum at the zone center in the commensurate high-eld phase consists of three dierent modes, whose polarization patterns were determined by examining dierent Q-positions. These results are fully consistent with the assumed eld-induced op of the spiral plane from bc to ab plane, which explains the giant magnetoelectric eect arising from the rotation of the electric polarization from P c to P a . One of the two observed cpolarized modes is the electromagnon of the multiferroic high-eld phase. These results indicate that the inverse Dzyaloshinski-Moriya coupling, see equation (1), which fully explains the temperature driven multiferroic coupling, also accounts for the eld-induced giant magnetoelectric eect.	2007-11-19T13:47:33.000Z	2007-11-19T00:00:00.000	{ "year": 2008, "sha1": "364541c24901a1a7f23fb0ac765d71ebca6e6495", "oa_license": null, "oa_url": "http://arxiv.org/pdf/0711.2930", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "364541c24901a1a7f23fb0ac765d71ebca6e6495", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
118464825	pes2o/s2orc	v3-fos-license	Range corrections for two-neutron halo nuclei in effective theory The range corrections to the universal properties and structure of two-neutron halo nuclei are investigated within an effective quantum mechanics framework. Treating the nucleus as an effective three-body system, we make a systematic improvement upon previous calculations by calculating the linear range corrections at next-to-leading order. Since the effective ranges for the neutron-core interactions are not known, we estimate the effective range to be set by the inverse of the pion mass. We investigate the possibility of excited Efimov states in two-neutron halo nuclei and calculate their mean square radii to next-to-leading order. We find that the effective range corrections are generally small and the leading order predictions are very robust. I. INTRODUCTION There is a considerable interest in the universal properties of physical systems with large scattering lengths [1][2][3][4]. The low-energy behavior of a few-body system with short-range interactions and scattering length \|a\| much larger than the range R of the underlying two-body interaction can be described in a model-independent way. This is achieved by formulating an effective theory that takes advantage of the separation of scales inherent in the system, thereby using the ratio R/\|a\| as a small expansion parameter. The theoretical uncertainty can then be systematically improved by including increasingly higher order terms in the expansion. These techniques can readily be applied to halo nuclei: a special class of nuclear systems consisting of a tightly bound core and a "halo" of lightly bound nucleons [5][6][7][8]. There is a growing history of studies which exploit the separation of scales found in halo nuclei to describe the system in an effective theory, assuming the core to be a structureless particle and treating the nucleus as a few-body system of core and valence nucleons [9][10][11][12][13][14][15][16]. In previous work [16], we explored the universal properties and structure of 2n halo nuclei to leading order (LO) in the expansion in R/\|a\| by describing the halo nuclei as an effective three-body system consisting of a core and two lightly bound valence neutrons. We constructed an effective potential made up of two-body contact interactions, and showed that this approach is equivalent to an effective field theory (EFT) framework. Our main focus was the possibility of such three-body systems to display the universal Efimov effect [17]. Assuming the nuclear ground state to be an Efimov state, we confirmed earlier model studies showing that 20 C is the only 2n halo nucleus candidate to display an excited Efimov state [9,10] if the 19 C binding energy is less than 165 keV. We also calculated the properties of this state. Moreover, we studied the structure of other known halo nuclei, calculating the matter density form factors and mean square radii (See also Ref. [14] for a previous study). In this paper, we improve upon our earlier calculation by including the momentum dependent next-to-leading order (NLO) term in the short-range effective potential. While the scattering of two particles at sufficiently low energy is determined to leading order by only the scattering length a, at NLO, the effective range r 0 of the two-body interaction comes into play. Including the linear range correction, we should be able to predict low-energy observables up to errors of order (r 0 /a) 2 ∼ (M low /M high ) 2 . Effective field theories with contact interactions have already been used to study the NLO range corrections to various few-body systems with large scattering lengths, mostly focused on the three-nucleon system. The range corrections to S-wave neutron-deuteron scattering in the doublet channel were calculated in perturbation theory in Ref. [18]. In Refs. [19][20][21], low-energy three-body observables were calculated up to next-to-next-to-leading order (N2LO) using a formalism that partially resums higher-order range effects. An analysis of three-and four-body observables to NLO within the resonating group model was carried out in [22]. For a more exhaustive list of such higher-order studies in the three-nucleon system, see Refs. [3,4] and references therein. There has also been interest lately in calculating the effective range corrections to the Efimov effect for three identical bosons. Thøgersen et al. [23] focused on the range corrections for ultracold atoms caught in an optical trap. Platter, Ji, and Phillips have explored the corrections to the Efimov spectrum linear in r 0 using once-subtracted momentum-space integral equations derived from effective field theory [24]. They showed that discrete scale invariance relates the relative corrections for different Efimov states and the bound state spectrum in the unitary limit is unaffected by the linear range corrections. In this work, we focus on the NLO correction to the properties of two-neutron (2n) halo nuclei arising from a non-zero effective range in the neutron-neutron and neutron-core interactions. In the next section, we discuss the theoretical framework for this extension. Our results are presented in Section III. We discuss the impact of this correction on the possibility of known 2n halo nuclei to have an excited Efimov state and on their binding energies. Moreover, we calculate the shift in the mean square radii of various halo nuclei due to the non-zero effective range. Our conclusions are given in Section IV. Finally, some details on the derivation of the Wigner bound and the calculation of the matter form factors are given in the appendices. II. THEORETICAL FRAMEWORK We want to extend the effective quantum mechanics framework used in Refs. [16,25] to next-to-leading order (NLO). This formulation is equivalent to using field-theoretic language for the problem at hand. The short-range interactions characteristic of halo nuclei are described using an effective interaction potential. Using the NLO effective potential, the low-energy behavior of the system will be reproduced up to errors proportional to the lowmomentum scale M low over the high-momentum scale M high squared. At this order, we require two coupling parameters C 0 and C 2 tuned to reproduce the scattering length a and effective range r 0 of the two-body interaction. The theory is valid up to a momentum scale M high at which the errors become of order one. For the large separation of scales involved in halo nuclei, zero-range interactions can be used in constructing the effective interaction potential. This leads to a separable potential made up of contact interactions in a momentum expansion. The two-body S-wave NLO potential can be written as where the dots indicate higher order interactions and g(p) is a regulator function (sometimes called the form factor). Of course, the low-energy observables must be independent of the regularization scheme, and one can choose the scheme most convenient for calculations. For this study, we use a strong cutoff regularization, in which the effective potential is set to zero, V ef f = 0, for momenta p, p ′ > Λ. This corresponds to the regulator function g(p) = θ(Λ − p) and allows for a simple inclusion of a three-body force term, as described below. A natural choice for the value of Λ is Λ ∼ M high , but observables are independent of Λ after renormalization, up to higher order corrections which scale with powers of 1/Λ. At this point it is worth commenting on the antisymmetry of the full wave function with respect to the exchange of nucleons in the core and the halo neutrons. In microscopic cluster models, such as the resonating group model [26] or fermionic molecular dynamics [27], the wave function is antisymmetric with respect to exchange of all nucleons. In our halo effective theory the wave function is only antisymmetric with respect to exchange of the halo nucleons since the nucleons in the core are not resolved. The error involved in this approximation is governed by the ratio of the binding momentum of the halo nucleons to the binding momentum of the core, i.e. M low /M high . Within the domain of validity of the effective theory such effects are subsumed in the effective range parameters of the neutroncore interaction and can be treated in perturbation theory. This can be understood as follows: The exchange of a nucleon from the core and a halo nucleon can only give a sizeable contribution if there is a spatial overlap between the wave function of the core and the wave function of the halo nucleon. The overlap of the wave functions, however, is determined by the ratio M low /M high . If this ratio is of the order 1/3, one can expect the antisymmetrization effects to be 30% at leading order and 10% at next-to-leading order. For a microscopic study of antisymmetrization effects in the neutron-alpha system, see Ref. [28]. The potential (1) is then used in the solution of the three-body Faddeev equations in terms of the spectator functions F i (q), which represent the dynamics of the core (i = c) and the halo neutron (i = n). To find the bound state of a halo nucleus composed of two valence neutrons and a core with spin zero, the resulting coupled integral equations are simply a generalization of the three-boson equation 1 (see [25,29] and references therein). In the remainder of the paper, we use units such thath = c = 1. For convenience, we set the nucleon mass m = 1. The resulting integral equations for the spectator functions F n (q) and F c (q) and the bound state energy B 3 > 0 are: The functionsG c andG n arise from the free three-body propagator. They are given bỹ where A is the number of nucleons in the core. The effects of the interactions are contained in the two-body T-matrices which are obtained by solving the Lippmann-Schwinger equation for the neutron-neutron and neutroncore interactions with the effective potential in Eq. (1). In the kinematics of Eqs. (2, 3), we have for the neutron-core T-matrix: 1 In fact, the equations are the same for any bound three-body system of two identical particles and a core with spin zero, which interact through the pair-wise zero-range potentials given in Eq. (1). and for the neutron-neutron T-matrix: withẼ A single index n, c indicates the spectator particle and a nn , r nn , and a nc , r nc are the n-n and n-c scattering lengths and effective ranges, respectively. For brevity we have defined the variables: Here, E ni is the two-body bound (virtual) state energy calculated from the pole of the twobody T-matrix at NLO in the limit Λ → ∞, with µ the corresponding reduced mass. In this limit, which implies a cutoff much larger than the momentum scales involved in the problem: Λ ≫ 1/\|a\|, Ẽ i , √ E ni , these T-matrices reproduce the usual effective range expansion at NLO. Therefore, the pole position can be found through the relation: At leading order (r ni = 0), this expression reduces to the universal formula for the twobody binding energy for systems with a large positive scattering length: E ni = 1/(2µa 2 ni ). Moreover, we have simplified the expressions for the two-body T-matrices (6,8) by defining the functions which approaches one asx becomes large and which vanishes for largex ni . The term proportional to h ni (x ni ) Λ generates the effective range term in the two-body T-matrices (6,8). Finally, the dimensionless function H(Λ) is a short-range three-body force. This term was included in the integral equations (2, 3) in analogy to the three-boson case which our equations reproduce in the limit of identical particles. It corresponds to a three-body contact interaction which is required for consistent renormalization [25,30]. For a given value of Λ, the three-body term H can be tuned to reproduce a given three-body observable and then other low-energy observables can be predicted using the same value H. The running of the three-body force H(Λ) with the cutoff Λ is governed by a limit cycle. As a consequence, it is always possible to find a value of the cutoff Λ at which the three-body force vanishes. Hence, at leading order Λ can simply be used as the three-body parameter in practical calculations [31]. The correct renormalization in the effective theory was explicitly verified [25]. Apart from the three-body observable, our input parameters at NLO are the n-c and n-n scattering lengths (or energies) and the corresponding effective ranges. The three-body binding energies are then given by the values of B 3 for which the coupled integral equations (2, 3) have a nontrivial solution. For most halo nuclei, the n-c scattering length is not as well known as the two-body bound (virtual) state energy. Therefore, we will generally use the two-body energy E nc as an input parameter. From E nc and r nc , we can calculate the corresponding a nc to be used in the two-body T-matrices through Eq. (11). The n-c effective ranges for most halo nuclei are not known experimentally. However, the range of the interaction between two nucleons is determined by the exchange of pions. We therefore use the inverse of the pion mass, m π = 140 MeV, to estimate the n-c effective range, r nc ≈ 1/m π = 1.4 fm. This is equivalent to setting the effective range to the natural low-energy length scale of the system. The value of the n-n effective range is known experimentally: r nn = (2.75 ± 0.11) fm [32]. Its size is comparable to the inverse of the pion mass scale. In our numerical calculations we use r nn ≈ r nc ≈ 1/m π = 1.4 fm. This choice is sufficient for our purpose of estimating the size of the range corrections and testing the robustness of the leading order results. Moreover, it allows a slightly larger range of cutoffs to be consistent with the Wigner bound. The Wigner bound is based on general arguments and constrains the value of the effective range that can be generated by short-range potentials. It has been shown that the renormalization of an effective potential made up of contact interactions in a momentum expansion can only be performed if certain constraints are placed on the effective range [33]. More specifically, the effective range must be negative in the zero-range limit of the potential. In our case, this corresponds to the limit Λ → ∞ since a finite cutoff generates a finite range. This constraint follows directly from a general bound on the derivatives of the phase shifts derived first by Wigner in 1954 [34]. Wigner derived this bound from the fundamental principles of causality and unitarity, holding that a scattered wave cannot leave the scatterer before the incident wave has reached it. Phillips and Cohen derived the relation between the Wigner bound on the derivatives of phase shifts with the constraint on the effective range for short-range potentials [35]. This bound applies even if the potential does not vanish exactly outside some finite radius but merely decreases fast enough for the wave function to approach the asymptotic solution sufficiently quickly. The Wigner bound for zero-range potentials was shown to hold true no matter how many terms in the momentum expansion are included in the potential [33]. A very recent study has generalized the Wigner Bound to arbitrary dimension and angular momentum [36]. An explicit derivation of the Wigner bound constraint for our effective potential is given in Appendix A. In our numerical results in the next section, the cutoff is always chosen in accordance with the Wigner bound. III. EFFECTIVE RANGE CORRECTIONS TO 2n HALO NUCLEI In this section we present numerical results for the effective range corrections to lowenergy observables of three-body halo nuclei composed of a core and two valence neutrons. We begin with the range corrections to the possibility of the Efimov effect in 2n halo nuclei, as well as to the binding energy of a possible Efimov excited state in 20 C. After that, we estimate and discuss effective range corrections to the mean square radii results. A. Range Corrections to 2n Halo Nuclei Binding Energies In Ref. [16], we have calculated boundary curves representing the existence of an excited Efimov state for various values of the core mass in the parametric region defined by the ratios (E nc /B (n) 3 ) 1/2 , where E nc and E nn are the n-c and n-n two-body energies related to the S-wave scattering length to LO. An analogous study was carried out previously in Ref. [10] within the renormalized zero-range model and motivated our investigation. It should be noted, however, that this type of analysis implicitly assumes that the ground state of a halo nucleus is an Efimov state. More general scenarios where only the excited state has universal character cannot be excluded. The curve itself is built up of the points for which the B (n+1) 3 binding energy is equal to the scattering threshold. This is equivalent to finding the critical scattering lengths of the three-body system. In similar studies looking at the range corrections to the Efimov effect in the three-boson system [23,24], it was observed that including a non-zero effective range shifted the positions of the critical scattering lengths. However, for all values of effective ranges r nc and r nn within the range of validity of our effective theory (r ni ≪ \|a ni \|, i = n, c), we find these shifts to be extremely small such that the boundary curves calculated with a non-zero effective range are indistiguishable from those at LO (See Fig. 2 of Ref. [16]). As a consequence, the main conclusion from [16] is only slightly changed by the NLO range corrections: if the n-18 C bound state energy satisfies E nc < 161 keV, 20 C will have an excited Efimov state. In the following, we investigate the dependence on the n-18 C bound state energy in more detail. In Ref. [16], we calculated the value of the possible excited state energies at LO as a function of E nc , using the standard value for a nn = (−18.7 ± 0.6) fm [37], and fixing the cutoff Λ to reproduce the experimental value of the ground state energy B (0) 3 = 3506 keV [38,39]. Now using the estimate r 0 = r nn = r nc ≈ 1/m π = 1.4 fm for the effective ranges along with the standard value for a nn given above, we calculate the excited state energies B 3 as a function of E nc to NLO, by fixing Λ and tuning the three-body term H to reproduce the experimental value of the ground state energy B Fig. 1, where the solid black line is the excited state energy to LO, the dotted-dashed red line to NLO, and the dashed line represents the scattering threshold. The inset graph shows the excited state energy relative to the scattering threshold. Whereas the excited state in the LO calculation exists when E nc < 165 keV, the state to NLO only exists when E nc < 161 keV. For larger values of E nc , the 20 C system moves across the scattering threshold, and the excited Efimov state disappears. We also see that the effective range corrections only lead to a recognizable shift toward smaller binding energies for E nc < 90 keV. For larger values of E nc there is a region where the effective range corrections lead to a shift towards larger binding energies. However, this shift is always very small, no greater than ≈ 0.5 keV over 3 = E nc . The inset shows the excited state energy relative to the scattering threshold. the whole range of possible E nc values shown, and always smaller than the NLO error bands. We have estimated this NLO error using the theoretical uncertainty of our effective potential. The uncertainty in binding energies calculated using the two-body effective potential of Eq. (1) is ≈ (r 0 /a) 2 . For effective ranges much smaller than the scattering length we know that 1/a 2 nc ≈ 2µ nc E nc . Therefore, we estimate the theoretical uncertainty in the binding energy of the excited state using the sum of the uncertainties from the n-n and n-18 C interactions: ∆B 3 ≈ (r nn /a nn ) 2 + r 2 nc (2µ nc E nc ). The TUNL nuclear data evaluation from 1995 [38,39] quotes the n-18 C bound state energy as E nc = (162 ± 112) keV. This value is based on experiments using strong breakup reactions [40][41][42][43] and would leave the possibility of an Efimov excited state in 20 C open. The more recent atomic mass evaluation AME2003 [44], however, quotes E nc = (580 ± 100) keV which excludes an excited Efimov state. This evaluation includes a recent measurement of E nc using Coulomb dissociation of 19 C that found E nc = (530 ± 130) keV [45]. Typel and Baur showed that the value of Ref. [45] is also consistent the distribution of the B(E1) continuum strength [46]. A review of the experiments was given in [47]. B. Range Corrections to 2n Halo Nuclei Mean Square Radii In this subsection, we will calculate the NLO corrections to the mean square radii of 2n halo nuclei. We will briefly review the necessary formalism and then present our results. A more detailed description of the formalism is given in Refs. [16,29]. For the calculation of the one-and two-body matter density form factors and mean square radii, we need the full three-body wave function, which can be reconstructed from the Faddeev spectator functions F n and F c . The relevant formulae are given in Appendix B. The mean square radii are then calculated from the matter density form factors in the low momentum transfer region, where the slope of the form factor determines the mean square radius r 2 : Of course, the mean square radius acquired depends on the choice of one-or two-body form factor. These matter density form factors are calculated from the full wave function of the three-body system through Eqs. (B8, B9, B10). The slope of the two-body form factor F ni (k 2 ), where i = n, c, will give the mean square distance between the two particles in the chosen two-body subsystem, either r 2 nn or r 2 nc . Analogously, the slope of the one-body form factor F i (k 2 ) will give the mean square distance of the spectator particle from the center of mass of the two-body subsystem, either r 2 c−nn or r 2 n−nc . However, it is more useful to calculate the distance of the individual particles from the center of mass of the three-body bound state. If b i is the slope of the one-body form factor F i (k 2 ) at k 2 = 0, the mean square radius of one of the bodies i from the three-body center of mass is given by: where m i is the mass of the desired constituent, i = n, c, and m n and m c are the neutron and core masses, respectively. We have extracted the radii by fitting a polynomial in k 2 to the form factor results for small k 2 . We have used polynomials of varying degree up to 5th order in k 2 in order to verify the stability and convergence of the fit. We have found a satisfactory stability in the slope when fitting to a polynomial to the fourth order in k 2 , up to a value of k 2 at which the form factor has dropped less than 10 percent. The extracted radii for known halo nuclei to both LO and NLO are shown in Table I. We show only a selection of the LO results found in Ref. [16], to give a general overview of NLO corrections to the LO results. As input we have used the standard value of the n-n scattering length, a nn = (−18.7 ± 0.6) fm [37], along with the experimental values of the n-c two-body energies E nc shown in the third column of Table I (negative values correspond to virtual energies). The effective range of both two-body subsystems is shown in the fourth column, indicating which results refer to LO (r 0 = 0 fm) or NLO (r 0 = 1.4 fm) calculations. As before, we use the inverse of the pion mass to estimate the effective range of both the n-n and the n-c interactions for all 2n halo nuclei. As a three-body input, the three-body term is tuned to reproduce the experimental ground state binding energy B Table I. These experimental values for the two-body and three-body energies are taken from the most recent results of the "Nuclear Data Evaluation Project" of TUNL [39], except where otherwise noted. The calculations are performed at a fixed cutoff Λ. Due to the Wigner bound, some care must be taken in choosing Λ. For the range of scattering lengths, or corresponding two-body energies, which we wish to explore, the Wigner bound leads to a maximum value for the cutoff for most of the 2n halo nuclei of approximately Λ < 630 MeV. For our calculations, we use a cutoff Λ = 580 MeV for all nuclei. This value is significantly larger than the typical momenta in halo nuclei which are well below the pion mass. The theoretical error for the NLO calculation is again estimated by the uncertainty of the two-body effective potential, Eq. (1), which is of order (r 0 /a) 2 , where r 0 is the effective range of the interaction, and a the scattering length. The uncertainty in the radii is then calculated exactly as was done in the previous subsection, using the sum of the uncertainties from the n-n and n-c interactions: ∆ r 2 / r 2 ≈ (r nn /a nn ) 2 + r 2 nc (2µ nc E nc ). We stress that our NLO results as well as their errors are based on our estimate of the effective range, r 0 = 1.4 fm. They do not include the uncertainty in the estimate of r 0 and therefore must be interpreted with some care. We will now discuss the results for the NLO corrections to the mean square radii due to the effective range of the interactions. For the Borromean halo nuclei 11 Li and 14 Be, in which all two-body subsystems are unbound, the general tendency is for a positive effective range to shift all mean square radii to smaller values. The only exception is the case of 11 Li using the central value of the n-c energy E nc = (−25 ± 15) keV [39]. This difference is due to the fact that this value of the virtual energy is very close to the resonant limit, E nc = 0.0, while the competing value E nc = (−800 ± 250) keV [48] is much larger in comparison. This can be seen more clearly if we look at the mean square radii over a range of E nc values. Using the central value of the three-body binding energy as input, B results, with error bands represented by the dashed lines [16]. The dotted-dashed red lines represent the NLO results, with solid colored error bands. We see that the mean square radii to NLO show the same behavior with changing E nc as the LO case, with a decrease in the magnitude of E nc leading to an increase in size, with a more rapid increase as the resonant limit is approached. However, for the NLO results, the increase is more rapid when E nc approaches zero, such that below some value of \|E nc \|, a positive effective range shifts the mean square radii to larger values. Otherwise, a positive effective range always shifts the mean square radii to smaller values. One can see that this shift is more drastic as the magnitude of \|E nc \| grows larger. This is most likely due to the fact that for these large values of \|E nc \|, the scattering length becomes smaller, nearing the order of magnitude of the effective range. For example, the virtual n-9 Li energy E nc = −1000 keV, along with the effective range r nc = 1.4 fm, corresponds to a scattering length a nc = −4.2 fm. The study by Marqués et al. [50] extracted experimental values for r 2 nn for Borromean halo nuclei using the technique of intensity interferometry along with the two neutron correlation function to study the dissociation at intermediate energies of two neutrons in halo nuclei. The result for 11 Li, r 2 nn exp = (6.6 ± 1.5) fm, is in close agreement with both the LO, r 2 nn = (6.8 ± 1.8) fm, and NLO, r 2 nn = (6.3 ± 0.5) fm, calculations found using the two-body virtual energy reported in [48], E nc = −800 keV. However, due to the large uncertainties in both the theoretical and experimental values for 11 Li, there exists a large range of E nc values which would produce a r 2 nn value in agreement with the experimental value of Marqués et al. [50]. The LO result for 14 Fermi below the the experimental value from Ref. [50], r 2 nn exp = (5.4 ± 1.0) fm, but the two values are consistent within one error bar. The NLO result, r 2 nn = (3.86 ± 0.09) fm, is even smaller and only consistent with the experimental value within two sigma errors. Recall, however, that the NLO results are found using only an estimate for the effective ranges. Our results indicate that the effective range in the n-12 Be-interaction is below this estimate. Furthermore, the large uncertainty in these experimental values is indicative of the need for more precise measurements of the mean square distances in 2n halo nuclei. Also, the recent work by Orr [51] discusses the care which must be taken in interpreting the experimental results of [50]. Specifically, the technique is sensitive to the population of states in the continuum by the dissociation process rather than being a true ground state measurement. For more details on this issue, see Ref. [51]. Turning now to the so called Samba halo nuclei 12 Be, 18 C, and 20 C, for which the n-c subsystem is bound, it can be seen that a positive effective range always shifts the mean square radii to larger values. We focus on the case of 20 C and look at the mean square radii over a range of E nc values. The results, using the central value for the three-body binding energy as input, B (0) 3 = 3506 keV, were calculated to LO in Ref. [16]. We have now calculated these values to NLO, using the inverse of the pion mass as an estimate of the effective range, and the results compared to the LO case can be seen in Fig. 3 mean square radii to NLO show the same behavior with changing E nc as the LO case, with an increase in E nc leading to an increase in size. Also we see more clearly the general shift toward larger radii due to the effective range corrections. As was shown in the previous subsection, there exists one Efimov excited state in 20 C for E nc < 165 keV. In Fig. 4, the mean square radii for this excited state to NLO are plotted over a range of E nc values, together with the LO results from Ref. [16]. Again we see that the general behavior of the mean square radii as a function of the two-body energy remains the same when calculated to NLO. At the endpoints, signifying the critical E nc where the excited state breaks up, the mean square radii diverge as the excited state is destroyed and the particles fly apart. For small values of E nc , there is a positive shift in the mean square radii for positive effective range, although this shift is relatively small, as compared to the relative shift in the ground state radii. However, as E nc grows, there is a region where a positive effective range causes a negative shift in the mean square radii. This is related to the region where the effective range causes a positive shift in the binding energy, as described in the previous subsection. In Table I, we have highlighted the results using two different values of the n-c two-body binding energy: E nc = (530 ± 130) keV [45] and the older value E nc = (162 ± 112) keV [39]. Moreover, we show results for E nc = 60.0 keV which would lead to an Efimov excited state. The rows marked by 20 C * give the results for the excited state. The three-body binding energy of this excited state is listed in the second column with theoretical uncertainty as calculated in Sec. III A. Within the error estimates our results generally agree with the values obtained previously by Yamashita et al. using the renormalized zero-range model [14]. Note, however, that this model does not include the effective range corrections and no error estimates are given. Finally, we note that we explicitly do not apply our method to 6 He. The n-4 He-interaction is dominated by strong P-wave resonance in the J = 3/2 channel, while our effective theory assumes that S-wave interactions dominate. A treatment of 6 He therefore requires an effective theory with a different counting scheme. While various schemes to treat such Pwave resonances in Effective Field Theory have been developed [12,13], their application to three-body systems remains to be worked out. IV. CONCLUSION In this paper, we have studied the next-to-leading order (NLO) effective range corrections to the universal properties of two-neutron halo nuclei. Our investigation was performed within an effective quantum mechanics framework using cutoffs of the order 500 MeV. Assuming that the halo nuclei have resonant S-wave interactions between the neutron and the core, the effective potential reduces to a separable S-wave potential. At NLO the potential is determined by the neutron-neutron and neutron-core energies and effective ranges plus one three-body parameter that can be fixed from any three-body datum. At next-to-next-toleading order in the expansion in M low /M high no new two-body parameters enter. Whether another three-body parameter enters at this order is still an open question that has not been completely resolved [19,21]. Our main focus was in investigating NLO corrections to the results for 2n halo nuclei which arise from a non-zero effective range. Because the neutron-core effective range is not known experimentally, we were only able to estimate the range corrections assuming the effective range to be of the order of the inverse pion mass, r 0 ≈ 1/m π = 1.4 fm. Based on this assumption, we found that the leading order results are very robust and range corrections are typically small. We investigated the possibility of excited Efimov states in halo nuclei assuming that the ground state is also an Efimov state. In particular, we found that the corrections to the parametric boundary curves, within which at least one excited Efimov state will occur were essentially unchanged for realistic values of the effective range. We found that the value of the critical n-18 C two-body energy required for such an excited state to exist in 20 C was decreased slightly from 165 keV at LO to 161 keV at NLO. While the TUNL nuclear data evaluation from 1995 [38,39] quotes this energy as E nc = (162 ± 112) keV, the most recent evaluation AME2003 [44] finds E nc = (580 ± 100) keV which excludes an excited Efimov state in 20 C. We also calculated the NLO shift in the excited state binding energy, finding that \|∆B 3 \| < 0.5 keV, a nearly negligible shift from the LO result. Moreover, we calculated the shift from the LO results in the mean square radii for known 2n halo nuclei. We found that for Borromean halo nuclei, the general tendency is for a positive effective range to shift all mean square radii to smaller values, unless the two-body n-c virtual energy is very close to threshold. The opposite was found to be true for the so called Samba halo nuclei, for which the n-c subsystem is bound. The positive effective range shifts the mean square radii of these halo nuclei to larger values. We also compared our results for the n-n mean square radius to the experimental data for the Borromean halo nuclei 11 Li and 14 Be [50]. While there is good agreement in the 11 Li system, the results for 14 Be only agree within two sigma errors. This indicates that the effective range in the n-12 Beinteraction is smaller than our naive estimate r 0 = 1.4 fm. For a better description within our effective theory, it will be necessary to determine the as yet unknown parameters, specifically the various neutron-core scattering lengths and effective ranges. Such information will also provide a solid basis for evaluating their universal behavior and determining the relevance of the Efimov effect as a possible binding mechanism for halo nuclei. If the Efimov effect is responsible for the binding of at least some three-body halo nuclei or their excited states it would open the possibility of a universal binding mechanism for higher-body halo nuclei as well. In Ref. [52], a pair of universal tetramer states associated with every Efimov trimer was predicted for the case of identical bosons and their binding energies were calculated in the vicinity of the unitary limit. Recently, the spectrum of these states was mapped out for all scattering lengths and their experimental signature in 4-body recombination was pointed out [53]. Soon after this prediction, the loss resonances from both tetramers in an ultracold gas of 133 Cs atoms were indeed observed [54]. Another calculation indicated that even higher-body cluster states with universal properties might exist [55], leading to an intriguing paradigm for a universal binding mechanism of weakly bound nuclei near the drip lines. Effective theories represent an ideal tool to explore this scenario in nuclei and predict its signatures in the structure and reactions of halo nuclei. Together with new precise experimental data from facilities such as FAIR and FRIB this will open the possibility to test this scenario. We note that this scenario does not require that the ground state of a halo nucleus is an Efimov state. More general situations where only excited states have universal character are also possible. spectator from the spectator functions we find: Ψ n (p, q) = G n 0 (p, q; B 3 ) + H(Λ) Λ 2 t n (q; B 3 )F n (q) Ψ c (p, q) = G c 0 (p, q; B 3 ) + where the two-body T-matrices t n and t c are given in Eqs. (6,8). The expressions for the propagators G n 0 and G c 0 are: Finally, the shifted momenta are: π ′ nc ≡π ′ nc (p, q) = p 2 + andπ ′ cn ≡π ′ cn (p, q) = p 2 + 1 4 q 2 − pqx. The three-body wave functions can be used to calculate other low-energy properties of the three-body bound state. With the Jacobi momentum states it is straightforward to calculate the Fourier transform of the one-and two-body matter densities with respect to the momentum transfer squared. These are defined as the one-and two-body matter density form factors F i (k 2 ) and F ni (k 2 ), respectively, where i = n, c. The derivation of the form factors from the three-body S-wave wave functions can be found in Refs. [16,29]. The resulting expression for the one-body form factors is: while the two-body form factors are: F nc (k 2 ) = dp p 2 dq q 2 1 −1 dx Ψ n (p, q)Ψ n ( p 2 + k 2 − 2pkx, q), and F nn (k 2 ) = dp p 2 dq q 2 1 −1 dx Ψ c (p, q)Ψ c ( p 2 + k 2 − 2pkx, q). (B10)	2010-02-25T12:51:26.000Z	2009-11-17T00:00:00.000	{ "year": 2010, "sha1": "4ef80088250df3369a5d6c8ccd26176e2a048eda", "oa_license": null, "oa_url": "http://arxiv.org/pdf/0911.3238", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "4ef80088250df3369a5d6c8ccd26176e2a048eda", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
247244913	pes2o/s2orc	v3-fos-license	Speech watermarking: a solution for authentication of forensic audio digital recordings In this paper we discuss the problem of authentication of forensic audio when using digital recordings. Although forensic audio has been addressed in several papers the existing approaches are focused on analog magnetic recordings, which are becoming old-fashion due to the large amount of digital recorders available on the market (optical, solid-state, hard disks, etc). We present an approach based on digital signal processing that consist of spread spectrum techniques for speech watermarking. This approach presents the advantage that the authentication is based on the signal itself rather than the recording support. Thus, it is valid for whatever recording device. In addition, our proposal permits the introduction of relevant information such as recording date and time and all the relevant data (this is not possible with classical systems). Our experimental results reveal that the speech watermarking procedure does not interfere in a significant way with the posterior forensic speaker identification. Introduction According to [Broeders 2001] rapid technical developments in the world of telecommunications in which speech and data are increasingly transmitted through the same communication channels may soon blunt the efficacy of traditional telephone interception as an investigative and evidential tool. The gradual shift from analogue to digital recording media and the increasingly widespread availability of digital sound processing equipment as well as its ease of operation make certain types of manipulation of audio recordings comparatively easy to perform. If done competently, such manipulation may leave no traces and may therefore well be impossible to detect. A promising development in the field of authenticity and integrity examinations of audio recordings in the analogue domain is the use of Faraday crystals as pioneered by a number of Russian scientists [Grechishkin 1996]. This potential gain is offset by the widespread availability of relatively inexpensive digital sound processing equipment. As part of the chain-of-custody process, audio recordings, like all digital data, are therefore increasingly required to be authenticated by means of checksums and hash codes or other methods to ensure their integrity. The formulation of standards for the forensic examination of audio recordings as undertaken by the AES [AES 1996] is a useful initiative, which may serve to improve standards across the whole field of forensic audio examination. However, it does not provide any technical solution. Digital technology has provided a large amount of benefits such as higher audio fidelity than analog recordings, the possibility to make exact copies of an original recording, minimize the effect of scratches and other physical defects by means of error correction codes (such as Reed-Solomon code used on CD-Audio). However, some drawbacks have also appeared such as the possibility to modify (insert and/or delete) some portions of a given recording in a transparent way that makes this alteration process inappreciable. Forensic applications require the study of the integrity and authenticity of recordings and this problem has remained unsolved for digital recordings. We believe that the solution to this problem appears changing the philosophy: instead of studying the recording support, some relevant information must be added to the audio signal, with the following desired properties: a) This added information must be imperceptible for a human listener. b) It must be impossible to remove or change this added information without destroying the signal itself. Thus, it cannot be a file header or a different associated file. c) It must be possible to determine the presence of this added information and its content in an easy and univoque way (without ambiguities). Approaches to speech authentication In the past few years, several approaches have been presented for the authentication of digital recordings which ought to be presented in court. Recently it has been proposed to use a electrical network frequency (ENF) criterion for determining the authenticity of a recording [Grigoras 2006]. This approach exploits the fact that even though the electrical network frequency is fixed at 50/60 Hz, a certain amount of variation is permitted. The pattern of those variations is unique for every given time instance. Furthermore the pattern is the same over a connected electrical supply net, e.g. the European power supply network. If a recording with a device connected to the power supply net is done -besides the intended signal -it also records the ENF signal. Even a battery powered appliance used in the range of the electromagnetic field of the power supply network records the ENF signal. So if one has reference data of the ENF at a certain instance of time the ENF signal of a questioned recording can be compared to this reference. The advantage of this method is, that the recording in question does not have to be prepared for this type of investigation. On the other hand, because the ENF information is recorded in a very narrow spectral band, which is usually not even used in human speech production, it can easily be removed by simple high-pass filtering. Another proposal was to use the serial copy management system (SCMS) which is implemented in the digital transmission standard of professional and consumer digital audio equipment [Cooper 2005]. The SCMS flags can be used to determine whether the recording presented is an original recording or whether it is possible that a forging attempt has been made. While this approach may work if the potential forger has only access to standard consumer type equipment, it does not help in case of a well equipped audio professional. Some professional sound-cards have full control over the SCMS settings, so any SCMS setting can be pretended. In addition using high quality D/A and A/D converters, will only result in unnoticeable degradation, but would also bypass the SCMS. The addition of meta-data to the sound signal data is also proposed. The meta-data can either be some hash code, which can be derived from the digital representation of the sound. It can also be content based information, i.e. the linear prediction coefficients of the speech signal. It has been suggested to either incorporate the meta-information attached to the signal (i.e. the wav audio format allows the embedding of meta-data in the file) [Alexander and McElveen 2006] . Alternatively the meta-information could be transmitted via an additional channel. Those approaches introduce the problem of authentication of the meta-data since with the appropriate tools this data can be easily edited. We believe that the short-comings of the described approaches can be overcome with watermarking technology, which will be introduced in the next section. Watermarking Fortunately, Forensics community is not the first one to face the problem described in section 1. Digital recording technology also permits facile data access and an increased opportunity for violation of copyright and tampering with or modification of content. This problem has been solved by means of data hiding [Bender 1996] and [Steinebach & Dittmann 2003]. Here the data of interest is directly encoded into the signal, rather than into a file header, so that it impossible or difficult to replace this information by an altered one. Another advantage is, that it is not dependent on the type of encoding of the signal. Introduction Data hiding and watermarking have received a lot of attention by different research communities during the past two decades. To avoid confusion about terminology we will provide a brief definition of terms and introduction. An extensive introduction would go beyond the scope of this article. We can point out introductory articles, which cover both ancient history and current watermarking approaches, are [Petitcolas et al. 1999 andHartung andKutter 1999]. Data hiding is the general term for hiding information on either a channel or in a medium. Often, it is also used synonymously with the term information hiding. The former is used for example by the military for covert communication or steganography (Greek, meaning "covered writing"). In contrast to encryption, where an unauthorized listener shouldn't be able to decode the information transmitted, the intruder shouldn't even be aware of a message sent. The latter would include information embedded in an image or an audio file, which an uninformed user should not be aware of. This is also a form of watermarking. Watermarking is the embedding of information in a medium, which functions as a host for this additional information. It can either be visible or invisible for the general user. Watermarking originates in the marking of paper by mills as a means of identifying origin and quality of a sheet of paper. This is still used today sometimes for high quality paper products. A very sophisticated form of paper watermarking is used as a security feature of money bills. A form of visible digital watermarking is sometimes found in images for copyright protection, where information about the copyright status is visibly embedded in the picture (see figure 1). In contrast invisible or transparent digital watermarks are desired not to interfere with the perception of the original multimedia content. This is the main focus of research for digital media watermarking. Among others, they are used for the purpose of copyright protection, identification, annotation and authentication of digital media contents. Figure 1: Visible watermarking. The copyright information is directly embedded in the image and visibly for the viewer of the image. There are several, often conflicting characteristics which are of interest for watermarking.  Quantity: Of course one wants to embed as much data as possible in a given media content. One important factor for the embedding capacity is the bandwidth of the host signal, i.e. one can be put much more additional data into a video stream, than on a telephone speech channel.  Robustness describes the need for invariance of the hidden data when a "host" signal is subject to a certain amount of distortion and transformation, e.g., lossy compression, channel noise, filtering, resampling, cropping, encoding, digital-toanalog (D/A) conversion, and analog-to-digital (A/D) conversion, etc.  Security: The embedded data should be immune to intentional modification attempts to remove or manipulate the embedded data. For authentication purposes this would be of high importance.  Fragility: Some applications, often used for data authentication do not allow any modification of the digital media content, so it is the opposite of a robust watermark. The embedded data is lost, as soon as any modification of the signal is performed. In case of a lost watermark the authenticity of the media cannot be guaranteed anymore  Transparency: This only relates to invisible watermarks, where the goal is to minimize the perceptual impact of the watermark on the host content. The host signal should be no objectionably degraded and the embedded data should be minimally perceptible.  Recovery: The embedded data should be self-clocking or arbitrarily re-entrant. This ensures that the embedded data can be recovered when only fragments of the host signal are available, e.g., if a sound bite is extracted from an interview, data embedded in the audio segment can be recovered. This feature also facilitates automatic decoding of the hidden data, since there is no need to refer to the original host signal. As mentioned before, some of those properties are competitive. For example, one can not both maximize security and data quantity. Depending on the application, for some characteristics a certain trade off has to be made in favor of more important properties. There has been a lot of research effort for image watermarking and to a lesser extent for music. The main commercial application so far is copyright protection. For instance, commercial products such as Corel Draw and Photoshop include an option for "watermarking" based on Digimarc technology. It is important to emphasize that this technique is not related to encryption, because watermarking cannot restrict or regulate access to the host signal, but rather to ensure that embedded data remain inviolate and recoverable. In addition, the encryption process is reversible. This means that once a hacker is able to decrypt a file, this file is not protected anymore in the future. On the other hand, watermarking is an irreversible procedure, and the watermark cannot be removed without altering the quality of the host signal that much, so it is worthless for any other future usage. Watermarking can provide protection of intellectual property rights, give an indication of content manipulation, and provide a means of annotation. In summary, a general watermarking system (see Fig. 2) consists of an embedding block, which overlays the host data with the additional information. The watermarked signal will the be transmitted on an arbitrary channel to the receiver of the message. On this channel both inoffensive modification, such a compression and malicious attacks, which aim at modification or removal of the watermark. On the receiver side, the embedded data can be extracted again and in the case of an authentication application should give information whether the content of the media is an original or a modified version. Wa te rma rk Embe dding While these properties are common for all digital contents, some particularities apply depending on the media type (image, video, text, music, voice, etc.). In this paper, we will focus on speech signals. Next section describes some characteristics of watermarking of speech signals. Watermarking of speech signals Audio watermarking research has focused on copyright protection for digital audio recordings. However, the lack of commercial applications for speech has slowed down the number of publications related to this topic. Watermarking for speech signals is different in some aspects than audio watermarking due to the much narrower signal bandwidth and the special properties of a speech signal. Compared to the 44.1 kHz sampling rate for CD-audio, telephony speech is usually sampled at 8 kHz. Therefore, less information can be embedded in the signal. For perceptual hiding usually the masking levels have to be calculated. The commonly used algorithms have their origin in audio compression, such as the widespread mpeg audio coder. They are optimized for CD-audio bandwidth and computationally very expensive, since compression usually does not need to be performed in real-time. Another difference is the expected channel noise. CD-audio recordings usually have very low noise. The audio signal looses its commercial value if the background noise rises beyond a certain threshold. On the other hand, the distortion allowed for a watermark signal is very low as well. Both artist and consumer would not trade in a reduced recording quality for better copyright protection. Speech, on the other hand, is very often transmitted over noisy channels, in particular true for air traffic control voice communication and telephone speech transmission. On the one hand, the channel noise is a disadvantage, on the other hand this allows much more power for the watermark signal, since the channel noise will cover it anyway. In some applications the listener expects a certain amount of noise in the signal. A summary of the differences can be seen in table 1. In our previous works we developed a watermarking system for air-traffic control [Hagmüller 2005] (speech communication among the airplane pilot and the air traffic controller at the airport), with the goal to improve air traffic security. It is based on a spread spectrum approach. This means that low bandwidth information is modulated with a broad-band pseudorandom signal, so it is spread over all the available bandwidth. Then we proposed in [Faundez-Zanuy 2006] an enhanced biometric security system by combining watermarking with biometric speaker recognition [Faundez-Zanuy 2005]. This proposal solves the problem of replay attacks that can suffer a biometric recognizer performing over a remote channel. In this case, we used the watermark for introducing an expiry date that makes worthless the use of a pre-recording of the genuine user. The watermarking system description can be found in [Faundez-Zanuy 2006]. An advantage of our proposed scheme is that the perceptual weighting hides the watermark below the formant peaks of the speech spectrum. Thus, the "noise" of the watermark is introduced on those portions of the spectrum where the signal has a considerable amplitude. This has two important effects: a) The perceptual quality is high for a human listener. b) The LPC envelope is well-preserved, so the posterior LPCC parameters used by the speech recognizer will be less corrupted than their counterpart without perceptual weighting. Figures 3, 4 and 5 illustrate this behaviour. Figure 3 shows the waveform representation of an original vowel fragment, and the same frame with and without perceptual weighting; it is clearly apparent that the weighted watermark introduces fewer artefacts. Looking at the LPC envelope ( fig. 4) or the periodogram ( fig. 5) we can see that the weighting procedure yields a signal closer to the original one. Thus, the LPCC parameters obtained by a recursion from the LPC coefficients [Deller 1993], will be less corrupted. The good results obtained in our previous work [Faundez-Zanuy 2006] motivated our interest for evaluating the watermarking techniques in forensic applications, as discussed in the next section. Watermarking of speech signals for forensic applications Recordings made by Law Enforcement Agencies in the exercise of their legal competences in order to prevent crimes or during crime investigations are carried out nowadays using digital technology. This technology allows us to have digital audio files, and there is a wide and varied collection of equipment, interfaces, protocols, and formats at hand in the market. The available IT security systems based on that technology make the detection of any fraudulent alteration of the original content of a recording almost impossible, reaching information protection levels never seen before with analogue technology. Public document authenticity has always been a concern for Public Administration regarding the management of official documents. Credibility or authenticity of those documents has been protected using complex physical or chemical procedures. By applying watermarking to digital technology, i.e. inserting it into digital files, we link two kinds of information, so that a speech or image file contains additional data (watermark) such as the owner name, date, data status (public, restricted), or any other relevant information in compliance with the criteria established by the author who marks the audio files. Watermarking can guarantee that the information stored in a file faithfully matches the purpose intended by their authors: to provide data reliability by including a signal certifying its origin. For instance, data were recorded using Police equipment. However, in particular, watermarking for forensic audio purposes should fulfil the following features:  Inaudible in order to reveal its presence only to those individuals legally entitled to know it;  Inserted in very short speech segments which could be used in any kind of situations;  Possibility of using automatic speaker recognition systems without a relevant efficiency loss;  Enough flexible to insert information with informative or identity value: timestamping, anagrams, encrypted information, etc.  Finally, the process must be irreversible to prevent the marking process from being reversed. Due to the fact that information gathered by Law Enforcement Agencies is submitted to other legally entitled partners (Civil, Military and Judicial Authorities, etc…), when this information involves digital data it has to be protected not only by a digital signature to guarantee its integrity and origin, but also taking into account that a part of the information contained in those protected files could be used in later processes such as copies aimed at authorised listeners. It should be possible to authenticate the origin of any digital information from Law Enforcement Agencies, regardless whether it was directly provided by these Agencies or when it involves partial or full copies of it. This goal can be fulfilled by means of watermarking, which makes possible to detect any insertion or erase processes implemented in the original recording. Therefore, watermarking improves the credibility of any copy made from the original recorded information. For the abovementioned reasons, digital signature and watermarking are two IT security systems that are very well matched for audio police recordings. Each one has its own functionality complemented by the other. We consider both useful to be applied in any kind of digital audio Police applications. High Court Jurisprudence requires Police Forces to submit the original recordings as a requirement to determine the unquestionable credibility of their content. This is a consequence of the presumption of innocence principle which establishes that the defendant is presumed innocent until proven guilty by the prosecution. Therefore, we consider strongly advisable to take IT security measures:  To guarantee the integrity of original recordings;  To make possible to detect non authorised alterations; and  To guarantee the origin of the information when segmentation of the original recording is needed and those segments are disclosed to third parties. Proposed speech authentication system 3.1 Description Our proposed system (see Fig. 6) uses watermarking technology, such es briefly introduced in section 2.2., to embed a time stamp information into the voice signal. This should be done directly in the recording device. Our system proposal embeds a watermark, which contains the exact time of the current recording every second. The embedding and reading of the time stamp can only be performed if a key, which determines the modulation sequence, is available. The watermarked signal can then be stored or distributed. In the case of e.g. the use of a voice sample in court the authenticity of the recording can be determined. If the recording has been tampered with, e.g. a word has been removed, replaced or inserted, the integrity of watermark at this time instance will be lost. Because of the high time resolution of the embedded time stamp, not only the general authenticity of the recording can be investigated, but also at which time instant a possible tampering attack has been made, which is where the continuous time stamp is broken. Next section is devoted to experiments about the interaction between speech watermarking and speaker identification for forensic applications. database description and experimental results All the speech material used in the experiment belongs to the B. ) and Tippet plots using test files, speaker models, and reference populations. In this experiment 300 test files have been used, each one 10 seconds long, spontaneous speech, GSM channel, all of them from legal phone tapings carried out from 2000 to 2004 and involving voices recorded on tapes. 10 files per speaker were necessary, with a total of 30 male speakers, all of them speaking Spanish, resulting in the above mentioned 300 files. Each utterance is independent, i.e. it is absolutely different from the other ones. 2 additional minutes of speech from each one of the 30 speakers were recorded in conversations independent from the abovementioned ones. These files were used to calculate the unquestioned acoustic model for each speaker. With regard to the reference population, it comprised 35 male Spanish speaking speakers different from the 30 earlier mentioned. The samples were obtained from legal phone tapings as well, under the same conditions. Comparing the Tippet (see figure 7) plot in the experiment, 300 target scores were calculated along with 8.700 non-target scores. The results from the original files are depicted in blue and in red those obtained using the watermarked files. The performance differences are irrelevant. The appropriate combination between noise and speech has avoided a substantial difference in performance. CONCLUSIONS In this paper we have discussed the problem of authentication of forensic audio. We have summarized the existing approaches and we have proposed a new one based on perceptual speech watermarking, which is independent of the recording device (optical, magnetic, solid state). In addition, we have performed a set of experiments on a forensic database and we have found that speech watermarking does not interfere with the posterior biometric speaker identification/verification.	2022-03-07T10:40:49.911Z	2022-01-01T00:00:00.000	{ "year": 2022, "sha1": "ef02578ee6b30491210e99750b50c70bf84a3246", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "ef02578ee6b30491210e99750b50c70bf84a3246", "s2fieldsofstudy": [ "Computer Science", "Engineering" ], "extfieldsofstudy": [] }
246277590	pes2o/s2orc	v3-fos-license	The 5th Annual Heart in Diabetes Conference (part 2) At the 5 Annual Heart in Diabetes (HiD) Conference, held in New York from 10 to 12 September 2021, a variety of topics were addressed related to the cardiovascular (CV) risks and CV complications of diabetes. This is the second of a two-part summary of some of the presentations at the meeting, reviewing approaches to therapy. Michael Farkouh, Toronto, Ontario, Canada, discussed the interaction of optimal medical therapy and revascularization strategies in patients with diabetes and coronary artery disease (CAD). After percutaneous coronary intervention (PCI) for CAD, nontarget lesions are frequent sites of atherosclerotic cardiovascular disease (ASCVD) events, suggesting that stent placement alone may not be sufficient treatment for persons with CAD. Farkouh reviewed the Future Revascularization Evaluation in Patients with Diabetes Mellitus: Optimal Management of Multivessel Disease (FREEDOM) study, comparing stent placement with coronary artery bypass graft (CABG) surgery and showing over 5 years 18.7% of enrolled patients with multivessel disease had myocardial infarction, stroke, or mortality after CABG, while these outcomes occurred in 26.6% of those having PCI. He noted that this and other studies suggest the importance of optimized medical therapy of CAD and acknowledged that future studies will need to include sodium glucose cotransporter 2 inhibitors (SGLT2i) and glucagon-like peptide 1 receptor agonists (GLP-1RA), but pointed out that even in these clinical trials there is lack of uniformity in achieving diabetes, lipid, and blood pressure goals, so that a major clinical challenge is the delivery of appropriate treatment to the large number of persons with diabetes and CAD. Interestingly, his colleague Lucas Godoy, Toronto, reviewed a meta-analysis performed with Farkouh of patients with diabetes undergoing PCI or CABG, followed for 4 years, showing similar outcome with low-density lipoprotein (LDL) cholesterol <70 and 70 to <100 mg/dL, with both groups having 23% lower event rates than those with LDL cholesterol ≥100 mg/dL, although another study from the group showed that after PCI there is a gradient of adverse outcome from LDL cholesterol below 70 to LDL between 70 and <100 to LDL of 100 and over, supporting that the LDL cholesterol goal is below 70 mg/dL for diabetes with ASCVD. Peter Grant, Leeds, UK, discussed antiplatelet and anticoagulant treatment approaches for persons with diabetes. In a study of more than 15 000 persons with diabetes not having evidence of ASCVD randomized to aspirin 100 mg daily or placebo, serious vascular events occurred in 1.1% fewer persons over a mean 7.4-year follow-up, but major bleeding (primarily gastrointestinal) occurred in 0.9% more, leading Grant to suggest that the choice of whether or not aspirin should be given in such a primary prevention setting should be personalized. In acute coronary syndrome, aspirin combined with one of the P2Y12 platelet adenosine diphosphate receptor inhibitors clopidogrel, prasugrel, or ticagrelor is recommended, with evidence that prasugrel leads in general to optimal outcome. The combination can be continued for up to 3 years unless there is high bleeding risk, after which P2Y12 inhibitors alone may lead to better outcome. At age over 75 years, or with prior stroke or transient ischemic attack, prasugrel increases cerebrovascular event rates, and clopidogrel or ticagrelor may be preferable. With peripheral arterial disease, a different approach may be preferred with low-dose rivaroxaban and aspirin. Silvio Inzucchi, New Haven, CT, discussed stroke prevention in diabetes, pointing out the association of worse glycemic control with risk of fatal stroke in the United Kingdom Prospective Diabetes Trial, with metformin associated with lower risk of stroke than conventional treatment, and as well with lower risk of stroke than insulin or sulfonylurea treatment. Pioglitazone was associated with 50% reduction in stroke risk among persons with diabetes and prior stroke in the Prospective Pioglitazone Clinical Trial in Macrovascular Events (PROactive) and with 25% reduction in the combined endpoint of myocardial infarction and stroke among persons with insulin resistance (but not diabetes) and prior stroke in the Insulin Resistance Intervention After Stroke (IRIS) randomized clinical trial. Among newer glucoselowering agents, GLP-1RA are associated with 14% reduction in stroke rate in clinical trials of these agents. DOI: 10.1111/1753-0407.13251 At the 5 th Annual Heart in Diabetes (HiD) Conference, held in New York from 10 to 12 September 2021, a variety of topics were addressed related to the cardiovascular (CV) risks and CV complications of diabetes. This is the second of a two-part summary of some of the presentations at the meeting, reviewing approaches to therapy. Michael Farkouh, Toronto, Ontario, Canada, discussed the interaction of optimal medical therapy and revascularization strategies in patients with diabetes and coronary artery disease (CAD). After percutaneous coronary intervention (PCI) for CAD, nontarget lesions are frequent sites of atherosclerotic cardiovascular disease (ASCVD) events, 1 suggesting that stent placement alone may not be sufficient treatment for persons with CAD. Farkouh reviewed the Future Revascularization Evaluation in Patients with Diabetes Mellitus: Optimal Management of Multivessel Disease (FREEDOM) study, comparing stent placement with coronary artery bypass graft (CABG) surgery and showing over 5 years 18.7% of enrolled patients with multivessel disease had myocardial infarction, stroke, or mortality after CABG, while these outcomes occurred in 26.6% of those having PCI. 2 He noted that this and other studies suggest the importance of optimized medical therapy of CAD 3 and acknowledged that future studies will need to include sodium glucose cotransporter 2 inhibitors (SGLT2i) and glucagon-like peptide 1 receptor agonists (GLP-1RA), but pointed out that even in these clinical trials there is lack of uniformity in achieving diabetes, lipid, and blood pressure goals, so that a major clinical challenge is the delivery of appropriate treatment to the large number of persons with diabetes and CAD. Interestingly, his colleague Lucas Godoy, Toronto, reviewed a meta-analysis performed with Farkouh of patients with diabetes undergoing PCI or CABG, followed for 4 years, showing similar outcome with low-density lipoprotein (LDL) cholesterol <70 and 70 to <100 mg/dL, with both groups having 23% lower event rates than those with LDL cholesterol ≥100 mg/dL, 4 although another study from the group showed that after PCI there is a gradient of adverse outcome from LDL cholesterol below 70 to LDL between 70 and <100 to LDL of 100 and over, 5 supporting that the LDL cholesterol goal is below 70 mg/dL for diabetes with ASCVD. Peter Grant, Leeds, UK, discussed antiplatelet and anticoagulant treatment approaches for persons with diabetes. In a study of more than 15 000 persons with diabetes not having evidence of ASCVD randomized to aspirin 100 mg daily or placebo, serious vascular events occurred in 1.1% fewer persons over a mean 7.4-year follow-up, but major bleeding (primarily gastrointestinal) occurred in 0.9% more, 6 leading Grant to suggest that the choice of whether or not aspirin should be given in such a primary prevention setting should be personalized. In acute coronary syndrome, aspirin combined with one of the P2Y12 platelet adenosine diphosphate receptor inhibitors clopidogrel, prasugrel, or ticagrelor is recommended, with evidence that prasugrel leads in general to optimal outcome. 7 The combination can be continued for up to 3 years unless there is high bleeding risk, after which P2Y12 inhibitors alone may lead to better outcome. At age over 75 years, or with prior stroke or transient ischemic attack, prasugrel increases cerebrovascular event rates, and clopidogrel or ticagrelor may be preferable. With peripheral arterial disease, a different approach may be preferred with low-dose rivaroxaban and aspirin. 8 Silvio Inzucchi, New Haven, CT, discussed stroke prevention in diabetes, pointing out the association of worse glycemic control with risk of fatal stroke in the United Kingdom Prospective Diabetes Trial, 9 with metformin associated with lower risk of stroke than conventional treatment, and as well with lower risk of stroke than insulin or sulfonylurea treatment. 10 Pioglitazone was associated with 50% reduction in stroke risk among persons with diabetes and prior stroke in the Prospective Pioglitazone Clinical Trial in Macrovascular Events (PROactive) 11 and with 25% reduction in the combined endpoint of myocardial infarction and stroke among persons with insulin resistance (but not diabetes) and prior stroke in the Insulin Resistance Intervention After Stroke (IRIS) randomized clinical trial. 12 Among newer glucoselowering agents, GLP-1RA are associated with 14% reduction in stroke rate in clinical trials of these agents. 13 Inzucchi concluded that diabetes treatment guidelines might differ in persons with history of stroke rather than other forms of ASCVD, an important point in view of the relative infrequency of current use of pioglitazone despite its apparent benefit in this setting. Pam Taub, San Diego, CA, discussed the question of optimal diets for prevention of cardiovascular disease (CVD). Evidence-based recommendations include appropriate calorie restriction based on age, sex, and activity level, for dietary fiber, for the Mediterranean and DASH diet approaches, and, perhaps, for time-restricted eating approaches. 14,15 She suggested that fasting can be considered a metabolic switch from metabolism of glucose to that of fatty acids and ketones, 16 with evidence of ketogenesis being associated with improvement in cardiac energetics. 17 Taub raised the interesting concept that a similar mechanism may underly the benefit of SGLT2 inhibitors, 18 although with the potential that fasting may be problematic with the use of these agents. Taub turned to a different topic, that of the amine oxide trimethylamine N-oxide (TMAO), produced by gut bacteria from choline, lecithin, and L-carnitine-rich foods such as fish, meat, eggs, and dairy products, associated with CVD and diabetes, with evidence that TMAO levels decrease with fasting, 19 a potential mechanism benefit of intermittent fasting approaches. Donna Ryan, New Orleans, LA, further addressed issues related to obesity, reviewing expected benefits, and current approaches to pharmacotherapy, noting the important difference between semaglutide, causing mean weight loss of 15% to 17%, and older medicines for appetite regulation such as phentermine/topiramate, naltrexone/bupropion, and liraglutide, all causing weight loss of 6% to 11%. Ryan noted the infrequent prescribing of antiobesity medications, only given to approximately 1% of eligible patients, in part because of financial barriers such as high co-pays and the need for prior authorization for prescriptions. 20 The Semaglutide Effects on Heart Disease and Stroke in Patients with Overweight or Obesity (SELECT) study is an ongoing randomized, double-blind, parallel-group trial of semaglutide 2.4 mg subcutaneously once weekly added to standard of care, which may show the beneficial effect of weight loss in reducing major adverse cardiovascular events in approximately 17 500 patients with established CVD and overweight or obesity but without diabetes. 21,22 Ryan also discussed the concept that weight normalization is not necessarily the goal, reviewing studies showing that substantial decrease in visceral fat, in intrahepatic fat, and in markers of glycemia, insulin sensitivity and action, liver function, dyslipidemia, and inflammation can be seen with weight loss to the degree found with semaglutide. 23 Indeed, in the Diabetes Prevention Program, 10% weight loss optimally reduced diabetes likelihood, 24 and in the Diabetes Remission Clinical Trial (DiRECT) of 306 persons with type 2 diabetes randomized to intensive diet intervention, those with weight gain vs losing 0 to 5 kg, 5 to 10 kg, 10 to 15 kg, and >15 kg had no, 7%, 34%, 57%, and 86% rates of diabetes remission without need for medications. 25 Furthermore, secondary analysis of the Look AHEAD randomized clinical trial of lifestyle intervention for persons with diabetes showed that those with ≥10% weight loss had 20% reduction in CVD outcomes. 26 Stephen Wiviott, Boston, MA, discussed the effect of SGLT2i on atrial fibrillation, reviewing the uncertainty as to the pathogenesis of this arrhythmia in diabetes, potentially involving alteration in the balance between parasympathetic and sympathetic tone due to autonomic neuropathy and insulin resistance. Certainly, the coexistence of diabetes with atrial fibrillation increases stroke risk. In analysis of a trial of more than 17 000 persons with diabetes, administration of dapagliflozin vs placebo was associated with 19% reduction in first event and a 33% reduction on subsequent events of atrial fibrillation. 27 Similar reduction in atrial fibrillation has been reported with canagliflozin 28 and in a meta-analysis of more than 50 000 persons in clinical trials of SGLT2i. 29 Wiviott noted a recent presentation suggesting reduction in ventricular arrhythmias with dapagliflozin and suggested that a variety of mechanisms may contribute to such antiarrhythmic effects of SGLT2i, including decongestive effect, blood pressure lowering, weight loss, A1c benefit, direct cardiac effects on remodeling, on sympathetic tone, on oxidative stress/inflammation, and on epicardial fat. Rajiv Agarwal, Indianapolis, IN, reviewed an analysis of the Finerenone in Reducing Kidney Failure and Disease Progression in Diabetic Kidney Disease (FIDELIO-DKD) study on the effect of this agent on CV outcomes in this study of 5674 persons with type 2 diabetes and diabetic kidney disease based either on having microalbuminuria with estimated glomerular filtration rate (eGFR) 25 to 59 or on having eGFR up to 74 with macroalbuminuria, 30 a group clinically recognized to be at extremely high risk of CVD complications. The composite CV outcome of CV death, myocardial infarction, stroke, or hospitalization for heart failure occurred in 13% of those receiving finerenone vs 14.8% of those receiving placebo, a significant 14% reduction, suggesting an important potential role of the agent in reducing CV events. The CV outcome benefit of finerenone appears particularly related to reduction in heart failure, 31 suggesting this, along with the SGLT2i, to be important additions to our therapeutic armamentarium. Sam Dagago-Jack, Memphis, TN, discussed an aspect of what he termed the "African American Paradox": that Blacks in the United States have higher blood pressure and rates of diabetes, but lower levels of LDL and triglyceride and higher levels of high-density lipoprotein cholesterol, yet their CVD mortality is higher than that in Whites. Part of the issue, he suggested, may be lesser degree of control of risk factors, 32 but he noted that there also appears to be lower provision of acute and effective optimized CV treatment. Dagago-Jack concluded by noting that the profile of SGLT2i "suggests that these agents may help reduce disparities in CKD, HF and CVD mortality," but he pointed out the underrepresentation of Blacks and Hispanics in CV outcome trials, with such broader enrollment needed to realize the promise of these and other novel agents. Zachary Bloomgarden Department of Medicine, Division of Endocrinology, Diabetes, and Bone Disease, Icahn School of Medicine at Mount Sinai, New York, NY, USA	2022-01-26T06:18:36.379Z	2022-01-24T00:00:00.000	{ "year": 2022, "sha1": "3191952b725d2fb36f1c9c892d4f2a05c09d48d1", "oa_license": null, "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/1753-0407.13251", "oa_status": "BRONZE", "pdf_src": "Wiley", "pdf_hash": "0d33e75d644cd6288e78687398bc6212167b1b0f", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
251771847	pes2o/s2orc	v3-fos-license	Analysis of KIR gene variants in The Cancer Genome Atlas and UK Biobank using KIRCLE Background Natural killer (NK) cells represent a critical component of the innate immune system’s response against cancer and viral infections, among other diseases. To distinguish healthy host cells from infected or tumor cells, killer immunoglobulin receptors (KIR) on NK cells bind and recognize Human Leukocyte Antigen (HLA) complexes on their target cells. However, NK cells exhibit great diversity in their mechanism of activation, and the outcomes of their activation are not yet understood fully. Just like the HLAs they bind, KIR receptors exhibit high allelic diversity in the human population. Here we provide a method to identify KIR allele variants from whole exome sequencing data and uncover novel associations between these variants and various molecular and clinical correlates. Results In order to better understand KIRs, we have developed KIRCLE, a novel method for genotyping individual KIR genes from whole exome sequencing data, and used it to analyze approximately sixty-thousand patient samples in The Cancer Genome Atlas (TCGA) and UK Biobank. We were able to assess population frequencies for different KIR alleles and demonstrate that, similar to HLA alleles, individuals’ KIR alleles correlate strongly with their ethnicities. In addition, we observed associations between different KIR alleles and HLA alleles, including HLA-B53 with KIR3DL2013 (Fisher’s exact FDR = 7.64e−51). Finally, we showcased statistically significant associations between KIR alleles and various clinical correlates, including peptic ulcer disease (Fisher’s exact FDR = 0.0429) and age of onset of atopy (Mann-Whitney U FDR = 0.0751). Conclusions We show that KIRCLE is able to infer KIR variants accurately and consistently, and we demonstrate its utility using data from approximately sixty-thousand individuals from TCGA and UK Biobank to discover novel molecular and clinical correlations with KIR germline variants. Peptic ulcer disease and atopy are just two diseases in which NK cells may play a role beyond their “classical” realm of anti-tumor and anti-viral responses. This tool may be used both as a benchmark for future KIR-variant-inference algorithms, and to better understand the immunogenomics of and disease processes involving KIRs. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-022-01392-2. Background Natural killer (NK) cells are an important component of the innate immune system that classically play an important role in the body's anti-tumor and anti-viral responses. In addition to their functions in these processes, recent research has further implicated their involvement in a much wider range of pathological processes that include cardiac, metabolic, oral, and gastrointestinal diseases [1][2][3][4]. While they represent only a small minority of circulating lymphocytes (10-15%), NK cells nonetheless are considered to be the immune cell subtype most effective at monitoring and clearing diseased cells from the body [5]. As one mechanism to distinguish healthy host cells from infected or tumor cells, NK cells employ killer immunoglobulin receptor (KIR) proteins on their membrane surfaces to bind to and recognize Human Leukocyte Antigen Class I (HLA-I) complexes on the surface of their target cells. Fifteen KIR genes and 2 KIR pseudogenes have been discovered [6]. These 15 genes may broadly be categorized into either activating KIRs, which promote NK cell activation and induce killing of the target cell on receptor stimulation, or inhibitory KIRs, which prevent NK cell activation and spare the target cell upon ligand binding. Inhibitory KIRs generally possess long cytoplasmic tails and are denoted with an L (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A/B, KIR3DL1, KIR3DL2, and KIR3DL3), whereas activating KIRs generally possess short cytoplasmic tails and are denoted with an S (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1); however, KIR2DL4 uniquely among the 15 KIR genes possesses both activating and inhibitory functions [7]. Modulation of NK cell activity, and thus susceptibility or resistance to various pathologies, likely depends strongly on the binding properties and interactions between KIR and HLA-I molecules. A high level of diversity in NK cell activity and its outcomes may be achieved largely through four different mechanisms: KIR recognition of highly distinct subsets of HLA-I allotypes, combination of KIRs into distinct haplotypes in different individuals, stochasticity of KIR expression on the surface of individual NK cells, and allelic polymorphism of individual KIR genes [8]. In this manuscript, we primarily explore the last of these mechanisms and its downstream effects on disease susceptibility by performing KIR allele inference using next-generation sequencing (NGS) data. Previous attempts to perform KIR genotyping at the individual gene level have either (1) relied on specially prepared primers and amplicon design, (2) required manual review as part of the algorithm, (3) utilized an experimental platform completely different from NGS, or (4) have merely assessed KIR gene presence or deletion rather than detected single-nucleotide-variants [9][10][11][12]. Given the rise and modern prevalence of NGS, especially with the recent releases of Whole Exome Sequencing (WES) data for large datasets including UK Biobank and The Cancer Genome Atlas (TCGA), there is a strong need for a fully automated pipeline that can detect single-nucleotide variants of these KIR genes using aligned WES data. In this work, we have developed and characterized the performance of a fully automated algorithm for accurate inference of KIR gene alleles from WES data: "KIR CaLling by Exomes" (KIRCLE). To demonstrate the utility of such an automated KIR genotyper, after running KIRCLE on 10,332 TCGA and 49,953 UK biobank exome samples, we discovered several novel correlations between KIR allele calls and other molecular and clinical features in these two datasets. Our work represents the first large-scale genetic analysis to elucidate pathologic and immunologic associations with human natural killer cells and provides an unprecedented resource for future investigations into the functionality of different KIR alleles. KIRCLE workflow description KIRCLE is an allele inference algorithm that uses aligned WES data in the form of a BAM or CRAM file to generate probability estimates for each KIR allele, as well as genotype predictions for each KIR gene. KIRCLE consists of 4 major steps: pre-processing, local alignment with BLAST, bootstrapped expectation-maximization, and thresholding ( Fig. 1a). Description of the KIRCLE methodology. a Flowchart describing the 4 steps of the KIRCLE algorithm as it processes a single KIR gene (KIR2DL1 as an example here). Inputs are green, computations are blue, and outputs are gold. KIRCLE hyperparameters are listed in parentheses where they are implemented. b Depiction of step 4 of KIRCLE (thresholding). Allele probabilities generated by expectation-maximization may lead to a homozygous solution, a heterozygous solution, or no solution at all, depending on the user-selected value of the threshold hyperparameter t. In the depicted example, running KIRCLE with t = 0.5 (blue) would generate a homozygous solution (2 copies of KIR2DL2003), whereas using t = 0.2 (green) would generate a heterozygous solution (1 copy each of KIR2DL2002 and KIR2DL2003). Using t = 0.8 (red) or t = 0.05 (purple) would have yielded no solution. c Depiction of one step of expectation-maximization. The initial allele-read matrix M t0 is collapsed into an expectation vector E t0 that is used to compute the next iteration of the matrix M t1 . This process is repeated until the convergence criterion is satisfied, at which point the final expectation vector represents an estimate of KIR allele probabilities Hyperparameter determination and validation for KIRCLE KIRCLE requires the use of 4 hyperparameters: α (the convergence threshold for expectation-maximization), n (the number of bootstraps to perform), p (the proportion of reads to use in each bootstrap), and t (the threshold used to convert KIR allele probabilities to binary KIR genotype calls). Of these, choices regarding p and t represent the greatest and most direct potential sources of variability in KIRCLE's accuracy. Using one randomly selected sample from UK Biobank, we were able to characterize KIRCLE's performance, as measured by the Shannon entropy of the inferred genotypes, across different values of p (from 0.2 to 0.8) and t (from 0.05 to 0.40). At each set of hyperparameters tested, we performed 500 iterations of KIRCLE on one arbitrarily selected sample in UK Biobank, collected the 500 genotype outputs, and empirically computed the log2 Shannon entropy of the genotype solutions for each KIR gene. An ideal genotype caller would be consistent and call the same solution for the same input, resulting in a "genotype-entropy" of 0. For many KIR genes, such as KIR2DL1 and KIR2DL4, contour maps of the resulting entropies revealed that KIRCLE was largely self-consistent, with little variability of output (genotype-entropy of 0) across a wide spectrum of hyperparameter values (Fig. 2a, b). This pattern was recapitulated in the majority of KIR genes, suggesting respectable consistency of KIRCLE output across multiple KIR genes ( Supplementary Fig. S1a-j). For all subsequent analyses in this manuscript, hyperparameter values of α=1e−5, n=100, p=0.5, and t=0.25 were used. Next, to establish the accuracy of our algorithm, we assessed the concordance of KIRCLE-generated genotypes between TCGA biological replicates. Of 10,332 exomes in TCGA, 1,062 were present twice as biological replicates and thus used in this analysis. We determined that 85.8% of genotype solutions called by KIRCLE across all KIR genes were concordant between replicates ( Supplementary Fig. S2a). We defined solutions to be concordant if the genotype inferred by KIRCLE in one sample was identical to that inferred in its replicate. Genes with the highest concordance between replicates were KIR2DS2 (98.3%), KIR3DL1 (92.1%), and KIR3DS1 (92.1%), whereas genes with the lowest concordance between replicates were KIR2DL2 (77.0%), KIR2DS5 (78.9%), and KIR3DL3 (81.0%) (Fig. 2c). Given the observed difference between KIRs with the highest concordance (e.g., KIR2DS2) and those with the lowest concordance (e.g., KIR2DL2), we sought to explain these differences in accuracy by analyzing the degree of sequence similarity between different alleles of each KIR. We assessed sequence similarity by performing a multiple sequence alignment (MSA) on all alleles of a KIR using Clustal Omega [13] and then measuring the mean phylogram distance between all coding variants (Supplementary Fig. S2b-c). We noted a strong positive correlation (Spearman's ρ=0.631) between the mean distance between alleles of a given KIR and the observed concordance between that KIR's genotype calls among TCGA biological replicates, indicating that the higher levels of sequence similarity among alleles of KIRs such as KIR2DL2, KIR2DS5, and KIR3DL3 are likely to account for their lower rates of observed concordance. Finally, we investigated whether KIRCLE is robust against differences in depth of sequencing. To do so, we compared the ambiguity of KIRCLE's output, quantified as the Shannon entropy of generated KIR allele probabilities, across TCGA samples with different depths of sequencing. Low ambiguity in KIR allele calling results in KIR allele probabilities of either 1 for a single allele and 0 for all other alleles (reflecting a homozygous genotype) or 0.5 for two different alleles and 0 for all other alleles (reflecting a heterozygous genotype), leading to entropies of either 0 or 1 respectively. Conversely, high ambiguity in KIR allele calling will lead to a more uniform distribution of KIR allele probabilities, leading to entropies higher than 1. For each TCGA sample, we measured both the average coverage and the KIR-allele-probability entropies for each KIR gene. Binning samples by their average coverages, we observed that allele probability entropies-and thus the ambiguity of KIR allele probabilities-are notably increased only at very low coverages (<20 average depth of coverage at the KIR gene locus). Furthermore, as negative controls, 20 "pseudo-BAMs" were generated by randomly sampling reads mapping to KIR gene loci from 50 randomly selected BAMs in TCGA. Pseudo-BAMs were generated with an average read depth commensurate with their constituent BAMs. After applying KIRCLE to these pseudo-BAMs, their resulting allele probability entropies were much higher (median=1.70; IQR 0.958-2.61) than a significant majority of actually observed entropies for all TCGA samples, regardless of the depth of coverage (Fig. 2d). Moreover, despite differences in average depth of coverage at different KIR gene loci, average KIR allele entropies between different KIR genes largely remained constant (Supplementary Fig. S2b). Overall, KIRCLE demonstrated a high level of consistency while being able to call a diverse set of KIR genotype solutions and is robust to the effects of low depth of coverage. KIR allele comparisons between TCGA and UK Biobank After benchmarking KIRCLE using internal quality control metrics, we assessed KIRCLE's performance by comparing its allele predictions in TCGA to its allele predictions in UK Biobank. We first compared the frequencies of different KIR alleles in TCGA with their frequencies in UK Biobank among Caucasian individuals in both datasets, in order to mitigate race as a confounding variable affecting observed allele frequency. For each KIR gene, we ranked its alleles by frequency in both TCGA and UK Biobank and then computed the Spearman correlation coefficient between the allele frequencies in the two datasets (Fig. 3a). We noted that all KIR genes displayed positive correlation coefficients and that the vast majority of KIR genes demonstrated highly similar distributions of allele frequencies between TCGA and UK Biobank (Spearman's ρ=0.802). Direct comparison of all KIR alleles ranked by frequency also demonstrated high consistency between the two cohorts' Caucasian subpopulations (Fig. 3b). Both UK Biobank and TCGA are largely composed of Caucasians (81.4% and 93.3% of the individuals analyzed in TCGA and UK Biobank respectively). Additionally, we were able to further validate observed KIR allele frequencies for certain KIR genes using allele frequency data from the US National Marrow Donor Program (NMDP), as reported by the Allele Frequency Net Database [14]. We used the NMDP dataset because the subjects in this cohort are predominantly Caucasians, similar to the TCGA patients. For KIR2DL4, the four most frequent KIR2DL4 alleles reported by the NMDP were KIR2DL4001, KIR2DL4008, KIR2DL4005, and KIR2DL4011 (34.6%, 30.2%, 19.9%, and 11.6% respectively). We were able to recapitulate these four alleles as the most frequent KIR2DL4 alleles in both TCGA and UK Biobank's Caucasian subpopulations, albeit in a different order for each dataset (Fig. 3c). In TCGA, KIR2DL4005 was the most frequent allele, followed by KIR2DL4001, KIR2DL4008, and KIR2DL4011 (44.4%, 23.5%, 14.4%, and 7.55%). In UK Biobank, this order was reversed with KIR2DL4011 being the most frequent allele, followed by KIR2DL4008, KIR2DL4001, and finally KIR2DL4005 (32.4%, 21.0%, 18.0%, and 13.9%). Further validation of allele frequencies against the NMDP was also performed for the alleles of KIR3DL2. The most frequent KIR3DL2 allele in a population of 75 Caucasians was KIR3DL2002 (26.1%), followed by KIR3DL2001 and KIR3DL2007 (21.0% and 18.8%) [15]. While KIR3DL2002 was found at similarly high frequencies in both TCGA (9.37%) and UK Biobank (9.27%) as the 4th and 3rd most frequent KIR3DL2 alleles respectively, KIR3DL2001 and KIR3DL2007 were much lower ranked at 9th and 13th in TCGA and 8th and 1st in UK Biobank respectively. However, these are still fairly well-represented alleles at 4.95% and 2.78% frequency in TCGA and 5.00% and 13.3% frequency in UK biobank respectively. Furthermore, considered overall, KIR3DL2 allele frequency ranks in TCGA and UK Biobank still demonstrate positive correlations with the allele frequency ranks observed in the NMDP ( Supplementary Fig. S2c). Despite slight numerical differences, confirmation of the status of the most frequent alleles in these two KIR genes increases our confidence in KIRCLE's ability to infer KIR alleles from WES data accurately. In addition to validating population frequencies of KIR alleles, we also examined patterns of KIR allele coexpression and dependence. As KIRCLE assesses for the presence of 535 KIR alleles over 15 KIR genes, the KIR genotype of each sample in TCGA and UK Biobank may be represented as a point in 535-dimensional "KIRspace. " We first used t-distributed stochastic neighbor embedding (t-SNE) to perform dimensionality reduction and thus visualize the distribution of individuals in TCGA in 2 dimensions [16]. When we colored this t-SNE map using individuals' SNP-inferred ethnicities [17], we observed that different ethnicities cluster together and are non-uniformly distributed (Fig. 3d). In particular, African Americans and-to a lesser extent-Asian Americans in TCGA formed clusters that were often very distinct from the Caucasian majority. Similar analyses performed in UK Biobank recapitulated this nonrandom distribution of KIR genotypes and confirmed the non-uniform distribution and clustering of those who self-identified their ethnicity as "Black" or "Asian" (Fig. 3e). Of particular note, the "Asian" population in TCGA comprises those of East Asian descent, whereas the "Asian" population in UK Biobank largely comprises those of South Asian descent (with major subcategories of Indians, Pakistanis, and Bangladeshis). However, both groups of Asians clustered distinctly and separately from the Caucasian majority to some extent in both datasets. KIR allele associations with HLA alleles As it is known that HLA and KIR bind to each other in an allele-specific way, we posited that strong correlations may also exist between KIR alleles and HLA alleles on the population level, due to a known co-evolution event in humans [18]. Using HLA types imputed by the [19], we observed 326 significantly associated pairs of KIR alleles with HLA alleles in the UK Biobank data (Fig. 4a, b, Supplementary Fig. S3a). Many of these associations belonged to a set of particularly common HLA alleles (e.g., HLA-B53) or KIR alleles (e.g., KIR3DL3005). Furthermore, we also note that the majority (73.0%) of significant associations are positive. We speculate that these associations reflect changes in direct physical interactions between HLA and KIR alleles, which result in co-selection due to an advantageous increase in fitness for individuals with these combinations of KIR and HLA alleles. Particularly visually striking examples of positive and negative associations between KIRs and HLAs include KIR3DL3005 with HLA-A74 (Fisher's exact FDR=7.43e−43; odds ratio=55.1) and KIR2DL3002 with HLA-A36 (Fisher's exact FDR=1.71e−12; odds ratio=0.0727), respectively (Fig. 4c). Additionally, when examining the t-SNE coordinates of individuals with HLA alleles such as HLA-B42, we observed a non-uniform distribution and clustering of these samples that closely mirrors the distribution of samples when labeled by ethnicity (Fig. 4d). While these findings may support the biological link between these two classes of molecules and shed additional light onto which particular HLA alleles may have evolved in parallel with particular KIR alleles, they also raise the possibility that our observed associations are driven by population stratification according to ethnicity. In order to disentangle the effects of this stratification on associations between HLA and KIR alleles, we reattempted the analysis using only Caucasian individuals in UK Biobank, while testing only KIR alleles with >1% allele frequency in UK Biobank (Fig. 4e). While this analysis unveiled a much smaller subset of HLA-KIR associations, we noted 3 significant associations: HLA-C17 with KIR2DS4016 and HLA-B41 with KIR2DL4011 and KIR2DS4016. Notably, both KIR2DS4 and KIR2DL4 have NK-cell-activating activity, and all three are affiliated with a negative odds ratio. These results indicate that HLA-C17 and B41 could be true activation ligands for KIR2DS4 and KIR2DL4, and their interactions may induce NK responses that impose negative selection pressure on individuals bearing both alleles. Although TCGA is a much smaller dataset than UK Biobank, we were able to use TCGA to discover a smaller set of correlations between HLA alleles and KIR alleles after filtering out KIR alleles with <1% allele frequency in TCGA to improve our Bonferroni correction factor ( Supplementary Fig. S3b). HLA allele calls for samples in TCGA were made using POLYSOLVER [20]. In particular, KIR2DL2003, KIR3DL2013, and KIR3DL3008 were strongly positively associated with HLA-B46, HLA-B53, and HLA-C15 respectively at the FDR < 0.25 level. The HLA-B53 association with KIR3DL2013, notably, was the most significant HLA-KIR association discovered in UK Biobank. However, when we re-attempted the analysis using only Caucasian individuals in TCGA to eliminate population stratification by ethnicity as a potential confounding factor, all significant associations between KIR and HLA alleles disappeared after Bonferroni correction. In summary, after correction of population stratifications, we found few significant associations between activating the KIR gene and HLA alleles. The absence of significant associations between inhibitory KIR genes and HLA alleles might suggest weaker selective pressure for KIR alleles, possibly due to the multiple redundant mechanisms inhibiting NK cell activation [21]. KIR allele associations with clinical correlates In addition to correlations with HLA alleles, we searched for KIR allele correlations with clinical features. We first examined KIR allele correlations with individuals' medical diagnoses documented in UK Biobank, as encoded by the 10th revision of the International Statistical Classification of Diseases (ICD10). To minimize the number of under-powered tests we performed, we attempted correlations only with KIR alleles represented at over 1% frequency in UK Biobank. Additionally, we excluded all diseases primarily associated with external causes, including accidents, injuries, and nutritional deficiencies, as well as obstetric and psychiatric diseases among others. Of note, this list of exclusions includes infectious diseases, which despite having a strong biological basis for association with KIR alleles, require exposure to a pathogen, which is largely driven by individuals' environmental circumstances. Strikingly, the only associations that remained significant at the FDR < 0.25 level were those associated with sickle-cell anemia (ICD10 D57) or with uterine leiomyomas (ICD10 D25), both diseases that disproportionately affect black people [22]. However, positing a direct biological mechanism behind these associations likely would represent a third-cause fallacy, as blacks are statistically more likely to possess both KIR alleles enriched in black populations as well as either the sickle-cell trait or uterine leiomyomas. Thus, we next narrowed our analysis to investigate only those individuals who self-identified as Caucasian. While the vast majority of correlations failed false-discoveryrate correction, we discovered a significant correlation between the KIR3DL3080 allele and ICD10 K25-peptic ulcer disease (PUD) (Fisher's exact test, FDR= 0.0429; Fig. 5a). Whereas those without KIR3DL3080 had merely a 1.04% chance of being diagnosed with PUD, patients with KIR3DL3080 had a 2.90% chance of being diagnosed with PUD, representing a 2.8-fold increase in (Fig. 5b). No significant association was found between KIR3DL3080 and usage of ibuprofen, which would predispose individuals toward developing PUD (data not shown). Thus, if KIR3DL3080 predisposes an individual toward PUD, it likely does so through an alternative mechanism. Significant correlations with ICD10 diagnosis codes in Black and Asian populations were not observed, likely owing to the lower statistical power these smaller populations had. Additionally, we explored correlations between KIR alleles with population frequency >1% and other clinical correlates besides ICD10 codes. When examining correlations with age of onset of several chronic diseases and conditions, we discovered that KIR3DL2107 was highly correlated with early age of onset of hay fever, rhinitis, or eczema in Caucasian individuals. Whereas individuals without KIR3DL2107 had an average age of onset of 24.7 years (IQR 12-35 years), those with at least one copy of KIR3DL2107 had an average age of onset of 22.4 years (IQR 11-30 years; two-sided Mann-Whitney FDR=0.0751; Fig. 5c). Moreover, an alternative allele of KIR3DL2, KIR3DL2062, was weakly associated with an increase in age of onset of hay fever, rhinitis, or eczema from 24.5 years (IQR 12-35 years) to 27.0 years (IQR 14-40 years; Mann-Whitney FDR=0.244; Fig. 5d). Later onset of these conditions was particularly pronounced in individuals with two copies of KIR3DL2062, with an average age of onset of 27.6 years (IQR 14-40 years). Together, these results suggest that polymorphisms in KIR3DL2 may play a key role in determining the age of onset of hay fever, rhinitis, and/or eczema. Moreover, hay fever and eczema, in conjunction with allergic asthma, more broadly represent manifestations of atopy, the genetic predilection to trigger IgE-mediated (type I) hypersensitivity reactions following allergen exposure with increased T H 2-driven responses [23]. Thus, we next attempted to generalize this association to encompass atopy more broadly by examining KIR3DL2107 and KIR3DL2062's associations with age of onset of either asthma or hay fever, rhinitis, or eczema, using the age of onset of whichever condition occurred earliest in life for each individual. We observed the same association: individuals with at least one copy of KIR3DL2107 had an average age of onset of 15.9 years (IQR 6-20.75 years), whereas those without any copies of KIR3DL2107 had an average age of onset of 19.0 years (IQR 8-28 years; Mann-Whitney p=0.012; Fig. 5e). Simultaneously, individuals with at least one copy of KIR3DL2062 (22.5 years; IQR 10.25-31.5 years), and particularly those with two copies of KIR3DL2062 (23.1 years; IQR 10.75-32.75 years), had later onsets of atopic reactions than those without KIR3DL2062 (18.7 years; IQR 7-27 years; Mann-Whitney p=0.019; Fig. 5f ). Together, these findings suggest a potential biological mechanism either delaying or hastening onset of atopic reactions like hay fever, eczema, or asthma that involves KIR3DL2, and the KIR3DL2107 and KIR3DL2062 alleles in particular. In addition to atopic reactions, we also observed significant associations of KIR alleles with other clinical correlates, including dental and oral health, quantitative blood analysis, and waist circumference, suggesting potentially broad impact of natural killer functions in affecting diverse human traits (Supplementary Fig. 4). Finally, to further explore the effects of KIR3DL2 polymorphism on age of atopy onset, we posited that each of the two aforementioned KIR3DL2 alleles follows either a dominant, semi-dominant, or recessive model of expression and then sought to determine which of these three models best explains the effect of KIR3DL2 genotype on age of atopy onset. In the recessive model, only a genotype homozygous for the KIR3DL2 allele in question contributes to a change in age of atopy onset from baseline. In contrast, in the dominant model, genotypes either homozygous or heterozygous for the KIR3DL2 allele in question contribute to changes in baseline age of atopy onset. Finally, in the semi-dominant model, homozygotes for the KIR3DL2 allele in question are twice as potent as corresponding heterozygotes in changing age of atopy onset from baseline. When assessed against each other using the UK Biobank data, the dominant model outperformed semi-dominant and recessive models of expression for KIR3DL2107, as measured by the Bayesian information criterion (−10.8145 versus −10.8151 and −10.8172, respectively). Meanwhile, expression patterns of KIR3DL2062 instead favored the recessive model over the semi-dominant and dominant models of expression for KIR3DL2062, as measured by the Bayesian information criterion (−10.8138 versus −10.8141 and −10.8146, respectively). In summary, our analysis indicated that KIR3DL2107 may "override" other alleles and thus present with a dominant phenotype, whereas KIR3DL2062 may be weaker than other KIR3DL2 alleles and thus present with a recessive phenotype. Discussion The fifteen KIR genes represent a polymorphic set of immune modulators with an array of potential effects on immune and clinical phenotypes. In this manuscript, we have developed, characterized, and implemented our algorithm KIRCLE, uncovering multiple correlations between KIR alleles and other features in TCGA and UK Biobank. KIR alleles associated with HLA alleles Class I HLAs represent well-known binding partners of KIRs. Thus, any change in either the KIR binding site or the HLA binding site that alters their affinities to each other may be expected to modulate NK cell activation or inhibition. However, as both HLA and KIR loci are highly polymorphic, it has historically been challenging to determine their matches through low-throughput experimental approaches or through small-scale computational analyses. By using KIRCLE and a large cohort of UK Biobank data, we were able to observe several statistically significant associations between KIR and HLA alleles. These observed KIR-HLA associations may be indicative of selective pressures for these receptors to co-evolve to maintain appropriate levels of NK cell activity. Specifically, we observed that Caucasian individuals in UK Biobank with KIR2DS4016 and KIR2DL4011, two KIR alleles with activating activity, have a lower frequency of HLA alleles HLA-C17 and HLA-B41 than individuals without these alleles. We may posit that this "anticorrelation" possibly represents evidence of a potential intolerance of lethal NK cell hyperactivity, leading to the observed underrepresentation of individuals with these particular KIR-HLA allele combinations. However, such an explanation is purely speculative, with further mechanistic studies required to validate the observed frequencies and either to confirm or to refute this hypothesis. Several factors affect the results of our analyses. In addition to observing differences in KIR allele frequencies among those with different HLA alleles, we also observed differences in KIR allele frequencies among different ethnic populations, which are already known to possess different HLA allele frequencies [24]. While this combination of observations may reflect common selective pressures historically experienced by these ethnic populations which then may have forced KIR and HLA alleles to co-evolve, it likely also points to ethnicity as a confounding factor in this purely correlative study. We attempted to account for this potential confounder by repeating our analysis on only Caucasian individuals within UK Biobank. Such an analysis yielded a much smaller set of statistically significant KIR-HLA associations. Finally, it is possible that the KIR allele cohorts as inferred by KIRCLE are not well-defined enough to fully elucidate the underlying KIR-HLA interactions driving any observed associations. Future work would be expected to resolve these cohorts more accurately and thus better detect KIR-HLA interactions as well as their effects on immune and cancer-related outcomes. KIR allele associations with peptic ulcer disease and atopic reactions Previous genome-wide association studies of PUD have largely been performed in East Asian populations and did not uncover any associations between KIR polymorphism and either PUD or H. pylori infection [25,26]. However, NK cells are known to be present in the gastric and duodenal mucosa and have been shown to be directly activated by H. pylori bacteria to produce IFN-γ and trigger an immune response [27]. Our result builds upon these existing known interactions and hypothesizes that KIR3DL3080 may increase susceptibility to PUD through modulating NK cells' natural response to H. pylori. Furthermore, we uncovered evidence for a potential association between age of presentation of atopy and two different variants of KIR3DL2: KIR3DL2107 and KIR3DL2062. At least one copy of one of these two variants is present in 7.89% of the Caucasian population in UK Biobank. Indeed evidence exists for NK cells' involvement in atopic and autoimmune diseases of the skin (i.e. eczema), even if the details of this involvement remain unclear [28], and increasing support has been seen for their role in allergen-specific immune suppression, Th1 cell generation, and Ig production [29]. Our finding that KIR3DL2107 is associated with earlier presentation of atopy and that presence of KIR3DL2062 associates with delayed presentation potentially further points to a role specifically for KIR3DL2 in regulating NK cell activity as it contributes to these diseases. One possible explanation for the opposite directions of impact on age of onset is that KIR3DL2107 is stronger than other alleles and thus presents with a dominant phenotype, whereas KIR3DL2062 is weaker than others and thus results in a recessive phenotype. As preliminary evidence supporting this explanation, we demonstrated that a dominant model of expression best fits KIR3DL2107, while a recessive model of expression best fits KIR3DL2062. However, it is worth emphasizing that even though our observed clinical correlations remained significant after correction for multiple hypothesis testing, it remains unclear to what extent the identified KIR alleles are the direct cause of these phenotypic changes. Moreover, NK cells in vivo utilize a combination of multiple KIRs and other receptors to interact with their target cells, whereas our current analyses examine bulk correlations with KIR genotypes and lack the single-cell resolution required to investigate the combinatorial complexity of actual KIR expression on the surface of NK cells in vivo. Thus, a more nuanced and more adequately powered study of the effects of multiple KIRs in combination at single-cell resolution may better resolve the biological interactions of KIRs that ultimately functionally cause these downstream clinical outcomes. Limitations and future directions While we were able to use biological replicates in TCGA to benchmark the accuracy of KIRCLE and compare our population-level estimates of KIR allele frequencies to prior estimates of KIR allele frequencies as reported by the US NMDP to validate our KIR genotype predictions, we were unable to carry out any experimental validation to benchmark its accuracy more directly. While our current analysis includes measures of the Shannon entropies of KIRCLE-predicted allele probabilities and concordance of predicted genotypes between biological replicates within TCGA as measures of the precision of our algorithm, such measures nevertheless remain susceptible to systematic biases in either the methodology or the nature of the datasets used (e.g., WES). In particular, our current analysis does not compare our KIR genotype predictions to gold-standard KIR genotypes as assessed by Sanger sequencing against each KIR gene's locus on chromosome 19 in live biological samples. Such comparison to gold-standard genotype predictions will be required to evaluate the overall accuracy of any KIR genotype inference algorithm more definitively. Additionally, after generating KIR genotype predictions, our downstream correlations all represented univariate analyses, due to the relatively low abundance of individual KIR alleles. While such a simplistic analysis is suitable for a first-pass search for potential direct associations, more nuanced future analyses of clinical associations with KIR alleles will need to account for confounding factors beyond human genetics to determine individuals' susceptibility to diseases, including individuals' living or occupational environments, full medical histories, lifestyles, and much more. Such analyses may help uncover any functional roles KIRs play in these processes and will likely become available with sufficient statistical power when UK Biobank fulfills its mission to sequence all 500,000 individuals. Meanwhile, the studied sample sizes of ~50,000 individuals in UK Biobank and ~10,000 individuals in TCGA are relatively underpowered to discover associations between all combinations of 535 KIR alleles with thousands of molecular and clinical correlates. As such, we employed a relatively liberal threshold of FDR < 0.25 in our downstream analyses. While an FDR threshold of 25% indicates that reported results are likely to be valid 3 out of 4 times and is appropriate in the context of exploratory, discovery-driven analyses, our reported findings in this manuscript nevertheless should be interpreted merely as interesting candidate hypotheses that require further validation in future work. Furthermore, Caucasian individuals are heavily overrepresented in both the TCGA and UK Biobank cohorts, and thus our downstream analyses have largely been suitably powered to investigate only those KIR alleles that are well represented among Caucasians. Future studies will be needed to use more racially diverse cohorts to analyze KIR alleles that are more frequently represented in other ethnicities. Finally, as mentioned at the outset, NK cell activity is modulated by a number of factors outside of individual KIR genes polymorphism, including the subset of HLA-Is they recognize, the distinct combinations of genes that constitute the individual's KIR haplotype, and stochasticity in KIR expression on the surface of individual NK cells. Indeed, over 40 distinct KIR haplotypes, each composed of at least seven KIR genes, have been documented in the human population [30]. Variation of any of these additional factors may further affect NK cell function and ideally would be explored in conjunction with KIR polymorphism at the individual gene level in future studies. In conclusion, our work has generated KIR allele predictions for TCGA and UK Biobank that will be invaluable for future studies of NK cells in these populations, uncovered multiple potential novel associations between KIR gene variants and clinical and molecular features, and has paved the way for future investigation into the role of KIRs in immunologic response and human disease. We hope that our algorithm can serve as a benchmark for future algorithms that will perform KIR genotyping and that others may use our algorithm to better understand the immunologic and pathologic processes surrounding KIR genes. Conclusions We have developed KIRCLE, a first-of-its-kind fully automated computational pipeline for the inference of germline variants of the highly polymorphic killer-cell immunoglobulin-like receptor (KIR) genes from whole exome sequencing data. We demonstrate the utility of such an algorithm by using KIRCLE to infer germline KIR variants in approximately sixty-thousand individuals in The Cancer Genome Atlas and UK Biobank and then discover novel molecular and clinical correlations with these variants. This work represents the first large-scale genetic analysis to elucidate immunologic and pathologic associations with human natural killer cells and will serve as a valuable resource for future investigations into the immunogenomics and disease processes involving KIRs. Allele inference using expectation maximization To infer allele probabilities from a set of read alignments to a database of KIR alleles, we use an expectation-maximization algorithm to aggregate the alignment data into an initial set of allele probability "expectations, " which is then used to further weight the alignment data in order to refine our estimates of KIR allele probabilities. Thus, given a bootstrap of read alignments with 100% identity to at least one KIR allele, KIR-CLE's EM algorithm iteratively generates probability estimates for each KIR reference allele. Let: m = the total number of alleles of this KIR gene n = the total number of reads in this bootstrap A = the set of KIR alleles {a 1 , a 2 , … , a m } R = the set of BAM reads {r 1 , r 2 , … , r n } x r = the number of alleles that read r aligns to with 100% identity t = each time step of the expectation maximization algorithm α = a heuristically chosen convergence threshold We first initialize an m x n "alignment matrix" M to encode our read alignments: Next, using M, we compute an initial expectation vector E t representing our rudimentary estimate of each KIR allele's probability in this sample: Then, at each time step t of the expectation-maximization algorithm, we update the values of our alignment matrix M in a Bayesian fashion using the previously generated expectation vector E t as our prior and M t as our likelihood: Subsequently, we may generate an updated expectation vector E t+1 using M t+1 in conjunction with Eq. (2) above. We continue to iterate through our expectation-maximization algorithm in this manner, computing E t from M t and then M t+1 from E t and M t , until we achieve our convergence criterion, defined as the sum of squared changes in M not exceeding a heuristically selected hyperparameter α: Ultimately, our final expectation vector E T is outputted as our vector of allele probability estimates. Allele coding region collapse Because we used WES data as our input, KIR variants that differed only at non-exonic sites were merged by summing their allele probability estimates. Furthermore, as we are primarily interested in the phenotypic effects of altered binding affinity to KIR domains, we merged variants that differ only by a silent mutation by summing their allele probability estimates as well. Thus, all KIR alleles subsequently are reported as a three-digit number following the KIR gene name (e.g., KIR2DL4*005). KIR allele correlations with molecular and clinical correlates Correlations between inferred KIR alleles and molecular and clinical correlates in TCGA and UK Biobank were performed by comparing the values of these correlates in samples with a given KIR allele versus those without that KIR allele. Comparisons were performed using two-sided Fisher's exact tests for categorical variables and two-sided Mann-Whitney U tests for continuous variables. Correction for multiple hypothesis testing in all analyses was performed using the Bonferroni method. Clinical effect model comparison To explore several correlations we discovered between KIR3DL2 genotype and the earliest age of atopy onset more deeply, we attempted to model the earliest age of atopy onset as a linear function of the KIR3DL2 genotype	2022-08-25T13:32:16.785Z	2022-08-24T00:00:00.000	{ "year": 2022, "sha1": "aabe707fce344c3a96fed52cda35d57857c9c2c8", "oa_license": "CCBY", "oa_url": "https://bmcbiol.biomedcentral.com/track/pdf/10.1186/s12915-022-01392-2", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "ae0757745481fd0ffd14b5bdac0df39952e52d92", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
8040159	pes2o/s2orc	v3-fos-license	Identification of Differentially Expressed Genes in RNA-seq Data of Arabidopsis thaliana: A Compound Distribution Approach Abstract Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product, which may be proteins. A gene is declared differentially expressed if an observed difference or change in read counts or expression levels between two experimental conditions is statistically significant. To identify differentially expressed genes between two conditions, it is important to find statistical distributional property of the data to approximate the nature of differential genes. In the present study, the focus is mainly to investigate the differential gene expression analysis for sequence data based on compound distribution model. This approach was applied in RNA-seq count data of Arabidopsis thaliana and it has been found that compound Poisson distribution is more appropriate to capture the variability as compared with Poisson distribution. Thus, fitting of appropriate distribution to gene expression data provides statistically sound cutoff values for identifying differentially expressed genes. 1. INTRODUCTION I n biological system, for better understanding of complex conditions, there is a need for advancements in identifying genes related to that trait. This can be achieved by enhancing our knowledge about gene expression through statistical models so as to perform statistical analysis of gene expression profiles. To measure gene expression (or transcript abundance), the sequencing reads obtained are aligned to a known reference genome sequence and the proportion of reads matching a given transcript is used as quantification of its expression level followed by statistical testing of difference in quantification values between samples (Bloom et al., 2009;Costa et al., 2010;Oshlack et al., 2010). RNA-seq is rapidly emerging as the method of choice for comprehensive transcript abundance estimation (Li and Xie, 2013). TopHat and Cufflinks (Trapnell et al., 2012) are free, open-source software tools for gene discovery and comprehensive expression analysis of high-throughput mRNA sequencing (RNAseq) data. Later on, Cuffdiff 2 (Trapnell et al., 2013) was developed, which is an algorithm that estimates expression at transcript-level resolution and controls for variability evident across replicate libraries. A gene is declared differentially expressed if a difference or change observed in read counts or expression levels/index between two experimental conditions is statistically significant. Transcription is the expression analysis of population of genes or analysis of differences in expression of gene populations under different environments, conditions, treatments, and stages. Several statistical methods are there for gene expression analysis. Statistical distributions are used to approximate the pattern of differential gene expression. Such genes are selected based on a combination of expression change threshold and score cutoff, which are usually generated by statistical modeling. The Poisson distribution and the negative binomial distribution are the most commonly used models. The main advantage associated with Poisson distribution is its simplicity, and it has only one parameter, but it has a constraint that variance of the model is equal to the mean. Several researchers have tested for differential expression using Poisson distributions (Marioni et al., 2008;Bullard et al., 2010;Wang et al., 2010). The Poisson assumption, however, does not account for biological variability in the data (Robinson and Smyth, 2007;Nagalakshmi et al., 2008). Biological replicates are more variable than technical replicates (McIntyre et al., 2011). Ignoring this issue on datasets with biological replicates will result in false-positive rates because of underestimation of sampling error (Anders and Huber, 2010). The negative binomial distribution has two parameters, encoding the mean and the dispersion, which allows modeling of more general mean-variance relationships. The negative binomial distribution, which requires an additional dispersion parameter to be estimated, is often used to deal with the biological variability in the data. Variations of negative-binomial-based differential expression analysis of count data have been proposed (Robinson and Smyth, 2007;Anders and Huber, 2010;Hardcastle and Kelly, 2010) along with models that extend the Poisson model by including overdispersion (Srivastava and Chen, 2010). Several R packages are available for expression analysis, like DEGseq (Wang et al., 2010). The Bioconductor software package edgeR (Anders and Huber, 2010;Robinson et al., 2010) has been developed to examine replicated gene count data using an overdispersed Poisson model. The statistical tests based on negative binomial distributions (DESeq, edgeR, and baySeq) had notably good control of false-positive errors with comparable specificity and sensitivity resulted from the tests (Rapaport et al., 2013). Soneson and Delorenzi (2013) conducted an extensive comparison of 11 methods for differential expression analysis of RNA-seq data. All methods are freely available within the R framework and take as input a matrix of counts. Different distributions have been fitted to the data as a whole and inferences drawn from it. These distributions do not handle different experimental sources of variation and the variability of the data is not properly addressed. Analysis of differences in expression of gene populations under different conditions, treatments, and developmental stages is required. Further, within a whole data set there are different subsets that possess different properties that can be modeled separately. The interest is to know whether there is statistically significant evidence that any of the genes under study exhibit a difference in expression across the groups/conditions/subpopulations. The concept of compound distribution has been used here for identification of differentially expressed genes. Compound distributions represent a useful way of describing heterogeneity in the distribution of a variable. In the present study, the focus is mainly to investigate the differential gene expression analysis for sequence count data based on compound distribution model as this model is able to capture extra variation. Compound mixture of Poisson-gamma distribution is used. The joint likelihood density function is obtained and the parameters of the model are estimated. MATERIALS AND METHODS It is very important to find statistical distribution to approximate the nature of differential gene expression data. It is found from the literature that, for differential expression analysis of count data, Poisson distribution is most commonly used. A discrete random variable X (number of reads per gene) is said to have a Poisson distribution with parameter k > 0 if it assumes only nonnegative values, and the probability mass function of X is given by 240 ANJUM ET AL. Here, mean = variance = k. The Poisson distribution has the advantage of simplicity and has only one parameter, k. Poisson distribution occurs when there are events that do not occur as outcomes of a definite number of trials of an experiment but that occur at random points of time and space wherein the interest lies only in the number of occurrences of the event, not in its nonoccurrences. However, it constrains the variance of the modeled variable to be equal to the mean. It has been noted that the assumptions of Poisson distribution are too restrictive: it predicts smaller variations that are observed in data. Therefore, the resulting statistical test does not control type 1 error (the probability of false discoveries). Overdispersion problem was solved in count data by using negative binomial distribution. A random variable X is said to follow a negative binomial distribution with parameters r and p if its probability mass function is given by The negative binomial distribution has two parameters, the mean and the dispersion, and hence allows modeling of more general mean-variance relationships. But the number of replicates in the data set of interest is normally too small to estimate both the parameters mean and variance reliably for each gene. For RNA-seq, it has been suggested that the Poisson distribution is well suited for analysis of technical replicates, whereas the higher variability between biological replicates necessitates a distribution incorporating overdispersion, such as the negative binomial. The theory of compound mixture distribution model has been attempted here to know whether there is statistically significant evidence that any of the genes under study exhibit a difference in expression across the groups/conditions/subpopulations. Compound distribution Consider a random variable X following a Poisson distribution with parameter k as given in Eq. (1), where the Poisson parameter k is itself a random variable, distributed according to a gamma distribution with parameters a and b; that is, The compound Poisson follows negative binomial distribution with parameters (b‚ p = a 1 + a ) with the following probability function: The mean of this compound Poisson distribution is b a and variance is b(1 + a) a 2 : The negative binomial distribution is thus here a mixture of a family of Poisson distributions with gamma mixing weights. Thus, the negative binomial distribution is known as a Poisson-gamma mixture. In case of a single biological sample (RNA extract), aliquots are taken to make technical replicates. These technical replicates will be distributed as Poisson. In case of multiple biological samples, multiple technical replicates out of each biological replicate will have multiple Poisson distributions for each biological replicate. The multiple Poisson distributions for each biological replicate can be described by a gamma distribution. Negative binomial as compound Poisson is more capable of capturing the variability as compared with Poisson distribution and hence identified more differentially expressed genes in case of RNA-seq data. RNA-seq data RNA-seq data of Arabidopsis thaliana have been considered for this investigation. The small size, simplicity, convenience and abundance, susceptibility to T-DNA insertions, short generation time, large number of progeny per plant, and small genome of A. thaliana make it attractive for molecular genetic analysis. The details of the RNA-seq data in terms of number of read counts of A. thaliana are given below: The steps followed for analyzing differential gene expression are as follows: 1. The expression data under the two conditions (hrcC and mock) for different genes are arranged. 2. The difference in read counts is taken over two conditions and is plotted. RESULTS The difference in read counts of A. thaliana is taken over two conditions (hrcC and mock) and is plotted. The changes in one direction in up-regulated gene expression can be clearly seen from the graphs in Figures 1 and 2. Poisson distribution was fitted to the data and the fitted plot is shown in Figure 3. ANJUM ET AL. It is seen that Poisson distribution is not able to capture the entire variability. Therefore, the mixture of Poisson distribution with gamma mixing weights (Poisson-gamma mixture) that results in negative binomial distribution was fitted to the up-regulated gene expression RNA-seq data. The fitted plot is shown in Figure 4. It is seen that it covers more variability and takes care of overdispersion. Similarly for the down-regulated gene expression values, changes in one direction are clearly seen (Figs. 5 and 6). Poisson distribution was fitted to the down-regulated gene expression data and the fitted plot is shown in Figure 7. It can be seen that Poisson distribution is not a good fit of the data, and hence the mixture of Poisson distribution with gamma mixing weights (Poisson-gamma mixture) was fitted to the down-regulated gene expression RNA-seq data. The fitted plot is shown in Figure 8. It is seen that it covers more variability and takes care of overdispersion. The parameter estimates of the Poisson and the compound Poisson were obtained as given in Table 1. Further, the likelihood ratio test was performed and the LRT was computed, which follows a chi-square with 1 degree of freedom. It is found that the null model of Poisson distribution is rejected in favor of the alternative model of compound Poisson distribution. The total number of genes identified as differentially expressed is shown in Table 2 based on the probability value p < 0.01 under the Poisson and compound Poisson distributions. DISCUSSION Here we have predicted differentially expressed genes by using the compound distribution approach, which is a way of mixing the distributions by assuming the parameter of the distribution to be random variable following certain distribution. It has been found that in case of RNA-seq data, compound Poisson distribution, which is a mixture of Poisson and gamma distribution, is more appropriate to capture the variability as compared with Poisson distribution and could identify the differentially expressed genes more accurately. Separate fitting was done for up-regulated and down-regulated genes. In case of up-regulated genes, out of 10,483 genes, 9649 genes were identified as differentially expressed based on the probability value cutoff with respect to Poisson distribution and 2081 were identified with compound Poisson distribution. Out of a total of 11,607 down-regulated genes, 10,357 were identified as differentially expressed by fitting Poisson distribution, whereas only 1954 were identified with compound Poisson distribution. It is seen that Poisson distribution is able to identify a large number of genes even for very small differences in read counts, which is not realistic. Table 3 lists few selected genes along with the read count values under two conditions (mock and hrcC). All the genes are differentially expressed using Poisson distribution irrespective of large or small differences. Gene numbers AT5G67640, AT1G01355, and AT5G67550 with a small difference of 2, 1, and 1, respectively, are identified as differentially expressed, whereas using compound Poisson these are not differentially expressed as p > 0.01. Hence, it can be seen that compound Poisson distribution, which is a mixture of Poisson and gamma, is able to identify the differentially expressed genes more accurately. Thus, fitting of appropriate distribution to gene expression data provides statistically sound cutoff values for identifying differentially expressed genes. R CODE For fitting the compound Poisson distribution to the data of RNA-seq, R codes have been written, which are given in the Supplementary File (Supplementary Material is available online at www.liebertonline.com/ cmb). The code also calculates the likelihood value for testing these models.	2018-04-03T00:00:34.947Z	2016-04-01T00:00:00.000	{ "year": 2016, "sha1": "328e990919b33271f7a7a6bf95728116473601fe", "oa_license": "CCBYNC", "oa_url": "https://www.liebertpub.com/doi/pdf/10.1089/cmb.2015.0205", "oa_status": "BRONZE", "pdf_src": "PubMedCentral", "pdf_hash": "328e990919b33271f7a7a6bf95728116473601fe", "s2fieldsofstudy": [ "Biology", "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Medicine", "Biology" ] }
1303943	pes2o/s2orc	v3-fos-license	Origin and Consequences of the Relationship between Protein Mean and Variance Cell-to-cell variance in protein levels (noise) is a ubiquitous phenomenon that can increase fitness by generating phenotypic differences within clonal populations of cells. An important challenge is to identify the specific molecular events that control noise. This task is complicated by the strong dependence of a protein's cell-to-cell variance on its mean expression level through a power-law like relationship (σ2∝μ1.69). Here, we dissect the nature of this relationship using a stochastic model parameterized with experimentally measured values. This framework naturally recapitulates the power-law like relationship (σ2∝μ1.6) and accurately predicts protein variance across the yeast proteome (r2 = 0.935). Using this model we identified two distinct mechanisms by which protein variance can be increased. Variables that affect promoter activation, such as nucleosome positioning, increase protein variance by changing the exponent of the power-law relationship. In contrast, variables that affect processes downstream of promoter activation, such as mRNA and protein synthesis, increase protein variance in a mean-dependent manner following the power-law. We verified our findings experimentally using an inducible gene expression system in yeast. We conclude that the power-law-like relationship between noise and protein mean is due to the kinetics of promoter activation. Our results provide a framework for understanding how molecular processes shape stochastic variation across the genome. Introduction Stochastic fluctuations in the biochemical processes that underlie gene expression produce cell-to-cell variation in protein levels, or ''noise'' [1][2][3]. Noise performs several biological functions. In unicellular organisms, noise improves fitness by generating phenotypic differences within clonal populations of cells, thus enabling a rapid response to fluctuating environments [4][5][6]. In multi-cellular organisms, noise plays a role in development, allowing identical progenitor cells to acquire distinct fates [7][8][9]. Because of its functional importance, a fundamental goal is to identify and dissect the molecular mechanisms that generate and control noise. Single-cell studies have connected pathway-specific (extrinsic) and gene-specific (intrinsic) factors to changes in protein variance [2,10,11]. These factors include the rate of transcript elongation [12], the presence of a TATA-box [2,4,13,14], nucleosome positioning at the promoter sequence [2,[15][16][17][18], fluctuating mRNA levels [19], translation rate [18,20,21], pathway-dependent fluctuations [11,19], and asymmetric partitioning at cell division [22]. However, it is unclear whether any of these processes evolved specifically to produce high levels of protein variance, or whether the observed variance is only a consequence of selective pressure on protein mean levels. This issue is complicated by the strong dependence of cell-to-cell protein variance on mean protein levels [11,19,20]. Several studies have revealed that a protein's cell-tocell variance is linearly related to its mean expression level when plotted on a log-log scale, suggesting this relationship can be approximated by a power-law (s 2 /m j ) [19,23,24]. This relationship is of paramount importance for investigations into the evolutionary origins and consequences of noise, because it allows to correctly normalize protein variances to identify proteins that display unexpectedly high single-cell variance given their mean levels. Although this relationship has been noted previously, two important questions have not yet been resolved. First, how does the process of gene expression specify this power-law relationship and consequently protein variance? Secondly, which molecular processes regulating gene expression have evolved to produce substantially higher protein variance than would be expected given the observed relationship? To answer these questions, we analyzed a dataset of protein variance using a stochastic model of gene expression parameterized with experimentally measured kinetic rates. This model recapitulated the relationship (s 2 /m 1.6 ) between mean and variance and accurately predicted protein variance on a proteome-wide scale (r 2 = 0.935). We find that this result is achieved under a general regime of promoter kinetics across the yeast genome characterized by slow promoter activation followed by rapid inactivation, resulting in mRNA production that is nearly a Poisson process (s 2 /m 1.1 ). However, the small non-linearity between RNA mean and variance is amplified during protein production, reproducing the observed power law. By further analyzing this model, we found that the kinetics of promoter activation dictate the exponent of the power-law. This finding allowed us to identify two distinct classes of processes that influence noise. Variables that influence processes downstream of promoter activation, such as the synthesis and degradation of mRNA and protein, increase variance by increasing mean levels, which then causes an concomitant change in protein variance in accordance with the power law. In contrast, variables that reduce the rate of promoter activation, such as promoter-positioned nucleosomes, increase variance by increasing the exponent of the power-law-like relationship linking protein mean and variance. Only the latter class of mechanisms generate protein variances that are significantly higher than expected from protein mean levels. In support of these conclusions, we performed experiments demonstrating that changing the rate of promoter activation, but not the rate of protein translation, modulates the exponent of the power-law and consequently the scaling between variance and mean. By providing a mechanistic interpretation of the power-lawlike relationship, our work provides the framework to achieve a better understanding of the molecular processes that lead to cellto-cell variation in gene expression. Results Protein mean and variance are connected by a powerlaw-like relationship To characterize the relationship between mean protein levels and cell-to-cell protein variance across the yeast genome, we analyzed a published dataset consisting of ,2200 S. cerevisiae GFP fusion strains for which protein levels had been measured at a single-cell resolution by flow-cytometry [13]. This dataset serves as a starting point to examine global trends between protein mean and variance as it represents an unbiased sampling of the yeast proteome. First, we performed a log-log regression analysis of cellto-cell protein variance as a function of the mean protein levels and observed a power-law-like relationship with an exponent of 1.69 (Figure 1a), in agreement with previous findings [23]. Ninetyseven percent of protein variance across the proteome can be explained solely by mean levels through this relationship, indicating that highly expressed genes naturally exhibit high cellto-cell variation whereas genes expressed at low levels are more uniformly expressed across different cells. Although the residual fraction of protein variance not explained by the power-law accounts for only 3% of the total variation, we found that, for certain genes, it increased protein variance up to 20-fold higher than expected (Figure 1b). In contrast, very few genes displayed smaller protein variances than expected given mean levels, as we observed, at most, a 2-fold reduction (see Figure 1b). Taken together, these results indicate that for most genes, protein variance is largely explained by the protein mean through a power-law-like relationship, except for a few notable cases in which protein variances are increased substantially beyond their expected values. A stochastic model of gene expression recapitulates the power-law-like relationship between protein mean and variance We next sought to understand the molecular origin of the relationship between protein mean and variance. One hypothesis is that this relationship originates purely as a consequence of stochasticity in the steps underlying gene expression [19]. Alternatively, this relationship could result from mechanisms that are independent of expression, such as asymmetric partitioning of protein and RNA molecules at cell division [22] or pathwaydependent fluctuations in trans-acting factors [11]. To distinguish between these two hypotheses, we tested whether a stochastic model based only on the processes involved in gene expression could recapitulate the observed power-law relationship. We applied a model [25] that describes cell-to-cell protein variance at steady-state as a function of kinetic parameters for promoter activation/inactivation events and mRNA and protein production/degradation (Figure 2a, Figure S1). For most parameter values, we used empirical measurements (see Supporting Information S1, section 1.2). This was not possible, however, for the rates of promoter activation and inactivation, which have only been measured in a few genes [26]. Since no high-throughput methods exist for measuring rates of promoter activation and inactivation, we assumed that the promoter kinetics would be similar across the genome and fit their values from the data (Supporting Information S1, section 1.3). The model converged to a regime in which promoter activation is an infrequent event that is quickly followed by promoter inactivation, a result supported by published experimental data [23] (Supporting Information S1, section 1.4, and Figure S1). We obtained a rate of promoter activation (Kon) of 0.59 min 21 , a value that agrees with empirically measured activation rate for the GLT1 gene in yeast (1.360.72 min 21 ) [26]. Using this value for Kon, the model naturally generates a power-law-like relationship between mean and variance that is similar to the one observed empirically (modeled relationship: s 2 /m 1.60 , observed relationship: s 2 /m 1.69 ). Furthermore, our framework correctly predicts protein variance across the genome (log space r = 0.962, p,2.210 216 ; linear space r = 0.839, p,2.210 216 , Figure 2b). We tested for over-fitting by performing 2-fold cross-validation 100 times and again found good agreement (r = 0.95760.018, p,2.210 216 ). Taken together, these results support the validity of our model and suggest that the power-law relationship between protein mean and variance depends solely on the kinetics of the processes that underlie gene expression. The power-law-like relationship between protein variance and mean depends on promoter kinetics We next sought to determine which of the processes involved in gene expression determine the exponent of the power-law-like relationship. Using our biophysical model, we randomly sampled transcription and translation rates, as well as degradation rates of mRNA and protein, while maintaining the same promoter activation regime we identified above (Kon = 0.59 min 21 ,, Koff). Virtually all permutations resulted in a power-law-like relationship between mean and variance that was nearly identical to the one observed experimentally (exponent = 1.61265.910 23 , 1000 permutations, Figure 3a). This result indicates that, when Kon ,, Koff, the exact form of the power-law-like relationship between mean and variance is independent of the rates of transcription, translation, and protein and mRNA degradation. In contrast, we found that the exponent of the power law was strongly dependent on promoter kinetics. Using the same modeling framework, we changed the parameters governing promoter transitions to enforce a slow kinetics regime (Kon and Koff ,, Km,Dm,Kp,Dp). We found that protein mean and variance followed a quadratic relationship (exponent = 1.97, Figure 3a), which differs substantially from our previous results and the observed power-law. Taken together these results suggest that the power-law relationship between protein mean and cell-to-cell variance is dictated by the kinetics of promoter activation, and is largely insensitive to downstream steps. The relationship between protein mean and variance identifies different sources of variance A strong prediction of our model is that perturbations that affect processes downstream of promoter activation should increase noise only through changes in mean protein level, which will then increase protein variance following the power law. In contrast, perturbations which affect the kinetics of promoter activation should increase protein variance by modulating the relationship between protein mean and variance. As a result, this class of perturbations are expected to show a much larger effect on protein variance once normalized to the general power-law relationship (s 2 /m 1.69 , Figure 1). Several variables have previously been correlated with increases in noise including changes in transcription [13] and translation rates [2,20,21], the presence of a TATA box [2,13,21] and promoter positioned nucleosomes [2,15,16,27]. Our model suggested that only variables involved in promoter activation should significantly increase protein variance when normalized to their mean levels, whereas variables affecting downstream processes would not. To test this hypothesis, we correlated the protein variance residuals with variables that reflect changes in promoter activation, and with variables that affect downstream processes. Genes with TATA boxes or promoter-positioned nucleosomes, factors which influence promoter activation, had high values of residual variance (Figure 3b), indicating that they increase noise by modulating the power-law. In contrast, differences in measured rates of mRNA synthesis and degradation [28], rates of protein degradation [29], measures of ribosomal occupancy [30], and the Codon Adaptation Index [31] showed little or no correlation with residual variance (Figure 3b). This result demonstrates that these variables, which affect processes downstream of promoter activation, influence cellto-cell protein variance almost exclusively by changing mean levels of gene expression. Taken together, the results support our hypothesis and suggest that positioned nucleosomes may account for a large portion of the residual variance. Promoter-positioned nucleosomes increase variance by slowing promoter activation kinetics Our model suggests that the increase in residual protein variance caused by positioned nucleosomes is the result of slower promoter activation in these genes. To test this hypothesis, we examined single-cell mRNA measurements performed for different genes in S. cerevisiae [32], since the relationship between mRNA mean and variance can be used to clearly distinguish groups of genes with different promoter kinetics [23] (see materials and methods). Our prediction is that genes without promoterpositioned nucleosomes (Figure 4b) will have fast promoter activation kinetics and thus display an approximately linear relationship between mean and variance ( Figure 4a, blue line, see Supporting Information S1, section 1.8). Indeed, this was observed in the single-cell mRNA data (see Figure 4a, red dots). In contrast, our model predicts that genes with promoter-positioned nucleosomes ( Figure 4c) will have slow promoter activation kinetics and will therefore display a quadratic scaling between mean and variance (Figure 4a, red line, see Supporting Information S1, section 1.8). This was again confirmed as genes lacking a nucleosome-free region displayed the predicted mean-variance relationship (Figure 4a, red dots). Experimental confirmation of the effects of promoter kinetics on the mean-variance relationship Finally, to obtain additional support for these findings, we experimentally tested whether changes in nucleosome occupancy could produce an increase in the mean-independent component of protein variance. Using in vivo nucleosome positioning data [33], we selected a set of S. cerevisiae TATA-containing genes whose promoters are nucleosome free in glucose but which acquire a positioned nucleosome in ethanol. A prediction of our analysis is that such genes would display increased residual variance when switched from glucose-containing medium to ethanol-containing medium. We measured the distribution of fluorescence of GFPtagged fusion strains [29] in both glucose and ethanol by flowcytometry, and computed the residual variance above what is expected from the mean-variance relationship. We observed a significant increase in residual variance as cells were shifted from glucose to ethanol relative to a control set of genes in which nucleosomes do not change between the two conditions (p-value , 0.05, T-test across 3 biological replicates, Figure 5a nucleosome occupancy set). Using this same gene set, we examined whether changes in protein translation rate affected the mean-independent component of the variance. Our model predicts that translation rate should not correlate with residual variance, and we did not observe any significant difference (p-value .0.4, T-test across 3 biological replicates, Figure 5a translation rate set). These results support our hypothesis that positioned nucleosomes are the major source of mean independent noise. We conclude that nucleosome bound promoters showed higher protein variance as a result of slowed promoter activation kinetics, which increases the exponent of the power-law-like relationship between protein mean and variance. These results can be summarized in a general model: most of the genes in S. cerevisiae exhibit promoter kinetics characterized by fast inactivation rate and as a result display a protein meanvariance scaling dictated by a general power-law relationship with exponent equal to 1.69 (Figure 5b, purple dots and line). In contrast, few genes characterized by slow promoter kinetics display approximate quadratic scaling between protein mean and variance (Figure 5b, orange dots and line). Changes in promoter kinetics induced by nucleosome positioning can affect this relationship, resulting in an increase in protein variance compared to the general power-law. Discussion Single-cell variance in protein levels plays a major role in generating phenotypic differences [4,5]. A fundamental property of protein variance is its dependence on mean protein levels through a power-law-like relationship. This relationship holds in yeast (s 2 /m 1.6 ), bacteria (s 2 /m 1.5 ) [24] and human T-cells (s 2 /m 1.7 ) [34], suggesting the processes that determine the power-law are common across different species. Using a stochastic model of gene expression parameterized with empirically measured kinetic rates [13,25,28], we found that the power-law is a natural consequence of the kinetics of transcription and translation, fundamental mechanisms shared between these three organisms. Through the same framework, we also were able to predict for the first time protein variance at a genomic scale. Molecular processes that differ significantly between these species, such as chromatin structure, nuclear export, or unequal partitioning during the cell cycle, were not required to explain the powerlaw nor to predict protein variance. These results were reached by fitting a global rate of promoter activation and assuming the same promoter kinetic regime across the whole genome. Although this is in fact an approximation as it would be unrealistic to expect all promoter to be activated at the same rate, we found this assumption to be largely true in promoter bashing experiments [14]. Furthermore, this result suggests a model where changes in promoter initiation arise mostly as a result in changes of promoter inactivation rather than activation, a result that has been empirically observed at a single gene level in different organisms [23,35]. The global regime of promoter initiation that we captured consisted in a fast promoter inactivation rate and slow activation rates, resulting in short burst frequency (0.59 min 21 ) and an average small burst size (0.104 transcripts per burst on average). These values are in agreement with the only direct empirical measure of transcriptional initiation in S. cerevisiae [26]. In this kinetic regime, most promoter transitions to the active state do not produce an mRNA transcript -for the ''average'' gene, approximately 89% are non-productive. Transitions that do produce a transcript typically only produce a single mRNA molecule (,9.4% of transitions, for the average of transitions, for transitions produce multiple transcripts (0.5%). In this regime, RNA production very nearly follows a Poisson process, with s 2 /m 1.1 . However, this small non-linearity between mean and variance is amplified at the protein level and the mean-variance relationship follows the s 2 /m 1.69 power-law-like relationship. One practical application of understanding the power law is that it allows to separate different mechanisms that contribute to the increase of protein variance. By using the power-law obtained under these rates, 97% of all protein variance across the genome can explained solely by mean protein levels, suggesting that this kinetic regime is a general feature of transcription in S. cerevisiae. The 3% of genes with excess variance (up to twenty-fold over the expected variance) is consistent with the occurrence of slow promoter kinetics, which our data suggests is caused for the most part by positioned nucleosomes on their promoters. The association of nucleosomes and chromatin related factors to increased promoter variance is not novel and it has been previously observed in several studies [2,15,16,27]. However, we find that nucleosome positioning is by far the dominant factor, explaining most of the excess variance. This result is even stronger when nucleosome occupancy is analyzed in the context of TATAcontaining genes, a notorious class of genes characterized by higher protein variance than the rest of the proteome [2,13,21]. Interestingly, a recent analysis of the effect of TATA-box using synthetic promoter libraries has revealed the TATA-box not to be sufficient to increase protein variance [14]. This suggests that perhaps an interplay between TATA and chromatin architecture is required to produce the observed increase in noise, a conclusion supported by our observation in genomic data as well as in promoter mutagenesis libraries [36]. In disagreement with previous observations [2,13,20,21], factors involved in molecular processes occurring after promoter initiation do not produce an excess of variance beyond what is expected. The analysis of the model explains this observation: factors modulating the kinetics of promoter initiation will produce an increase in the exponent of the power-law for that particular gene, which will result in an apparent excess of protein variance. In contrast, factors operating downstream will produce an increase in variance solely through an increase in mean following the power-law exponent specified by the kinetics of the controlling promoter. Our work therefore suggests that the power-law is a universal feature of protein expression whose particular shape is determined by the rates at which promoters transition between their active and inactive states [37,38]. The performance of our model and the conclusions of our analysis pertain only to the intrinsic, or gene specific [10] portion of protein variance, as the dataset that we analyzed minimized the effect of global or extrinsic factors through gating [13]. The reduced extrinsic component of this dataset may also explain the absence of association of translation specific factors to excess protein variance, as previous genetic dissection revealed their enrichment among factors modulating global variance changes [18]. Finally, we did not observe any genes with variances significantly below that expected from the power-law. Reducing protein variance may be difficult for the cell due to physical constraints that render this process energetically dis-advantageous. A theoretical analysis on the limits of suppression of molecular fluctuations [39] supports this observation. Alternatively, it is possible that cells have evolved regulatory networks with intrinsic robustness to molecular fluctuations [40], suggesting that even if achievable, noise reduction may not be necessary. Identifying the sources of noise and their underlying mechanisms is an important step in determining their role in increasing fitness. The work presented here provides a way to isolate meanindependent effects from protein variance and to connect them to their biophysical origins. A long-standing question regarding Figure 5. Modulating promoter kinetics changes protein mean-variance scaling. a) Increment in residual variance from glucose to ethanol in genes that show increased occupancy in ethanol (orange set: test) and genes with unaltered occupancy (orange set: control) compared to the same genes ranked by high (purple set: test) or low (purple set: test) increase in translation rate (purple set) (* indicates p,0.05, t-test). b) Diagram connecting the power-law exponent to promoter kinetics: most genes in S. cerevisiae exhibit promoter kinetics characterized by fast inactivation rate (purple dots) and display protein mean-variance scaling dictated by a power-law with 1.69 exponent (purple line). A small set of genes (orange dots) exhibit slow promoter kinetics and consequently present protein mean-variance scaling dictated by a quadratic scaling (orange line). doi:10.1371/journal.pone.0102202.g005 stochastic gene expression is its role in fitness [4]. Through this framework, it will be possible to completely decouple the role of protein variance from the mean, allowing a better understanding of the functional and evolutionary constraints that shape gene expression variance. Data Sources We used single-cell protein mean and variance values from flow-cytometry measurements on S. cerevisae GFP-fusion strains grown in YPD for ,2000 genes from Newman et al. [13]. mRNA level measurements in YPD and YPEtOH were obtained from Gasch et al. [41]. We acquired mRNA synthesis and degradation rates from Miller et al. [28]. mRNA single-cell measurement data were obtained from Gandhi et al. [32]. Nucleosome occupancy was assessed from mnase-seq datasets in YPD and YPEtOH from Kaplan et al. [33]. We used protein mean and variance from synthetic promoter libraries from the work of Mogno et al. [14]. Definition of TATA-containing and TATA-less were obtained from Basehoar et al. [42]. We obtained in vivo ribosome occupancy profiles for each mRNA species measured in YPD from Ingolia et al. [30]. Data and source code generated and used in this work can be found at http://cgs.wustl.edu/,fvallania/ 5_noise_2011/5_noise_website/ NOISE_Project_supporting_materials.html. Analysis of the relationship between protein mean and variance Using single-cell protein mean and variance values in S. cerevisiae [13], we assumed that the underlying relationship between mean and variance could be non-linear and exponential in nature. This formulation can be generally expressed as s 2~k m J where k is a scaling factor and J is the exponential index. In logspace, this equation transforms into where J can now be directly calculated as the slope of a linear regression. We estimated the fraction of variance explained by the mean as the r 2 of the regression. Variance residuals originated from this fit were defined as mean-independent variance. Regression analysis was performed using the R programming language. Stochastic modeling of protein and mRNA variance To model mRNA and protein variance in S. cerevisae, we used analytical stochastic models derived from the solution of a system of stochastic differential equations as previously described [25]. This model describes the steady-state value of mRNA and protein variance as a function of the kinetic rates for protein activation and inactivation (Kon and Koff), mRNA synthesis and degradation (Km and Dm), and protein translation and degradation (Kp and Dp). The model for mRNA variance is expressed as whereas for protein variance, the equation is: In order to predict genome-wide protein variance in S. cerevisiae, we assumed Kon and Koff to be uniform across the genome and fit their values. Fitting, prediction and crossvalidation were computed in Perl. Analysis of the fit was performed in R. (for complete explanation see Supporting Information S1). Correlation analysis between mean-independent variance and molecular properties We compared mean-independent variance to mRNA synthesis rate, mRNA degradation rate, ribosomal occupancy and CAI (Codon Adaptation Index). CAI was computed as previously described [31]. To determine the amount of variation of noise explained explained by each property, we correlated meanindependent variance with the log of the measure of each property and calculated the Pearson's correlation coefficient. We used a linear regression in log scale to avoid any non-linear effects. Regression analysis was performed in R. Regression model between mean-independent variance and nucleosome occupancy We computed the Pearson's correlation coefficient between mean-independent variance and nucleosome occupancy at a single base resolution for each base ranging from 21000 to +600 relative to the transcription start site of each gene in S. cerevisae for which we had both nucleosome data and residual mean-independent variance. For each base, we obtained a correlation value, which was plotted as a function of its position relative to the TSS. We repeated this analysis focusing on TATA-containing and TATAless genes only. In order to estimate the amount of variation explained by nucleosome occupancy on TATA-containing genes, we applied a linear model to predict residual mean-independent variance as a function of nucleosome occupancy. We performed a forward-regression strategy to determine the positions in the promoter sequence to be used as predictive features for our model followed by leave-one-out cross-validation to assess over-fitting (Supporting Information S1 for details). Regression analysis was performed in R. Experimental measurement of mean-independent variance as a function of nucleosome occupancy We selected 15 yeast genes that acquired a nucleosome when grown in YPEtOH compared to YPD using genome-wide nucleosome occupancy data [33] (YAL054C, YBL015W, YBL075C, YBR139W, YBR145W, YDL097C, YER081W, YFL021W, YGL040C, YGL197W, YLR042C, YMR315W, YNL241C, YOL143C, YOR084W, YPR127W). We constructed a second set (control set) of 15 genes either stable nucleosomebound or nucleosome-free promoters (YBR066C, YBR092C, YER056C-A, YJL200C, YKL071W, YLR177W, YNL112W, YOR355W, YAL060W, YDR055W, YDR495C, YDR533C, YDL222C, YER054C). For each gene in each set, we grew a corresponding GFP-fusion S. cerevisiae strain [29] in YPD and YPEtOH to log phase and measured single-cell protein levels using a Beckmann-Coulter Cytomics FC500 MPL flow-cytometer (Beckmann Coulter, Fullerton, CA) as previously described previously [13]. We calculated residual variance from mean and variance as described above and, for each gene we computed differential residual mean independent variance between YPEtOH and YPD. We then tested for increase in residual variance between the test and control set using one-sided t-test. Additionally, we computed the translation rate for each gene in both conditions (described in Supporting Information S1, section 1.2) and computed the differential translation rate (DKp) between conditions (defined as Kp YEtOH -Kp YPD ). We then ranked the genes by decreasing DKp and tested for increase in residual variance between the top and bottom half of this set using one-sided t-test. Statistics were performed in R. (a) Experimental de-sign: We selected 15 genes that acquired a nucleosome when grown in YPEtOH compared to YPD using genome-wide nucleosome occupancy data. A control set of equal size was also built with genes with stable nucleosomes across the two conditions. For each gene in each set, we grew a corresponding GFP-fusion S. cerevisiae strain in YPD and YPEtOH to log phase and measured single-cell protein levels by flow-cytometry. (b) Representative results of 3 yeast strains from the test group. For each strain, the distribution of fluorescence intensity is shown in YPD (cyan) and YPEtOH (purple) respectively. The amount of residual variance (labeled as MIV or mean-independent variance) is displayed under each histogram. (c) Same as in (b) but for representative strains from the control group. 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167210734	pes2o/s2orc	v3-fos-license	Insufficient iodine nutrition status and the risk of pre-eclampsia: a protocol for systematic review and meta-analysis Introduction Pre-eclampsia is one of the leading causes of maternal and perinatal morbidity and mortality worldwide. Although subclinical hypothyroidism (SCH) in pregnancy is one of the established risk factors for pre-eclampsia, the link between iodine deficiency, the main cause of hypothyroidism and pre-eclampsia remains uncertain. About two billion people live in areas with iodine insufficiency. The increased renal blood flow during pregnancy leading to increased renal iodine clearance together with the increased placental transfer of iodine to the fetus leads to further iodine deficiency in pregnancy. Iodine is one of the most potent exogenous antioxidants whose deficiency is associated with oxidant imbalance and endothelial dysfunction, one of the mechanisms associated with increased risk of pre-eclampsia. Methods and analysis A systematic search of published literature will be conducted for case–control studies that directly determined the iodine nutrition status of women with pre-eclampsia and appropriate normotensive controls. A similar search will be conducted for cohort studies in which the incidence of pre-eclampsia among pregnant women with adequate and inadequate iodine nutrition status was reported. Databases including MEDLINE, EMBASE, Google Scholar, SCOPUS and Africa Wide Information will be searched up to 31 December 2018. Screening of identified articles and data extraction will be conducted independently by two investigators. Risk of bias of the included studies will be assessed using a Newcastle-Ottawa Scale. Appropriate meta-analytic techniques will be used to pool prevalence and incidence rates, odds and relative risk of pre-eclampsia from studies with similar features, overall and by geographical regions. Heterogeneity of the estimates across studies will be assessed and quantified and publication bias investigated. This protocol is reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analysis protocols (PRISMA-P) 2015 guidelines. Ethics and dissemination Since the proposed study will use published data, there is no requirement for ethical approval. This review seeks to identify the risk of pre-eclampsia associated with insufficient iodine nutrition in pregnancy. This will help to ascertain whether insufficient iodine intake may be an independent risk factor for pre-eclampsia. This will advise policy makers on the possibility of maximising iodine nutrition in pregnancy and reproductive age as one of the remedies for prevention of pre-eclampsia among populations at risk of inadequate iodine intake. This review is part of the thesis that will be submitted for the award of a PhD in Medicine to the Faculty of Health Sciences of the University of Cape Town. In addition the results will be published in a peer-reviewed journal. PROSPERO registration number CRD42018099427. Introduction Pre-eclampsia is one of the leading causes of maternal and perinatal morbidity and mortality worldwide. Although subclinical hypothyroidism (SCH) in pregnancy is one of the established risk factors for preeclampsia, the link between iodine deficiency, the main cause of hypothyroidism and pre-eclampsia remains uncertain. About two billion people live in areas with iodine insufficiency. The increased renal blood flow during pregnancy leading to increased renal iodine clearance together with the increased placental transfer of iodine to the fetus leads to further iodine deficiency in pregnancy. Iodine is one of the most potent exogenous antioxidants whose deficiency is associated with oxidant imbalance and endothelial dysfunction, one of the mechanisms associated with increased risk of pre-eclampsia. Methods and analysis A systematic search of published literature will be conducted for case-control studies that directly determined the iodine nutrition status of women with pre-eclampsia and appropriate normotensive controls. A similar search will be conducted for cohort studies in which the incidence of pre-eclampsia among pregnant women with adequate and inadequate iodine nutrition status was reported. Databases including MEDLINE, EMBASE, Google Scholar, SCOPUS and Africa Wide Information will be searched up to 31 December 2018. Screening of identified articles and data extraction will be conducted independently by two investigators. Risk of bias of the included studies will be assessed using a Newcastle-Ottawa Scale. Appropriate meta-analytic techniques will be used to pool prevalence and incidence rates, odds and relative risk of pre-eclampsia from studies with similar features, overall and by geographical regions. Heterogeneity of the estimates across studies will be assessed and quantified and publication bias investigated. This protocol is reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analysis protocols (PRISMA-P) 2015 guidelines. Ethics and dissemination Since the proposed study will use published data, there is no requirement for ethical approval. This review seeks to identify the risk of preeclampsia associated with insufficient iodine nutrition in pregnancy. This will help to ascertain whether insufficient iodine intake may be an independent risk factor for pre-eclampsia. This will advise policy makers on the possibility of maximising iodine nutrition in pregnancy and reproductive age as one of the remedies for prevention of pre-eclampsia among populations at risk of inadequate iodine intake. This review is part of the thesis that will be submitted for the award of a PhD in Medicine to the Faculty of Health Sciences of the University of Cape Town. In addition the results will be published in a peer-reviewed journal. PrOsPErO registration number CRD42018099427. IntrOduCtIOn Pre-eclampsia is one of the leading causes of maternal and perinatal morbidity and mortality worldwide. 1 Although the actual cause of pre-eclampsia remains unknown, the risk factors of pre-eclampsia are multifactorial. [2][3][4] Subclinical hypothyroidism (SCH) in pregnancy is one of the established risk factors for pre-eclampsia. [5][6][7] Given that iodine deficiency is the leading cause of hypothyroidism, 8 9 iodine deficiency could be an independent risk factor of pre-eclampsia especially in settings with endemic iodine deficiency. Although some studies have reported an association between iodine nutrition status and pre-eclampsia, 10 11 it is not yet certain whether this association is consistent across different settings around the world. The WHO estimates that about two billion people live in areas with iodine insufficiency. 12 In addition, iodine deficiency is on the rise in areas originally thought to be iodine sufficient. 13 This is due to inadequate iodisation strengths and limitations ► To our knowledge, this is the first systematic review and meta-analysis that is aimed at ascertaining the relationship between insufficient iodine nutrition status and pre-eclampsia. ► This review may however be limited by the small number of eligible studies and small sample sizes that may make it liable to a considerable degree of heterogeneity. ► The eligible studies may have varied research designs that may potentially preclude the pooling of the test results. Open access in the background of increased amount of perchlorate and thiocyanate in water sources and the diet. 14-16 Both perchlorate and thiocyanate significantly diminish the uptake of iodine by the thyroid gland especially in states of increased thyroid stimulation by thyroid stimulating hormone (TSH) secondary to iodine deficiency. 15 16 rationale Iodine deficiency has been shown to be associated with defective trophoblast proliferation and migration, which are some of the mechanisms proposed in the aetiology of pre-eclampsia. 17 18 The placenta is a highly metabolic organ with potential for production of reactive oxygen species. It is also one of the organs with high physiological concentrations of iodine whose antioxidant effect reduces lipid peroxide formation and may ensure normal placentation and function. [19][20][21] During pregnancy, there is increased renal perfusion with increased iodine filtration and urinary iodine excretion in addition to increased transfer of iodine to the fetus. 22 Hence women with inadequate iodine intake are at risk of developing iodine deficiency in pregnancy and possibly at increased risk of developing subclinical or overt hypothyroidism and pre-eclampsia. [21][22][23] Furthermore, the elevation in serum TSH that occurs in overt or SCH, is associated with hyperstimulation of the thyroid gland excessive superoxide production among individuals with iodine deficiency, which when released into the circulation, causes endothelial dysfunction and atherosclerosis. 24 25 These are known pathological pathways of pre-eclampsia. 26 Objectives This systematic review and meta-analysis is intended to ascertain whether insufficient iodine nutrition status is associated with increased risk of pre-eclampsia. review questions The purpose of this review is to address the following questions: 1. Do pregnant women with insufficient iodine nutrition status have an increased risk of pre-eclampsia compared with pregnant women with adequate iodine nutrition status? 2. Is there a difference in the urinary iodine concentration (UIC) of pregnant women with pre-eclampsia versus that of normotensive pregnant women? MEthOds This protocol is developed following the Preferred Reporting Items for Systematic reviews and Meta-Analysis protocols (PRISMA-P) 2015 Guidelines. 27 Eligibility criteria Inclusion criteria The selection of studies for inclusion in the review will be guided by the Population, Intervention/exposure, Comparison and Outcome protocol as stipulated below: 1. Population: pregnant women. 2. Exposure: the exposure is insufficient iodine nutrition status during pregnancy for both case-control and cohort studies. In this systematic review this will be defined according to the WHO/International Council for Control of Iodine Deficiency Disorders (ICCIDD) classification of iodine intake of populations using median UIC. 28 For pregnant women, a urine iodine concentration (UIC) <150, 150-249, 250-499 and >500 µg/L is considered an estimate of, respectively, insufficient, adequate, more than adequate and excessive iodine nutritional status. 3. Comparator: for both case-control and cohort studies the comparator will be the participants with sufficient iodine nutrition status (UIC >150 µg/L) during pregnancy. 4. Outcome: the outcome is the prevalence (for casecontrol studies) and the incidence (for cohort studies) of pre-eclampsia among women with and without adequate iodine nutrition status in pregnancy from which the ORs will be determined. Pre-eclampsia has been defined as new onset hypertension after 20 weeks of amenorrhoea characterised by elevated systolic blood pressure of 140 mm Hg or diastolic blood pressure of 90 mm Hg or more or both, measured twice with a gap of 4 hour or one measurement of systolic blood pressure of ≥160 mm Hg or diastolic blood pressure of ≥110 mm Hg or both accompanied by one of the following: proteinuria in 24 hour-urine ≥300 mg or protein/creatinine ratio ≥0.3 or urine protein measured by dipstick ≥1+; or thrombocytopenia (platelets less than 150 000/µL), kidney insufficiency (concentration of creatinine in serum above 97 µmol/L), decreased liver function (enzyme activity of Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) twice higher than the upper limit of the referential interval), compromised pulmonary function or pulmonary oedema or visual or other symptoms and signs of deficient cerebral function. 29 There may be considerable heterogeneity if pre-eclampsia has been variably defined in different studies that are eligible for inclusion in the current systematic review. Exclusion criteria ► Studies in which none of the following parameters was computed: means, medians, ORs, incidence and prevalence rates and with absence of data to compute them. ► Eligible studies that are missing some critical data where after repeated attempts to contact an author via email for relevant information, no response is gotten. ► Letters to editors, reviews, commentaries, editorials and any publication without primary data. ► Duplicate publications from the same study. For studies published in more than one journal/conference, the most recent and comprehensive publication will be used. ► Studies not performed in human participants. ► No language restriction will be applied. Patient and public involvement The public or patients were not involved in the development of this protocol. search strategy for study identification Electronic searches We will search PubMed MEDLINE, Google Scholar, SCOPUS, ISI Web of Science (Science Citation Index) databases for all published studies on iodine deficiency and pre-eclampsia up to 31 December 2018. This search shall be conducted using a predefined comprehensive and sensitive search strategy combining relevant terms and synonyms which are variably used to denote abnormally high blood pressure in pregnancy and insufficient iodine intake or iodine deficiency. Table 1 depicts the main search strategy to be employed for MEDLINE database that will also be adapted for searches in other electronic databases. We will search reference lists of relevant citations for articles of interest. Grey literature We will contact experts in the field, research organisations, conference websites and conference proceedings that dealt with micronutrient deficiency and pre-eclampsia, for any relevant data. study records Data management We will use an appropriate citation management software to remove duplicates from the references articles that will have been gathered. Prior to screening of studies, we will create a set of standardised questions according to the inclusion criteria which will then be pretested on a sample of eligible studies. Screening Two investigators will independently select studies that meet inclusion criteria. Citations and abstracts will be screened for possible inclusion, and duplicate citations will be excluded. Titles and abstracts will then be screened following inclusion criteria described above, following which the full texts of potentially eligible articles will be obtained. These full texts will be screened using a standardised and pretested form to include eligible studies. Disagreements will be resolved by consensus, with consultation of a third author (when resolution cannot be achieved). Corresponding authors of potentially eligible studies that did not report data that are relevant to our study analysis will be contacted. Reasons for exclusion of non-eligible studies will be documented. The whole review process will be summarised in a flowchart. Data extraction Two investigators will independently extract data from included studies, using a standardised and pretested data extraction form. Any inconsistencies or disagreement shall be resolved by consensus or consultation with the third investigator. Data items Data will include the geographic region and country where study was conducted, the year study was carried out and year of publication, the language of publication, demographic characteristics of participants, time of measurement of exposure (such as first, second or third trimester of pregnancy or preconception), study design, setting (rural or urban, health-facility or community-based), sample size and the criteria used for determination of the iodine intake. For cross-sectional and case-control studies, the median (25th-75th percentiles) and or mean (SD) UIC will be extracted. The overall mean UIC for cases and controls together with the mean UIC difference will be determined. The proportions of women with insufficient iodine nutrition among cases and controls will be extracted or determined based on the standard definition by WHO and the ICCIDD. Then the odds or data required to compute the odds of insufficient iodine nutrition among the cases will be extracted. Where applicable adjusted ORs will be extracted or computed so account for traditional risk factors for pre-eclampsia such as obesity, primigravida, advanced maternal age or diabetes. For cohort studies, the incidence rates and relative risk of pre-eclampsia according to baseline status for iodine nutrition will be extracted. Assessment of methodological quality and risk of bias Two reviewers will independently score the quality of included studies. The risk of bias in individual studies will be assessed using the Newcastle-Ottawa scale 30 Open access Cochrane guidelines available in Review Manager V.5.3 (http:// tech. cochrane. org/ revman). Discrepancies will be resolved by consensus or by consulting the third investigator. Inter-rater agreement on screening, data abstraction and methodological quality (selection, comparability of groups and ascertainment of exposure/outcome) will be assessed using Cohen's κ coefficient. 31 Data synthesis, analysis and assessment of heterogeneity For data unsuitable for meta-analysis, we will provide a narrative description of major study characteristics and findings. For outcomes of interest consistently reported across studies, random effects model meta-analyses will be used to pool estimates across those studies. 32 In process, the Freeman-Tukey double Arcsine and squareroot transformation will be applied, respectively, to stabilise the variances of prevalence and incidence rates prior to meta-analysis, and estimates back-transformed for reporting. The degree of heterogeneity across studies will be assessed using the Cochrane Q statistic and inconsistency index (I 2 ) statistic and will be classified respectively as low: I 2 <25%; moderate: 25%-50%; high: I 2 >50%). 33 For studies with high heterogeneity subgroup and metaregression analyses will be performed to investigate the sources of heterogeneity. The following grouping variables will be used where appropriate: time of measurement of exposure (first, second or third trimester), study setting (rural vs urban, health-facility vs community based), geographical region (continental and or endemicity of iodine deficiency), high income versus low or middle-income status, and study quality. The Begg test and Egger funnel plot will be used to check for the publication bias. 34 35 sensitivity analysis Sensitivity analysis will be carried out to check the effect of every study on pooled estimates by removing one study at a time and assessing the effect on pooled estimates and heterogeneity statistics. The Duval and Tweedie trim-andfill will be used to adjust estimates for the effects of potential publication bias. Data analyses will use the 'meta' package of the statistical software R (V.3.3.3 [2017-03-06], The R Foundation for statistical computing, Vienna, Austria), and the 'meta' package. Confidence in cumulative evidence We will assess the strength of evidence provided by studies included in the review, using the Grading of Recommendations Assessment, Development and Evaluation approach. This assessment of the quality of evidence would include risk of bias, consistency and publication bias. Studies in which further research is unlikely to change effect estimates, or likely to have a considerable impact on effect estimates, or capable of changing the effect estimates, or those in which there is uncertainty in effect estimates, will be respectively described as 'high', 'moderate', 'low' or 'very low' qualities. Reporting of this review The proposed systematic review will be reported following the PRISMA guidelines. 36 We intend to publish a PRISMA checklist alongside the final report. Potential amendments We do not intend to make any amendments to the protocol, to avoid the possibility of outcome reporting bias. However, any amendments that do prove necessary will be documented and reported transparently. COnClusIOn Iodine deficiency whose degree is exacerbated by pregnancy has recently been associated with oxidative imbalance and endothelial dysfunction which are some of the pathophysiological mechanisms that precede the clinical manifestation of pre-eclampsia. Although iodine deficiency is the the most common cause of hypothyroidism, one of the established risk factors of pre-eclampsia, the association between iodine deficiency in pregnancy and pre-eclampsia is still uncertain. This review aims to decipher if iodine deficiency in pregnancy, which can be corrected through supplementation, increases the risk of pre-eclampsia in various settings worldwide. Possible limitations of this study would include very few studies with small sample sizes and uncertain quality that may generate significant heterogeneity precluding further analysis. Ethics and dissemination The current study is based on published data, and hence does not require ethical approval. This review is part of the thesis that will be submitted for the award of a PhD in Medicine to the Faculty of Health Sciences of the University of Cape Town. The final report of this review in the form of a scientific paper will be published in a peer-reviewed journal. Findings will also be presented at conferences and submitted to relevant health and policy authorities. We also plan to update the review in the future to monitor any progressive changes on the subject.	2019-05-28T13:10:05.596Z	2019-05-01T00:00:00.000	{ "year": 2019, "sha1": "9a84b47196034fc694cd26d24b9645ce7de8b405", "oa_license": "CCBYNC", "oa_url": "https://bmjopen.bmj.com/content/bmjopen/9/5/e025573.full.pdf", "oa_status": "GOLD", "pdf_src": "Highwire", "pdf_hash": "34f74a470a985d8ed60642ab6a04dd0e4ffa211c", "s2fieldsofstudy": [ "Medicine", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
236162738	pes2o/s2orc	v3-fos-license	Traces of Semantization, from Episodic to Semantic Memory in a Spiking Cortical Network Model Abstract Episodic memory is a recollection of past personal experiences associated with particular times and places. This kind of memory is commonly subject to loss of contextual information or “semantization,” which gradually decouples the encoded memory items from their associated contexts while transforming them into semantic or gist-like representations. Novel extensions to the classical Remember/Know (R/K) behavioral paradigm attribute the loss of episodicity to multiple exposures of an item in different contexts. Despite recent advancements explaining semantization at a behavioral level, the underlying neural mechanisms remain poorly understood. In this study, we suggest and evaluate a novel hypothesis proposing that Bayesian–Hebbian synaptic plasticity mechanisms might cause semantization of episodic memory. We implement a cortical spiking neural network model with a Bayesian–Hebbian learning rule called Bayesian Confidence Propagation Neural Network (BCPNN), which captures the semantization phenomenon and offers a mechanistic explanation for it. Encoding items across multiple contexts leads to item-context decoupling akin to semantization. We compare BCPNN plasticity with the more commonly used spike-timing-dependent plasticity (STDP) learning rule in the same episodic memory task. Unlike BCPNN, STDP does not explain the decontextualization process. We further examine how selective plasticity modulation of isolated salient events may enhance preferential retention and resistance to semantization. Our model reproduces important features of episodicity on behavioral timescales under various biological constraints while also offering a novel neural and synaptic explanation for semantization, thereby casting new light on the interplay between episodic and semantic memory processes. Introduction Episodic and semantic memory were originally proposed as distinct systems that compete in retrieval (Tulving, 1972). More recent studies suggest, however, that this division is rather vague (McCloskey and Santee, 1981;Howard and Kahana, 2002;Renoult et al., 2019), as neural correlates of episodic and semantic retrieval overlap (Weidemann et al., 2019). Episodic memory traces are susceptible to transformation and loss of information (Tulving, 1972), and this loss of episodicity can be attributed to semantization, which typically takes the form of a decontextualization process (Viard et al., 2007;Habermas et al., 2013;Duff et al., 2020). Baddeley (1988) hypothesized that semantic memory might represent the accumulated residue of multiple learning episodes, consisting of information which has been semanticized and detached from the associated episodic contextual detail. Extensions of the classical Remember/Know (R/K) behavioral experiment demonstrated that item-context decoupling can occur rapidly (Opitz, 2010). In these experiments, items were presented either in a unique context, or across several contexts. Low context variability improved the recollection rate, whereas context overload led to decontextualization and "Know" type of responses, i.e., recognition of item-only information without any detail about episodic context (Opitz, 2010;Smith and Manzano, 2010;Smith and Handy, 2014). To the best of our knowledge, there have not been any computational hypotheses proposed to offer mechanistic insights into this item-context decoupling effect. Several computational spiking neural network models of cortical associative memory have previously been developed and used to investigate mechanisms underlying working memory maintenance and recall (Lundqvist et al., 2010(Lundqvist et al., , 2011Herman et al., 2013). A similar model enhanced with a Bayesian-Hebbian learning rule (Bayesian Confidence Propagation Neural Network; BCPNN) representing synaptic and intrinsic plasticity was then used to study one-shot memory encoding (Fiebig and Lansner, 2017), and more recently, it was extended into a multinetwork cortical model to examine a novel "indexing theory" of working memory (Fiebig et al., 2020). In the present study, relying on a similar spiking neural network model with identical modular architecture we propose and evaluate a Bayesian-Hebbian hypothesis about synaptic and network mechanisms underlying memory semantization and qualitatively match model output to available behavioral data. We show that associative binding between items and contexts becomes weaker when an item is presented across multiple contexts (high context variability). This gradual trace transformation relies on the nature of Bayesian learning, which normalizes and updates weights over estimated presynaptic (Bayesian-prior) as well as postsynaptic (Bayesian-posterior) spiking activity. We compare these findings with an analogous model that features the more well-known spike-timing-dependent plasticity (STDP) instead of the BCPNN learning rule, and demonstrate that no memory semantization effect can be reproduced, regardless of the degree of context variability. Notably, there have been earlier modeling attempts at semantization using STDP or other learning rules but this memory phenomenon has been interpreted differently involving slow memory consolidation (requiring sleep, repeated exposures, or systems consolidation) or extraction of semantic relations (a.k.a. prototype learning) among a group of episodic memories sharing statistical similarities (Deperrois et al., 2021;Remme et al., 2021). We argue that our hypothesis is more generic as it does not assume any statistical structure of the memory object representations. Finally, we also show how selective plasticity neuromodulation of one-shot learning (tentatively modeling effects of attention, emotional salience, and surprise on plasticity) may delay or prevent decontextualization. In contrast to existing computational models of episodic memory (Norman and O'Reilly, 2003;Wixted, 2007;Greve et al., 2010), our model bridges behavioral outcomes with neural and synaptic mechanisms. It reproduces episodic memory phenomena on behavioral time scales under constrained network connectivity with plausible postsynaptic potentials, firing rates, and other biological parameters. Neuron and synapse model We use adaptive exponential integrate-and-fire point model neurons, which feature spike frequency adaptation, enriching neural dynamics and spike patterns, especially for the pyramidal cells (Brette and Gerstner, 2005). The neuron model is an effective model of cortical neuronal activity, reproducing a wide variety of electrophysiological properties, and offers a good phenomenological description of typical neural firing behavior, but it is limited in predicting the precise time course of the subthreshold membrane voltage during and after a spike or the underlying biophysical causes of electrical activity (Gerstner and Naud, 2009). We slightly modified it for compatibility with the BCPNN synapse model (Tully et al., 2014) by integrating an intrinsic excitability current. Development of the membrane potential V m and the adaptation current I w is described by the following equations: Vm ÀV t Dt À I w 1I ext 1I syn (1) dI w dt ¼ ÀI w t Iw 1 bd ðt À t sp Þ: Equation 1 describes the dynamics of the membrane potential V m including an exponential voltage dependent activation term. A leak current is driven by the leak reversal potential E L through the conductance g L over the neural surface with a capacity C m . Additionally, V t is the spiking threshold, and D T shapes the spike slope factor. After spike generation, membrane potential is reset to V r . Spike emission upregulates the adaptation current by b, which recovers with time constant t I w (Table 1). To simplify the model, we have removed subthreshold adaptation, which is part of some AdEx models. Besides a specific external input current I ext , model neurons receive synaptic currents I syn j from conductance based glutamatergic and GABAergic synapses. Glutamatergic synapses feature both AMPA/NMDA receptor gated channels with fast and slow conductance decay dynamic, respectively. Current contributions for synapses are described as follows: The glutamatergic synapses are also subject to synaptic depression and augmentation with a decay factor t D and t A , respectively (Table 1), following the Tsodyks-Markram formalism (Tsodyks and Markram, 1997). We have chosen those time-constants from the plausible range of computational fits made on the basis of electrophysiological recordings of cortical pyramidal cells (Wang et al., 2006). The utilization factor u represents the fraction of available resources used up by each transmitted spike (a proxy of synaptic release probability), whereas x tracks the fraction of resources that remain available because of transmitter depletion (synaptic depression): Spike-based BCPNN plasticity We implement synaptic plasticity of AMPA and NMDA connection components using the BCPNN learning rule (Lansner and Ekeberg, 1989;Wahlgren and Lansner, 2001;Tully et al., 2014). BCPNN is derived from Bayes rule, assuming a postsynaptic neuron employs some form of probabilistic inference to decide whether to emit a spike or not. Despite that it accounts for the basic Bayesian inference, it is considered more complex than the standard STDP learning rule (Caporale and Dan, 2008), and as such, it reproduces the main features of STDP plasticity. The BCPNN synapse continuously updates three synaptic biophysically plausible local memory traces, P i , P j , and P ij , implemented as exponentially moving averages (EMAs) of preactivation, postactivation, and coactivation, from which the Bayesian bias and weights are calculated. EMAs prioritize recent patterns, so that newly learned patterns gradually replace old memories. Specifically, learning implements exponential filters, Z, and P, of spiking activity with a hierarchy of time constants, t z , and t p , respectively [the full BCPNN model implements additional eligibility E traces (Tully et al., 2014), which are not used here]. Because of their temporal integrative nature, they are referred to as synaptic (local memory) traces. To begin with, BCPNN receives a binary sequence of presynaptic and postsynaptic spiking events (S i , S j ) to calculate the traces Z i and Z j : f max denotes the maximal neuronal spike rate, e is the lowest attainable probability estimate, t spike denotes the spike duration while t z i ¼ t z j are the presynaptic and postsynaptic time constants, respectively (t z ¼ t AMPA ¼ 5 ms for AMPA, and t z ¼ t NMDA ¼ 100 ms for NMDA components; Table 1). P traces are then estimated from the Z traces as follows: The parameter k adjusts the learning rate, reflecting the action of endogenous modulators of learning efficacy (i.e., activation of a D1R-like receptor). Setting k = 0 freezes the network's weights and biases, though in our simulations the learning rate remains constant (k = 1) during encoding (see Results, Semantization of episodic representations in the BCPNN model and Item-context interactions under STDP). However, we trigger a transient increase of plasticity in specific scenarios to model preferential retention of salient events (see Results, Preferential retention; Table 1). Finally, P i , P j , and P ij are used to calculate intrinsic excitability b j and synaptic weights w ij with a scaling factor b gain and w syn gain respectively (Table 1): Spike-based STDP learning rule In our study, we examine the impact on semantization when the STDP learning rule replaces BCPNN associative connectivity in the same episodic memory task. Synapses under STDP are developed and modified by a repeated pairing of presynaptic and postsynaptic spiking activity, while their relative time window shapes the degree of modification (Ren et al., 2010). The amount of trace modification depends on the temporal difference (D t ) between the time point of the presynaptic action potential (t i ) and the occurrence of the postsynaptic spike (t j ) incorporating a corresponding transmission delay (t d ) from neuron i to neuron j: After processing Dt, STDP updates weights accordingly: Here, l corresponds to the learning rate, a reflects a possible asymmetry between the scale of potentiation and depression, t 6 control the width of the time window, while m 6 [ {0,1} allows to choose between different versions of STDP (i.e., additive, multiplicative; Morrison et al., 2008). Synapses are potentiated if the synaptic event precedes the postsynaptic spike and get depressed if the synaptic event follows the postsynaptic spike (Van Rossum et al., 2000). Associative weights w ij are initialized to w 0 , and their maximum allowed values are constrained according to w max to ensure that synaptic weights are always positive and between [w 0 ; w max ] ( Table 2). The resulting associative weight distributions are generally comparable in strength to the BCPNN model weights, but to make them match, we adjust w max in conjunction with a reasonably small learning rate l . The maximum allowed weight (w max ) is a necessary standard parameter of the default STDP we use in NEST (see below, Code accessibility). To obtain a stable competitive synaptic modification, the integral of Dw ij must be negative (Song et al., 2000). To ensure this, we choose a = 1.2, which introduces an asymmetry between the scale of potentiation and depression along with a symmetric time window resulting in a ratio of at À =t 1 .1.0 (Ren et al., 2010). We set m 6 = 1 resulting in multiplicative STDP (in-between values lead to rules which have an intermediate dependence on the synaptic strength). Two-network architecture and connectivity The network model features two reciprocally connected networks, the so-called Item and Context networks. For simplicity, we assume that Item and Context networks are located at a substantial distance accounting for the reduced internetwork connection probabilities (Table 3). Each network follows a cortical architecture with modular structure compatible with previous spiking implementations of attractor memory networks (Lansner, 2009;Lundqvist et al., 2011;Tully et al., 2014Tully et al., , 2016Fiebig and Lansner, 2017;Chrysanthidis et al., 2019;Fiebig et al., 2020), and is best understood as a subsampled cortical layer 2/3 patch with nested hypercolumns (HCs) and minicolumns (MCs; Fig. 1A). Both networks span a regular-spaced grid of 12 HCs (Table 3), each with a diameter of 500 mm (Mountcastle, 1997). In our model, items are embedded in the Item network and context information in the Context network as internal well consolidated long-term memory representations (cell assemblies), supported via intranetwork weights derived using prior MCs. We preload abstract long-term memories of item and context representations into the respective network, in the form of distributed cell assemblies with weights establishing corresponding attractors. Associative plastic connections bind items with contexts. The network features lateral inhibition via basket cells (purple and blue lines) resulting in a soft winner-take-all dynamics. Competition between attractor memories arises from this local feedback inhibition together with disynaptic inhibition between HCs. B, Weight distribution of plastic synapses targeting pyramidal cells. The attractor projection distribution is positive with a mean of 2.1, and the disynaptic inhibition is negative with a mean of À0.3 (we show the fast AMPA weight components here, but the simulation also includes slower NMDA weight components). C, Weight matrix between attractors and competing MCs across two sampled HCs. The matrix displays the mean of the weight distribution between a presynaptic (MC pre ) and postsynaptic MC (MC post ), within the same or different HC (black cross separates grid into blocks of HCs, only two of which are shown here). Recurrent attractor connections within the same HC are stronger (main diagonal, dark red) compared with attractor connections between HCs (off-diagonals, orange). Negative pyramidal-pyramidal weights (blue) between competing MCs amounts to disynaptic inhibition mediated by double bouquet cells. Research Article: New Research MCs per HC (;100 MCs per HC in biological cortex). The high degree of recurrent connectivity within (Thomson et al., 2002;Yoshimura and Callaway, 2005) and between MCs links coactive MCs into larger cell assemblies (Stettler et al., 2002;Binzegger et al., 2009;Muir et al., 2011;Eyal et al., 2018). Long-range bidirectional internetwork connections (item-context bindings or associative connections) are plastic (shown in Fig. 1A only for MC1 in HC1 of the Context network), binding items, and contextual information (Ranganath, 2010). On average, recurrent connectivity establishes 100 active plastic synapses onto each pyramidal cell from other pyramidals with the same selectivity, because of a sparse internetwork connectivity (cp PPA ) and denser local connectivity (cp PP , cp PPL ; connection probability refers to the probability that there is a connection between a randomly selected pair of neurons from given populations; in Fig. 1A, connection probabilities are only shown for MC1 in HC1 of the Context network). The model yields biologically plausible EPSPs for connections within HCs (0.45 6 0.13 mV), measured at resting potential E L (Thomson et al., 2002). Densely recurrent nonspecific monosynaptic feedback inhibition mediated by fast spiking inhibitory cells (Kirkcaldie, 2012) implements a local winner-take-all structure (Binzegger et al., 2009) among the functional columns. IPSPs have an amplitude of À1.160 mV (60.003) measured at À60 mV (Thomson et al., 2002). These bidirectional connections between basket and pyramidal cells within the local HCs are drawn with a 70% connection probability. Notably, double bouquet cells shown in Figure 1A are not explicitly simulated, but their effect is nonetheless expressed by the BCPNN rule. A recent study based on a similar single-network architecture (i.e., with the same modular organization, microcircuitry, conductancebased AdEx neuron model, cell count per MC and HC) demonstrated that learned mono-synaptic inhibition between competing attractors is functionally equivalent to the disynaptic inhibition mediated by double bouquet and basket cells (Chrysanthidis et al., 2019). Parameters characterizing other neural and synaptic properties including BCPNN can be found in Table 1. Figure 1B shows the weight distributions of embedded distributed cell assemblies, representing different memories stored in the Item and Context networks. Attractor projections can be further categorized into strong local recurrent connectivity within HCs, and slightly weaker long-range excitatory projections across HCs (Fig. 1C). Axonal conduction delays Conduction delays (t ij ) between a presynaptic neuron i and a postsynaptic neuron j are calculated based on their Euclidean distance, d, and a conduction velocity V (Eq. 11). Delays are randomly drawn from a normal distribution with a mean according to distance and conduction velocity, with a relative SD of 30% of the mean to account for individual arborization differences, and varying conduction speed as a result of axonal thickness and myelination. In addition, a minimal delay of 1.5 ms (t syn min ; Table 3) is added to reflect synaptic delays because of effects that are not explicitly modelled, e.g., diffusion of neurotransmitters over the synaptic cleft, dendritic branching, thickness of the cortical sheet and the spatial extent of columns (Thomson et al., 2002). Associative internetwork projections have a 10-fold faster conduction speed than those within each network, reflecting axonal myelination: Stimulation protocol Noise input to pyramidal cells and fast spiking inhibitory basket cells is a zero-mean noise, generated by two independent Poisson generators with opposing driving potentials. Pyramidal cells coding for specific items and contexts are stimulated with an additional specific excitation during encoding and cued recall (all parameters in Table 3). Item-context association encoding is preceded by a brief period of background noise excitation to avoid initialization transients. Attractor activation detector We detect and report cued recall of items or contexts by using an attractor activation detection algorithm based on EMAs of spiking activity. Pattern-wise EMAs are calculated using Equation 12, where the d function d denotes the spike events of a pattern-selective neural population of n pop = 30 pyramidal cells. The filter time constant t = 40 ms is much larger than the sampling time interval DT = 1 ms: Pattern activations are detected by a simple threshold (r th ) at about 10-fold the baseline activity with a small caveat: to avoid premature offset detection because of synchrony in fast spiking activity, we only count activations as terminated if they do not cross the threshold again in the next 40 ms. This method is highly robust because of the explosive dynamics of recurrent spiking activity for activated attractors in the network. Any attractor activation that crosses this threshold for at least 40 ms is considered a successful recall. Semantization of episodic representations in the BCPNN model An episodic memory task simulated in this work is inspired by a seminal memory effect shown in an experimental study by Opitz (2010). We deliberately abstract away some details of Opitz (2010) experimental design to provide a qualitative proof of principle with as few task assumptions as possible. This approach also offers a more generalized computational framework for studying the interplay of synaptic learning and its outcomes. In the same spirit, the systems architecture of our model is reduced to the Item and Context networks storing item and context information, respectively, as internal long-term memory representations ( Fig. 1; for details, see Two-network architecture and connectivity and Table 3). We stimulate some items in a single context and others in a few different contexts establishing multiple associations (Fig. 2). Stimulus duration during encoding is t stim = 250 ms with a T stim = 500 ms interstimulus interval, and a test phase occurs after a 1-s delay period, which contains brief t cue = 50-ms cues of previously learned items (Table 3). Figure 3A illustrates an item-context pair, established by an associative binding through plastic bidirectional BCPNN projections (dashed lines). Item and context attractors (solid red lines) are embedded in each network and remain fixed throughout the simulation, representing well-consolidated long-term memory. We show an exemplary spike raster of pyramidal neurons in HC1 of both the Item and Context networks reflecting a trial simulation (Fig. 3B). Herein, item-3 (blue) establishes a single association, while item-4 (yellow) is encoded in four different contexts (Figs. 2A, 3B). We find clear evidence for strong item-context decoupling. The yellow item-4 (but not the blue item-3) is successfully recognized when cued but without any corresponding accompanying activation in the Context network (Fig. 3B). Figure 3C demonstrates that this itemcontext decoupling effect holds true also for the multi trial average as the performance of contextual retrieval when items serve as cues deteriorates with a higher context overload. Successful item recognition without any contextual information retrieval accounts for a "Know" response, as opposed to "Remember" judgments, which are accompanied by context recall. In fact, episodic loss in our network implies that no context is recalled despite the item memory activation. To elucidate this observed progressive loss of episodicity with the higher context variability (Fig. 3C), we sample and analyze the learned weight distributions of itemcontext binding recorded after the association encoding period (Fig. 3D). The item-context weight distribution in the one-association case is significantly stronger than in the two-association, three-association, or four-association case (p , 0.001, Mann-Whitney, N = 2000). This progressive weakening of weights leads to significantly lower EPSPs for the associative projections (p , 0.05 for one vs two associations; p , 0.001 for two vs three and three vs four associations, Mann-Whitney, N = 300; Fig. 3D, see inset). To measure EPSPs, we stimulate individually all the neurons in HC1 of an item which forms one, two, three, or four Fig. 2A). Each item is drawn with a unique color, while contexts inherit their coactivated item's color in the raster (i.e., the yellow pattern in the Item network is repeated over four different contexts, forming four separate associations marked with the same color). The testing phase occurs 1 s after the encoding. Brief 50-ms cues of already studied items trigger their activation. Following item activation, we detect evoked attractor activation in the Context network. C, Average cued recall performance in the Context network (20 trials). The bar diagram reveals progressive loss of episodic context information (i.e., semantization) over the number of context associations made by individual cued items (compare Fig. 2A). D, Distribution of plastic connection weights between the Item and Context networks (NMDA component shown here). Weights are noticeably weaker for items which participate in multiple associations. The distributions of synaptic weights exhibit a broader range for the items with multiple context associations, as the sample size is larger. The inset displays the distribution of EPSPs for the binding between Item and Context networks. The EPSP distributions follow the trend of the associative weights. The amplitudes (,1 mV) are lower for higher context variability. E, The distribution of intrinsic excitability currents of pyramidal cells coding for specific context representations. The intrinsic excitability features similar distributions because each context is activated exactly once, regardless of whether the associated item forms multiple associations or not. F, Average cued recall performance in the Item network (20 trials). Decontextualization over the number of associations is also observed when we briefly cue episodic contexts instead (compare Fig. 2B). G, Distribution of strength of plastic connections from the contexts to their associated items. Analogously to D, synapses weaken once an item is encoded in another context. H, Intrinsic plasticity distribution of cells in the Item network. Intrinsic excitability distributions are higher for pyramidal cells coding for repeatedly activated items; *p , 0.001 (Mann-Whitney, N = 20 in C, F). Error bars in C, F represent SDs of Bernoulli distributions. Distributions of one, two, three, and four associations in D, G, H show significant statistical difference (p , 0.001, Mann-Whitney, N = 2000). associations and record the postsynaptic potential onto their associated context neurons. EPSPs are in general below 1 mV (Thomson et al., 2002;Song et al., 2005), measured at resting potential E L (Table 1), after item-context association encoding phase. Therefore, we attribute the loss of episodicity to a statistically significant weakening of the associative weight distributions with the increasing number of associated contexts. The associative weight distributions shown here refer to the NMDA component, while the weight distributions of the faster AMPA receptor connections display a similar trend (data not shown). The gradual trace modification we observe is a product of Bayesian learning, which normalizes and updates weights over estimated presynaptic (prior) as well as postsynaptic (posterior) spiking activity (see below, BCPNN and STDP learning rule in a microcircuit model for details). Our simulation results are in line with related behavioral studies (Opitz, 2010;Smith and Manzano, 2010;Smith and Handy, 2014), which also reported item-context decoupling as the items were presented across multiple contexts. In particular, Opitz (2010) concluded that repetition of an item across different contexts (i.e., high context variability) leads to item-context decoupling, which is in agreement with our study. Furthermore, Smith and Manzano (2010) demonstrated in an episodic context variability task configuration, that recall deteriorates with context overload (number of words per context). Mean recall drops from ;0.65 (one word per context) to 0.50 (three words per context), reaching ;0.33 in the most overloaded scenario (fifteen words per context). In Figure 3E, we show the distribution of intrinsic excitability over units representing different contexts. Pyramidal neurons in the Context network have a similar intrinsic excitability, regardless of their selectivity because all the various contexts are encoded exactly once. Next, analogously to the previous analysis, we show that item-context decoupling emerges also when we briefly cue contexts rather than items during recall testing (Fig. 2B). In agreement with experimental data (Smith and Manzano, 2010;Smith and Handy, 2014), we obtain evidence of semantization as items learned across several discrete contexts are hardly retrieved when one of their associated contexts serves as a cue (Fig. 3F). We further sample and present the underlying associative weight distribution, between the Context and the Item networks (Fig. 3G). The distributions again reflect the semantization effect in a significant weakening of the corresponding weights. In other words, an assembly of pyramidal neurons representing items encoded across multiple contexts receives weaker projections from the Context network. At about four or more associations, the itemcontext binding becomes so weak that it fails to deliver sufficient excitatory current to trigger associated representations in the Item network. At the same time, intrinsic excitability of item neurons increases with the number of associated contexts corresponding to how much these neurons were active during the encoding phase ( Item-context interactions under STDP In this section, we contrast the results obtained with the BCPNN synaptic learning rule with those deriving from the more commonly used STDP learning rule in the same episodic memory task ( Fig. 2; Spike-based STDP learning rule). The modular network architecture as well as neural properties and embedded memory patterns remain identical, but associative projections between networks are now implemented using a standard STDP synaptic learning rule (Morrison et al., 2008). The parameters of the STDP model are summarized in Table 2. Figure 4A shows an exemplary spike raster of pyramidal cells in HC1 of both the Item and the Context networks, based on the first variant of the episodic memory task described in Figure 2A. As earlier, items are encoded in a single or in multiple different contexts and they are briefly cued later during recall. A successful item activation may lead to a corresponding activation of its associated information in the Context network. We detect these activations as before (see Materials and Methods, Attractor activation detector) and report the cued recall score over the number of associations (Fig. 4B). Unlike the BCPNN network, we observe no evidence of semantization for high context variability. Instead, recollection is noticeably enhanced with an increase in the number of associations, which is in fact the opposite of what would be needed to explain item-context decoupling. STDP generates similarly strong associative binding regardless of context variability (Fig. 4C). The enhanced recollection in high context variability cases stems from the multiplicative effect of synaptic augmentation in the Tsodyks-Makram model on the Hebbian attractor weights. Items stimulated multiple times (e.g., four times) have a higher likelihood of being encoded near the end of the task, leading to more remaining augmentation during testing, thus, effectively boosting cued recall (Fig. 5A). This recency effect diminishes after removing synaptic augmentation from the model as attractor weights in the Item network have comparable distributions leading to similar cued recall performance regardless of context variability (Fig. 5B,C). As far as the context-cued variant of the task is concerned (Fig. 2B), there are also no signs of item-context decoupling for high context variability (Fig. 4D). The associative projections between Context and Item networks again have distributions with comparable means over context variability (Fig. 4E). Overall, decontextualization is not evident in either variant of the episodic memory task under the STDP learning rule. BCPNN and STDP learning rules in a microcircuit model To better elucidate the emergent synaptic changes of the BCPNN and STDP model, we also apply these learning rules in a highly reduced microcircuit of spiking neurons. To this end, we now track the synaptic weight changes continuously. The neural and synaptic parameters (and most importantly all the plasticity parameters) used for the highly reduced BCPNN and STDP model are identical to the ones used for the large scale BCPNN and STDP model, respectively (see Tables 1, 2). First, we apply the BCPNN learning rule to the microcircuit model. We consider two separate item neurons (ID = 1 and 2), which form two or three associations with context neurons (ID = 3, 4, or 5, 6, 7), respectively (Fig. 6A). We display the synaptic strength development of the synapse between item neuron-1 and context neuron-3 (two associations, green), as well as the synapse between item neuron-2 and context neuron-5 (three associations, red) over the course of training these associations via targeted stimulation. BCPNN synapses get strengthened when the item-context pairs are simultaneously active and weaken when the item in question is activated with another context. Therefore, synapses of the item neuron that is encoded in three different contexts converge on weaker weights (Fig. 6A, 12 s), than those of the item neuron with two associated contexts. Weight modifications in the microcircuit model reflect the synaptic alterations observed in the large-scale network. BCPNN weights are shaped by traces of activation and coactivation (Eqs. 7, 8; Materials and Methods), which also get updated during the activation of an item within another context. For example, the item neuron-1 and context neuron-3 are not . Network model where associative projections are implemented using standard STDP synaptic plasticity. A, Spike raster of pyramidal neurons in HC1 of both the Item and Context networks. B, Average item-cued recall performance in the Context network (20 trials). Episodic context retrieval is preserved even for high context variability (as opposed to BCPNN; compare Fig. 3C). C, Distribution of NMDA receptor mediated synaptic weights between the item and context neural assemblies following associative binding. The distributions of item-context weights have comparable means at ;0.065 nS regardless of how many context associations a given item forms. Bins merely display a higher count for the four-association case as the total count of associative weights is more extensive compared with items with fewer associations. D, Average cued recall performance in the Item network when episodic contexts are cued (20 trials). E, Distribution of NMDA component weights between associated context and item assemblies; p , 0.001 (Mann-Whitney, N = 20 in B, D). Error bars in B, D represent SDs of Bernoulli distributions. stimulated together between 6 and 8 s, but neuron-1 and context neuron-4 are. Thus, the P traces of the item activation (P i ) increase, while the ones linked to context-3 (P j ) decay with a time constant of 15 s (Table 1). Since the item and context neuron (ID = 1, 3) are not stimulated together, their coactivation traces (P ij ) decay between 6 and 8 s. Overall, this leads to a weakening of the weight and hence to a gradual decoupling (Eq. 8; Materials and Methods). In the same manner, we keep track of weight change in a microcircuit with the STDP learning rule (Fig. 6B). Unlike the microcircuit with BCPNN presented in Figure 6A, the STDP weights corresponding to the associations made by both item neurons converge to similar values, although they are associated with different number of contexts. As before, the synapse between an item neuron and an associated context neuron strengthens when this pair is simultaneously active, but remains stable when the item neuron is encoded in another context. For instance, the synapse between item neuron-2 and context neuron-5 strengthens when this pair is encoded (0-2 s), yet remains unaffected when item neuron-2 is activated in another context (i.e., context neuron-6, 4-6 s). This synaptic behavior explains the observed differences between the BCPNN and STDP largescale model. Preferential retention Several studies propose that one-shot salient events promote learning, and that these memories can be retained on multiple time scales ranging from seconds to years (Frankland et al., 2004;Petrican et al., 2010;Gruber et al., 2016;Panoz-Brown et al., 2016;Eichenbaum, 2017;Sun et al., 2018). Hypothetical mechanisms behind these effects are dopamine release and activation of DR1 like receptors, resulting in synapse-specific enhancement (Otmakhova and Lisman, 1996;Kuo et al., 2008), and systems consolidation (McClelland et al., 1995; Fiebig and Neural and synaptic parameters correspond to those in the scaled model. In both cases, two item neurons (ID = 1,2) are trained to form two or three associations, respectively (dashed connections are simulated but their weight development is not shown here). During training, neurons are stimulated to fire at 20 Hz for 2 s. We display the developing synaptic weight between specific item-context pairs (ID = 1 and 3 in the 2-association scenario) and (ID = 2 and 5 in the 3-association scenario), and compare the converged weight values between the two-association and three-association case under both learning rules, following a final readout spike at 11 s. . Overall, salient or reward driven events may be encoded more strongly as the result of a transient plasticity modulation. Recall from long-term memory is often viewed as a competitive process in which a memory retrieval does not depend only on its own synaptic strength but also on the strength of other components (Shiffrin, 1970). In view of this, we study the effects of plasticity modulation on encoding specific items within particular contexts, with the aim of investigating the role of enhanced learning for semantization in our model. Using the same network and episodic memory task as before ( Fig. 2A), we modulate plasticity during the encoding of item-1 (red) in context-E via k = k boost (Eq. 7; Materials and Methods; Table 1). This results in an increased cued recall probability for the item associated with three episodic contexts relative to the unmodulated control (Fig. 7A, Normal vs Biased scenario, three associations). Episodic retrieval improves from 0.6 (Normal; Fig. 7A, left) to 0.8 (Biased, modulated plasticity; Fig. 7A, right) when item-1 is cued, which now performs more similarly to item with just two associated contexts. We further analyze and compare the recall of each context when its associated item-1 is cued (Fig. 7B, three associations). The control scenario (Normal; Fig. 7B, left) without transient plasticity modulation shows that the three contexts (ID = A, E, and J) are all recalled with similar probabilities. In contrast, encoding a specific pair with enhanced learning (upregulated k = k boost ) yields higher recall for the corresponding context. In particular, the plasticity enhancement during associative encoding of the context-E (with item-1) results in an increased recall score to 0.8 (0.25 control), while the other associated contexts, ID = A and J, are suppressed (Fig. 7B), primarily because of soft winner-take-all competition between contexts (Fig. 1A). We attribute these changes to the stronger weights because of enhanced learning (Fig. 7C, dark red distribution, g ). Weights between unmodulated item-context pairs (item-1 and context-A,-J) show mostly unaltered weight distributions (a,b , light red), while the biased associative weight distribution between item-1 and context-E is now comparable to the weight distribution of the one-association case. Performance does not exactly match that case though because of some remaining competition among the three contexts. Overall, these results demonstrate how a single salient episode may strengthen memory traces and thus impart resistance to semantization (Rodríguez et al., 2016). Discussion The primary objective of this work was to explore the interaction between synaptic plasticity and context variability in the semantization process. To cast new light on the episodic-semantic interplay, we built a cortical memory model of two spiking neural networks that feature the same modular architecture. The networks are coupled with plastic associative connections, which collectively represent distributed cortical episodic memory. Our results suggest that some forms of plasticity offer a synaptic explanation for the cognitive phenomenon of semantization, thus bridging scales and linking network connectivity and dynamics with behavior. We use a spiking neuronal network model combined with BCPNN, which allows us to directly compare it with a standard Hebbian STDP learning rule. In particular, we demonstrated that with Bayesian-Hebbian (BCPNN) synaptic plasticity, but not with standard Hebbian STDP, the model can reproduce traces of semantization as a result of learning. Notably, this was achieved with biologically constrained network connectivity, postsynaptic potential amplitudes, and firing rates compatible with mesoscale recordings from cortex and earlier models. Nevertheless, our hypothesis of the episodic-semantic interplay at a neural level requires further experimental study of the synaptic strength dynamics in particular. As mentioned, quantitative data on cortical synaptic plasticity is still quite limited, and while STDP has been shown to offer explanation for Figure 7. Plasticity modulation of a specific item-context pair enhances recollection and counteracts semantization. A, Context recall performance. One of the pairs (context-E, item-1) presented in the episodic memory task (compare Fig. 2A) is subjected to enhanced plasticity during encoding, resulting in the boosted recall rate (3 associations, Normal vs Biased, 20 trial average). B, Individual context retrieval contribution in the overall recall (3 associations). Retrieval is similar among the three contexts since plasticity modulation is balanced (left: Normal, k = k normal ; compare Table 1). However, when context-E is encoded with enhanced learning (with item-1), its recall increases significantly (right: Biased, k = k boost ; compare Table 1) some associative memory phenomena (Pokorny et al., 2020), any specific plasticity rule is insufficiently validated experimentally. Yet our results with BCPNN offer a possible explanation and testable behavioral predictions. The spiking version of this plasticity rule has repeatedly been shown to be compatible with detailed, biologically constrained network activity and structure. Importantly, our simulations clearly demonstrate how cognitive phenomena such as semantization could be produced and thus explained by microscopic plasticity processes. In particular, BCPNN solves the issue of decontextualization by its information-theoretical principle, not by being hand-crafted to do so. Like any Bayesian estimator, BCPNN trades-off synaptic strength (weights) for increased intrinsic excitability (bias) in highly active neurons, thus decreasing synaptic strength of neurons that are highly active outside of a specific spiking correlation. Unlike many other conceivable learning rules that might achieve this effect, BCPNN is working only on locally available information, and is thus also biologically plausible. Our study conforms to related behavioral experiments reporting that high context variability or context overload leads to item-context decoupling (Opitz, 2010;Smith and Manzano, 2010;Smith and Handy, 2014). These studies suggest that context-specific memory traces transform into semantic representations while contextual information is progressively lost. Traces of item memory representations remain intact but fail to retrieve their associated context. Semantization is typically described as a decontextualization process that occurs over time. However, several experiments, including this study, proposed that exposures of stimuli in different additional contexts (rather than time itself) is the key mechanism advancing semantization (Opitz, 2010;Smith and Manzano, 2010;Smith and Handy, 2014). In fact, simple language vocabulary learning implies that learners encode words in several different contexts, which leads to semantization and a definition-like meaning of the studied word (Beheydt, 1987;Bolger et al., 2008). Although our network model is limited to a simple item-context decoupling scenario, the proposed plasticity mechanistic explanation for the observed item-context decoupling effect may be generalized to support semantization in more complex scenarios in which other mechanisms may synergistically interact and contribute to decontextualization. Admittedly, our hypothesis does not exclude other seemingly coexisting phenomena and mechanisms supporting memory retrieval that may facilitate semantization over time, e.g., reconsolidation or systems consolidation because of sleep or aging (Friedrich et al., 2015). Further, our model does not feature any higherorder mechanisms allowing a neutral stimulus (lacking prior pairing) to evoke the same contextual memory response as a conditioned stimulus does despite their prior pairing. In other words, each stimulus has to be independently coupled with its context(s). We also demonstrated (Results, Preferential retention) how a transient plasticity modulation, reflecting known isolation effects, may preserve episodicity, staving off decontextualization. Semantization may also be overcome by accumulating additional evidence regarding an episode. In our simulations, we typically used single context cues to retrieve an item during cued recall (e.g., item-4 forms four associations, but only one of its associated contexts was cued, compare Figs. 2B and 3F). However, an interesting question is whether providing multiple context cues that share the same target item boosts its recall (Fig. 8A). Figure 8B shows the result where we sequentially stimulated all the four different contexts in the fourassociation case. The 4-fold contextual information considerably increases the likelihood of retrieval of a nearly fully semanticized item (compare Figs. 3F and 8B, four Figure 8. Average cued recall performance in the Item network after sequentially cueing all the contexts that are associated with the item that forms four associations. A, Spike raster of pyramidal neurons in HC1 of both the Item and Context networks. The cue paradigm during test for the one-association, two-association, and three-association case remains identical to the control case (compare Fig. 2B). However, in particular for the four-association case, we sequentially cue all the four available contexts that share the same target item. B, Average cued recall performance in the Item network (20 trials). The bar diagram reveals progressive loss of item information over the number of context associations, but not for the four-association case at which all the available contexts were cued during test. Thus, providing more evidence via different sources boosts retrieval (;95%) recovering a nearly decontextualized item (compare Fig. 3F, four associations, single cue, 25% accuracy score); **p , 0.001 N = 20). Error bars represent SDs of Bernoulli distributions. associations). These results are relatively intuitive yet novel from a modeling point of view and in line with behavioral studies reporting enhanced cued recall with multiple cues compared with a single one (Rubin and Wallace, 1989;Broadbent et al., 2020;Pearson and Wilbiks, 2021). To our knowledge, there is no other spiking computational model of comparable detail that captures the semantization of episodic memory explored here, while simultaneously offering a neurobiological explanation of this phenomenon. Unlike other dual-process episodic memory models, which require repeated stimulus exposures to support recognition (Norman and O'Reilly, 2003), our model is able to successfully recall events learned in "one shot" (a distinctive hallmark of episodicity). We note that the attractor-based theory proposed in this study does not exclude the possibility of a dual-process explanation for recollection and familiarity (Yonelinas, 2002;Yonelinas et al., 2010). Related models of familiarity and recollection Perceptual or abstract single-trace dual-process computational models based on signal detection theory explain episodic retrieval but the potential loss of contextual information is only implied as it does not have its own independent representation (Wixted, 2007;Greve et al., 2010). These computational models often aim to explain traditional R/K behavioral studies. As discussed earlier, participants in such studies are instructed to give a "Know" response if the stimulus presented in the test phase is known or familiar without any contextual detail about its previous occurrence. Conversely, "Remember" judgments are to be provided if the stimulus is recognized along with some recollection of specific contextual information pertaining to the study episode. This results in a strict criterion for recollection, as it is possible for a subject to successfully recall an item but fail to retrieve the source information (Ryals et al., 2013). Numerous studies suggest that recollection contaminates "Know" reports because recalling source information sensibly assumes prior item recognition (Wais et al., 2008;Johnson et al., 2009). Mandler (1979Mandler ( , 1980 and Atkinson and Juola (1973) treat familiarity as an activation of preexisting memory representations. Our results are compatible with this notion because our model proposes to treat item-only activations as "Know" judgments, while those accompanied by the activation of context representations best correspond to a "Remember" judgment. Item activation is a faster process and precedes context retrieval (Yonelinas and Jacoby, 1994), and our model reflects this finding by necessity, as item activations are causal to context retrieval. We assume that familiarity recognition is simply characterized by lack of contextual information, yet the distinction we make between the Context and Item networks is arbitrary. Memory patterns stored in the Context network are referred to as contexts and those in the Item network as items. From the perspective of the network's architecture, items and contexts have representations of the same nature, nonoverlapping sparse distributed patterns. While sparse internetwork connectivity is sufficient for our model's function, both networks may just as well be part of the same cortical brain area. The actual physical separation of the two networks (which incurs connection delays commensurate with the axonal conduction speed) is motivated by our assumption that items and contexts are not necessarily represented within the same network. A more specific scenario might assume that items and contexts share part of the same local network. In principle, our model should be capable of replicating similar results in that case. Biological plausibility and parameter sensitivity We investigate and explain behavior and macroscale system dynamics with respect to neural processes, biological parameters of network connectivity, and electrophysiological evidence. Our model consequently builds on a broad range of biological constraints such as intrinsic neuronal parameters, cortical laminar cell densities, plausible delay distributions, and network connectivity. The model reproduces plausible postsynaptic potentials (EPSPs, IPSPs) and abides by estimates of connection densities (i.e., in the associative pathways and projections within each patch), axonal conductance speeds, typically accepted synaptic time constants for the various receptor types (AMPA, NMDA, and GABA), with commonly used neural and synaptic plasticity time constants (i.e., adaptation, depression). The model synthesizes a number of functionally relevant processes, embedding different components to model composite dynamics, hence, it is beyond this study to perform a detailed sensitivity analysis for every parameter. Instead, we provide insightful observations for previously unexplored parameters that may critically affect semantization. Importantly, a related modular cortical model already investigated sensitivity to important short-term plasticity parameters (Fiebig and Lansner, 2017). After extensive testing we conclude that the model is generally robust to a broad range of parameter changes and its performance only gradually degrades in terms of the effect size. We expect even lower sensitivity to parameter variations in a network approaching biological scales. Further, it is worth reporting that the model's function is preserved across a wide range of sizes of a cortical column as long as the number of pyramidal cells is not excessively low. The same holds for the population of the inhibitory basket cells provided that the rough total inhibitory synaptic current is maintained by controlling feedback synaptic strength (g BP ; Table 3). The P trace decay time constant, t p , of the BCPNN model is critical for the learning dynamics modelled in this study because it controls the speed of learning in associative connections and the resulting weight amplitude. High values of t p imply long-lasting but weaker memory traces and therefore lead to slower, more inertial learning (more resistance to change and encoding new information as well as forgetting) with overall lower weights and hence weaker binding. Varying t p by 630% does not change the main outcome, i.e., episodicity still deteriorates with a higher context variability. At the same time, as mentioned, slower weight development results in weaker associative binding and overall lower recall (and vice versa for faster learning). To compensate for this loss of episodicity, an additional increase in the unspecific input is usually sufficient to trigger comparable recall rates. Alternatively, the recurrent excitatory gain can be amplified to complete noisy inputs toward discrete embedded attractors. Unspecific background input during recall plays a critical role as well. In general, we use a low background noise input into the two coupled networks. However, for the enhanced noise by 140% the model operates in a free recall regime with spontaneously reactivating memories without any external cues. As we explained in the investigation of the reduced microcircuit model, semantization is an inherent property of BCPNN-driven weight dynamics, derived from Bayesian logic. However, countervailing forces in local microcircuits contribute to the generation and maintenance of our associated memories, Bayesian weight development drives semantization while intrinsic plasticity counteracts it. In consequence, it is possible to lessen the relative impact of this synaptic weight-dependent effect by making intrinsic plasticity more prominent: frequently activated items (in varying contexts) become more excitable because of memory recency effect. Conversely, we can maximize the semantization effect by making the bias current weaker, though fully removing it (b gain = 0) is hard to justify from a biological perspective. By manipulating the strength of intrinsic plasticity (b gain ) to diminish signs of decoupling as described above, the capacity, i.e., the number of retrievable item-context associations, can increase beyond three associations (compare Fig. 3). Other key factors that can enhance model capacity for itemcontext associations (resistance to semantization over many established episodic associations) are larger network size, higher associative binding connection probability (e.g., increase in cp PPA from 2% to 4%; Table 3), and elevated background unspecific noise during the cue-response association period. Strengthening associative binding by upregulating w syn gain can also enhance the model's capacity for item-context associations (Table 1). Still, there is an upper limit to w syn gain as extreme values can lead to implausible EPSPs. This study also demonstrates how a selective transient increase of plasticity can counteract semantization. The plasticity of the model can be modulated via the parameter k (Eq. 7; Materials and Methods). Typically, k is set to 1 (k = k normal ; Table 1), whereas we double plasticity (k = k boost ; Table 1), when modeling salient episodic encoding. We notice that by selectively tripling or quadrupling plasticity (relative to baseline) during encoding of a specific pair whose item component forms many other associations, the source recall improves progressively (data shown only for k = k boost in Results, Preferential retention). Finally, in Results, BCPNN and STDP learning rule in a microcircuit model, we compare STDP and BCPNN plasticity in a highly reduced model. We bind items with contexts to form different number of associations and keep track of the weight development per time step. STDP plasticity generates same magnitude item-context binding regardless of how many associations an item forms. A detailed parameter analysis for every critical synaptic parameter (630%) did not yield any behaviorally significant changes to the converged weights. Semantization over longer time scales Source recall is likely supported by multiple independent, parallel, interacting neural structures and processes since various parts of the medial temporal lobes, prefrontal cortex and parts of the parietal cortex all contribute to episodic memory retrieval including information about both where and when an event occurred (Gilboa, 2004;Diana et al., 2007;Watrous et al., 2013). A related classic idea on semantization is the view that it is in fact an emergent outcome of systems consolidation. Sleep-dependent consolidation in particular has been linked to advancing semantization of memories and the extraction of gist information (Payne et al., 2009;Friedrich et al., 2015). Models of long-term consolidation suggest that retrieval of richly contextualized memories become more generic over time. Without excluding this possibility, we note that this is not always the case, as highly salient memories often retain contextual information (which our model speaks to). Instead, our model argues for a much more immediate neural and synaptic contribution to semantization that does not require slow multiarea systems level processes that have yet to be specified in sufficient detail to be tested in neural simulations. It has previously been shown, however, that an abstract simulation network of networks with broader distributions of learning time constants can consolidate memories across several orders of magnitude in time, using the same Bayesian-Hebbian learning rule as used here (Fiebig and Lansner, 2014). That model included representations for prefrontal cortex, hippocampus, and wider neocortex, implementing an extended complementary learning systems theory (McClelland et al., 1995), which is itself an advancement of systems consolidation (Squire and Alvarez, 1995). We consequently expect that the principled mechanism of semantization explored here can be scaled along the temporal axis to account for lifelong memory, provided that the plasticity involved is itself Bayesian-Hebbian. Our model does not advance any specific anatomic argument as to the location of the respective networks (Yonelinas, 2002;Diana et al., 2007). The model purposefully relies on a generic cortical architecture focused on a class of synaptic plasticity mechanisms which may well serve as a substrate of a wider system across brain areas and time. In conclusion, we have presented a computational mesoscopic spiking network model to examine the interplay between episodic and semantic memory with the grand objective to explain mechanistically the semantization of episodic traces. Compared with other models of episodic memory, which are typically abstract, our model, built on various biological constraints (i.e., plausible postsynaptic potentials, firing rates, connection densities, synaptic delays, etc.) accounting for neural processes and synaptic mechanisms, emphasizes the role of synaptic plasticity in semantization. Hence it bridges micro and mesoscale mechanisms with macroscale behavior and dynamics. In contrast to standard Hebbian learning, our Bayesian version of Hebbian learning readily reproduced prominent traces of semantization.	2021-07-22T13:19:36.847Z	2021-07-19T00:00:00.000	{ "year": 2022, "sha1": "12081d285b06a79d7a051e3bd6c8fad1a12e2284", "oa_license": "CCBY", "oa_url": "https://www.eneuro.org/content/eneuro/early/2022/07/08/ENEURO.0062-22.2022.full.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "562093190fee72b4d5ef8f0654a30ce50700c093", "s2fieldsofstudy": [ "Psychology", "Computer Science", "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
49230929	pes2o/s2orc	v3-fos-license	Instrumentation for Millimeter-wave Magnetoelectrodynamic Investigations of Low-Dimensional Conductors and Superconductors We describe instrumentation for conducting high sensitivity millimeter-wave cavity perturbation measurements over a broad frequency range (40-200 GHz) and in the presence of strong magnetic fields (up to 33 tesla). A Millimeter-wave Vector Network Analyzer (MVNA) acts as a continuously tunable microwave source and phase sensitive detector (8-350 GHz), enabling simultaneous measurements of the complex cavity parameters (resonance frequency and Q-value) at a rapid repetition rate (approx. 10 kHz). We discuss the principal of operation of the MVNA and the construction of a probe for coupling the MVNA to various cylindrical resonator configurations which can easily be inserted into a high field magnet cryostat. We also present several experimental results which demonstrate the potential of the instrument for studies of low-dimensional conducting systems. I. Introduction Anisotropy, or low-dimensionality, plays a fundamental role in many areas of contemporary condensed matter physics, both pure and applied. For example, much of the technology which has revolutionized the microelectronics industry in recent years has resulted from the development of low-dimensional semiconductor devices. More recently, research into bulk layered materials such as the transition metal oxides, 1 organic conductors, 2 semiconductor superlattices, 3 magnetic nanostructures, 4 etc., has resulted in the discovery of a range of new physical phenomena, e.g. high temperature superconductivity, 5 colossal magnetoresistance, 6 and a novel form of the quantum Hall effect. 7 Many of these discoveries challenge our basic understanding of condensed matter physics while, at the same time, they hold the key to future technologies. The use of microwave techniques to probe the electrodynamic properties of metals is not new. However, due to recent interest in a range of novel conducting systems in which the characteristic energy scales (e.g. electronic bandwidths, or energy gaps) coincide with the millimeter and sub-millimeter-wave spectral ranges, this field is undergoing a renaissance. Frequency dependent transport measurements in the presence of (strong) magnetic fields − the topic we call magnetoelectrodynamics, in analogy of magnetooptics − are expected to yield significant new insights into the physics of low-dimensional conductors, just as conventional magnetooptics and magnetotransport measurements were essential decades ago in leading to our present understanding of simple metals. To date, few experiments on highly conducting materials have combined high magnetic fields and millimeter-wave spectroscopy. 8 The reasons for this can be attributed to a range of factors. First, there are few commercially available sources and detectors working in this frequency range and those that can be used tend be highly priced, cumbersome to use, and possess either low power or an unstable output. Second, spectroscopy in this frequency range is complex, particularly in the case of metals: high sample reflectivity requires extreme sensitivity in order to detect small changes in conductivity; and the radiation wavelengths are typically larger than available samples, thus rendering conventional reflection spectroscopy useless. The inclusion of a magnetic field complicates these techniques considerably, since good optical coupling between the spectrometer (source and detector) and the sample under investigation is essential, and this is obviously difficult to achieve through the small bore (comparable to λ) of a large magnet. In this paper we describe instrumentation which has been developed at Montana State University (MSU) for carrying out sensitive spectroscopy of low-dimensional conductors in the frequency range between 40 and 200 GHz, and in magnetic fields up to 33 tesla which are available at the National High Magnetic Field Laboratory (NHMFL) in Tallahassee, FL. In order to a) maximize sensitivity, and b) achieve the required control over the electromagnetic field distribution in the vicinity of the sample, we utilize an enclosed cavity perturbation technique (section II-B). 8,9 The wide frequency coverage and outstanding dynamic range (signal-to-noise) are achieved using a Millimeter-wave Vector Network Analyzer (MVNA) 10,11,12 as a source and detector (see section II-A). The resulting instrumentation offers unique experimental possibilities in the area of metals physics in high magnetic fields. The paper is organized as follows: section II contains a detailed technical description of the system; in section III, we discuss the performance of the instrument; and we conclude with a summary of the paper in section IV. II. TECHNICAL DESCRIPTION A. The MVNA As a source and detector, we employ a Millimeterwave Vector Network Analyzer (MVNA) 10,11,12 to monitor the phase and amplitude of millimeter-wave radiation transmitted through a resonant cavity containing the sample under investigation. The MVNA 8-350, which employs purely solid-state electronics, allows measurements over an extended frequency range (8 -350 GHz) 13 through harmonic multiplication of a sweepable centimeter source, S 1 , which provides a nominally flat output power in the range F 1 = 8 -18 GHz. The mm-wave signal used in our experiments is extracted from a Shottky diode (harmonic generator − HG) which has been optimized to produce the desired harmonic N of the sweepable cm source, i.e. F mm = N × F 1 . Detection is then achieved by mixing the mm-wave signal (F mm ) with the signal from a second cm source, S 2 , at a second Schottky diode (harmonic mixer − HM). The beat frequency, F beat , which preserves the phase and amplitude information of the mm-wave signal relative to the local oscillator S 2 , is then sent to a heterodyne vector receiver (VR). The low noise floor of the MVNA is achieved by defining the frequency difference between the two cm sources, S 1 and S 2 , using a main oscillator. If one then uses the same harmonic rank on the source side (HG), and on the detection side (HM), the phase noise associated with the cm sources cancels in the beat signal (F beat ) which is sent to the VR. Thus, the phase reference of the VR can be taken directly from the main oscillator. 14 With the noise characteristics of the analyzer optimized, its dynamic range is limited only by the harmonic conversion efficiency of the Schottky diodes. At MSU, three pairs of Schottky diodes are used, operating in the V (∼ 45 − 70 GHz, N = 3 and 4), W (70 − 110 GHz, N = 5 and 6) and D-bands (110− > 200 GHz, N = 7 to 12). The V-band diodes are nominally flat broadband, while the W and D band diodes are mechanically tunable and require optimization each time the source frequency (F 1 ) is changed. Operating in this mode, it is possible to perform bench-top tests up to 350 GHz. Table I lists the optimum dynamic ranges achieved at MSU (MVNA 8 − 350 − 1 − 2) 15 in each frequency band, and for each harmonic, up to 200 GHz (N = 12). Similar analyzers are in use at several high magnetic field laboratories around the world; 16 in particular, the national magnet labs in the US and The Netherlands have MVNA 8-350 analyzers (with ESA options) 13 which are available to external users. The MVNA at the NHMFL, which has been used for some of the studies discussed in this paper, additionally operates in the Q (30 − 50 GHz, N = 3), K (16 − 32 GHz, N = 2) and X (8 − 18 GHz, N = 1) bands. Although the cm sources (S 1 and S 2 ) are phase (frequency) locked to each other, their absolute frequencies must be stabilized also. The frequency precision and stability provided by the MVNA 8-350 is not adequate for narrow-band cavity perturbation measurements when the bandwidth of the cavity is less than about 100 MHz. For this reason, it is common to phase-lock one of the sources (i.e. both) to a quartz standard. At MSU and at the NHMFL, EIP 575 source-locking frequency counters 17 are used, which provide both the stability and precision necessary for the measurements described in this paper. One other mode of operation involves phase locking the source (S 1 ) directly to the high−Q cavity resonator used for the experiment. The counter is also useful in this case for recording changes in frequency resulting from any changes in dispersion within the cavity. Comparisons between the two frequency locking techniques are discussed in section III-B. The dynamic ranges listed in Table I represent ideal values for the MVNA 8-350 assuming there are no insertion losses between HG and HM. Due to the considerable size of a typical magnet cryostat and, in particular, the dimension separating the magnetic field center and the top of the cryostat (∼ 1.5 m in our case), a considerable insertion loss is unavoidable. Furthermore, it is essential to keep a reasonable distance (∼ 2 m) between the MVNA and the magnet, since the cm sources (YIG oscillators) are sensitive to stray magnetic fields of more than a few Gauss. The Schottky diodes (HG and HM), on the other hand, are not field sensitive and may be placed closer to the experiment. Thus, the cm and beat frequencies are propagated between the MVNA and Schottky diodes through flexible coaxial cables, which introduce a combined insertion loss of 4 to 5 dB. Meanwhile, the mm-wave signal is propagated from the HG to the cavity within the magnet cryostat, and back to the HM, using a pair of rigid waveguides. These waveguides account for a major part of the insertion loss of the system; minimizing these losses is the subject of section II-B.1. A schematic of this arrangement is shown in Fig. 1. In addition to its use as a source and detector for solid state spectroscopy, the network analysis capability of the MVNA is crucial during the developmental stages of a measurement system. The frequency sweeping capability can be used to determine the precise location of impedance mis-matches and/or microwave leaks from the system, and for the characterization and optimization of different resonator designs. B. The sample probe As outlined in the introduction, our aim is to be able to conduct sensitive cavity perturbation measurements, at low temperatures, high magnetic fields and over a broad frequency range. In this section, we describe an experimental scheme − the "sample probe" − for coupling the MVNA to various enclosed cylindrical resonator configurations which can easily be inserted into a high field magnet cryostat. The "sample probe" refers to the passive microwave hardware which has been developed at MSU and is compatible with the MSU and NHMFL magnet systems. It consists of the following components: two long waveguides for propagating mm-waves from the HG, into the magnet cryostat (incidence waveguide), and back to the HM (transmission waveguide); a demountable cavity, of which we have developed two standard types; a coupling between the cavity and the incidence and transmission waveguides; a vacuum tube to isolate the probe from the surrounding liquid cryogens; and electronics for controlling the sample temperature, and for magnetic field modulation. Fig. 1 shows a schematic of the probe situated within the superconducting magnet cryostat at MSU, and Fig. 2 shows an equivalent LRC circuit representation of the key microwave components; we defer explanation of these figures until following sections, where various aspects of the probe design are discussed in detail. From the outset, we established two key experimental objectives: i) it should be possible to make measurements at several well separated frequencies without the need to interfere with, or warm up, the sample probe; and ii) the sensitivity, dynamic range and mechanical stability of the measurement system should be optimized as far as possible. The flexibility in choice of frequency is important for experiments at the NHMFL, where magnet time is often limited. There are also other advantages to this, e.g. by not having to interfere with the sample, it is possible to ensure that it sits in an identical electromagnetic field distribution for a given series of cavity modes. The second goal is fairly self explanatory, nevertheless, high sensitivity comes at the expense of some flexibility. We have, therefore, opted for a rigid construction, i.e. no insitu rotation of either the cavity or the sample within the cavity is possible. Our experience has shown that such mechanisms result in radiation leakage from the cavity and diminished sensitivity. Reducing losses in the waveguides The success of our recent measurements owes as much to reductions in the insertion losses associated with our sample probe, as it does to the optimization of the cavity designs. In this section, we discuss the technical details of how to propagate mm-wave radiation from the Schottky diodes into a low temperature cryostat (pumped 4 He) within the bore of a high field magnet system where the resonant cavity containing the sample is situated. As illustrated in Fig. 1, the MSU setup utilizes a separate inner cryostat to control the temperature of the experiment. This cryostat, which sits within the bore of the superconducting magnet, draws liquid helium from the main reservoir into its tail, which is thermally isolated from the main helium reservoir, thus allowing good control over the sample temperature. The microwave probe is based on V-band rectangular waveguides which are terminated at their source/detector ends with UG385/U flanges for convenient connection to the Schottky diodes. 18 V-band waveguide cuts off below ∼ 45 GHz, which is sufficiently low for most of the intended applications, and is also the lowest frequency band currently available at MSU. Furthermore, the narrow bores of the magnets at the NHMFL 19 essentially rule out the possibility of using waveguides below Q-band (cut-off at 33 GHz) for transmission measurements. Commonly used low loss, high conductivity, waveguide materials such as copper or silver are not ideal for our purposes. Unacceptable heat flow down the waveguides and into the cryostat leads to excessive liquid helium boil-off and limits the ability to cool the cavity/sample to pumped 4 He temperatures (∼ 1.5 K). For cryogenic purposes, Stainless Steel (SS) waveguide offers an attractive alternative: 18,20 it has both a low thermal conductivity, 21 and it is possible to use thinner walled waveguide material. 22 Unfortunately the microwave losses in SS waveguides are severe, as illustrated in Fig. 3, which shows a comparison between the insertion losses of SS and Ag, as measured using the frequency sweeping capability of the MVNA; note that, in this range, the loss in SS exceeds 10 dB/m, whereas the loss in Ag is less than 2 dB/m. 23 The solution, therefore, is to construct composite waveguides from highly conducting waveguide sections isolated by SS sections. Although this necessitates several joints in the waveguide assembly, we have found that these do not significantly impair the functionality of the waveguide. Placement of the SS waveguide sections is crucial to the cryogenic performance of the system, as illustrated in Fig. 1. The largest temperature gradient occurs in the upper part of the magnet dewar where the radiation baffles are situated. It is in this region that we first use SS. We again use short SS sections at the lower end of the probe in order to thermally isolate the cavity/sample from the long Cu/Ag 24 sections which run from just below the baffles, through the liquid 4 He reservoir, and into the bore of the magnet. The cavity and sample are, therefore, more-or-less isolated from the main 4.2 K helium reservoir, enabling control over the temperature of the cavity/sample from about 1.35 K, up to 40 or 50 K (see section II-B.4). The lower SS waveguide sections have been gold plated 25 for reasons which are discussed in the following section. One unforeseen benefit of the composite waveguide construction, which greatly improves the mechanical stability of the probe, is its insensitivity to the liquid helium level in the magnet cryostat. We assume that the Cu/Ag section maintains a fairly uniform temperature (∼ 4.2 K) over its entire length due to its high thermal conductivity. Thus, the temperature gradient in the SS section does not depend on the liquid helium level. In contrast, the thermal gradient in a single SS section immersed in liquid helium would depend on the level of the liquid, causing the waveguide to expand slightly over time, thereby affecting the microwave phase stability of the system. The SS and Cu/Ag rectangular waveguide sections, which have different outer dimensions, 22 are coupled together using specially machined clamps which screw tightly around the waveguides. 26 The joints are staggered so as to minimize cross talk between the waveguides due to any microwave leakage from the joints (see following section). Thus, although both waveguides pass through each clamp, only one of the waveguides is joined at each clamp. This construction is extremely rugged, yet it easily permits modifications in the overall probe length, and in the placement of the SS sections. Therefore, the same waveguide assembly can easily be re-configured for use at other magnet facilities such as the NHMFL. Finally, the entire sample probe fits tightly inside a 19.05mm OD (0.4mm wall) vacuum jacket. The integrity of the vacuum is maintained across the waveguides at their room temperature ends using mylar windows clamped between standard flanges (UG385/U) which have been modified to hold rubber 'O'-rings. Again, we offset the vacuum seals on the incident and transmission waveguides, and wrap them in steel wool to minimize cross-talk. A hermetically sealed connector provides 19 electrical feedthroughs for thermometry, field modulation coils, and for powering a heater. Minimizing undesirable instrumental effects The basic principal behind the cavity perturbation technique is relatively simple, and involves measuring changes in the complex cavity parameters (i.e. resonance frequency f o and Q−value) upon insertion of a sample. 8,9 Relating these changes to the complex electrodynamic properties of the sample can be a formidable task, and we refer the reader to a series of three articles 27,28,29 dealing with this analysis for precisely the types of materials which we set out to study in high magnetic fields, i.e. highly anisotropic crystalline conductors. However, in order to reliably apply such an analysis in our case, it is essential that we first consider how each element in the sample probe affects our ability to extract the relevant information from the cavity mounted at the end of the probe. As we shall show, this is a formidable task in itself, which is complicated considerably by the restricted access into the bore of a typical high field magnet cryostat, and by the need for the probe to accommodate cavities resonating over a broad range of frequencies. The task of optimizing the probe design is best tackled by considering an equivalent AC circuit (Fig. 2). Each component of the probe may be modeled as a self contained LRC circuit which is inductively coupled to the next. The Schottky diodes, HG and HM, attach to the upper ends of the incidence and transmission waveguides, which have impedances Z I and Z T , respectively. The coupling between HG (HM) and the incidence (transmission) waveguide is modeled as a coupling mutual inductance m HG (m HM ). The cavity, which is mounted at the lower end of the two waveguides and has impedance Z C , is coupled to each of the waveguides through coupling mutual input (m in ) and output (m out ) inductances. Ideally, the incidence and transmission waveguides should be coupled solely through the cavity. However, a leak signal between the incidence and transmission waveguides, in parallel with the cavity, is inevitable; this is modeled as a direct coupling mutual inductance, m l , between the two waveguides. It is changes in Z C , caused by the insertion of a sample into the cavity, which one would like to measure in a cavity perturbation experiment. Since the MVNA measures phase and amplitude, it is important to see how Z C affects both of these parameters. Before, considering the probe as a whole, i.e. the entire circuit in Fig. 2, we examine the cavity by itself. Variations in the amplitude and phase of a wave transmitted through a cavity, as the frequency (f ) is swept across a resonance, are given by and where f o is the center frequency and Γ is the FWHM of the resonance; these expressions have been normalized so that A = 1 and φ = 0 at resonance. As f is swept from 0 to ∞, A and f sweep out a circle in the complex plane, where the real and imaginary amplitudes are given by and the more familiar expressions for a Lorentzian. Such a circle is shown in Fig 4a, for a resonance at 60 GHz, with a Q of 5000. Each point in the figure corresponds to a different frequency, and the frequency interval between each point is 800 kHz. Points closest to the origin correspond to f ≪ 60 GHz and f ≫ 60 GHz. The resonance frequency (f o ) lies along the real axis (φ = 0) where the spacing between points is greatest, i.e. when the phase rotates most rapidly with frequency. Clockwise rotation around the circle represents increasing frequency. The corresponding changes in Figs 4b and c, respectively. In a locked frequency experiment, changes in dispersion (Im{Z C }) cause the cavity to go off resonance, i.e. the point corresponding to f o in Fig. 4a will move away from the real axis. Provided that this effect is weak (a perturbation), the dominant result is a change in the phase of the signal transmitted through the cavity, and no appreciable change in amplitude. Conversely, changes in dissipation (Re{Z C }) within the cavity will result in a reduction in the amplitude of the signal transmitted through the cavity and, hence, a reduction in the diameter of the circle in Fig. 4a. Dissipation alone does not move f o away from the real axis and, therefore, does not affect the phase of the wave. However, dispersion can affect both the amplitude and the phase of the wave if f o moves appreciably off the real axis. For this reason, it is often desirable to conduct a locked phase experiment, which completely decouples these two effects. With a phase lock, the cavity stays on resonance (on the real axis in Fig. 4a), and dispersion affects the resonance frequency only, which we can measure with a frequency counter. Meanwhile, changes in dissipation again affect the amplitude of the transmitted signal only. Unfortunately, each of the additional circuit elements required to link the MVNA to the cavity, and/or improper coupling between these components, has the potential to seriously distort the simple relationships between dissipation, dispersion, amplitude and phase discussed above. Ideally, the sample probe should be passive, low loss, insensitive to temperature and magnetic field and, with the exception of the cavity, should have a flat broad-band frequency response. In practice, this is never actually possible to achieve. Nevertheless, by conducting a thorough characterization and optimization of each element in the microwave circuit ( Fig. 2), it is possible to minimize these instrumental effects to negligible levels. The MVNA performs a pivotal role in this hardware developmental process. The following paragraphs discuss various undesirable instrumental characteristics, their potential effect on a measurement, and the steps we have taken to eliminate these sources of error. A leak wave bypassing the cavity directly through to the transmission waveguide has two adverse effects. First, it diminishes the useful dynamic range − ideally 100% of the signal reaching the detector should pass through the cavity. Second, if the leak amplitude is comparable to the amplitude of the signal passing through the cavity, the resonance may become severely distorted, making it extremely difficult to distinguish between dissipative and dispersive effects within the cavity. As illustrated in Fig. 5, a leak wave adds a complex vector to the signal transmitted through the cavity. By minimizing the leak, one can control its amplitude. However, it is not possible to control the phase of the leak wave. Consequently, big leaks lead to an arbitrary vector translation of the circle in Fig. 4a; this is a pure translation, i.e. each point on the circle is translated by the same vector. Hence, the line joining the resonance frequency, f o , and the f = 0 & ∞ points, remains parallel to the real axis. As a result, the transmitted amplitude on resonance is not necessarily the maximum amplitude; indeed, it can take on any value from zero to one plus the leak amplitude. This is illustrated in Fig. 5 for an arbitrary translation of the circle in Fig. 4a, together with the corresponding variations in phase and amplitude, plotted versus frequency. It is apparent from this figure that the phase of the leak signal is entirely responsible for the way in which dissipation and dispersion affect the phase and amplitude of the signal transmitted through the cavity. Thus, an appreciable leak signal is intolerable, and we have taken every step to reduce the leak in our probe to at least 20 dB below the typical signal transmitted through the cavity on resonance, as discussed above. Standing waves in the waveguides are unavoidable and, without proper attention, can cause considerable problems, especially when operating in the phase-locked mode in which the incident mm-wave frequency is locked to the cavity resonance frequency. Changes in this frequency will result in changes in the phase and amplitude of the mm-wave signal incident upon the cavity. Thus, it becomes impossible to distinguish between the intrinsic cavity response and spurious effects due to the standing waves. Furthermore, the phase is no longer truly locked to the cavity resonance under these circumstances, but rather to the coupled response of the entire circuit in Fig. 2. Standing waves should not be ignored altogether in the frequency locked mode either, particularly when the cavity is well coupled to the waveguides. Under these circumstances, changes in Z C influence the impedance matching between the cavity and the waveguides (i.e. m in and m out ) and, therefore, affect the standing waves. There is little to be gained from trying to eliminate the standing waves completely. This would require precise impedance matching of each component in Fig. 2, which is only possible to achieve over a narrow frequency range and would, therefore, defeat the purpose of the probe, which is intended to work over a fairly broad frequency range. Instead, we concentrate on minimizing the influence of the standing waves on a measurement at any given frequency. This is achieved by reducing the frequency bandwidth of the measurement to well below the periodicity of the standing wave pattern, i.e. so that the response of the waveguides is essentially flat over the relevant frequency interval. In this way, a range of cavities or cavity modes may be utilized, covering an extremely broad frequency range in comparison to the standing wave periodicity. Meanwhile, each cavity mode samples only a minute portion of the waveguide spectrum, over which its response is essentially flat. The fastest standing wave period is governed by the longest dimension of the sample probe, which is 3m in our case (HG to HM, via the cavity). This gives rise to a standing wave periodicity of about 100 MHz which, in turn, requires cavity filling factors of less than 10 −4 , so that the frequency shift in any given measurement never exceeds about 10 −4 of the measurement frequency, i.e. ∆f o < 10 MHz for f < 100 GHz. Cavity filling factors of between 10 −5 and 10 −4 are typical for the types of samples we study, so standing wave problems do not force this restriction upon us. Nevertheless, to compensate for the small filling factors, it is essential to have cavity Q−factors on the order of 10 4 . In order to attain such high Q−values, the cavity must necessarily be coupled weakly to the waveguides, which further reduces problems associated with standing waves. P hase instability, and/or a strong frequency dependence of the phase reaching the VR, can lead to a variety of problems. Although the internal sources within the MVNA are phase locked, additional phase jitter can arise in the mm-wave signal due to mechanical and/or thermal instabilities in the sample probe. Mechanical vibrations do not pose any problems, either at MSU or at the NHMFL. However, thermal stability is essential for achieving the best results. Due to extremely high cavity Q−values, minor temperature fluctuations can lead to significant phase instabilities, particularly when studying samples with a strongly temperature dependent electrodynamic response. For this reason, active temperature control is necessary (see section II-B.4). Long term thermal stability of the entire probe, including the 1.5m waveguide sections, is also desirable and is best achieved using the composite waveguides described in the previous section. A strong frequency dependence of the phase reaching the VR occurs when the distances between the sources S 1 and S 2 and the HM are very different. The signal originating at S 1 has to travel the extra 2 × 1.5 m into, and out of, the magnet cryostat. For this reason, it can be beneficial to compensate for the extra distance between S 1 and the HM by adding 3 m of coaxial cable 30 between S 2 and the HM, especially when performing phase-locked or frequency swept measurements, even though this introduces an extra 2 − 3 dB insertion loss. Spurious magnetic resonances caused by paramagnetic contamination of either the cavity, or the waveguides close to the magnetic field center, will give rise to both sharp and broad instrumental features (Electron Paramagnetic Resonances -EPR) in the transmission versus magnetic field response of the system. These spurious resonances can be hard to distinguish from the genuine response of the sample within the cavity, and should be eliminated so that the magnetic field response of the unloaded probe (i.e. with no sample) is as flat as possible. The SS waveguide sections cause the most severe problems, which we suspect is due to the presence of small traces of Fe 3+ (rust) at the surface of the metal. For this reason, we have gold plated the SS sections which couple directly to the cavity. This not only reduces the insertion loss due to these sections, but also completely eliminates a broad instrumental resonance, leaving an extremely flat response (∆A < 0.05 dB), as shown in Fig. 6. A number of other measures have been taken to avoid contaminating the lower end of the probe (i.e. close to the field center) with paramagnetic impurities. These include clamping all components together rather than using adhesives or solders, and avoiding the use of cutting oils when machining the resonators. We have also gold plated some of the cavities to prevent tarnishing. Nevertheless, electrolytic OFHC Cu cavities work extremely well without plating (see Fig. 6). The cavities To date, our efforts have focussed on the use of enclosed cylindrical resonators for cavity perturbation measurements. There are several compelling reasons to do so. To begin with, the simple design concept we have developed, in which the cavities are assembled from relatively few easily machinable components (see Figs. 7 and 8), enables us to fabricate a range of cavities and, therefore, switch frequency range/coverage with relative ease. 31 More importantly, relative to rectangular cavities, it is straightforward to achieve extremely high Q−values (> 10 4 ) for the TE01n (n = 1, 2, 3...) modes of cylindrical resonators. The reason for this has to do with the fact that no AC currents flow between the end and side walls of a cylindrical cavity excited in its TE01n mode. Consequently, joints at these ideal locations (see Figs 7 and 8) do not diminish the cavity Q−factor. These modes also possess electromagnetic field geometries which are highly desirable for the measurements described in this paper. Axial cavities For the most part, we use axial cavities of the type shown in Fig. 7, i.e. ones in which the cavity axis is coincident with the axis of the superconducting solenoid. This is by far the most versatile design, though it does have some limitations, especially when it is necessary to tilt the sample with respect to the applied DC magnetic field. The cavity is constructed from OFHC electrolytic copper in three pieces -a blank end plate, a coupling plate and a cylindrical barrel. This assembly bolts onto the under side of a copper housing which clamps around the incidence and transmission waveguides. These bolts also provide the pressure for reproducibly clamping the cavity assembly tightly together; no adhesives or solders are used. The cavity may be disconnected from the waveguides in a matter of seconds for easy sample insertion/removal. The housing which clamps around the waveguides is also easily interchanged with a separate housing for a transverse cylindrical cavity (see below). Coupling between the waveguides and the cavity is achieved by means of small circular apertures in the thin coupling plates. Since these plates terminate the waveguides and the cavity, there are no transverse microwave electric (Ẽ-) fields at the locations of the apertures. Thus, it is the microwave magnetic (H-) fields in the waveguides and the cavity which should be matched. For the TE01n modes, theH-fields flow radially at the cavity ends and, therefore, theH-fields in the waveguides should do so also. For this reason, the incoming waveguides are oriented with their shortest edges closest together (see Figs 1,7 and 8). 32 We can control the degree of coupling between the waveguides and the cavity (m in and m out ) by means of the dimensions of the coupling apertures (diameter and thickness). There is an important trade off here between i) strong coupling (large apertures), which ensures good power throughput from the source to the detector and, hence, a large dynamic range, and ii) weak coupling (small apertures), which limits radiation losses from the cavity, resulting in higher cavity Q−values and increased sensitivity, at the expense of some dynamic range. We have found empirically that the optimum coupling apertures should be small for our setup, with diameter ∼ λ/4. It is also necessary for the coupling plate to be very thin (∼ λ/20), since the signal is obviously attenuated as it passes through the apertures, which are way below cutoff. The relatively weak coupling to the cavity makes it all the more important to reduce any/all other losses in the sample probe and, hence, preserve a reasonable dynamic range, i.e. without taking steps to reduce losses in the waveguides (see section II-B.1), we would be forced to increase the coupling, resulting in reduced sensitivity. The resonance frequency of a particular cavity is determined by the length and the inner diameter of the cylindrical barrel (see Fig. 7). Because of the simplicity of machining this section, it is no great task to construct a large number of cavities, providing many TE01n modes covering a wide range of frequencies. However, because of the wavelength dependence of the coupling between the waveguides and the cavity, it is also necessary to construct several different coupling plates − roughly one for each frequency band, i.e. V-, W-, D-, etc.. Because the inner diameters of the cavities generally get smaller at higher frequencies, the positioning of the apertures is also critical. In all cases, the input and output coupling apertures are located diametrically opposite each other, and at the same radial distance from the axis of the cavity. In order to minimize the number of these coupling plates, we have limited the cavity diameters to four standard sizes. For V-band (cavity dia. = 9.52 mm), the coupling apertures are located at the optimum positions, both with respect to the cavity and the waveguides, i.e. in the middle of the waveguides and half way between the cavity axis and its perimeter. For the higher frequency bands, a compromise between these positions is made. A clear gap of 0.51mm is maintained between the incidence and transmission waveguides, which terminate flush with the under side of the cavity housing. The coupling plate is machined with a 0.51mm wide, 0.75mm high, ridge running perpendicular to the line joining the coupling apertures, and intersecting the mid point between them (see Fig. 7). This ridge locates inside a matching groove on the under side of the cavity housing and, therefore, in between the incidence and transmission waveguides, as shown in Fig. 7. We have found that it is this joint between the cavity and the waveguides which is most susceptible to microwave leaks, and that the ridge in the coupling plate dramatically reduces the leak amplitude. The leak may be reduced further still by applying a small amount of Indium into the groove before assembling the cavity. For a perfectly cylindrical cavity, the TE01n modes are degenerate with the TM11n modes. It is a trivial task to lift this degeneracy without diminishing the Q−values of the TE01n modes. We achieve this by drilling a small indent in the center of the blank end plate where essentially no currents flow for the TE01n modes; this hole may also be used to hold a quartz pillar for mounting a sample on axis above the cavity end plate (see Fig. 9). Consequently, the TM modes are shifted to lower frequencies by up to 200 MHz, they are also weaker and have lower Q−values than the TE modes. Transverse cavities Recently, we have developed a transverse cavity optimized to work in V-band (shown in Fig. 8). Although the construction of this cavity is a little more complex than the axial cavities, it offers a major advantage for experiments in which it is necessary to tilt the sample relative to the applied DC magnetic field. The major difference between this cavity and the axial one is that the mm-waves are coupled through apertures drilled directly through the side wall of the cylindrical barrel, i.e. a separate coupling plate is not employed. This requires removing material from one side of the cylindrical barrel so that the side wall of the cavity is sufficiently thin in the vicinity of the coupling apertures. This flat surface also facilitates attachment to the underside of a specially designed cavity housing, which again clamps around the incidence and transmission waveguides. The cavity end plates simply bolt onto either end of the cavity barrel; one of these end plates has machined slots at the bolt circle radius so that it may be rotated about the cavity axis. As with the axial cavity, we again implement a ridge/groove arrangement between the waveguides and the cavity, in order to minimize any microwave leakage at this position. The locations of the coupling apertures in the side wall of the cavity have been optimized for exciting the TE012 mode, i.e. at 1 / 4 h and 3 / 4 h from the ends of the cavity, where h is the cavity height. Nevertheless, these positions provide good coupling to the TE011 mode and, to a lesser extent, the TE013 mode. Sample positioning, and sample rotation Using one or other of the two cavity types, it is possible to subject a sample to virtually any combination of AC E-andH-field polarizations, relative to the applied DC magnetic field (B o ) orientation, e.g. at anH node with E//B o , or at anẼ node withH⊥B o , etc.. However, it turns out that for studies of highly anisotropic conductors, two convenient locations are sufficient for most experiments utilizing TE01n modes. 8,9,28 In the "end plate" configuration, the sample is placed on the blank cavity end plate, exactly half way between the cavity axis and its perimeter, as shown in Fig. 9a. In the "quartz pillar" configuration, the sample is mounted atop a thin quartz pillar (dia. = 0.71mm) on the axis of the cavity, as shown in Fig. 9b. In both of these configurations, the sample sits in anH-field antinode for the TE011 mode, the polarization of which is radial for the end plate configuration and axial for the quartz pillar configuration (see Figs 9a and b). For the end plate configuration, all TE01n modes have radialH-field antinodes at the sample location. This is not the case for the quartz pillar configuration; for example, if the sample is mounted precisely mid-way between the cavity end plates, the even n modes have bothẼ andH-field nodes at this location. Careful forethought as to the positioning of the sample can rectify this problem to a certain extent. However, whenever positioning the sample away from the mid-point (H max -point) of the cavity, sensitivity is compromised. Indeed, even the end plate position is appreciably less sensitive than the cavity mid-point. 28 To understand how it is that the end plate and quartz pillar locations within a TE01n cavity can be sufficient for studying anisotropic systems, we consider the electrodynamics of a quasi-two-dimensional (Q2D) conductor. We assume a quasi-static approximation and consider the FaradayẼ F -field resulting from the time varying H-field, i.e. an oscillatoryẼ F -field which curls around the polarization of theH-field. In an isotropic conductor, this Faraday field would induce circulating currents in a plane perpendicular to theH-field. Indeed, this is approximately what happens if theH-field is polarized perpendicular to the highly conducting planes of a Q2D conductor, as illustrated in Fig. 9c. However, because of the high in-plane conductivity, these induced currents are damped within the interior of the sample, i.e. currents only circulate within the skin layer at the edge of the sample. Consequently, the in-plane complex surface impedance, Z S = R S + iX S , governs the electrodynamic response of the sample in this situation, where R S and X S are the surface resistance and reactance respectively. 27,28,29 If, instead, theH-field is polarized parallel to the highly conducting layers,Ẽ F will induce both in-plane and inter-layer currents. As before, the in-plane currents will only flow at the edges of the sample, whereas the inter-layer currents flow throughout the bulk of the sample, due to the poor conductivity in this direction. Consequently, the complex inter-layer conductivity dominates the electrodynamic response of the sample under these conditions, as depicted in Fig. 9d. 27,28,29 Of major interest for studies of low-dimensional conductors is the possibility to rotate the applied DC magnetic field, B o , with respect to the sample. Since the cavities are rigidly connected to the sample probe, the orientation of B o cannot be adjusted relative to the cavity, i.e. B o is fixed parallel to the cavity axis for the axial cavity, and perpendicular to the axis for the transverse cavity. Thus, the axial cavity essentially limits investigation to only two orientations of B o relative to the sample, namely parallel or perpendicular to the Q2D layers, which we denote B // and B ⊥ respectively. One could tilt the sample away from either of these geometries within the cavity. However, this would result in a misalignment of theH-field polarization relative to the sample. This is the main reason for developing the transverse cavity. Because of the cylindrical symmetry of the TE01n modes, one can rotate the sample about the cavity axis by means of rotating the cavity end plate to which it is attached, without affecting the polarization ofH relative to the sample; this works for both the end plate and quartz pillar configurations, as illustrated in Fig. 9e. Temperature and magnetic field control Being constructed from a block of high conductivity copper, the cavity makes an excellent heat reservoir for controlling the temperature of the sample, which is kept in good thermal contact with the cavity at all times, either by directly attaching it to the cavity end plate, or by mounting it on a quartz pillar; Silicone grease is used to hold the sample in place. A coil of high resistance wire is used as a heater. This coil is wound around a pillar which screws into the cavity housing and is, therefore, easily interchanged between the various cavity geometries (not shown in Figs 7 and 8). Low pressure helium gas is admitted into the 19.05mm vacuum jacket for exchanging heat between the sample probe and the surrounding pumped liquid helium cryostat. Thus, the cavity and the sample can accurately and controllably be maintained at any temperature in the range from 1.35 K up to about 50 K. The temperature is stabilized according to a calibrated Cernox resistance thermometer embedded in the walls of the copper cavity. Cernox thermometers have negligible magnetoresistance above 4.2 K, and we can correct for a weak magnetoresistance below 4.2 K. Magnetic fields at MSU are generated using a standard commercial superconducting solenoid and power supply. This magnet routinely operates to 8 T, and will go to 9 T at pumped liquid helium temperatures. At the NHMFL, the strongest magnetic fields are produced in axial Bittertype water cooled resistive magnets powered by 20 MW supplies. Presently, the highest available continuous field is 33 T. 33 Details of the NHMFL magnets are published elsewhere. 34 Fig. 10 shows a frequency sweep across the TE011 mode of an axial cavity (cavity A, height = 6.7mm, dia. = 9.52mm, see Table II); the cavity is loaded with a sample of κ-(BEDT-TTF) 2 Cu(SCN) 2 (approx. dimensions 0.5 × 0.50 × 0.2 mm 3 ) 35 in the end plate configuration, and the temperature is 4.2 K. In the upper panel, we plot linear amplitude versus phase. The data points (squares) form a perfect circle passing through the origin, indicating negligible leak vector. The frequency interval between each point is approximately 250 kHz and the solid line is a fit to the data. In the lower panel, we plot both phase and linear amplitude (normalized) versus frequency. This resonance is perfectly symmetric due to the fact that the leak amplitude is 34.5 dB below the amplitude on resonance. The loaded Q−value of the cavity is 19,000 and, thus, the resonance width (2.34 MHz) is considerably less than the standing wave period, which is on the order of 100 MHz. The absolute value of the phase returned by the VR is arbitrary, 36 which is why the phase on resonance is 64 o rather than 0 o (see Fig. 4a). In any subsequent experiment, we would null the phase on resonance, and interpret changes in the complex parameters of the signal returned to the VR according to the procedure described in section II-B.2. It should be noted that this data is about as good as one could expect were the cavity mounted on the bench top and the HG and HM connected directly to the cavity, i.e. the influence of the intervening waveguides has been completely eliminated. III. PERFORMANCE A. Tests Next, we consider the influence of the nearby TM111 mode on measurements made at the TE011 resonance frequency. Fig. 11 shows two such resonances obtained at liquid helium temperature (cavity A): the main part of the figure plots linear amplitude versus frequency; the inset shows the circles in the complex plane obtained for each of the resonances. The TM111 mode has been shifted 230 MHz below the TE011 mode, which corresponds to almost 100 times the width of the TE011 mode (Γ T E011 = 2.42 MHz) and about 40 times the width of the TM111 mode (Γ T M111 = 5.73 MHz). The resonance amplitude of the TM111 mode is about 60% of the TE011 resonance amplitude. However, more importantly, the power of the TM111 signal (Obtained by extrapolation of a Lorentzian fit) is 44.5 dB below the power of the TE011 signal when the TE011 mode is at resonance. Thus, for all intents and purposes, we can rule out any interference between these modes. Even if there were a slight mixing, both modes haveH-fields perpendicular to the applied DC field (B o ) for the end-plate configuration, and the TM111 mode has anH-field node at the center of the cavity where the sample is usually placed in the quartzpillar configuration; this has been verified experimentally using electron paramagnetic resonance standards. 37 Q−values as high as 24,900 have been obtained at liquid helium temperatures for the loaded axial cavities excited in TE011 modes. In general, higher n (higher f o ) TE01n modes have reduced Q−values. In addition, the shorter wavelengths associated with the higher frequencies slightly increases the leak amplitude relative to the signal transmitted through the cavity. These facts, together with the diminished dynamic range of the spectrometer (see Table I) at higher frequencies, make it harder to observe TE01n (n > 1) resonances of comparable quality to the TE011 modes. Nevertheless, the data in Fig. 10 far exceeds the criteria discussed in section II-B for making successful cavity perturbation measurements. Consequently, we have been able to make reliable measurements at frequencies up to 130 GHz. Above about 130 GHz, we have less confidence in the mode assignment of the resonances. However, by following the frequency dependence of data containing distinct features which also depend strongly on the polarization of the AC-fields within the cavity (see following section), we have been able to characterize and use axial cavity modes all the way up to 180 GHz. Fig. 12 shows several higher n TE01n axial cavity modes, together with selective higher frequency resonances. Table II lists the frequencies, Q−values, leak amplitudes and dynamic ranges associated with these modes, as well as some parameters for the transverse cavity (all at 4.2 K). These figures clearly demonstrate the potential of the system for cavity perturbation measurements. It should also be noted from Fig. 12 that many of the resonances were obtained using a single resonator (cavity A), which was one of the main objectives for this system from the very outset. 31 In the following section, we show real data obtained over the frequency range covered by this single cavity. Finally, Fig. 13 illustrates the importance of using active temperature stabilization: the upper panel shows magnetic resonance data taken at the base temperature of the cryostat ( 1.4 K); the upper panel shows the same data obtained using active temperature stabilization at 1.8 K. A clear drift in the unlocked temperature data is observed throughout the course the up and down sweeps of the magnetic field. B. Experimental examples By eliminating the influence of all components of the sample probe (aside from the cavity) on the signal returned to the VR, it is possible to record changes in the complex cavity parameters in real time, i.e. the vector (either amplitude and phase, or amplitude and frequency) recorded at the VR is directly related to the impedance of the cavity, Z C . Here, once again, we see the power of the MVNA. Using a scalar detection scheme, it would be necessary to modulate the frequency in order to extract the complex cavity response. 28 This would inevitably result in a much longer time for recording each data point. The MVNA essentially returns phase and amplitude information at the detection frequency of the VR, which is approximately 10 kHz. 10,38 This aspect of the instrument described in this paper makes it highly suited to measurements in high magnetic fields, which can be expensive to run for long periods. A distinct advantage of conducting fixed frequency optical measurements, as a function of magnetic field, is that the spectral features which are under investigation may be expected to change with field, i.e. the magnetic field is the variable which is used to tune the electronic excitation spectrum of the material under investigation. If, for example, this induces a change from an insulating state, to a metallic state, then the optical response at frequencies comparable to the gap will reflect this change. A beautiful example of this can be seen from data obtained at the NHMFL, (Fig. 14) which shows the complex electrodynamic response of the organic superconductor (TMTSF) 2 ClO 4 , at low temperatures, as the magnetic field is swept to 30 tesla. 39 A rich behavior is observed; each of the more pronounced features may be attributed to modifications in the electronic configuration of the system which are known to occur in the field ranges from 5 to 10 tesla (Field-Induced-Spin-Density-Waves -FISDW), and from 20 to 30 tesla (a phase line within the final FISDW phase). 40 Magneto-quantum-oscillations are also observed at high fields. The data displayed in Fig. 14 were obtained using a phase lock while recording changes in amplitude (A) and the resonance frequency (f o ) of an axial cavity excited at 47.2 GHz. The sample was oriented with its c* axis parallel to the applied DC magnetic field, B o , and currents were excited in the ab plane of the sample. Each field sweep took less than 10 minutes. The quality of this data is, at worst, comparable to an equivalent DC measurement. From the combined information obtained from changes in dissipation (∆A) and dispersion (∆f o ) within the cavity, we can obtain valuable information about the electrodynamics of the bound and free carrier systems in this fascinating material. 39,40 The above example is quite different from a zero-field measurement where data are usually taken at many frequencies in order to investigate a particular spectral feature. Such experiments are generally plagued by poor dynamic range (signal-to-noise), especially in the millimeter and sub-millimeter spectral ranges. This is because identical coupling between the sample and the spectrometer cannot be guaranteed at each frequency, resulting in a large scatter of the data. This is unfortunate, since the cavity perturbation technique is inherently sensitive and, through the use of a suitable spectrometer (e.g. the MVNA), provides plenty of dynamic range at any given frequency. The instrument described in this paper is, instead, optimized at each frequency in order to detect minute changes in the optical conductivity of a sample as a function of magnetic field. These changes may subsequently be normalized to the zero-field conductivity using an appropriate instrument. Whether to choose a phase lock or a frequency lock depends on the experiment. A frequency lock generally provides better stability because of the higher Q−value of the quartz frequency reference, as compared to the cavity. Fig. 15 shows an example of separate measurements using both techniques. The sample under investigation is the purple bronze η−Mo 4 O 11 . 9,41 Fig. 15a shows variations in amplitude for both up and down sweeps of the magnetic field. There is a slight hysteresis, which is well documented for this material. However, the transmitted amplitude is considerably lower at high magnetic fields for the frequency locked measurement. This can be attributed to the cavity going well off resonance, as evidenced by a phase change of 25 o in the first 7 or 8 tesla (Fig 15b). Inspection of Fig. 4 clearly illustrates that a 25 o phase shift will lead to such a reduction in amplitude, even if there is no change in the dissipation within the cavity. Indeed, the ratio of the amplitudes obtained by each technique scales nicely with the cosine of the phase shift, as shown in the inset to Fig. 15b. Consequently, the frequency lock is inappropriate in this case, because of considerable mixing of the dissipative and dispersive responses of the sample in the phase and amplitude re-turned to the VR. Fig. 15c shows the frequency shift observed using the phase lock. Fig. 16 shows Periodic Orbit Resonances (PORs − related to cyclotron resonance) 42 observed through the inter-layer conductivity of the quasi-two-dimensional organic conductor α−(BEDT-TTF) 2 TlHg(NCS) 4 . 43,44 The frequencies, which are indicated above each trace in the figure, were obtained using a single axial cavity, and range all the way from 44 GHz to 182 GHz. The sample was mounted in the end-plate configuration. Not all of the frequencies correspond to TE01n modes. However, from the shapes of the resonances (∼Lorentzian peaks), we can be certain that the sample sits in the same electromagnetic environment for all of the modes, i.e. with the polarization of the oscillatoryH-field parallel to the conducting layers, resulting in the excitation of inter-layer currents (see Fig. 9d). This can be confirmed by studying the same sample in the TE011 quartz pillar configuration, as shown in Fig. 17, which is the conventional geometry for observing cyclotron resonance. Because of a high in-plane conductivity, it is the in-plane surface resistance of the sample that governs the dissipation in the cavity (see Fig. 9c). 27,45 This is the reason for the rather unconventional lineshape, i.e. an inflection rather than a symmetric dip or peak. The data in Figs 16 and 17 are exceptional in their quality when compared to earlier attempts to measure cyclotron resonance in organic conductors. 46 Furthermore, the resonance line shapes agree precisely with theory, thereby providing absolute confidence in the ability of the technique to discriminate between in-plane and interlayer transport phenomena in quasi-two-dimensional conductors. This has traditionally been problematic using conventional DC resistivity probes, because of uncertainties in the current paths within the samples. This opens up a huge range of possibilities for tackling issues concerning the role of electronic dimensionality in the physical properties of low-dimensional systems. IV. SUMMARY We have described an instrument for conducting millimeter-wave cavity perturbation measurements over a continuously tunable frequency range (40 − 200 GHz). The system is compatible with magnets both at Montana State University (up to 9 tesla) and at the National High Magnetic Field Laboratory (up to 33 tesla) in Tallahassee, FL. The utilization of a Millimeter-wave Vector Network Analyzer enables simultaneous measurements of the complex cavity parameters (resonance frequency and Q−value) at a rapid repetition rate (∼ 10 kHz). Several experimental examples are presented which demonstrate the potential of this system for studying the magnetoelectrodynamics of low-dimensional conducting systems. The first column lists the frequency band and the MVNA harmonic. The second column lists the cavity (letter) and end plate used (number), as well as the mode (if known); cavities A, B and C have dimensions (length × diameter) 6.7mm × 9.52mm, 8.73mm × 9mm, and 5.54mm × 5.64mm respectively, cavity T is the transverse cavity (10.16mm × 7.75mm), and end plates 1, 2 and 3 have coupling hole diameters of 1.32mm, 0.84mm and 0.62mm, respectively. The next three columns list the resonance frequencies (fo), Q−values and dynamic ranges (signal-to-noise) for each mode. The final column lists the contrast in dB between the transmitted amplitude on resonance [A(fo)] and the leak amplitude (A l ). The dynamic range is somewhat lower at 45 GHz than in the main part of the V-and W-bands due to the fact that the V-band wave-guide is so close to cut off at these low frequencies. 2. An equivalent LRC circuit representation of the key microwave components of the sample probe. The coupling between HG (HM) and the incidence (transmission) waveguide is modeled as a coupling mutual inductance mHG (mHM ). The cavity is coupled to each of the waveguides through coupling mutual input (min) and output (mout) inductances. A leak signal between the incidence and transmission waveguides, in parallel with the cavity, is modeled as a direct coupling mutual inductance, m l , between the two waveguides. Fig. 4. The complex leak vector translates the resonance in the complex plane, as shown by the arrow in a). The result is that the resonance frequency, fo, no longer lies along the real axis (φ = 0). Thus, fo no longer corresponds to the maximum amplitude, resulting in the distorted Lorentzian lineshape shown in b) and c). Magnetic field (tesla) FIG. 6. A comparison between the background (empty Cu cavity) response of the sample probe using Gold plated and unplated stainless steel sections at the lower end of the probe; these data were obtained at 76.7 GHz and 4.2 K. The broad dip is attributed to an EPR absorption due to small quantities of Fe 3+ (rust) at the surface of the SS. Professional gold plating (solid line) clearly eliminates this contamination, which is evident both in the unplated data (short dash) and the data obtained after our efforts to plate the SS at MSU (long dash). The quartz pillar configuration, showing the sample at the heart of a TE011 cavity atop a quartz pillar, and the axialH-field at the position of the sample. c) Excitation of in-plane currents at the edges of a Q2D conductor. d) Excitation of inter-layer currents within the bulk of a Q2D conductor. In both c) and d), the low conductivity direction is parallel to the normal to the disc shaped sample. e) Sample rotation for the quartz pillar configuration in the transverse cavity − note that the orientation of the axial ACH-field does not change relative to the sample upon rotation. 13. A comparison between magnetic field sweeps with the temperature unlocked (upper panel) and actively controlled (lower panel). The broad dip corresponds to the Fe 3+ resonance seen in Fig. 6. Note: this data was obtained prior to Au plating the lower SS section of the probe. FIG. 15. a) A comparison between the amplitude variation versus magnetic field obtained using frequency and phase locking techniques. The sample is the purple bronze η−Mo4O11, and the observed changes are due to the excitation of inter-layer currents in this quasi-two-dimensional conductor. b) Shows the phase variation during the frequency locked measurement (absolute microwave frequency fixed at 44.272 GHz), while c) shows the frequency variation during the phase locked measurement (absolute microwave phase across the sample probe locked). The inset to b) compares the ratio of the amplitudes obtained for the two techniques in a), with the cosine of the phase change in the main part of b). The good correspondence between these quantities confirms that the difference between the traces in a) can be attributed to the large phase shift during the frequency locked measurement. Thus, a phase lock should be applied in this situation. axial cavity in the end plate configuration; the temperature is 1.8 K. The data appear noisier at higher frequencies due to the reduced dynamic range of the spectrometer and due to the reduced sensitivity (lower Q−values) of the higher order cavity modes. The resonances have been scaled so as to appear the same strength, and have been offset for the sake of clarity. The sharp spikes observed slightly above the main peak positions are Electron Paramagnetic Resonance signals due to a DPPH sample placed in the cavity.	2018-06-16T10:26:36.527Z	1999-07-20T00:00:00.000	{ "year": 1999, "sha1": "952ffae856e1ac56c92e549690c407a32f03ad69", "oa_license": null, "oa_url": "http://arxiv.org/pdf/cond-mat/9907310", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "e424ef7d089c71c17920f84892afce1b01ee202d", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
54160494	pes2o/s2orc	v3-fos-license	Immunotargeting of Melanoma The main task of the immune system is to defend the body against pathogens. The ability of the immune system to recognize and eliminate cancer cells is the basis for cancer immunotherapy. There is ample evidence of how important role plays the immune system to fight cancer: (i) spontaneous remission in patients with certain cancers; (ii) the presence of specific cytotoxic T lymphocytes in the environment of the tumor or regional lymph nodes, (iii) the presence of monocytes, lymphocytes and plasma cells infiltrating the tumor, (iv) increased incidence of some malignancies in immunosuppressed patients, (v) documented remissions of the disease after use of immunomodulators. Better understanding of the molecular and cellular mechanisms that control the immune system has enabled the development of many innovative and promising therapeutic strategies that modulate the immune response. It seems that in the next 5 to 10 years past surgery, radiotherapy and chemotherapy, immunotherapy will find a permanent position in the treatment of cancer modalities. Introduction The main task of the immune system is to defend the body against pathogens. The ability of the immune system to recognize and eliminate cancer cells is the basis for cancer immunotherapy. There is ample evidence of how important role plays the immune system to fight cancer: (i) spontaneous remission in patients with certain cancers; (ii) the presence of specific cytotoxic T lymphocytes in the environment of the tumor or regional lymph nodes, (iii) the presence of monocytes, lymphocytes and plasma cells infiltrating the tumor, (iv) increased incidence of some malignancies in immunosuppressed patients, (v) documented remissions of the disease after use of immunomodulators. Better understanding of the molecular and cellular mechanisms that control the immune system has enabled the development of many innovative and promising therapeutic strategies that modulate the immune response. It seems that in the next 5 to 10 years past surgery, radiotherapy and chemotherapy, immunotherapy will find a permanent position in the treatment of cancer modalities. reduced expression/absence of expression of major histocompatibility complex antigens class I and II -(MHC I and II) (Resifo et al., 1993;Garrido et al., 1993;Ward et al., 1990) -loss of tumour antigens (Knurth et al., 1989;Uyttenhove et al., 1983;Ward et al., 1989) -improper intracellular antigen processing to prepare antigens for presentation (defects in proteosome function or adenosine-tri-phosphatate (ATP) dependent proteins transporting TAP peptides (Selinger et al., 1998) -reduction/loss of costimulatory signals of e.g. B7 molecules on the surface of tumour cells suppressed expression of adhesion molecules inhibited expression of the Fas receptor and/or Fas ligand resulting in the apoptosis of cytotoxic T lymphocytes (CTL) and/or natural killer (NK) cells (Strand et al., 1996;Hahne et al., 1996;Walker et al., 1997) -synthesis and secretion of immunosuppressive factors, e.g. interleukin (IL)10, TGF-beta, prostaglandine E 2 suppressing immune response (Boon et al., 1995;Schmidt-Wolf et al., 1995) -expression of TRIAL (TNF-related apoptosis inducing ligand) on the tumour cell surface leading to T lymphocyte apoptosis (Baker et al., 1998) -prolonged stimulation of specific T cells resulting in clonal exhaustion or AICD (activation-induced cell death) (Overwijk et al., 2001) The term "cancer immunosurveillance" is no longer sufficient to precisely describe the difficult interactions that arise among a developing tumor and the immune system of the host. It was believed that cancer immunosurveillance was protecting the host by the adaptive immune system in the early phase of cell transformation. Dunn and coworkers described that not only the adaptive but also innate immune system plays a role in the process and serve not only to defend the host from tumor development but also to sculpt, or edit, the immunogenicity of tumors that may finally form. The authors use a wider term -"cancer immunoediting" to more suitably stress the double roles of immunity in not only preventing but also shaping neoplastic disease. According to this theory cancer immunoediting comprises of three phases: elimination, equilibrium and escape. Elimination phase is based on the original concept of cancer immunosurveillance. If during this phase the developing tumor can be successfully eliminated the immunoediting process is completed without progression to the subsequent phases. If the neoplasm circumvents the immune surveillance system, it may enter the next subclinical stage, termed the equilibrium phase, in which tumour tries to establish itself and withstand the growing pressure exerted by the immune system. Although lymphocytes and the secreted IFN-gamma attack the tumour cell, its genetic instability and multiple mutations protect it against destruction. The equilibrium phase is a long-term process and may last for years. The next stage is the escape phase which can stem from the exhaustion or suppression of the immune system, reduced expression of type I MHC on neoplastic cells or decreased sensitivity to interferon (IFN) gamma. The equilibrium phase does not occur in non-immunogenic tumours and the modified cell automatically enters the escape phase. This clinical stage is associated with fast growth and progression of the tumour (Dunn et al., 2002(Dunn et al., & 2006. In tumours induced by viruses and chemical substances it has been found that tumourrelated antigens are immunogenic and trigger specific cellular and humoral responses. Cytotoxic cells, including CD8+ CTL, NK cells and CD4+ T helper cells, are presumed to www.intechopen.com play a decisive role in the process. Cellular immune response also involves neutrophil granulocytes (Cavallo et al., 1992) and macrophages. Humoral response is executed by antibodies which are directed against tumour antigens produced by activated B lymphocytes -plasmocytes. The process leading to the lysis of tumour cells in this mechanism may comprise activation of the complement system or induction of ADCC (Antibody Dependent Cell-mediated Cytotoxicity). The role of different subpopulations of helper T lymphocytes (Th cells) has not been fully explored yet (Nishimura et al., 1999). Cytokines secreted by helper T cells activate humoral or cellular anti-tumour immune response. Depending on the profile of secreted cytokines, Th cells are divided into two sub-groups: Th1 producing IL-2, IFN-gamma and IL-12 inducing cellular immune response; and Th2 secreting IL-4, IL-5, IL-6 and IL-10 which stimulate the humoral response and suppresses the cellular response (Swain et al., 1995). Moreover, the subpopulation of regulatory T cells CD4+/CD25+ hihg Foxp3 may inhibit immune response by paracrinous secretion of immunosuppressive cytokines IL-10 and TGFbeta. Th17 lymphocytes belong to the most recently described CD4+Th . A characteristic feature of these cells is the secretion of interleukin-17 but also IL-22, IL-26, IL-6, TNF-alfa. So far, no unequivocal influence of Th17 lymphocytes for the development of cancer has been described. It was shown that IL-17 may promote tumor cell growth by inducing tumor vascularization or enhancing inflammation. Meanwhile, a lot of data suggests the antitumor activity of Th17 cells. It therefore appears that Th17 cells may have different effects on tumor growth depending on its immunogenicity, clinical stage (different role in the early and late stages), as well as the origin of cancer and the influence of inflammation and angiogenesis in tumor microenvironment (Hus et al., 2010). Effective anti-tumour response elicited by the immune system consists of two phases, induction and effector. The induction phase is marked by stimulation of specific antitumour response, while the effector phase involves a selective elimination of tumour cells. The following mechanisms are sequentially activated in the induction phase: 1. Presentation of tumour antigens in the context of HLA (Human Leukocyte Antigens) class I to CD8+ lymphocytes, or HLA class II to CD4+ lymphocytes; 2. Providing the costimulatory signal for T lymphocytes, e.g. binding of B7.1 molecules (CD80, CD86) with the CD28 receptor on the surface of T lymphocytes (Janeway et al., 1994) 3. Providing the proliferation signal for immune cells in the area of tumour antigen presentation (typically cytokines or growth factors) (Pardoll et al., 1995) Induction of immune response is initiated by antigen presentation by dendritic cells (DCs) (Banchereau et al., 1998). DCs phagocyte antigens from necrotic or apoptotic tumour cells and then migrate into regional lymph nodes, where they undergo maturation. In the lymph nodes, they present the antigen on their surface to T lymphocytes CD8+ (in the context of HLA I) and to T lymphocytes CD4+ (in the context of HLA II). This is where CD8+ and CD4+ CTLs are formed together with antibodies directed against specific tumour antigens. The stimulation of humoral response requires a presentation of tumour antigen by a B lymphocyte to the specific CD4+T lymphocyte. Direct lymphocyte interaction, as well as production of cytokines by CD4+, triggers transformation of the B lymphocyte into a plasma cell and secretion of antibodies. The effector phase of anti-tumour response may include the following cell destruction mechanisms by: www.intechopen.com 1. Specific activated CD8+ and CD4+ CTLs 2. Activated NK cells 3. Tumour-infiltrating granulocytes and macrophages 4. Specific antibodies determining activation of the complement system or ADCC 5. Inhibition of tumour neoangiogenesis by cytokines, such as IFN-gamma secreted by activated T lymphocytes (CD8+ and CD4+) into the tumour microenvironment. Absence of one or several components listed above makes it possible for tumour cells to escape immune surveillance, which leads to tumour progression. Mechanisms involved in the induction and effector phases of anti-tumour response which have been identified so far makes it possible to apply immunotherapy to restore the capacity to recognise and eliminate tumour cells in immune defence mechanisms (Nanda et al., 1995). Tumour immunotherapy Tumour immunotherapy is a treatment strategy based on intentional interference with the human immune system in order to enhance or modify the body's defence mechanisms against the developing tumour. Immune therapy can be divided into two major categories: passive and active. Within each category the therapy might be either specific or non-specific. Passive non-specific immunotherapy Passive non-specific immunotherapy involves transfer of factors or activated effector cells to elicit a non-specific activation of the immune system provoking anti-tumour response. Immunotherapeutic approaches may use e.g. cytokines or LAK (Lymphokine Activated Killers) cells. Cytokines are low molecular weight proteins which play a vital role in all phases of immune response, both humoral and cellular. In order to display biological activity, cytokine must target a specific receptor on imune cells (T and B lymphocytes, NK cells, monocytes/macrophages and granulocytes). Various cytokines may demonstrate an antagonistic, agonistic, additive or synergistic effects in biological processes. Known antitumour effects of cytokines include (i) direct cytotoxic effect (TNF-sensitivity of cancer cells to cytotoxic effects of various biological or chemical factors (IFN-gamma, TNF-alpha), (ii) inhibition of tumour cells proliferation (IFN-alpha, IFN-gamma) and (iii) activation of NK cells (Granulocyte-Macrophate Colony-Stimulating Factor -GM-CSF, IL-2, IL-6). The first recombinant cytokine registered for clinical application was IFN-alpha which has been used in the treatment of hairy-cell leukaemia, chronic myeloid leukaemia, melanoma, Kaposi's sarcoma, metastatic renal cell cancer and malignant lymphoma. Several randomised phase III trials evaluating IFN-alfa-2a and IFN-2b in low, medium and high dose have been conducted. Only in two of them statistically significant improvement of OS (overall survival) was observed. High dose IFN-alfa-2b (Intron®) has been approved by the U.S. FDA (Food and Drug Administration) based on the results of ECOG 1684 trial. Intron is indicated in patients after resection of high-risk melanoma (stage IIB and stage III). In the registration trial 287 patients after surgical removal of melanoma were randomised into two study arms: INF-alfa-2b vs observation. At a median follow-up of 6.9 years, a statistically significant improvement in survival was demonstrated for the patients treated with IFNalfa-2b. However at 12,6 years of follow-up, OS was not significantly different between the two study groups, even though there was a significant benefit for relapse free survival (RFS). About 80% of patients developed grade 3 and 4 toxicity (Kirkwood et al., 1996). In another phase III study (ECOG 1694) efficacy of high-dose IFN-alfa-2b was compared with an experimental vaccine GM2-KLH21. After 2 years of median follow-up the median RFS and OS were significantly longer in patients treated with IFN-alfa-2b (Kirkwood et al., 2000). Concerns raising the vaccine control group used in ECOG 1694 lead to initiation of another randomized phase III trial (EORTC 18961). The study which enrolled 1314 patients with stage II melanoma evaluated efficacy of GM2-KLH21 compared with observation. The trial was closed early by the data monitoring committee because of longer survival in the observation arm (Eggermont et al., 2008a). Recently (March 2011) pegylated-IFN-alfa-2b (Sylatron®) has been approved for the treatment of patients with melanoma with microscopic or gross nodal involvement after definitive surgical resection including complete lymphadenectomy. The approval was based on the results of EORTC 18991 trial published in Lancet in the year 2008. The study enrolled 1256 patients with resected stage III melanoma to either observation or pegylated-IFN-alfa-2b. The estimated median RFS was 34.8 months (95% confidence interval (CI): 26.1, 47.4) and 25.5 months (95% CI: 19.6, 30.8) in the Sylatron and observation arms, respectively [hazard ratio (HR) 0.82 (95% CI: 0.71, 0.96); unstratified log-rank p = 0.011]. Unfortuntly, there was no difference in OS between the Sylatron and the observation arms [HR 0.98 (95% CI: 0.82, 1.16)]. The grade 3 and 4 adverse events were less frequent in patients treated with pegylated-IFN-alfa-2b than observed in subjects receiving IFN-alfa-2b in earlier trials (Eggermont et al., 2008b). Another cytokine registered in the USA for palliative treatment of renal cancer and melanoma is IL-2, however, it has recently been demonstrated that high doses of IL-2 stimulate Treg lymphocytes, what in fact suppresses immune response (Ahmadzadeh et al., 2006). IL-2 was approved by FDA in 1998 for the treatment of patients with metastatic melanoma. Overall objective response rates in patients treated with high dose IL-2 was 17% (Rosenberg et al., 1994a). In a highly selected patient population with metastatic melanoma (270 patients) complete response (CR) was observed in 6% with median duration of response over 59 months. Partial response (PR) was seen in 10% of IL-2 treated patients. Disease did not progress in any patient responding for more than 30 months. Grade 3 and 4 toxicity was very frequent (Atkins et al., 1993(Atkins et al., & 1994. In another trial 684 patients with metastatic melanoma received high-dose IL-2 either alone or in combination with a variety of melanoma vaccines. The overall objective response rates were highest in patients treated with gp100:209-217(210M) peptide vaccine (22%) compared to IL-2 (13%) alone (P=0.01) . Number of clinical trials evaluating the efficacy of biochemotherapy (combination of chemotherapy and biological agents) have been conducted. In a small phase III trial the investigators compared sequential CVD (dacarbazine, cisplatin, vinblastine) chemotherapy with IL-2 and interferon alfa with CVD alone. Patients treated with biochemotherapy responsed more frequently (48%) to the treatment than receiving chemotherapy alone (25%). The median OS was 11,9 months vs. 9,2 months for biochemotherapy and CVD alone respectively (Eton et al., 2002). In another similar phase III randomized trial (E3695) the observation seen in the previous study was not confirmed -no improvement in OS between both study arms (CVD + IL-2 + IFN-alfa-2b vs CVD) (Atkins et al., 2008). A recent meta-analysis of 18 trials involving 2621 patients with metastatic melanoma showed that biochemotherapy improves overall response rate without benefit of survival (Ives et al., 2007). At the American Society of Clinical Oncology (ASCO) 2010 annual meeting Lawson et al. presented results of a phase III trial (E4697) evaluating efficacy of GM-SCF in the adjuvant www.intechopen.com treatment in patients with resected high risk melanoma (stage III and IV). Patients were injected with 250 µg of GM-CSF or placebo s.c. daily for 14 consecutive days in a 4 week intervals. The treatment duration was 1 year. Median disease free survival (DFS) of patients treated with GM-CSF was significantly longer than of patients receiving placebo -11,8 vs. 8,8 months (p=0,034). Median OS was 72,1 vs. 59,8 months respectively in the study arm evaluating GM-CSF and placebo, but the difference was not statistically significant (P=0,551). Toxicity was consistent with known effects of GM-CSF (Lawson et al., 2010). Another type of passive non-specific immunotherapy is the transfer of LAK cells. In this method, mononuclear cells are isolated from the blood of a patient, stimulated with IL-2 ex vivo and injected back with a simultaneous administration of high doses of IL-2 for continuous lymphocyte stimulation. LAK-based therapy was used by Rosenberg in a clinical trial of renal cancer and melanoma. It was found, however, that the therapeutic effect was rather achieved by IL-2, but not LAK cells, which is why further trials were discontinued (Rosenberg et al., 1985(Rosenberg et al., & 1993. Passive specific immunotherapy Passive specific immunotherapy is a treatment method based on the administration of factors or effector cells targeting specific tumour cells. Examples include application of antibodies directed against antigens present on tumour cells or cell therapies using tumourinfiltrating lymphocytes (TIL) which are isolated, cultured, activated and then transferred back into the patient. High expectations are currently held for the recently developed therapy based on modification of autologous lymphocytes isolated from PBLs (Peripheral Blood Lymphocytes) (Morgan et al., 2006). Passive immunotherapy employing antibodies was first described as far back as 100 years ago. However, it was not until the development of a viable technique of generating monoclonal antibodies (mAb) (Kohler et al., 1975) that the method found more extensive applications in tumour therapy. The dynamic development of genetic engineering techniques has enabled generation of humanised and human mAb (technology using transgenic mice -Xenomouse®) (Green, 1999) deprived to the maximum extent possible of toxic effects associated with the induction of HAMA (Human Anti Murine Antibody) reaction. Modified specific mAb used in immunotherapy act by binding directly to the tumour antigen, activate ADCC and CDC (Complement Dependent Cytotoxicity). Monoclonal antibodies can also block receptors on tumour cells, e.g. growth factor receptors. Antibodies conjugated with radioisotopes, cytostatics, enzymes, cytokines or toxins immediately destroy cells which they target. The first mAb registered by the FDA in 1997 was Rituximab (MabThera®) used in the treatment of patients with low malignancy B-cell non-Hodgkin lymphomas. To date there are several mAb (trastuzumab, cetuximab, panitumumab, bevacizumab, ibritumomab, tiuxetan, tositumomab, gemtuzumab, ozogamicin, alemtuzumab) approved for the treatment of various malignancies. Ipilimumab, anti-CTLA4 (Cytotoxic T-lymphocyteassociated antigen 4) mAb has been recently registered for the treatment of metastatic melanoma (described in section: active non-specific immunotherapy). Angiogenesis is of profound importance in melanoma development (Streit et al., 2003). Malignant melanocytes excrete vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) (Kurzen et al., 2003, Lacal et al., 2000, Lev et al., 2003. The VEGF is the fundamental regulator of new vessels formation in the tumour and its expression is www.intechopen.com related to poorer prognosis in melanoma patients (Ugurel et al., 2001). Bewacizumab is a mAb directed against VEGF. Its effectiveness has been proven in the treatment of colon and kidney cancer (Saltz et al., 2008, Melichar et al., 2008. In the treatment of advanced melanoma its efficacy was evaluated in combination with low dose interferon-alfa-2b in a phase II trial which did not show extension of survival in studied patients (Varker et al., 2007). In another phase II trial enrolling 62 metastatic melanoma patients, the effectiveness of bewacizumab in combination with temozolomide was tested. Objective clinical responses were observed in 26% of patients, while 30% of the patients developed SD (stabilization disease) lasting for 1.5-7.5 months (median of 3 months) (Von Mos R et al., 2007). In a subsequent phase II trial with 214 randomized patients, the therapy with bevacizumab in combination with carboplatin and paklitaxel (first line treatment) improved effectiveness as the median OS was significantly longer (12.3 months) than in the control group (8.6 months) treated with chemotherapy alone. The toxicity of treatment was similar in both study arms (the number of grade 3-5 adverse events was 2% greater in the group treated with bevacizumab) . At the ASCO 2009 annual meeting, preliminary results were presented on the effectiveness of treatment with bevacizumab in combination with nab-paklitaxel in 41 patients with non-resectable III and IV stage melanoma (50% in M1c stage). Nab-paklitaxel is composed of paklitaxel and albumin. This combination alleviates the adverse events caused by administration of paklitaxel alone. The percent of 6 and 12months survival was 91 and 83%, while the median OS was not reached (Boasberg et al., 2009). The preliminary results of a phase II trial evaluating the efficacy of bevacizumab in combination with everolimus in first line treatment of advanced melanoma were presented at ASCO 2009 conference. Fifty six patients were qualified to the study, but the preliminary analysis included 31 subjects. PR was observed in 1 patient, SD in 19 patients and the median PFS was 3.5 months. The most frequent adverse event was mucositis. Grade 3 toxicity was observed in 13% of patients, other grade 3 adverse events were noted in less than 10% of subjects (Peyton et al., 2009). α v integrin family such as α v b3 and α v b5 are related to the promotion of angiogenesis in the tumor and contribute to its growth. Human mAb CNTO 95 (intetumumab) directed against integrin has been found to show anticancer and antiangiogenic activity in animal models (Trikha et al., 2004). At the ASCO 2009 annual meeting results of the phase II trial with 129 metastatic melanoma patients enrolled, were reported. The patients were randomised into four study arms: (i) intetumumab 5 mg/kg; (ii) intetumumab 10 mg/kg; (iii) intetumumab 10 mg/kg + DTIC; (iv) placebo + DTIC. Median OS of patients respectively in the abovementioned groups was 9.8; 14; 10.9 and 7.6 months. The treatment was well tolerated and the percent of patients with serious adverse events was the highest in the arm treated with dacarbazine (DTIC) plus placebo (Loquai et al., 2009). Volociximab is a chimeric monoclonal antibody that combines with α5β1 integrin, inhibiting angiogenesis in tumors by damaging the bonds between endothelial cells and fibronectin in the intracellular cytoplasm. Effectiveness of volociksimab was evaluated in a phase II trial which enrolled 19 patients diagnosed with advanced melanoma after progression during the first line treatment. Objective clinical responses were observed only in 5% of the patients. Responses to the treatment were associated with the expression of integrin α5β1 in patients tumor samples.The treatment was relatively well tolerated. Grade 1 and 2 toxicity (nausea, vomiting, diarrhoea) occurred in 68% of patients (Linette et al., 2008). Overexpression of glycoprotein NMB (GPNMB) was observed in many malignancies including melanoma. CRO11-vcMMAE is a human mAb targeting extracellular domain of www.intechopen.com GPNMB, combined with the tubulin destabilising factor -monomethyl auristatin E (MMAE). The whole complex is stable in the blood, while MMAE is released within the tumour (Tse et al., 2006). CRO11-vcMMAE was evaluated in phase I/II clinical trials. At ASCO 2009 the phase II study results were reported (MTD 1.88 mg/kg); this trial enrolled 36 patients with metastatic melanoma treated earlier with systemic therapy. 4 PR and 19 SD were observed, while the median PFS was 4 months. The most often noted grade 3 and 4 included neutropenia (22% patients) and rash (19%). Rash grade 2 and higher correlated with longer PFS (Hwu et al., 2009). Another therapeutic strategy of specific immunotherapy is the use of TIL cells which are extracted from patients tumors and incubated ex vivo in the presence of IL-2. Next they are transferred back into corresponding patients with a simultaneous administration of IL-2. In 1994, et al. observed objective clinical responses in 34% of patients with metastatic melanoma (Rosenberg et al., 1994b). However, therapeutic benefit was assigned to i.v. injected IL-2 which stimulated lymphocytes. To increase the efficacy of adoptive cell therapy, prior to TIL infusion, chemotherapy and/or total body irradiation (in order to obtain limfodepletion -elimination of suppressor T-cells) followed by administration of IL-2 was applied. Although effectiveness of this strategy was limited in the treatment of leukemia (Curti et al., 1998;Schultze et al., 2001) some activity in the therapy of melanoma was observed (Oble et al., 2009). Patients with metastatic melanoma prior to TIL and IL-2 infusion received a limphodepleting dose of cyclophosphamide and fludarabine (Dudley et al., 2005). In 51% (n=35) of treated patients objective response rate was observed. A very high overall response rate (70%) was seen in another trial where in melanoma patients prior to TIL transfusion a total body irradiation with a dose of 1200 cGy (in 3 fractionated doses) was obtained (Rosenberg et al., 2008). Clinical trials, which tested peripheral blood lymphocytes stimulated with cytokines did not produce the desired result due to low numbers of antigen-specific lymphocytes. In order to increase the number of antigen-specific lymphocytes an in vitro stimulation with DC loaded with particular antigen can be obtained. In a phase I study patients with metastatic melanoma were treated with CD8+ T cell isolated from peripheral blood and incubated ex vivo w i t h m a t u r e a u t o l o g o u s D C p u l s e d w i th melanoma antigen Melan-A. Objective responses were seen in 3 out of all 11 patients. (Mackensen et al., 2006). It has been proven that administration of CD4+ T-cells instead of CD8+ lymphocytes may turn out to be more effective in adoptive cell therapy (Perez-Diez et al., 2002). Remission was observed in a patient with metastatic melanoma who received autologous CD4 + T cells specific for the melanoma antigen NY-ESO-1 (Hunder et al., 2008). Morgan et al. isolated PBLs which were then modified ex vivo with genes coding for TCR specific for a number of tumour-associated antigens (MART-1, gp100, NY-ESO-1 or p53). They demonstrated that modified PBLs recognise, in the context of HLA-A2, the abovementioned tumour antigens present on melanoma, lung and breast cancer cells. The interaction of TCR and tumour peptides was associated with the production of a large quantity of IFN-gamma. In the clinical trial, Morgan et al. enrolled 31 patients with advanced melanoma resistant to IL-2 therapy. All subjects received autologous PBLs engineered with the anti-MART-1 TCR gene. Two patients showed a prolonged remission lasting more than 20 months. Patients who achieved a clinical response also exhibited high levels of modified circulating lymphocytes after 1 year from the injection of transduced PBLs. No toxicity was found to be associated with this type of therapy (Morgan et al., 2006). www.intechopen.com Active non-specific immunotherapy Active non-specific immunotherapy is a therapeutic method based on stimulation of the immune system, cell-mediated immune response in particular, with antigens that are not present in tumour cells. Historically, the therapy has involved microorganisms, microbial fragments, enzymes and hormones. In recent years, due to the development of advanced genetic engineering technology and growing understanding of immune mechanisms involved in tumour growth, immune response-modulating mAbs were constructed. Substances which contribute to the stimulation of immune processes include non-specific immunostimulators and immunomodulators. Immunostimulators include (i) intact microorganisms (living BCG -Bacillus Calmette-Guérin, killed Corynebacterium parvum), (ii) cell wall elements (BCG, Nocardia), (iii) microbial glycoproteins derived from Klebsiella, (iv) synthetic components, e.g. endotoxins (lipopolysaccharides). Immunomodulators include (i) thymus extracts (TPI, THF, TFX), (ii) synthetic thymus hormones (thymosin alpha-1, thymopoietin), (iii) tuftsin, (iv) enkephalins and endorphins (v) lymphocyte extracts (transfer factor, immunogenic RNA) (Hersh et al., 1991). Some of the substances listed above, when injected directly into the tumour, have triggered local inflammatory reaction associated with the infiltration of APCs, neutrophils, and T and B lymphocytes. Regression of injected tumours was often accomplished, however, the method was not capable of producing a specific systemic anti-tumour responses. No active non-specific immunotherapy modality has entered routine clinical practice as yet. Passive non-specific immunotherapy includes also monoclonal antibodies modulating the immune system. CTLA-4 is an immune checkpoint molecule that is up-regulated on activated T-cells, which suppresses further activation of specific CD4+ and CD8+ T-cells by interaction with DC or directly as a result of a contact between suppressor and effector T lymphocytes. The anti-CTLA4 monoclonal antibody by blocking the interaction of CTLA-4 with CD80/86 switches off the mechanism of immune suppression and enables continous, unrestrained stimulation of T-cells by DC (J. Mackiewicz & Kwinta, 2010). Two IgG monoclonal antibodies directed against CTLA-4 -ipilimumab and tremelimumab have been tested in number of clinical trials in patients with melanoma. In 2011 (March) ipilimumab (Yervoy®) has been approved by FDA for the treatment of metastatic melanoma patients that failed previous systemic therapy. The approval was based on the results of a randomized phase III trial which included 676 HLA-A*0201-positive patients with unresectable stage III or IV melanoma. Patients enrolled were treated previously with IL-2 or chemotherapy and were randomly assigned for administration of ipilimumab plus glycoprotein (gp)100 vaccine (403 patients), ipilimumab alone (137), or gp100 alone (136). Ipilimumab, at a dose of 3 mg per kilogram of body weight, was administered with or without gp100 every 3 weeks for up to four treatments. Patients receiving ipilimumab plus a peptide vaccine had a median survival of 10 months, compared with 6.4 months of patients receiving the gp100 alone (P<0.001). Subjects treated with ipilimumab alone had a nearly identical median survival -10.1 months -in the 3-group clinical trial (P < 0.003). Immune related adverse events (grade 3 and 4) occurred in 10-15% of patients treated with ipilimumab and in 3% treated with gp100 alone (Hodi et al., 2010) Another monoclonal antibody targeting CTLA-4 is tremelimumab (administered in the dose of 15 mg/kg every 3 months), which effectiveness has been evaluated in a phase II trial in previously treated patients. From among 256 patients with metastatic melanoma enrolled into the study, objective clinical responses were observed in 8.3% patients, while the median OS was 10.2 months (Kirkwood et al., 2008). The above results inclined the next phase III www.intechopen.com trial in which 643 patients were treated with tremelimumab in monotherapy or in combination with DTIC/TMZ in the first line setting. Analysis of preliminary results did not show the advantage of tremelimumab over the standard therapy (OS 11.8 vs 10.7) and the trial was terminated (Ribas et al., 2008). The effectiveness of treatment with tremelimumab in combination with high doses interferon-alfa-2b was evaluated in phase II trial in which from among 16 patients with inoperable stage III and IV melanoma, clinical response was observed in 19%. The most frequent grade 3 and 4 adverse events included: neutropenia (3 patients, 19%), elevated level of the liver enzymes (2, 13%), fatigue (6, 38%), anxiety (2, 13%) (Tarhini et al., 2008). Another human mAb modulating the immune system is MDX-1106 (Medarex) directed against PD-1 (a molecule close to that of CTLA-4), which undergoes expression on activated T lymphocytes. Results of the phase I trial have shown regression of the tumours in patients with advanced melanoma and low toxicity of the treatment (Brahmer et al., 2008). The monoclonal antibody BMS-663513 targeting co-stimulating molecule CD137 (4-1BB) acts according to a different mechanism. Binding of the ligand or anti-CD137 antibody with 4-1BB receptor on the surface of T lymphocytes provides a co-stimulating signal enhancing the cell's activation and triggering its proliferation. The phase I trial enrolling 54 patients with solid tumors has shown acceptable toxicity level and a certain clinical activity of BMS-663513 (Sznol et al., 2008). We look forward to the results of large randomised phase II study which has just been completed (US National Institutes of Health [NIH], 2008a). CP-870.893 is a human agonistic mAb against co-stimulating molecule CD40 that is up-regulated on the surface of the APC. Phase I trial has shown PR in 4 (27%) out of 15 patients with advanced melanoma and 1 CR lasting 18 months after single administration of the drug (Vonderheide et al., 2006). Currently, the trial evaluating the efficacy of CP-870,893 in combination with carboplatin and paclitaxel has been completed and the results probably will be disclosed soon (US National Institutes of Health [NIH], 2008b). Active specific immunotherapy Active specific immunotherapy is a method of treatment which stimulates immune response to antigens specific for a given tumour type. Active specific immunotherapy includes therapeutic cancer vaccines which encompass cell and non-cell based products. Cell based vaccines comprise: cancer cell lysates, whole cancer cells with adjuvants, gene modified whole cancer cells, DCs pulsed with DNA, RNA, peptides, proteins or cell lysates, pulsed DCs modified with immune stimulators, fused cancer cells with DCs cells or B-lymphocytes. Non-cell based vaccines include DNA vaccines (naked, plasmid), peptide vaccines, protein vaccines, viral-vector vaccines, anti-idiotypic antibody vaccines, particle based vaccines (Table 1) (J. Mackiewicz & A. Mackiewicz, 2009). Therapeutic cancer vaccines As early as in 1883, a New York surgeon William Colley was the first to make an attempt at administering a cancer vaccine. Colley injected bacterial toxins into sarcoma patients and observed disease remission. First-generation cancer vaccines were created from irradiated autologous or allogeneic tumour cells. Cell lysates or natural cell-surface tumour antigens (such as gangliosides GD-2 and GM-2) were also used. In first-generation vaccines, injected cells or antigens were phagocytosed and degraded by mononuclear cells which are then presented to T and B www.intechopen.com lymphocytes in the context of MHC molecules class I and II. Second generation vaccines include gene-modified tumour vaccines (GMTV) which use (autologous, allogeneic or mixed) tumour cells modified with genes encoding tumour antigens, immunostimulating factors, e.g. cytokines. Their functions include supply and presentation of tumour antigens together with providing co-stimulatory signal for the stimulation of specific anti-tumour mechanisms. Vaccine type Vaccine Reference Glycoprotein 100 (gp100) The strategy of immunisation with genetically unmodified autologous DCs is based on ex vivo preincubation of DCs with tumour antigens, followed by administration to patients. This type of immunotherapy activates an antigen-specific cell-mediated response. Non-modified cell vaccines Vaccines based on whole tumour cells and stimulating factors (adjuvants) were one of the first and fundamental specific tumour immunotherapy strategies. Berd et al. (1990) evaluated the effects of immunisation of forty late-stage melanoma patients with a vaccine consisting of irradiated autologous melanoma cells mixed with BCG. Objective clinical response was observed in 5 patients, whereas median survival time was 10 months. The next stage was the use of established cell lines (allogeneic vaccines) which present antigens specific for a given tumour type. Their immunogenicity was enhanced by response to alloantigens present on vaccine cells. Allogeneic vaccines have superseded autologous vaccines due to the difficulties in obtaining the sufficient number of cells for repeated www.intechopen.com vaccinations. A vaccine consisting of three established allogeneic melanoma lines (Cancervax®) and BCG as an adjuvant was developed by Morton et al. (1992). The ensuing phase II trial involved 157 advanced melanoma patients. Objective clinical response was observed in 15-20% of trial subjects (Chan et al., 1998). On the other hand, the outcome of randomised phase III clinical trials of patients treated with Cancervax failed to confirm prolonged survival of patients in comparison with the control group which received only BCG (Morton et al., 2007). Melacine is a melanoma tumor cell lysate vaccine consisting of two allogeneic melanoma cell lines (MSM-M-1 and MSM-M-2) combined with Detox® adjuvant (Vaishampayan et al., 2002). After encouraging early phase studies the vaccine failed the phase III trial. Though, retrospective analysis showed that patients receiving Melacine and expressing at least two of five HLA antigens present on the vaccine cells developed longer RFS and OS (p =.0.0002 and p = 0.0001, respectively). For that reason, the HLA pattern of the patient served here as a biomarker and allowed stratification of patients who would respond to the treatment. The Melacine may serve as an example of personalized therapeutic vaccine (J. . Wallach et al in a multicenter randomized phase III trial assessed the efficacy of VMO -Vaccinia Melanoma Oncolysate. This preparation is a melanoma cell lysate (four cell lines) derived by infection of these lines by the vaccinia virus. The study enrolled 217 patients after resection of metastases to the lymph nodes. Test results showed no significant differences in RFS and OS (Wallack et al., 1998). Intracellular gene transfer Systems of intracellular gene transfer can be broadly divided into non-viral (physical) and viral. The former category includes (i) electroporation (mechanical introduction of DNA in the electric field), (ii) "gene gun" (injection of DNA-coated gold beads into cells by means of pressurised helium). Non-viral chemical strategies are based on modifications of cell membrane permeability for macromolecules under the influence of cationic compounds or enabling penetration of liposome-encapsulated genes marked by high affinity to cell membranes. The most common gene transfer systems used in genetic therapy of human cancer nowadays are based on viral vectors: retroviral, adenoviral, adeno-associated viruses (AAV)) and lentiviral. Recombinant retroviruses are predominantly based on Moloney murine leukaemia virus (MoMLV). They are used to transduce cells both in vitro and in vivo. Retroviruses transfer genetic material to dividing cells, with viral DNA becoming permanently incorporated into the host's genome, thus producing constant expression of the therapeutic gene in target cells and their descendants. Retroviruses are vectors of choice for constructing cellular cancer vaccines. When administered in vivo to humans, murine-enveloped retroviruses are quickly eliminated via complement activation. Consequently, human vectors or human-enveloped vectors were constructed. Adenoviral vectors are used to modify tumour cells in vivo. A typical feature of adenoviral vectors is highly efficient transduction of target cells. On the other hand, as there is no interaction of genetic material with the host's genome, gene expression is trasient. In the case of serial administration of adenoviral vectors, they are eliminated from the human body due to the presence of specific adenoviral antibodies in the human serum. The main feature distinguishing adenoviral carriers from retroviral vectors is the ability to deliver genes into non-dividing cells. Adeno-associated vectors (AAV) have a low capacity and an ability of episomal (extrachromosomal) replication of genetic material and simultaneous integration of inserted genetic material with the host cell's DNA. Initially, AAV vectors contained "contaminants" in the form of immunogenic adenoviral particles necessary for AAV packaging, however new techniques have recently been developed to purify AAV vectors. Lentiviral vectors are based on the human immunodeficiency virus type 1 (HIV-1). They have a capacity of permanent integration of genetic material with dividing and non-dividing cell genomes and are thus an efficient tools used to transduce early CD34+ stem cells (approximately 40%). Stimulation of CD4+ and CD8+ cells IL-6+sIL6R Genetically modified cell vaccines Stimulation of CD4+, CD8+ and NK cells, DCs maturation, presentation of cryptic antigens by DCs, inhibition of Treg formation, induction of GM-CSF secretion by lymphocytes B7 Co-stimulatory signal for T cells HLA-B7 As an allogeneic HLA molecule -stimulation of local production of interferons and cytokines in the tumour microenvironment gene-modified tumour cell vaccines (GMTV). GMTV vaccines are based on whole tumour cells that can be modified with genes encoding (i) immunostimulatory cytokines (such as IL-2, IL-4, IL-6, IL-7, IL-12, IFN-gamma, TNF) (Table 2), (ii) costimulatory molecules (CD80, CD86), (iii) adhesion molecules, (iv) histocompatibility (MHC) antigens. The aim of genetic modification of cancer cells is to augment their immunogenicity, e.g. via phenotype modifications (increased expression of MHC I and II) or activation of effector mechanisms of the immune system via the delivery of costimulatory signals (J. . GMTV have been tested in many early phase clinical trials in the adjuvant and therapeutic settings ( In both studies patients were vaccinated with AGI-101 composed of two irradiated melanoma cell lines modified to express Hyper-IL-6 -a fusion protein composed of interleukin 6 (IL-6) and soluble IL-6 receptor . AGI-101 (5 x 10 7 cells per dose) was administered 8 times at 2-week intervals (induction phase) and then monthly (maintenance phase). At disease progression the induction phase (+/-surgery) was restarted, followed by a second maintenance phase. At progression 43 (Trial 3) and 39 (Trial 5) patients were re-induced +/-surgery followed by a second maintenance phase; of those 11 and 16 patients respectively are alive following re-induction. The 5-year survival in Trial 3 was 66,7%, 43,8% and 26,1% respectively in stage IIIB, IIIC and IV. In Trial 5 the 5-year survival was as follows 56,3%, 39,8% and 41,2% correspondingly in stage IIIA/B, IIIC and IV. The OS observed in trial 3 was 4,4 years and 3,1 year in trial 5 (A. . The vaccine was well tolerated as no vaccine related toxicity of CTC>2 was detected. Intensive research of melanoma vaccines is currently curried out in a number of countries worldwide. However, no vaccine, has been approved by regulatory authorities so far. Vaccines based on dendritic cells Extensive research of DCs has shown that they are the most efficient APCs (Banchereau et al., 1998;Hart et al., 1997). DCs play a vital role in inducing immune response. They are the only representatives of APCs that are capable of inducing primary response of virgin T lymphocytes. The use of DCs for antigen presentation offers an opportunity to trigger www.intechopen.com immune response even to weakly immunogenic tumour antigens and break immune tolerance. Human DCs are generated by isolation of immature DCs from blood (Fong et al., 2000) and differentiation ex vivo in the presence of IL-4 and GM-CSF, myeloid progenitor cells (CD34 +) or monocytes (CD14 +) (Sallusto et al., 1994). Obtained immature DCs can be pulsed with tumor cell lysates (Nair SK et al. 1997) or synthetic peptides (Gitlitz et al., 2003), modified with genes encoding tumor antigens or tumor cells RNA (Ashley et al., 1997). For immunization are also used hybrids of DCs with tumor cells (Avigan et al., 2004). In a small trial, a group of 11 patients with advanced-stage melanoma were immunised with autologous DCs previously incubated with the MAGE-3 peptide presented by HLA-A1. Regression of cancerous lesions in the skin, lungs and the liver was achieved in 6 cases (Thurner et al., 1999). In one of the few randomized phase III studies, Schadendorf et al injected metastatic melanoma patients with autologous DC pulsed with peptides presented in the context of HLA class I and II. However, preliminary analysis has not demonstrated superiority of vaccine over dacarbazine (control arm) and the study was terminated (Schadendorf et al., 2006). Nevertheless, only 53 patients in the vaccine group and 55 in the control arm were participating in the trial and the vaccine was administered depending on the amount of DC cells, usually only from two to several times. Though, subsequent analysis showed that immunized patients with HLA-A2 +/HLA-B44 haplotyp lived longer than those treated with dacarbazine (Engel-Noerregaard et al., 2009). Peptide-pulsed DCs have certain limitations as well, including (i) short period of antigen presentation, dependent on the half-life of the MHC-peptide complex, (ii) the fact that using a given peptide is only limited to patients with an appropriate MHC haplotype (Amoscato et al., 1998). It is believed that the strategy of modified DCs can be both more efficient and more universal. DCs can also be loaded with genes encoding tumour antigens, immunostimulatory factors or cytokines. Furthermore, DCs derived from different patients can be modified with the same genetic sequence (no need to match appropriate MHC haplotypes), while expression of a given introduced sequence is long-term. Metharom et al. immunised mice with dendritic cells transduced with the mTRP-2 gene (tumour antigen of B16 murine melanoma cells). Tumour regression was observed in 4 out of 7 mice (Metharom et al., 2001). In a phase I/II trial, melanoma patients were immunised with autologous DCs transduced with tumour-derived mRNA, which enables lymphocytes to present -beside shared melanoma antigens -a wide range of tumour antigens that are unique for a given patient, as well as previously unknown antigens. The trial showed that the vaccine is completely safe. Response of T lymphocytes against tumour antigens encoded by tumour-derived mRNA in vivo was observed in the majority of cases (Kyte et al., 2006). Melanoma DC vaccines tested to date, despite encouraging results noted in phase I and II trials, have not proven effective in phase III randomised trials. Cancer immunotherapy clinical trial design Results of many promising early-phase clinical studies evaluating the effectiveness of immunotherapy have not been confirmed in phase III trials. It is becoming clear that these failures could be related to the design of clinical trials (Finke et al., 2007). At present, clinical trials design of new therapeutic strategies including immunotherapy are based on criteria developed for cytotoxic drugs. However immunotheraputics, especially of active immunotherapy have very different clinical characteristics, mechanism of action and toxicity prifile than chemotherapeutic agents. Accordingly, new clinical immunotherapy trial design paradigm was developed . Here we outline some aspects which should be considered while designing clinical trials for the assessment of the effectiveness of immunotherapy. Patients qualified to earlier clinical trials evaluating the efficacy of immunotheraputics were in late stage of the disease often treated previously with chemo-or radiotherapy. However, the highest clinical benefit was observed in patients with minimal residual disease and such patient population should be considered as candidates for immunotherapy trials (mainly cancer vaccine trials). Clinical trial end points evaluating chemotherapeutic agents drugs may not be adequate for immunotherapy trials. The Response Evaluation Criteria in Solid Tumors (RECIST) in the assessment of objective clinical response to chemotherapy may not reflect the benefit from immunotherapy. The time points between tumor assessments in trials evaluating immunotherapeutics should be longer (in contrast to chemotherapy trials) giving time for the immune system to mount response to the treatment. In a recent phase II metastatic melanoma study, patients treated with AGI-101H (genetically modified melanoma vaccine secreting Hyper-IL-6) developed clinical response usually after 3-4 months of treatment. However, in many patients tumor shrinkage was observed after several months or even years following SD (Nawrocki & A. Mackiewicz, 2007, Nawrocki et al., 2000. Nevertheless, in many studies assessed tumors tend to enlarge first due to infiltration of inflammatory cells and then shrink. Furthermore immunotherapy may fall to induce tumor decrease, but yet still be effective in slowing the rate of progression giving the patient benefit in OS. The cancer Vaccine Clinical Trial Working Group (CVCTWG) propose that patients after initial clinically not significant progression should not be excluded from the treatment and following tumor regression their response rate could be scored based on the largest tumor volume measured after the start of treatment, not necessarily from baseline tumor volume . Based on the observations from phase II clinical trial evaluating earlier described ipilimumab in metastatic melanoma patients' new guidelines for evaluation of immune-related response criteria (irRC) have been developed. In some patients treated with ipilimumab responses after initial increase in total tumor burden in the presence of new lesions were observed. Those patterns were associated with favorable survival. According to irRC: (i) new, nonmeasurable lesions do not define progression; (ii) new measurable lesions are not defined as progression but are incorporated into tumor burden; (iii) progression of the disease has to be confirmed by a repeat consecutive assessment no less than 4 weeks from the first documented date (Wolchok, 2009). OS is a "gold standard" used for efficacy evaluation of new drugs and is a primary end point of choice in phase III clinical studies. OS is a very good end point, but might be affected by subsequent therapies, is time consuming, and requires large samples of patients. In efficacy randomized phase II trials using adoptive component might be required. DFS and PFS used respectively in adjuvant and metastatic disease setting are acceptable surrogate primary end points which can shorten the time of randomized efficacy trials. However, definitions of DFS and PFS might not be consistent for immunotherapy trials. While as mentioned before patient's immune system needs time to develop clinical activity and even patients after progression of disease still may benefit from treatment. In terms of classical DFS/PFS description patients with relapse or progression of the disease could be excluded from the trial to early. Modifications of these definitions have been proposed: www.intechopen.com confirmation of progression after at least second tumor assessments; not taking into account early progression within a defined time-interval (eg, three month from the beginning of the treatment). In patients who developed early progression with subsequent response to study treatment, the time of DFS and PFS should be calculation from the first day of drug administration . Moreover, in some malignancies like melanoma where there is no optional effective second line treatment, immunotherapy should be continued. In patients developing progression, changes in immunotherapy schedule (induction phase) or metastasectomy may need to be performed. In our own melanoma study patients treated with AGI-101H vaccine received induction phase (8 doses in 2 week interval), with subsequent maintenance phase (1 dose monthly). After developing progression induction phase schedule followed by maintenance was performed. We observed that patients undergoing re-induction benefit from the treatment (Nawrocki & A. Mackiewicz, 2007. Similar observation was seen in the ipilimumab phase III study conducted in metastatic melanoma patients. Ipilimumab was administered four times in 3 week intervals (induction phase). Patients with SD for 3 months duration after week 12 or a confirmed PR or CR were offered reinduction after developing progression. Among 31 patients given reinduction therapy with ipilimumab, a CR, PR or SD was achieved by 21 patients. A properly designed immunotherapy clinical trial is very important, while these agents in contrast to chemical agents or small molecules like tyrosine kinases inhibitors might cure patients with immunogenic malignancies (eg. melanoma, renal cell carcinoma) even when the disease is disseminated. In a highly selected patient population with metastatic melanoma (270 patients) treated with IL-2, CR was observed in 6% with median duration of response over 59 months. PR was seen in 10% of IL-2 treated patients. Disease did not progress in any patient responding for more than 30 months (Atkins et al., 1993(Atkins et al., & 1994. Another example is a study evaluating ipiliumumab in metastatic melanoma patients, where best overall response (CR+PR) rate of only 10.9% was observed. However 60.0% of patients developed an objective response for at least 2 years (26.5 to 44.2 months[ongoing]) (Hodi et al., 2010). Conclusions and further directions of the development Recent approval of DC based prostate cancer vaccine -Stipuleucel-T or immunostimultory antibody -Yervoy prove the potential of active immunotherapeutic approaches to treat cancer. Recent better understanding of immune tolerance mechanisms and their braking, development of vaccine design and the paradigm of immunotherapy clinical trials design will lead to the boosting of progress in the field of active cancer immunotherapy. Since examples already include melanoma, certainly immune targeting especially specific active immunotherapy approaches such as melanoma therapeutic vaccine will continue to be tested and finally successful. Most likely immunotherapy will need to be combined with other modalities such as for ex. small molecules, but certainly without support of the immune system elimination of cancer may not be possible.	2018-12-02T19:39:54.788Z	2011-09-22T00:00:00.000	{ "year": 2011, "sha1": "2e58197e7138824a364976636817fe5a0ffecc81", "oa_license": "CCBYNCSA", "oa_url": "https://www.intechopen.com/citation-pdf-url/20599", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "b65cdae28846bd5635b830e3b02d6d0d3e514b2f", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
237328551	pes2o/s2orc	v3-fos-license	Microbiota-Associated Metabolites and Related Immunoregulation in Colorectal Cancer Simple Summary In the past decade, the interaction between intestinal microbiota and colorectal cancer has been an active research area. Microbial metabolites, which could act locally and systematically, have a significant impact on the development of colorectal cancer, especially by inciting immune responses. In this paper, we systematically reviewed the recent insights on microbial metabolites and their immunoregulation on colorectal cancer and discussed the controversial role of some metabolites, hoping to provide a different understanding of the role of bacterial metabolites in colon carcinogenesis. Abstract A growing body of research has found close links between the human gut microbiota and colorectal cancer (CRC), associated with the direct actions of specific bacteria and the activities of microbiota-derived metabolites, which are implicated in complex immune responses, thus influencing carcinogenesis. Diet has a significant impact on the structure of the microbiota and also undergoes microbial metabolism. Some metabolites, such as short-chain fatty acids (SCFAs) and indole derivatives, act as protectors against cancer by regulating immune responses, while others may promote cancer. However, the specific influence of these metabolites on the host is conditional. We reviewed the recent insights on the relationships among diet, microbiota-derived metabolites, and CRC, focusing on their intricate immunomodulatory responses, which might influence the progression of colorectal cancer. Introduction The microbiome contains a metagenome 100 times larger than that of the human host [1], playing an increasingly critical role in human health, including digesting indigestible macronutrients and producing vitamins, defending against pathogens, and maintaining immune homeostasis [2]. Increasing data suggest that the microbiota has an impact on the etiology of cancer [3]. The large intestine is where humans are most exposed to microorganisms; therefore, it is not surprising that the interplay between the gut microbiome and colorectal cancer (CRC) has been an active research field. CRC is one of the most common cancers and the second leading cause of cancer-related deaths worldwide [4]. The established risk factors of CRC include genetic mutations, inflammation, microbiota, and diet [5][6][7][8]. Although a direct causal relationship between the microbiota and CRC remains a matter of debate, it is proposed that bacteria can function as a 'driver' in tumorigenesis, complementing the genetic 'adenoma-carcinoma sequence' model partly through the production of DNA-damaging compounds and persistent inflammation [9]. CRC is closely associated with a western high-fat, low-fiber diet [6,10,11]. Diet can influence the composition and metabolism of the intestinal flora (Box 1), which helps to produce metabolites that link the microbiota with the host by reprogramming enterocyte Acetate, propionate, and butyrate are readily absorbed by the host; however, their subsequent distribution and influences differ. Butyrate is consumed locally as the preferred energy source of colonocytes, whereas the other two absorbed SCFAs are drained into the portal vein. Most propionate metabolism occurs in the liver, resulting in acetate being the most abundant SCFA in the peripheral circulation [27]. Thus, acetate and propionate have more systemic effects on metabolic syndrome [23], while the butyrate has a significant impact on colon health, especially tumor development. SCFAs as Signaling Molecules and Related Effects on Immunity Increasing evidence shows that SCFAs exert an important influence on preventing carcinogenesis via their anti-inflammatory properties, the induction of apoptosis of cancerous cells, and anti-proliferative activities [28][29][30][31][32]. What are the underlying molecular mechanisms? SCFAs, which act as both extracellular and intracellular signal molecules, play a significant role in the differentiation and functions of host cells, especially immune cells, at least partly through epigenetic modification and receptor-mediated signaling ( Figure 1). SCFAs Act as Histone Deacetylase Inhibitors Once inside the cell, SCFAs can act as inhibitors of histone deacetylases (HDACs) to facilitate the hyperacetylation of histones in colonocytes and immune cells [23]. For example, when exposed to SCFAs, the production of proinflammatory cytokines, such as interleukin-6 (IL-6) and nuclear factor-κB (NF-κB), in mononuclear cells and macrophages decreased, similar to the behavior observed when exposed to HDAC inhibitors [33,34]. Propionate and butyrate also block the differentiation of bone marrow stem cells into dendritic cells (DCs) via HDAC inhibition, thereby reducing undue inflammatory responses [35]. Apart from their effects on innate immune cells, SCFAs also influence the homeostasis of peripheral T cells via HDAC inhibition, particularly facilitating the differentiation of regulatory T cells (Tregs), which is essential to the containment of intestinal inflammation and subsequent tumorigenesis [36][37][38]. Specifically, by promoting hyperacetylation at the promoter and conserved non-coding sequence 1 (CNS1) of the forkhead box P3 (FOXP3) locus, butyrate and propionate increased the expression of the FOXP3 gene, expanded the peripheral pool size of colonic FOXP3 + Tregs, and induced functional IL-10-producing colonic Tregs [37,39]. Notably, acetate, previously not recognized as an inhibitor of HDAC (HDACi), was also found to inhibit HDACs, but only in active immune responses [40]. Significantly, He et al. recently report that butyrate directly enhanced cytotoxic CD8 + T cell response through ID2-dependent IL-12 signaling via its HDAC inhibitory activity, thus promoting the antitumor efficacy of oxaliplatin and anti-PD-L1 therapy [41]. SCFAs, as HDAC inhibitors, can also act directly on cancer cells. The metabolic products of Faecalibaculum rodentium, mainly SCFAs, directly inhibited colon tumor growth SCFAs, mainly consisting of acetate, propionate, and butyrate, act as histone deacetylase (HDAC) inhibitors and ligands of G-protein-coupled receptors (GPCRs), leading to the expansion and differentiation of FOXP3+ regulatory T (Treg) cells, accompanied by upregulation of immunosuppressive IL-10 and transforming growth factor-beta (TGF-β), downregulation of proinflammatory cytokines in macrophages and neutrophils, and inhibition of differentiation towards T helper type 17 (Th17) cells, thereby suppressing inflammation and carcinogenesis. Significantly, SCFAs, especially butyrate, could influence the antitumor responses of CD8+ T cells by regulating signaling pathways in dendritic cells (DC), involving IL-12, IL-27, and IFN-β, which also have an impact on the combination therapy of tumor. Additionally, SCFAs directly inhibited tumors by inhibiting HDAC. Regarding their other protective properties, SCFAs could increase the secretion of mucin, enhance epithelial integrity by activating inflammasome, and activate the peroxisome proliferator-activated receptor-γ (PPAR-γ) pathway, which maintains anaerobic conditions. See the text for details. EC, epithelial cell; GC, goblet cell; NF-κB, nuclear factor κB; IFN-β, interferon β. SCFAs Act as Histone Deacetylase Inhibitors Once inside the cell, SCFAs can act as inhibitors of histone deacetylases (HDACs) to facilitate the hyperacetylation of histones in colonocytes and immune cells [23]. For example, when exposed to SCFAs, the production of proinflammatory cytokines, such as interleukin-6 (IL-6) and nuclear factor-κB (NF-κB), in mononuclear cells and macrophages decreased, similar to the behavior observed when exposed to HDAC inhibitors [33,34]. Propionate and butyrate also block the differentiation of bone marrow stem cells into dendritic cells (DCs) via HDAC inhibition, thereby reducing undue inflammatory responses [35]. Apart from their effects on innate immune cells, SCFAs also influence the homeostasis of peripheral T cells via HDAC inhibition, particularly facilitating the differentiation of regulatory T cells (Tregs), which is essential to the containment of intestinal inflammation and subsequent tumorigenesis [36][37][38]. Specifically, by promoting hyperacetylation at the promoter and conserved non-coding sequence 1 (CNS1) of the forkhead box P3 Figure 1. SCFAs and their Immunomodulatory Functions. SCFAs, mainly consisting of acetate, propionate, and butyrate, act as histone deacetylase (HDAC) inhibitors and ligands of G-protein-coupled receptors (GPCRs), leading to the expansion and differentiation of FOXP3+ regulatory T (Treg) cells, accompanied by upregulation of immunosuppressive IL-10 and transforming growth factor-beta (TGF-β), downregulation of proinflammatory cytokines in macrophages and neutrophils, and inhibition of differentiation towards T helper type 17 (Th17) cells, thereby suppressing inflammation and carcinogenesis. Significantly, SCFAs, especially butyrate, could influence the antitumor responses of CD8+ T cells by regulating signaling pathways in dendritic cells (DC), involving IL-12, IL-27, and IFN-β, which also have an impact on the combination therapy of tumor. Additionally, SCFAs directly inhibited tumors by inhibiting HDAC. Regarding their other protective properties, SCFAs could increase the secretion of mucin, enhance epithelial integrity by activating inflammasome, and activate the peroxisome proliferator-activated receptor-γ (PPAR-γ) pathway, which maintains anaerobic conditions. See the text for details. EC, epithelial cell; GC, goblet cell; NF-κB, nuclear factor κB; IFN-β, interferon β. SCFAs as GPCR Ligands Extracellular SCFAs can affect the immune system via meaningful interactions with some G-protein-coupled receptors (GPCRs). There are three main types of GPCRs related to SCFAs, GPR41 (FFAR3), GPR43 (FFAR2), and GPR109A [23], which are expressed on a variety of host cells, including colonocytes and immune cells [37,42]. GPR43 has a higher affinity for acetate and propionate; the ligands of GPR41 are mainly propionate and butyrate [19], while GPR109A only interacts with butyrate and niacin [43]. Smith et al. [36] found that acetate and propionate could induce Tregs in a GPR43-dependent manner to protect against colitis. However, there are some doubts about the GPCR-based mechanism because some studies indicated that T cells did not functionally express GPR41 and GPR43 [40]. Nevertheless, Sivaprakasam et al. [44] confirmed the critical role of SCFAs- GPR43 interaction in the control of inflammation and carcinogenesis. They found that GPR43 was downregulated in human colon cancer tissues compared with that in matched healthy tissues. Consistent with that, GPR43 −/− mice suffered more severe colitis and increased numbers of polyps, even on a fiber-plus supplemented diet. In contrast, the expression of inflammatory cytokines, including IL-1β and IL-17A, and tumor loads in the colons of wild-type mice significantly decreased, accompanied by an increase in Bifidobacterium species [44]. As for the possible protective mechanisms, for one thing, the expression of GPR43 is necessary for SCFAs-mediated downregulation of the inflammatory response of innate immune cells, such as the infiltration and activation of neutrophil [45]; for another, the activation of GPR43 on the DCs decreased frequency of IL27-producing DCs, which could conduce to the CD8 + T-cell exhaustion, and reduced the number of colon tumors in the Apc Min/+ mice treated with DSS [46]. Butyrate-mediated activation of GPR109A could upregulate anti-inflammatory effector molecules in colonic DCs and macrophages, which promoted the differentiation of IL-10-producing CD4 + T cells and Tregs, and inhibited the development of IL17-producing T cells, thereby preventing colitis and colon carcinogenesis [43]. Besides, the butyrate-GPR109A signaling pathway could block the activation of NF-κB and induce apoptosis in a manner independent of HDAC inhibition [47]. Indeed, sometimes, SCFAs can act as both HDAC inhibitors and GPCR ligands simultaneously. A recent study showed that butyrate could promote IL-22 secretion from CD4 + T cells through both GPR41 and inhibition of HDAC to protect intestines from inflammation [48]. Although there is a long way to go from bench to bedside, some findings from animal models have been tested in clinical experiments. Patients with mild-to-moderate ileocolonic Crohn's disease, the critical risk factor for CRC, were supplemented with 4 g of butyrate daily. After eight weeks, 69% of the patients responded to the treatment, accompanied by downregulation of mucosal inflammatory cytokines [49]. Furthermore, a recent study also found that oral supplementation with butyrate could ease the inflammatory phenotype of monocytes in the blood [50]. Other Antitumor Properties of SCFAs A healthy intestinal epithelial barrier is undoubtedly crucial because of its functions of maintaining mucosal immunity and preventing the translocation of bacteria and bacterial components. In contrast, a leaky barrier permits an increased level of blood lipopolysaccharide (LPS), which triggers continuously low-grade inflammation [8]. SCFAs can enhance the gut epithelial barrier function, for example, by increasing the secretion of mucus. Bacteroides thetaiotaomicron, which produces high levels of acetate, increased mucus production by inducing goblet cell differentiation and the expression of mucus-related genes [26]. Studies also proposed that SCFAs activated the NLR family pyrin domain containing 3 (NLRP3) inflammasome, thereby upregulating the secretion of IL-18, which is protective for epithelial integrity, and ameliorating DSS-induced colitis [51]. Moreover, certain strains of Bifidobacterium longum, which are high producers of acetate, defended against fatal infection with enterohaemorrhagic Escherichia coli O157:H7 by enhancing epithelial integrity and inhibiting the translocation of lethal toxins into the systemic circulation [52]. Besides, SCFAs have other significant antitumorigenic effects. Intestinal expression of the vitamin D receptor could be restored by butyrate to reduce dysbiosis. The autophagy of butyrate-activated Paneth cells was beneficial to suppress DSS-induced inflammation [53]. Activation of PPAR-γ by butyrate promoted β-oxidation to maintain anaerobic conditions, contributing to intestinal homeostasis [54]. The Butyrate Paradox Mounting evidence has shown that butyrate is beneficial to protect against CRC; however, some studies reached the opposite conclusion called the "butyrate paradox" [55]. Indeed, butyrate can either enhance or dampen tumor-suppressive responses depending on a specific treatment. For example, in both MC38 and B16F1 tumor models, butyrate Cancers 2021, 13, 4054 6 of 21 abrogated the anti-cancer effect of ionizing radiation by inhibiting the upregulation of STING-activated IFN-I in DCs, which is required for tumor-specific cytotoxic T cell function [56]. As we discussed earlier, SCFAs represented by butyrate could facilitate the differentiation towards functional Tregs, which are potent anti-inflammatory cells, to suppress the chronic inflammation and subsequent tumorigenesis [43]. However, Tregs and other anti-inflammatory cells also mediate tumor immune evasion. Researchers found that bacterial extracts from the microbiota of hepatocellular carcinoma, containing a high level of SCFAs, elicited an immunosuppressive phenotype, featured by expanding Tregs and diminution of CD8 + T cells, which may be related to immunotherapy resistance and poor prognosis [57]. Besides, Belcheva et al. reported that butyrate fueled aberrant hyperproliferation of colon epithelial cells in Apc Min/+ Msh2 −/− mice [58], which is inconsistent with the situation that in tumor cells, butyrate accumulates as an HDACi because of the Warburg effect [59], thereby contributing to the growth inhibition of tumor cells. Researchers argued that this disparity might result from the host's genetic background and age [60]. Interestingly, butyrate also impacts tumorigenesis by regulating intestinal stem cells (ISC), the cells of origin of intestinal cancer [61,62]. A recent study showed butyrate suppressed the intestinal stem cell proliferation in crypts surrounding the ulcers in a mouse model of colitis by acting as an HDACi to promote the expression of the negative cell-cycle regulator Foxo3 [63]. Although delaying proper wound repair may cause detrimental influence, butyrate prevents stem cells from dividing following exposure to genotoxic luminal contents after mucosal injury, thereby reducing the risk of cancerous transformation of ISC. Together, these data suggest that butyrate exerts an intricate influence on the crosstalk between the microbiota and the host, especially the immune system. The difference in conclusions of studies might root in the different dose of butyrate, the host genetic background, the tumor stage, and the specific combination therapy, which indicates that the role of butyrate in tumorigenesis will still require further investigation. More importantly, findings from experimental studies need to be tested in large-scale human studies to seek more therapeutic opportunities. The Impact of Phytochemicals In addition to SCFAs, there are some metabolites from phytochemicals of fruit, vegetables, and grains, which have antioxidant, anti-inflammatory, and anticarcinogenic properties [64][65][66]. Studies have suggested that all fermentation products together exert better inhibition on colonic cancerous cells than SCFA mixtures alone [67], demonstrating the importance of phytochemicals. Phytochemicals, present in complex glycosylated forms, mainly accumulate in the large intestinal lumen, where they undergo enzymatic transformation by the gut microbiota, such as dehydroxylation or decarboxylation, to be transformed into more bioactive metabolites [64,68]. As widely distributed phytochemicals, dietary polyphenols, especially those in tea, grapes, and coffee, have been investigated intensively. Recently, the immunoregulation of polyphenols related to tumorigenesis is intriguing. It is reported that the tea extracts significantly decreased the production of proinflammatory cytokines (IL-6 and tumor necrosis factor-α (TNF-α))and increased the anti-inflammatory IL-10 in RAW264.7 macrophages and colitis mice [69]. Additionally, ellagitannin-rich cloudberries feeding decreased both the size and number of adenomas in Apc Min/+ mice, along with a smaller ratio of intraepithelial to all mucosal CD3 + T lymphocytes than that in the control group and attenuation of the mucosal inflammation [70]. In Azoxymethane (AOM)/Dextran sodium sulfate (DSS)-treated mice, the commonly used CAC mouse model, cocoa reduced the colon tumor burden, accompanied by decreased macrophage infiltration and inhibition of IL-6/STAT3 signaling pathways [71]. In addition to suppressing inflammation, polyphenols also prevent tumor-induced dysfunction of effector T cells and restore the immunological surveillance, thus repressing carcinogenesis [72]. However, the antitumor activity of polyphenols seemly depends on settings. Researchers discovered that treatment with gallic acid strikingly facilitated Cancers 2021, 13, 4054 7 of 21 tumorigenesis in the proximal gut of Apc Min/+ mice that express mutant p53, not wildtype p53 [73]. Coincidently, AOM/DSS mice treated with Streptococcus gallolyticus could degrade tannin to gallic acid and had more colorectal tumors with high malignancy. Mechanistically, Streptococcus gallolyticus selectively recruited tumor-infiltrating myeloid cells and thus maintained an immune-suppressive microenvironment [74]. Nevertheless, whether gallic acid mediated the tumor-promoting effect of Streptococcus gallolyticus will need further exploration. There is an interactive relationship between intestinal flora and phytochemical-derived metabolites. Specific bacterial species promote the biotransformation of phytochemicals, which in turn regulate the composition of the gut microbiota and related microbial metabolism [75]. For instance, supplementation with black raspberry anthocyanins reversed the unbalanced composition of the gut microbiota in AOM/DSS-treated mice, inhibiting the pathogenic Desulfovibrio sp. and Enterococcus spp., and restoring probiotics such as Faecalibacterium prausnitzii and Lactobacillus, thus partly preventing the carcinogenesis [76]. Oxyberberine, transformed from berberine by intestinal microflora, appreciably suppressed TLR4-MyD88-NF-κB innate immune signaling pathway and ameliorated DSS-induced colitis, implicated in normalizing the dysbacteriosis [66]. Interestingly, highmolecular-weight polysaccharides (>300 kDa) derived from the fungi Hirsutella sinensis selectively enriched Parabacteroides goldsteinii in high-fat diet (HFD)-fed mice. Of note, oral administration of HFD-fed mice with live P. goldsteinii reproduced the benefits of H. sinensis, including augmenting intestinal integrity and reducing levels of inflammation [77]. While shaping the landscape of gut microbiota, phytochemical-derived metabolites also influence its metabolism, for example, by promoting the production of SCFAs. More specifically, indole-3-carbinol selectively increased butyrate-producing Roseburia through up-regulating IL-22. The increment of butyrate effectively suppressed the proinflammatory Th17 cells and induced anti-inflammatory Tregs in the mesenteric lymph nodes of colitis mice [78]. Besides, phytochemical-derived compounds could impact the development of cancer by altering the colonic bacterial enzyme activities. Rosmarinic acid, one of the major components of polyphenol, significantly decreased the activities of bacterial β-glucosidase and mucinase, which respectively hydrolyzed 1,2-dimethylhydrazine (DMH) to toxic methylazoxymethanol and degraded protective mucin, thereby restraining the colon carcinogenesis in DMH treated rats [79]. Collectively, phytochemicals perform protection against colonic inflammation and tumorigenesis together with SCFAs, consolidating the evidence that a balanced diet containing fruits and vegetables is beneficial to health. Proteolytic Fermentation and Related Metabolites Generally, elevated proteolytic fermentation of a high-protein diet in the gut produces some potentially toxic by-products, such as amines and hydrogen sulfide [15,80]. In the following part, we focus on some representative metabolites and their controversial immunoregulation on the progression of colonic carcinogenesis (Figure 2). cial to health. Proteolytic Fermentation and Related Metabolites Generally, elevated proteolytic fermentation of a high-protein diet in the gut produces some potentially toxic by-products, such as amines and hydrogen sulfide [15,80]. In the following part, we focus on some representative metabolites and their controversial immunoregulation on the progression of colonic carcinogenesis (Figure 2). Indole and Its Derivatives Indole and its derivatives are major bacterial metabolites produced from diet tryptophan. Indole can be transformed by a variety of Bacteroides and Enterobacteriaceae with tryptophanase activity, while indole derivatives are only produced by a few commensal species, including Peptostreptococcus spp. and Lactobacillus spp. [81]. Both of them are the main ligands for the aryl hydrocarbon receptor (AhR) [42]. Being a cytosolic transcription factor, AhR is induced by ligands and expressed on epithelial cells, immune cells, and tumor cells [13]. Given that AhR signaling is a pivotal component of the immune response at barrier sites, the interplay between the metabolites and AhR has a significant influence Indole and Its Derivatives Indole and its derivatives are major bacterial metabolites produced from diet tryptophan. Indole can be transformed by a variety of Bacteroides and Enterobacteriaceae with tryptophanase activity, while indole derivatives are only produced by a few commensal species, including Peptostreptococcus spp. and Lactobacillus spp. [81]. Both of them are the main ligands for the aryl hydrocarbon receptor (AhR) [42]. Being a cytosolic transcription factor, AhR is induced by ligands and expressed on epithelial cells, immune cells, and tumor cells [13]. Given that AhR signaling is a pivotal component of the immune response at barrier sites, the interplay between the metabolites and AhR has a significant influence on maintaining intestinal homeostasis, inhibiting infection with pathogens [82], and ameliorating DSS-induced colitis [83]. Those protective mechanisms are mainly mediated by IL-22, for example by reprogramming the differentiation of CD4 + T cells to Tregs [84] and enhancing the gut barrier function [85,86]. Researchers found that mice lacking Card9, a gene related to susceptibility to inflammatory bowel disease (IBD) in humans and encoding caspase recruitment domain family member 9, were more prone to develop colitis accompanied by dysbiosis, deficient indole derivatives, and decreased IL-22 levels, which was similar to IBD patients deficient in CARD9 [87]. AhR activation also exerts a cancer-preventive effect that does not rely solely on suppressing inflammation. Indole-3-aldehyde produced by Lactobacillus reuteri D8 activated STAT3 to restore the function of Lgr5 + ISC via AhR-IL-22 signaling, thus recovering the regeneration of intestinal epithelial following inflammatory assault [88]. Furthermore, IL-22 also protected stem cells against malignant transformation in the presence of genotoxic stress via regulating components of the DNA damage response [89]. Additionally, mice exhibiting dysregulated AhR signaling developed large tumors throughout the colon within 4 months of AOM application, whereas no tumors were observed in wild-type mice. However, the effective activation of AhR by dietary ligands prevented colonic tumorigenesis via restoring the dysregulated Wnt-β-Catenin pathway, which indicated the unrestricted proliferation of intestinal stem cells [82]. Moreover, the interaction between Indole 3-propionic acid (IPA) and the pregnane X receptor likewise contributed to enhancing the gut barrier function and easing intestinal inflammation [90]. Collectively, indole metabolites have protective effects on the development of tumors in the colon in an AhR-dependent manner, including inhibiting inflammation, promoting barrier function, and restraining the hyperproliferation of intestinal stem cells. Polyamines and Associated Metabolism Polyamines are arginine derivatives, metabolized from host tissues and the gut microbiota, including putrescine and spermine [19]. Generally, they are implicated in a variety of biological functions. Polyamines maintain intestinal health by enhancing the intestinal barrier through the expression of Toll-like receptor 2, secretion of mucin, and induction of Ecadherin [91,92]. Unexpectedly, Levy et al. found that spermine was overrepresented in the colons of NLRP6-deficient mice, which caused decreased secretion of IL-18, thereby weakening the gut barrier [93]. Additionally, polyamines perform anti-inflammatory functions. Myeloid-specific deletion of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, promoted M1 immune response and therefore significantly enhanced gastritis during Helicobacter pylori infection, but alleviated DSS-induced colitis [94,95]. Consistent with that, polyamines downregulated LPS-induced IL-1 and IFN-γ, upregulated the production of IL-10 in macrophages [96], and suppressed the activation of proinflammatory M1 macrophages [94]. Moreover, as natural reactive oxygen species (ROS) scavengers, they could also protect DNA from oxidative stress [97]. Nevertheless, dysregulated levels of polyamines are related to cell dysfunction. Aberrant polyamine metabolism is linked to cancer development [97]. Elevated polyamine levels in cancer contribute significantly to immunosuppression in the tumor microenvironment (TME), such that the polyamine blocking therapy restricted tumor growth and enhanced the antitumor efficacy of PD-1blockade through increasing tumor-specific cytotoxic T-cells while decreasing myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM), which are characterized by high levels of arginase 1 (ARG1) and therefore deprived of arginine that is essential for T cell activation [98,99]. Consistently, loss of ODC in macrophages protected mice from colon carcinogenesis in the AOM/DSS model by increased M1 response against tumors [95]. Besides, polyamines favor the immunosuppressive tumor microenvironment by rendering DCs immunosuppressive dependent on indoleamine 2,3-dioxygenase 1 (IDO1) [100] and promoting M2 polarization in macrophages through inducing mitochondrial oxidative phosphorylation by eIF5A hypusination [101]. Noteworthily, spermidine improved tumor chemotherapy in an immune-dependent fashion by stimulating autophagy [102], which suggests the role of polyamines in tumors varies with conditions. As for the other effects of polyamines on tumors, Johnson et al. found that upregulation of N 1 ,N 12 -diacetylspermine, the end-product of spermine metabolism, within biofilm-positive colon cancer tissues was further enhanced, indicating that polyamine metabolites are associated with the development of CRC [103]. Although it has been argued that polyamines promote the formation of carcinogenic biofilms [103], the relationship between polyamine metabolites within the biofilm and CRC remains to be further investigated. Another representative example of aberrant polyamine metabolism acting on tumorigenesis is associated with Enterotoxigenic Bacteroides fragilis (ETBF), a widely supported contributor to intestinal tumorigenesis [55]. ETBF was reported to be upregulated polyamine catabolism, thereby exaggerating inflammation and tumorigenesis via ROS-related DNA damage [104]. The relationship between polyamines and cancer has been investigated for several decades. Polyamines appear to be both anti-carcinogenic and pro-tumorigenic, depending on the context, such as the concentration, the products of metabolism, the developmental stage of the tumor, and combination therapy. Based on the important role of the polyamine pathway in carcinogenesis, targeting it might lead to tumor prevention and curative treatment. Certainly, the intricate interplay between microbial and host polyamine metabolism and the development of colon cancer deserves further exploration. In addition to metabolites discussed above, Hydrogen sulfide (H 2 S), produced by thiogenic bacteria by reducing inorganic sulfur or fermenting sulfur-containing amino acids, also contributed to colon carcinogenesis partly through its proinflammatory property [105]. For instance, one study discovered a significant expansion of Bilophila wadsworthia and more severe inflammation in IL10 −/− mice supplemented with taurocholate instead of glycocholate [106], which indicated the important role of taurine-derived sulfide in colitis. Taken together, proteolytic metabolites have an immunoregulation effect on the development of CRC. Notably, the effects of a high-protein diet on CRC in humans could be even much more complicated when it comes to the specific sources of protein and chemical reactions of other dietary components consumed in addition to protein. For example, the heme in red and processed meats promoted the formation of carcinogenic N-nitroso compounds and aldehydes and elicited epithelium damage and hyperproliferation, which could be facilitated by H 2 S via breaking the gut mucin barrier [107], thus H 2 S and heme have a synergistic effect on carcinogenesis. Bile Acid Metabolism Bile acids (BAs) contain primary bile acids and secondary bile acids (Figure 3), associated with the consumption of a high-fat and low complex carbohydrate diet, and primarily help absorb dietary fats and fat-soluble vitamins [108]. Primary bile acids, mainly including cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized from cholesterol within hepatocytes and are secreted after being combined with glycine or taurine. Approximately 5% of them escape enterohepatic circulation and then enter the colon, where they are subjected to microbial metabolism to form secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA) [20]. The ratio of primary/secondary BAs depends on the situation. For healthy people, the ratio in the duodenal fluid is about 5 to 6 [109,110], the ratio in feces is around 4, with primary BAs accounting for 80% [111] and the ratio of DCA metabolites to CA metabolites in serum is close to 5 [112]. However, for patients with colorectal cancer or polyps, the proportion of secondary BAs significantly increased [113], especially the LCA. Altogether, the above data might suggest a relationship between CRC and secondary BAs. BAs generally act on receptors, including G protein-coupled bile acid receptor 1 (GPBAR1; also known as TGR5) and the farnesoid X receptor (FXR) [114]. These receptors are highly represented in innate immune cells and have pivotal roles in mediating anti-inflammatory effects, which involve inhibition of the NLRP3 inflammasome; downregulation of proinflammatory cytokines in innate immune cells, partly through inhibiting the NF-KB signaling pathway [114][115][116][117]; and the preservation of the intestinal barrier [118,119]. A recent study confirmed that supplementation with LCA and DCA, which are reduced in pouches of ulcerative colitis (UC), mitigated inflammation, partly relying on TGR5 activation on immune cells [120]. Although the action of BAs on the adaptive immune system remains poorly characterized, researchers found that 3β-hydroxydeoxycholic acid (isoDCA) expanded the population of colonic RORγ+ Tregs by counteracting FXR signaling in DCs, thereby minimizing the severity of colitis. The bile acid-vitamin D receptor signaling axis plays an important role in maintaining immunological balance in the colon [121,122]. with LCA and DCA, which are reduced in pouches of ulcerative colitis (UC), mitigated inflammation, partly relying on TGR5 activation on immune cells [120]. Although the action of BAs on the adaptive immune system remains poorly characterized, researchers found that 3β-hydroxydeoxycholic acid (isoDCA) expanded the population of colonic RORγ+ Tregs by counteracting FXR signaling in DCs, thereby minimizing the severity of colitis. The bile acid-vitamin D receptor signaling axis plays an important role in maintaining immunological balance in the colon [121,122]. Figure 3. Immunoregulatory effects of bile acids on host cells. Bile acids consist of primary bile acids (such as cholic acid and chenodeoxycholic acid) and secondary bile acids, including deoxycholic acid (DCA) and lithocholic acid (LCA). The latter is metabolized from the former by gut microbiota. In the healthy situation, both types of bile acids can inhibit the activation of the NLRP3 inflammasome in epithelial cells (ECs), downregulate proinflammatory cytokines, such as IL-6 and IL-1, in macrophages and dendritic cells (DCs), and promote peripheral-induced regulatory T (pTreg) cell expansion in a vitamin D receptor (VDR)-dependent manner. IsoDCA, a secondary bile acid, contributes to pTreg cell expansion by counteracting farnesoid X receptor (FXR) in DCs. Additionally, they promote the secretion of mucin in goblet cells (GCs), which helps to enhance the gut barrier. However, when there is a metabolic disorder caused by a high-fat diet, the concentration of bile acids, especially secondary bile acids, are elevated, resulting in a high level of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and increased epithelial permeability, which causes translocation of bacteria and their metabolites and persistent inflammation. Despite the above evidence suggesting that bile acids have anti-inflammatory effects, some studies argue that metabolic disorders of bile acids can contribute to inflammatory diseases and carcinogenesis [123][124][125]. Indeed, African Americans with a higher risk of CRC have a higher concentration of secondary bile acids in their feces in comparison with Figure 3. Immunoregulatory effects of bile acids on host cells. Bile acids consist of primary bile acids (such as cholic acid and chenodeoxycholic acid) and secondary bile acids, including deoxycholic acid (DCA) and lithocholic acid (LCA). The latter is metabolized from the former by gut microbiota. In the healthy situation, both types of bile acids can inhibit the activation of the NLRP3 inflammasome in epithelial cells (ECs), downregulate proinflammatory cytokines, such as IL-6 and IL-1, in macrophages and dendritic cells (DCs), and promote peripheral-induced regulatory T (pTreg) cell expansion in a vitamin D receptor (VDR)-dependent manner. IsoDCA, a secondary bile acid, contributes to pTreg cell expansion by counteracting farnesoid X receptor (FXR) in DCs. Additionally, they promote the secretion of mucin in goblet cells (GCs), which helps to enhance the gut barrier. However, when there is a metabolic disorder caused by a high-fat diet, the concentration of bile acids, especially secondary bile acids, are elevated, resulting in a high level of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and increased epithelial permeability, which causes translocation of bacteria and their metabolites and persistent inflammation. Despite the above evidence suggesting that bile acids have anti-inflammatory effects, some studies argue that metabolic disorders of bile acids can contribute to inflammatory diseases and carcinogenesis [123][124][125]. Indeed, African Americans with a higher risk of CRC have a higher concentration of secondary bile acids in their feces in comparison with rural Africans [11,126]. Emerging experimental evidence supports that a high level of secondary bile acids is carcinogenic to the colon, for example, by driving malignant transformations in cancer stem cells [127,128]. As an example, the HFD diet appreciably increased the levels of tauro-β-muricholic acid (T-βMCA) and DCA in Apc Min/+ mice, which markedly impaired intestinal integrity and promoted tumor growth. Mechanistically, T-βMCA and DCA antagonized the function of FXR, thus inducing the proliferation and genomic instability in Lgr5-expressing cancer stem cells, fueling the progression of colorectal cancer [129]. BAs-TGR5-SRC/YAP signaling in Lgr5 + cells are essential to maintain the regeneration of intestinal epithelium after DSS damage and thus curtailed colitis in mice [130]. Regarding direct cancer-promoting effects of BAs, researchers found that among 18 wild-type mice given DCA orally for 8-10 months, 17 developed colon tumors, of which 10 suffered from cancer, whereas tumor formation could be reversed by the addition of the antioxidant, chlorogenic acid [131]. This was consistent with the fact that the carcinogenic properties of secondary bile acids are implicated in inducing ROS and RNS, thus causing DNA damage and inflammatory damage [132]. Moreover, elevated levels of secondary bile acids or their abnormal proportion are believed to increase epithelial permeability. For instance, upregulation of certain highly hydrophobic secondary bile acid levels and reduction of ursodeoxycholic acid (UDCA) levels is related to epithelial apoptosis [133,134], resulting in bacterial translocation and persistent low-grade inflammation, which eventually promoted carcinogenesis. Besides, DCA at high concentrations (100 µM and greater) was reported to activate the NLRP3 inflammasome and induce high levels of IL-1β in LPS-primed macrophages [135]. Taken together, whether bile acids function as carcinogenic agents or tumor-suppressors depends on many factors, such as the concentrations and the specific types of bile acid, the cell type being exposed, and the interactions with other metabolites. Although the complicated roles of bile acids in regulating intestinal immunity and carcinogenesis requires further elucidation, animal experiments have demonstrated that activation of TGR5 and FXR with synthetic agonists, or their reactivation in colon tumors, protected against inflammation and tumorigenesis, which provide good prospects for IBD and CRC therapy [118,136]. Specific Pathogens Associated with CRC So far, we have discussed the role of bacterial metabolites in the etiology of CRC. Indeed, certain specific microorganisms are involved in promoting CRC. Accumulating metagenomic analyses have shown a higher abundance of Fusobacterium (F.) nucleatum in CRC tissues versus matched normal tissues [137,138]. Moreover, liver metastases of CRC were reported to contain fusobacterium strains and their associated microbiome that are highly similar to the primary tumors [139], suggesting a strong association between F. nucleatum and the metastasis of CRC. A previous study argued that F. nucleatum could promote inflammation and oncogenesis by activating the β-catenin signaling pathway [140]. Noteworthily, F. nucleatum appears to be implicated in reforming the TME. The interaction between Fap2, a microbial protein of F. nucleatum, and the inhibitory receptor T cell immunoreceptor with Ig and ITIM domains (TIGIT) of immune cells, and the infiltration of myeloid cells in intestinal tumors of Apc Min/+ mice driven by F. nucleatum, could suppress effector killer cells and promote immune evasion, favoring the survival and growth of the tumor [141,142]. Besides, the enrichment of F. nucleatum correlated negatively with CD3 + T cell density in CRC tissue [143,144]. Other mechanisms contributing to carcinogenesis by F. nucleatum are related to promoting proliferation and mediating chemoresistance via activating autophagy [145,146]. Although inflammatory response genes such as IL-6, COX-2, and TNF were upregulated in CRC patients with a high abundance of F. nucleatum [141], whether F. nucleatum contributes to colitis-associated carcinogenesis is controversial because F. nucleatum could not exacerbate colitis or inflammation-associated intestinal carcinogenesis [141]. Nevertheless, ETBF and Escherichia (E.) coli are thought to promote colitis-associated cancer. Recently, a metagenomic analysis from four cohorts of patients with CRC found that Bacteroides fragilis exists consistently in the gut microbiota across populations [147]. The gene encoding B. fragilis toxin (BFT), accounting for ETBF pathogenicity, is more prevalent in the colonic mucosa of patients with CRC [148]. ETBF could rapidly induce colonic inflammation and tumors in a mouse model of multiple intestinal neoplasias depending on IL-17 signaling through inciting the infiltration of immunosuppressive myeloid cells in TME [149][150][151]. E. coli adhering to and invading IECs has been observed increasingly in patients with IBD and CRC [152]. The polyketide synthase (PKS) genotoxic island encodes genes that synthesize the colibactin genotoxin, which could cause DNA damage in vivo and in vitro, thereby partly explaining the pro-tumorigenesis role of pks + E. coli [153]. Furthermore, exposure of human intestinal organoids to pks + E. coli induced specific mutational signatures that were also detected in genomes from patients with CRC [154]. Notably, interactions between microorganisms might have a synergistic contribution to inflammation and tumorigenesis. For example, the invading biofilm in the colonic mucosa of patients with familial adenomatous polyposis (FAP) predominantly comprises pks + E. coli and ETBF. Colon tumorigenesis was reinforced in tumor-prone mice co-colonized with both strains compared with mono-colonization with either bacterium. Mechanistically, mucus degradation induced by ETBF promoted the colonization of pks + E. coli, resulting in enhanced DNA damage, which might contribute to tumorigenesis [153]. Peptostreptococcus (P.) anaerobius, significantly enriched in the feces and tissues of CRC patients, has also been investigated. Researchers found that, by acting on TLR2 and TLR4, P. anaerobius could facilitate colonic tumorigenesis through promoting cholesterol biosynthesis in a ROS-dependent manner [155]. Beyond that, the surface protein of P. anaerobius, putative cell wall binding repeat 2 (PCWBR2) had been identified. Firstly, PCWBR2 directly interacts with α2/β integrin, a receptor generally overexpressed in human CRC tumors and cell lines, which might explain why P. anaerobius preferentially colonizes the CRC tumors [156]; and second, P. anaerobius activated the α2/β1-PI3K-Akt-NF-κB signaling cascade to trigger the inflammation in Apc Min/+ mice. Besides, P. anaerobius expanded immune-suppressive MDSCs, TAMs, and granulocytic TANs, to promote tumor progression [156]. Collectively, P. anaerobius facilitated the tumor progression by promoting inflammation, modifying the immune cells in TME, and regulating the metabolism of cancerous cells. Conclusions and Perspectives The experimental and clinical evidence discussed above indicates that specific pathogens and microbial metabolites play a critical role in the interactions between the microbiome and immune responses regarding tumorigenesis. Significantly, despite few studies about the role of the microbiota on the efficacy of CRC immunotherapy, researchers found that inosine derived from Bifidobacterium pseudolongum activated antitumor T cells via the adenosine A2A receptor, thereby enhancing checkpoint inhibitor immunotherapy [157]. Shalapour and Karin proposed that intestinal barrier disruption was the origin of tumorpromoting inflammation, combined with cancer-initiating mutations, contribute to the formation of a tumor [158]. Correspondingly, the Western diet, with high-fat levels and low complex carbohydrate levels, disturbs gut microbiota homeostasis, resulting in impaired biogenesis of protective metabolites, a compromised gut barrier, continuous inflammation, and eventual carcinogenesis. Except for the microorganisms and metabolites mentioned above, other less investigated bacteria, fungi, archaea, viruses, and metabolites synthesized de novo by gut microbes also influence the host (reviewed elsewhere [10,13]). To give an example, Nod1 stimulation by bacterial peptidoglycan-derived muramyl peptides (MPs) induced monocytic MDSCs infiltration and TAM, which drove carcinogenesis in AOM/DSS treated mice and Apc Min/+ mice [159]. The latest studies uncovered that Streptococcus thermophilus and Faecalibaculum rodentium exerted anti-cancer effects by their metabolites, which reminds us that the role of the bacteria and their metabolites strongly reduced during carcinogenesis is worth exploring to seek therapy opportunity [32,160]. Although there are some controversial findings because of discordance in experimental models, specimens, or contexts, we still see the prospective benefits of screening, diet interventions, therapy, and predicting the prognosis of CRC based on large-scale epidemiological investigations and clinical studies (Table 1). Although most studies have been well performed, further studies to determine the biological mechanism are needed to go beyond simple correlations. Given that data on diet intervention are primarily derived from observational or retrospective studies, to finally determine causality, further larger-scale longitudinal intervention studies on specific metabolites, as well as new technological advances, are required [13]. Conflicts of Interest: The authors declare no conflict of interest.	2021-08-28T06:17:16.852Z	2021-08-01T00:00:00.000	{ "year": 2021, "sha1": "6b71d12193dde543643e78dce8ecb44875e71a29", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2072-6694/13/16/4054/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "c0a8875b9cc163f3bcf5ca7c865714a8b79b9b19", "s2fieldsofstudy": [ "Medicine", "Biology", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
46986914	pes2o/s2orc	v3-fos-license	Expression of phospholipase A2 receptor and IgG4 in patients with membranous nephropathy Objectives The aims of this study were to detect the expression of M phospholipase A2 receptor (PLA2R) in the kidney tissue of patients with idiopathic membranous nephropathy (IMN), secondary membranous nephropathy (SMN), and the nonmembranous nephropathy (non-MN), to evaluate the value of PLA2R in the kidney tissue and serum anti-PLA2R antibody in the diagnosis of membranous nephropathy (MN), and to explore the relationship between PLA2R of the kidney tissue or serum anti-PLA2R antibody and clinical features of MN. Methods The kidney tissue was collected by kidney biopsy. Immunofluorescence assay was used to detect the level of PLA2R and IgG4 antigen in kidney tissue. Furthermore, the level of the PLA2R was detected using the enzyme-linked immunosorbent assay (ELISA). The positive and negative rates of PLA2R and IgG4 in different diseases and the sensitivity and specificity, were calculated using the statistical method. The specificity and coincidence rate of PLA2R or anti-PLA2R used in the differential diagnosis of IMN and SMN were evaluated. Results The expression intensities of anti-PLA2R antibody and IgG4 were significantly higher in patients with IMN than in patients with SMN but are not non-MN. There was no significant difference in anti-PLA2R antibody and IgG4 in patients with SMN and non-MN. The coincidence rate of serum anti-PLA2R antibody and PLA2R in kidney tissue was 100%. Conclusion The expression of PLA2R and IgG4 antibody had great significance in the pathological diagnosis of MN. The detection of the serum anti-PLA2R antibody had great diagnostic value in diagnosing MN. Introduction Membranous nephropathy (MN) is one of the most common pathological types of the nephrotic syndrome in China. With the gradual improvement of the utilization rate of pathological diagnosis technology in the diagnosis of kidney diseases, the increasing incidence of MN has been reported in many different medical centers 1 and has become the most common cause of nephrotic syndrome. Usually, the MN refers to the idiopathic MN (IMN), which is also named the primary MN. Approximately 80% of patients were diagnosed with nephrotic syndrome characterized by large number of proteinuria and hypoalbuminemia, complicated with infection, thrombosis, and embolism events. Without a timely and effective diagnosis and treatment, patients may progress to end-stage renal failure after 5-10 years. Therefore, it is of high urgency to improve the diagnosis and treatment effect. In the past many authors have studied how to improve the accurate diagnosis rate of MN. In 2009, Beck et al 2 first discovered that the extracellular domain of phospholipase submit your manuscript \| www.dovepress.com 104 Liu et al A2 receptor (PLA2R) was used as the mutant antigen to activate autoimmunity response. Combined with the PLA2R antibodies produced in the body, PLA2R forms the insitus immune complex, resulting in the injury of basement membrane, which was the major pathogenic factor of the majority of IMN patients. Many literature reported that the detection of peripheral blood in patients with serum, results positive rate of anti PLA2R antibodies in patients with IMN was 50-80%, [3][4][5] and was about 20% 6 in patients with secondary MN (SMN), in the patients with non-MN and normal population was almost to zero. 7 Recent studies have pointed out that the titers of anti-PLA2R antibodies in peripheral blood were associated with the disease activity. [8][9][10] Thus, the detection of serum anti-PLA2R antibodies had great significance for diagnosis, treatment, and prognosis of the disease. In recent years, more and more studies have confirmed that the immunoglobulin IgG subtypes play an vital role in the identification of MN caused by different antibodies, such as IgG4 mainly in IMN kidney tissue deposits 11 and IgGl and IgG3 mainly in SMN. 12,13 Beck et al 2 first identified IgG4 as the major component of anti-PLA2R antibody in IMN patients, which is consistent with kidney pathology. Thus, the detection of glomerular IgG4 expression could serve as the reference index in the diagnosis and differential diagnosis of kidney diseases. In this study, we analyzed the data of patients with MN diagnosed in our hospital and the expression of PLA2R and IgG subtype in blood and kidney tissue, respectively. The presence of IgG subtype and PLA2R in the identification of IMN and non-MN was assessed, and the values of both expressions were also evaluated. Inclusion and exclusion criteria for patients Patients aged between 12 and <65 years and those who satisfied the standard diagnostic criteria of chronic kidney disease (CKD) were included in the study. The diagnosis of CKD was made when one of the following two criteria was met: 1) structural or functional abnormalities of the kidney lasting for at least 3 months, and 2) glomerular filtration rate (GFR) <60 mL/min/1.73 m 2 for at least 3 months. 14 Patients were excluded based on the following two criteria: 1) patients who were not willing to perform kidney pathology, and 2) patients who had contraindications to kidney pathology. The risk of the kidney pathology examination is higher in patients older than 65 years, hence, the examination was not done in these patients. Inclusion and exclusion criteria for IMN and SMN The inclusion criteria for IMN were as follows: the pathological diagnosis of the kidney tissue was MN, except for clinical factors such as systemic lupus erythematosus (SLE), hepatitis B virus (HBV), tumor, and drug-induced secondary membranous nephropathy. The inclusion criteria for SMN were as follows: 1) membranous lupus nephritis (MLN): ≥ SLE classification revised by Systemic Lupus International Collaborating Clinics (SLICC) in 2012 and the pathological diagnosis of the kidney tissue was MN; 2) HBV-related glomerulonephritis (HBV-GN): serum HBsAg was positive, except for clinical factors such as SLE, tumor, and drugs, the pathological diagnosis of the kidney tissue was MN, and hepatitis B surface antigen (HBsAg) and/or hepatitis B core antigen (HBcAg) in the kidney tissue were positive. 15 Clinical data Age, gender, serum creatinine, serum albumin, and 24 h protein excretion were used for this study. Kidney histopathology examination The kidney tissues were collected from the patients, and the pathological specimens examined under light immunofluorescence microscopy were sent to KingMed Diagnostics. Kidney pathology and immunofluorescent examination Immunofluorescent staining was performed for PLA2R and IgG4 on 3 μm sections of formalin-fixed tissue using a kit, according to manufacturer's instructions. Briefly, the rabbit antihuman PLA2R polyclonal antibody (1:100 dilution; Sigma-Aldrich Co., St Louis, MO, USA) was added to the sections. After 1 h of incubation, the goat antirabbit IgG (Abcam, Cambridge, UK), which had been diluted to 1:50, was added to the sections. After 1 h of incubation, the sections were washed with water, dried, glycerol mounted, and then observed under fluorescence microscopy. The method and steps of IgG4 detection were as follows: the first antibody Serum anti-PLA2R antibody detection According to the kit instructions (Euroimmun, Lübeck, Germany), the blood was extracted from the peripheral vein and the serum was detected by ELISA after centrifugation. The ELISA detector measured the absorbance value at the 450 nm wavelength. The serum antibody level was calculated according to the curve equation, with 0-14 RU/mL as the standard reference value of the serum antibody. Statistical analysis Normally distributed data are presented as the mean ± standard deviation. The data were statistically analyzed using the SPSS 17.0 software package (SPSS Inc., Chicago, IL, USA). Parametric data were analyzed using one-way analysis of variance and Student's t-test. The sensitivity and specificity, negative predictive value, and positive predictive value were calculated by Fisher's exact test. P<0.05 was considered as a statistically significant difference. Clinical data and biochemical parameters There were 39 patients enrolled in this study from August 2016 to December 2016. All patients underwent kidney biopsy, including 27 males and 12 females, with mean age 45.8±13.6 years, and were divided into the following three groups according to the pathological results: primary MN group (IMN; 27 patients), SMN group (five patients), and non-MN group (seven patients). No statistically significant differences were observed for serum albumin and 24 h urine protein in the three groups. There was one case of acute renal injury and one case of severe lupus in the non-MN group; the serum creatinine concentration of two patients was significantly higher than normal, so there was a significant difference compared with IMN and SMN groups (P<0.01) ( Table 1). PLA2R and IgG4 expressions in the kidneys of the three groups The positive expressions of PLA2R (88.89%) and IgG4 (81.48%) were higher in the IMN group than in the SMN group and non-MN group, and the difference was statistically significant (P<0.01). There was no significant difference in PLA2R-positive expression and IgG4-positive expression between the SMN group and non-MN group (P>0.05) ( Table 2). Comparison of PLA2R between kidney tissue and serum in IMN patients The results showed that 24 patients whose kidney tissue PLA2R were positive and their anti-PLA2R antibodies in serum were also detected positive. Three cases of IMN kidney tissue PLA2R-negative patients with anti-PLA2R antibodies in serum were also negative, suggesting that serum Evaluation of indicator of PLA2R and IgG4 expressions in the kidney tissue According to the results, the detection of the PLA2R and IgG4 expression in the kidney tissue had higher sensitivity compared with SMN group and non-MN groups. The detection of the PLA2R or IgG4 had higher specificity compared with SMN group and non-MN groups, which may be due to the specificity of expression of PLA2R in the non-IMN group. Compared with the sensitivity, specificity, negative predictive value, positive predictive value, and coincidence rate in the three groups, no statistically significant difference was observed (P=0.459, 0.222, 0.387, 0.718, and 0.843). This was a significant limitation in our study; the number of patients included was small due to the shorter research period. We will include more cases in the future to improve research persuasiveness (Table 4). Immunofluorescence changes in the three groups Immunofluorescence showed that there were significant differences in the fluorescence values among the three groups ( Figure 1). Immunofluorescence-labeled immunocomplex was more abundant in the glomerular capillary loops in the IMN group but not in the SMN and non-MN groups. Discussion MN is a common pathological type of nephrotic syndrome, and the incidence increases year by year. Some causes such as the hepatitis, drug, high incidence of cancer, environmental pollution and so on could cause MN. [17][18][19][20] In the present study, we collected all patients who underwent kidney pathology within 5 months in our hospital were collected; the statistical results showed that the incidence of MN was up to 69.2%, and the incidence of MN was higher than IgA nephropathy, which had been the first cause in chronic glomerular disease. Phospholipase A2 (PLA2) is a group of enzymes distributed in multiple organs of the human body. It has many subtypes and can bind to PLA2R to form an immune complex, which plays a key role in regulating cell proliferation, adhesion, and activity factor secretion in the inflammatory reaction process. PLA2R was overexpressed in renal tissue of IMN patients, and anti-PLA2R antibody produced by its expression was also observed. A series of studies confirmed that anti-PLA2R antibody levels in IMN patients were significantly elevated compared with the normal and non-IMN patients. 21,22 Anti-PLA2R antibodies bind to PLA2R on kidney podocytes to cause complement activation, podocyte damage, and basement membrane damage. 23,24 In this study, we found that the PLA2R from kidney tissue was positive in 88.89% of IMN patients, while PLA2R from kidney tissue was positive in 40 and 0% of SMN and non-MN patients, respectively. Beck et al reported that IgG4 was mainly anti-PLA2R antibodies in IMN patients. Many studies showed that the pathogenesis of IMN is because the recognition of IgG4 recognition and the glomerular podocyte PLA2R, the formation of in situ immune complexes, and the activation of the complement system, which led to immune injury, podocyte morphological change, proteinuria, hypoproteinemia, and other clinical symptoms. 25 Our study showed that the positive rate of IgG4 expression in kidney tissue of IMN patients was 81.48%, while the positive rate of IgG4 expression in kidney tissue of SMN and non-MN patients was 20 and 14.9%, respectively. 26 2) the combined detection of PLA2R and IgG4 could not completely exclud the secondary causes; and 3) the mechanism of MN was not completely clear and the explanation of the role of PLA2R and IgG4 in patients with MN was difficult. 27 Still, we have reasons to believe that the positive rate of IgG4 in kidney tissue consistent with PLA2R, and the detection of IgG4 combined with PLA2R improved the diagnostic accuracy of MN. Therefore, IgG4-related disease (IgG4-RD) is recently gaining a lot of attention. It is worth mentioning that IgG4 found in IMN is not IgG4-related disease. In 2012, IgG4-related MN was proposed by Alexander et al. 28 In patients with MN secondary to IgG4-RD, an immunofluorescence assay showed granular deposits of C3 and IgG, of which IgG4 was the dominant subclass, along the glomerular basement membrane. In these patients, the kidney tissue is typically negative when staining with anti-PLA2R antibodies, similar to the serum anti-PLA2R staining. Currently, there is still no uniform diagnostic standard for MN secondary to IgG4-RD. The diagnosis of MN secondary to IgG4-RD should be made in the context of IgG4-RD in other organs or IgG4related tubulointerstitial nephritis (IgG4-TIN). 29 Conclusion IgG4 and PLA2R detection in kidney tissue was used as an important diagnostic tool. For those patients who were not or refused to perform kidney pathology, anti-PLA2R antibody detection in serum was used as a convenient and rapid detection method to provide guidance for clinical diagnosis. In this study, the number of patients included was less because of the shorter period of study. We will continue this study and include more patients to improve the scientific nature of this study.	2018-06-12T02:04:40.258Z	2018-05-29T00:00:00.000	{ "year": 2018, "sha1": "9c2d96207287e62603c7602a83c7b340d0016ef4", "oa_license": "CCBYNC", "oa_url": "https://www.dovepress.com/getfile.php?fileID=42357", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "bee4ed9d3101e86b92bf8f2c2359867a9fc998fe", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
257574030	pes2o/s2orc	v3-fos-license	The sexual behavior of young people living with a disability: Findings from the KAP study in Northern Uganda Introduction Young people living with disability form one of the most vulnerable population groups globally. There is limited information on the use of SRH services among young people living with a disability. Methods This analysis is based on household survey data among young people. Drawing on a sample of 861 young people living with a disability (15 -24 years), we investigate the sexual behaviour, and identify the risk factors associated with sexual behavior of young people living with a disability. Multilevel logistic regression was used. Results Results indicate that risky sexual behaviour was associated with alcohol consumption (aOR = 1.68; 95%CI: 0.97, 3.01), having limited knowledge of HIV and STI prevention methods (aOR = 6.03; 95%CI: 0.99, 30.00), and low life skills (aOR = 4.23; 95%CI: 1.59, 12.87). The odds of not using a condom at last sex were significantly higher among in-school young people than out of school young people (aOR = 0.34; 95%CI: 0.12, 0.99). Discussion Targeted interventions aimed at reaching out to young people living with a disability should consider their sexual and reproductive health needs, barriers, and facilitators. Interventions can also promote self-efficacy and agency of young people living with a disability in making informed sexual and reproductive health choices. Introduction The Convention on the Rights of Persons with Disabilities (CRPD) advocates for enjoyment of all human rights and fundamental freedoms among people living with disabilities (1). Disability occurs as a result of an impairment of cognitive, developmental, emotional, physical, mental, sensory or a combination of all that affects one to fully participate in activities (2). Goal Three of the Sustainable Development Goal (SDG) calls for universal access to health including access to sexual and reproductive health (SRH) services and information (3)(4)(5). Young people living with a disability (YPWD) form one of the most vulnerable population groups globally (6). YPWD form a significant proportion of young people in developing countries; with nearly 80 percent of 180-220 million YPWD globally living in developing countries (7,8). In Uganda, the recent 2016 Uganda Demographic and Health Survey reports that 16% of young people aged (10-19 years) were living with a disability (9). Access to SRH services among YPWD is a public health issue (10). There is limited information on the use of SRH services among this population group (11). While sexual behavior may be regarded as a universal aspect for every human being (12, 13), some literature has pointed to misconceptions surrounding sexual behavior among YPWD (14). For example, there is a view that YPWD do not engage in sexual activity or that they are asexual (15) or that having quality sexual behavior is less important compared to providing medical attention or rehabilitation needed for them (16). These views assume that YPWD do not need information regarding prevention risky sexual behavior or how to maneuver around it. The quality of life for every individual including YPWD is an important dimension (17), which improves wellbeing (18). Further, YPWD can have unsafe sex (19), or engage in sexual behavior, but may miss the necessary skills, negotiation power and information to engage in safer sexual activity (8,20). As a result, YPWD are often marginalized, discriminated, and relegated to the background or neglected when it comes to accessing SRH services (21-24). Moreover, health workers often miss some information regarding the sexual behavior of YPWD and are unable to advise them on some prevention strategies against STIs (25). YPWD are unable to receive SRH services or information in the form of contraception to prevent unwanted pregnancies (26), or even prevent STIs because of the misconception that YPWD do not have sex (27). Other challenges associated with limited information or services among YPWD include physical barriers (28), transport challenges (29), long waiting times (30), lack of confidentiality (31), need for an escort (32) and disability-related stigma (33) or negative attitudes (34). While these challenges are common across the board, they tend to be exacerbated among YPWD because they are categorically different, and with different needs. For example, the young people with mental disability would have different requirements from those with physical disability. In general, given the challenges associated with limited access to SRH services, YPWD tend to engage in risky sexual behavior with far reaching negative consequences (35). For example, YPWD tend to experience child marriage (35), unintended pregnancies (36), induced abortion (21), sexually transmitted infections (STIs) including HIV (37) because of risky sexual behavior. While access to SRHR services is a general problem, YPWD tend to be more vulnerable than their counterparts. YPWD are more likely to engage in risky sexual behavior or abused than young people who are not living with a disability (38). Most interventions are tailored towards young people without taking into consideration the vulnerable groups such as YPWD (39). This paper therefore seeks to understand the sexual behavior of YPWD, and the determinants associated with sexual behavior of YPWD. The results from such analyses can inform the design of interventions on reproductive health among YPWD. Moreover, the results can contribute to improving the quality of SRH services among YPWD. Methods The current analysis is based on household survey data collected for the baseline knowledge, attitudes and practices (KAP) study of the UNFPA's supported program on Advancing Sexual Reproductive Health and Rights (ANSWER) in Northern Uganda. The study population was young people (15-24 years). Data were collected between August and September 2021. The household KAP survey was based on a stratified two-stage cluster design with stratification on districts, cross-stratified on the ruralurban residence. In refugee-hosting districts, cross-stratification was also done on refugee communities and host communities. In the first stage, a probability proportional to the size sample of villages was taken from each stratum. In the second stage, a probability systematic sample of households with young people (15-24 years) was taken. The outcome variables in this study included: involvement in risky sexual behavior (RSB) in the past 12 months, that is multiple sexual partnerships, transactional sex and non-use of condoms at last sex with a non-marital partner. The explanatory variables included age, education, marital status, nationality, alcohol consumption, household wealth, religion, exposure to media, attitudes towards teenage pregnancy and condom use, knowledge of sexually transmitted infections, HIV and their prevention methods, life skills, self-efficacy, gender norms, type of disability. The assessment of attitudes toward teenage pregnancy was based on a proxy indicator, where respondents were asked to justify having a child whilst still a teenager. These included: (a) having a baby to love, (b) moving out of parent's house, (c) getting married early, (d) proving one's fertility, (e) enjoying the child/children growing up, (f) having a partner to love, and (g) proving you are a man/woman. However, we note that we are likely to miss out on the bad/negative attitudes especially from teenagers who have ever been pregnant since the response options available are only about the positive/good things. Household asset index as a proxy measure for the economic wellbeing of a household was constructed through Principal Component Analysis model of household owned domestic items (radio, television set, sofa sets, mattress, solar/electricity for lighting, access to running in the house or yard), transport assets (bicycle, motorcycle, car), and productive assets (computer, mobile phones) and has an income generating activity. Generally, based on the pca scores, households were classified as having high household assets index if they had at least 6 of these 12 items assessed. Knowledge of preventing HIV/AIDS and STIs, and their treatment was obtained from alpha factoring of six items. Respondents were asked to affirm the following: (a) having and being faithful to only one sexual partner is an effective way of preventing HIV, (b) a person can reduce their chances/risk of getting HIV by not having sex, (c) a person can reduce their chances/risk of getting HIV by using condoms when having sex, (d) the HIV/AIDS virus can be transmitted by mosquito bites, (e) the HIV/AIDS virus can be transmitted by supernatural means, (f) a person can become infected by sharing food with a person who has the AIDS virus, (g) a girl or boy cannot get HIV the first time she/he has sexual intercourse, and in addition to knowledge at least two other STIs in addition to HIV/AIDS, and source of their treatment. The score for gender and social norms was computed from the responses to the following questions, with Likert scale options: (a) boys and girls have equal abilities, (b) Giving a bath and feeding kids are the mother's responsibility, (c) Woman's role is taking care of her home and family, (d) a man should have the final word about decisions in the home, (d) preventing pregnancy is a woman's responsibility, (a) Young people like you/me should not be allowed to use contraceptive services; (b) It is wrong for young girls who are sexually active to use contraceptives, (c) Women who use contraception may become promiscuous. Life skills score for self-efficacy to avoid risky sex, including using a condom was measured by asking the respondents to affirm to the following statements: (a) I am confident I can get the person with whom I have sex to use a condom, even if he/she doesn't want me to use a condom, (b) I am confident If my partner and I do not have a condom, I can say no to sex, (e) I make smart decisions to avoid unsafe sex. Self-efficacy was measured by asking the respondents to affirm to the following statements: (a) I am confident if I did not want to have sex, I would be able to refuse sex with a person who has power over me, like a teacher, employer, relative, etc., (b) I am confident I can get the person with whom I have sex to use a condom, even if he/she doesn't want me to use a condom, and (c) I am confident If my partner and I do not have a condom, I can say no to sex. Positive attitude condom use was measured by asking respondents to respond to the following statements: (a) condoms reduce sexual pleasure, (b) condoms can slip off a man's penis and enter a woman's body, (c) it is too embarrassing for someone like you to buy or obtain condoms, and (d) if a girls suggested to her partner to use a condom it would mean that she does not trust him. An additional module based on the Short Set of questions developed by the Washington Group on Disability Statistics to estimate the prevalence of disabilities was also included in the Household Questionnaire. The Short Set questions address six core functional domains-seeing, hearing, communication, cognition, walking, and self-care. The analysis is based on 861 young people living with disability, from among the 6,056 young people aged 15-24 years. These were mainly individuals with some difficulties with their sight (42%), hearing (25%), and memory (17%), and 27% had physical disabilities. We note that this sample size, based on sample size formula by Cochran (1963), is adequate for estimating of a proportion of YPWDs involved in risky sexual behaviour within the two standard errors with 95% certainty. This holds for population level proportions of at least 10% of YPWDs engaging in risky sexual behaviour. At the bivariate analysis level, F-test was used to test for associations. Variables with p < 0.10 at the bivariate level and those contextually important (sex, age, schooling status and marital status) were included in multilevel logistic regression model for each of the three outcome variables. In addition, inverse probabilities of selection were included in the models as weights to further account for the complex sample survey design features. Analyses were executed in Stata V15. In multivariable regression models, adjusted model coefficients and their 95% confidence intervals (95% CI) were computed using the stepwise approach. Hosmer-Lemeshow test statistics were conducted to check model goodness of fitness. This study was approved by Uganda National Counsil for Science and Technology (UNCST) (HS1079ES). Sample characteristics of respondents Of the 861 respondents, 45.4% were female, 44.4% were aged 20-24 years, and 50.9% were out of school (Table 1). Further, 10.2% were refugees (10.2% males vs. 10.2% females), 61.7% were Roman Catholics (60.6% males vs. 63.1% females), and 33.8% were living in a marital relationship (37.2% males vs. 29.6% females), and only 21.8% had a secondary school education (24.5% males vs. 18.5% females). Sixty-three percent reported exposure to media at least once a month, notably radios, newspapers, tabloids, television, and social media. Most of the respondents (75.8%) were from households with less than four of the 12 household assets assessed, and less than 1% were from households that had at least 10 assets. About 12% reported alcohol consumption within the last week. The distribution of age groups was similar between male and female respondents but there were slightly more females married than males (33.8% vs. 29.6%), and more males consume alcohol than females (16.0% vs. 6.4%). Sexual behaviour of respondents Of the 861 individuals, 48.2% (40.3% of females and 54.8% of males) reported having ever had sex, and 17.1% (9.7% of females and 23.2% of males) reported having been involved in risky sexual behavior (RSB) in the past 12 months preceding the survey, i.e., multiple sexual partnerships, transactional sex and no use of condom use in the last sex act with a non-marital partner. Fourteen percent (16.9% of the females and 11.5% of the males) had sex before turning 15 years of age, and 53.3% of females and 56.4% of males had sex before the age of 18 years. Equivalently, among all the YPWD, 6.6% (6.3% of the male and 6.8% of the female) and 26.6% (32.2% of the male and 22.0% of the female) had sex before the ages of 15 years and 18 years, respectively. Nearly, one in four young women and girls reported their last sexual partner in the past 12 months to have been at least five years older than them. The current use of modern contraceptives (including consistent use of male condoms) was reported by 33.1% of the young people while 40.4% of the girls and young women reported their last pregnancy as unintended. Factors associated with risky sexual behavior Engagement in multiple sexual partnerships in the past 12 months: The involvement in multiple sexual partnerships within the past 12 months was mostly reported by men and boys, the YPWD who consume alcohol, and those negative gender norms. A higher proportion of individuals who consume alcohol reported multiple sexual partners than the proportion of individuals who do not consume alcohol (41.9% vs. 25.8%, F = 4.99; pval = 0.027). Moreover, a significantly higher proportion of individuals with negative gender norms than that of individuals with equitable gender norms reported multiple sexual partnerships (32.7% vs. 24.0%, F = 2.89, pval = 0.049). Condom use in the last risky sex act in the past 12 months: Results in Table 2 show that up to 32% of male respondents and 27% of female respondents did not use a condom in their most recent risky sex act. At bivariate analysis, secondary education level, knowledge of HIV prevention methods, knowledge of other STIs (in addition to HIV/AIDS), knowledge of where to obtain contraceptives (including condoms), positive gender norms, high score of life skills and self-efficacy, and unfavorable attitudes toward teen pregnancy were associated high percentages using a condom. Eighty-seven percent of respondents with secondary school education as compared to only 23.5% of those without formal education reported condom use in the last risky sex act (F = 7.10; pval = 0.049). Similarly, a higher percentage of individuals with good knowledge of HIV prevention methods (74.4%) used a condom as compared to 47.1% of those with limited knowledge (F = 3.951; pval = 0.036). More individuals (76.2%) with positive gender norms as compared to 63.0% of those with negative gender norms used a condom (F = 2.93, pval = 0.049), and 73.1% of individuals with high self-efficacy to avoid risky behavior as compared to 20.9% of individuals with low self-efficacy used the condom (F = 5.945; pval = 0.001). However, positive, or negative attitudes toward condom use did not matter. Involvement in risky sexual behavior (composite score) among sexually active young people in the last 12 months: Bivariate analysis results in Table 2 show that a lower proportion of female YPWD (24.0%) engaged in RSB than the male YPWD (42.3%) (F = 11.06; pval = 0.001); and lower proportion of out of school YPWD (31.7%) than the proportion of in-school YPWDs (53.2%) reported RSB in the past 12 months preceding the survey (F = 5.55, pval = 0.019). The results also show that a higher percentage of individuals with good knowledge of HIV prevention methods engaged in risky sexual behavior as compared to those with limited knowledge (39.5% vs. 22.3%; F = 6.96; pval = 0.009). Involvement in risky sexual behavior (composite score) among all young people in the last 12 months: Among all YPWD (sexually active and non-sexually active), only 17.2% (23.2% of male and 9.7% of female) of young people were involved in risky sexual behaviour (RSB) within the last 12 months ( Table 2). Bivariate analysis presented in Table 2 show that having secondary school education, being out of school, consuming alcohol, and holding inequitable gender norms were associated with RSB within the past 12 months. Twenty percent of individuals with inequitable gender norms as compared to 14.5% of individuals with equitable gender norms were engaged in RSB (F = 3.19; pval = 0.045); and 34.2% of individuals who drink alcohol as compared to only 14.8% of those who do not reported RSB. Overall, being in school was protective of engagement in RSB as only 9.0% of the in-school YPWD engaged in RSB as compared to 24.8% of the out of school young people (F = 17.00; pval < 0.001). Taken together with results in the previous section, this implies that schooling is a protective factor to RSB but the few in-school young people who become sexually active face higher risks of pregnancy, and STIs than the out of school young people. Sexual debut before 18 years of age: The results are presented in Table 2. At bivariate analysis, factors associated with early initiation of sexual debut were being male (32.2% vs. 22.0% of females; F = 11.25, pval = 0.004); being out of school (41.4% vs. 13.2% of in-school; F = 45.29; pval < 0.001); alcohol consumption Multivariable analysis of factors associated with risky sexual behavior The odds of involvement in RSB relative to other sexually active young people were independently increased by alcohol consumption (aOR = 1.68; 95%CI: 0.97, 3.01) and higher among boys and men than girls and women (see Table 3). The odds of engaging in RSB was low among young people with life skills; young people who reported below average scores of life skills had more than four times the odds of engaging in RSB than those with above average scoresabove vs. below average had (aOR = 0.23; 95%CI: 0.07, 0.79). Similarly, the involvement in RSB with respect to the whole population of YPWD was also driven by these same factors, in addition to age group. For some of the individual components of RSB such as the odds of not using a condom in a high-risk sexual act were driven by alcohol consumption (aOR = 2.63; 95%CI: 1.11, 6.19), limited knowledge of HIV and STI prevention methods (aOR = 6.03; 95%CI: 0.99, 30.00), and low life skills score (or low selfefficacy) (aOR = 4.23; 95%CI: 1.59, 12.87). The odds of not using a condom was significantly higher among in-school young people than out of school young people (aOR = 0.34; 95%CI: 0.12, 0.99). Discussion Our findings demonstrate that on average about two in ten YPWD engage in high risky sexual behavior such as multiple sexual partnerships, transactional sex and non-use of condom in the last sex act with a non-marital partner. Studies conducted in other low-and middle-income countries have found similar results that point to risky sexual behaviour among YPWD (20,(39)(40)(41)(42). For example, Oladunni (2012) found that about a third of adolescents with a disability in Nigeria had multiple sexual partners at a time and engage in multiple sexual relationships and about 12 percent do not take any action to prevent pregnancy. Results show early initiation of sex before 15 years (sexual debut) and by the age of 18 years more than a quarter of YPWD have had sex, and cross-generational sex was high among female YPWD. These results are in conformity with those from other studies in Africa and from other low-income countries (21, 41). For example, Oladunni (2012) found that more than half of adolescents with a disability had sex by the age of 15 years. Similarly, a study conducted in Ghana established that young people with hearing and vision loss had a higher likelihood of engaging in casual sex (42). This further reemphasizes the need to prioritise and integrate disability in programming for SRHR (43). Consistent use of male condoms was low among YPWD, and this may explain the very high rate of unintended pregnancies that affects four in five girls and young women. Results confirm those of other studies conducted in relatively similar contexts in Africa and low-income countries (36). For example, in a study carried out in Ethiopia among YPWD, Kassa and colleagues (2014) found that only a third of them used contraceptives (21). Individuals who consume alcohol had almost twice the odds of engaging in RSB than those who do not. These results are similar to a number of studies that underscore alcohol consumption as a risk factor for engagement in risky sexual behaviour for YPWD (44)(45)(46). For example, Marshal (2014) found that alcohol use and other risk-taking behaviours and risky sexual behaviour emerge in adolescence and tend to cluster together. Conclusion This study has demonstrated that YPWD are sexually active and engage in sex as early as 15 years. The YPWD are at risk of unintended pregnancies and acquiring of sexually transmitted infections, and associated consequences. The risks are heightened by low life skills, limited agency, and alcohol consumption. The study underscores the importance of schooling and access to sexuality education. This means policies and programmes for government and nongovernment entities should be intentional in promoting access and completion of schooling for YPWD. This further emphasizes the need to address barriers of access to education for YPWD including social and gender norms that create stigma and downplay education for them and the other structural barriers related to infrastructure, skilled human resources especially teachers and other service providers as well as the required equipment. Interventions designed to promote safe sexual practices and negative SRH outcomes should integrate addressing alcohol as a risk factor. The results also highlight the need for interventions targeting YPWD starting early in their adolescence to ensure they have the appropriate sexuality education, life skills and agency to make informed choices related to engaging in safe sex to prevent undesirable reproductive health outcomes. The YPWD should also be engaged in co-design of interventions and services designed to meet their SRH needs and to promote safe sexual practices. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. Ethics statement The studies involving human participants were reviewed and approved by Makerere University School of Public Health Research and Ethics Committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.	2023-03-17T15:22:29.754Z	2023-03-15T00:00:00.000	{ "year": 2023, "sha1": "921a66e958abf7f76ba57ec81418d3c8665904ad", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/frph.2023.1065437/pdf", "oa_status": "GOLD", "pdf_src": "Frontier", "pdf_hash": "cc3f5cbe5b4d92ecff8249d9ba685f5ca46e2422", "s2fieldsofstudy": [ "Medicine", "Psychology" ], "extfieldsofstudy": [ "Medicine" ] }
3228765	pes2o/s2orc	v3-fos-license	Patient preferences for chemotherapies used in breast cancer Correspondence: Kathleen Beusterien 7315 Wisconsin Ave, Ste 250W, Bethesda, MD 20814, USA Tel +1 301 841 3858 Fax +1 240 482 0043 Email kathy.beusterien@ oxfordoutcomes.com Background: Therapies for invasive breast cancer may be associated with an incremental survival advantage that should be weighed against the risk of toxicities when making treatment decisions. The objective of this study was to elicit patient preferences for a comprehensive profile of attributes associated with chemotherapies for breast cancer. Methods: This was a cross-sectional study of 121 patients with stage I-IV breast cancer who completed an internet-based conjoint survey that assessed the following attributes: ten grade III/IV toxicities, survival advantage, and administration regimen. Literature and expert input were used to identify descriptions for each attribute and respective levels (eg, different risks of toxicities). Subjects rated the attribute levels on a series of scales and indicated preferences in pair-wise comparisons of two hypothetical treatments differing in attribute levels. Ordinary leastsquares regression was used to calculate utilities (preference weights) for each attribute level. Results: Of the twelve attributes, survival was the most important; specifically, a survival advantage of 3 months versus no survival advantage was most influential in the perceived value of chemotherapy. Among toxicities, the differences in the risks of neutropenia with hospitalization, diarrhea, nausea, and fatigue had the most impact on preferences; the risk differences of myalgia, stomatitis, and hand-foot syndrome had the least. In general, a more convenient administration regimen was less important than a 13% chance or more of severe toxicities, but more important than a 10%–12% chance of severe toxicities. Conclusion: Breast cancer patients place high value on small incremental survival advantages associated with treatment despite the risk of serious toxicities. Introduction Breast cancer is the second most common cancer among women in the United States, with approximately 54,010 new cases of carcinoma in situ (noninvasive cancer) and 207,090 cases of invasive cancer diagnosed per year. In addition, breast cancer is the second leading cause of cancer death in women, with about 39,840 deaths occurring per year. 1 Five-year survival rates vary by stage: the 5-year survival rate for patients with stage 0 through stage IIA breast cancer ranges from 92 to 100%, while the 5-year survival rate for patients with stage IV metastatic breast cancer is approximately 20%. 1 Thus, the goal of therapy for invasive breast cancer is primarily palliative -that is, aimed at alleviating and controlling symptoms as well as improving quality of life. 2 Treatments in common use for invasive breast cancer include cytotoxic chemotherapy, hormonal treatments, and targeted therapies/biologics, which can be administered as single agents or in combination with each other. 3 Given that current chemotherapies have advantages or disadvantages relative to each other, it would be useful to understand how these influence patient preferences for treatment. Some studies have examined patient preferences with respect to chemotherapy toxicities. For example, one recent study evaluated preferences for health states associated with chronic lymphocytic leukemia treatments using the standard-gamble method. 4 Other studies have also examined patient preferences for select groups of chemotherapy toxicities. 5,6 However, to date, no study has evaluated the importance of toxicities relative to other characteristics such as efficacy and administration. Conjoint analysis, which is increasingly being used in evaluating medical interventions, involves respondents making trade-offs among product features (attributes) such as mode of administration and risk of adverse events. 7,8 The resulting data, or utilities, enable the assessment of the relative importance of each treatment attribute; specifically, they show the influence that each attribute has on overall treatment preferences. The goal of the present study was to use conjoint analysis to capture patient preferences for attributes associated with chemotherapies for invasive breast cancer. Specifically, the survey aimed to assess the trade-offs that breast cancer patients are willing to make among chemotherapies according to different toxicity, regimen, and efficacy profiles. Methods This was a cross-sectional, internet-based survey assessing the chemotherapy treatment preferences of women with breast cancer. Study participants were recruited through newspaper advertisements and online breast cancer support forums. All participants were women with United States residency; at least 18 years of age; had a diagnosis of stage I through IV breast cancer; had received chemotherapy treatment within the past 5 years; and provided informed consent and Health Insurance Portability and Accountability Act-compliant Authorization. 9 Participants completed the survey at home and worked at their own pace. However, research team members were available via telephone or email to answer questions or provide technical help if necessary. Participants were compensated US$30.00 for their time. The study followed the tenets of the Declaration of Helsinki, and the protocol was approved by a commercial institutional review board (Independent IRB; Plantation, FL). This research was implemented following published methodological guidelines for conjoint studies. 10 The survey design and analysis are detailed below. Survey design Twelve attributes of eight invasive breast cancer chemotherapy treatments were identified from a comprehensive literature review, the Common Toxicity Criteria grading system, a detailed assessment of breast cancer forum discussions, and consultation with clinical experts. These attributes included selected grade III/IV toxicities, efficacy (survival advantage), and regimen. Attributes were described in lay terminology so that they could be easily understood by patients. Each attribute was presented in different levels that represented the full range of possibilities across available breast cancer treatments. Each toxicity attribute had three levels. Based on the literature and clinician input, potential ranges for the incidence of each of the toxicities were identified, and these were used as the most favorable and least favorable levels, respectively. The regimen attribute had six levels, each describing a different chemotherapy regimen. Table 1 presents the attributes and the respective levels used in the survey. An adaptive conjoint analysis (ACA) approach was used to design the survey. This involves a hybrid approach, in which the data are collected in phases. 11,12 The respondent completes the ACA computer-assisted questionnaire, and the questionnaire is modified during the responding process on the basis of the respondent's previous choices. Thus the interview can focus on just those attributes that the respondent considers most important and those attribute levels regarded as most relevant. In the first phase, respondents were asked to rate the levels of each attribute in terms of acceptability on a 7-point scale, from 1, "not at all acceptable", to 7, "acceptable". The second phase (paired comparison questions) elicited treatment preferences by asking respondents to make trade-offs among attributes and choose from a pair of hypothetical treatments. In each of these questions, the profiles of two hypothetical treatments -labeled simply chemotherapy A and chemotherapy B -were presented with different levels of the same three attributes (no chemotherapies were named in the survey). Respondents used a 7-point scale to indicate not only their preference, but the strength of their preference; response options ranged from "strongly prefer A" to "strongly prefer B". The profiles presented to respondents in this second phase were customized based on responses to previous questions. The final section of the survey contained demographic and clinical questions. The survey was pilot-tested with respect to wording and comprehension in a sample of four breast cancer patients; no major changes were necessary. submit your manuscript \| www.dovepress.com Grade III/IV sensory neuropathy Chance of severe numbness, sharp tingling (pins and needles), and burning sensation in arms and legs, interfering with daily tasks like holding a pen, dressing, or cooking; may last 6 months after receiving chemotherapy. 0% 7% 13% Grade III/IV motor neuropathy Chance of feeling very weak, shaky, and unsteady, making it difficult to hold objects and requiring a cane or other assistance; may last 6 months after receiving chemotherapy. 0% 4% 10% Grade III/IV myalgia/arthralgia Chance of severe joint/muscle aches, pain, and stiffness, making it difficult to move; may last up to 4-7 days after receiving chemotherapy. 0% 4% 15% Grade III/IV nausea and vomiting Chance of severe nausea and/or vomiting requiring intravenous fluids at the doctor's office or ER; may last 2-3 days after receiving chemotherapy. 0% 4% 15% Grade III/IV fatigue Chance of severe fatigue, making it difficult to perform daily activities; may last 6 months after receiving chemotherapy. 0% 8% 24% Grade IV neutropenia resulting in hospitalization Chance of having a fever or infection requiring hospitalization with intravenous antibiotics for 3-5 days. Grade III/IV mucositis/stomatitis Chance of sores and blisters in mouth that make eating and drinking painful; may last 1-2 weeks after receiving chemotherapy. 0% 5% 10% Grade III/IV hand-foot syndrome Chance of tightness of the skin, peeling, sores and blisters on hands and feet making it very painful to use them; may last 7-14 days after receiving chemotherapy. 0% 5% 12% Grade III/IV diarrhea Chance of more than 6 episodes of diarrhea per day, requiring intravenous fluids at the hospital; may last 2-5 days after receiving chemotherapy. Efficacy Has not shown an additional survival benefit Has shown an additional survival benefit of 1 month Has shown an additional survival benefit of 3 months Regimen: frequency and duration of chemotherapy administration; Six levels • 21-day cycle; oral tablets taken twice daily for first 2 weeks Analysis The conjoint data were analyzed using Sawtooth Software SSI Web (v 6.4; Sawtooth Software, Sequim, WA). Analysis of ACA data involved the combination of the initial rating (the acceptability questions in the first phase) and the paired comparison questions. Specifically, using the information from the initial rating questions, prior utilities were calculated. The average for each attribute was subtracted to center its values at zero. For example, desirability values 3, 2, and 1 would be converted to 1, 0, and -1, respectively. These initial estimates are part-worths, where within each attribute the values have a mean of zero, and differences between values are proportional to differences in desirability ratings or rank orders of preference. With respect to the paired comparison questions, a column vector was created for the dependent variable as follows: the respondents' answers were zerocentered, where the most extreme lowest value was given -4, and the most extreme highest value was +4. Interior ratings submit your manuscript \| www.dovepress.com 281 Treatment preferences in breast cancer were fitted proportionally within that range. Each pair's question contributed a row to both the independent variable matrix and dependent variable column vector. Ordinary least-square estimates of the n attribute levels were computed by regression of the dependent variable column vector on the matrix of independent variables. The part-worth estimates based on the prior and paired comparison phases were normalized to have equal sums of differences between the best and worst levels of each attribute across all attributes. The two vectors of part-worths were added together, yielding final utilities for each attribute level for each respondent. The absolute values of the final utilities were arbitrary; what was important was the magnitude of difference between them. These weights were "zero-centered," whereby the utility weight of the attribute level falling in the middle had a value that approximated zero and the more favorable level was higher, or positive, and the less favorable level was lower, or negative. Finally, the Bayes approach was applied to the data to further refine the precision of the utility estimates. The utilities enable the calculation of the relative importance of each attribute for each respondent in influencing treatment decisions. Specifically, the relative importance was calculated for each respondent by dividing the range for each attribute (utility of highest level-utility of lowest level) by the sum of ranges of all attributes for the respective individual and multiplying it by 100. These estimates indicate how much the difference in importance between the best and worst levels of each attribute affects the decision to choose a treatment. These are ratio data, meaning for example that an attribute with an importance of 10% is twice as important as an attribute with an importance of 5%. Using the mean utility weights for the attribute levels, we compared the percentages of patients who would prefer the following scenarios differing with respect to selected attributes, while holding all other attributes constant: (1) three levels of efficacy (no additional survival, 1 month additional survival, and 3 months additional survival); (2) Results Of 121 participants who completed the conjoint survey, 108 (89%) were included in the final data analysis. Thirteen participants were excluded because their responses in the first section of the survey were illogical (eg, a participant rating a 24% chance of fatigue as more acceptable than a 0% chance) in at least two instances. Table 2 reports the demographic and clinical characteristics of the study population who were included in the analysis. The mean age was 50 years, with a mean time of 80 months since cancer diagnosis; 60% had attained at least a college-level education. With respect to treatment experience, about 35% of patients were on chemotherapy at the time of their participation in the study, and the mean time since the sample's last chemotherapy administration was approximately 19 months. The mean utilities for each attribute level are presented in Table 3 (the actual values are arbitrary; the magnitude of the differences among them are what should be evaluated). In general, the utilities for each attribute level were ordered in the direction that was expected, where the most favorable attribute level had the highest utility and the least favorable level had the lowest. The efficacy attribute had the highest utility value for its most favorable level ("additional survival benefit of 3 months") and lowest utility value for its least favorable level ("no additional survival benefit"), with means of 97.05 and -83.77, respectively. This finding indicates that patients view a change in survival advantage from 0% to 3 months as more important than a change from the least favorable to the most favorable level for all other attributes. Figure 1 presents the "percentage importance" estimates for each attribute. Overall, the most important attributes in driving treatment preferences were survival advantage, neutropenia/hospitalization, and toxicities such as nausea/ vomiting, fatigue, and diarrhea. Least important attributes were the toxicities of mucositis/stomatitis, myalgia, and hand-foot syndrome. In general, the difference between the most inconvenient to the most convenient administration regimen was less important than a 13% chance or more of severe toxicities, but more important than a 10%-12% chance of severe toxicities. The exception to this finding was myalgia, which had a maximum level of 15% chance of occurring; the difference between 0% versus 15% chance of myalgia was still less important than the difference between the most inconvenient and most convenient regimen (percentage importances = 8.8% versus 3.2%). In sensitivity analyses, we created scenarios for each toxicity (ie, each attribute that was a side effect) in which we compared preferences for a 0% risk of that toxicity versus a 10% risk of that toxicity, holding the risk of all other toxicity attributes constant. Specifically, we obtained preference estimates for two product profiles in which one toxicity differed submit your manuscript \| www.dovepress.com 283 Treatment preferences in breast cancer by 10% and the remaining attributes were held constant; we repeated this for each side effect. Figure 2 shows the percentages of patients who would prefer a treatment given a 0% chance versus a 10% chance of each toxicity occurring. The findings show that patients most preferred to avoid neutropenia/hospitalization, diarrhea, nausea/vomiting, and sensory neuropathy (ie, they most preferred no risk of these toxicities relative to no risk of the other toxicities). An additional analysis found no substantial differences in relative importance estimates of toxicities between those who had experienced the side effect versus those who had not (data not shown). Figure 3 presents the percentages of patients preferring each of three regimens that differ in efficacy (survival Hand-foot syndrome, 5.5% Alopecia, 6.5% Figure 1 relative importance of attributes. Note: ratio data: 10% is twice as important as 5%. advantage) and the percentages of patients preferring three regimens, ie, an oral regimen, a regimen somewhat in "the middle" with respect to convenience (28-day cycle; 6-10 minute infusion on days 1, 8, and 15), and the most inconvenient (21-day cycle; 3-hour infusion on days 1, 8, and 15). The findings show that the difference between a 1-month and 3-month survival advantage substantially influenced patient preferences; the percentages of patients preferring each of these regimens increased from 5.7% to 93.7%, respectively. Also, an oral regimen was highly preferred; 76.6% of patients would choose an oral regimen versus 18.9% who would choose the "middle" regimen. Discussion To the best of our knowledge, this study is the first to assess patient preferences for invasive breast cancer treatments considering a variety of treatment features. Specifically, this study assessed the trade-offs that breast cancer patients are willing to make among the risks of severe toxicities, administration regimen, and efficacy when choosing a chemotherapy. Among the chemotherapy attributes evaluated in this study, those that were most influential in driving patient preferences for treatment were improved survival, the risk of neutropenia leading to hospitalization, and the risks of severe fatigue, nausea and vomiting, and diarrhea. Least important were the risks of severe myalgia, mucositis, and hand-foot syndrome. The study findings are useful in better understanding patient preferences in oncology and may enhance the medical decision-making process. For most patients, the difference between having no additional survival advantage and having a survival advantage of 3 months was about three times more important than an approximate 10% versus 0% risk of experiencing several severe toxicities, including severe myalgia, mucositis, hand-foot syndrome, and motor neuropathy. In addition, the incremental improvement in survival was one-and-a-half times as important as an approximate 15%-20% versus 0% risk of having severe nausea and vomiting, fatigue, diarrhea, or sensory neuropathy. The finding of the high relative importance of improved efficacy is consistent with previous conjoint analyses of medical therapies 13,14 and a review of previous literature, 15 which found that patients are willing to accept the risk of serious adverse events in exchange for improved efficacy. For example, Johnson et al 12 evaluated the treatment perceptions among patients with multiple sclerosis, and they found that most patients indicated they are willing to accept risks of life-threatening adverse events in exchange for improvements in their health outcomes. This study builds upon previous work that has examined rankings among chemotherapy side effects. 16 Specifically, Sun et al 16 evaluated preferences for 27 different side effects among patients with ovarian cancer. They found that severe nausea and vomiting was the least preferred toxicity and that "numbness in hands/feet" was among the most 18 Figure 3 Percentages of patients preferring treatments with differences in survival advantage and differences in regimen convenience, holding all other attributes constant. submit your manuscript \| www.dovepress.com Dovepress Dovepress distressful side effects. Similarly, the current study showed that patients would most want to avoid the chance of severe nausea and vomiting and the chance of severe numbness in the arms and legs (sensory neuropathy) among the various potential toxicities evaluated, assuming that they each had an equal chance of occurring. Our study found no substantial differences in the relative importance of toxicities between those who had experienced the toxicity versus those who had not. This finding differed from a previous study that examined utility (time trade-off) estimates for chemotherapy-related ototoxicity, nephrotoxicity, and neurotoxicity among ovarian cancer patients. 17 Specifically, the study found that the most favorable assessment of a particular toxicity was reported from individuals who experienced the selected toxicity. This may be attributable to the fact that patients were asked to assume that the toxicity was permanent; as such, patients with experience may have felt they could better tolerate the effect than naïve patients. In contrast, in the current study, each of the side effects had a specified duration, and thus this may not have been significant enough to translate into differences between those experienced versus not experienced with each toxicity. Moreover, our findings were consistent with the finding of no substantial differences observed in relative importance of eleven of 12 side effects between HIV treatment-experienced, who may have experienced treatment side effects, versus treatment-naïve patients. 18 The current study also found that a more convenient administration regimen was more important to patients than severe side effects when the respective risk was less than 13%. Similarly, improved convenience has been found to be influential in treatment choices in other studies. 13,14 For example, in a study of patients with idiopathic thrombocytopenic purpura, patients were willing to accept significant treatment-related risks in exchange for improvements in treatment efficacy and convenient administration. 13 Our study was not without limitations. First, each of our toxicity attributes was described using durations from a few days to 6 months after treatment end, and these durations represented clinical averages. These average durations may not be reflective of individual experiences, however, as our discussions with clinicians revealed that side effect experience is highly variable between patients. In addition, the participants in our sample did not represent the full range of breast cancer patients; the majority were well-educated, most were Caucasian, and they were all healthy enough to complete a computer-based survey at home. It is unclear whether the preferences of severely ill cancer patients (eg, those who are bedridden) or other ethnic groups would be different from those expressed by patients in our sample. Because of these limitations, while our study has yielded important findings with implications for the medical community, future research is necessary to evaluate the generalizability of our conclusions. Conclusion In summary, this survey evaluated a comprehensive set of severe toxicities and other features that are observed with chemotherapies for invasive breast cancer. Our study showed that, despite the risk of serious toxicities, a small incremental survival advantage is highly influential in patient preferences for chemotherapy. The findings from this research may be useful in incorporating patients' views into medical decision making processes, patient education, cost resource allocations, and drug development. A better understanding of patients' preferences may help to improve patient satisfaction and compliance with treatment regimens. Disclosure Kathy Beusterien and Jessica Grinspan are employed by Oxford Outcomes, Inc, an ICON plc company, which consults for Eisai, the study sponsor. Thomas Tencer is a former employee of Eisai, the study sponsor. Adam Brufsky and Constance Visovsky report no conflicts of interest in this work.	2018-01-24T17:25:33.048Z	2011-05-20T00:00:00.000	{ "year": 2011, "sha1": "4a8f52ae7890f098aa71f44bc0ac0dadba3ff356", "oa_license": "CCBYNC", "oa_url": "https://www.dovepress.com/getfile.php?fileID=13118", "oa_status": "GREEN", "pdf_src": "Anansi", "pdf_hash": "2553734d665804e80ad13ed186ae100c1950e446", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
229014985	pes2o/s2orc	v3-fos-license	Application of Computer Technology in English Translation Network Teaching The development of modern educational technology and computer network technology will bring translation teaching into a new era, and a new translation teaching model will emerge. Computer network technology is completely suitable for translation teaching, because the type and characteristics of translation teaching determine this applicability. The scientific and artistic nature of translation can enable the learning subject to implement a high degree of self-control and independent learning in the network environment, and actively display creativity and individuality. The teaching subject can also adopt a combination of individualized and collaborative teaching methods to achieve "double "The main teaching mode" means to play the leading role of teachers and the cognitive subject role of students at the same time. The new translation teaching model based on computer network technology will certainly arouse the general attention of the domestic translation teaching circle and be widely used. Theoretical basis and feasibility analysis of audiovisual teaching model Judging from the current status and development trend of college English listening and speaking teaching in my country, the author believes that the visual listening and speaking model is highly feasible in undergraduate English teaching for the following reasons: Source conditions From the perspective of constructivism, human knowledge is actively constructed by individuals on the basis of existing experience rather than transmitted by others. Different students' original cognitive structure will lead to great differences in their English learning ability, cognitive style, cognitive strategies and learning strategies. Therefore, the essence of English learning is an individualized and individualized process. In other words, the pros and cons of the English cognitive structure formed by the learners in the middle and high schools will directly affect the implementation of the audiovisual teaching model at the undergraduate level [1] . With the continuous deepening of the English teaching reform in the middle school stage, the English quality of freshmen is improving year by year, which provides a reliable source of students for the practice of audiovisual teaching mode. For freshmen with a solid foundation in English, with a little training, they can adapt to this teaching model that requires the learners to give full play to their own subjective initiative. If we continue to use the old teaching model and continue to "lay the foundation" in the limited hours, it will inevitably make college English listening and speaking teaching "lag behind". Learning purpose and motivation Constructivism believes that learning is the process by which learners construct their own knowledge structure.This means that learners are not passively accepting stimuli from the outside world, nor are they mechanically moving knowledge from the outside world to memory, but on the basis of their original experience, they actively select and process external information. In other words, whether it is language knowledge or language skills [2] , students must take the initiative to learn. If the students themselves are unwilling to take the responsibility of learning, no matter how the teacher guides them, it will be useless. The 21st century emphasizes the comprehensive cultural literacy of talents and puts forward higher requirements for the ability to use English to communicate with foreigners. This undoubtedly makes the learning goals of college students more clear, and their learning motivation is significantly enhanced. All of these provide conditions for the implementation of the audiovisual teaching model, and also make it possible to perform specific operations at the undergraduate level. The development of multimedia technology As we all know, all school curriculums are ultimately designed to meet the needs of society. Under the influence of globalization, English has become an indispensable tool for future communication between countries [3] . This means that modern English listening and speaking teaching should not only be limited to the learning level of students listening to the recorded materials and teachers' retelling the original texts, but should also start with the cultivation of the comprehensive skills of correct use of English for daily communication. Therefore, English listening and speaking teaching has become a complicated task [4] .The rapid development of modern technology (especially computer technology) has made multimedia-assisted teaching as a new language input, creating a better language environment for students from various perspectives, and providing the importance of successful language acquisition. Application in learning After the Higher Education Department of the Ministry of Education promulgated the Teaching Requirements for College English Courses (Trial) in 2004, Tsinghua University Press and other units have successively introduced a college English online teaching system, and conducted trials in 180 colleges and universities across the country. The author unit selected the "New Era Interactive English" published by Tsinghua University Press in 2005-audiovisual,listening, reading and writing translation textbooks and its network software system. At the same time, the English teaching reform project team was established. A total of 1614 freshmen from 56 classes of the Eastern Science and Technology College of Hunan Agricultural University in 2005 were pilot teaching subjects, and began to explore a new model of college English audio-visual teaching based on computer and multimedia technology. The specific implementation steps are as follows: Preliminary preparation Active research. In order to ensure the smooth progress of the teaching reform, the school has successively conducted research and study in the first batch of 180 colleges and universities, such as Beijing Jiaotong University and Beijing Institute of Technology, to conduct teaching reform pilot projects, and learn from their experience. He also participated in relevant education reform Because the computer-based and network-based audio-visual mode is a brand-new teaching mode, it requires teachers to change the old teaching ideas and challenge the traditional teaching methods; it requires students to abandon the old learning ideas and cultivate the ability to learn independently. In order to achieve this goal, the project team invited staff who participated in the development and research of Tsinghua University Press "New Era Interactive English Audiovisual Network Software System" to give all the faculty and staff of the author's unit a comprehensive training for college English audiovisual listening The reform of teaching mode laid the ideological foundation.The "New Era Interactive English" published by Tsinghua University Press-audio-visual, listening, reading and writing translation textbooks and its network management platform were selected to provide hardware support for the practice of audio-visual and speaking teaching mode. Teaching arrangements The pilot teaching consists of three parts: student self-learning (2 hours a week), teacher Q&A (synchronized with student self-study), and oral lessons (irregular).Self-directed learning by audiovisual and listening. The new model abandons the traditional concept that only through teachers imparting knowledge to students, requires students to independently arrange their learning progress according to their own knowledge level and cognitive style, and better reflects the learner's dominant position.The teacher answers questions.The learner does not acquire knowledge through the teacher's systematic teaching, but transforms knowledge into his own internal expression through his own experience interpretation of the knowledge unit.For those inexperienced learners, in this process, there will definitely be some wrong judgments of knowledge. At this time, teachers need to guide students to acquire correct cognition through error correction and answering questions.Appointment in oral English. In this link, try to involve students.Knowledge. Second, practice activities based on the content of each unit of audiovisual and listening. The purpose of this activity is to familiarize students with the knowledge points of each unit and train their listening and speaking skills. Third, the communicative practice activities related to the theme of each unit of audiovisual and listening.This type of activity requires students to use the language they have learned to communicate their ideas truthfully and freely, and jointly complete the task of using language knowledge to communicate [5] . Analysis of teaching effect In order to understand the teaching effect of this model, the English teaching reform project team of the author randomly selected 149 students from the experimental subjects as the experimental group (76 girls and 73 boys), and randomly selected 141 students from the non-experimental subjects for comparison. The group (41 girls and 100 boys) organized an oral test in mid-December 2005. In order to ensure the fairness and impartiality of the test, the students in the experimental group and the comparison group were randomly combined, and the list of candidates issued to the invigilator did not indicate the department of each candidate. The results of this oral test are shown in the following From the above test results, it can be seen that through one semester of audio-visual mode pilot teaching, the average scores of girls and boys in the experimental group are higher than those of the control group, which shows that the audio-visual teaching mode can quickly improve non-English major college English learners Oral level.Taking into account the correlation between listening and speaking, the author concludes that the audiovisual teaching model can improve the listening level of learners.In addition, the average scores of girls in the experimental group and the comparison group are higher than those of boys, which reflects the gender differences in learners. Several key changes in the implementation of audiovisual teaching mode Although the audiovisual teaching model has many advantages, in the process of implementation, due to the influence of the subjective factors of teachers and students and the objective environmental factors, some problems also arise. For example, teachers and students have different levels of recognition of college English teaching reform, the actual operating ability of computers and networks is uneven, and the supporting facilities such as computer hardware configuration and local area network connection cannot meet the teaching requirements. In order to solve these problems and ensure the effectiveness of audiovisual teaching, several key changes must be grasped in practice. Guiding ideological change That is to change from continuing the teaching of language ability to highlighting the cultivation of practical communication ability. Emphasize the "use of English for the development of communication skills" and emphasize the transformation of traditional learners from listening and not speaking into a combination of listening and speaking in the process of English audio-visual teaching; the traditional learners passively receiving the language knowledge taught by the teacher will be transformed into learning Through the interpretation of his own experience of the knowledge unit, the reader transforms it into his own internal expression, and then gradually constructs a cognitive structure using English listening and speaking skills to achieve daily communication in the process of interacting with the environment. This is the basic guiding ideology and starting point of the audiovisual teaching model [6] . Teacher training changes The reform of audio-visual teaching mode based on computer and network is actually an innovation of traditional teaching methods, teaching methods and traditional concepts. Therefore, teachers must be regularly trained in modern education concepts, network skills, information technology knowledge, subject education and teaching theory and subject development trends, information technology and educational psychology theory and practice. Teaching equipment update To ensure the normal operation of this new teaching model, the traditional language room can no longer meet our requirements. Therefore, how to upgrade and update the original hardware equipment has become a problem that we must face, which will directly affect the implementation of the audiovisual teaching model.	2020-11-05T09:07:46.066Z	2020-10-01T00:00:00.000	{ "year": 2020, "sha1": "149f41a5aab9d6d214d2ccd261d6ba0577f055f2", "oa_license": null, "oa_url": "https://doi.org/10.1088/1742-6596/1648/2/022124", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "dff1a63a1a433acb4a284687cf0332c87e7acb49", "s2fieldsofstudy": [ "Computer Science", "Education", "Linguistics" ], "extfieldsofstudy": [ "Physics", "Computer Science" ] }
253000631	pes2o/s2orc	v3-fos-license	The Correlation between the Triglyceride-Glucose Index and Coagulation Markers in Patients with Recent Acute Myocardial Infarction Background Metabolic abnormalities and hypercoagulability seem to have an important predictive role in patients with coronary artery disease (CAD). The triglyceride-glucose (TyG) index has emerged as a good marker for metabolic syndrome with predictive value for cardiovascular events. Overall haemostatic potential (OHP) is a reliable global haemostatic essay to identify hypercoagulability in CAD patients. The aim of our study was therefore to evaluate a possible correlation between the TyG index and haemostatic derangements in patients with CAD. Methods Consecutive patients referred for the first follow-up visit after acute myocardial infarction between December 1, 2018, and March 31, 2020, and did not meet exclusion criteria were included. We determined OHP, overall coagulation potential (OCP), overall fibrinolytic potential (OFP), fibrinogen, D-dimer, and von Willebrand factor from peripheral blood samples. The TyG index was calculated with the previously described and validated formula. Linear regression models were constructed for the multivariate analysis. Results A total of 117 patients (mean age 56 ± 10 years, 20% women) were included. A correlation was found between TyG index and OCP (r = 0.229, p = 0.026), TyG index and OHP (r = 0.202, p = 0.050), and TyG index and fibrinogen (r = 0.271, p = 0.005). In the multivariate model which accounted for sex, age, and BMI, the correlation between TyG index and OCP (R2 0.108; ANOVA for regression p = 0.035; beta 2.08 [0.79-4.01], p = 0.042) and between TyG index and fibrinogen (R2 0.11; ANOVA for regression p = 0.015; beta 0.35 [0.08-0.62], p = 0.012) emerged as statistically significant. Conclusion The TyG index, a marker of metabolic syndrome, has a strong correlation with a hypercoagulability state in CAD, as determined by the OCP and higher fibrinogen levels. Our findings suggest that metabolic syndrome may be an important driver of atherothrombotic risk in patients with CAD. Introduction Coronary artery disease (CAD) is a form of atherosclerotic vascular disease associated with significant morbidity and mortality despite mounting efforts of prevention, early diagnosis, and aggressive management [1]. CAD is characterized by progressive atherosclerotic plaque build-up, which is prone to rupture, and atherothrombotic coronary artery occlusion, yielding myocardial infarction. On the one hand, atherosclerosis progression is associated with cardiovascular risk factors. While some risk factors, such as smoking, have been stagnating or declining in western populations, others-especially cardiometabolic risk factors, such as obesity, diabetes, and dyslipidaemia-have been on the rise [2,3]. On the other hand, atherothrombotic events are associated with a procoagulant state [4], which may discern stable chronic CAD from unstable acute coronary syndromes. Obesity and insulin resistance are notable hallmarks of cardiometabolic risk, suggesting underlying derangements in glucose and lipid metabolism. However, recent studies suggest that cardiometabolic abnormalities can be detected in nonobese individuals as well [5,6], and identification of cardiometabolic risk beyond apparent clinical characteristics-possibly through biomarkers-is being pursued. One such biomarker is the triglyceride-glucose (TyG) index, which has been proposed as a marker of metabolic syndrome and has been associated with carotid atherosclerosis, coronary artery calcification, and higher risk of cardiovascular disease [7][8][9]. Moreover, recent studies have identified the TyG index as an independent predictor of coronary calcification progression, of clinical outcomes in patients with stable CAD and premature CAD, and of cardiovascular outcomes in acute coronary syndromes [7,[10][11][12][13]. In addition to risk factors-driven coronary atherosclerosis progression, haemostatic derangements play an important role in the development of myocardial infarction. CAD has been associated with a procoagulant state with mounting evidence of high haemostatic factor activity-such as increased fibrinogen levels, a procoagulant platelet response, and thrombogenicity [4,[14][15][16][17]. Procoagulant activity is associated with both the severity of chronic CAD and the likelihood of its progression to myocardial infarction [4,18]. Since coagulation is a complex and multifactorial process, global haemostatic essays-as opposed to the measurement of individual procoagulant and fibrinolytic factors-have recently gained popularity [19]. Overall haemostatic potential (OHP) is a global haemostasis essay [20,21], which has been validated in healthy individual as well as various groups of patients [22][23][24]. In CAD patients, OHP detected a hypercoagulability state, driven primarily by impaired fibrinolysis [23,25]. Clinical features of the metabolic syndrome are associated with a prothrombotic state. Several coagulation markers are increased, and fibrinolysis is impaired in patients with metabolic syndrome, which may contribute to the adverse atherothrombotic pathophysiology and high-risk features of CAD in patients with dysmetabolic profiles [26,27]. In the present study, we sought to appraise a possible association between the TyG index, a marker of the metabolic syndrome beyond traditional clinical features, and the OHP, an essay for haemostatic derangements, in patients with CAD. Study Design and Population. Consecutive patients referred for the first follow-up visit after acute myocardial infarction between December 1, 2018, and March 31, 2019, were screened for inclusion. Inclusion criteria were 18-70 years of age and recent myocardial infarction (within 90 days from inclusion). Patients with advanced heart failure (New York Heart Association classes III and IV), patients on anticoagulation treatment, and pregnant women were excluded. The study was conducted in accordance with the Declaration of Helsinki (1964) and approved by the National Medical Ethics Committee (approval number 0120-77/2020/3). After signing informed consent, patients were included in the study and had a fasting (>12 hours after the last meal) blood sample drawn from the cubital vein; blood was collected into 4.5 mL 0.109 M sodium citrate tubes. Citrated blood was then centrifuged for 20 minutes at 2000 × g; the extracted plasma was then stored at -70°C for further analysis. Statistical Analysis. Baseline characteristics are expressed as the mean (±standard deviation) for normally distributed continuous variables, as median (interquartile range) for nonnormally distributed continuous variables, and as frequency (%) for categorical variables. Between-group differences were assessed by the t-test for normally distributed variables and by the Mann-Whitney U test for nonnormally distributed variables; proportions were compared using the χ 2 test. Correlations were explored with Pearson's tests. Linear regression models were constructed for the multivariate analysis; results are expressed as beta with corresponding 95% confidence intervals (CI). A 2-tailed p < 0:05 was considered significant. Statistical analysis was carried out using SPSS Statistics version 23 (SPSS Inc, Chicago, USA). Results A total of 143 patients were assessed for eligibility; 26 patients were excluded (20 did not meet the inclusion criteria and 6 declined to participate), 117 patients after a recent myocardial infarction were included, and no participant had a previous history of arterial or venous thrombotic events. Baseline characteristics and risk factor presence are reported in Table 1. Medical therapy, prescribed at the time of the event, is listed in Table 1. The median triglyceride value was 115 mg/dL (IQR 88), median glucose value was 100 mg/dL (IQR 14), and median TyG index value was 8.68 (IQR 0.5). We divided participants according to the median value of the TyG index and compared the two groups: BMI and total cholesterol were found to be significantly increased and HDL cholesterol to be significantly decreased in the group with the TyG index above median (Tables 1 and 2). A comparison between the lowest and highest tertile of the TyG index was performed (Table 3); a significant difference was found in diabetes prevalence and a trend to significance for BMI. Among coagulation markers, OCP and OHP were found to be significantly higher in patients with the TyG index above median (Table 2). To further explore the association between coagulation biomarkers and TyG index, a ROC analysis was performed. Both OCP and fibrinogen showed a modest predictivity for the TyG index (ROC area 0.60, 95% CI 0.480-0.713 and ROC area 0.59, 95% CI 0.484-0.701, respectively). Discussion In the present study, we have shown a significant association between the TyG index (a biomarker of cardiometabolic risk) and haemostatic abnormalities in patients after myocardial infarction. The finding suggests a possible link between cardiometabolic and coagulation derangements beyond traditional clinical features of the metabolic syndrome. To the best of our knowledge, ours is the first study to appraise a strong positive correlation between a procoagulant state (OCP and fibrinogen levels) and the TyG index, suggesting a pathophysiological interplay between cardiometabolic abnormalities and atherothrombotic potential in patients after a recent coronary event. The metabolic syndrome is a known predictor of cardiovascular events in patients with established CAD [28][29][30]. Recent research has identified the TyG index as a reliable marker of metabolic syndrome and a strong predictor of CAD, premature CAD, and adverse events in CAD patients [10][11][12][13]28]. In our study, values of the TyG index were comparable to those from previous studies in patients with CAD, wherein TyG index values were also associated with an increased event risk [11]. Our data complete these observations by proposing a pathophysiological link between the TyG index and haemostatic derangements; as the majority of CAD events represent thrombotic complications of atherosclerosis, an association between cardiometabolic and haemostatic derangements provides an important further insight in the unfavourable atherothrombotic prognostic role of the metabolic syndrome. Previous studies have shown that insulin resistance and metabolic syndrome may confer a procoagulant state through several mechanisms. Adipose tissue may impair fibrinolysis through direct production of coagulation factors (such as PAI-1 and thrombin activatable fibrinolysis inhibition), dysmetabolism-associated nonalcohol liver steatosis may alter the hepatic synthesis of haemostatic factors, and adipokines may affect platelet function; insulin resistance-associated lowgrade inflammation and endothelial dysfunction may modulate the genetic expression, activity, and interactions of haemostatic factors [27,[31][32][33]. In our study, we detected a strong correlation between known markers of metabolic disorder and TyG index and the association of the TyG index with a procoagulant state. The traditional assessment of a procoagulant state relies on measuring individual coagulant factors but may fail to capture the complexity of the overall haemostatic processes, and even in our study, D-dimer and von Willebrand factor did not show a significant correlation with the TyG index, but a strong correlation, independent of BMI, was found for fibrinogen, a known marker of coagulation. The finding supports coagulation activation in patients with metabolic syndrome and CAD. Global haemostatic assays for net yields of coagulation and fibrinolysis have recently been proposed as more suitable indicators for overall haemostasis appraisal [19]. They have been extensively validated in diverse populations, including patients with CAD [20,24,25,34]. In our study, OCP levels-indicating hypercoagulability-were comparable to other studies of patients with CAD [23,25] and significantly increased in patients with an elevated TyG index. Hence, our results corroborate that CAD confers a Disease Markers procoagulant state, which is especially pronounced in the CAD subpopulation with metabolic syndrome. Moreover, the association between TyG index and procoagulation was found to be independent of BMI. While clinical features of the metabolic syndrome (i.e., obesity, blood pressure, and metabolic dyslipidaemia) represent pivotal characteristics for the diagnosis and management of the condition, preclinical cardiometabolic biomarkers-such as the TyG index-may provide an independent, additional tool for early and meaningful detection of dysmetabolic derangements. Hence, our results suggest that the TyG index is an independent marker of both cardiometabolic and haemostatic derangements. In this respect, an independent association of the TyG index and a procoagulant state in patients after a recent myocardial infarction may confer an important starting point for future risk assessment strategies for patients with CAD. Limitations of our study primarily pertain to its realisation in a single centre and to its cross-sectional observational design and the study of surrogate biomarkers. Firstly, the single-centre nature of our study is somewhat counterbalanced by the fact that it was carried out at a national referral centre gathering a wide and varied patient population. In fact, the characteristics and risk factor presence in our participant group are similar to data from the literature, and hence, our results can be fairly generalized to the population of patients with CAD after a recent myocardial infarction. Secondly, the cross-sectional design can carry some measurement bias. Our measurements were in line with those reported in the literature for participants with CAD. Thirdly, we were able to include in the analysis only BMI as a marker of excess body fat. Including waist circumference or ectopic fat deposition would have added a deeper layer of understanding about the interplay between various components of the metabolic syndrome and coagulation markers. Fourthly, ours was a study to find an association; while it provides interesting and potentially important insights into the pathophysiological interplay between cardiometabolism and haemostasis in CAD, further prospective cohort studies are needed to establish a potential clinical relevance of our findings. Conclusions In conclusion, the TyG index represents an important and potentially relevant pathophysiological link between cardiometabolic and haemostatic derangements in patients with CAD. The TyG index is a reliable marker of metabolic syn-drome and has a strong correlation with a procoagulant state. Our findings suggest that metabolic syndrome may be an important driver of atherothrombotic risk in patients with CAD. Data Availability The data used to support the findings of this study are available upon request to the corresponding author. Disclosure The authors alone are responsible for the content and writing of the paper. Conflicts of Interest The authors declare no conflict of interest. Figure 1: (a) Correlation between triglyceride-glucose index and overall coagulation potential; (b) correlation between triglyceride-glucose index and fibrinogen. TyG: triglyceride-glucose index; OCP: overall coagulation potential.	2022-10-20T11:19:37.853Z	2022-10-20T00:00:00.000	{ "year": 2022, "sha1": "6d949dbbc6423f9f85ce9f219a2cfa1b0d28c921", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/dm/2022/6206802.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "2e1652e7cdf612142c40ef1c53dc99ee01ea8d11", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
228906270	pes2o/s2orc	v3-fos-license	Investigate Tourist Behavior through Mobile Signal: Tourist Flow Pattern Exploration in Tibet : Identifying the tourist ﬂow of a destination can promote the development of travel-related products and e ﬀ ective destination marketing. Nevertheless, tourist inﬂows and outﬂows have only received limited attention from previous studies. Hence, this study visualizes the tourist ﬂow of Tibet through social network analysis to bridge the aforementioned gap. Findings show that the Lhasa prefecture is the transportation hub of Tibet. Tourist ﬂow in the eastern part of Tibet is generally stronger than that in the western part. Moreover, the tourist ﬂow pattern identiﬁed mainly includes “(diverse or balanced) di ﬀ usion from the main center”, “clustering to the main center”, and “di ﬀ usion from a clustered circle”. is generally stronger than that of the western part in Tibet. The tourist ﬂow pattern identiﬁed for di ﬀ erent prefectures in Tibet mainly includes “(diverse or balanced) di ﬀ usion from the main center,” “clustering to the main center,” and “di ﬀ usion from a clustered circle.” In addition, future studies can be extended to other countries and regions to investigate tourist ﬂow patterns to promote sustainable development by balancing regional economic development. The present study has three limitations. First, positioning-related errors may exist through tracking tourist ﬂow by a mobile signal. In addition, the present study only tracked ﬂow patterns of tourists who used the China Unicom telecommunication service, but those tourists who used other mobile telecommunication companies were not tracked. Moreover, the present study only investigated tourist ﬂow patterns in each of the prefectures in Tibet, and tourist ﬂow patterns that cross di ﬀ erent prefectures were not considered. Hence, future research can track the ﬂow patterns of tourists who use di ﬀ erent mobile telecommunication companies and compare the di ﬀ erences in tourist ﬂow patterns who use di ﬀ erent mobile telecommunication companies. Future studies can further explore the di ﬀ erent preferences of tourists from di ﬀ erent countries or origins and examine tourist ﬂow patterns that cross regions to provide accurate implications for tourism practitioners regarding regional tourism development. Introduction Tourist flow refers to the spatial distribution of tourists, reflecting the travel patterns of tourists in a certain region [1]. Understanding the spatial distribution of tourist flows and the movement patterns of tourists can provide practical implications to tourism practitioners in terms of resource allocation, infrastructure construction, and effective tourism planning for a destination [2][3][4][5]. Tourist flow can also assist in the management of tourism's environmental and cultural impacts [4]. Cox found three movement patterns of humans; namely, distance-, direction-, and connection-based movement patterns [6]. The distance-biased movement pattern denotes the distance-related intensity of movement. The direction-based movement pattern reflects the direct movement of tourists. Last, the connection-biased movement pattern pays attention to the connection points during movement, reflecting the important role of connection in determining the characteristics of the movement. Oppermann revealed that "trip itineraries" can be used to reflect travel patterns and tourist flows. In addition, Oppermann proposed two categorizations of tourist movements; namely, tourist movement among different locations and tourists' stays in different locations [7]. Zhong, Zhang, and Li pointed out the disadvantages of conventional directional bias, such as the inability to identify the emitted and the attracted tourist flows, and proposed a new concept of "functional tourism region" [8]. Previous investigations of tourist flows are mostly based on scale. For example, Liu, Zhang, Zhang, and Chen categorized 31 provincial destinations in China into national and regional tourist centers and common and marginal destinations [9]. Jin, Xu, Huang, and Cao investigated tourist flows in different attractions in Nanjing, the inner city of China [10]. In summary, the identification of movement patterns of tourists seems to shift from complex to simple, and the investigation of the scale of the spatial distribution of tourists ranges from the country to the city level [2,11]. Familiarity with tourist flow can guide tourism practitioners to achieve effective and harmonious coordination in different areas of a destination and ultimately promote sustainable tourism development [9]. Tourist flows can also assist tourism practitioners in identifying the potential of a particular destination and promoting balanced tourism development. With the enhanced accessibility brought by transportation, tourist travel is no longer restricted to the most commonly recommended or popular tourist attractions. Tibet, a remote and autonomous region of China, has been developing rapidly because of enhanced accessibility and China's national development strategy of turning the plateau into a tourism destination [12]. Tourist arrivals and tourism revenues in Tibet have increased by more than 20% since the opening of the Qinghai-Tibet railway in 2006 [13,14]. In addition, the rapid development of the Internet increases Tibet's opportunities to connect with the outside world [5]. Hence, Tibet may further develop its tourism industry. As a large destination with different prefectures, the present study selected Tibet as a case to generate tourist flow patterns from a regional perspective, together with the detailed tourist flow patterns in each of its prefectures. Hence, Tibet is selected as a case to help tourism practitioners come up with corresponding strategic plans for a balanced and sustainable tourism development [12]. Although previous studies examined tourist movement patterns or categorized tourism destinations from a country or city level [9,10], the implications of the country-or city-level tourist flow is either very general or specific. The detailed implications of tourist flow to a regional-level perspective are also limited, to a certain extent. In addition, although previous studies explored tourists' movement patterns or itineraries, tourist inflows and outflows received limited attention. Knowledge of tourist inflows and outflows can greatly assist destinations in balancing regional tourism development and developing their tourism effectively. Hence, the present study uses the theory of social network analysis (SNA) to identify the tourist flows in each of Tibet's prefecture. Specifically, the present study aims to visualize tourist flow in Tibet and examine the differences among tourist inflows, outflows, and total tourist flow. Moreover, this study aims to summarize the patterns of tourism flows and provide practical implications for effective future tourism planning and development. Tourist Flow or Movement Investigations on tourist flow have begun since the late 1980s to 1990s, mainly focusing on economic impacts. For example, Japan received limited tourism inflows despite its massive tourism outflows. Buckley, Mirza and Witt indicated that further attention should be paid to the structural adjustment to attract foreign travelers to Japan and balance the payment of deficit items [15]. Kulendran adopted a cointegration analysis to estimate quarterly tourist flows to Australia from countries such as the US and Japan, to investigate the economic impacts of tourist flows. Moreover, Kulendran considered the factors affecting travel, such as income and airfare, and found that the estimated long-run elasticity of the relative price variable for the UK and Japan is close to unity but is greater than one for the US and New Zealand [16]. Jansen-Verbeke and Spee examined the interregional and intraregional tourist flows within Europe and found that competition does not exist among countries but among regions [11]. Coshall adopted spectral analysis to detect cycles within and between time-series datasets and found that the leading cycles of dependencies rely on tourist flows and exchange rates [17]. Dimoska and Petrevska considered the net flows of tourism services by analyzing the balance of payments in Macedonia and revealed possible ways to increase tourism inflows, such as attracting foreign tourists [18]. In addition, Patuelli, Mussoni, and Candela used an econometric model and found that regions with world heritage sites in Italy can enhance tourist inflows, considering all else being equal [19]. Degen examined nine main stations of the Beijing-Shanghai High-Speed Railway through SNA and identified the destination choice of tourists, spatial distribution, and travel time [1]. Then, Degen compared the time-space distribution of tourist flow and found that tourism origins-Beijing, Shanghai, and Nanjing-are strengthened as tourism centers, and tourism flow resulted in the "Matthew effect" [1]. Here, the "Matthew effect" reflects the cumulative advantage of main cities as tourism centers. Lew and McKercher [4] identified the factors that could influence the movement patterns of tourists and found that such patterns are influenced by four types of territorial and three linear path models. Specifically, the four types of tourist movement behaviors in a destination are "no movement," "convenience-based movement," "concentric exploration," and "unrestricted destination wide movement" [4]. They identified three movement pattern variations of tourists; namely, "point-to-point," "circular," and "complex patterns" [4]. Zhong et al. studied the overnight travel patterns of Chinese tourists and categorized tourists flow into pointing of tourist flow and inertial state of pointing [8]. Three basic types of pointing were identified, namely, "city," "seaside," and "sun-lust pointing." Based on the extended contemporary urban transportation model, Zhong et al. also identified the movement patterns of tourists to examine and enhance the attractiveness of a destination [5]. Data about tourist movement or flow in a tourist destination are not enough to provide detailed implications regarding tourism development in a destination. Hence, the flow patterns of tourists in a certain region must be explored further to provide valuable implications for sustainable tourism development. Theory of SNA and Its Extension in Tourism Diverse analytical methods are used to study tourists' movement patterns, including co-integration analysis, spectral analysis, and SNA [1,16,17]. The present study selects SNA because it can evidently reflect the nodes during the changes or movements of tourists, which provides an overall picture of the movement or flow patterns rather than the characteristics of individuals only [20]. The SNA concept appeared before the 1930s, and researchers gradually built the concept of social structure and recognized its importance as a social "fabric" and "web" [21]. Wasserman and Faust [20] noted that SNA examines the relationships among people, organizations, or other related information and is normally reflected as links or nodes to form a network. Otte and Rousseau also confirmed that SNA can be used to investigate social structures [22]. Shih investigated the travel routes of drive tourists from 16 destinations in Nantou, Taiwan, and provided the structural patterns of connected systems by applying SNA to tourism [23]. The findings show that necessary facilities should be provided to different locations in Nantou, Taiwan [23]. Leung et al. used SNA to examine 500 online trip diaries and found tourist movement patterns during the Olympics in Beijing in 2008. The findings revealed that international tourists are mostly interested in well-known traditional attractions, and their activities are within the central areas of Beijing [24]. Raisi, Baggio, Barratt-Pugh, and Willson analyzed 1515 tourism websites and visualized the network structure using SNA. They found that the SNA structure tends not to be hierarchical, and the communities tend to be formed on the basis of the geographical locations [25]. David-Negre, Hernández, and Moreno-Gil examined the spending pattern of tourists through SNA [26]. Jin et al. found and summarized three movement patterns of tourists; namely, "diffusion from a single center," "clustering to a single center," and "balancing between multiple centers" [10]. The literature has been constantly paying attention to the economic impacts of tourist flow since the 1990s. Most previous studies only investigated tourist flow at the country level, and detailed implications to a region or a destination are lacking to a certain extent. If the scale of the destination is small with few attractions, then movement patterns of tourists/tourist flow can be easily identified. By contrast, valuable practical implications can be provided for large-scale destinations by identifying the movement patterns of tourists or tourist flow, which is more difficult in this context than that of small-scale destination [27]. Although Lew and McKercher comprehensively indicated the movement patterns of tourists, the current study focused on patterns between the locations of tourists' accommodations and attractions [4]. Lew and McKercher listed different types of tourist movements and pointed out the factors, such as demand and supply and transportation networks [4]. However, empirical studies lack data on tourist flows, which can help visualize how different regions interact and compete with one another [19]. The present study visualizes and examines the tourist flow in different prefectures in Tibet to provide not only an overall trend of tourist flow but also the flow patterns of tourists in each of the prefectures. The objective is to promote sustainable tourism development and maximize tourism revenue. Methodology Most previous studies adopted questionnaire surveys or secondary data to explore tourist flows or patterns in a destination [19,26]. For instance, Zeng identified the characteristics of tourist flow of Chinese tourists visiting Japan by retrieving 430 travel itineraries from travel agencies in China and 458 itineraries of independent tourists from their trip diaries [28]. Nevertheless, the aforementioned traditional data collection methods, such as questionnaire surveys or secondary data, lack efficiency and completeness. Toha and Ismail discussed the applicability of various tracking technologies, such as global positioning system or land-based tracking technologies, to track the movement of tourists in historical cities of Melaka. However, an empirical investigation is still absent [29]. Hence, considering the shortages of traditional data collection, such as questionnaire surveys, the present study employed the concurrent data collection method through the mobile signal to obtain rich data to gain comprehensive information of tourist flow. Recently, along with the wide adoption of smartphones by tourists during travel, large volumes of user-generated data have become available to generate rich data [5]. The present study tracked the movements of tourists in Tibet by retrieving data via mobile phone signals of tourists. That is to say, mobile phone signal information from China Unicom telecommunication service was retrieved. In other words, for tourists who used the telecommunication service from China Unicom, their movements in Tibet were tracked. Specifically, once tourists entered Tibet, their mobile phone signals were identified and tracked through their points of entry. Similarly, once tourists left Tibet, points of exit were recorded. In the meantime, data were encrypted to protect the privacy of tourists, and the encryption assures that the personal information of tourists was kept confidential as no party can read or obtain this information. As a result, only their movements in Tibet were tracked and recorded. In summary, in October and November 2018, tourist inflows and outflows of different prefectures (including domestic and international tourists) in Tibet were retrieved. October was selected because China's National Day is in this month, which has a long holiday and is the peak season of tourism in Tibet. On the contrary, compared with October, November is the off-peak season for tourism in Tibet. Thus, the present study selected the representative data in October and November as an example to investigate the tourist flow pattern in peak and off-season in Tibet. The tourist flows of six regions (i.e., from west to east Tibet); namely, Ngari, Nagqu, Xigaze, Lhasa, Shannan, and Nyingchi prefectures, were retrieved based on the administrative regions in Tibet. Qamdo prefecture is not included because it received lower tourist flows compared with that in other prefectures of Tibet. Figure 1 depicts the directions of tourist inflows, outflows, and the total tourist flow, including the intensity of tourist flows, using software UCIENT and NETDRAW. The light blue circles represent 59 districts among six prefectures in Tibet, whereas the three dark blue circles signify tourist inflows, outflows, and the total tourist flow. The degree of the thickness of the lines represents the intensity of the tourist flow. The findings show that Lhasa prefecture (i.e., Tibet) is Tibet's transportation hub. In addition, Nagqu (i.e., Nagqu) and Xigaze (i.e., Samzhubze) prefectures are considered secondary transportation hubs. Furthermore, Nyingchi (i.e., Nyngchi) and Ngari (i.e., Gar) prefectures are supportive transportation hubs. Specifically, compared with other prefectures, the Lhasa prefecture received the most tourist flows, followed by Nagqu, Xigaze, Nyingchi, Shannan, and Ngari prefectures. The following sections will provide detailed information about the number of tourist inflows or outflows, present the number of total tourist flows in each prefecture in Tibet, compare the differences of tourist inflows and outflows, and identify the flow patterns of tourists. Specifically, compared with other prefectures, the Lhasa prefecture received the most tourist flows, followed by Nagqu, Xigaze, Nyingchi, Shannan, and Ngari prefectures. The following sections will provide detailed information about the number of tourist inflows or outflows, present the number of total tourist flows in each prefecture in Tibet, compare the differences of tourist inflows and outflows, and identify the flow patterns of tourists. Table 1 shows the number of tourist inflows and outflows and the total number of tourist flows in the Ngari prefecture in Tibet. Compared with other prefectures in Tibet, the Ngari prefecture has relatively fewer tourist flows. The Gar district is a central place that connects the northern and southern parts. In addition, no significant differences are found among tourist inflows, outflows, and the total number of tourist flows in the Ngari prefecture in October and November of 2018. Located in the western part of Tibet, the Ngari prefecture generally receives fewer tourists than its eastern parts. The tourist flow pattern in the Ngari prefecture is very simple which is indicated by "a three-point line." Hence, tourism practitioners should consider the selling point of the Ngari prefecture, market its attractions, further encourage the exploration of tourists, and increase its tourist flow in different parts of the Ngari prefecture gradually. Table 2 lists the number of tourist inflows and outflows and the total number of tourist flows of the Nagqu prefecture in Tibet. Nagqu generally plays a central role in connecting different parts of the Nagqu prefecture. Specifically, the Shuanghu district in the western part of the Nagqu prefecture receives fewer tourist flows than other areas in the Nagqu prefecture. In addition, tourist flows are concentrated in the central and eastern parts of Nagqu and are scattered in western parts, such as the Xainze district. Through paired samples t-test, significant differences (p = 0.041) are found between October 2018 and November 2018 regarding tourist inflows of the Nagqu prefecture. They are significant at the 95% confidence interval. Specifically, the number of tourist inflows of the Nagqu prefecture in October 2018 is higher than those in November 2018. This result indicates that, for the Nagqu prefecture, compared with November, tourists prefer to visit Nagqu in October. In other words, efforts can be made by the Tibet tourist bureau to attract more tourists to visit Tibet in November by creating special themes, as an example. Findings and Discussion Significant differences are found regarding tourist inflows of the Nagqu prefecture, whereas no significant difference is found between tourist outflows and the total number of tourist flow. The tourist flow of the Nagqu prefecture is characterized and reflected by the primary flows around the city center, along with the secondary flows between the core and the minor nodes (i.e., Nagqu-Amdo-Nyainrong; Nyainrong-Baqen-Nagqu). The tourist flow in Nagqu prefecture is generally indicated by the structure of "diffusion from the main center" and "clustering to the main center." Patuelli et al. [19] indicated that an increase in world heritage sites cannot only lead to a 4% increase in tourist inflows but also helps a certain region gain competitive advantages over other regions or districts. Hence, taking advantage of the Nagqu center and highlighting the appeal of attractions in nearby districts to extend the primary flows and increase the secondary flows can be considered. Lew and McKercher suggested that tourists can venture further as they become familiar with a region, thereby helping a destination increase secondary flows [4]. Table 3 reveals the number of tourist inflows and outflows and the total number of tourist flows in the Xigaze prefecture. The number of total tourist flow in the Xigaze prefecture indicates that the Samzhubze district has the highest tourist flow, whereas the Gamba district has the lowest number of tourist flows. In addition, the Samzhubze district acts as a central place connecting all the other districts in the Xigaze prefecture. Moreover, the tourist flows in the eastern and western parts are relatively the same. Through paired sample t-test, significant differences (p = 0.017) are found for tourist outflows in the Xigaze prefecture between October 2018 and November 2018. They are significant at 95% confidence. Regarding the total number of tourist flows of the Xigaze prefecture, the finding shows that the p value is 0.062 and is significant at the 90% confidence interval. Specifically, tourist outflows and the total number of tourist flows of the Xigaze prefecture in November 2018 is greater than those in October 2018. With the Tingri district as a center, tourist flows in the Xigaze prefecture are generally reflected by the primary flows among core nodes, the tertiary flows between the core and the minor nodes, and the normal moves among minor nodes. Thus, the Lhaze, Samzhubze, Bainang, and Tingri districts are the centers in western, central, eastern, and southern Xigaze, respectively. In summary, the tourist flow in the Xigaze prefecture is indicated by the structures of "balanced diffusion from the main center or diffusion from multiple centers." Although Lew and McKercher [4] indicated that certain tourists prefer time-efficient travel routes, the number of and the attractiveness of the attractions can effectively encourage tourists to extend their exploration in a certain region to achieve a balanced tourism development in different parts of the prefecture. Table 4 indicates tourist inflows and outflows and the total number of tourist flows in the Lhasa prefecture. The Chengguan district in the Lhasa prefecture can be regarded as a distribution center for tourists, whereas the Nyemo district receives the least tourists among all areas in the Lhasa prefecture. Moreover, tourist flows are concentrated in the Chengguan and Doilungdeqen districts and are scattered in three different directions (i.e., north, west, and eastern parts). Damxung, Quxu, and Maizhokunggar districts represent the North, West, and East, respectively. A paired sample t-test shows that significant differences (p = 0.094) exist between October 2018 and November 2018 regarding tourist outflows of the Lhasa prefecture. They are significant at the 90% confidence interval. Specifically, the number of tourist outflows of the Lhasa prefecture in November 2018 is greater than that in October 2018. Significant differences are generally found on the tourist outflows of the Lhasa prefecture, whereas no significant difference is found on tourist inflows of the Lhasa prefecture and the total number of tourist flows. In summary, the tourist flow of the Lhasa prefecture is reflected in the concentrated center with scattering in different directions. In other words, scenic tourist spots (i.e., Potala Palace and Jokhang Temple) in the city center play dominant roles in influencing the overall tourist flow. The tourist flow pattern in the Lhasa prefecture is indicated by the structure of "diffusion from the main center." The central tourist flow is similar to the findings of Leung et al. [24], that tourist activities were within the center area of Beijing during the Olympics in Beijing in 2008. However, the tourist flow pattern in Tibet has further expanded in different directions. Table 5 reveals the number of tourist inflows, outflows, and the total number of tourist flows in the Shannan prefecture. In general, the Nedong district is a central place connecting the western, southern, and eastern parts of the Nyingchi prefecture. This district also has the most tourist flows, whereas Comai has the lowest number of tourist flows. Furthermore, Nedong and its nearby districts receive more tourist flow, whereas the western, eastern, and southern parts receive less tourist flow. Paired sample t-test shows significant differences (p = 0.020) between tourist outflows and the total number of tourist flows (p = 0.015) of the Shannan prefecture from October to November 2018. They are significant at the 95% confidence interval. Specifically, tourist outflows and the total number of tourist flows in the Shannan prefecture in November 2018 is greater than that in October 2018. In general, significant differences are found between tourist outflows and the total number of tourist flows. The tourist flow in the Shannan prefecture is generally indicated by the structures of "clustering from the main center" and "diffusion from a clustered circle." The identified tourist flow is considered a relatively balanced tourist flow, reflecting the primary flows between core nodes and the secondary flows scattered in different directions. Contemporary urban transportation models assume that the majority of people will take the most efficient route in a tourist destination if possible [4,30]. However, the findings of the present study reflect that a region may achieve such a balanced tourist flow by considering the convenience of transportation and the attractiveness of the attractions. Thus, transportation is considered an important factor affecting the spatial distribution of tourist flow [4,31]. Table 6 shows tourist inflows and outflows and the total number of tourist flows in the Nyingchi prefecture. The Nyingchi district in the Nyingchi prefecture is a central place that connects the areas in three different directions in the Nyingchi prefecture. The Nyingchi district has the highest tourist flow, whereas Nangxian has the lowest tourist flow. Furthermore, Nyingchi and its nearby districts receive more tourist flows than the districts that are remote to the Nyingchi district. In other words, districts that are far away from the Nyingchi district receive less tourist flows than nearby districts. The paired sample t-test shows significant differences (p = 0.041) in tourist outflows in the Nyingchi prefecture between October 2018 and November 2018. They are significant at the 90% confidence interval. Specifically, tourist outflows of the Nyingchi prefecture in November 2018 were more than those in October 2018. Tourist flow is generally concentrated in the Nyingchi district, and the flow is scattered in the western and eastern parts. Gongbo'gyamda and Nangxian districts represent the western direction, and the Zayu district indicates the eastern direction. In contrast to other prefectures, tourist flow in the Nyingchi prefecture is different in attracting more tourist flow in the western part than that in the eastern part. Similar to the tourist flow pattern identified in the Lhasa prefecture, the tourist flow pattern in the Nyingchi prefecture is also indicated by the structure of "diffusion from the main center" but with less tourist flow directions. In conclusion, from a regional perspective, tourist flow in the western parts is lower than that in the eastern parts of Tibet. Ma and Wu found that the spatial structure of a destination is not in a state of equilibrium, and tourists tend to prefer the products in the eastern part of Xi'an, China [3]. The total number of tourist flows in different prefectures in November is generally more than that in October 2018. Jin et al. [10] stated that the tourist flow pattern is characterized by "diffusion from a single center," "clustering to a single center," and "balancing between multiple centers." Findings reveal that the tourist flow patterns of the Nagari, Lhasa, and Nyingchi prefectures mainly belong to the "(diverse) diffusion from the main center." The tourist flow pattern in Nagqu prefecture extends the identified tourist flow pattern by adding "clustering to the main center" to "diffusion from the main center." By contrast, Xigaze and Shannan prefectures reflect different tourist flow patterns despite what is identified by previous studies. The tourist flow pattern of the Xigaze prefecture is indicated by "a balanced diffusion from the main center or balancing between multiple centers," and that of the Shannan prefecture is reflected by "diffusion from a clustered circle." Furthermore, the tourist flow patterns in different prefectures in Tibet are characterized by primary, secondary, and tertiary flows [10]. Implications and Conclusions The present study uses the SNA theory to visualize tourist flow and specifically examine tourist inflows, outflows, and the total number of tourist flow, thereby identifying tourist flow patterns in each of the different prefectures in Tibet. The findings show that the Lhasa prefecture has the most tourist flow among other prefectures in Tibet. Specifically, the Lhasa prefecture attracts the largest number of tourist flow, followed by Nagqu, Xigaze, Nyingchi, Shannan, and Ngari prefectures. Similar to the concept of distance decay [32,33], the findings of the present study reveal that distance also plays a vital role in determining the amount of tourist flow in Tibet. The overall structure of the tourist flow pattern is spreading from the center to outer parts, and the tourist flow in eastern parts is stronger than that in western parts. Zhong et al. detected regional disparity and found that China's eastern economic belt continues to have tourism-related benefits [8]. The present study extends SNA by integrating tourist flow into the movement patterns of tourists to identify tourist flow pattern. The findings of the present study not only provide an overall picture of the tourist flow in a certain region (i.e., Tibet) but also indicate the detailed tourist flow pattern in each of the prefectures in Tibet. Furthermore, they contribute to the literature by providing tourist flow pattern from a regional perspective and extending the identification of the structures of tourist flow pattern identified by previous studies. The findings of the present study also provide valuable practical implications to tourism practitioners regarding the infrastructure construction of a certain region. Becken et al. [2] pointed out that the information about international visitor arrivals to New Zealand can provide sufficient information at a geographic level for infrastructure-related decision-making. The findings suggest that the Tibet tourism bureau must consider increasing the tourist flow in the western part to balance the development between eastern and western parts. The Ngari prefecture has the lowest tourist flow. Thus, tourist practitioners must come up with corresponding measures, such as infrastructure construction and transportation consideration, to attract more tourists. Among all different prefectures in Tibet, the Shannan and Xigaze prefectures reflect a relatively balanced tourist flow that helps promote healthy and sustainable tourism development. In other words, "balancing between multiple centers" can be considered to facilitate the balanced tourism and economic development of different areas in a region. In conclusion, although previous studies have identified either movement patterns or itineraries of tourists [4,5], considerations of tourist inflows and outflows are lacking. Hence, the present study identifies the inflows and outflows of tourists in Tibet based on the SNA to provide implications to balance its regional economic development and promote its sustainable tourism development. Tourist flows in different prefectures in Tibet are identified and analyzed by retrieving data generated by the mobile phone signal of China Unicom. The findings show that the Lhasa prefecture is the transportation hub of Tibet. Tourist flow in the eastern part is generally stronger than that of the western part in Tibet. The tourist flow pattern identified for different prefectures in Tibet mainly includes "(diverse or balanced) diffusion from the main center," "clustering to the main center," and "diffusion from a clustered circle." In addition, future studies can be extended to other countries and regions to investigate tourist flow patterns to promote sustainable development by balancing regional economic development. The present study has three limitations. First, positioning-related errors may exist through tracking tourist flow by a mobile signal. In addition, the present study only tracked flow patterns of tourists who used the China Unicom telecommunication service, but those tourists who used other mobile telecommunication companies were not tracked. Moreover, the present study only investigated tourist flow patterns in each of the prefectures in Tibet, and tourist flow patterns that cross different prefectures were not considered. Hence, future research can track the flow patterns of tourists who use different mobile telecommunication companies and compare the differences in tourist flow patterns who use different mobile telecommunication companies. Future studies can further explore the different preferences of tourists from different countries or origins and examine tourist flow patterns that cross regions to provide accurate implications for tourism practitioners regarding regional tourism development. Conflicts of Interest: The authors declare that they have no conflict of interest.	2020-11-05T09:09:18.587Z	2020-11-03T00:00:00.000	{ "year": 2020, "sha1": "b66a986e951397ebd98ea8ab6ace17ad45f914ec", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2071-1050/12/21/9125/pdf", "oa_status": "GREEN", "pdf_src": "ScienceParsePlus", "pdf_hash": "980f2bf4202c87dd618876fc128b1b68623d2eea", "s2fieldsofstudy": [ "Geography", "Business" ], "extfieldsofstudy": [ "Geography" ] }
67309989	pes2o/s2orc	v3-fos-license	Detection Hand Motion on Virtual Reality Mathematics Game with Accelerometer and Flex Sensors Montessori method is a learning method using props. One of the developments props is to use the game as a medium of learning. The examples Game media as learning is the use of Virtual Reality or VR Technology. By using the VR, players will be brought into the virtual world as if the player is in the real world. The weakness of the VR game is the limited interaction with the outside world. Interaction uses only buttons and joysticks. In this paper we use Flex sensor and accelerometer sensor to detect hand movements for VR mathematic game. The result is VR games are more interactive and interesting with hand motion. Introduction Montessori Method is a learning method using props in teaching [1].This method continues to development, one of them is using the game.Examples of games as a medium of learning is game 'berhitung', 'sinau boso jowo' and so on.The game is effective as a learning medium because the game involves players in the game [2]. Game technology has many developments, one of them is virtual reality technology.Virtual reality is a technology that describes a three-dimensional environment in which the 3D environment is simulated by computer [3].In Virtual Reality, players can interact with the environment. By using VR technology, the Montessori method will be more easily implemented because VR becomes a real learning tool.Players will be brought into the virtual world like in the real world.The player will be more easily accepted the value.This accordance with the level of the learning model on the Edgar Dal's pyramid.With doing the real thing, the lesson will be easier to remember.There is showing in the Figure 1 conical media learning Edgar dale.This figure provides an overview of the learning method according to Edgar Dal's pyramid [4].However.VR games are still a lot of weakness, which is this VR games has limited movement.This VR games depending on the head or the environment around the eye.If a player wants to go forward or backward, the player must use certain movements on his head like a bump to go forward or look up to retreat.This will reduce the appeal of play.Though according to Edgar Dale learning will be more remembered where player using all body movement. For this reason, hand motion sensor is implemented on the VR games interface.This hand motion sensor will detect hand movements.By using hand gestures into VR games, the VR games will be more interactive and learning will be easier to remember.Hand motion sensor use a flex sensor and accelerometer sensor ADXL 335. Related on Work Brenda, Frade uses VR games for Math learning.They use of VR games as a medium of learning mathematics is still limited to the use of VR head sheets and have not used another interface [5], [6].Barmpoutis uses Kinect sensors in learning.This method produces effective learning [7].While Yulia, Conn, and Lee use glove hand sensors in virtual 3D [8][9][10].they use accelerometer and gyroscope sensors to detect hand gestures.Tageldeen use it also, Tageldeen using accelerometer and gyroscope sensors on VR to detect hand movements.Tangeldeen uses it for body arm rehabilitation [11].However, applications they made do not use finger detection or flex sensors.Flex sensors are usually used to detect finger movements.Vibol uses finger movements to be brought into the virtual world.A sensor used Vibol very sensitive because based on pressure and frequency vibration [12].Motion sensor using the flex sensor and accelerometer sensor is widely used to moving the robot's hand.Flex sensor and accelerometer are connected with Arduino.Then, Arduino sends signals to robot hand through xbee, direct cable and RF signal [13][14][15]. Literature Review 3.1. Virtual Reality The term Virtual Reality (VR) was introduced by Jaron Lanier.At that time Lanier founded a company for VR business.Virtual Reality (VR) is a computer technology linked to the surrounding environment to connect with a simulation in a virtual world where it is a 3D image.VR is connected in real time and can connect with other users.In addition, this technology is pseudo-natural immersion or the user can move naturally, so the movement in the virtual world through the sensor components is just like the movement in the real world.Virtual Reality Elements [3]: a. Virtual world, i.e. the content of a given medium that can be either screen play or script.In this element, the user will experience the virtual world.b.Immersion, the sensation of existence in an environment.Immersion is divided into several types' i.e. mental immersion, physical immersion, and mentally immersion.c.Sensory feedback, which is information displayed to the senses of the user in the virtual world, sensory feedback can be visual, audio or touch.d.Interactivity, the virtual world that responds to user actions in real time. Flex Sensor The usual analog phenomena measured in the internal robot system relate to position, velocity, acceleration and inclination.From outside the robot system, the measurement is closely related to the determination of the robot coordinating position on the reference workspace [16].Figure 2 shows is the dimension of Flex Sensor.The following Figure 3 is the basic circuit of the Flex sensor. Flex Sensor is a flexible sensor that has a length of 4.5 inches.This bending sensor is patented by Spectra Symbol.This flexible sensor resistance changes when the metal pads are out of bending.Specification: 1. Temperature coverage: -35o C to + 80o C. ADXL 335 ADXL335 is a 3-dimensional motion/acceleration sensor with 13-bit resolution (213=8194 precision level) that can detect movement at range up to 16g (16 x 9.81 m/s 2 ~±157 m/s 2 ).This sensor is able to detect the tilt by monitoring changes in static pull and dynamic acceleration arising from movement and impact [17].Measurement method of ADXL 335 using rotation is illustrated in Figure 4. What is detected is the rotation of the sensor.It is not a shift of x, y, z but the rotation of: Φ-Roll-X rotation θ-Pitch-Y rotation Ψ-Yaw-Z rotation Research Method Figure 5 shows that there are 2 sensors applied i.e. flex sensor to detect the movement of fingers, and ADXL335 to detect the movement of the wrist.All sensors will be controlled by Arduino.Arduino will send it to the VR game application on android based mobile phone using Bluetooth.The Bluetooth application used is MH-04.Serial data will be captured by VR games.Engine Game applications utilize game engine unity. Results and Analysis The variation of flex sensor is compared with the output of digital data with flex sensor deflection position.Figure 6 shows Graph of voltage measurement comparison with flex sensor deflection.The result of accelerometer sensor testing can be shown in the following Figure 7, where the accelerometer output in digital data is compared with changes in 3 dimensions (x, y and z).The resulting measurement will be converted into digital data by Arduino, where the all sensors will be inputting on analog input in Arduino.Analog data will be converted into digital data and then sent to android smartphone via Bluetooth signal using component mh-04.The data are transmitted in serial form and the sampling data every 0.5 second.By android, the serial input data from Bluetooth is used as a hand drive in the VR game.The serial data is then converted to the existing layout position in the VR game.The weakness of accelerometer sensor only detects hand movement and does not detect the position of the hand.As a result, the hand sensor cannot follow the movement of the head or the eyes.To resolve this problem, the hand drawing on 3D will be made state following the head movement.The weakness of this app is hand gestures not really like the real state.Or in other words, the movement of the hand that can be detected under whereas the VR is on top.The settings for hand motion detection are as follows in Table 1.VR games are designed for math lessons in grade 1 elementary school children.The design of this math game is selecting the shape of the specified image, for example choosing the shape of the cube.The player will choose the shape of the displayed pictures.The player has 3 chances then the game ends.The player will get points for each correct answer.The appeared spatial forms are selected by hand.Figure 8 shows the VR game. Conclusion Real hand motion will control hand motion in virtual reality games.Hand movement is detected by using a sensor where the output is a different voltage.Variable voltage will be converted to digital by Arduino and then sent to VR game to control arm in 3D of VR world.Flex sensor to detect the movement of the finger while the accelerometer to detect hand motion.The use of the accelerometer only detects hand motion and does not detect the position of the hand, to resolve this problem; the hand drawing on virtual reality is made fixed Figure 5 . Figure 5. Design of motion sensor Figure 6 .Figure 7 . Figure 6.Graph of voltage measurement comparison with flex sensor deflection Figure 8 . Figure 8. Display on VR game Table 1 . Setup Motion Detection	2019-02-17T14:20:14.393Z	2018-10-01T00:00:00.000	{ "year": 2018, "sha1": "c2a1114a2ec52d4748251b73b64f095eaa456fb2", "oa_license": "CCBY", "oa_url": "https://zenodo.org/record/3982200/files/44%207594.pdf", "oa_status": "GREEN", "pdf_src": "ScienceParseMerged", "pdf_hash": "fa5e7dc17bae6d07cce24d41672a55d01b67d44b", "s2fieldsofstudy": [ "Computer Science", "Mathematics" ], "extfieldsofstudy": [] }
271104480	pes2o/s2orc	v3-fos-license	Research Progress on Ammonia Sensors Based on Ti3C2Tx MXene at Room Temperature: A Review Ammonia (NH3) potentially harms human health, the ecosystem, industrial and agricultural production, and other fields. Therefore, the detection of NH3 has broad prospects and important significance. Ti3C2Tx is a common MXene material that is great for detecting NH3 at room temperature because it has a two-dimensional layered structure, a large specific surface area, is easy to functionalize on the surface, is sensitive to gases at room temperature, and is very selective for NH3. This review provides a detailed description of the preparation process as well as recent advances in the development of gas-sensing materials based on Ti3C2Tx MXene for room-temperature NH3 detection. It also analyzes the advantages and disadvantages of various preparation and synthesis methods for Ti3C2Tx MXene’s performance. Since the gas-sensitive performance of pure Ti3C2Tx MXene regarding NH3 can be further improved, this review discusses additional composite materials, including metal oxides, conductive polymers, and two-dimensional materials that can be used to improve the sensitivity of pure Ti3C2Tx MXene to NH3. Furthermore, the present state of research on the NH3 sensitivity mechanism of Ti3C2Tx MXene-based sensors is summarized in this study. Finally, this paper analyzes the challenges and future prospects of Ti3C2Tx MXene-based gas-sensitive materials for room-temperature NH3 detection. Introduction NH 3 is a colorless, alkaline gas with a pungent odor that is now widely used in industry, agriculture, and other fields such as refrigerant and nitrogen fertilizer production.Excessive NH 3 emissions are closely related to the biological environment and the health of humans, such as the formation of acid rain and some human diseases [1,2].According to the Occupational Safety and Health Administration (OSHA), when the human body is exposed to 35 ppm of NH 3 for more than 15 min, this will endanger human health, and when the concentration of NH 3 reaches 500 ppm, people will suffer from acute toxicity, eye pain, shortness of breath, and other symptoms, as well as the risk of asphyxiation [3,4].In addition, NH 3 can be a marker gas for human diseases such as kidney, liver, or H. pylori infections [5], and NH 3 can be used for the early prevention of diseases and monitoring of the disease process via changes in the concentration of exhaled NH 3 in the human body [6]. A gas sensor is a sensitive device capable of converting the gas concentration that needs to be measured into an electrical signal.By analyzing the electrical signal, the sensor can obtain information such as the gas concentration present in the environment [7][8][9].In recent decades, traditional commercialized NH 3 gas-sensitive materials have focused on metal oxide semiconductors (MOS) such as SnO 2 , TiO 2 , WO 3 , ZnO, and other materials.Scientists have conducted many studies on them because they have high gas-response values [10].However, most of the MOS-based NH 3 sensors operate at temperatures above Sensors 2024, 24, 4465 2 of 22 100 • C, and the higher operating temperature and their consumption of large amounts of energy limit their applications at room temperature and the flexible wearable applications of NH 3 [11].Therefore, the development of a sensing material that can realize the real-time and efficient detection of NH 3 , which exhibits strong sensitivity and a rapid response and can work at room temperature, is urgently needed for production safety and human health [12]. Two-dimensional (2D) transition metal carbons and nitrides (MXenes) are emerging 2D materials with a graphene-like layered structure with the general formula M n+1 X n T x , where the "M" stands for the early transition metals, such as Ti, V, Ta element, etc.; the "X" is the C or N element; the "n" is 1, 2, or 3; and the "T" is the surface functional group, such as hydroxyl (-OH), oxygen (-O), fluorine (-F), chlorine (-Cl), and so on [13].MXenes were first discovered by Prof. Yury Gogotsi in 2011 [14].Their excellent electrical conductivity, large specific surface area, and rich surface functional groups that provide more active sites for gas adsorption and reaction have attracted widespread attention and led to their use in energy storage, photovoltaic devices, supercapacitors, and gas sensors [15][16][17].Ti 3 C 2 T x is one of the first and most extensively studied MXene materials [18].It has been shown both theoretically and experimentally that Ti 3 C 2 T x MXene has excellent NH 3 sensitivity at room temperature [19].Density functional theory (DFT) calculations have shown that the -F, -O, and -OH groups on the surface of Ti 3 C 2 T x MXene materials have high adsorption energies with NH 3 .It was also shown that Ti 3 C 2 T x MXene materials are effective at sensitizing gases to NH 3 [20,21]. However, it is the high adsorption energy between Ti 3 C 2 T x MXene and NH 3 that also leads to the resistance drift and long recovery time from the NH 3 environment to the air.NH 3 sensors based on pure Ti 3 C 2 T x MXene often exhibit poor selectivity, slow response and recovery times, and low response values [22,23].So, in order to obtain an excellent NH 3 -sensitive performance at room temperature, scientists have paid a lot of attention and conducted a lot of research focused on the choice of synthesis method, composite modification, and systematic sensitization mechanism of Ti 3 C 2 T x MXene-based ammonia sensors. In this context, this paper begins with Ti 3 C 2 T x MXene synthesis methods, provides a classified overview of the most recent research progress in Ti 3 C 2 T x MXene synthesis, which is primarily classified as HF etching, in situ HF etching and alkali solution etching, and summarizes the benefits and drawbacks of the various etching techniques.In addition, several types of composites, including metal-oxide semiconductors, conductive polymers, and 2D material composites, are briefly discussed in order to enhance the NH 3 -sensitive behavior of NH 3 sensors based on pure Ti 3 C 2 T x MXene.These Ti 3 C 2 T x MXene composites are further analyzed to prove that the primary mechanisms of the enhanced NH 3 -sensing performance of these sensors are the synergistic effects between the composites, such as the formation of a heterojunction of the composites, adsorption energy and charge transfer, chemical sensitization and electron sensitization, and the increase in specific surface area and adsorption sites due to their unique morphology [24,25]. Finally, this paper reviews the research progress regarding the sensitization mechanism of NH 3 sensors based on Ti 3 C 2 T x MXene, then summarizes and discusses the two current sensitization mechanisms that can explain the p-type response of MXene to either electron acceptor or electron donor gases: (1) MXene is metallic in nature, and the number of carriers decreases and the electrical resistance increases after the gas adsorption.When they exhibit semiconductor properties, the charge transfer mechanism follows the Wockenstein model [26][27][28].(2) When the MXenes are exposed to the gas, the interlayer expansion hinders electron transfer and the resistance increases [16].Finally, the paper summarizes the key challenges and potential paths forward for NH 3 sensors based on Ti 3 C 2 T x MXene.We expect that this review will provide novel ideas for the development of high-performance, room-temperature Ti 3 C 2 T x MXene NH 3 sensors. Synthesis of Ti 3 C 2 T x MXene The majority of Ti 3 C 2 T x MXene materials are produced by selectively etching the precursor MAX phase (a hexagonal layered structure with the common structural formula M n+1 A n X n , where the "n" = 1, 2, or 3; examples of such materials are Ti 2 AlC, Ti 3 AlC 2 , Ti 2 SiC, Ti 4 AlN 3 , etc.; the "A" is a group IIIA or IVA element [29,30]).The A atomic layer is more easily etched away during the reaction with the etchant, leaving a 2D layered structure with alternating M and X layers because the M-A bonds in the MAX phase are stronger than the M-X bonds in terms of bond strength. The functional groups -OH, -O, and -F in the etching system are easily attached to the surface of this alternating layer structure, eventually forming a 2D layered structure, Ti 3 C 2 T x MXene [31,32].Ti 3 AlC 2 is most used in the MAX phase, as shown in Figure 1 Sensors 2024, 24, 4465 3 of 23 Synthesis of Ti3C2Tx MXene The majority of Ti3C2Tx MXene materials are produced by selectively etching the precursor MAX phase (a hexagonal layered structure with the common structural formula Mn+1AnXn, where the "n" = 1, 2, or 3; examples of such materials are Ti2AlC, Ti3AlC2, Ti2SiC, Ti4AlN3, etc.; the "A" is a group IIIA or IVA element [29,30]).The A atomic layer is more easily etched away during the reaction with the etchant, leaving a 2D layered structure with alternating M and X layers because the M-A bonds in the MAX phase are stronger than the M-X bonds in terms of bond strength. The functional groups -OH, -O, and -F in the etching system are easily attached to the surface of this alternating layer structure, eventually forming a 2D layered structure, Ti3C2Tx MXene [31,32].Ti3AlC2 is most used in the MAX phase, as shown in Figure 1, which presents a schematic diagram of the etching process of Ti3AlC2 by HF, where the Ti3AlC2 structure consists of a single Ti3C2 layer separated by Al atoms.The Al atom layers between the Ti3AlC2 layers are eliminated by the HF treatment, and the subsequent loss of metallic bonds causes the individual Ti3Al2C layers to separate, producing Ti3C2Tx.The reaction process is shown in Equations ( 1)-( 3): (1) The substitution of the -F functional group represented by Equation ( 3) is similar to that of -OH; therefore, its reaction mechanism can be considered to regard -OH in Figure 1 as -F.The different etching methods and the choice of etchant can change the types and ratios of the functional groups at the end of the Ti3C2Tx MXene material, thus affecting the adsorption behaviors of Ti3C2Tx MXene and NH3 and influencing the NH3-sensitive performance of the sensors.In the current study, most Ti3C2Tx-based NH3-sensing materials are synthesized using HF etching and in situ HF etching.In addition, there are a small number of research papers that mention the alkaline solution etching method.The substitution of the -F functional group represented by Equation ( 3) is similar to that of -OH; therefore, its reaction mechanism can be considered to regard -OH in Figure 1 as -F. The different etching methods and the choice of etchant can change the types and ratios of the functional groups at the end of the Ti 3 C 2 T x MXene material, thus affecting the adsorption behaviors of Ti 3 C 2 T x MXene and NH 3 and influencing the NH 3 -sensitive performance of the sensors.In the current study, most Ti 3 C 2 T x -based NH 3 -sensing materials are synthesized using HF etching and in situ HF etching.In addition, there are a small number of research papers that mention the alkaline solution etching method.Therefore, this chapter will briefly describe these three etching methods and their effects on ammonia gas-sensing. HF Etching HF etching is a useful approach for preparing Ti 3 C 2 T x MXene, which can be created by etching the MAX phase.In 2011, Naguib et al. [14] prepared the first 2D Ti 3 C 2 phase MXene by etching Ti 3 AlC 2 powder with high concentrations of HF. Figure 2a shows an HFtreated SEM image of Ti 3 AlC 2 , revealing that the Ti 3 C 2 T x basal plane is accordion-like with a fan-like dispersion.The energy band structure of the synthesized MXene (Ti 3 C 2 ) terminated with the −OH and −F end groups was calculated, and the results displayed that the mechanism of the electrical conductivity of the Ti 3 AlC 2 material changed from metallic to semiconducting after etching due to a change in its surface chemistry (Figure 2b).Due to the simplicity of the HF etching method, it remains one of the most commonly used etchants for synthesizing Ti 3 C 2 T x MXene.Therefore, this chapter will briefly describe these three etching methods and their effects on ammonia gas-sensing. HF Etching HF etching is a useful approach for preparing Ti3C2Tx MXene, which can be created by etching the MAX phase.In 2011, Naguib et al. [14] prepared the first 2D Ti3C2 phase MXene by etching Ti3AlC2 powder with high concentrations of HF. Figure 2a shows an HFtreated SEM image of Ti3AlC2, revealing that the Ti3C2Tx basal plane is accordion-like with a fan-like dispersion.The energy band structure of the synthesized MXene (Ti3C2) terminated with the −OH and −F end groups was calculated, and the results displayed that the mechanism of the electrical conductivity of the Ti3AlC2 material changed from metallic to semiconducting after etching due to a change in its surface chemistry (Figure 2b).Due to the simplicity of the HF etching method, it remains one of the most commonly used etchants for synthesizing Ti3C2Tx MXene.The specific surface area of Ti3C2Tx MXene has a great influence on its gas-sensitive properties.The gas-sensitive properties can be improved by increasing the specific surface area of Ti3C2Tx Mxene through intercalation and layering.For the further intercalation and delamination of Ti3C2Tx obtained by HF etching, Lian et al. [33] reported a method involving 2D Ti3C2Tx nanosheets synthesized by intercalation and a delamination reaction in tetramethylammonium hydroxide (TMAOH) solution after dilute HF etching.They added a quantitative amount of Ti3AlC2 powder to a 5% concentrated HF solution, which was magnetically stirred at room temperature for 24 h.Then, the suspension was washed with deionized water until the pH of the supernatant was 5-6 and centrifuged.After that, the precipitate was put in a 25% solution of tetramethyl hydroxide (TMAOH) and The specific surface area of Ti 3 C 2 T x MXene has a great influence on its gas-sensitive properties.The gas-sensitive properties can be improved by increasing the specific surface area of Ti 3 C 2 T x Mxene through intercalation and layering.For the further intercalation and delamination of Ti 3 C 2 T x obtained by HF etching, Lian et al. [33] reported a method involving 2D Ti 3 C 2 T x nanosheets synthesized by intercalation and a delamination reaction in tetramethylammonium hydroxide (TMAOH) solution after dilute HF etching.They added a quantitative amount of Ti 3 AlC 2 powder to a 5% concentrated HF solution, which was magnetically stirred at room temperature for 24 h.Then, the suspension was washed with deionized water until the pH of the supernatant was 5-6 and centrifuged.After that, the precipitate was put in a 25% solution of tetramethyl hydroxide (TMAOH) and Sensors 2024, 24, 4465 5 of 22 magnetically agitated for another 24 h to allow for more intercalation and delamination.Afterward, the precipitate was centrifuged to obtain the two-dimensional, less-layered Ti 3 C 2 T x nanosheets.Figure 2c displays the XRD spectra of Ti 3 AlC 2 and Ti 3 C 2 .After HF etching, the (104) diffraction peak of Ti 3 AlC 2 at 2θ ≈ 39.1 • almost disappeared, which indicated that the Ti 3 C 2 was completely stripped [34,35].The SEM image of the Ti 3 C 2 nanoflakes intercalated with TMAOH is shown in Figure 2d.A high level of delamination is indicated by the two-dimensional structure, semi-transparent, and wrinkled textures of the Ti 3 C 2 nanoflakes.In future research, we can increase the specific surface area of Ti 3 C 2 T x MXene-sensitive materials through the intercalation and delamination of Ti 3 C 2 T x to increase the adsorption sites on the surface of the materials to promote their adsorption and gas reaction with ammonia molecules, ultimately aiming to achieve the purpose of improving the sensitivity of Ti 3 C 2 T x MXene. In-Situ HF Etching Although the HF etching process is easy to operate, it has obvious disadvantages, such as a longer etching time and a more dangerous operation [36].Furthermore, the Ti 3 C 2 T x MXene obtained by this process has more shortcomings: HF is dangerous to humans and the environment, and fluorine-containing functional groups are inert, degrading their properties.In 2014, Ghidiu et al. [37] presented a method to synthesize Ti 3 C 2 T x MXene by etching the Ti 3 AlC 2 phase in situ with LiF and HCl solution.Specifically, LiF was added to a 6 M HCl solution and stirred to dissolve before slowly adding Ti 3 AlC 2 powder and maintaining this at 40 • C for 45 h.After that, the mixture was washed until the pH of the supernatant was 6.The obtained deposit formed a clay-like paste, which was rolled between the roller mill's permeable membranes to produce pliable, freestanding thin films within a few minutes.When diluted, it can be applied as an ink to deposit or print MXene on various substrates. During the reaction process, Li + can spontaneously insert into the interlayer of Ti 3 C 2 T x MXene, and due to the hydrophilicity of Ti 3 C 2 T x MXene, water molecules can easily enter the interlayer, leading to a much larger lattice parameter for Ti 3 C 2 T x MXenes obtained by LiF/HCl etching than for Ti 3 C 2 T x MXenes obtained by HF etching.This process produces large-component monolayer Ti 3 C 2 T x flakes with a high yield, large lateral size, and excellent quality.More importantly, using LiF and HCl to etch MAX avoids the use of concentrated HF, which is very corrosive and poisonous, while also reducing the nanoscale defects generated by direct etching with HF.Furthermore, the simplicity of this method contrasts with previous films produced through laborious insertion, layering, and filtration techniques [38].It is noteworthy that MXenes can still be obtained when H 2 SO 4 is used instead of HCl.However, fine-tuning the etching reagent might affect the surface chemistry or the intercalation ions, which needs more exploration.Currently, in situ etching by LiF and HCl solutions is widely used in the synthesis of Ti 3 C 2 T x MXene materials. Han et al. [39] compared the ammonia sensitivity of Ti 3 C 2 T x MXene materials synthesized by HF etching and LiF/HCl etching.They found that the Ti 3 C 2 T x materials prepared by the LiF/HCl etchant had better gas sensitivity than that of HF etchant.They could detect a wide range of NH 3 at room temperature with higher sensitivity and stability.The higher sensing performance of the Ti 3 C 2 T x MXene materials obtained by the LiF/HCl etching method was analyzed to be due to the high ratio of -O and -OH functional groups on the surface of Ti 3 C 2 T x materials prepared by this method. Alkali Solution Etching Yang et al. [40] improved the humidity and NH 3 -sensing properties of organ-like Ti 3 C 2 T x MXene synthesized from HF solution via alkalization.Organ-like Ti 3 C 2 T x MXene is a class of Ti 3 C 2 T x MXene materials that can provide a fast pathway for charge and ion transfer, thereby preventing the reduction in specific surface area due to the restacking of layers.Firstly, Ti 3 C 2 T x MXene powder was prepared through HF acid etching using Ti 3 AlC 2 and a 45 wt% HF solution.Subsequently, the obtained Ti 3 C 2 T x MXene powder was put into a 5 M NaOH solution and subjected to continuous magnetic stirring at room temperature for 2 h, and the alkalized Ti 3 C 2 T x powder was finally obtained.The alkali treatment embeds Na + in the interlayer of Ti 3 C 2 T x , which plays a crucial role in regulating water for humidity sensing.During stage 1 of Figure 3, several water molecules are coupled with a single Na + ion, forming a stable [Na(H 2 O) m ] + cluster structure.Meanwhile, in stage 2 of Figure 3, the alkaline treatment enhances the N-Ti bonding sites due to the increased -O terminus, resulting in higher NH 3 adsorption.The alkalized Ti 3 C 2 T x devices have an improved sensing performance for humidity and NH 3 compared to non-alkalized Ti 3 C 2 T x , due to the embedding of Na + and the higher oxygen-to-fluorine atomic number ratio ([O]/[F]), and have the opposite response signal.materials using only alkali etching, this method allows for the preparation of Ti3C2Tx MXene with a large number of oxygen-containing functional groups, is simple to perform, and does not contain inert groups -F.Therefore, it is expected to be applicable to the synthesis of Ti3C2Tx MXene-based ammonia-sensitive materials in the future and to obtain sensitive materials with a strong ammonia-adsorption capacity. Ti3C2Tx MXene synthesized by conventional fluoride-containing methods usually uses fluoride ion-containing solutions as an etchant, which is harmful to the environment.In addition, highly reactive fluoride ions can penetrate into the human body and cause fatal damage to body tissues [41,42].It has been demonstrated that fluoride-based etching procedures generate a large number of -F-terminal functional groups [43,44], which substantially limit charge transfer and diminish chemically active sites, hence compromising the electrochemical characteristics of Ti3C2Tx MXene [45][46][47].Therefore, modulating the terminal functional groups of Ti3C2Tx MXene can affect its electrochemical properties and NH3 sensitivity [48].Furthermore, the popular HF and LiF/HCl etching methods are typically time-consuming [49].Therefore, it is important to develop environmentally friendly and F-free synthesis routes for NH3 sensor-based Ti3C2Tx MXene in the future.Although there is no report on the synthesis of Ti 3 C 2 T x MXene ammonia-sensitive materials using only alkali etching, this method allows for the preparation of Ti 3 C 2 T x MXene with a large number of oxygen-containing functional groups, is simple to perform, and does not contain inert groups -F.Therefore, it is expected to be applicable to the synthesis of Ti 3 C 2 T x MXene-based ammonia-sensitive materials in the future and to obtain sensitive materials with a strong ammonia-adsorption capacity.Ti 3 C 2 T x MXene synthesized by conventional fluoride-containing methods usually uses fluoride ion-containing solutions as an etchant, which is harmful to the environment.In addition, highly reactive fluoride ions can penetrate into the human body and cause fatal damage to body tissues [41,42].It has been demonstrated that fluoride-based etching procedures generate a large number of -F-terminal functional groups [43,44], which substantially limit charge transfer and diminish chemically active sites, hence compromising the electrochemical characteristics of Ti 3 C 2 T x MXene [45][46][47].Therefore, modulating the terminal functional groups of Ti 3 C 2 T x MXene can affect its electrochemical properties and NH 3 sensitivity [48].Furthermore, the popular HF and LiF/HCl etching methods are typically time-consuming [49].Therefore, it is important to develop environmentally friendly and F-free synthesis routes for NH 3 sensor-based Ti 3 C 2 T x MXene in the future. Ti 3 C 2 T x MXene-Based Nanocomposites Material Gas Sensors for NH 3 Ti 3 C 2 T x Mxenes are a typical new 2D material with high selectivity for ammonia at room temperature.Dillon, Lipatov et al. [35,50] measured the high electrical conductivity of Ti 3 C 2 T x MXene to be 6500 S cm −1 and 4600 ± 1100 S cm −1 .Halim et al. [51] have also reported that Ti 3 C 2 films exhibit metal conductivity at a temperature of about 100 K. Ti 3 C 2 T x MXenes is considered to have good application prospects for ammonia detection at room temperature due to its excellent electrical conductivity, unique structure, and the large number of adsorption sites provided by functional groups such as -O and -OH on the surface [52][53][54].Due to the low response values, serious base-resistance drift, and poor stability of pure Ti 3 C 2 T x MXenes when utilized as ammonia-sensing materials, it is essential to combine Ti 3 C 2 T x MXene with other sensitive materials to enhance their sensing capabilities [25,55,56].In this chapter, a brief overview will be provided of the most-researched methods of compositing several sensitive materials with Ti 3 C 2 T x MXenes to improve their sensing performance.These methods include combining them with MOS materials, conductive polymer materials, and certain 2D materials. Metal Oxide Modification MOS materials such as TiO 2 , SnO 2 , In 2 O 3 , WO 3 , and Ti 3 C 2 T x MXene usually use synergistic effects such as the Fermi energy level effect (Fermi level bending, carrier separation, depletion layer regulation, and increase in interfacial barrier energy) between the composites, the formation of heterojunctions, and the specific morphology, etc., to enhance the gas-sensitive performance of the sensors [57][58][59].In particular, for TiO 2 /Ti 3 C 2 T x MXene nanocomposites, in addition to the introduction of TiO 2 material in the matrix, due to the instability of Ti 3 C 2 T x MXene, TiO 2 can be synthesized through partial oxidization to Ti 3 C 2 T x MXene, leading to in situ derivatization using techniques such as high-temperature sintering. When a semiconductor material is irradiated by UV light whose optical energy is larger than its band gap, photocarrier pairs are generated at the interface of the sensitive material [60,61], and the additionally generated photogenerated electrons and holes can promote the adsorption and desorption processes and redox reactions of gases, thus improving the response value and shortening the response recovery time of the sensor [62,63].Therefore, using the UV irradiation of gas sensors to obtain highly NH 3 -sensitive Ti 3 C 2 T x MXene-based sensors operating at room temperature is a feasible strategy. To overcome the limitations of Ti 3 C 2 T x MXene as a room-temperature ammonia gas sensor, such as limited its sensitivity and selectivity, Zhang et al. [64] proposed growing TiO 2 on Ti 3 C 2 T x MXene in situ.They also utilized UV light (the light energy of the used 365 nm UV light source is 3.4 eV, which is larger than the band gap 1.6 eV of (001)TiO 2 /Ti 3 C 2 T x ) to boost the performance of the (001) TiO 2 /MXene heterostructure ammonia gas sensor.The Ti 3 C 2 T x powder was prepared by removing the Al layer from MAX (Ti 3 AlC 2 ) with a 50% HF solution.NaBF 4 (0.1 mol/L, 8 mL) was introduced as a control agent for the crystal surface in the HCl solution of Ti 3 C 2 T x .The mixture was then hydrothermally treated at 160 • C for 8, 12, 16, and 32 h.The hydrothermal reaction promoted the formation of (001) planes during the crystal growth process, converting Ti in Ti 3 C 2 into hydrated Ti 3 + ions and binding F ions to the (001) planes with the aid of NaBF 4 as an inducer.As shown in Figure 4a, the T-T-12 h TiO 2 /MXene sensor that undergoes a hydrothermal reaction for 12 h is more sensitive to ammonia, with a lower detection limit than that of the pure Ti 3 C 2 T x -based sensor, and also exhibits good durability in terms of its response/recovery time, repeatability, and selectivity.Analyzing the energy band diagram schematic of the T-T-12 h TiO 2 /MXene-based sensor demonstrated in Figure 4c, it can be concluded that the enhanced gas-sensitive performance of NH 3 is obtained because Ti 3 C 2 T x greatly facilitates the separation of electron-hole pairs by storing holes through the Schottky junctions formed at the interfaces with TiO 2 , which enhances the ammonia-sensing performance.In Figure 4d, we can see that adding UV light to the highly active (001) crystalline TiO 2 makes the charge separation of the T-T-12 h TiO 2 /MXene sensor when exposed to UV light even better, which leads to a better gas-sensing performance.Figure 4b also shows a two-fold increase in sensitivity compared to when UV is absent.The TiO 2 /MXene sensor type T-T-12 h exhibited 34 times more sensitivity to 30 ppm of ammonia compared to pristine Ti 3 C 2 T x .Density functional theory reveals that the (001) side of the TiO 2 and Ti 3 C 2 T x composite has the greatest attraction in terms of ammonia adsorption [64]. To further improve the sensing performance of pure Ti To further improve the sensing performance of pure Ti3C2Tx MXene for NH3, Kan et al. [65] combined surface-functionalized In2O3 nanotubes with Ti3C2Tx nanosheets.The composite was further loaded onto thermoplastic polyurethane (TPU) foam, a dualfunctional sensing platform constructed based on Ti3C2Tx/In2O3 nanocompositesand modified TPU foam sensors.The produced nanocomposites exhibited an improved ammonia-sensing performance and pressure-sensitive properties, utilizing the strong synergistic effect of the sensing of In2O3 nanotubes and high conductivity of Ti3C2Tx nanoflakes, as well as the foam substrate's pressure-sensitive and gas-permeable capabilities.Ti3C2Tx nanoflakes are negatively charged on the surface due to the terminal functional groups such as -OH and -F.At this time, the modification of In2O3 with the cationic surfactant (3-aminopropyl) triethoxysilane (APTES) can make the surface of In2O3 positively charged, and the two can be compounded to obtain Ti3C2Tx/In2O3 composites via simple ultrasonication with electrostatic adsorption, which was shown to have higher NH3 response values than pure Ti3C2Tx nanoflakes.In2O3 is an n-type sensing material, which has a bandgap in the range of 3.55-3.75eV [66] and a work function of about 4.28 eV [67]; Ti3C2Tx MXene behaves as a p-type sensing material, which has a bandgap of 0.19 eV [68] and a work function of about 4.5 eV [69].Two main factors are used to investigate the enhanced sensing ability: First, n-type In2O3 has a high Fermi energy level, and electrons will be transferred from the In2O3 nanotubes to the Ti3C2Tx nanoflakes until the Fermi energy levels of the two reach equilibrium, thus forming a space charge layer at the Ti3C2Tx and In2O3 interface.At the same time, the energy bands at the interface are bent on both sides, generating a potential barrier that increases the base value resistance of the sensor, thus increasing the gas response.Second, the In2O3 nanotubes work as isolators to inhibit the re-stacking of Ti3C2Tx nanoflakes, thereby increasing the layer space and effectively facilitating the diffusion and permeation of gases in the sensing layer.In addition, the sensor achieves the flexible and interference-free detection of complex exhaled environments at room temperature, with a memory function for detecting NH3 gases down to 1 ppm, realizing the dual-mode detection of NH3 gases.To address the limited sensitivity of Ti 3 C 2 T x MXene gas sensors, Guo et al. [70] reported a Ti 3 C 2 T x /WO 3 composite resistive sensor that had excellent NH 3 sensitivity at room temperature.Ti 3 C 2 T x /WO 3 composites with WO 3 nanoparticles anchored on Ti 3 C 2 T x nanosheets were prepared using an ultrasonic technique.As demonstrated in Figure 4e,f, the detection sensitivity of the Ti 3 C 2 T x /WO 3 -50% sensor was up to 22.3%, which was 15.4 times higher than that of the pure Ti 3 C 2 T x sensor, at 1 ppm NH 3 at room temperature.The improved NH 3 -sensing performance is mainly attributed to the increase in the aspect ratio of the composite, the increase in the active sites provided by WO 3 , and the more effective charge transfer bestowed by the formed heterojunction (Figure 4g).It was demonstrated that the Ti 3 C 2 T x /WO 3 -50% sensor remained effective over a wide range of relative humidity (RH) conditions (3.5%~72.9%RH), and low concentrations of NH 3 were still detected even at high humidity.As RH increases, the sensor response to NH 3 sensing gradually decreases.Humidity compensation methods, which are often used in practical applications, can help to solve the humidity effect's limitations. Table 1 outlines the gas-sensitive properties of different MOS/Ti 3 C 2 T x MXene composites towards NH 3 at room temperature in recent years.The acronym "LOD" in the table refers to "the limit of detection". Conductive Polymer Addition Conducting polymers, such as polyaniline (PANI), polypyrrole (PPy), polydioxothiophene (PEDOT), and their derivatives, possess the advantages of high flexibility, the ability to work at room temperature, and a high response to ammonia.As a result, they are receiving increasing attention for their use in NH 3 sensing at room temperature [77,78].To address the issues of Ti 3 C 2 T x MXene resistance drift, the long recovery time, the low response value, and susceptibility to mechanical deformation, it has been proposed to hybridize Ti 3 C 2 T x MXene with conductive polymers.This approach aims to enhance the sensitivity and wearable performance at room temperature of Ti 3 C 2 T x MXene through the heterojunctions formed between the two materials and the high flexibility of the conducting polymers [79,80]. Polyaniline (PANI) has been widely used in room-temperature ammonia sensors due to its strong electrical conductivity, superior selectivity to ammonia, and room-temperature workability [81,82].Yang et al. [82] used electrostatic spinning to combine polyaniline with Ti 3 C 2 T x nanosheets to construct polyaniline/Ti 3 C 2 T x composite nanofibers with excellent ammonia responsiveness for room-temperature flexible ammonia sensors.The SEM image in Figure 5a demonstrates that the synthesized Ti 3 C 2 T x exhibits a distinct layer structure, and the Tyndall phenomenon can be seen in the inset.Figure 5b is an SEM image of PANI/Ti 3 C 2 T x , which reveals a 3D network fiber structure with random fiber orientation.Figure 5c shows that the PANI/Ti 3 C 2 T x flexible sensor has a greater NH 3 -sensing response at 25 • C (2.3 times the response value at 20 ppm compared to pure PANI), with good selectivity, repeatability, and long-term stability.Figure 5f demonstrates that by adjusting the bending angle (0 • -150 • ) and the number of bending times (up to 3200), a high sensing performance at 20 ppm NH 3 and enduring flexible bending stability can be attained.PANI is a conductive p-type semiconductor with a band gap of about 2.47 eV [83]; the resistance of the pure Ti 3 C 2 T x sensor, exhibiting a metallic nature, was tested to be about 24 Ω [79], and the Ti 3 C 2 T x band gap was reviewed in the literature to be about 0.19 eV [68].In the UPS results of the composites in Figure 5d,e, the figures of merit for Ti 3 C 2 T x and PANI/Ti 3 C 2 T x composite nanofibers are 2.99 eV and 3.44 eV, respectively (UV is He I, 21.22 eV).Therefore, the formation of the Schottky junction at the interface between Ti 3 C 2 T x and PANI enhances the resistance modulation of the flexible sensor.The gas sensitivity of the PANI/Ti 3 C 2 T x flexible sensor was shown to be enhanced because the composite of the PANI and Ti 3 C 2 T x nanosheets constitutes a Schottky junction, the degree of PANI protonation is increased, and the two are hybridized to form a special three-dimensional reticulated fibrous structure, which has great potential for the construction of flexible ammonia gas sensors. In addition, electrostatic spinning has been widely used in conductive polymer sensors as an easy, efficient, low-cost nanofiber preparation technique.Compared with the traditional synthesis method, the electrostatic spinning method can greatly increase nanomaterials' specific surface area volume ratio [84].By integrating polymers with nanofiber structures into resistive gas sensors, porous materials with a high surface area and large porosity can be prepared to enhance the adsorption of ammonia molecules by the sensors and improve the sensitivity of the sensors, as well as to further optimize the properties of the polymer-sensitive materials, such as their hydrophobicity, conductivity, and flexibility [85,86].In addition, these sensors also showed fast response and recovery times.Zhang synthesized polyaniline and polymethyl methacrylate (PMMA) nanocomposite fibers using electrostatic spinning [87].This sensitive material was shown to have a fast response and high sensitivity to NH 3 , with a sensitivity of 4.5% to 5 ppm NH 3 and a response/recovery time of about 5 s/12 s.On this basis, through the composite of different conductive polymer materials and Ti 3 C 2 T x MXene, as well as the optimization of the electrostatic spinning process conditions, the development of NH 3 -sensitive materials with a high sensing performance is expected.This is essential for further optimizing the ammonia sensor performance of the conducting polymer/Ti 3 C 2 T x MXene composites. Poly (3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) has attracted a lot of attention in the field of gas sensing because of its good electrical conductivity, low bandgap, environmental friendliness, capacity to detect dangerous chemicals at low operating temperatures [88][89][90][91], and environmental stability [92,93].Jin et al. [94] created PEDOT:PSS/Ti 3 C 2 T x MXene composites by combining 3,4-ethylenedioxythiophene (PE-DOT) and poly (4-styrenesulfonate) (PSS) on a Ti 3 C 2 T x MXene material on a polyimide (PI) substrate through in situ polymerization.Figure 5g,h show the SEM images of Ti 3 C 2 T x MXene and PEDOT:PSS/Ti 3 C 2 T x MXene composites, respectively, where the Ti 3 C 2 T x exhibits a monolayered or few-layered lamellar structure after the introduction of the PEDOT:PSS, where the surface of Ti 3 C 2 T x MXene is covered by micro-and submicrometer PEDOT:PSS plates.Comparing Figure 5i,j reveals that the introduction of PEDOT:PSS increases the gap between the layers of the composite material.The gas sensitivity of NH 3 achieved the highest response value (Figure 5k) when Ti 3 C 2 T x MXene contained 15 wt% and the fastest recovery/response with 36.6% response to 100 ppm NH 3 at room temperature, a 116 s response time, and a 40 s recovery time.Furthermore, Figure 5l shows a bending test of the sensor at a maximum angle of 240 • , and the gas response of the device does not change with the change in the bending angle, indicating that it has good mechanical stability. achieved the highest response value (Figure 5k) when Ti3C2Tx MXene contained 15 wt% and the fastest recovery/response with 36.6% response to 100 ppm NH3 at room temperature, a 116 s response time, and a 40 s recovery time.Furthermore, Figure 5l shows a bending test of the sensor at a maximum angle of 240°, and the gas response of the device does not change with the change in the bending angle, indicating that it has good mechanical stability.The mechanism for analyzing the gas-sensitive performance enhancement of the PEDOT:PSS/Ti3C2Tx MXene composite mainly involves the redox reaction between the composite and the analyte, the charge transfer between the composite and the analyte, and synergistic effect of the increase in the specific surface area of the composite materials.Compared with pure PEDOT:PSS and Ti3C2Tx MXene-based sensors, the composite sensors have a high gas response, fast response/recovery, a low detection limit, good reproducibility, high selectivity, and excellent mechanical stability.A number of studies have demonstrated that the NH3 sensors prepared from Ti3C2Tx MXene and conductive polymer hybrid materials have a low detection limit, flexibility, and an excellent sensing performance [95]. Table 2 outlines the gas-sensing properties of different conductive polymer /Ti3C2Tx MXene composites towards NH3 at room temperature that were determined in recent years.The mechanism for analyzing the gas-sensitive performance enhancement of the PEDOT:PSS/Ti 3 C 2 T x MXene composite mainly involves the redox reaction between the composite and the analyte, the charge transfer between the composite and the analyte, and synergistic effect of the increase in the specific surface area of the composite materials.Compared with pure PEDOT:PSS and Ti 3 C 2 T x MXene-based sensors, the composite sensors have a high gas response, fast response/recovery, a low detection limit, good reproducibility, high selectivity, and excellent mechanical stability.A number of studies have demonstrated that the NH 3 sensors prepared from Ti 3 C 2 T x MXene and conductive polymer hybrid materials have a low detection limit, flexibility, and an excellent sensing performance [95]. Table 2 outlines the gas-sensing properties of different conductive polymer /Ti 3 C 2 T x MXene composites towards NH 3 at room temperature that were determined in recent years. Other 2D Material Hybrids In addition to MXene, graphene, as well as various emerging 2D materials such as transition metal disulfide compounds (TMDs), reduced graphene oxide (rGO), and black phosphorus (BP), have been rapidly developed for NH 3 -sensing due to their graphenelike layered structure, large specific surface area, semiconducting properties, and roomtemperature operation.It has been demonstrated that the heterojunction constructed by compositing Ti 3 C 2 T x MXenes with these 2D materials can obtain complementary electrical and adsorption properties, which enhances their sensing performance [95]. It has been demonstrated that when MXenes are combined with TMDs, such as molybdenum disulfide (MoS 2 ), tin disulfide (SnS 2 ), tungsten disulfide (WS 2 ), and other transition metal disulfide compounds, their charge transfer and adsorption abilities are enhanced, which leads to better sensing effects [100,101].In addition, the composite of TMDs and MXene has almost no lattice mismatch [102], and is expected to replace the traditional metal oxide composite with MXene to construct high-performance ammonia gas-sensitive materials [103,104].In order to overcome the difficulty of detecting ultralow concentrations of ammonia at room temperature in chemical-based gas sensors, He et al. [56] developed an MXene/SnS 2 heterojunction-type chemoresistive sensor SM-5 (the nominal weight ratio of MXene/SnS is 1:5), as shown in Figure 6a,b, which exhibits an excellent gas-sensitive performance for ammonia at sub-ppm at room temperature.The SM-5 sensor can detect NH 3 concentrations of as low as 10 ppb at room temperature.In addition, Figure 6c shows the excellent long-term stability of the sensor, with a decrease in response value of about 3.4% in 20 days.Meanwhile, the SM-5 sensor showed good selectivity to a wide range of possible interfering gases, such as HCHO, C 2 H 5 OH, CH 3 OH, C 3 H 6 O, C 6 H 6 , and NO 2 .The sensing mechanism of the MXene/SnS 2 -based sensor is closely related to the formation of heterostructures.DFT calculations show that the higher sensitivity and selectivity may be due to the more effective charge transfer bestowed by the formed heterostructure, the better catalytical activity, and the stronger NH 3 adsorption of the formed MXene/SnS 2 composite material. Sensors 2024, 24, 4465 14 of 23 stability in detecting NH3.At 100 ppm ammonia, the composite gas sensor had 1.79 and 2.75 times higher response values than the pristine Ti3C2Tx MXene and MoS2.Furthermore, at room temperature, the Ti3C2Tx MXene@TiO2/MoS2 nanocomposite gas sensor exhibited high selectivity for triethylamine, trimethylamine, n-butanol, acetone, formaldehyde, and nitrogen dioxide.As shown in Figure 6g, MoS2 is an n-type sensing material, which has a bandgap in the range of 1.2-1.9eV [105] and a work function of about 3.9 eV [106].Ti3C2Tx MXene behaves as a p-type sensing material, which has a bandgap of 0.19 eV [68] and a work function of about 4.5 eV [69].The heterojunction of the p-type Ti3C2Tx MXene and n-type MoS2 was the primary reason for the sensor's improved gas-sensitive performance for NH3.Bader charge analysis can further obtain the adsorption between sensitive materials and gases by evaluating the amount of charge transfer between gas molecules and sensitive material models [107].Bader analysis further confirmed that ammonia molecules increase charge transfer in the heterojunction, which enhances the interaction of Ti3C2Tx MXene@TiO2/MoS2 nanocomposites with ammonia.Graphene fibers (GFs) have a high mechanical flexibility, electrical conductivity, and wear ability and have enormous potential for application in wearable electronic devices [108,109].Lee et al. [19] reported a simple, scalable, and effective strategy for wet-spinning MXene/graphene-based hybrid fibers using a wet-spinning process to obtain metal-free binder Ti3C2Tx MXene/graphene hybrid fibers.These hybrid fibers have excellent mechanical and electrical properties, making them suitable for flexible, wearable gas sensors.The synergistic effect of the electronic properties and gas adsorption capacity of MXene/graphene resulted in a high NH3 gas sensitivity at room temperature.Figure 7a compares the response values of MXene, rGO fiber, and MXene/rGO fiber sensors, and shows that the Ti3C2Tx MXene/graphene hybrid fiber exhibited a significantly improved Our group [30] demonstrated a Ti 3 C 2 T x MXene@TiO 2 /MoS 2 nanocomposite gas sensor, which successfully realized the detection of 500 ppb NH 3 at room temperature.MoS 2 nanosheets were grown in situ on the etched Ti 3 C 2 T x MXene material via the hydrothermal method, and then some of the Ti 3 C 2 T x MXene was converted into rectangular TiO 2 particles through hydrothermal reaction.The SEM images of Ti 3 C 2 T x MXene@TiO 2 /MoS 2 at different scales are shown in Figure 6e; it can be observed that rectangular TiO 2 nanoparticles are attached between the layers of the material and on the surface, which increase the spacing of the layers of the composite material and can provide more reaction sites for the target gas.As shown in Figure 6f, this novel gas sensor (MTM-0.2) based on a layered structure exhibits the advantages of a fast response and high stability in detecting NH 3 .At 100 ppm ammonia, the composite gas sensor had 1.79 and 2.75 times higher response values than the pristine Ti 3 C 2 T x MXene and MoS 2 .Furthermore, at room temperature, the Ti 3 C 2 T x MXene@TiO 2 /MoS 2 nanocomposite gas sensor exhibited high selectivity for triethylamine, trimethylamine, n-butanol, acetone, formaldehyde, and nitrogen dioxide.As shown in Figure 6g, MoS 2 is an n-type sensing material, which has a bandgap in the range of 1.2-1.9eV [105] and a work function of about 3.9 eV [106].Ti 3 C 2 T x MXene behaves as a p-type sensing material, which has a bandgap of 0.19 eV [68] and a work function of about 4.5 eV [69].The heterojunction of the p-type Ti 3 C 2 T x MXene and n-type MoS 2 was the primary reason for the sensor's improved gas-sensitive performance for NH 3 .Bader charge analysis can further obtain the adsorption between sensitive materials and gases by evaluating the amount of charge transfer between gas molecules and sensitive material models [107].Bader analysis further confirmed that ammonia molecules increase charge transfer in the heterojunction, which enhances the interaction of Ti 3 C 2 T x MXene@TiO 2 /MoS 2 nanocomposites with ammonia. Graphene fibers (GFs) have a high mechanical flexibility, electrical conductivity, and wear ability and have enormous potential for application in wearable electronic devices [108,109].Lee et al. [19] reported a simple, scalable, and effective strategy for wet-spinning MXene/graphene-based hybrid fibers using a wet-spinning process to obtain metal-free binder Ti 3 C 2 T x MXene/graphene hybrid fibers.These hybrid fibers have excellent mechanical and electrical properties, making them suitable for flexible, wearable gas sensors.The synergistic effect of the electronic properties and gas adsorption capacity of MXene/graphene resulted in a high NH 3 gas sensitivity at room temperature.Figure 7a compares the response values of MXene, rGO fiber, and MXene/rGO fiber sensors, and shows that the Ti 3 C 2 T x MXene/graphene hybrid fiber exhibited a significantly improved NH 3 -sensing response (∆R/R 0 = 6.77%).The hybrid fiber exhibits excellent mechanical flexibility.Figure 7b depicts a schematic diagram of the device used to detect the stability of the fiber optic sensor by bending it repeatedly and detecting the change in the resistance of the sensor.As shown in Figure 7c, the resistance fluctuation remains small, ±0.2%, even after more than 2000 bends.In addition, the highly flexible MXene/rGO hybrid fiber was woven/knitted into the lab coat using a simple conventional weaving procedure, demonstrating a dependable sensing capability.The synergistic effect of the optimized bandgap and the enhanced atomic oxygen content at the MXene end of the MXene/rGO hybrid fiber significantly improved the NH 3 -sensing response performance of the MXene/rGO hybrid fiber with low power consumption. Yotsarayuth et al. [25] successfully synthesized Ti 3 C 2 T x Mxene/GO/CuO/ZnO nanocomposites by mixing common NH 3 -sensing materials such as graphene oxide (GO), copper oxide (CuO), and zinc oxide (ZnO) into Ti 3 C 2 T x Mxene via a simple and low-cost hydrothermal method.Figure 7d shows the SEM images of the composites, and the Ti 3 C 2 T x Mxene/GO/CuO/ZnO nanocomposites exhibit a highly uniform two-dimensional stacked structure.A comparison with the SEM image of pure Ti 3 C 2 T x MXene in Figure 7e reveals that the composites have a high surface roughness, which enhances the active sites for the adsorption of NH 3 gas molecules.Figure 7f depicts Ti 3 C 2 T x MXene, GO, CuO, and ZnO figures of work functions of 4. 35, 4.78, 4.7, and 5.14 eV [19,110-112], respectively, with Ti 3 C 2 T x MXene having the lowest figure of work function.As a result, electrons are transferred from Ti 3 C 2 T x MXene to the other three materials to achieve Fermi energy level equilibrium, forming multiple p-n heterojunctions at the interfaces of the different materials.Ti 3 C 2 T x MXene/GO/CuO/ZnO exhibits a 59.9% response to 100 ppm NH 3 at room temperature, with a response time of 26 s and a recovery time of 25 s.The Ti 3 C 2 T x MX-ene/GO/CuO/ZnO exhibits excellent selectivity, high responsiveness, good repeatability, strong stability, a quick response recovery time, and humidity independence.The Ti 3 C 2 T x MXene/GO/CuO/ZnO gas sensor exhibits a remarkable NH 3 -sensing performance.This is because of the inherent features and properties of the nanocomposites, including their functional groups, their bonding, the strong intermolecular attraction between NH 3 molecules, the nanocomposites, and the formation of a p-n heterojunction.Yotsarayuth et al. [25] successfully synthesized Ti3C2Tx Mxene/GO/CuO/ZnO nanocomposites by mixing common NH3-sensing materials such as graphene oxide (GO), copper oxide (CuO), and zinc oxide (ZnO) into Ti3C2Tx Mxene via a simple and low-cost hydrothermal method.Figure 7d shows the SEM images of the composites, and the Ti3C2Tx Mxene/GO/CuO/ZnO nanocomposites exhibit a highly uniform two-dimensional stacked structure.A comparison with the SEM image of pure Ti3C2Tx MXene in Figure 7e reveals that the composites have a high surface roughness, which enhances the active sites for the adsorption of NH3 gas molecules.Figure 7f depicts Ti3C2Tx MXene, GO, CuO, and ZnO figures of work functions of 4.35, 4.78, 4.7, and 5.14 eV [19,[110][111][112], respectively, with Ti3C2Tx MXene having the lowest figure of work function.As a result, electrons are transferred from Ti3C2Tx MXene to the other three materials to achieve Fermi energy level equilibrium, forming multiple p-n heterojunctions at the interfaces of the different materials.Ti3C2Tx MXene/GO/CuO/ZnO exhibits a 59.9% response to 100 ppm NH3 at room temperature, with a response time of 26 s and a recovery time of 25 s.The Ti3C2Tx MXene/GO/CuO/ZnO exhibits excellent selectivity, high responsiveness, good repeatability, strong stability, a quick response recovery time, and humidity independence.The Ti3C2Tx MXene/GO/CuO/ZnO gas sensor exhibits a remarkable NH3sensing performance.This is because of the inherent features and properties of the nanocomposites, including their functional groups, their bonding, the strong intermolecular attraction between NH3 molecules, the nanocomposites, and the formation of a p-n heterojunction. Table 3 outlines the gas-sensitive properties of different 2D materials/Ti3C2Tx MXene composites towards NH3 at room temperature in recent years.The unique properties of terminating functional groups on the surface of MXenes make them ideal for ammonia-sensitive materials.Bhardwaj et al. [115] found that the effective adsorption energy of Ti 2 CO 2 with NH 3 was −0.37 eV using DFT calculations, and the charge transfer between monolayer Ti 2 CO 2 , which has a similar structure to that of Ti 3 C 2 O 2 and NH 3 , was 0.174 e.Similarly, Atkare et al. [95] discovered that MXenes, including monolayers of Ti 3 C 2 O 2 , Ti 2 C(OH) 2 , and others, exhibit a significant affinity to NH 3 , resulting in a charge transfer of 0.117 e. At room temperature, the reaction mechanism of Ti 3 C 2 T x MXene to NH 3 molecules can consist of two parts: the reaction between NH 3 molecules and oxygen molecules on the surface of the sensitive material and the reaction between NH 3 molecules and specific functional groups at the end of Ti 3 C 2 T x MXene [18,57].First, the adsorption of O 2 molecules on the surface of the sensitive material traps electrons in the form of oxygen ions, mainly in the form of O 2− .When the sensor is exposed to a reducing gas such as NH 3 , the O 2− will react with the NH 3 molecules to form NO and H 2 O while releasing electrons.The resistance decreases when these electrons return to the conduction band of Ti 3 C 2 T x MXene and its composites.The reaction process is shown in Equations ( 4)-( 6): The gas-sensing mechanism of Ti 3 C 2 T x /In 2 O 3 composites to NH 3 shown in Figure 8b can be used as a reference.In addition to the electron transfer process between oxygen ions and NH 3 molecules on the surface of Ti 3 C 2 T x MXene-sensitive materials, Lee et al. [57] mentioned that the reaction of -O and -OH on the surface of Ti 3 C 2 T x with NH 3 resulted in hole-electron complexation and a subsequent increase in resistance, and the conjectured mechanism diagram is shown in Figure 8a.The reaction formulae are shown in Equations ( 7) and ( 8) as follows: The mechanism for the enhanced gas sensitivity of Ti3C2Tx MXene composite materials to NH3 is generally achieved by the synergistic effect between the two materials, such as the formation of a heterojunction of the composites, adsorption energy and charge transfer, chemical sensitization and electron sensitization, unique morphology, etc. [11,61].When heterojunctions are formed, the gap in the work function between Ti3C2Tx MXene The mechanism for the enhanced gas sensitivity of Ti 3 C 2 T x MXene composite materials to NH 3 is generally achieved by the synergistic effect between the two materials, such as the formation of a heterojunction of the composites, adsorption energy and charge transfer, chemical sensitization and electron sensitization, unique morphology, etc. [11,61].When heterojunctions are formed, the gap in the work function between Ti 3 C 2 T x MXene and its composite material causes electrons to transfer directionally from the lower to the higher work function.This transfer occurs due to the equilibrium of the Fermi energy levels when the materials come into contact with each other.This results in the formation of an electron depletion layer and an electron accumulation layer on the low-work-function material.The electron accumulation layer promotes oxygen adsorption on the surface of the sensing material.In contrast, the electron depletion layer increases the potential energy barrier at the interface, while the height of the intergranular barrier hinders the transport of carriers, resulting in an increase in the initial resistance [116] and a higher gas response.As an illustration, Zhou et al. [57] synthesized Ti 3 C 2 T x /In 2 O 3 nanocomposites.In 2 O 3 is classified as an n-type semiconductor, while Ti 3 C 2 T x is regarded as a metallic phase because of its excellent conductivity.When Ti 3 C 2 T x comes into contact with In 2 O 3 , it exhibits a high work function.As a result, electrons migrate from In 2 O 3 to Ti 3 C 2 T x , forming a Schottky barrier and a depletion layer at the interface of In 2 O 3 and Ti 3 C 2 T x (as shown in Figure 8c,d).As a result, the charge transfer is blocked, and the base-value resistance of Ti 3 C 2 T x /In 2 O 3 becomes high in the air, resulting in a significant increase in the gas-sensitive response [22].In addition, the improved gas-sensitive performance of the sensitive material is also related to its lattice mismatch, and the oxygen vacancies generated at the heterojunction due to the lattice mismatch will also provide additional active sites for the sensitive material [117]. The most significant characteristic of MXenes is their demonstration of similar metallic properties to their precursor MAX, with a fixed electron density near the Fermi energy level.In fact, the gas-sensing mechanism of MXene is notably more complex than the classical charge transfer model [95].Lee et al. [18] were the first to report the gas-sensing mechanism of Ti 3 C 2 T x MXene.They observed increased resistance in Ti 3 C 2 T x sensors when exposed to four electron donor gases (ethanol, methanol, acetone, and ammonia).Conversely, the resistance of Ti 3 C 2 T x films decreased without the presence of these gases.Therefore, they inferred that Ti 3 C 2 T x films exhibit p-type sensing behavior.The analysis suggests that the p-type semiconductor nature of Ti 3 C 2 T x MXene may be due to the many molecules introduced during the Al etching process acting as p-type dopants to Ti 3 C 2 T x , such as water and oxygen. It is well known that gas sensors for semiconductor materials exhibit either a positive or negative resistance change depending on the gas type [118].Specifically, an n-type semiconductor-sensitive material exposed to oxidizing gases decreases in resistance due to the loss of electrons and increases due to the gain in electrons when exposed to reducing gases, and the opposite is true for p-type semiconductor-sensitive materials.In contrast, Kim et al. [118] found that Ti 3 C 2 T x exhibits a positive resistance change under oxidized or reduced gas conditions, indicating that the carrier transport of Ti 3 C 2 T x will be blocked when it adsorbs gases.In other words, the sensing mechanism of Ti 3 C 2 T x is different from that of semiconductor materials such as metal oxides.Therefore, the mechanism for the universal p-type response of Ti 3 C 2 T x was proposed.Due to the metallic conductivity of Ti 3 C 2 T x MXene [119], the gas adsorption reduces the number of carriers and therefore increases the channel resistance.While Koh et al. [120] investigated the effect of the interlayer swelling of Ti 3 C 2 T x MXene films upon gas action on the gas-sensitive properties, the change in the interlayer space of Ti 3 C 2 T x MXene upon the introduction of gas was studied by in situ XRD measurements, which found that the degree of swelling of Ti 3 C 2 T x MXene films was consistent with the gas response.Therefore, another mechanism is proposed: gas-phase molecules are inserted into the MXene interlayer instead of surface adsorption, and the interlayer expansion induced to reduce the conductivity due to the metallic nature of Ti 3 C 2 T x MXene is one of the reasons why MXene generally exhibits a p-type response to various gases. Both mechanisms currently explain MXene's p-type response to the electron acceptor gas and the electron donor gas.To date, the gas-sensing mechanism of Ti 3 C 2 T x MXene and its composites has not been explained in a unified manner.The electron transfer model of the adsorbed gas molecules on the surface of Ti 3 C 2 T x composites may be more complex than reported and thus needs to be further developed and refined through experimental validation. Conclusions and Outlook Ti 3 C 2 T x MXene, as the earliest discovered MXene material, which has been extensively studied in the field of ammonia gas sensing, has been regarded as an excellent room-temperature NH 3 -sensing material due to its graphene-like two-dimensional lamellar structure, which confers a large specific surface area, good room-temperature sensitivity, and a high adsorption capacity between its surface-rich functional groups and NH 3 .This review describes the current research progress in modulating the ammonia gas-sensing properties of Ti 3 C 2 T x MXene by means of its preparation method and composite modification.Ti 3 C 2 T x MXene with differences in its morphology, surface functional groups, electrochemical properties, nano-defects, and stability can be obtained through improvements in the preparation method to achieve the desired performance, which in turn affects the NH 3 -sensing behavior of the sensors.Ti 3 C 2 T x MXene-based composites incorporating additional composites can demonstrate enhanced response values, lower detection limits, quicker response recovery, and an improved stability compared to pure Ti 3 C 2 T x MXene. Although significant progress has been made in the design and modification of Ti 3 C 2 T x MXene materials for ammonia gas sensors in recent years, there are still many challenges and much room for further development in optimizing and improving the sensing performance.Since the gas-sensing mechanism of MXenes is much more complex than the traditional semiconductor classical charge-transfer model, further calculations and investigations regarding the charge transfer and adsorption-desorption are needed in terms of the NH 3 -sensing principle of Ti 3 C 2 T x MXene.Meanwhile, a large number of studies have demonstrated that the surface-functional groups of Ti 3 C 2 T x MXene have a significant effect on its electrochemical properties and stability.Therefore, synthesizing Ti 3 C 2 T x MXene materials with controllable surface-functional groups is significant for its application in room-temperature ammonia gas sensors.Furthermore, Ti 3 C 2 T x MXene's composite structure can be further optimized to improve its NH 3 -sensing performance.In the next step, we could design Ti 3 C 2 T x MXene sensors with excellent long-term stability and a response value that is less impacted by humidity to widen their applications.In the future, we should broaden our research on Ti 3 C 2 T x MXene in these fields to develop higher-performance Ti 3 C 2 T x MXene-based room-temperature ammonia sensors. Funding: This research received no external funding. , which presents a schematic diagram of the etching process of Ti 3 AlC 2 by HF, where the Ti 3 AlC 2 structure consists of a single Ti 3 C 2 layer separated by Al atoms.The Al atom layers between the Ti 3 AlC 2 layers are eliminated by the HF treatment, and the subsequent loss of metallic bonds causes the individual Ti 3 Al 2 C layers to separate, producing Ti 3 C 2 T x .The reaction process is shown in Equations (1)-(3): Figure 1 . Figure 1.Ti3AlC2 is etched as Ti3C2Tx.(a) Structural schematic diagram of Ti3AlC2, (b) schematic of the process by which -OH replaces Al atoms after HF treatment, and (c) hydrogen bond breaking and nanolayer separation after sonication treatment [14]. Figure 1 . Figure 1.Ti 3 AlC 2 is etched as Ti 3 C 2 T x .(a) Structural schematic diagram of Ti 3 AlC 2 , (b) schematic of the process by which -OH replaces Al atoms after HF treatment, and (c) hydrogen bond breaking and nanolayer separation after sonication treatment [14]. Figure 2 . Figure 2. (a) SEM image of the Ti3C2Tx MXene after HF treatment; (b) by comparing the single-layer band structures of Ti3C2(OH)2, Ti3C2F2, and Ti3C2, it can be seen that Ti3C2Tx exhibits a change from metal to semiconductor due to changes in surface functional groups [14]; (c) XRD pattern of Ti3AlC2 and as-prepared Ti3C2Tx; (d) SEM images of Ti3C2 nanoflakes after exfoliation by TMAOH [33]. . Figure 2 . Figure 2. (a) SEM image of the Ti 3 C 2 T x MXene after HF treatment; (b) by comparing the single-layer band structures of Ti 3 C 2 (OH) 2 , Ti 3 C 2 F 2 , and Ti 3 C 2 , it can be seen that Ti 3 C 2 T x exhibits a change from metal to semiconductor due to changes in surface functional groups [14]; (c) XRD pattern of Ti 3 AlC 2 and as-prepared Ti 3 C 2 T x ; (d) SEM images of Ti 3 C 2 nanoflakes after exfoliation by TMAOH [33]. Figure 3 . Figure 3. Adsorption process of H2O and NH3 molecules on the surface of alkalized Ti3C2Tx [40].Figure 3. Adsorption process of H 2 O and NH 3 molecules on the surface of alkalized Ti 3 C 2 T x [40]. Figure 3 . Figure 3. Adsorption process of H2O and NH3 molecules on the surface of alkalized Ti3C2Tx [40].Figure 3. Adsorption process of H 2 O and NH 3 molecules on the surface of alkalized Ti 3 C 2 T x [40]. 3 C 2 T x MXene for NH 3 , Kan et al. [65] combined surface-functionalized In 2 O 3 nanotubes with Ti 3 C 2 T x nanosheets.The composite was further loaded onto thermoplastic polyurethane (TPU) foam, a dualfunctional sensing platform constructed based on Ti 3 C 2 T x /In 2 O 3 nanocompositesand modified TPU foam sensors.The produced nanocomposites exhibited an improved ammoniasensing performance and pressure-sensitive properties, utilizing the strong synergistic effect of the sensing of In 2 O 3 nanotubes and high conductivity of Ti 3 C 2 T x nanoflakes, as well as the foam substrate's pressure-sensitive and gas-permeable capabilities.Ti 3 C 2 T x nanoflakes are negatively charged on the surface due to the terminal functional groups such as -OH and -F.At this time, the modification of In 2 O 3 with the cationic surfactant (3-aminopropyl) triethoxysilane (APTES) can make the surface of In 2 O 3 positively charged, and the two can be compounded to obtain Ti 3 C 2 T x /In 2 O 3 composites via simple ultrasonication with electrostatic adsorption, which was shown to have higher NH 3 response values than pure Ti 3 C 2 T x nanoflakes.In 2 O 3 is an n-type sensing material, which has a bandgap in the range of 3.55-3.75eV [66] and a work function of about 4.28 eV [67]; Ti 3 C 2 T x MXene behaves as a p-type sensing material, which has a bandgap of 0.19 eV [68] and a work function of about 4.5 eV [69].Two main factors are used to investigate the enhanced sensing ability: First, n-type In 2 O 3 has a high Fermi energy level, and electrons will be transferred from the In 2 O 3 nanotubes to the Ti 3 C 2 T x nanoflakes until the Fermi energy levels of the two reach equilibrium, thus forming a space charge layer at the Ti 3 C 2 T x and In 2 O 3 interface.At the same time, the energy bands at the interface are bent on both sides, generating a potential barrier that increases the base value resistance of the sensor, thus increasing the gas response.Second, the In 2 O 3 nanotubes work as isolators to inhibit the re-stacking of Ti 3 C 2 T x nanoflakes, thereby increasing the layer space and effectively facilitating the diffusion and permeation of gases in the sensing layer.In addition, the sensor achieves the flexible and interference-free detection of complex exhaled environments at room temperature, with a memory function for detecting NH 3 gases down to 1 ppm, realizing the dual-mode detection of NH 3 gases.Sensors 2024, 24, 4465 9 of 23 active (001) crystalline TiO2 makes the charge separation of the T-T-12 h TiO2/MXene sensor when exposed to UV light even better, which leads to a better gas-sensing performance.Figure 4b also shows a two-fold increase in sensitivity compared to when UV is absent.The TiO2/MXene sensor type T-T-12 h exhibited 34 times more sensitivity to 30 ppm of ammonia compared to pristine Ti3C2Tx.Density functional theory reveals that the (001) side of the TiO2 and Ti3C2Tx composite has the greatest attraction in terms of ammonia adsorption [64]. Figure 4 . Figure 4. (a) Dynamic response curves of TT8 h-, TT12 h-, TT16 h-and TT32 h-based sensors; (b) dynamic response curves with UV illumination on T-T-12 h and without UV illumination on TT12 h; (c) schematic illustration of band diagrams of Ti3C2Tx and (001) TiO2; (d) schematic illustration of ammonia gas-sensing mechanism under UV irradiation [64].(e) The transient response curves of the composite sensors with different WO3 contents to 1 ppm NH3 at room temperature; (f) the Figure 4 . Figure 4. (a) Dynamic response curves of TT8 h-, TT12 h-, TT16 h-and TT32 h-based sensors; (b) dynamic response curves with UV illumination on T-T-12 h and without UV illumination on TT12 h; (c) schematic illustration of band diagrams of Ti 3 C 2 T x and (001) TiO 2 ; (d) schematic illustration of ammonia gas-sensing mechanism under UV irradiation [64].(e) The transient response curves of the composite sensors with different WO 3 contents to 1 ppm NH 3 at room temperature; (f) the dependence of the composite sensor response on the WO 3 content; (g) schematic illustration of the gas-sensing mechanism and the energy band structure diagram of Ti 3 C 2 T x /WO 3 before and after exposed NH 3 [70]. Figure 5 . Figure 5. (a) SEM image of Ti 3 C 2 T x MXene; (b) SEM image of PANI/Ti 3 C 2 T x ; (c) PANI/Ti 3 C 2 T x composite nanofiber sensor for the detection of NH 3 at room temperature; (d) UPS spectra of pure Ti 3 C 2 T x sensor and (e) PANI/Ti 3 C 2 T x composite sensors; (f) the PANI/Ti 3 C 2 T x -based flexible sensor bent at different angles [82].FESEM images of (g) Ti 3 C 2 T x MXene (inset is Ti 3 C 2 T x MXene on an AAO membrane) and (h) PEDOT:PSS/Ti 3 C 2 T x MXene composites.The cross-sectional FESEM images of (i) Ti 3 C 2 T x MXene films and (j) PEDOT:PSS/Ti 3 C 2 T x MXene films, (k) effect of the Ti 3 C 2 T x MXene content in PEDOT-PSS on the sensor response toward 100 ppm NH 3 at room temperature (27 • C), and (l) gas response of the PEDOT:PSS/Ti 3 C 2 T x MXene composite-based sensor against 100 ppm NH 3 bent at different angles [94]. Figure 6 . Figure 6.The resistance variation curves of the SM-5 sensor to (a) ppm-level NH 3 concentration from 2 to 100 ppm and (b) ppb-level NH 3 concentration from 10 ppb to 1 ppm at 18 • C; (c) the long-term stability of the SM-5 sensor to 1 ppm NH 3 for 20 days [56].(d,e) Ti 3 C 2 T x MXene@TiO 2 /MoS 2 ; (f) dynamic sensing performance of the sensor-based Ti 3 C 2 T x MXene to NH 3 at a room temperature of 27 • C and an RH of 43%; (g) energy band diagrams of Ti 3 C 2 T x MXene@TiO 2 /MoS 2 sensors [30]. Sensors 2024 , 24, 4465 15 of 23 hybrid fiber significantly improved the NH3-sensing response performance of the MXene/rGO hybrid fiber with low power consumption. Figure 7 . Figure 7. (a) Comparison of the gas response of MXene film, rGO fiber, and MXene/rGO hybrid fiber; (b) schematic illustration of the fiber bending test.The "M" stands for multimeter.(c) Cyclic bending fatigue versus resistance difference of the rGO fiber and MXene/rGO hybrid fiber [19].(d) FE-SEM images of Ti 3 C 2 T x MXene/GO/CuO/ZnO nanocomposite and (e) pristine Ti 3 C 2 T x Mxene.(f) Schematic diagram of the energy band structure of the Ti 3 C 2 T x MXene/GO/CuO/ZnO heterostructure [25]. ) 23 Figure 8 . Figure 8.(a) Schematic diagram of the possible gas-sensing mechanisms of the Ti3C2Tx MXene for NH3 [18].(b) The gas-sensing mechanism diagram of the Ti3C2Tx/In2O3 composite materials.(c,d) Schematic diagram of the electron transfer at the interface of Ti3C2Tx/In2O3 composite materials in the air and NH3 [57]. Figure 8 . Figure 8.(a) Schematic diagram of the possible gas-sensing mechanisms of the Ti 3 C 2 T x MXene for NH 3 [18].(b) The gas-sensing mechanism diagram of the Ti 3 C 2 T x /In 2 O 3 composite materials.(c,d) Schematic diagram of the electron transfer at the interface of Ti 3 C 2 T x /In 2 O 3 composite materials in the air and NH 3 [57]. Table 1 . The gas-sensitive properties of various MOS/Ti 3 C 2 T x MXene nanocomposites to NH 3 at room temperature. Table 2 . The gas-sensitive properties of various conductive polymer/Ti 3 C 2 T x MXene nanocomposites to NH 3 at room temperature. Table 3 outlines the gas-sensitive properties of different 2D materials/Ti 3 C 2 T x MXene composites towards NH 3 at room temperature in recent years. Table 3 . The gas-sensitive properties of various 2D material /Ti 3 C 2 T x MXene nanocomposites to NH 3 at room temperature.	2024-07-12T15:06:08.762Z	2024-07-01T00:00:00.000	{ "year": 2024, "sha1": "dd3a1896464dca4a7d4c549b4ce34627c40a4847", "oa_license": "CCBY", "oa_url": "https://doi.org/10.3390/s24144465", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "1d62ab23a864b7bf65fff4f2adffbf406374c630", "s2fieldsofstudy": [ "Materials Science", "Chemistry" ], "extfieldsofstudy": [ "Computer Science", "Medicine" ] }
238428244	pes2o/s2orc	v3-fos-license	Research on Data Integrity Verification Technology Based on Blockchain With the rapid development of Internet of Things technology, it has changed People’s Daily production and life to a great extent, but the large number of applications of Internet of Things devices affect the security level of data to a certain extent. In order to ensure data integrity in the Internet of Things environment, it is necessary to conduct in-depth research on blockchain-based data integrity verification technology, analyze the authenticity structure based on data sources, and carry out data integrity verification exploration according to data source identification structure. The experimental system for data integrity verification is proposed. In this way, the effectiveness of blockchain-based data integrity verification technology can be guaranteed, and the integrity of data can be guaranteed under the condition of decentralization, while the scattered storage of data and data privacy can be protected. Introduction At present, the application of computer technology is becoming more and more common, especially in the era of big data and cloud, computer has become an important basic equipment for processing data and information. However, in the process of using computer to carry out data processing and application, the confidentiality, integrity and availability of data may be damaged due to various factors. The integrity of the data is damaged, it can not be restored to the original data, resulting in permanent loss of data. This will seriously affect the application value of the data, and may even lead to the loss of enterprises or individuals. In order to ensure the integrity and security of data in the information age, it is necessary to conduct in-depth discussion on the data integrity verification technology based on block chain and design a data integrity verification scheme based on block chain. Thus, the data source integrity verification technology is widely used. Research status of data integrity verification technology based on block chain Blockchain in the application process can provide inherent data storage space, to ensure that the existing transaction data will not be modified or deleted. Moreover, in the application of blockchain technology for transaction verification, the authenticity and credibility of information data can be analyzed without any third-party authentication. Historical transactions are imtamperable and can ensure the authenticity and effectiveness of the stored data. In the application process of blockchain technology, the application of encryption and digital signature identity authentication and authenticity technology is more common, which can highlight the advantages of block chain read and write access control, and further improve the security of block chain technology in the application process. At present, with the continuous development of information technology, the application of blockchain technology has gradually matured and improved. The characteristics of non-tampering and Effective security prevention and management of data collection, transmission and stored process in the Internet of Things environment is an important measure to improve the level of data security. If effective security measures are not taken in the process of data processing and application, data may be lost, leaked or expired. All these problems will have a certain impact on data integrity and harm the application value of data in the Internet of Things. Therefore, it is necessary to effectively apply and utilize the blockchain technology. The unique decentralized and anonymous trust management mechanism of blockchain technology can verify the integrity of data without the need to transfer the complete data to the network server, which is conducive to solving the data integrity verification problem with privacy protection requirements [1]. Figure 1 Block structure diagram Data integrity verification mechanism is the research focus in the current data management process. Data integrity verification mechanism can ensure the safe storage of local data and the security of remote data at the same time. At present, the research on data integrity verification mechanism is continuously developing, and the more common integrity verification mechanisms include the following: First, some researchers put forward the data ownership proof, which is mainly to complete the integrity verification process of data in the file block. Although this mechanism can effectively identify the integrity of remote data, it cannot effectively identify the availability of remote data. Second, some researchers put forward the theory of data recovery. The recoverable proof mechanism based on sentinel can not only accurately judge whether the remote data is damaged, but also recover the damaged data to a certain degree. Third, open validation model validation scheme. This verification mechanism requires the use of third-party verification institutions to verify the integrity of data, and can also use data structures to complete dynamic data verification. Fourth, the application of random source technology, this technology can not only verify the integrity of the data, but also protect the privacy of the data, can hide the data information that needs to be verified in the cloud server, to prevent third-party verification agencies from obtaining the data content. Fifth, secure cloud storage service architecture DIAAS. This architecture requires a dedicated data integrity service to perform integrity verification. Sixth, the data integrity verification 3 mechanism based on block chain technology. In the continuous development of blockchain technology, some researchers envision a verification mechanism based on the hashing method. This mechanism is similar to the theory of third-party verification of the integrity of data records. In addition, there are other blockchain data integrity verification methods based on audit tracking method, which pay more attention to the privacy of data and emphasize the non-repudiation of privacy audit logs. At present, some researchers have proposed a data integrity verification framework based on blockchain. In the construction process of the framework, data verification work should be carried out based on the data integrity verification protocol of the own system. In the process of research on this verification scheme, it is necessary to conduct in-depth exploration on the validity of the data integrity verification scheme based on the Internet development environment [2]. Data integrity verification mechanism In the process of further research on blockchain-based data integrity verification mechanisms, it is necessary to understand that the overall goal of proving data integrity is consistent, to ensure that the data is recorded as expected, to ensure that it is the same as the original record in subsequent retrievals. Therefore, the main purpose of data integrity is to prevent abnormal changes in information. If the system can provide an integrity system, it needs to ensure that the data is sent to the receiver from an authenticated data source and that the data has not been tampered with. This ensures that the data is nonrepudiation. The integrity verification protocol of data storage is not mainly guaranteed by replication technology, because in the process of data storage, the data integrity may be affected by accidental errors or intentionally damaged data by the data storage party. In the process of designing the data integrity verification protocol, it is necessary to ensure the security of local data storage, remote data storage and cloud data storage. Only in this way can the integrity and authenticity of the data be truly guaranteed. At present, in the research process of data integrity verification mechanism, the P2P storage environment data integrity protocol has been studied deeply. The protocol mainly includes the following contents: during the operation of the protocol, the owner of the data will complete the data replication and store the copy in the data store. The data store and the data owner negotiate the storage time of the data, which is checked periodically by the data validation. In the verification protocol operation, the interactive form of knowledge proof should be used to enable the data storekeeper to prove the stored data protocol to the data verifier, which mainly includes query and response message. The data verifier needs to periodically send query messages to the data store. The data store can calculate the corresponding response message based on the data and the label and send the message to the data verifier. The data verifier makes accurate judgment on the integrity of the data based on the metadata. Metadata is an important part of the validation message, which is generally calculated by the data owner and sent to the data verifier [3]. of the data, to ensure that the server can have the original data. In order to achieve this goal, a message authentication code symmetric encryption, and the application of other methods are more common, in the file before send to distrust cloud server, user metadata can be stored in the file, the user will be sent to the corresponding file cloud servers, can be sent via metadata and data comparison, the integrity of the data can be validated. The restorability verification method with CAPTCHA can also be used for remote validation of language server data without even needing to recover from the cloud server to the local disk. Proof of data ownership and proof of recoverability can be applied when verifying semitrusted and untrusted server data [4]. Overall system design In the design process of data integrity verification scheme based on block chain, the design work should be carried out according to the main characteristics of block chain. Blockchain has the characteristics of tamper-proof, decentralization and redundant storage. The participating computing nodes are peer to peer, and there is no central node. As a result, can block chain data network as a peer-to-peer network, all nodes can save a copy of the block chain, each node area through the block header information to ensure the consistency of the copy, each block by the hash value and the connection between the previous block can effectively prevent data information is malicious tampering. If you want to modify a block, you must recalculate the hash values of all previous blocks in front of the block, which will greatly increase the complexity of calculation, so as to prevent information tampering. This is the main advantage of the application of blockchain technology in secure storage systems. In addition, the use of blockchain distributed storage system can also prevent a single point of failure. Once the central node is down or fault, the distributed storage can use blockchain to complete the recovery of key data metadata. In particular, the tamper-proof feature of blockchain can ensure the integrity and validity of the data stored on the blockchain to the greatest extent. This is also a necessary condition for the construction of a secure storage system. The specific design framework is shown in Figure 3, which mainly includes Internet of Things devices, blockchain, smart contract, database, etc. First, the database is a traditional distributed storage system, whose main function is to complete the data storage of Internet of Things devices and the corresponding metadata storage. In order to provide reliable support for data integrity verification. Second, blockchain. The main function of the blockchain is to ensure the global state of the blockchain in the data storage area, and all server nodes can query to the blockchain. In this way, the state of the local blockchain is synchronized to ensure that the state of the blockchain is unified. In addition, the metadata stored by the blockchain is read-only. If the data stored by the user changes, new metadata can be generated, and the newly generated metadata can be stored in the blockchain through the smart contract, which can prevent malicious tampering of information data to the greatest extent. Third, smart contracts. Smart contracts allow IoT devices to communicate with the blockchain network. After the smart contract between IoT devices and the production chain, they interact with the database in the overall design architecture network. The corresponding function in the smart contract can complete the registration and deletion of the corresponding Internet of Things devices in the Ethernet address, and can complete various functions such as data storage and data integrity verification [5]. System development environment design In the process of designing the data integrity verification scheme based on block chain, the system development environment must be reasonably selected. In this experiment, the hardware environment is Intel(R)Core(TM) I7-8550UCPU (1.80GHz), and the RAM is 8GB. The experimental running system is Ubuntu16.04LTS 64-bit operating system. The language of smart contracts is solidity. The blockchain environment uses the Solidity programming language, the development framework is Truffle, and the visual private chain client Ganache. System function design and implementation In the design process of integrity verification experimental system based on block chain, its function design mainly includes two functions: data storage function and data integrity verification function. In the design of the data storage function, the main role of the blockchain node device is to collect various metadata additional information such as the hash value of the data, the time and digital signature of the data, and the owner of the data. So as to construct a new block, chain. When designing the data verification function, the main role of the blockchain node is to complete the request to receive the verification, update the blockchain copy, retrieve the relevant metadata of the given authenticity identification, and return the retrieval result. To ensure the smooth implementation of the data integrity verification scheme. There are two types of entities in the design process of functional modules. In the design of entity components, it mainly includes IoT nodes and blockchain nodes. Function modules include authenticity identification function, data storage function, blockchain consensus function and data integrity verification function. In the specific design process, it is necessary to ensure that IoT nodes correspond to IoT devices in the IoT structure, whose main role is to collect various data in the IoT environment. In the process of functional module design, it is necessary to ensure that the blockchain node can correspond to the peer node in the network system. The specific functional design includes the following contents: First, the authenticity of the identification function module design. In this module design, it is necessary to ensure that the blockchain nodes can provide the data information collected under the Internet of Things environment. Before the data is stored on the blockchain, this feature needs to be utilized to preprocess the data to build a data structure that identifies the authenticity of the data source. In the application of the data integrity verification experimental system based on the complete block chain in the Internet of Things environment, the authenticity of the data source and the storage space of the block chain need to be fully considered. In the design process of this functional module, the main content is to decide which data to put on the blockchain and which data to put off the blockchain. Second, the design of data storage function module. In this module design, the data structure that needs to be stored should be provided to the blockchain node, the corresponding script should be used to perform the storage operation, and the validity of the storage operation should be verified, and finally the data information should be stored in the blockchain. This function is similar to the transaction function in the network blockchain. Verification of transactions is a unique mechanism in the blockchain network, and all blockchain nodes must verify transactions accurately. Third, block chain consensus function module design. In this functional design process, the block construction and the block forming function of the chain in the block chain should be completed. All data stores and records must be verified by the consensus function module before they can be assembled into blocks. After validation of the block, the block can be linked to the latest block of the blockchain, which is the core content of the blockchain structure and is similar to the consensus protocol in the network blockchain. Fourth, the design of data integrity verification function module. In the design process of this module, relevant data need to be obtained from the blockchain and compared with the metadata. Thus, data integrity verification results are obtained. Blockchain nodes should provide authenticity identification of acquired data, and use corresponding scripts to perform data acquisition operations to verify the consistency between acquired data and data provided by IoT nodes. This function is similar to the transaction function of block chain technology in the use process [6]. Conclusion In a word, in the design process of blockchain-based data integrity verification system, the corresponding functions must be comprehensively analyzed to ensure the reliability and effectiveness of the data integrity verification system. In addition, after the completion of the design of the data integrity verification system, the operation effect of the whole system needs to be analyzed, and the results of functional test and benchmark test can be statistically compared. The final test results are obtained by executing the corresponding test procedures for different test cases. This ensures the correctness of the data storage and validation to the maximum extent possible, and helps to ensure the integrity of the data. In the context of the continuous development of information technology, it is necessary to further explore and study the data integrity verification system based on block chain. In the	2021-10-08T20:05:59.182Z	2021-09-01T00:00:00.000	{ "year": 2021, "sha1": "35ac4317972d536da1215ca5250866318a678114", "oa_license": null, "oa_url": "https://doi.org/10.1088/1742-6596/2035/1/012017", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "35ac4317972d536da1215ca5250866318a678114", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Physics" ] }
227073219	pes2o/s2orc	v3-fos-license	Expression profiling on subclasses of primary parotid gland carcinomas Introduction: The underlying molecular mechanisms of parotid gland carcinomas (PGC) are still unknown. Knowledge about the tumor-driving signaling pathways is necessary either for diagnostics or developing new therapeutic options in this heterogeneous and rare entity. Material and Methods: 94 matching RNA formalin-fixed and paraffin-embedded tissue samples from PGC and the corresponding non-tumor area, RNA quality and quantity were sufficient for gene expression profiling of 770 genes using the NanoString's nCounter technology. Oncogenic and tumor suppressor genes were examined in the three common PGC tumor entities: adenoid cystic carcinoma (ACC), adenocarcinoma NOS (AC-NOS), and mucoepidermoid carcinoma (MEC). Results: Expression profiling and subsequent hierarchical cluster analysis clearly differentiated between non-tumor gland tissue samples and PGC. In addition expression pattern of all three entities differed. The extensive pathway analysis proved a prominent dysregulation of the Wnt signaling pathway in the three PGC entities. Moreover, transcript upstream analysis demonstrated a pronounced activation of the PI3K pathway in ACC and MEC. Discussion: Our findings revealed divergent molecular expression profiles in MEC, ACC and AC-NOS that are presently studied for their potential application in PGC diagnostics. Importantly, identification of Wnt and PI3K signaling in PGC revealed novel options of PGC therapy. INTRODUCTION Salivary gland carcinomas account for 1-5% of all head and neck tumors worldwide and, according to the World Health Organization (WHO), can be divided into a total of 22 subgroups [1][2][3]. Despite their lower incidence compared to other carcinomas, they represent a scientifically interesting and little-researched tumor group to date due to their diversity of entities. The most frequent histological types are adenocarcinomas, adenoid cystic carcinomas and mucoepidermoid carcinomas [4]. The anatomical location and the potential local spreading pattern of salivary gland cancer (SGC)-especially the parotid gland (PGC)-make these tumors, on the one hand, surgically demanding, and on the other the psychosocial role for the patient quite vital, given that the surgery may involve extensive cosmetic changes to the patient's face. The development of salivary gland tumors is still largely unexplained. The treatment of first choice for salivary gland malignancies is surgical tumor excision. Depending on the degree of differentiation, primary tumor extent (T classification) and lymph node status (N classification), surgical therapy should include a more-or-less extensive neck dissection [5]. Adjuvant radio (chemo) therapy is additionally recommended in patients with high T classification, positive resection margins, pN+, perineural invasion or a high-grade subtype, especially in MEC. The benefit of additional chemotherapy has not been conclusively clarified, the main reason being, in particular, the lack of data on effectiveness. There are only limited studies on systemic therapy concepts that could be considered in palliative situations [6][7][8]. Recently, the developments in molecular pathology diagnostics open up manifold possibilities, which provide new diagnostic, prognostic and especially therapeutic approaches as well as points of targeted therapy. Due to the overall lower incidence compared to other tumors, the more precise molecular examination of tumor DNA-and especially the tumor RNA-is a hurdle, as an eventual therapeutic relevance is not determinable due to the often-missing large cohorts of SGC [9,10]. Over the past decade, molecularly targeted therapies that block important oncogenic pathways have made remarkable progress [11]. A personalized tumor therapy conceptas applied in the case of colon or lung carcinomas-is not yet established, due to limited data of tumor driving pathways, which could then be targeted by future individualized therapy approaches [12][13][14]. Recent study demonstrated high frequencies of activating PIK3CA and HRAS mutations as well as deactivating TP53 mutations in epithelial myoepithelial, salivary duct, squamous cell, oncocytic and in large cell undifferentiated carcinoma as well as in basal cell adenocarcinoma [15]. However, these tumor driving genetic alterations were not found in mucoepidermoid (MEC) and adenoid cystic (ACC) parotid gland carcinoma or on adenocarcinoma not otherwise specified (AC-NOS). On the one hand, these entities should be investigated due to their frequency and the hitherto poorly understood tumor genesis. Novel diagnostic marker genes and therapeutic targets for these entities are urgently needed. Therefore, we focused on these entities and compiled a sample collective from a total of 61 PGC patients. Importantly, comprehensive expression profiling on histologically well-classified MEC, ACC, and AC-NOS carcinoma defined divergent expression patterns between the entities. Moreover, we studied tumor driving pathways and demonstrated a common activation of the PI3K/AKT pathway. Most notably, prominent stimulation of Wnt signaling was observed in most MEC, ACC, and AC-NOS. Clinical characteristics To access the deregulation of gene expression in the most frequent parotid gland carcinoma entities, MEC, ACC, and AC-NOS, we collected 61 PGC samples and the corresponding non-tumor tissues. Macrodissection and RNA extraction were performed from all matching FFPE samples but there was only sufficient RNA available from 47 matching samples for expression profiling from both, tumor tissue and normal tissue. Clinicopathological characteristics of the 47 patients, considered in our study, are summarized in Table 1. Genetic differences within tumor entities in regard to gene expression A total of 730 highly cancer relevant genes involved in 13 tumor driving signaling pathways were studied (Supplementary Table 2). Hierarchical cluster analysis showed a clear difference in the expression pattern of the cancer associated, in comparison to the non-tumor associated, gene sets ( Figure 1). Moreover, we observed entity specific gene clusters. In particular, in ACC tumors distinct gene expressions were shown ( Figure 1). Detailed comparison of the expression profiles of MEC, ACC, and AC-NOS confirmed that the differences in expression clusters of ACC in comparison to MEC and AC-NOS (Supplementary Figure 1A-1C), whereas MEC and AC-NOS differ only slightly from each other (Figure 2, Supplementary Figure 1D-1E). Importantly in ACC, genes of inflammatory pathways were upregulated such as TNF1 as well as interleukin 1 and 6 and mediators (e.g., IRAK 2, 3, IL1B, JAK3, STAT1, STAT4) (Supplementary Figure 1C). In MEC and AC-NOS, for example, the subunits of phospholipase C, D, FGF12 and the chemokine CXXC4 were highly expressed (Supplementary Figure 1E). www.oncotarget.com In a further hierarchical cluster analysis, the tumor entities were examined in detail, including primary tumor progression (pT) and locoregional lymph node metastasis (pN). Figure 2 shows the differences in gene expression patterns between the entities MEC, ACC and AC-NOS. With the exception of a few individual samples, the samples were grouped according to their entities and gene expression profiles. A grouping due to tumor progression or lymphogenic metastasis across the entities was not apparent. The grouping was based on similarities in gene expression was therefore based more on membership of an entity than on the degree of progression or metastasis. It should also be noted that these tumors generally have a higher T and N classification. Tumor-relevant gene expression signature in mucoepidermoid carcinoma The divergent gene expression pattern between tumor and non-tumor area of the mucoepidermoid carcinomas shows, as demonstrated in Figure 3A and Table 2A, a clear upregulation of COMP. Similarly, some Wnt pathway genes such as WNT7B, FZD10 and SFRP4 are significantly overexpressed, whereas WIF1, CXXC4, PLCB1 and 4 are downregulated. Upregulation is also found in matrix proteins such as COL1A1 and COL11A1. As shown in Figure 4A, the analysis of the corresponding tumor and non-tumor samples also revealed upregulation of COMP, matrix proteins such as COL5A1, COL11A1, and FN1, and genes of the Wnt pathway (WNT7B, FZD10, SFRP2), while PLCB1, CAMK2B, PLCB4 and WIF1 are downregulated (Table 3A). Tumor-relevant gene expression signature in adenoid cystic carcinoma The divergent gene expression pattern between tumor and non-tumor area of ACC showed, as demonstrated in Figure 3B and Table 2B, also a significant upregulation of COMP, BMP7 and MYB. An upregulation Oncotarget 4126 www.oncotarget.com is also found in matrix proteins such as COL2A1 and COL5A1. As shown in Figure 4B, Tumor-relevant gene expression signature in adenocarcinoma NOS The divergent gene expression pattern between tumor and non-tumor areas of the adenocarcinoma NOS also demonstrated a distinct upregulation of COMP as shown in Figure 3C and Table 2C. An upregulation is also found in matrix proteins like COL11A1, COL1A1 and FN1. All AC-NOS showed an overexpression of COMP, in ACC all tumors showed an overexpression except one, in MEC there were two tumor samples that showed no expression. Thus, an overexpression of 44/47 (93.6%) was found. Genetic differences within tumor entities in regard to signaling pathways (advanced analysis) Most notably, the overall expression studies and a detailed analysis of the expression pattern of matching tumor versus non-tumor samples, followed by advanced pathway analysis, proved a prominent dysregulation of the Wnt signaling pathway in the three SGC entities (see Figure 5). Moreover, transcript upstream analysis demonstrated a pronounced activation of the PI3K pathway in AC-NOS and MEC, which is a main target of current cancer therapeutic strategies (see Figure 5). DISCUSSION In the present study, a gene expression analysis of more than 700 genes in salivary gland carcinomas and their corresponding non-tumor tissue samples was performed. Salivary gland carcinomas of the three most common entities were considered. The gene expression profiles showed clear differences between tumor and non-tumor tissue across all entities. In addition, the entities showed a different gene profile when compared to one another. The Wnt signaling pathway and the PI3K signaling pathway were particularly striking in the individual examination of dysregulated genes and signaling pathways. These could represent a starting point for further research to deepen the understanding of the molecular genetic processes of salivary gland carcinomas and the development of new diagnostic and therapeutic options. In recent decades, recurrent and therapy-relevant gene changes have been demonstrated for various organs. For example, a therapy with tyrosine kinase inhibitors in treatment protocols has been implemented in EGFRmutated pulmonary carcinomas [13,14]. EGFR-directed treatment strategies were introduced in colorectal adenocarcinomas wild-type for NRAS and KRAS [12] and BRAF inhibitor treatment finds application in BRAF V600E mutated malignant melanomas [16]. A structured evaluation of the genetics of the tumor tissue in a high number of salivary gland cancers has not been performed yet [15]. This is due on the one hand to the rarity of this tumor entity and on the other hand to the heterogeneity of the tumors in salivary glands. Grünewald et al. were able to carry out mutation analyses of 84 tumors in 13 subtypes of parotid carcinomas. Using a next-generation sequencing methodology, mutations could be shown in 22 different genes; mostly affected were TP53, followed by RAS genes, PIK3CA, SMAD4 and members of the ERB family. However, these tumors driving genetic alterations were not found in MEC, ACC and AC-NOS [16]. Across all entities, it can be concluded from the data of this study that numerous genes of the Wnt signaling pathway are dysregulated in the examined salivary gland tumors (Tables 2 and 3). The strong and significant downregulation of WIF1 is consistent between the three entities. The significantly reduced expression of the genes PLCB4 (MEC and AC-NOS), PLCB1 (MEC), CAMK2B (MEC) and WNT5A (MEC and ACC) was also found in the investigated entities (Table 3). Pathological activation of the Wnt signaling pathway in colon carcinomas and melanomas has been described more than 20 years ago. Further evidence can be found for prostate cancer, leukemia and medulloblastoma [17]. The importance of the Wnt signaling pathway for salivary gland tumors was also highlighted as early as 1988. From mouse models, it is sufficiently proven that a constant activation of the Wnt signaling pathway contributes to hyperplasia and oncogenesis of the salivary glands. An increased expression of the WNT1 gene led to an increased incidence of breast and salivary gland tumors in mice [18]. In addition, it became clear that upregulation of the β-catenin gene (CTNNB1) and β-catenin target genes through inappropriate activation of transcription factors can trigger proliferation and de-differentiation of epithelial cells of the salivary glands and thus contribute to benign and malignant tumor formation [19][20][21][22][23]. The www.oncotarget.com expression of further genes of the Wnt signaling pathway was downregulated in this study. These are WNT5A, which is strongly and significantly decreased in MEC and ACC. In a review by Tabatabai et al., it is stated that an increased expression of WNT5A had a tumor-inhibiting effect in various tumors such as neuroblastomas, breast carcinomas, thyroid carcinomas and leukemia [24]. WIF1, which was significantly downregulated in all entities, was recently reported in several human carcinomas, correlating with aberrant Wnt/β-catenin signaling, including salivary gland ACC by Wang et al. [25]. In all entities COMP was highly upregulated. Cartilage oligomeric matrix protein (COMP) is an extracellular matrix protein expressed via different tumors and contributes to the progression of various malignant diseases. COMP is a 524 kDa Ca2+-binding glycoprotein indicated primarily in cartilage tissue and plays an important role in the formation of the extracellular matrix. In addition, it regulates the activation and inhibition of the complement system, which is an important system of innate immunity as the first defense against invasion by microorganisms and altered cells. COMP (also called TSP-5) belongs to the thrombospondin family and is dependent on Ca2+ in its structure and function. Mutations in the Ca2+-binding region of COMP lead to skeletal dysplasia [26]. COMP has been shown in recent studies to be a promising new marker for breast cancer [27]. It was shown that up to 79% of malignant breast tumors express COMP more strongly and that the expression is significantly related to factors such as low survival rate, larger tumors in vivo, increased invasion in vitro, protection against stress-induced apoptosis, more frequent metastasis and the Warburg effect. The large tumor volume is not caused, as previously assumed, by COMP-induced extracellular matrix formation in the tumor stroma, as the tumor mass is mainly composed of tumor cells and less of stroma cells. COMP-expressing cells appear to have a greater capacity for proliferation, invasion and metastasis [27]. A high proportion of COMP in breast cancer and in salivary gland tumors may indicate an analogy of tumor structure and tumorigenesis. Triantafyllou et al. demonstrated parallels of the pathology of salivary and breast cancers with respect to the composition of myoepithelial cells, stromal components, analogues of mucosa-associated lymphoid tissue, steroid receptors and intraparenchymal cells of monocytic origin [28]. This results in interesting questions for future projects regarding the analogy of tumor development of the significantly more frequent breast cancers and salivary gland carcinomas. In addition to COMP, all entities show dysregulated gene expression compared to normal tissue in numerous components of the PI3K pathway (Tables 2 and 3). Particularly in AC-NOS, upregulation of a large number of genes that are transcriptional target genes for activation of the PI3K pathway became evident. Interestingly, these target genes include genes, coding for extracellular matrix proteins such as FN1, SSP1, COL1A1 or LAMC2. Studies have shown that PI3K plays an important role in growth regulation in both healthy and malignant degenerate cells of many tumors, and that genes of the PI3K/AKT pathway are the most frequently altered genes in human carcinomas [29]. This signaling pathway could also be suitable as a diagnostic agent and therapeutic target domain. Various PI3K inhibitors are currently undergoing clinical trials, in combination or monosubstance therapy, in solid and hematological cancers [30]. Due to the multitude of genetic changes within this signaling pathway (p110, p85, AKT, mTOR, PTEN, etc.) there would be various molecular targets for a therapy whose further research seems promising. In addition, it is important to identify the key elements of the signaling pathway in order to achieve the most effective therapy possible. The demonstrated results suggested that in ACC, genes of inflammatory pathways were upregulated such as TNF1 as wells as interleukin 1 and 6 receptors and mediators (e.g., IRAK 2, 3, IL1B, JAK3, STAT1, STAT4) (Supplementary Figure 1C). This result seems unusual, as ACC has been described as less immunogenic in different studies [31]. ACCs showed a suppressed immune system in Tumor microenvironment in a study by Linxweiler. Characteristic were however the presence of M2-polarized macrophages and myeloid suppressor cells and a low Tumor mutational burden [31]. Especially macrophages release TNF and interleukin 1 and 6. Thus, the data do Another interesting result was also that the entity of AC-NOS, which can histologically represent a very heterogeneous entity, showed a rather homogeneous group in the evaluation of the nanostring analysis. Surely the largest limitation of the study is the low number of cases of the individual tumor entities. Multicentric studies should be pursued in order to be able to put the data presented here on a broader basis. Likewise, to limit it, this study involved only 3 entities, albeit frequent ones. No validation cohort could be measured due to the small number of cases of the individual tumors and in portions of insufficient tumor material. Corresponding measurements are being planned on a multicenter basis in subsequent projects. The PanCancer pathways covers a wide range of genes, there may be clinically relevant genes that were not assessed. The study does not address intratumoral heterogeneity. It would be conceivable that gene expression in the area of the invasion front differed from those in the solid tumor area. The status of the fusion genes, especially in ACC and MEC, were not known in the patients and were not included in the study. These could have an influence on the results. Further studies should investigate a correlation. In the group of AC-NOS is an entity of adenocarcinomas, which cannot be divided further. Thus, certain heterogeneity of the tumors is assumed. It may thus be difficult to identify a consistent pattern of gene expression in this entity. In future studies, the results of the gene analytical studies should also be correlated with clinical factors such as survival rates, response to therapy, metastasis or recurrence rates. Despite these limitations, the present work contributes to the knowledge of the genetic mechanisms Oncotarget 4135 www.oncotarget.com of this rare form of tumor to a large extent and offers a multitude of starting points for more intensive research due to the high number of genes analyzed. Patient specimens and clinical data The analysis was conducted according to the Declaration of Helsinki on biomedical research involving human subjects. A total of 60 PGC patients with MEC (n = 20), ACC (n = 20) and AC-NOS (n = 20) were applied to the study. All patients were treated for PGC with primary surgery between 1990 and 2014 at the Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Cologne, Germany. All specimens were obtained from the tissue bank of the Institute for Pathology at the University Hospital of Cologne (Cologne, Germany) and were used in accordance with the policies of the local institutional review board of the hospital. All specimens used for gene expression profiling were reviewed and selected by two experienced pathologists (J. M., U. D.). The data that support the findings of this study are available from the corresponding author upon reasonable request. Patients who were staged clinically as being negative for lymph node metastasis (cN0) had been treated with selective neck dissection at least for levels I-III. Patients with clinically positive cervical lymph node status (cN1-3) had received a modified radical neck dissection. In cases of high-grade carcinoma of MEC (G3 or G4 based on the AJCC Cancer Staging Manual) incomplete resection margins, loco-regional neck lymph node metastasis and perineural invasion adjuvant RT were additionally administered. Tumor and corresponding non-tumor areas from formalin-fixed and paraffin-embedded tissues were macrodissected from formalin fixed and paraffinembedded tissues and used for extraction. After RNA quantification, 94 matching tumor-non-tumor samples from a total of 47 cases (Supplementary Table 1 and Table 1) including 14 cases of ACC, 20 cases of AC-NOS and 13 patients suffering from MEC from which sufficient RNA was isolated-were chosen for expression profiling of oncogenic and tumor suppressor genes by means of NanoString's nCounter technology. Microdissection and RNA isolation Sections were prepared and stained with hematoxylin and eosin (HE) according to the standard protocol. Histological examination using HE-sections provides a targeted assessment of the areas that have the highest-at least 70-80%-tumor content. Corresponding to the sample hematoxylin and eosin staining, tumor and non-tumor areas were collected via macrodissection as described before the sections were scraped off with a scalpel and collected into plastic tubes [32]. The total RNA was then isolated using the RNA FFPE kit from Promega on the Maxwell ® 16 LEV (Promega AS1260, Heidelberg) according to the manufacturer's instructions. RNA concentration was quantified by the QuantiFluor ® RNA System, as recommended by the supplier (Promega, E6090). After concentrating the RNA to 150 ng in 5 µL by means of a speed vac, the samples were taken for the Nanostring analysis. N-Counter hybridization and gene expression analysis 150 ng RNA from each specimen were used for N-Counter hybridization technology using the PanCancer Pathways Panel (Nanostring, Seattle, WA, USA) (Supplementary Table 2) following the manufacturer´s recommendations. Briefly, the RNA extracts to be tested are denatured at 85°C and incubated with the reporter and capture probes in a thermocycler overnight (12 hours) at 65°C. Fluorochrome barcoded hybrids were captured and purified using the Prep Station platform (Nanostring) and subsequently quantified by the Digital Analyzer (Nanostring). For normalization of expression profiles, 35 housekeeping genes were selected using the GeNorm algorithms (Supplementary Table 3). Expression profiles were then studied using the NSolver 4 software from Nanostring. The analysis was carried out according to the manual of the NSolver 4 software (Gene Expression Data Analysis Guidelines, MAN-C0011-04, https:// www.nanostring.com/support/data-analysis/nsolverdata-analysis-support). Background signals, determined as the mean values of all system negative controls, were subtracted from probe signals. Hierarchical clustering was performed using the algorithm of Euclidean distance calculation. For pathway analysis, either the Nanostring pathway annotation or GO analysis was carried out. In addition, we studied gene expression of the matching tumor and non-tumor samples, considering only genes that were divergently expressed more than twofold in tumor vs non-tumor of greater than 66.7% of the respective PGC entity. CONCLUSIONS Gene expression analysis revealed a clear difference in the gene expression profile between tumor tissue and non-tumor tissue in MEC, ACC and AC-NOS. In addition, the entities can be distinguished from each other by their differential gene profile. In addition to a large number of dysregulated genes, the expression pattern of individual genes manifests a cross-entity dysregulation in the Wnt signaling pathway, in particular a downregulation of	2020-11-12T09:06:07.065Z	2020-11-10T00:00:00.000	{ "year": 2020, "sha1": "00101b9d73340cc9ed7f00ecf0538119c682e8dc", "oa_license": "CCBY", "oa_url": "https://www.oncotarget.com/article/27797/pdf/", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a65f1d438a02fedd013f04fb2a8671ecb7bb768f", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
56021378	pes2o/s2orc	v3-fos-license	A simulation-based Data Envelopment Analysis (DEA) model to evaluate wind plants locations Article history: Received July 10, 2014 Accepted December 31, 2014 Available online January 3 2015 As the world is getting overpopulated and over polluted the human being is seeking to utilize new sources of energy that are cleaner, cheaper, and more accessible. Wind is one of these clean energy sources that is accessible everywhere on the planet earth. This source of energy cannot be stored for later use; therefore, environmental circumstances and geographical location of wind plants are crucial matters. This study proposes a model to decide on the optimum location for a wind farm among the demand area. To tackle the uncertainty related to the geographical position of the nominated location such as wind speed; altitude; mean temperature; and humidity; a simulation method is applied on the problem. Other factors such as the time that a plant is out of service and demand fluctuations also have been considered in the simulation phase. Moreover, a probability distribution function is calculated for the turbine power. Then Data Envelopment Analysis (DEA) performs the selection between all the nominated locations for wind farm. The proposed model takes into account several important elements of the problems. Elements such as land cost; average power received from the wind blowing; demand point population etc. are considered at the same time to select the optimum location of wind plants. Finally, the model is applied on a real case in order to demonstrate its reliability and applicability. Growing Science Ltd. All rights reserved. 5 © 201 Introduction Wind energy, the world's fastest growing energy source is a free, clean, renewable, never-ending energy source that is totally accommodated with the environment.The uneven heating of the atmosphere by the sun, the irregularities of the earth's surface, and the rotation of the earth cause winds (http://windeis.anl.gov).Wind is available in large quantities and does not make pollution.The point that mostly makes this source of energy important is that no matter how much it is used today; there will still be the same supply in the future.Considering these facts, utilizing this kind of energy is rapidly increasing in the world.Despite all the benefits of wind power, there are some challenges related to it.The major challenge to use wind as a source of energy is that it is intermittent and does not always blow when electricity is needed.Besides, not all kind of winds can be harnessed to meet the timing of electricity demands.Moreover, wind energy cannot be stored.However, electricity generated by wind can be stored, if batteries are used.Additionally, efficient wind sites are often located in locations that are far from areas that electric power is in demand (such as factories and cities).Having all these deficiencies in the mind, locating the wind power facilities comes to immense importance.An ideal locating of the wind plants can cope with most of these deficiencies.For instance, if a set of wind plants be located in different locations with different wind characteristics and demand fluctuation in a way that they cover their out of service time, low blowing time and so on; it would diminish the effect of wind intermittency.In Addition, an appropriate locating can help to find less costly land and more usable places.Nevertheless, recently there are wind plants placed in offshore, however it is not always reachable and feasible to build a wind plant in water. Location decisions are used in any field of facility establishment.The term "location" refers to the modeling, formulation, and solving of a class of problems that can best be described as setting facilities in some given space.There are four components that characterize location problems (Bhatnagar & Sohal, 2005): 1) Customers, who are presumed to be already located at points or on routes, 2) Facilities that will be located, 3) A space in which customers and facilities are located, and 4) A metric that indicates distances or times between customers and facilities Applications of location problems vary from gas stations and fast food outlets to landfills and power plants.In a basic perspective, the facility location problem consists of a set of potential facility sites 'L' where a facility can be opened or not, and a set of demand points 'D' that must be serviced.The goal is to pick a subset of facilities 'F' to be open, to minimize the sum of distances from each demand point to its nearest facility, plus the sum of opening costs of the facilities.In recent years, an increasing tendency has been shown to work on the location optimization in the field of new and clean energies (Aytun Ozturk & Norman, 2004;Azadeh et al., 2008;Bowling et al., 2011;Rentizelas et al., 2009).This study contributes to the literature by proposing a model that performs to find the most appropriate location for establishing a wind farm.The model is a combination of discrete event simulation (DES) method and data envelopment analysis (DEA).The simulation phase calculates some factors that directly influence the efficiency of the location that a plant should be built in.This study proposes a probability density distribution function for the power produced by a wind turbine that helps the simulation phase to be more precise.Measures such as altitude, annual mean temperature, and humidity that affect the probability distribution are considered in the paper.The results of the simulation phase are input to the DEA phase that combines the simulation results with the other factors that are deterministic.Factors such as cost of land; proximity to demand points; weather situation and so on that are discussed in detail in the section five; are directly considered in the DEA model.Finally, DEA ranks all nominated locations.Overview of the research is shown in Fig. 1. The rest of the paper is organized as follows: First, an overview of the literature of the subject is presented in section 2. Then the method of the paper is discussed in two parts in section 3. The first part of the method section deals with DEA and the second part contribute to discrete event simulation.The third section presents the model.A numerical example has been presented in the fourth section.Finally, the conclusion of the paper is discussed in section five. Simulation Input Parameters Calculating turbine power Probability Distribution Simulation Phase Simulation Output Measures DEA Phase Final Ranking of the Nominated Locations DEA Indicators Fig. 1.Overview of the research Literature Review In this section, we review some of important studies that are related to this study.First, the body of literature that is related to the studies that include facility location problem using simulation or DEA is overviewed.Then a review of studies that have utilized the combination of aforementioned methods is presented.Certainly, the most related study to this work is done by Azadeh et al. (2011).They proposed an integrated hierarchical data envelopment analysis to optimize the location of wind plants.Moreover, they used two multivariable methods namely Principal Component Analysis (PCA) and Numerical Taxonomy (NT) to validate the results of the DEA model.They also introduced several indicators that are suitable for assessing a candidate wind plant's location.The indicators they presented were population and human labor; distance of power distribution networks; land cost; the average wind blow; the intensity of natural disasters occurrence; quantity of proper geological areas and quantity of proper topographical areas.In another work, Azadeh et al. (2008) presented a model for deciding on location of solar plants.They utilized an integrated hierarchical approach by DEA, PCA, and NT.They used PCA and NT to validate the results of the DEA.Klimberg and Ratick (2008) developed location modeling formulations to find optimal and efficient facility location/allocation patterns.They utilized data envelopment analysis efficiency measures to execute the model.Their model simultaneously considered the interaction of spatial efficiency of different location patterns using least cost objective and the facility efficiencies at those sites by DEA.Evaluating performance and efficiency of 22 seaports in East Africa and the Middle East was done by Al-Eraqi et al. (2007).They used DEA to perform their model.Mitropoulos et al. (2012) suggested a methodology to consider health service provider efficiencies based on multiple measures.They took advantage of DEA and Integer Programming (IP) location allocation models to do so.Guo (2009) proposed a model to evaluate the efficiencies of objects with fuzzy input and output data.He utilized fuzzy DEA for solving the problem.The primary factors that he used in the paper's case study were rent of establishment; traffic amount; level of security; consumer consumption level and competition level. Andor and Hesse (2011) used a Monte Carlo experimentation to analyze the optimal approach for determining individual efficiency scores.They also compared the two most popular estimation methods, DEA and Stochastic Frontier Analysis (SFA).McMullen and Frazier (1999) tested several heuristics to select the best assembly line balancing strategy by using data envelopment analysis.They obtained several output performance measures from the simulation. These measures were used as outputs for DEA.In a recent study, to select a site for wind farms, Chatterjee and Bose (2013) proposeed a COPRAS (COmplex PRoportional ASsessment) based multicriteria decision-making (MCDM) methodology under fuzzy environment with the help of multiple decision makers.Their study's purpose was to focus the applicability of COPRAS-F as a strategic decision making tools to handle the group decision-making problems. Despite the researches which have taken nonparametric and probabilistic methods into account, numerous researches have applied mathematical modeling methods in order to address location problem (Arvan et al., 2014;Gendron et al., 2013;Gollowitzer & Ljubić, 2011;Marín, 2011). Data envelopment analysis DEA is a linear programming based method.First, the idea of DEA initiated by Farrell (1957) but later it developed by Charnes, Cooper, and Rhodes (1978).DEA method has been increasingly used since the pioneering work of Charnes et al. (1978) in different fields to evaluate efficiency of a set of homogenous DMUs.DEA commonly applies in different fields such as electrical industries; hospitals; university departments; refineries; location problems and manufacturing enterprises etc. in order to measure the efficiency.DEA is a nonparametric method so it can be used for determining the quantity of inefficiency in each input relative to each output.In this section, a brief review of the salient features of DEA is done.For more investigation the reader is referred to Abdollahi et al. (2014), Andersen and Petersen (1993), Charnes (1994) and Ganley and Cubbin (1992). Generally, there are two basic models for DEA: Constant Returns-to-Scale (CRS) or CCR model and Variable Returns-to-Scale (VRS) or BCC model.Both models could be either input-orientated or output-orientated.In input oriented, the outputs are assumed constant and accordingly DEA method defines the frontier by looking for the maximum possible proportional reduction in the input usage.In output-oriented, input levels are fixed and DEA method seeks to maximize the proportional increase in output value.The efficiency score when there are multiple inputs and outputs is defined as (Talluri, 2000): Let (X1, X2, … , Xm) be the input values, (V1, V2, … ,Vm) weights assigned to them, (Y1, Y2, … , Ys) be the output values and (U1, U2, … , Us) weights assigned to outputs with n decision-making units, models are as follows (Cooper et al., 2011): A basic input oriented CCR model: , 0 A basic input oriented BCC model: , 0 The main difference between CCR and BCC models is existence of W in BCC model that defines return to scale measure (Cooper, 2011). Discrete Event Simulation: Simulation allows one to specify a system accurately by using the logically complex, and often nonalgebraic, variables and constraints.Complex and stochastic systems can therefore be modeled through simulation (Abdollahi, Arvan, Omidvar, & Ameri, 2014).In the field of simulation, a discrete-event simulation (DES) models the operation of a system as a discrete sequence of events in time.Each event occurs at a particular instant in time and marks a change of state in the system (Robinson, 2004).The position of discrete event simulation in the different systems is shown in Fig. 2. Continuous A discrete-event simulation model is defined by three attributes (Bossel, 1994): 1 . Stochastic: at least some of the system state variables are random; 2. Dynamic: the evolution time of the system state variables is important; 3. Discrete-event: events that occur at discrete time instances only make significant changes in the system state variables. By definition, the nature of discrete-event simulation is the way that one does not actually experience with or modify an actual system.Instead, one develops and then works with a discrete-event simulation model.This matter is also declared in this paper.Since the probability density, distribution of the wind speed is apparently a continuous one so a method is proposed to transform the probability density distribution of the wind speed to probability density distribution of turbine power.The transformation is done in a way that we consider the rotation of the wind turbines made by the wind that is blowing.It is known that the power of the wind directly affects the speed of turbines so by testing the speed of turbines due to winds with different powers we can estimate the probability distribution function of the wind turbine power. Simulation Design In this section, a method to reach the probability distribution function of the wind turbine power is presented and the factors that affect the simulation are reviewed.Finally, the simulation model is proposed in this section.Based on (Grogg, 2005) total energy that may come to an imaginary area A during the time t is: where  is the density of the air that is in kilograms per cubic meter; v is the wind speed based on meter per second; AVt is the volume of the air passing through area A. In this formulation, A is considered perpendicular to the direction of the wind. 2 1 2 v  is the kinetic energy of the moving air per unit volume. Nevertheless, we know that power is energy per unit time so the wind power flowing into area A is: If A is equal to the rotor area of the wind turbine then the power that performs to the turbine is: In this formulation, r is the radius of the rotor based on the meter.When the wind blows into the turbine it affects the wind speed and this fraction cause lower efficiency in transforming the wind energy to the turbine.Albert Betz in 1919 proved that a turbine could take advantage of maximum 59 percent of the wind that blows on its way so coefficient  is added to the Eq. ( 16). 2 3 In Eq. ( 17), the unit of power is the watt.Fig. 3 demonstrates a typical wind turbine power output with wind speed.As it is shown in Figure 3, the output power is kept constant when wind speed is higher than the rated wind velocity, even though the wind turbine has the potential to produce more power.This matter happens because there is a risk of damage to the rotor.As a result, a braking system is employed to bring the rotor to a standstill.This limit is called the rated power output speed and is usually around 14 meters per second (Billinton & Bai, 2004;Wang & Nehrir, 2008).Moreover, it can be observed that after wind speed exceeds 25 meters per second the system is taken out of operation to protect its components.This limit is called cutout speed and is usually around 25 meters per second.However, it is a matter of common observation that the wind is not steady.Thus in order to calculate the power delivered by a wind turbine from its power curve, it is inevitable to recognize the probability density distribution of the wind speed.To estimate the frequency of wind speeds at a particular location, a probability distribution function often fits the observed data.However, different locations have different wind speed distributions but based on researches wind speed distributions of hourly/tenminute in many locations closely follows the Weibull model (Dorvlo, 2002;Seguro & Lambert, 2000).Moreover a Rayleigh distribution can be used to simply estimate the probability distribution function of the wind speed that is equivalent to a Weibull distribution with k=2 (Celik, 2004). The probability distribution function of a Weibull random variable x is: where 0 k  is the shape parameter, 0   is the scale parameter of the scale parameter of the distribution.The mean and variance of Weibull distribution function can be expressed as: In which In addition, the cumulative distribution function is as below: We already proved that the power output from a wind turbine is a function of the speed that a turbine can get (Eq.( 17)).Clearly, based on Figure 3 the speed of a turbine varies with steady wind speed. It is proved that if x is a random variable on  set and g is a function so that : To calculate the probability density function of y in case function g is invertible (means that 1 g  exists), and also increasing, then equation ( 23) can be extended to: (24) If we substitute v in Eq. ( 17) with its probability function that is considered to be a Weibull probability function.Then, provided Eq. ( 17) being an invertible and non-decreasing function based on v, Eq. ( 24) can be used to calculate the probability function of turbine power.Since  it can simply be transformed into: That is a unique function and clearly, when p increases v increases subsequently; thus, this is an increasing invertible function.Note that as already mentioned when v exceeds the rated output wind, the system remains in a constant output and when it exceeds the cutout speed, the system stops operating.Then, to calculate the turbine power probability function 1 ( ) g y  is replaced by equation ( 24) and the cumulative distribution function F would be a Weibull that is calculated in equation ( 22).Therefore, we have: This equation equals to: Calculating the integral in Eq. ( 27) results in Eq. ( 28) that is the turbine power cumulative distribution function. By differentiating the right side of Eq. ( 28) with respect to p, the probability density functions of turbine power can be calculated: Thus, the probability density functions of turbine power would be: In the second row of Eq. ( 30) rated wind speed is considered so that when the wind speed is less than 14 meters per seconds, the probability density function of turbine power can be used, otherwise the probability of   14 25 V   should be calculated.In this probability, the system works steadily and by increasing the wind speed the power does not increase.For   is taken out of operation in order to protect its components. After these calculations, this function can be used to generate random variables related to turbine power. In this function, the parameter that is changeable by the place of turbine is  the density of the air. Note that  changes for different kind of turbines but the place of the turbine does not directly affect this parameter; therefore, we do not consider  as a variable of the simulation. The mass per unit volume of atmospheric gases is called density of air.Air density and altitude have a reverse connection, so that decreasing the attitude causes increasing air density.It also changes with variation in temperature or humidity.According to International Standard Atmosphere (ISA), at sea level and at 15 °C the air has a density of approximately 1.225 kg/m3 (0.0023769 slugs/ft3). Since in this paper nominated locations to locate wind plants are in different geographic positions; thus, they differ in terms of attitude, mean temperature during the year and humidity factors.Therefore, these factors are considered in simulation model in addition to wind speed. Based on Table 1 increasing the temperature decreases the quantity of  .Moreover, increasing altitude decreases density of air (ρ).All the items that results from the simulation phase are listed in Table 2. Items 1, 2 and 3 in Table 2, affect the parameter  (density of the air) in the probability distribution function of produced power.The density of the air is determined Based on the formulation below (Davis, 1992;Mujumdar, 1996): The final formulation that would be used to determine density of the air in the probability distribution function of turbine power is: Item number 4 in Table 2 responds to the time that the system is unavailable and maintenance should be done on the turbines.By experience, the distribution of the turbine failure is considered to be exponential. Item 5 considers demand fluctuations during a day.A day is divided into three intervals.First interval is from 8 AM to 16 PM that the demand and consumption of electricity is high due to industrial and office usage.Second interval is from 16 PM to 24 PM that has the highest demand among three parts because of the need for electricity in purpose of illumination.The last interval that has the lowest consumption rate is from 24 PM to 8 AM. Item 7 deals with the wind speed characteristics that are demonstrated in wind Weibull distribution function.Parameter k is the shape parameter and  that is the scale parameter and in our problem is equal to 0, but because this make the probability function infinite a very small number is determined for this parameter. The output measures of the simulation phase that are the input for the DEA phase are listed in Table 3. Table 3 The output measures of the simulation phase It is assumed that at each nominated location, 25 wind plants are installed in a wind farm and they have 500 kW turbines. In the simulation model, it is assumed that the power produced by wind can be stored for 10 minutes so if there is no demand for that power it will be wasted.Note that the usage of wind-generated electricity has priority toward other resources.The simulation phase has been carried out in AweSim simulation software for 100 repetitions in which each repetition was about simulating the model for 720 hours equal to one month.The simulation has been applied for every location candidate and the results are stored in Table 4. DEA Structure From the viewpoint that some indicators and measures to select the best location for the wind plant are deterministic; thus, the DEA phase should consider these measures as well as the measures that have resulted from the simulation phase.Azadeh et al. (2011) in their study take account of location optimization of the wind plants by an integrated hierarchical data envelopment analysis.They introduced some indicators that were adapted from (Aras et al., 2004) work.These indicators are as follows:  Population and human labor: Every industrial unit should have a low distance to the population who that industry is going to serve.Wind plant follows this matter too.Therefore, the closer the plant is to a mass population, the better.As a result, the nearest population to the scenario location is considered as an indicator. Distance of power distribution networks form the demand point: Proximity to the demand point has always been an important factor in location optimization problem.The transportation cost of materials for constructing the plant and the maintenance cost are directly related to the distance of power distribution networks form the demand point. Land cost: Apparently this indicator effectively influence the cost of constructing a wind plant. The intensity of natural disasters occurrence: Natural disasters such as earthquake, storm and fluid can harm the plant easily.Therefore, in selecting the optimum location to build a plant, this indicator should highly be regarded. Quantity of proper geological areas: A wind plant cannot be built in every ground.The ground slope, material and grade of soil and many more items affect building a plant on that ground. Some of these indicators are used in this study as well. Computational Experiment In this section, we apply location optimization of wind plants in 24 nominal locations in Iran to examine the applicability of the model.The locations are the DMUs of the DEA problem.Table 4 presents the values of simulation phase parameters the for these locations.This information is gathered from http://www.weatherbase.com/and http://www.suna.org.irwebsites.Table 5 shows the results of the simulation phase for these locations.Results of simulation phase are consisted of time out of service (second per year) for each plant that is calculated based on the maintenance time of each plant; satisfied demand through wind plants (GWh/day) that is the average amount of demand directly satisfied by the plant; satisfied demand from other resources (GWh/day) that is the amount of demand satisfied through other power resources; wasted power (GWh/day) that since the electricity power can't be stored for a long time should be considered and average power generated (GWh/day) that is the average amount of electricity power generated by the plant in a day. Moreover, the values of indicators that were discussed in previous section are listed in Table 6.The indicators are population and human labor; distance of power distribution networks form the demand point; land cost; the intensity of natural disasters occurrence; and quantity of proper geological areas.These data are adapted from (Azadeh et al., 2011).Efficiency scores and ranking the nominated locations is presented in Table 7. Conclusion In this study, a model was proposed to find the optimum location for building a wind plant.The electric power generated by the wind plants is highly affected by the wind speed; therefore, a simulation approach has been proposed in the first phase of the model to handle the uncertainty of the wind speed.Moreover, a probability density function has been calculated for the output power of wind plant respect to the wind speed.In addition, for density of the air () that is a critical parameter in the before mentioned probability function, a formulation has been presented.This formulation calculates the  based on attitude, temperature and humidity.The density of the air directly affects the power generated by the turbines.In the second phase of the model, DEA deals with the selection and ranking the nominated locations.In this phase, input data of the DEA are two folded: some are derived from the simulation phase and some enter directly to the DEA.The indicators that were derived from the simulation phase were time out of service; the amount of demand that is satisfied through other power resources rather than wind plants; the amount of power that is wasted in wind plant due to lack of demand for it (since it is inefficient to save electricity for a long time); the amount of demand that is satisfied by the wind plants and the average power received from wind speed.The indicators that enter directly to DEA phase are quantity of proper geological areas; quantity of proper topographical areas; population and human labor; the intensity of natural disasters occurrence. For future research, one can consider the simulation model with different turbine models.Moreover, a hierarchy model could be proposed to handle the connections between indicators.The fluctuations of the weather also can be a good area to work on in the simulation model.Using Fuzzy Data Envelopment Analysis (FDEA) for ranking the locations in a fuzzy environment can also be considered for future development of this paper. Fig. 2 . Fig. 2. The position of discrete event simulation in system structure A basic input oriented BCC model: Fig. 3 . Fig. 3. Typical wind turbine power output with wind speed of water vapor or partial pressure (Pascals) P  Total air pressure (Pascals) v R =Gas constant for water vapor R= Universal gas constant that is 8314.32 d M =Molecular weight of dry air that is 28.964 (gm/mol) v M = Molecular weight of water vapor that is 18.016 (gm/mol) T= Temperature, deg K= deg C+273.15Byreplacing P with formulation below, we reach a function for density of the air based on humidity, temperature, and attitude above sea level(Aras, Erdoğmuş, & Koç, 2004 Table 1 Density of air sensitivity against Temperature and Altitude Table 2 Items Considered in Simulation Table 4 Values of parameters of simulation phase Table 5 Results of simulation phase that are considered as input and output measures for DEA phase Input Output Azadeh et al. (2011)t location or city for placing a wind plant is Manjil, after that Zabol has the best rank and Neyshabour, Kerman and Zahedan come next.Previous studies in Iran reveal that Manjil, Sabzevar and Zabol have the best potential to place a wind plant.Moreover the study byAzadeh et al. (2011)reports Manjil, Birjand, Zabol and Sabzevar as the best candidates.These results demonstrate that the results of this study are similar to other studies.	2018-12-05T02:58:51.698Z	2015-01-01T00:00:00.000	{ "year": 2015, "sha1": "0c96eee7326fb39a9848bd204465d1793b0ffbcf", "oa_license": "CCBY", "oa_url": "https://doi.org/10.5267/j.dsl.2015.1.001", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "0c96eee7326fb39a9848bd204465d1793b0ffbcf", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Engineering" ] }
15414405	pes2o/s2orc	v3-fos-license	Local moment approach to multi-orbital Anderson and Hubbard models The variational local moment approach (V-LMA), being a modification of the method due to Logan {\it et al}., is presented here. The existence of local moments is taken from the outset and their values are determined through variational principle by minimizing the corresponding ground state energy. Our variational procedure allows us to treat both fermi- and non-fermi liquid systems with many orbitals as well as insulators without any additional assumptions. It is proved by an explicit construction of the corresponding Ward functional that the V-LMA belongs to the class of conserving approximations. As an illustration, the V-LMA is used to solve the multi-orbital single impurity Anderson model. The method is also applied to solve the dynamical mean-field equations for the multi-orbital Hubbard model. In particular, the Mott-Hubbard metal--insulator transition is addressed within this approach. Introduction The single impurity Anderson model (SIAM) is one of the most investigated models in condensed matter physics [1]. This model is regarded as a prototype to understand and describe: i) properties of metals with magnetic atoms [16], ii) charge transport through quantum dots [24], iii) Mott-Hubbard metal-insulator transitions (MIT) within the dynamical mean-field theory (DMFT) [23,6,14,28,30,27], and iv) a crossover between weak and strong coupling limits and confinement phenomena. The SIAM consists of a term describing band electrons coupled by hybridiza-tion to a term corresponding to a single impurity where the local Coulomb interaction is taken into account [1]. In the featureless hybridization limit the SIAM is solved exactly within the Bethe ansatz or conformal field theory techniques so the ground state and the whole excitation spectrum as well as thermodynamics are exactly known [16]. Unfortunately, these methods cannot in practice provide dynamical quantities, for example one-particle spectral functions or dynamical susceptibilities, for all interesting energies. Also the (asymptotic) exact solvability is not possible for a general hybridization term. For practical applications of the SIAM one has to rely on either a numerically exact or an analytical but approximate solution. Numerically exact methods, like the numerical renormalization group (NRG) [5] or the determinant quantum Monte Carlo (QMC) [14] are very time (CPU) consuming. In particular, the CPU is very long when the number of orbitals is large in the NRG case and when the temperature is low in the QMC case. Also to extract dynamical quantities is a rather tricky task [17]. Reliable analytical methods are therefore needed. One of such methods, which recovers properly both weak and strong coupling limits, is a local moment approach (LMA) invented recently by Logan et al. [26]. The LMA is a perturbative method around an unrestricted Hartree-Fock solution with broken symmetry, i.e. with a non-zero local magnetic moment. The broken symmetry is restored at the end by taking the average of the solutions corresponding to different directions of the local magnetic moment [26]. In the present contribution we describe the LMA method and our implementation of it, which is different from the original one [26] by the way of how the value of the local moment is determined. Namely, we use the variational principle demanding that the ground state energy is minimized by the physical value of the local moment. Therefore we use the name variational local moment approach (V-LMA) for this method. Such a procedure allows us to easily generalize the V-LMA for multi-orbital models as well as for finite temperatures and systems with disorder [8,7,9,10]. We also discuss the Luttinger-Ward generating functional for the V-LMA and claim that this method belongs to the class of conserving approximations. The application of LMA for studying the electron flow through quantum dots and the Mott-Hubbard MIT is addressed at the end of the contribution. Local moment method in one orbital SIAM The single impurity Anderson model is given by the Hamiltonian where the conduction electrons are described by where ε k is an energy (a dispersion relation) for an electron in a state k and spin σ = ±1/2, the impurity electrons with the local Coulomb interaction U are represented by with n dσ = d † σ d σ , and the hybridization between conduction and impurity electrons is All local (on impurity site) properties are expressed by the hybridization function and not by ε k and V k separately. This can be proved by tracing out the noninteracting conducting electrons. Mean field solution of the single impurity Anderson model The Hartree-Fock mean-field solution of the SIAM is obtained by factorizing the interacting term n d↑ n d↓ ≈ n d↑ n d↓ + n d↑ n d↓ − n d↑ n d↓ [1]. For the interaction U above U c and corresponding impurity electron densitiesn d the mean-field solution is unstable toward the local moment formation with non-zero moment µ ≡ n d↑ − n d↓ . The solution is doubly degenerate because of two equivalent directions of the local moment µ = ±\|µ\|, which give the same energy of the system. The local (impurity) Green function within the Hartree-Fock solution is where the static Hartree-Fock self-energy Σ HF σ = U nσ and δ → 0 + . Since there are in principle two possible signs of the local moment, there are two different possible Hartree -Fock Green functions denoted by G A σ (ω) HF and G B σ (ω) HF that differ only by the sign of the local moment and depend parametrically on its value \|µ\|. The fundamental deficiency of the Hartree-Fock approximation is that it leads to a broken symmetry solution which cannot persist in the thermodynamic limit, i.e. a single impurity cannot lead to the magnetic solution in the infinite system. Also this solution does not recover the singlet ground state known from the exact Bethe ansatz solution. Nevertheless it turns out to be useful as a starting point in the further perturbative calculation combined with the symmetry restoration. Two self-energy description The two Hartree-Fock Green functions G A,B σ (ω) HF are used in the time-dependent many-body perturbation expansion. Within the random phase approximation (RPA) the polarization diagrams are and correspond to spin flip processes as represented by the Feynmann diagrams in Fig. 1. For each type of the mean-field solution G A,B σ (ω) HF we have the corresponding self-energy depending on frequency and parametrically on \|µ\| as well. The full RPA-Green functions G A,B σ (ω) are constructed by using the Dyson equation separately for A and B solutions. Note that G A,B σ (ω) depends parametrically on still unknown \|µ\|. Symmetry restoration ansatz To restore the spin-rotational symmetry Logan et al. [26] proposed the following ansatz for the full symmetrized Green function Within the LMA the physical Green function is an average of the two solutions with equal probabilities. Although each G A,B σ (ω) is determined within the renormalized perturbation scheme the final Green function turns out to capture nontrivial nonperturbative physics as was shown by Logan et al. [26] and is also reproduced below. In particular, the LMA is able to recover the Kondo peak in the spectral function correctly with the exponential width. Determining the value of local moment The value of the local moment is a free parameter and must still be determined. In the original approach, Logan et al. [26] imposed the Fermi liquid condition to determine \|µ\| at zero temperature. This condition might be too restrictive at finite temperatures or in the multi-orbital cases. Therefore we decided to find the physical solution to the problem by minimizing the relevant thermodynamical potential with respect to \|µ\| [20]. At zero temperature the relevant potential is just the ground state energy of the system, i.e. where in the case away of half-filling the particle density n must also be determined. The variational method reproduces the Fermi liquid properties where they are expected. Ground state energy in the Anderson impurity model The ground state energy of the SIAM is given by E G = 0\|H\|0 . This quantummechanical average consists of two parts: the bulk, which is proportional to the system volume and is independent of the local moments, and the impurity part, which depends explicitly on \|µ\|. The impurity part of the ground state energy, expressed by the local Green function G σ (ω) and the hybridization function ∆ (ω), is equal to [21] where the contour integral is over the half circle in the upper complex plane. LMA as a conserving approximation According to Kadanoff and Baym [3] any approximate theory is conserving if there exists a Luttinger-Ward functional Φ[G] for this theory. It is necessary that this functional: i) is universal, i.e. it dependents only on the full propagator G σ (ω) and not on the atomic properties of the system and ii) has a functional derivative with re-spect to G σ (ω) which is by definition equal to the self-energy of the system. It can be shown [20] that the LMA is a conserving approximation and we can construct explicitly the Luttinger-Ward functional where Tr = T ∑ σ ∑ iω n e i0 + and the functionals Φ A,B RPA are represented diagrammatically by the RPA diagrams with G A,B σ (ω) respectively. The constraint that G = 1 2 (G A + G B ) must be satisfied. Finally, the free energy functional is given by and the stationarity condition δ Ω [G]/δ G gives the Dyson equation and the physical solution for G. The fact that the LMA is a conserving approximation, as we proved above, makes this theory reliable in describing correlated electron systems, in particular in the intermediate regimes of parameters. Local moment approach for the multi-orbital SIAM In reality the magnetic impurities in metals are atoms with partially field d-or forbitals. Such orbitals have degenerate levels. Even when a particular environment which decrease the symmetry and leads to e g and t 2g split levels, partial degeneracy between orbitals remains. The appropriate model to describe such situations is the multi-orbital single impurity Anderson model. It describes a single impurity with many orbital levels α, which can be degenerate or split depending on the singlebody matrix element ε α . In this case the electrons can interact via direct (densitydensity) type of the interaction and via the exchange (Hund) interaction. Microscopically, the single impurity Anderson model with many orbital levels is given by the Hamiltonian: where the direct U and U ′ as well as exchange J interactions between the electrons of spin σ and on orbitals α or β are taken into account. This multi-orbital version of the SIAM is also of interest in quantum dot physics, where dots with a few orbitals can be prepared and investigated experimentally. One of the interesting aspect of such system is the possibility to observe the orbital Kondo effect [18]. LMA generalization In the mean field approximation of the multi-orbital SIAM we also encounter a doubly degenerate solution, where the two possible Green functions differ only by the sign of the impurity magnetic moment. Within the LMA, we introduce for each pair of orbital indices α and β the two Green functions G αβ ,A σ (ω) HF and G αβ ,B σ (ω) HF that correspond to the two possible directions of the total magnetic moment on the impurity. These Hartree-Fock Green functions depend now parametrically on values of local moments on each of the orbitals µ α . Next we use the RPA approximation to obtain two Green functions G αβ ,A σ (ω) and G αβ ,B σ (ω), which are parametrically dependent on the local moments on each orbitals µ α . Symmetry restoration and determining the local moment values The symmetry restoration in the multi-orbital case is a straightforward generalization of the previous ansatz, i.e. except that now the symmetrized Green functions G αβ σ (ω) depend explicitly on local moments on all of the orbitals, i.e. \|µ α \|. The parameters \|µ α \| have to be determined independently. They are found by the minimization of the ground state energy of the impurity with respect to both local moment values on orbitals µ α and particle number on each of the orbitals n α As mentioned above, the variational procedure allows us to extend the LMA on the multi-orbital cases, where the Luttinger (Fermi liquid) condition for each orbital is absent. Also the possibility of non-Fermi liquid solution is naturally included within present generalization of the LMA [20], i.e. the variational local moment approach. Application to multilevel quantum dots A single quantum dot with many atomic-like levels coupled to leads are described by a multi-orbital single impurity Anderson model: where H dot is the local impurity part of the SIAM Hamiltonian, H leads corresponds to the conduction electron part of the SIAM Hamiltonian, and H dot−leads is equal to the hybridization term in SIAM [24]. V-LMA in quantum dots The properties of transport in a quantum dot in equilibrium, i.e. with infinitesimally small bias voltage between the leads, are determined by the spectral functions on each of the orbitals. Examples of the spectral functions are presented in Fig. 2 for the one-orbital case (left panel) and for the two orbital case (right panel). In the two orbital case the atomic levels are shifted such that one of the orbitals is at half filling (dashed line) and the other is away of half filling (solid line). The Kondo peak in the symmetric case is suppressed by the exchange (Hund) interaction (J = 0), which favors parallel spin orientations. In the asymmetric case the Kondo peak survives due to the presence of uncompensated magnetic moment and is shifted toward the lower Hubbard band. Further investigation of multilevel quantum dots including transport properties will be presented elsewhere [20]. Application to the multi-orbital Hubbard model The generalized variational LMA is also applied to solve the multi-orbital Hubbard model where the local part is a lattice sum of the terms which are of the same form as the atomic part in the SIAM. This model is solved within the DMFT where the selfconsistency condition relates the local matrix Green functions with the matrix of the self-energies [14]. In this way the lattice problem is mapped onto the Anderson impurity problem which has to be solved for different hybridization functions until self-consistency is achieved. In order to solve the Hubbard model within DMFT we need to solve the SIAM for arbitrary hybridization functions. The self-consistency condition simplifies greatly for the Bethe lattice which is used in this contribution. V-LMA method in DMFT In the recent few years the orbital-selective Mott-Hubbard metal-insulator transition has been the subject of extensive studies [22,25,13,4,2]. Using the V-LMA to obtain the solution of the SIAM in each of the DMFT loops the spectral functions for two-orbital Hubbard model at zero temperature were found. As an example, Fig. 3 shows the results for the case with different bandwidths and non-zero Hund coupling J. Since one of the spectral function is metallic-like (finite at ω = 0) and the other is insulating-like (vanishes at ω = 0) we conclude that the orbital selective MIT occurs in this model system. At the end we discuss the V-LMA in perspective to other methods used to solve the impurity problem and DMFT equations. The V-LMA belongs to the class of approximate, analytical methods like for example the iteration perturbation theory (IPT) [15], the non-crossing approximation (NCA) [12], or slave-boson theory (SB) [29], and various extenstions of these methods. As we showed here, the V-LMA is a conserving approximation, contrary for example to the IPT, and correctly describes high-and low-energy parts of the spectra, recovering the Kondo peak and Luttinger pinning. We tested this theory at zero temperature but there is no conceptual obstacle why the V-LMA should not work at finite temperatures as well. The V-LMA is not numerically exact like the quantum Monte Carlo method [14], the numerical renormalization group (NRG) [5], dynamical matrix renormalization group (DMRG) [31], or exact diagonalization (ED) [11]. However, each of the numerically exact methods suffers from principal obstacles in practical applications, in particular when the temperature is too low (QMC) or too high (NRG), or number of orbitals increases (NRG, DMRG, ED). Therefore we conclude that the V-LMA is a method of choice for solving the DMFT equations and can be used as a relatively fast and accurate impurity solver. The only technical difficulty in the variational LMA is to compute with high accuracy the system energy and to find its minimum. This should be performed with a great care. Summary The generalized variational LMA to the multi-orbital SIAM allows us to efficiently solve the problems of correlated electron systems such as multilevel quantum dots and the Hubbard model within the DMFT. In particular it is relatively easy to address the problems of different band widths and also the removing of the orbital degeneracy [19]. We experienced that the local moment approach is an efficient method in studying these problems, in particular, when the number of the orbitals is larger than two.	2009-02-02T14:59:30.000Z	2009-02-02T00:00:00.000	{ "year": 2009, "sha1": "784731643ca2a6026a8ebb7bb7f079b33f9a05ae", "oa_license": null, "oa_url": "http://arxiv.org/pdf/0902.0296", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "784731643ca2a6026a8ebb7bb7f079b33f9a05ae", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
266743290	pes2o/s2orc	v3-fos-license	Diagnostic status and epidemiological characteristics of community-acquired bacterial meningitis in children from 2019 to 2020: a multicenter retrospective study Community-acquired bacterial meningitis (CABM) is the main cause of morbidity and mortality in children. The epidemiology of CABM is regional and highly dynamic. To clarify the diagnostic status and epidemiological characteristics of children with CABM in this region, and pay attention to the disease burden, so as to provide evidence for the prevention and treatment of CABM. By retrospective case analysis, the clinical data of 918 CABM cases in children aged 0–14 years in Zhejiang Province from January, 2019 to December, 2020 were collected. The etiological diagnosis rate of CABM in children was 23.1%, the annual incidence rate 4.42–6.15/100,000, the annual mortality rate 0.06–0.09/100,000,the cure and improvement rate 94.4%, and the case fatality rate 1.4%. The total incidence of neuroimaging abnormalities was 20.6%. The median length of stay for CABM children was 20(16) days, with an average cost of 21,531(24,835) yuan. In addition, the incidence rate was decreased with age. Escherichia coli(E.coli) and group B Streptococcus agalactiae(GBS) were the principal pathogens in CABM infant<3 months(43.3%, 34.1%), and Streptococcus pneumoniae(S. pneumoniae) was the most common pathogen in children ≥ 3 months(33.9%). In conclusion, the annual incidence and mortality of CABM in children aged 0–14 years in Zhejiang Province are at intermediate and low level. The distribution of CABM incidence and pathogen spectrum are different in age; the incidence of abnormal neuroimaging is high; and the economic burden is heavy. Introduction Community-acquired bacterial meningitis (CABM), an inflammation of meningitis influencing the pia, arachnoid, and subarachnoid space induced by bacteria and bacterial products, is a major cause of morbidity and mortality in children [1].The epidemiology of CABM is regional and highly dynamic, affected by factors such as vaccines, climate, latitude, population movement, viral infections, and poverty.For example, in well-resourced areas, the incidence of meningitis is decreased to less than 0.5-1.5 per 100,000 population; while in the Sahel region of Africa, epidemic meningitis induced by Neisseria meningitidis and Streptococcus pneumoniae (S. pneumoniae) persists, with an incidence of 1000 per 100,000 cases [2].In addition to geographical differences in the incidence of CABM, the prognosis also varies with the age of onset, regions, and causative agents [1,3]. Studies have shown that the case fatality rate of CABM in children is 30%, and 50% of survivors suffer neurological complications [3].These complications include seizures, focal neurological deficits, subdural effusions, hydrocephalus, hearing loss, cognitive impairment, and epilepsy, among others.Therefore, prompt assessment and immediate empirical treatment of CABM can reduce the likelihood of death outcomes and chronic neurological sequelae [3,4].Studies showed mortality rates of untreated BM approaching 100% [1].An important basis for empirical treatment is the epidemiological characteristics of CABM in the region.At present, there is a relative lack of data from epidemiological studies of CABM in children in Zhejiang Province.Consequently, in this study, we employed a multicenter and large-sample retrospective case analysis to clarify the epidemiological characteristics of CABM children in Zhejiang Province.And this study could not only guide empirical treatment in the early clinical stage, but also provide an etiological basis for the immunization program of meningitis vaccine. Study design This study was a multi-center retrospective study with 50 hospitals as participators.The 50 hospitals included in the study covered almost all the hospitals with children's inpatient departments in Zhejiang province.These hospitals were distributed in 11 prefecture-level cities in Zhejiang province, including 42 tertiary hospitals and 8 secondary hospitals.The clinical data of 918 children aged ≤ 14 years with discharge diagnosis of "bacterial meningitis (BM)", "purulent meningitis" or "intracranial infection" from January 1, 2019 to April 30, 2021 were collected for retrospectively analysis.By referring to the electronic medical records, the clinical data of cases that met the inclusion criteria were collected, including demographics, length of hospital stay, hospitalization costs, clinical prognosis, blood and cerebrospinal fluid (CSF) sampling results (microbiologic results, CSF routine, CSF biochemistry, etc.), and head imaging results (ultrasound, CT, and magnetic resonance; all included cases completed at least one of the above imaging data).Inclusion criteria: Patients aged ≤ 14 years (including neonates) with "BM", "purulent meningitis" or "intracranial infection" were discharged from the hospital from January 1, 2019 to April 30, 2021.Exclusion criteria: (1) Cases whose clinical data did not meet the diagnostic criteria for suspected bacterial meningitis.(2) Intracranial infections induced by laboratory-confirmed nonbacterial pathogens, including viral encephalitis, fungal encephalitis, intracranial infections with mycoplasma, rickettsia and parasitic.(3) Nosocomial BM (craniotomy, CSF leakage, intracranial catheter infection, etc.), infection secondary to traumatic brain injury, and secondary intracranial infection such as tumor, transplantation or chemotherapy in patients with impaired immune system.(4) Discharged from hospital in 2019, with the onset of illness in 2018; Cases that started in 2021.(Fig. 1) Case classification criteria Grouping criteria were based on the WHO recommended case definition [5]. Pathogen confirmed (Confirmed) group: Clear pathogens were determined in CSF or blood with one of the following methods: (1) culture; (2) non-culture methods: antigen detection, Gram staining smear, nucleic acid detection.Clinical diagnosis case (Clinical) group: Typical clinical manifestations; typical changes in CSF; no pathogen found in CSF.Probable case (Probable) group: Atypical changes in CSF; no pathogen observed in CSF; and at least one of the following items were met: (1) prodromal suppurative infection lesions; (2) anatomical factors; (3) peripheral white blood cells ≥ 15 × 10 9 /L and/or high sensitive C-reactive protein ≥ 40 mg/L and/or serum procalcitonin ≥ 1 ng/ml.Typical clinical manifestations of children with clinical syndromes of suspected BM were shown as follows: fever (usually > 38.5 ° C rectal or 38.0 ° C axillary), drowsiness, confusion, severe headache, convulsions, projectile vomiting, bulging anterior fontanel, nuchal rigidity, etc.Typical changes in CSF: (A) turbid or rice soup alike appearance, and the pressure was increased (200-500 mm CSF); (B) up-regulated white blood cell count, often ≥ 1000 × 10 6 /L, and the classification was dominated by multinucleated cells (80 -90%); elevated protein concentration (> 100 mg/dl), and low CSF glucose concentration (< 40 mg/dl) [6].Atypical changes of CSF (only 1-2 abnormalities in the following 3 items): white blood cell count of CSF was slightly high (tens to hundreds); CSF glucose was mildly low or protein was a little elevated. Clinical prognostic criteria Cure: full course of anti-infection, disappearances of clinical symptoms and signs, and normal CSF indicators.Improved: full course of anti-infection, disappearances of clinical manifestations and signs, normal CSF white blood cell count; and only CSF protein and/or glucose concentration did not return to normal level.Not cured: Discharge with insufficient course of anti-infection or sufficient course but accompanied by serious complications (brain abscess, secondary epilepsy, cognitive impairment, etc.).Death. Statistical analysis Spss20.0 statistical software was applied in this report.Continuous variables exhibited a skewed distribution and were expressed as M (IQR).And continuous variables between two groups and among three groups were compared through Mann Whitney U test and Kruskalwallis test respectively.Categorical variables were displayed as number of cases n (%), and Chi-square test or Fisher's exact test was applied to check the outcomes.P < 0.05 was considered as significant difference.Incidence rate = the number of new cases of a disease in a population in a certain period/the number of exposed population in the population during the same period *K.K = 100,000/100,000.Number of exposed population: people in the population of a certain area who were likely to develop the observed disease during the observation period.Therefore, individuals with the disease were not included.However, because it was difficult to divide in practical work, the average population of the area during the observation period was used as the denominator.In this study, the average population during the observation period was used as the denominator.Mortality rate = total number of deaths (due to a disease) in a period/average population in the same period ×100%.In this study, the population base of Zhejiang Province aged 0-14 years was based on the data released by the 7th national census in 2020, and the newborn population was collected from the Statistics Bureau of Zhejiang Province.The 95% confidence interval (95% CI) of the rate was calculated using the following formula: p ± 1.96 pq n , q = 1-p.The 95% CI of the rate difference was calculated as follows: Diagnostic status A total of 918 children with CABM were included in this study, including 212 pathogen confirmed cases, 275 clinically diagnosed cases, and 431 probable cases.In this study, the etiological diagnosis rate was 23.1% (212 cases, 95% CI: 20.4,25.8).To be specific, the positive rates of blood and CSF culture were (122 cases, 13.3%, 95% CI:11.1, 15.5) and (138 cases, 15.0%, 95%CI: 12.7, 17.3) respectively.In addition, seven pathogens were detected in CSF by metagenomic next-generation sequencing (mNGS).This study also observed that the positive rate of pathogen culture in septic shock children (55.9%, 19 cases) was much higher than that in children without shock (21.0%, 186 cases), the difference was statistically significant (P < 0.01). General clinical data Of the 918 cases, the gender ratio between male and female was 177.9:100.Neonates accounted for the largest proportion of all cases, reaching 44.4%, and only 12.3% were 1-14 years old.The median length of hospital stay of all children was 20 ( 16) days, with an average cost of 21,531 (24,835) yuan.In comparisons with the clinical and probable groups, confirmed group exhibited longer length of hospital stay and higher average cost.The overall cure and improvement rate (867 cases, 94.4%, 95% CI: 93.0, 95.9) as well as case fatality rate (13 cases, 1.4%, 95% CI: 0.7, 2.2) was observed.The cure and improvement rate of confirmed group (188 cases, 88.7%) was lower than that of clinical (264 cases, 96.0%) and probable groups (415 cases, 96.3%) (Table 1). Clinical distribution and outcome of confirmed cases There were 212 children in the confirmed group.Among them, 119 cases were detected with Gram-positive bacteria (G +) and 93 with Gram-negative bacteria (G −).The onset was commonest in neonates (G + 62 cases, G − 58 cases).The cure and improvement rates of positive and negative bacteria were (108 cases, 90.8%, 95% CI: 85.5, 96.0) and (80 cases, 86%, 95% CI: 78.8, 93.2) respectively, and the case fatality rates were (1 case, 0.8%, 95% CI: -0.8, 2.5) and (5 cases, 5.4%, 95% CI: 0.7, 10.0) respectively.There was no statistically significant difference in the compositions of clinical cure, improvement, and other outcomes (including death and not healed) between the groups of G + and G-(P > 0.05).Besides, statistical differences between the two groups were not observed in the length of hospital stay and average cost (Table 2). Pathogen distribution and detection rate A total of 267 bacterial pathogens were detected from the blood and CSF of all children, including 147 G + and 120 G −. 2).Furthermore, the pathogen detection rate in the newborns group accounted for the highest proportion in all age groups, reaching 29.41%.And there was a statistically significant difference in the pathogen detection rate among different age groups (P < 0.01) (Table 4). The age of patients with subdural fluid collection/ empyema consisted of 66 cases (90.4%) under 1 year old, and 41 cases (56.2%) were < 3 months, including 17 cases of newborns (23.3%).The incidence of subdural fluid/ empyema was different between age groups, and the incidence in children aged 3-12 months with confirmed pathogen was 36%.For comparisons of hydrocephalus, the incidence in the confirmed group (10.4%) and the clinical group (7.6%) were remarkably superior to that in the probable group (0.7%).There was no significant difference in the incidence of hydrocephalus in both G + and G − groups (P > 0.05).The age composition of children with hydrocephalus was less than 1 year of age in 40 cases (87.0%) and < 3 months in 32 cases (69.6%), including newborns in 21 cases (45.7%).The incidence of hydrocephalus varied between 4.8% and 5.3% in different age groups. Discussion Meningitis is an sever infectious disease syndrome in childhood, with a huge disease burden and large regional incidence variations [4,7].The Global Burden of Disease study in 2016 revealed that the incidence of meningitis was as high as 207.4 per 100,000 in South Sudan and 0.5 per 100,000 in Australia [7].Nearly 3% of deaths in children below 5 years of age worldwide are attributed to meningitis [7].The global mortality rate of meningitis/encephalitis in children below 5 years old ranges from 21.28/100,000 to 28.1/100,000, and the case fatality rate of BM fluctuates between 3% and 7% [8,9].From September 2006 to December 2009, the incidence of BM in children under 5 years old ranged from 6.95/100,000 to 22.30/100,000 in four provinces of China (Shandong, Hubei, Hebei, and Guangxi) [10].Our study revealed that the incidence of CABM was 4.42/100,000-6.15/100,000 in children aged 0-14 years and 12.33/100,000-16.91/100,000in children under 5 years in Zhejiang Province from 2019 to 2020.And the incidence in Zhejiang Province is at the middle and low level in China. As this study demonstrated that, the mortality rate of CABM (0.14/100,000-0.27/100,000) and case fatality rate (1.4%) in children under 5 years of age in Zhejiang Province from 2019 to 2020 were lower than in the foreign level.When it comes to the cost, the confirmed group was the highest, with an average cost of 35,824 yuan per time.The average cost and length of hospital stay of CABM in children in Zhejiang Province from 2019 to 2020 were higher than those of pneumonia in China (5,026.76yuan; 7.63 days) [11].And it has revealed that the disease economic burden of CABM in children is heavy. The incidence of BM varies according to age group.In this study, the incidence of CABM in newborns in Zhejiang Province was 30.13/100,000-39.12/100,000from 2019 to 2020, the children aged 1-2 months was 16.72/100,000-18.80/100,000,and aged 5-14 years was decreased to 0.35/100,000-0.61/100,000.Also, USA exhibited the incidence of 81/100,000 in children under two months of age from 2006 to 2007, while 0.4/100,000 in children aged 11-17 years [3].Infants aged ≤ 2 months are high risk population with CABM, and newborns incidence is the highest.The immature immune system of infants under 2 months of age may be responsible for the above phenomenon.On the one hand, there is a lack in maternal immunoglobulin that crosses the placenta before 32 weeks of gestation, and on the other hand, phagocytic ability of neutrophils and monocytes is limited [12].In addition, it was discovered by our study that the incidence of CABM in children in 2020 in Zhejiang Province was remarkably lower than that in 2019.And this result was considered to be correlated with the measures such as reducing population aggregation, wearing masks, quarantine and isolation after the outbreak of COVID-19.At the same time, some studies had shown that SARS-CoV2 caused a 20-30% reduction in the incidence of meningitis during the COVID-19 pandemic [2]. Due to the high mortality rate, CABM requires immediate assessment and prompt empirical therapy [4].A total of 918 children were included in this study, involving 46.9% probable cases.The clinical manifestations or laboratory data findings were atypical in probable group and there was a great risk of missed diagnosis.However, with the case fatality rate approaching 100% for untreated BM, suspected cases need immediate actions for specific diagnosis and empirical antimicrobial therapy [1].Importantly, early recognition and use of appropriate antibiotics are essential to minimize deaths and complications caused by BM [2].This study enrolled the children with probable meningitis to increase clinicians' attention to their diagnosis and treatment.The pathogenic diagnosis rate of this study was 23.1%, while the positive rate of CSF culture was only 15%.The children in this study conducted blood culture in the early stage of the disease and the CSF of a few children received mNGS detection, improving the diagnostic yield of the pathogen.Therefore, early aseptic body fluid culture is recommended for BM children in clinical practice.For another, non-culture tests should also be performed for patients who require early detection of pathogens or have received antibiotic treatment to identify the pathogen to guide treatment as soon as possible [1]. The distribution of common pathogens of CABM in children varies among age groups.A global META analysis of BM etiology manifested that S. pneumoniae infection was the most common cause of BM in all pediatric groups [13].In this study, the top two pathogens in newborns and infants < 3 months in Zhejiang Province were E.coli (43.3%) and GBS (34.1%), and S. pneumoniae was the most common pathogen in children ≥ 3 months (33.9%).A study by Shenzhen Children's Hospital reported that GBS and E.coli were the main pathogens in newborns, and S. pneumoniae was mainly observed in older children [14].The similar results of the two studies indicated a descending trend in the incidence of E.coli and GBS, and an ascending trend in S. pneumoniae with the increase of the age. In this study, subdural fluid collections/empyema and hydrocephalus were common brain imaging changes in children with CABM.The incidence of subdural fluid collections/empyema was 8% and didn't appear obvious differences in G + and G-infections; but the incidence in the confirmed group (16%) was much higher than that in the clinical group (8%) and the probable group (3.9%).The above findings suggested that the children with positive pathogen culture at early stage suffered higher pathogen loading, more severe host inflammatory response, and were prone to subdural fluid collections/empyema, while had little correlation with the G + or G-pathogen.Compared with older children, subdural fluid collections are most commonly observed in infants (< 1 year of age) [15].In this paper, 90.4% children with subdural fluid collections/empyema were younger than 1 year old, and the incidence of subdural fluid collections/empyema was the highest among children aged 3-12 months in the confirmed group.In addition, the incidence of hydrocephalus in CABM patients was 5%, which is significantly lower than the results of other studies (15 -18.8%) [16,17].And this difference was mainly caused by the inclusion of children with probable BM in this study.The incidence of hydrocephalus in the probable group was only 0.7%, much lower than 10.4% and 7.6% in the confirmed and clinical groups.There was no significant difference in the incidence of hydrocephalus between different age groups in this study, but some study showed that hydrocephalus is more common in neonates and infants (the incidence in patients aged < 3 months reaches 14 -27%) 18].Zhou Wei et al. revealed a higher incidence of hydrocephalus in meningitis newborns with G- [19], while the incidence was not significantly different between G + and G-in children aged 0-14 years in this study. In this article, the overall cure and improvement rate of CABM in children in Zhejiang Province from 2019 to 2020 was 94.4%.The prognosis of the confirmed group was worse than that of the clinical group and the probable group, suggesting that the prognosis of CABM in children was related to the bacterial loading.The main experimental method to confirm the diagnosis in our study was bacterial culture and its positive rate was correlated with bacterial loading [6].Children with negative blood culture had lower level of bacterial loading in their blood compared to infants with positive one [20].Bacterial loading is associated with the outcome of severe infection in the host [21].In this study, the positive rate of bacterial culture in children with septic shock was much higher than that in those without shock, indicating that children with positive bacterial culture were more likely to suffer severe infections.Besides, the overall prognosis of CABM in children in the confirmed group was not significantly different between G + and G-.This conclusion was consistent with the results of a domestic study based on the prognosis of newborns with BM [19].In summary, the prognosis of CABM in children is linked to bacterial loading, with little correlation with the G + and G-. Limitations There were some limitations in this study.First, it lacked a long follow-up, and further improvement in the evaluation and follow-up of long-term neurological complications was needed.Furthermore, the non-culture detection method of bacterium-free fluids in the early stage of the disease should be pay more attention.Finally, in calculating the annual incidence, the local population during the observation period was used as the denominator.The inclusion of individuals with preexisting conditions in the denominator may have led to an underestimation of incidence. Conclusions To sum up, our study assessed the annual incidence and mortality of CABM in children aged 0-14 years in Zhejiang Province.In this study, children with CABM were classified according to their laboratory data and clinical manifestations, including pathogen confirmed group, clinical diagnosis group and probable case group.Neuroimaging abnormalities were more common in the confirmed group, and the clinical prognosis of these children was poor.In addition, our study demonstrated that infants, especially newborns, were at high risk of CABM in children.E.coli, GBS and S. pneumoniae are the top three pathogens of CABM in children in Zhejiang Province.Subdural fluid collections/empyema and hydrocephalus are the most common brain imaging complications.Clarifying the epidemiological characteristics of CABM in children in the region can not only guide early clinical empirical treatment, but also provide an etiological basis for the immunization program of BM. Fig. 1 Fig. 1 Flow diagram for the selection of subjects in the retrospective cohort study of CABM in children aged 0-14 years in Zhejiang province from 2019 to 2020 Note: 1).Some critical cases died without lumbar puncture.This type of case could be diagnosed by typical clinical manifestations and brain imaging abnormalities, such as subdural effusion/empyema, meningeal thickening, or enhanced echo of the brain ependymal.2) The first atypical CSF change required dynamic observation of changes in indicators. Fig. 2 Fig. 2 Composition of 267 pathogens detected by CABM in children aged 0-14 years in Zhejiang Province from 2019 to 2020 Table 2 Clinical characteristics of CABM in 212 confirmed cases of children in Zhejiang from 2019 to 2020 G +: Gram-positive bacteria; G-: Gram-negative bacteria Table 3 Comparison of the incidence and mortality of CABM in children aged 0-14 years in different age groups in Zhejiang from 2019 to 2020 Table 4 Detection rate of pathogens in 918 children with CABM. Table 5 Analysis of brain imaging results of 918 CABM children aged 0-14 years in Zhejiang Province from 2019 to 2020 Table 6 Analysis of brain imaging results of 212 pathogen confirmed CABM children aged 0-14 years in Zhejiang Province from 2019 to 2020	2024-01-04T14:21:28.199Z	2024-01-04T00:00:00.000	{ "year": 2024, "sha1": "50aa085f91769fd019d268a1d8d05988147007f1", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "377a29b5cdc739f53c6a504e8106dde68fc2529b", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
231615193	pes2o/s2orc	v3-fos-license	Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi American trypanosomiasis, or Chagas disease, is transmitted by both domestic and sylvatic species of Triatominae which use sensory cues to locate their vertebrate hosts. Among them, odorants have been shown to play a key role. Previous work revealed morphological differences in the sensory apparatus of different species of Triatomines, but to date a comparative functional study of the olfactory system is lacking. After examining the antennal sensilla with scanning electronic microscopy (SEM), we compared olfactory responses of Rhodnius prolixus and the sylvatic Rhodnius brethesi using an electrophysiological approach. In electroantennogram (EAG) recordings, we first showed that the antenna of R. prolixus is highly responsive to carboxylic acids, compounds found in their habitat and the headspace of their vertebrate hosts. We then compared responses from olfactory sensory neurons (OSNs) housed in the grooved peg sensilla of both species, as these are tuned to these compounds using single-sensillum recordings (SSRs). In R. prolixus, the SSR responses revealed a narrower tuning breath than its sylvatic sibling, with the latter showing responses to a broader range of chemical classes. Additionally, we observed significant differences between these two species in their response to particular volatiles, such as amyl acetate and butyryl chloride. In summary, the closely related, but ecologically differentiated R. prolixus and R. brethesi display distinct differences in their olfactory functions. Considering the ongoing rapid destruction of the natural habitat of sylvatic species and the likely shift towards environments shaped by humans, we expect that our results will contribute to the design of efficient vector control strategies in the future. (1) Response: Thank you for your general positive feedback and your highly useful comments! A parametric unpaired t test was chosen after assessing the normal distribution of the data with a Shapiro-Wilk test, which allows analysis of small sample sizes. (2) In Methods section basiconic sensilla are mentioned. However, later in the MS they seem to be called thin-walled trichoid. The nomenclature used should be clarified, citing previous papers and indicating the different names previously given to the same sensillum type (this is to allow the reader compare the information from this and other papers on the same sensillum type). I would suggest using the nomenclature by (2) Response: We apologize for being not consistent regarding the nomenclature of the different sensilla types studied in our MS. As suggested by the reviewer, we adopted the nomenclature by Shanbhag et al (1999), and referred to it in the text (lines 263ff, 279ff, 291ff) and corresponding figure ( (3) Response: Thank you for the remark, the formula was corrected. Results: (4) In Fig 3 I did not understand which responses were significantly different from the negative control. Responses to CO 2 have been found but nothing is said about those responses. The resolution of figures 4, 5 should be improved. They are difficult to read. (4) Response: In Figure 3, the filled bars represent significant responses compared to solvent control. This is stated in the Figure legend and now also added to the Results section (line 305). We apologize for not commenting on the response to CO 2 in the previous version of our MS. We now refer to the CO 2 responses in the Results and Discussion sections, as well as in the Methods section where we describe how the delivery of CO 2 was implemented. Moreover, we have thoroughly changed the arrangements of Figs. 4-6 and added Fig. 7 in order to improve the text flow and to make the figures easier to read and follow. Editorial suggestions/ minor modifications: (5) In line 264 the following reference should be added: May-Concha, et al 2016. Infec. Genet. Evol. 40, 73-79 (5) Response: Thank you for providing the reference, which is indeed very relevant. It has now been added. (6) In line 100 (or 106 or 104) the following reference should be added: Guidobaldi & Guerenstein 2016 Journal of medical entomology 53, 770-775 (6) Response: We apologize for missing this very important reference. It has now been added. (7) There is a mistake in line 302. Aldehydes have also been tested (see your Ref 27). (7) Response: The text has been amended accordingly. (8) Check line 122 , it seems to contradict a previous sentence. Line 59 Replace "its" for "a" Correct TrYpanosoma in Author summary (8) Response: The author summary has been revised and corrected. Summary and General Comments: (9) This MS deals with a comparison of the morphology and physiology of the olfactory system of two triatomine species, one considered domestic, the other sylvatic. A higher density of two types of olfactory sensilla in the sylvatic species was found compared to the domestic one. Also, the sylvatic species presented overall higher and broader olfactory responses than its domestic relative. (It is suggested that domestic species present a decreased olfactory function, in relation to the limited relevance of this sensorial input in their particular environment with limited olfactory cues.) In addition, knowledge about the olfactory responses on the antenna of triatomines has been extended, and this includes responses to "new" compounds and a better understanding of functional subtypes. The morphology work is adequate and the experiments are correctly carried out. It would be interesting to continue this work, for example, by studying the dose-response characteristic of the ORNs studied. Also, an antennal response to CO 2 is reported for the first time in triatomines. It would be interesting to study this further. In conclusion, this MS contributes new information on triatomines and in general add up to our knowledge on the olfactory system of hematophagous insects. (9) Response: Thank you for your positive feedback! We agree with the reviewer that it would be interesting in future experiments to determine dose-response curves for a selected group of odorants, which would allow to characterize the responses of these ORNs further. We also agree that the antennal response to CO 2 is an interesting observation, as it still remains unknown which receptor mediates its detection. In preparatory experiments, application of CO 2 to the grooved peg sensilla did not result in a response. However, these suggested experiments are very time-consuming and should therefore be rather part of further investigations. We added these ideas regarding future studies to the discussion section of our manuscript (lines 536-538). Methods: (1) The objectives are clearly articulated and the study design is appropiate to address the stated objectives. (1) Response: Thank you for the positive feedback and your highly constructive comments to our study, which we all incorporated into the revised version of our manuscript. Results: (2) The reading in this section was difficult. Probably because the Figures are quite messed up in the text, which makes reading tough. Therefore, I suggest (as a possibility) to group data differently. For example, data of figure 4A and those of figures 6C and D could be on the same template, where the averaged responses are shown (without function subtype discrimination). And, graphs of Figure 4B could be included along with data of Figure 6A-B. If so, this would need rewriting the text. (2) Response: We thank the reviewer for suggesting to present and group the data in a different way. We have thoroughly revised the manuscript by changing the order and arrangements of the different figures and modifying the different sections, especially the results section, to improve text flow, comprehension and readability of the MS. We changed the arrangement and order of Figs. 4-6 and added Fig. 7. Conclusions: The authors could certainly further develop the limitations of the study and give alternative hypotheses to those provided (which I detail below). (3) Response: We have thoroughly revised our hypotheses according to the reviewer's suggestions. Below we explain and respond in detail to each comment of the reviewer. Summary and General Comments: (4) In the manuscript "Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi", the authors analyze morphological and physiological differences in the olfactory antennal sensilla between R. prolixus and R. brethesi. They found differences across species in the number of certain morphological types of olfactory sensilla (i.e. thin-and thick-walled trichoid sensilla) and also differences in the olfactory response profile of grooved peg (GP) sensilla. Whereas GP sensilla were found in similar numbers in both species, they showed different olfactory tuning responses. The results here provided are solid, novel and are a useful contribution to the sensory physiology of triatomine insects. (4) Response: Thank you for your positive feedback! (5) However, I have major concerns about the construction of the hypothesis and the hypothesis of this work. First, the authors claim that R. prolixus is a domiciliated species, which is not accurate. Whereas R. brethesi until now was only found in sylvatic environments, R. prolixus depending on the region can be found in sylvatic (living in palm trees, e.g. Colombia, Ecuador, Brazil, etc.) or in domestic environments (living in human dwellings, e.g. Guatemala). Therefore, R. prolixus does not represent the best example of an exclusively domiciliated species. An interesting comparison would be to analyze the antennal morphology and physiology of domiciliated and sylvatic R. prolixus. (5) Response: Indeed, R. prolixus presents a broad distribution in Colombia, Ecuador, Nicaragua, however it presents a higher degree of domiciliation than other species of the genus Rhodnius (as evidenced by the number of houses infested by the insects), especially compared to R. brethesi. However, we agree that the differences we observe cannot solely and clearly be attributed to a possible domiciliation, as these species are also found in sylvatic areas and, as the reviewer points out, sylvatic species might also live in a restricted environment, like R. brethesi. To avoid over interpretation of our results, we restricted the hypothesis to a comparison between two species of triatomines, one being sylvatic and the other having a broader distribution in sylvatic, domestic and peridomestic areas. Furthermore, we agree that a comparison between domestic and sylvatic triatomines would be interesting to assess intraspecific differences in sensory perception, and would certainly increase our knowledge about the role the environment has in shaping olfactory detection in triatomines. Our study, however, was aimed at examining inter-specific differences between triatomines, which might result from speciation. We decided to focus on R. prolixus as it is broadly distributed and is one of the triatomine species that received most attention in the past. Based on previous work, we expanded, not without limitation, the knowledge we have about this species. Our choice of R. brethesi was based on the fact that this species has been found only in sylvatic environments. We asked ourselves: how different are the responses from this sylvatic species, compared to those ones of R. prolixus? We hope that the modifications of the text and our hypotheses now capture our research question more precisely. (6) Second, the authors postulate that sylvatic species (R. brethesis) have higher number of sensilla and a larger olfactory tuning of sensory neurons than domicialiated species (R. prolixus) due to their need for an active search for hosts, a requirement that would be higher in sylvatic species than in domiciliated ones. This statement, on which the general hypothesis of the work is based, is weak. In fact, we know that sylvatic species also live in close association with their hosts, probably spending their whole lifecycle in the same vertebrate nest. In these nests they form large colonies and several cohorts coexist. Therefore, asserting that sylvatic triatomines explore and search more actively than domiciliated species, and consequently their need for a more complex sensory machinery than domiciliated ones, is not a solid argument or hypothesis for triatomine species as it might be for other insect species. The differences found with respect to the sensory tools could simply be attributed to species-specific differences related to different host preferences. (6) Response: We agree that the differences found could also be attributed to speciesspecific differences in hosts. We hope our thorough revision of the text now reflects this hypothesis. Still, it remains as an open question whether sylvatic insects need to discriminate specific olfactory cues against a background containing a higher amount of volatiles, even if they live in close association with a host. (7) Lastly, reading was difficult. Probably because the Figures are quite messed up in the text, which makes reading tough. Therefore, I suggest (as a possibility) to group the data differently. For example, data of Figure 4A and those of Figures 6C and D could be on the same template, where the averaged responses are shown (without function subtype discrimination). And, graphs of Figure 4B could be included along with data of Figure 6A-B. If so, this would need rewriting the text. (7) Response: We are sorry for the difficulties caused, and thank the reviewer for the constructive suggestions. The figures and text have been thoroughly revised. We decided to re-arrange the figures in a different manner. What used to be Figure 6C, D is now Figure 4, Figure 5A is now Figure 5, Figure 4A is now under Figure 6 and we combined Figures 4B,C, Figure 6A, B and Figure 5B into a new figure (i.e. Figure 7). We hope that this new arrangement improves the legibility of the manuscript. The text has been accordingly rewritten. (8) lines 47-48 "American Tripanosomiasis, also known as Chagas disease, is a disease which no one speaks out…." This affirmation sounds weird when the WHO produces annual reports informing to the scientific and non-scientific community about the statistics of the disease (% of people affected, transmission rates, preventive actions and policies, etc..). Chagas disease is probably less known in Europe or Asia, but it is not the case in the Americas. Moreover, the Center for Disease Control and Prevention (CDC) of the United States considered the Chagas disease as 1 of 5 parasitic infections to be targeted as priority for public health (CDC, 2018). (8) Response: We have revised the author summary and avoided to mention that Chagas disease represents a hardly known disease, as it has been well characterized and documented. (9) line 52 "… and to get their vital blood meal, while infecting them at the same time" this is not precise but is confusing and can be misunderstood. If kissing bugs do not defecate during blood intake, there are no chances of infecting the host, given the parasites are only present in feces and not in the salivary glands. Thus, they can take a small blood meal and leave the host without defecating. I suggest editing the text, to avoid confusion and misunderstandings. (9) Response: We are sorry for the phrasing and confusion. The author summary has been corrected. (10) line 66 replace for Chagas disease (10) Response: Thank you for the remark; it has been amended. We also decided to uniformly use "Chagas disease" throughout the text. (11) Response: We apologize for our phrasing that understated previous significant work in the field. The text has been revised accordingly and the missing references have been added (lines 104-108). The intention was to point out that most of SSR studies were done in T. infestans and not in R. prolixus. As we discuss, there might be differences in responses between these two species. (12) lines 109 -111 This is not precise either. Like domestic triatomines sylvatic species also live in close association with their hosts. Sylvatic species live in vertebrate nest, where they can spend in the same nest their entire lifecycle, forming large colonies. Therefore, I disagree with the authors in claiming that sylvatic triatomines explore and search for hosts more actively than domiciliated species. (12) Response: The comment is accurate and we agree with the reviewer that sylvatic species can also live in restricted habitats and present very narrow host choices. Such is indeed the case for R. brethesi. We have revised this part to avoid confusions (lines 109ff). (13) line 278. You stated that you've identified 2 new types of sensilla: 1-a peg-in-pit sensillum and a type of coeloconic sensillum. Please specify whether it was an eventual finding or not. If not, please specify in how many preparations you found them. (13) Response: The finding of new sensilla types was indeed an eventual finding, which is now specified in the text as suggested. (14) lines 294-295: In figure 2, which is the point of the micrographs shown above the bars of density quantification of sensilla? Please refer to them or exclude them. (14) Response: The micrographs show the confocal scans that were used to quantify the sensillum density. However, following the reviewer's suggestion, we decided to exclude them, as they did not significantly contribute to the understanding of the sensilla quantification. (15) It is shown in Fig.3 that you've tested the response to CO2, please detail in Mat & Met how did you offer the CO2 to the insects, and which was the concentration offered? (15) Response: We apologize for not explaining how CO2 was exactly applied and what concentration was used in our study. We have now added these details to the Materials and Method and Results sections (lines 157-158, 170-172, 313-315). (16) lines 344-346 : You stated "While only 37% of the odor-sensillum combinations in R. prolixus yielded responses >15 spikes s-1 above solvent response, all combinations did in R. brethesi (Fig. 4, Fig. 5). " However this is not easily to visualize, on the contrary, it looks the opposite. To facilitate cross-species comparisons, I strongly suggest, in Figure 5, putting the Y axes on the same scale. (16) Response: As the absolute number of odor-sensillum combinations vary between species, we have revised Figure 5 using the same scaling for both species, as suggested by the reviewer, and the corresponding total percentages (i.e. cumulative sum) have been corrected. (18) lines 492 -494 You stated "However, it is conceivable that these chemicals are detected by organs other than the antenna, as the odorant co-receptor orco is expressed also in tarsi, genitalia and rosti". No olfactory sensilla have been identified in tarsi, genitalia or rostrum of triatomine bugs. The presence of odorant-related receptors in these appendages may not necessarily be related to an olfactory function. Please rephrase. (18) Response: We agree with the reviewer's comment and have rephrased this sentence (lines 483-486). However, we would like to point out that in Rhodnius prolixus not only RproOrco but also RproOR13, RproOR20 and RproOR104 were found to be expressed in rostri, tarsi, tibial pads and ovipositor or genitalia of females and males, respectively	2021-01-16T14:20:05.462Z	2021-01-08T00:00:00.000	{ "year": 2021, "sha1": "f5c88f244f907d551392db7acee6a1dcf614cabd", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0009098&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "1b9d897a13ffab07c256bad1a337dec18f3fdfdf", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology" ] }
255824660	pes2o/s2orc	v3-fos-license	Conservative surgical approach towards placenta accreta spectrum disorders for uterine preservation Objective We previously described a technique for repair of the myometrial defect at repeat Caesarean section which increases residual myometrial thickness thereby potentially reducing future niche-related complications. Here we describe how this technique can be modified for use for placenta accreta spectrum disorders, in line with emerging evidence that this is more a disorder of myometrial deficiency than morbid adherence. Design The surgical performance of peripartum hysterectomy was compared with that of the modified technique in all women having repeat Caesarean delivery for placenta accreta spectrum disorder in a tertiary unit in Singapore between December 2019 and October 2021. Methods Modification of the original technique involved the systematic delivery of the placenta starting from its most posterior attachment after uterine exteriorization. This is followed by the identification, mobilization, and apposition of the boundaries of myometrial defects as described previously. Results Ten women had Caesarean hysterectomy and ten had Caesarean section using the modified approach. Age and gestational age at delivery were similar for the two groups. Women in the modified technique group had had fewer prior Caesarean sections and had a lower body mass index. Operating time, estimated blood loss and need for transfusion were all lower in the myometrial repair group but without statistical significance. There were no visceral injuries in the repair group but there was one bladder injury in the hysterectomy group. Conclusion The modified approach provides an effective alternative to peripartum hysterectomy with favourable surgical profile and allows uterine conservation with restoration of myometrial thickness. likelihood of a niche and the larger the defect [2]. Niche development may be accompanied by formation of new vascular connections within the scar area, which along with in-migration of a variety of inflammatory cells contributes to healing [3]. During future pregnancies the niche has the potential to become an isthmocele leading to complications including uterine rupture and placenta accreta spectrum disorders (PASD). As the niche deepens, the residual myometrial thickness (RMT) gets less, with the RMT further thinning during pregnancy as the niche widens. The longer and deeper the initial scar area, the larger the decrease in RMT seen in pregnancy and the larger the resultant defect [4]. The likelihood of developing complications in future pregnancy is related to the RMT, as it reflects the amount of muscle remaining. An RMT of less than 2 mm carries a significant risk of scar rupture [5]. The frequency of PASD has markedly increased over the last few years because of the increased Caesarean section rate. The risk is higher when placenta praevia complicates a pregnancy following previous CS, especially if the primary indication mandated a higher uterine scar and rises with each subsequent CS [6]. Historically treatment options centered round intentional retention of the placenta (IRP) with or without hysterectomy but latterly several techniques have been developed aimed at conserving the uterus including the Triple P [7] and Naussica [8] procedures. Recent evidence of PASD being a disorder of defective decidua and uterine dehiscence rather than destructive trophoblastic invasion [9] suggests that a conservative approach with effective manual removal of placenta, when combined with peri-operative prophylactic haemostatic measures can be an optimal approach. We have previously described a technique (Siraj et al.) for repair of the myometrial defect or isthmocele at the site of the previous scar at the time of repeat Caesarean Sect. [10]. The repair increases the RMT and reinforces the posterior uterine wall which is often thin in these cases. We now describe how this technique can be extended for use in cases of suspected PASD with focus on control of the major hemorrhage, in part due to the neovascularization around the scar which can be offered as an alternative to peripartum hysterectomy in women who request uterine conservation. This approach has the added advantage of offering opportunity to reconstitute the uterine wall by contemporaneous correction of the niche, potentially allowing future pregnancy and avoiding other niche-related complications. Methods Our conservative surgical approach comprises the following: Preoperative assessment and peri-operative prophylactic haemostatic measures For antenatally diagnosed cases of PASD the surgery is pre-planned and done by a dedicated team. The patient is counseled on surgical options and associated risks, an anesthetic review is performed, and insertion of internal iliac artery catheters (IIAC) is offered prophylactically to reduce peri-operative blood loss and blood is available. IIAC is a helpful but not essential part of the technique. For patients presenting as an emergency, IIAC insertion was only offered it time permitted. For an on-table diagnosis, IIAC insertion was not used. Management depends on patient preference towards a conservative approach and needs to be established preoperatively as the surgery is done under general anesthesia (GA). The method presented here is suitable for women wishing to avoid the prolonged follow up and potential risks associated with IRP but who wish for uterine preservation. Consent needs to include bleeding risk potentially necessitating hysterectomy, possible visceral injury, and potential for PASD recurrence or future scar pregnancy if uterine conservation is successful without ligation. Incision Sites of both abdominal and uterine incisions need careful consideration when performing any CS for suspected PASD. A longitudinal skin incision which can initially be placed sub umbilically has advantages. It aids access both for adhesiolysis especially if the uterus is adherent to the anterior abdominal wall and for peripartum hysterectomy if required. It can also protect against inadvertent bladder injury if the bladder is drawn up. Extension offers access to the upper uterus should a classical or fundal uterine incision be chosen for placental avoidance, which itself allows placental retention should there be no signs of separation and uterine preservation is requested. However, a longitudinal skin incision may be less cosmetically acceptable and associated with more postoperative morbidity than a transverse wound. A classical uterine incision on the upper segment may obscure the degree of placental separation leading to delay in controlling blood loss and difficulty in gaining access to the retracted muscle to repair the defect obligating a second transverse incision to be made for visualization, potentially increasing the risk of uterine rupture in any subsequent pregnancy. A transverse incision in the lower segment or isthmocele almost inevitably disturbs the placenta which can initially cause heavy bleeding and rules out placental retention. However, the use of a single uterine transverse incision through the previous scar has the advantage of allowing the placenta to be delivered in a systematic way under direct vision from its attachment on the posterior uterine wall first followed by removal from the neovascularized anterior wall while tracing the boundary of the sheared posterior myometrial defect prior to repair. This moderates the initial high blood loss from the neovascularised isthmocele associated with the more orthodox anterior placental separation and helps in the management of the often unrecognized bleeding from the posterior myometrial defect and the bleeding from the anterior inferior muscle close to the level of the internal os prior to repair. We advocate entry through a pre-existing skin incision, usually transverse and suprapubic followed by a transverse incision through the upper third of the isthmocele above the level of the uterovesical fold through which the fetus and placenta are delivered. This avoids unnecessary dissection of the bladder and risk of renal tract injury as well as disruption of troublesome bridging vessels which run over the isthmocele and in the bladder serosa but does have the drawback of the patient being subject to a higher peripartum blood loss. Delivery of baby Once the uterus has been entered, delivery is conducted in the usual way but needs to be expeditious. After delivery of the baby, the IIA balloons are inflated, and the uterus is exteriorized. This improves visualization of the operative field and provides access to the posterior uterine wall aiding both placental detachment and identification of the retracted posterior retracted muscle. It also allows manual compression of the uterine arteries helping reduce the blood loss while the myometrial rings are clamped. Delivery of placenta A low-lying placenta may be situated within the isthmocele or attached to the previous scar depending on its level (within or above the endocervical canal). If the placenta is not adherent, it will separate spontaneously with or without uterotonics. If it is adherent, manual removal will be required. Regardless of method of placental delivery, both leave a non-retractile isthmocele which may be bleeding profusely from the retracted muscle rings at its boundary and the numerous bridging vessels lying in the serosa. We recommend a posterior rather than an anterior starting approach for the manual removal of the placenta to reduce the risk of bleeding from the aberrant vessels present anteriorly where the tissue planes are obscured. This has the added advantages of reducing risk of bladder injury and helping in identification of the posterior myometrial defect as described in the original method. If manual removal is required, we advocate the following steps: 1. After uterine entry the operator's right hand is inserted through the incised isthmocele and directed towards the upper border of the most posterior aspect of placental attachment (Fig. 1) from where detachment is initiated, working laterally, bilaterally, to expose the posterior uterine wall defect (Fig. 2). 2. The exposed retracted inferior and superior posterior muscles are then grasped with Green Armytage clamps (Fig. 3). In some instances, the sheared posterior defect may be large and the inferior myometrial boundary difficult to identify. Recognition may be aided by the assistant's right hand raising the outer aspect of the uterus below the utero sacral ligament facilitating application of the Green Armytage clamps to the lower posterior retracted muscle within. 3. Once the lateral aspects of the placenta are reached the operator's left index and middle fingers are inserted into the endocervical canal (after digital dilatation, if necessary,) to the anterior fornix ( Fig. 4) to lift the cervix (Fig. 5) and allow the retracted anterior inferior muscle edges to be identified and clamped, before detaching any residual attached placenta. This muscle ring is normally found at the level of the internal cervical os and needs to be isolated prior to application of clamps to avoid damage to the bladder base. Once the anterior ring of inferior muscle has been caught securely, the bleeding starts to slow, and the bladder can be separated safely from the lower seg- Fig. 6, is then repaired as previously described [10]. Success of this technique involves correct recognition of the retracted muscles at the boundary of the myometrial defect and repair of the muscle edges. As the posterior myometrial defect is closed and the uterine angles are secured the bleeding slows further allowing completion of the repair. Any redundant fascia forming the isthmocele, if not already ruptured, is incorporated into the anterior repair to build up the anterior uterine wall at the site of the scar and reduce the bleeding from the overlying aberrant vessels which can be difficult to control. Control of blood loss Blood loss is at its highest immediately following placental delivery. This can be reduced by inflating the internal iliac balloons or applying a paracervical tourniquet, if feasible, while the edges of the myometrial defect are identified (Fig. 6) and secured. Uterine artery ligation can even be considered if necessary. Other measures which can be employed include the standard uterotonic drugs, balloon tamponade, blood transfusion, cell salvage and tranexamic acid. We examined a consecutive series of twenty cases of PASD managed over a period of twenty-two months between December 2019 and October 2021. The study was conducted in a tertiary obstetric referral hospital in Singapore which has around 12,000 deliveries per year and a Caesarean section rate of 31-32%. The study was reviewed and approved by the SingHealth Centralised Institutional Review Board (IRB Approval Reference number -2011/711/D). The surgical method adopted for each case was mainly governed by patient choice. A low-lying placenta was identified for all women on a mid-trimester pelvic ultrasound scan with characteristic ultrasonic features of PASD being identified on subsequent scans. Most of the patients also had an MRI. All deliveries were performed under GA and pre-operative intra iliac balloon catheterization was offered to all women having elective delivery and any being delivered as an emergency, if time permitted. We compared demographic data and peri-operative details as well as outcomes between groups. We looked at surgical time, estimated blood loss, need for transfusion, visceral injury, admission to the Intensive Care Unit and length of stay for our patients. Results Of the twenty consecutive patients, ten had a Caesarean section with peripartum hysterectomy and ten were managed by Caesarean section using our technique. None of the woman had a Caesarean hysterectomy for failed conservative management. Five women in each group had the procedure performed electively between 34 and 36 weeks. The remainder needed emergency delivery for an intervening complication. All but one of the women who had had previous myomectomy and was delivered by CS with myometrial repair, had had at least one previous CS. The groups were similar with respect to age and gestational age at delivery (Table 1). More women having hysterectomy had had two or more previous CS compared to women having CS and repair. Patients opted for hysterectomy had more caesarean section and it was their choices as family completed. The BMI was higher in the hysterectomy group although this did not reach statistical significance. The hysterectomy group had a longer surgical time (p = 0.05). Estimated blood loss (EBL) and need for transfusion were lower in the myometrial repair group but these did not reach statistical significance. There was one bladder injury in the hysterectomy group but no visceral injury in the conservative myometrial repair group. Main findings All clinical options for PASD after delivery of the fetus carry potential complications including major hemorrhage. IRP can lead to sepsis and secondary hemorrhage requiring delayed hysterectomy. Hysterectomy results in permanent inability to have more children regardless of family planning wishes although even with uterine conserving techniques contemporaneous sterilization is often offered to avoid recurrence. Peripartum hysterectomy carries additional surgical morbidity related to the need to remove the cervix. It has been associated with a 7% ureteric injury and 15% rate of bladder injury [7]. The Triple-P procedure, which involves delivery of the fetus above the placenta and resection of the myometrium with attached placenta after pelvic devascularization, was found to have fewer bladder injuries and no ureteric injury compared with hysterectomy but was complicated by delayed primary hemorrhage requiring embolization or re-laparotomy for intra-abdominal bleeding from neovascularization of the bladder serosa [7]. Other conservative surgical techniques are time consuming and associated with high operative risk most notably bleeding and visceral damage. Our conservative procedure, with favorable surgical outcomes, differs from the Triple-P and other procedures by focusing on formal recognition and repair of the pre-existing myometrial defect whose muscle edges are actively bleeding after delivery to restore tissue integrity. Blood loss is further controlled using several maneuvers including expeditious exteriorization of the uterus, applying manual compression at the level of the uterine arteries, and systematically delivering and detaching the placenta from posterior wall first and working anteriorly; Grading of PASD by Histology Accreta (2) Increta ( all of which can be supported using IIA balloons and other measures to achieve haemostasis. The technique, which focuses on conservation and restitution of the uterus rather than excision, provides an alternative to other conservative surgical approaches for placenta accreta spectrum disorder and has a comparable surgical profile. It has the added advantage of addressing the myometrial niche with implications for patient's future symptomatology. Strengths and limitations Our conservative surgical approach for placenta accreta spectrum disorder has a favourable surgical outcome but does depend on a full understanding of the pathophysiology of the placental condition and the steps required for optimal surgical correction of the myometrial deficiencies. The approach has the strength of conserving the uterus with adequate myometrium thickness, reducing the risks of gynaecological and obstetrical complications later. The limitations relate to the need for a comprehensive multidisciplinary team with experience in addressing large blood loss, careful fluid management and anaesthetic input, a practice which may be difficult to achieve in a non-tertiary setting, especially if case numbers are small. In such a situation, a regional network could be beneficial. Our study is also a small consecutive series. We acknowledge the inevitable selection bias in this study produced by the choice of surgical approach being made by the individual patient after objective discussion of their options. Although there may be some uncertainty of the PAS diagnosis for women who underwent myometrial repair and data for one woman in each group is incomplete, all the other women had at least one diagnostic information suggestive of PAS, either from ultrasound scan, MRI or histology strongly supportive of the diagnosis; and several had all three. Interpretation (in light of other evidence) There has been recent evidence of PASD being a disorder of defective decidua and uterine dehiscence rather than destructive trophoblastic invasion [9]. When there is a high index of suspicion for PASD, there is often preoperative uncertainty around the true extent of the problem due to lack of sensitivity of screening tools including obstetric ultrasound and pelvic MRI, for both detection and determination of extent of uterine wall involvement. In our experience, in patients with previous Caesarean section with PASD, a low-lying placenta in the current pregnancy is invariably found to be occupying the isthmocele, rather than to be adherent to it, consistent with PASD being a disorder of defective decidua and uterine dehiscence rather than a disease of trophoblastic invasion. The integrity of the myometrium in the area of placental attachment in the anterior wall, rather than the placentation itself, impedes safe access to avascular dissection planes. Thus, our starting approach from the posterior uterine wall for removal of placenta is aimed at improving the speed and safety of the procedure. Our results supported this approach. Future research, in the form of a larger prospective study, is required to further assess its morbidity, performance relative to other conservative surgeries and the subsequent reproductive and gynaecological outcomes. Conclusion This surgical approach involving delivery of baby through the incised isthmocele, expeditious uterine exteriorization and systematic manual removal of the placenta from posterior uterine wall to anterior, combined with our previously described technique of myometrial defect repair, can optimally conserve the uterus while reducing the likelihood of future niche complications in cases of PASD. It is performed within the uterine boundary which reduces the risk of perioperative complications. As it is designed for use in high-risk situations, where alternatives are equally fraught with risk, including life threatening bleeding, training, practice, and experience in the technique is obligatory. Fundings The study is supported by Singapore Duke-NUS Benjamin Henry Sheares Professorship in Obstetrics & Gynecology and Integrated Platform for Research in Advancing Metabolic Health Outcomes of Women & Children (IPRAMHO) Study Group (NMRC CGAug16C008). The funding was used to cover the costs of medical illustration and open access submission. PASD Placenta accreta spectrum disorders CS Caesarean section AS Anterior superior muscle AI Anterior inferior muscle PS Posterior superior muscle PI Posterior superior muscle	2023-01-16T05:07:06.050Z	2023-01-14T00:00:00.000	{ "year": 2023, "sha1": "13126856573aeb275672436408bfbc4397bbd9b9", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "13126856573aeb275672436408bfbc4397bbd9b9", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
212697243	pes2o/s2orc	v3-fos-license	Chaste: Cancer, Heart and Soft Tissue Environment Chaste (Cancer, Heart And Soft Tissue Environment) is an open source simulation package for the numerical solution of mathematical models arising in physiology and biology. To date, Chaste development has been driven primarily by applications that include continuum modelling of cardiac electrophysiology (‘Cardiac Chaste’), discrete cell-based modelling of soft tissues (‘Cell-based Chaste’), and modelling of ventilation in lungs (‘Lung Chaste’). Cardiac Chaste addresses the need for a high-performance, generic, and verified simulation framework for cardiac electrophysiology that is freely available to the scientific community. Cardiac chaste provides a software package capable of realistic heart simulations that is efficient, rigorously tested, and runs on HPC platforms. Cell-based Chaste addresses the need for efficient and verified implementations of cell-based modelling frameworks, providing a set of extensible tools for simulating biological tissues. Computational modelling, along with live imaging techniques, plays an important role in understanding the processes of tissue growth and repair. A wide range of cell-based modelling frameworks have been developed that have each been successfully applied in a range of biological applications. Cell-based Chaste includes implementations of the cellular automaton model, the cellular Potts model, cell-centre models with cell representations as overlapping spheres or Voronoi tessellations, and the vertex model. Lung Chaste addresses the need for a novel, generic and efficient lung modelling software package that is both tested and verified. It aims to couple biophysically-detailed models of airway mechanics with organ-scale ventilation models in a package that is freely available to the scientific community. Chaste is designed to be modular and extensible, providing libraries for common scientific computing infrastructure such as linear algebra operations, finite element meshes, and ordinary and partial differential equation solvers. This infrastructure is used by libraries for specific applications, such as continuum mechanics, cardiac models, and cell-based models. The software engineering techniques used to develop Chaste are intended to ensure code quality, re-usability and reliability. Primary applications of the software include cardiac and respiratory physiology, cancer and developmental biology tissues ('Cell-based Chaste'), and modelling of ventilation in lungs ('Lung Chaste'). Cardiac Chaste addresses the need for a high-performance, generic, and verified simulation framework for cardiac electrophysiology that is freely available to the scientific community. Cardiac chaste provides a software package capable of realistic heart simulations that is efficient, rigorously tested, and runs on HPC platforms. Cell-based Chaste addresses the need for efficient and verified implementations of cell-based modelling frameworks, providing a set of extensible tools for simulating biological tissues. Computational modelling, along with live imaging techniques, plays an important role in understanding the processes of tissue growth and repair. A wide range of cell-based modelling frameworks have been developed that have each been successfully applied in a range of biological applications. Cell-based Chaste includes implementations of the cellular automaton model, the cellular Potts model, cell-centre models with cell representations as overlapping spheres or Voronoi tessellations, and the vertex model. Lung Chaste addresses the need for a novel, generic and efficient lung modelling software package that is both tested and verified. It aims to couple biophysically-detailed models of airway mechanics with organ-scale ventilation models in a package that is freely available to the scientific community. Chaste is designed to be modular and extensible, providing libraries for common scientific computing infrastructure such as linear algebra operations, finite element meshes, and ordinary and partial differential equation solvers. This infrastructure is used by libraries for specific applications, such as continuum mechanics, cardiac models, and cell-based models. The software engineering techniques used to develop Chaste are intended to ensure code quality, re-usability and reliability. Primary applications of the software include cardiac and respiratory physiology, cancer and developmental biology The software Chaste is available on GitHub https://github.com/Chaste/Chaste, and the current stable release is version 2019.1. Please see the Readme.md file on the Github repository for links to the Chaste wiki and install guides. Previous publications about Chaste have detailed the rationale for, and design principles behind, Chaste (Pitt-Francis et al., 2009), as well as the main application areas of Chaste up to 2013 (Mirams et al., 2013). Chaste places an emphasis on reproducibility and verification and, as such, extensive automated testing is used to ensure software quality and reliability. A series of test suites must all pass before any commit is considered a release-candidate. Most testing is performed on Long Term Support (LTS) versions of Ubuntu Linux, with unit tests additionally being run on macOS. Testing includes compilation of all libraries with GCC, Clang and Intel C++ compilers; extensive unit testing; performance profiling to identify any slowdowns over time; memory testing with valgrind; verification of code coverage; and running unit tests with different combinations of dependencies to ensure portability. The output of these tests is available at https://chaste.cs.ox.ac.uk/buildbot/. Since 2013, Chaste has substantially changed to modernise its infrastructure and to enable new science. In terms of infrastructure, Chaste now uses a modern CMake build system, the C++14 language standard, and makes extensive use of BuildBot for continuous integration. In terms of science, Lung Chaste is entirely new and allows the use of Chaste in a new scientific domain. In Cardiac Chaste, we can now create algebraic Jacobians for CellML ODE systems, which can improve speed of simulation for cardiac action potential and tissue simulations (Cooper, Spiteri, & Mirams, 2015), and metadata annotations of CellML files have replaced manual specification of variables in configuration files. Cell-based Chaste has been overhauled to improve flexibility. Changes include hierarchies of simulation modifiers, information writers, cell-cycle models, subcellular reaction network models, and numerical methods that allow new customisation points in almost every area of all cell-based simulations. In addition, simulation output has been standardised to use VTK, a standard and powerful visualisation framework, and some cell-centre simulations now run in parallel using MPI. Comparison with other software Chaste provides substantial common infrastructure enabling a wide range of applications across multiple disciplines. Common elements include meshing, solving differential equations, input/output and continuum mechanics, and these form a platform for Cardiac, Cell-based and Lung Chaste. A key goal of Chaste is to enable the implementation of many different modelling frameworks. This not only allows a user to select the most appropriate tool for their research but, importantly, enables the comparison of different modelling frameworks to better understand the benefits and drawbacks of each (Osborne, Fletcher, Pitt-Francis, Maini, & Gavaghan, 2017). This is an explicit design goal of Chaste, which focusses on the flexibility of implementing multiple models rather than (for example) building a graphical user interface. See Table 1 for a comparison of alternatives to Chaste in specific domains, with all other software tools implementing a single modelling framework. Installation Installation of Chaste has been greatly simplified through the development of a Docker image https://github.com/chaste/chaste-docker. Docker is a lightweight, open-source virtualisation technology for running encapsulated applications ('containers') on all major operating systems at near-native speed. This enables Chaste (including all dependencies, environment settings, convenience scripts and the latest precompiled release) to be downloaded and installed with just a single command. Isolating Chaste within a container also means that its dependencies and those installed on the user's host system can coexist without interference or version conflicts. In addition to simplifying the set-up and execution of Chaste, importantly this also enhances its reproducibility by providing a homogeneous computational environment regardless of the underlying operating system and hardware. Not only is the Chaste source code version-controlled, but so too are the dependencies, configuration settings and environment variables used to build and run it. This means that collaborators and reviewers can easily and consistently reproduce results (to within machine precision) on any platform while developers can seamlessly migrate and scale-up their simulations from a laptop to a workstation or HPC cluster. Example usage Chaste has tutorials to walk users through basic functionality for each application area. Tutorial examples are bundled for each specific release version, and examples for this release are available at https://chaste.cs.ox.ac.uk/chaste/tutorials/release_2019.1. Tutorials take the form of C++ header files that each define 'tests' in the Chaste testing infrastructure. These tests must be compiled and run to produce an output, which can be visualised using ParaView. In the following sections we showcase a specific tutorial for each of cardiac, cell-based, and lung Chaste, with minimal commands necessary to reproduce the output shown. Cardiac example Here we demonstrate how to run and visualise a three-dimensional monodomain cardiac simulation. This follows the tutorial TestMonodomain3dRabbitHeartTutorial which simulates the result of an electrical stimulus being applied to a realistic rabbit heart geometry. Assuming that 1 Chaste has been installed on Ubuntu Linux (or is running within a Docker container), 2 the Chaste source code exists at $CHASTE_SOURCE_DIR, 3 the environment variable $CHASTE_TEST_OUTPUT is set to a valid directory, a minimal set of commands to build and run the tutorial is as follows: mkdir build && cd build cmake $CHASTE_SOURCE_DIR make TestMonodomain3dRabbitHeartTutorial ctest -R TestMonodomain3dRabbitHeartTutorial This will produce output in the following directory: $CHASTE_TEST_OUTPUT/Monodomain3dRabbitHeart To view the results evolving over time as an animation in ParaView it is necessary to postprocess the results with the following command: Cell-based example Here we demonstrate how to run and visualise a cell sorting simulation using Chaste's vertex model implementation. This follows the tutorial This will produce output in the following directory: $CHASTE_TEST_OUTPUT/TestVertexBasedDifferentialAdhesionSimulation To visualise the simulation, open the file results.pvd in ParaView, choose to colour by'Cell types', and display 'Surface With Edges'. Lung example Here we demonstrate how to run and visualise the lung airway generation tutorial. This follows the tutorial TestAirwayGenerationTutorial which statistically generates lung airways given initial geometry segmented from a CT scan. Assuming that 1 Chaste has been installed on Ubuntu Linux (or is running within a Docker container), 2 the Chaste source code exists at $CHASTE_SOURCE_DIR, 3 the environment variable $CHASTE_TEST_OUTPUT is set to a valid directory, a minimal set of commands to build and run the tutorial is as follows: mkdir build && cd build This will produce output in the following directory: $CHASTE_TEST_OUTPUT/TestAirwayGenerationTutorial To visualise the generated airway geometry, open the file example_complete_conducting_ airway.vtu in ParaView. Application of an 'Extract Surface' filter followed by a 'Tube' filter allows the centreline and radius information to be viewed as a series of tubes. Recent publications enabled by Chaste Since our last publication on Chaste, over 70 peer-reviewed publications have been enabled in the areas described below. Publications using Cardiac Chaste have included scientific studies relating to: basic mechanisms of cardiac electrophysiology and tissue structure in healthy and diseased settings; the sources and consequences of inter-subject electrophysiological variability; predicting the effects of drugs on cardiac activity, including safety assessment and the development of associated web-based tools (Cooper, Scharm, & Mirams, 2016). Other studies enabled by Cardiac Chaste have advanced technical methodologies for parameter identifiability and inference, model selection and uncertainty quantification in cardiac electrophysiology models (Johnstone et al., 2016); and for the verification and efficient numerical simulation of cardiac models (Green, Bohn, & Spiteri, 2019). The continuummechanics solvers in Chaste have been used for studies of cardiac electromechanics (Carapella et al., 2014). The Cardiac Chaste code has also been applied to gastric electrophysiology, in particular focusing on the interstitial cells of Cajal network (Sathar, Trew, & Cheng, 2015). Publications enabled by Cell-based Chaste have focused on: the cellular mechanisms and dynamics of intestinal homeostasis and carcinogenesis; the mechanisms underlying vascular tumour growth and response to therapy in the Microvessel Chaste project (Grogan et al., 2017); the biomechanical characterization of epithelial tissue development and wound healing; the organisation and proliferation of stem and pluripotent cells in development; the spread of sexually-transmitted infections; vascular remodelling (Osborne & Bernabeu, 2018); cell-based model calibration and parameterisation (Kursawe, Baker, & Fletcher, 2018); and their efficient numerical solution (Cooper, Baker, & Fletcher, 2017). Papers on Lung Chaste describe its use for patient-specific airway tree generation and flow modelling (Bordas et al., 2015).	2020-03-14T07:17:47.561Z	2020-03-13T00:00:00.000	{ "year": 2020, "sha1": "25e9a0b5ea6b76f01dc5c49e5039e567400e13c0", "oa_license": "CCBY", "oa_url": "https://joss.theoj.org/papers/10.21105/joss.01848.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "2f005c662c5056948c5059a30c403709407e8db5", "s2fieldsofstudy": [ "Medicine", "Engineering" ], "extfieldsofstudy": [ "Medicine", "Computer Science" ] }
219738131	pes2o/s2orc	v3-fos-license	Association of mental and physical health of first year undergraduate students with ABO blood groups in an Indian urban medical college Background: Medical students tend to have greater stress levels than the general population. Present study aims to explore mental and physical health of first year medical undergraduate students and association with different ABO blood groups. Methods: In this cross-sectional study, 99 first year students were segregated on the basis of ABO blood groups. Mental health was assessed using validated General Health Questionnaire (GHQ-12). For physical health, height and weight of each student were measured and Body Mass Index (BMI) was calculated. Results: Study shows higher prevalence of stress (GHQ-12 scores) of 54.6%. There was no statistically significant association of stress levels (GHQ-12 scores) and BMI with various ABO Blood groups using Pearson Chi Square test. On comparison GHQ-12 scores were highest for O blood group students and BMI values were greater in blood group B and O, but not significant. AB blood group had least GHQ-12 scores and BMI. On correlation of GHQ-12 scores with BMI, there was weak positive correlation. Conclusions: Blood groups have no association with mental and physical health status. Interventions in early phase to reduce stress in medical students will reduce incidence of obesity and related health disorders in future. INTRODUCTION Stress is the body's reaction to changes that requires physical, mental, or physiological adjustment or response. 1 It is observed that medical students undergo tremendous stress during their course. 2 Most of the studies show first year as the most potential target for stress during medical training. 3,4 Research in various countries has reported high incidence of emotional disorders like anxiety and depression among medical students. 5 It is postulated that blood groups can predict personalities and some personalities are more prone to stress. Cattell et al, showed significant association of Premsia-tenderminded versus Harria-toughminded personality trait. A blood type were more premsic, and O, B, and AB were more harric. 6 Many studies shows that mental illness such as personality disorders, mania, eating disorders, nervous tension are related to blood groups. 7 Stress is also related to declining physical function like obesity over time. 8,9 Obesity has been prevalent and one of the most important metabolic concerns in the recent years that can be a predisposing factor for the development of many cardiovascular, endocrine, and malignant ailments. 10 Increased BMI pose increased risk of additional health problems. 12 Some common conditions related to overweight and obesity include cardiovascular diseases, high blood pressure, diabetes, osteoarthritis and some cancers. The ABO blood group system is not only important in blood transfusions, organ transplantation, cardiovascular diseases, erythroblastosis in neonates, but also one of the strongest predictors of national suicide rate and a genetic marker of obesity. 13,14 Research on ABO group system has been of immense interest due to its medical importance as non-modifiable attributing factor in causation of different diseases. Considering the lacunae in literature, present study is proposed to explore mental and physical health of the first year medical students in association with different ABO blood group types. A Cross Sectional Study was carried out in Terna Medical College, Nerul, Navi Mumbai during period of one year from September 2017 to August 2018 after obtaining approval from Institutional Ethical Committee. Inclusion criteria  First MBBS students in the age group of 18-20 years of either sex consenting to participate were included in the study. Exclusion criteria  Students having any significant history of major illnesses like hypertension, diabetes, neurological, endocrine, psychiatric disorders or reporting any addiction and drug abuse on history taking were excluded from the study. Total 99 students participated in the study willingly, of which 50 students were male and 49 students were female. Students were explained about the objectives of the study and were assured confidentiality of the information collected. To evaluate mental health of the students, validated General Health Questionnaire (GHQ-12) was used. It is an extensively used tool to quantify stress levels. It is used as screening instrument for emotional disorders. GHQ-12 consists of 12 items, each assessing the severity of a mental problem over the past few weeks. GHQ-12 items were rated under four categories of responses; more so than usual, same as usual, less than usual and much less than usual for 6 positive items and not at all, no more than usual, more than usual and much more than usual for 6 negative items. Data collection was conducted 6 months after the commencement of the academic year. This period measured the natural stress level and avoided the examination period which might introduce biasness to the measurement. Lecture hall was chosen as the study location since all the students normally gathered at this location for lecture session. Students were requested to rate each event based on the problems they encounter for the past few weeks by choosing from four responses. The scoring for GHQ-12 was done by modified binary scoring method whereby the two least symptomatic answers score 0 and the two most symptomatic answers score 1 i.e 0-0-1-1 for positive items and 0-1-1-1 for negative items. 15 The minimum CGHQ-12 total score was 0 and the maximum score of 12. The sensitivity and specificity of GHQ-12 score at cut-off point of 4 were 81.3% and 75.3% respectively with positive predictive value of 62.9% and therefore participants who scored GHQ-12 equal to 4 and above were considered as having significant stress and taken as case in this study. 16 Higher the score, more is the psychological distress. The severity of stress was graded on the basis of scoring in the GHQ-12 scale: GHQ-12 score of 1-3 as no stress, score of 4-6 as mild stress, score of 7-9 as moderate stress and score of 10-12 as severe stress. Age, sex, and anthropometric parameters: height (meters) and weight (kg) were noted for each student. Height was measured using a measuring scale whose least count was 0.1cm. Height of each student was converted in unit of metres. Weight was measured using weighing machine whose least count was 0.5kg. As physical health parameter for diagnosing obesity or malnutrition and related health risks, BMI of each student was calculated using Quetelet's index: BMI= Weight (kg)/Height 2 (m). BMI was classified according to the proposed criteria of the WHO, where BMI of the following values: <18.5kg/m 2 , 18.5-24.9kg/m 2 , 25-29.9kg/m 2 , and ≥30kg/m 2 , is categorized as underweight, normal weight, overweight, and obese respectively. 17 ABO blood grouping of each student was done by agglutination method using Anti-A and Anti-B antisera for each student in the Physiology department Haematology laboratory. Students were grouped into A, B, AB and O blood groups depending on the results. Statistical analysis All data was presented in table form. The data was analysed using Statistical Package for Social Sciences (SPSS) version 19. Percentage of frequency of occurrence for each parameter was calculated. Pearson Chi Square test was applied and p value was obtained to find the significance of difference between different study groups. For comparison between groups, mean and standard deviation (SD) was calculated. One-way analysis of variance (ANOVA) and Post-hoc by Tukey-Honestly significant difference (HSD) test was used for analysis. p value less than 0.05 was considered to be statistically significant. Correlation of GHQ-12 scores with BMI was done using Karl-Pearson Correlation Coefficient analysis test. RESULTS Out of total 99 students, 31 (31.3%) students belong to blood group A, 30 (30.3%) belong to blood group B, 9 (9.1%) belong to blood group AB and 29 (29.3%) belong to blood group O. Table 1 depicts high prevalence of stress (GHQ-12 score) among first year medical students. Out of total 99 students, 57.6% students had stress and 42.4% had no stress. 38.4% of students had mild stress, 16.2% had moderate stress and 3% had high stress levels. There was no statistically significant difference between various blood groups. Stress prevalence was highest in blood group O (65.5%) and least for AB blood group (0%). Among 99 students, 17.2% belonged to overweight and obese group, 15.1% in underweight and 67.7% in normal BMI group. There was no statistically significant difference between various blood groups. However, overweight and obese group subjects mainly belonged to blood group O (20.7%) followed by blood group A (20%) and least in AB blood group (11.1%). Table 2 depicts that on comparison between various blood groups, there was no statistically significant difference in GHQ-12 score for most of the groups except between blood group B and O there was statistically significant difference in female group. The mean GHQ-12 score was highest for blood group 'A' subjects followed by blood group 'O' and least for blood group AB. In males, highest score was for blood group A and in females, for blood group O. There was no statistically significant difference on comparison of BMI between various blood groups as shown in Table 2. BMI values were higher in males than females. Higher mean values of BMI were observed for blood group B and O students in total and female group. BMI values were least for AB blood group students. On correlation of stress (GHQ score) with BMI in different groups, there was mild degree of positive correlation but not statistically significant as shown in Table 3. DISCUSSION Present study shows that out of 99 first year medical students in an Indian urban medical college, 57.7% students had stress with mean GHQ-12 score of 8.82±2.70 indicating significant mild to moderate level of psychological distress. This finding is similar to other studies which showed first year as the most potential target for stress. 3,4 Although minimal amount of stress is desirable and is necessary to spark in a healthy competitive spirit, undue stress has undesirable impact on health of the students. 18 There was no significant difference of GHQ-12 score between different ABO blood groups. This finding is consistent with previous study which concluded that there is no association between blood type and psycho wellness of individual. 19 In this study, stress prevalence was highest in O blood group subjects. Mean GHQ-12 score was higher for blood group A followed by blood group O though not statistically significant. Most of the studies show variable results in this regard. A study indicates that subjects with blood group O perceived more stress as compared to subjects of blood group A. 20 Boyer demonstrated that blood type might influence psychiatric symptoms. He showed that subjects with blood type A scored higher than those with type O on the obsessivecompulsive and psychoticism factors. 21 Neumann et al, found that individuals of blood type A demonstrated higher levels of depression, anger, and anxiety than those of blood type O. 22 It is postulated that blood type A had higher initial levels of very low density lipoprotein (LDL) toxicity preventing activity (TxPA), cortisol but quicker stress recovery rates than type O group. 23 O blood group has poorer recovery from stress as compared to A blood group, thus leading to stress related duodenal ulcers. 6,24 O type blood has also been found to occur more frequently in manic-depressive patients. 24,25 There was no significant difference of BMI between different ABO blood groups. This finding is consistent with previous study that showed that no particular blood type was more predisposed to overweight, obesity and other health disorders like hypertension and diabetes. 26 Most of the studies show no association between anthropometric measures and ABO blood groups. 27,28 In this study, mean BMI values were higher for blood group B and O in total and female group, but not statistically significant. This finding is in agreement with the study that observed the B blood group was more susceptible to hypertension and obesity followed by blood group O, A and AB blood group had less chance of getting hypertension and obesity. 29,30 In another study Group O has been linked to obesity, diabetes and cardiovascular risk. 31 Different persons respond differently to the overwhelming stress, some lose their appetite, while others start eating more. These changed eating habits may lead to changes in the weight and BMI. 32 In present study, mild degree of positive correlation was observed between stress (GHQ-12 score) and BMI for all study groups. This finding was contrary to the studies that say that perceived stress scores are not related to body weight but consistent with findings of other studies. 30,34 Shet et al, and Rizvi et al, have studied perceived stress with BMI and stress in software professionals in different settings and in both the studies, there was a positive association of stress with BMI. 11,34 The positive association between stress level and BMI points out that prompt measures should be taken to control obesity and in turn prevent the occurrence of life-threatening diseases such as hypertension and diabetes in young medical undergraduates, as academic stress is inevitable part of medical curriculum. Students should be encouraged to adopt stress copying skills in an early phase of medical education. Inclusion of stress and time management in foundation course of new competency based undergraduate curriculum by Medical council of India for newly admitted medical students is likely to bring change in their holistic health status as needed by future Indian physicians. 35	2020-05-28T09:16:27.629Z	2020-05-26T00:00:00.000	{ "year": 2020, "sha1": "79f5cab7194fb204b6a92b503ab536b8b31536b2", "oa_license": null, "oa_url": "https://www.msjonline.org/index.php/ijrms/article/download/8137/5736", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "3dcca9b931f3f5babe591092baadb7c9702b4fd6", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
267148317	pes2o/s2orc	v3-fos-license	THE INFLUENCE OF THE HELLO ENGLISH APPLICATION TOWARDS STUDENTS ’ LEARNING ACHIEVEMENT IN VOCABULARY MASTERY The objective of this research was to determine the impact of the Hello English application on students' vocabulary acquisition. This study adopted a quasi-experimental design and quantitative research methodology. Data were obtained from 30 eighth-grade students, 15 of whom were in the experimental class and 15 of whom were in the control class. The instrument for data collection was vocabulary tests that were intended to determine if the Hello English program had an effect on students' vocabulary knowledge. The findings of the data analysis indicate that the experimental class's mean score on the pre-test was only 34 but climbed to 57.3 on the post-test. In contrast, the mean pre-test score for the control group was 43, whereas the post-test score was just 39.6. As a result, the class adopting Hello English as the treatment saw a considerable improvement. INTRODUCTION We must have a strong vocabulary to articulate our thoughts and comprehend what others are saying (Alqahtani 2015).Therefore, vocabulary is the cornerstone for assisting the language acquisition of international students.On the other hand, students learning a foreign language confront some major challenges, one of which is the difficulty of growing their vocabulary (Zalmansyah 2013).In addition, there are still many educators who are unaware of the significance of vocabulary development for young language learners.Priyono in Ginting (2019) said that pupils' restricted vocabulary has been their greatest obstacle in the EFL environment while learning English.This issue may be related to how instructors and students see vocabulary teaching and learning and how vocabulary is taught and learned in ELT.In addition, pupils had difficulty comprehending the lecture and lacked enthusiasm to learn English owing to a lack of vocabulary competence.Southerland said that junior high school instructors must capture the interest of their pupils since adolescents at this age are quickly bored (Southerland 2011). In addition, the online learning method led to declining student success.Learning achievement is the degree of a student's success in learning the subject matter in school, as measured by scores on topic-specific assessments (Tari 2021).In this moment and context, mobile learning is essential for instruction.In addition, the pupils represent the Z generation.The Z generation was born after the advent of technology.This situation makes this generation's society technologically advanced, adaptable, more intelligent, and tolerant (Rastati 2018).As an alternative to teaching and learning English, several programs are available, notably for teaching vocabulary, such as Hallo English.It was launched in October 2014 by Culture Alley.It has 475 interactive lessons, games, live classes with a professional teacher, trending news and articles, role-play (conversation), tip of the day, word of the day, and a helpline (ask teachers).This app is available on iOS, Windows, the web, and Android.It makes it easy to use for everyone and everywhere. Below, the writers highlight recent studies on the Hello English app.Maimanah (2020), in her research, stated that the Hello English app made it easier for students to memorize vocabulary.Besides, it helped students pronounce the vocabulary correctly and find new vocabulary creatively.The Hello English app positively impacted and motivated them to learn English.The result of the research was collected through interviews about the students' higher education perceptions.Another researcher, Fendiasari (2020), investigated the effectiveness of the Hello English app.Using a quasi-experimental design, she found that the average of the experimental class became higher after using the Hello English app.It means there is a significant difference between the application and conventional methods, and it is effective in teaching English in senior high school, particularly vocabulary.Ginting (2019) also described and supported the previous explanation that the Hello English app has significant effect on the students' vocabulary mastery.Liana, Wahyudin, and Hanoum ( 2018), in their research, they used the Hello English app to improve student's learning achievement, especially in improving listening skills.Their experiment research found that using Hello English improved the students' learning from under 50% to 82.16%.This result shows that the Hello English app could increase students' learning achievement in learning English, particularly in listening skills.The research by Puspitaloka et al. (2017) aimed to improve young learners' vocabulary using the Hello English app.The research was implemented for four weeks using the Hello English app as an Android-based educational game.Besides, the research used experimental and controlled classes.The result showed that there was a significant difference between the experimental class and the control class after the intervention was implemented.It improved students' learning competence in learning vocabulary, and the game encourages active participation during classroom activities.Using an android based educational game was one of the fun ways to teach and learn English, and it stimulated students' creativity.Furthermore, the implementation of Hello English for Teachers discovered that this was a recommended method for improving their teaching ability. The distinction between this research and the other research in this study was made using online learning due to the COVID-19 pandemic.This study can be an alternative way for teachers and students to learn English in a fun way.The other research was conducted before online schooling.Therefore, it has a different situation.The writers intended to do further research about using Hello English to improve students' learning achievement.Thus, based on the situation, this study investigated the influence of Hello English on students ' Design This quantitative study used a quasi-experimental method to get reliable data and compared the results to show the cause-and-effect relationship between the variables.The researcher involved two groups in this research.They are the class that did the experiment and the class that did not.Before giving either group treatment, the researcher gave each of them a pre-test.The Hello English app was then given to the experimental class to help them learn English and vocabulary.On the other hand, the Hello English program was not used in the control group.After the treatment, there was a test to see if the Hello English app had any effect. Population and Sample The population in this study was eighth graders.Moreover, the sample was obtained through cluster random sampling.The sample was split between two distinct groups.These are the experimental and control groups.Fifteen pupils in the experimental class received therapy by learning English using Hello English.However, the control class, which served as the experimental class's comparison group, did not give any treatment.There were 25 pupils in this class who were not given any therapy. Research Instruments A research instrument is necessary for the study.It is needed to obtain and collect relevant information for the research (Wilkinson and Birmingham 2003).It helped the writers do the research and get the data needed to find the result of the study.To collect the data for this study, the writers need tools to collect them.It is referred to as an instrument.The research instrument used in this study was a test.The researcher did the test twice.The first test was called the pre-test.The test intended to see the students' English level before the writers applied the treatment. On the other hand, the control group does not receive any treatment; they are subjected to all of the tests.The second test is called the "post-test" or "final test."This test aimed to see the value after the treatment was applied.The pre-test and the post-test all contain 20 numbers of multiple-choice and 5 points for each correct answer.The students get a score of 100 if they get the correct answer for all the questions. Furthermore, the instrument used by the author has been validated to determine whether or not the test is valid.According to Ghozali in Armin (2020), the value of the r count is compared to the value of the r table to calculate the validity test.If r count > r, a table with the significance level is displayed, and the value is less than 05, the statement in the test is invalid.Besides the validation, the test was also analyzed for reliability.A reliability test is a way to determine how reliable a test is to predict a variable or concept.A test is said to be reliable if the same person always gives the same answer to the same question.After they did a validity test, the writers did a reliability test on Microsoft Excel. In collecting data, the writers used pre-test and post-test.The test was multiple-choice and consisted of 20 questions.The questions were selected from questions or materials in the Hello English application.Each question has five points for the correct answer.Therefore, if the students get 20 correct answers, they get 100.The pre-test and post-test questions were different yet still had the same level and composition.This technique was intended to get more valid data.In the pre-test, the test was given to both classes.The test aimed to analyze the students' English levels.Besides, it was used as a data comparison for the posttest score.After giving the pre-test, the next procedure was to continue treating the experimental class.The treatment was conducted over eight meetings.In addition, students in the experimental class learned English through the Hello English application online twice weekly.Every time the students used the Hello English application, they completed two missions from the app (for each meeting).For the next meeting, the students continued with the next mission from the Hello English application.In the post-test, both classes took a test.The questions for the post-test are different from the pre-test questions, but they still have the same context and level as the previous pre-test's questions.This post-test aimed to see whether there was a difference between the two classes and whether there was a substantial improvement in the student's learning achievement. Data Analysis Data analysis was a step the researcher took after collecting data to answer the problem statement and verify the hypothesis by calculating data groupings from the respondents and variables.Present the data based on the variable from all the responses (Sugiyono 2011).Besides, the technique of data analysis in this study used SPSS 22.The data was collected and then analyzed with the normality test using a paired T-test, which is the dissimilarity of two groups with a normal distribution.The researcher used Shapiro-Wilk as the formula for the normality test by using the SPSS 22 application due to the small sample size (below 50 samples).A homogeneity test was also used to look at the data.As the assumption from the independent T-test shows, there must be a homogeneity test.This homogeneity test aims to find out if all of the different data types in a population have the same variation.Even though the test result for homogeneity is not uniform, the independent test can still be done.A paired T-test is also needed to make a research decision.After the normality and homogeneity tests are done, it can be seen whether there is a significant change in the paired sample.The pre-test and post-test from each class were used to test the data.If the significant level is less than .05,then the Hello English app's use affects how well the students learn.If the significant level is more than.05,using Hello English doesn't change how well students learn.The hypothesis test was the last part of the analysis.The goal was to find out if the information strongly supported the hypothesis about the characteristics of the population. Findings After completing the investigation, the writers uncovered two categories of data.There were both experimental class (VIII-C) and control class (VIII-C) data (VIII-B).The pre-test and post-test of the examination yielded quantifiable results.During the first meeting, the pre-test was administered before the therapy.In contrast, the post-test was administered at the last meeting after the therapy.The outcome of the pre-test was as follows: The above graph shows how the two classes did on their pre-tests.In the experimental class, one student's grade ranged from 0 to 19 points.Four students in the experimental group and two students in the control group got between 20 and 29 points.Five students in the group and two students in the control group got a score between 30 and 39.Three students in the experimental group and five students in the control group got grades between 40 and 49.In the experimental group, only one student had a score between 50 and 59.This was not the case in the control group.Two students in the control group and one student in the experimental group got scores between 60 and 69.The results of both courses were almost the same, which shows that their vocabulary levels are similar.On the other hand, here's what happened in the post-test: Based on the post-test results, the above graph shows how the values of the two groups compare.The numbers showed that the four students in the control group with the worst scores were all in the control group (20-29).In the meantime, there was no one in the experimental class.Then, in the experimental class, there was a student with a score between 30 and 39.On the other hand, the control group had only three people.Four students in the experimental class got between 40 and 49 points.Three people did it in the control class.Also, more people with scores between 50 and 59 were in the experimental class.Five students in the experimental class and two people in the control class got scores between 60 and 69.Two students in the experimental class and one student in the control class got 70-79.The highest score on the test was between 80 and 89.Students with this score are in the experimental class, which has only one student, while the control class has no one with this score. After describing the data findings from the tests, this study also spelled out the findings of the analysis of the collected data.The writers analyzed the data in three ways in this study.First, using normality and homogeneity tests on the preliminary data.Second, calculating the hypothesis test results.Finally, the research concluded by counting the statistical hypotheses.First, in the normality test, the data was analyzed using Saphiro-Wilk.The result of the test is as follows: Teaching English is also very suitable for these students because the learning is smartphonebased, and they are the Z generation.They were born and grew up with and love technology, so they can use this app easily.Referring to the findings and the data analysis, Hello English influenced students' learning achievement, particularly vocabulary.It can be seen from the value of the experimental class, which increased significantly after this method was applied, while the value of the controlled class did not increase significantly. CONCLUSION After researching and analyzing the data, it was feasible to conclude that Hello English impacted students' vocabulary mastery.As evidence, the experimental group's post-test scores increased after the treatment was given.The mean score in the experimental pre-test class is only 34; nevertheless, it rises to 57.3 on the post-test.The control group's mean pretest score was 43, and their post-test score was just 39.6.If the result of the independent sample t-test sig.(2-tailed) less than .05,then there was a substantial difference between the classes.As a result, sig (two-tailed) proved .002and was less than .05.This presented that Ha was accepted. Refers to the above conclusion, this study is hoped to be useful and persuade readers to use attractive media to learn English.One of the media is the Hello English application.Furthermore, it is hoped that the teacher will be able to use an enjoyable method or tool in teaching English, particularly in online learning.The writers also hoped this study would serve as a resource for future researchers interested in this topic.	2024-01-24T18:14:28.439Z	2023-10-07T00:00:00.000	{ "year": 2023, "sha1": "97495be74d4f81f1f5e95071d0db495ad64a8fa1", "oa_license": "CCBY", "oa_url": "https://foster.pbi-iainpalopo.ac.id/index.php/contents/article/download/125/71", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "3910e5be87dc8f2e244674662ff44136b354ebbb", "s2fieldsofstudy": [ "Education" ], "extfieldsofstudy": [] }
25153705	pes2o/s2orc	v3-fos-license	Identification of ING4 (Inhibitor of Growth 4) as a Modulator of Docetaxel Sensitivity in Human Lung Adenocarcinoma Resistance to docetaxel (DTX) usually occurs in patients with lung adenocarcinoma. To better elucidate the underlying molecular mechanisms involved in resistance to DTX-based chemotherapy, we established a DTX-resistant lung adenocarcinoma cell line (SPC-A1/DTX). By gene array analysis, the expression of ING4was found to be significantly downregulated in SPC-A1/DTX cells. Additionally, the decreased expression of the ING4 gene was induced upon DTX treatment of SPC-A1 cells. Overexpression of ING4 reverses DTX or paclitaxel resistance of DTX-resistant lung adenocarcinoma cells (SPC-A1/DTX or A549/Taxol) by inducing apoptosis enhancement and G 2 /M arrest, and small interfering RNA–mediated ING4knockdown renders DTX-sensitive lung adenocarcinoma cells more resistant to DTX or paclitaxel. Also, overexpression of ING4 could enhance the in vivo sensitivity of SPC-A1/DTX cells to DTX. The phenotypical changes of SPC-A1/DTX cells induced by overexpression of ING4might be associated with the decreased ratio of Bcl-2/Bax, which resulted in the activation of caspase-3. The level of ING4expression in tumors of nonresponding patients was significantly lower than that in those of responders, suggesting that the expression of ING4was positively correlated with tumor response to DTX. Our results provide the first evidence that ING4might be essential for DTX resistance in lung adenocarcinoma. Thus, ING4will be a potential molecular target for overcoming resistance to DTX-based chemotherapies in lung adenocarcinoma. INTRODUCTION Non-small cell lung cancer (NSCLC) has been one of the most lethal malignancies around the world. Adenocarcinoma of the lung is the most common type of lung cancer and accounts for 30% to 35% of primary lung tumors (1). Currently, systemic chemotherapy is still an important treatment option for patients with advanced lung adenocarcinoma (2). Docetaxel (DTX) has become an integral part of several commonly used chemotherapy regimens in NSCLC (3). However, the development of intrinsic or acquired resistance to DTX remains the greatest obstacle to the successful treatment of patients with lung adenocarcinoma. Thus, elucidating the mechanisms by which DTX resistance arises in lung adenocarcinoma remains a critical issue for overcoming and predicting DTX resistance in NSCLC. Recently, a wealth of novel insights into molecular targets and mechanisms of cancer chemosensitivity and resistance have yielded much progress in genomic and proteomic studies (4). DTX (Taxotere TM ) is a novel microtubule-stabilizing agent that has been synthesized from a precursor extracted from a renewable natural source, the needles of the European yew, Taxus baccata (5). This agent can enhance microtubule assembly and inhibit the depolymerization of tubulin. In the clinical setting, DTX has been used for adjuvant therapy after resection of localized NSCLC and in combination with radiation for locally advanced NSCLC and treatment of patients with advanced NSCLC (6,7). However, the therapeutic results in some patients with advanced NSCLC have been unsatisfying, as in cases of intrinsic or acquired chemoresistance. The dysregulation of oncogenes such as Bcl-2 family members or tumor suppressors such as PUMA (p53 upregulated modulator of apoptosis) has been found to be associated with DTX resistance of tumor cells (8)(9)(10). However, the molecular mechanisms of DTX resistance are very complicated and require further elucidation. Previously, we successfully established a DTX-resistant lung adenocarcinoma cell line (SPC-A1/DTX) from a DTXnonresistant lung adenocarcinoma cell Identification of ING4 (Inhibitor of Growth 4) as a Modulator of Docetaxel Sensitivity in Human Lung Adenocarcinoma line (SPC-A1). To gain further insight into the mechanisms of DTX resistance and explore novel potential therapeutic targets for reversing the DTX resistance of lung adenocarcinoma, we performed a microarray analysis on lung adenocarcinoma cells using the Affymetrix U133A microarray, which showed that a total of 2332 genes that were differentially expressed between the SPC-A1 and SPC-A1/DTX cell lines. Among these genes, ING4 (inhibitor of growth 4) was found to be significantly downregulated in the SPC-A1/DTX cell line in comparison with the parental SPC-A1 cell line. ING4, a novel member of the inhibitor of growth (ING) gene family, has attracted much attention as a tumor suppressor because of its ability to suppress tumor growth, angiogenesis and invasion (11). In our previous studies, we also showed that downregulation of ING4 was associated with poor prognosis of patients with lung adenocarcinoma (data not published). However, there have been no reports about the association of ING4 expression with DTX sensitivity of lung adenocarcinoma. In the present study, we attempted to investigate the roles of ING4 in docetaxel-induced drug resistance and its possible molecular mechanisms. Herein, we report our finding that restoration of ING4 expression could reverse the resistance of NSCLC cells to DTX both in vitro and in vivo by inducing apoptosis enhancement and cell cycle G 2 /M arrest. Also, the expression of ING4 in advanced lung adenocarcinoma might be positively correlated with the response of patients to DTX. Taken together, our results indicate that ING4 might be a key regulator of DTX resistance in lung adenocarcinoma cells and has the potential of being a therapeutic target for chemosenstization of lung adenocarcinoma. Cell Lines and Chemotherapeutic Reagents The human lung adenocarcinoma cell lines (SPC-A1 or A549) and taxolresistant human lung adenocarcinoma cell line (A549/Taxol) were purchased from the Shanghai Institute of Cell Biology (Shanghai, China). The DTXresistant lung adenocarcinoma cell line (SPC-A1/DTX) was established and preserved in our lab. The DTX-resistant SPC-A1 cell line was selected by continuous exposure to increasing concentrations of DTX. DTX was added into exponentially growing cultures of SPC-A1 cells at a concentration of 0.008 μg/L and allowed to remain in the culture until cell growth resumed. The cultures were then split and treated again with progressively higher concentrations of DTX. Over the course of selection, the DTX concentration was increased to 5.0 μg/L. The resulting subline was designated as SPC-A1/DTX cells (SPC-A1/DTX). The taxol-resistant A549 cell line (A549/ Taxol) was preserved in a 0.2-μg/mL final concentration of taxol according to the manufacturer's instruction. All cell lines were cultured in RPMI 1640 (GIBCO-BRL, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin in humidified air at 37°C with 5% CO 2 . DTX and paclitaxel and pancaspase inhibitor (Z-VAD-FMK) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Stock solutions of DTX and paclitaxel (1.0 μg/mL) were prepared with dimethyl sulfoxide and diluted with phosphate-buffered saline (PBS) to the required concentrations before each experiment. Microarray Analysis Total RNA from the lung adenocarcinoma cell line (SPC-A1) or the corresponding DTX-resistant lung adenocarcinoma cell line (SPC-A1/DTX) was isolated by use of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The quality and quantity of the RNA samples were assessed by standard electrophoresis and spectrophotometry methods. Complementary DNA (cDNA) microarray analysis was performed with reagents and according to protocols provided by Affymetrix (Santa Clara, CA, USA). Briefly, we prepared double-stranded cDNA using the One-Cycle cDNA synthesis kit. The GeneChip IVT labeling kit was then used to synthesize biotinlabeled cRNA, which was then fragmented prior to hybridization. The labeled, fragmented cRNA samples were then hybridized to Affymetrix U133A microarrays, and the array was washed, stained and scanned with the Affymetrix GeneChip Scanner 3000. The acquired image was analyzed by the Affymetrix GeneChip operating software version 1.0. Differential cDNA expression was determined with a two-sided t test on a single cDNA basis. Differentially detected signals were generally accepted as true when the ratio of the p value was < 0.05 and were then selected for cluster analysis. Real-Time Reverse Transcription-Polymerase Chain Reaction Assay Total cellular RNA was prepared using TRIzol reagent (Invitrogen) and reversely transcribed according to the manufacturer's instruction. Real-time polymerase chain reaction (PCR) products were detected with SYBR Green I dye by using a Light Cycler instrument (Roche, Basel, Switzerland). The GAPDH gene was amplified as an internal control. Relative quantitation was done by using the ΔCt (threshold cycle) method by taking the difference (ΔCt) between the Ct of GAPDH and ΔCt of each transcript and computing 2 -ΔCt . We obtained the sequences of primers for ING4 by referring to Tzouvelekis et al. (12). The sequences of primers for the ING4 gene were as follows: sense 5′-AGCTT GCCAT GCAGA CCT-5′; reverse 5′-GCGCA CGAGC TTTAA CTT-3′. Ct values were normalized to the reference gene GAPDH. The sequences of primers for the GAPDH gene (sense 5′-GAAGG TGAAG GTCGG ATGC-3′; reverse 5′-GAAGA TGGTG ATGGG ATTTC-3′) were designed by using the Primer Premier 5.0 software package. Transfection and Stable Selection The parental SPC-A1 or A549 and resistant SPC-A1/DTX or A549/Taxol cells were seeded into 6-well plates at 2.0 × 10 4 cells/well, respectively, and cultured overnight to 80% confluence prior to transfection. We performed transfection Flow Cytometric Analysis of Apoptosis An annexin V-fluorescein isothiocyanate apoptosis detection kit (Oncogene Research Products, Boston, MA, USA) was used to detect apoptosis according to the manufacturer's instructions. Flow Cytometric Analysis of Cell Cycle Cells were harvested at the 70% confluent stage and fixed in 70% ethanol at -20°C. After being washed with PBS, the cells were treated with PBS containing RNase A (100 mg/mL) at 37°C for 30 min. After centrifugation, the cells were resuspended in PBS containing propidium iodide (50 μg/mL) and stained at room temperature for 30 min. DNA content was evaluated by use of a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) and CellQuest software (Becton Dickinson). Assay of Caspase-3 Activity Caspase-3 activity was determined by use of the colorimetric CaspACE Assay System (Promega Corp., Madison, WI, USA) following the manufacturer's instructions, as previously reported (14). Each determination was performed in triplicate. In Vivo Chemosensitivity Assay Animal studies were performed according to institutional guidelines. Approximately 5.0 × 10 6 SPC-A1/DTX/ control or SPC-A1/DTX/ING4 cells were suspended in 100 μL PBS and injected subcutaneously into the right side of the posterior flank of female BALB/c athymic nude mice (Department of Comparative Medicine, Jinling Hospital, Nanjing, China) at 5 to 6 wks of age. Tumor growth was examined every other day with a vernier caliper. Tumor volumes were calculated by using the equation: , wherein A is the largest diameter, and B is the perpendicular diameter. When the average tumor size reached about 50 mm 3 , DTX was given through intraperitoneal injection with a concentration of 1.0 mg/kg, 1 dose every other day with 3 doses totally. After 2 wks, all mice were killed, and necropsies were performed. The primary tumors were excised, paraffin embedded and formalin fixed. Then we performed hematoxylin and eosin staining and immunostaining analysis for PCNA protein expression and analyzed the apoptosis with a TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) apoptosis detection kit (KeyGEN, Nanjing, China) according to the manufacturer's instructions. Patients and Tissue Samples A total of 18 lung adenocarcinoma tissues were collected from patients with advanced lung adenocarcinoma who received chemotherapy at the Department of Medical Oncology, Jinling Hospital, between March 2005 and September 2006. Patients met all of the following criteria: suffering from primary lung adenocarcinoma; a histological diagnosis of lung adenocarcinoma with at least one measurable lesion; a clinical stage of IIIB-IV; first-line chemotherapy either with DTX 75 mg/m 2 and cisplatin 100 mg/m 2 or DTX 75 mg/m 2 and carboplatin AUC (area under the curve) 6 mg/mL/min administered every 3 wks for a maximum of 5 cycles; and availability of sufficient tumor tissue in paraffin blocks for assessment by immunohistochemistry. Tumor response was examined by computed to-mography and evaluated according to the Response Evaluation Criteria in Solid tumors (RECIST) as a complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD), as described previously (16). Written permission to use human tumor tissues was obtained from the patients. Statistical Analysis Experimental data were expressed as the mean ± SD of at least three independent assays. Statistical analyses were carried out using one-way ANOVA and Student t test to evaluate the continuous variables. Progression-free survival (PFS) was assessed from the first day of chemotherapy administration to the date of objective disease progression. The probability of survival was plotted by the Kaplan-Meier method and compared by the log-rank test. Differences between groups were considered significant at p < 0.05. All statistical analyses were per-formed using the SPSS 13.0 statistical software. All supplementary materials are available online at www.molmed.org. Analysis of Growth or Cytotoxicity of DTX in DTX-Nonresistant or -Resistant Lung Adenocarcinoma Cells The DTX-resistant cell line (SPC-A1/ DTX), was developed from the DTXnonresistant cell line (SPC-A1). As observed by optical microscopy (Figure 1A), the morphology of the SPC-A1 and SPC-A1/ DTX cells was significantly different and the SPC-A1/DTX cells appeared as large swellings or spindle-or rhombus-shaped cell forms. The doubling time of the SPC-A1/DTX cell line (34.0 h) was significantly longer than that of the SPC-A1 cell line (26.5 h) (p < 0.05; Figure 1B). With the relative resistance (as a resistance factor) calculated via the ratio of the half maximal inhibitory concentration (IC 50 )-resistant variant/IC 50 of the parental cell line, the SPCA-1/DTX cell line was 5.37-fold resistant to DTX (13.58 μg/L) and was 3.46-fold resistant to paclitaxel (0.61 μg/L) ( Figure 1C), suggesting that SPC-A1/DTX cell line acquired resistance to cross-resistant to paclitaxel. Colony formation assays also showed significant DTX resistance in the SPC-A1/ DTX compared with the SPC-A1 cell line ( Figure 1D). Therefore, the sensitivity of DTX was shown to be significantly different between the SPC-A1 and SPC-A1/ DTX cell lines, a finding that might provide us better cell models to investigate the molecular mechanisms of DTX resistance in lung adenocarcinoma. DTX Resistance of Lung Adenocarcinoma Cells was Correlated with Loss of the G 2 /M Checkpoint in the Cell Cycle To investigate whether resistance to DTX was correlated with drug-induced apoptosis and/or cell-cycle distribution, we performed a flow cytometry assay to detect the changes in apoptosis and cell cycles. In the SPC-A1/DTX cell line, there was a significant decrease in apoptosis on exposure to various doses of DTX in comparison with the parental SPC-A1 cell line (Figure 2A). Likewise, following treatment with various doses of paclitaxel, a significant reduction in the extent of cell death was also seen by comparison of parental SPC-A1 and SPC-A1/DTX cells. Next, the perturbation in cell cycle following exposure to DTX (1.0 μg/L) was analyzed. After 24-h exposure to DTX in parent SPC-A1 cells, the percentage of G 2 /M-phase cells was significantly increased (p < 0.05), and an apoptosis peak (sub-G1) could be seen before the G 1 phase. However, in DTX-resistant SPC-A1/ DTX cells, there were no obvious changes in the sub-G1 population. Furthermore, the percentage of G 2 /M-phase cells was obviously decreased in resistant SPC-A1/DTX cells relative to parental SPC-A1 cells (p < 0.05; Figure 2B). Therefore, it was concluded that loss of the G 2 /M cell-cycle checkpoint function and abrogation of apoptosis might be involved in the acquired DTX resistance of lung adenocarcinoma cells. Microarray Analysis of Differentially Expressed Genes Associated with DTX Resistance of Lung Adenocarcinoma Cells To investigate the molecular mechanisms of DTX resistance in lung adenocarcinoma cells, we performed a microarray analysis on lung adenocarcinoma cells using the Affymetrix U133A microarray. The microarray data showed that a total of 2332 genes were differentially expressed between the SPC-A1 and SPC-A1/DTX cell lines (Supplementary Figure S1A; p < 0.05). By the fold-change analysis, we found that 338 or 31 of the 29,187 flagged cDNAs in SPC-A1/DTX cells showed at least a 4.0-or 15.0-fold change in expression level compared with parental SPC-A1 cells. Compared with SPC-A1 cells, 12 genes were significantly downregulated in SPC-A1/DTX cells, whereas the other 19 genes were significantly upregulated in SPC-A1/ DTX cells (Table 1). Then, a real-time quantitative reverse transcription (RT)-PCR assay was employed to validate the top three downregulated and top three upregulated genes (PDE1A, SLC7A7, ALDH2, SERPINB5, LUM, ING4) between the two cell lines. The expression of the 6 genes showed concordance with the microarray data ( Supplementary Figure S1B). The Expression of ING4 Gene is Downregulated in SPC-A1/DTX and A549/Taxol Cells Our gene expression data showed that ING4 was one of the top three Figure 4B). Therefore, a decreasing level of ING4 expression oc-curred in lung adenocarcinoma cells in response to DTX. Overexpression of ING4 Leads to the Increased Chemosensitivity of SPC-A1/DTX or A549/Taxol Cells to DTX To investigate whether ING4 expression affects the sensitivity of lung adenocarcinoma cells to DTX, a pcDNA/ING4 vector expressing ING4 was stably transfected into SPC-A1/ DTX and A549/ Taxol cells. Compared with parental SPC-A1 or SPC-A1/DTX/ control cells, the level of ING4 mRNA or protein expression in SPC-A1/DTX/ ING4 cells was significantly increased (Figures 5A, B). Likewise, the level of ING4 mRNA or protein expression in A549/Taxol/ING4 cells was also significantly increased compared with A549 or A548/Taxol/ control cells (Figures 5A, B). Then, the IC 50 value of DTX or paclitaxel in SPC-A1/DTX or A549/Taxol cells was determined using an MTT assay. Compared with SPC-A1/ DTX/ control cells, the IC 50 value of DTX or paclitaxel in SPC-A1/ DTX/ING4 was significantly decreased by 58.6% or 36.8%, respectively ( Figure 5C). Compared with A549/Taxol cells, the IC 50 value of DTX or paclitaxel in A549/ Taxol/ ING4 cells was significantly decreased by 64.3% or 45.6%, respectively ( Figure 5C). Next, we analyzed cellcycle distribution in SPC-A1/ DTX/ ING4 or A549/Taxol/ING4 cells. Compared with the SPC-A1/DTX/control or A549/Taxol cell line, the SPC-A1/DTX/ ING4 or A549/ Taxol/ING4 cell line triggered an accumulation of cells at the G 2 /M stage, whereas the numbers of cells in S-phase and G 0 /G 1 phase accordingly decreased ( Figure 6A). Then, we examined the changes in apoptosis. Compared with SPC-A1/DTX/ control cells, the apoptotic rate of SPC-A1/ DTX/ ING4 cells was significantly enhanced by approximately 16.11% (p < 0.05; Figure 6B). These results strongly suggest that overexpression of ING4 could reverse the resistance of SPC-A1/ DTX cells to DTX or paclitaxel by induc- ing a G 2 /M-phase arrest and apoptosis enhancement. Downregulation of ING4 Leads to the Decreased Chemosensitivity of SPC-A1 or A549 Cells to DTX To extrapolate the finding that overexpression of ING4 could reverse the chemoresistance of SPC-A1/DTX or A549/Taxol cells, we employed RNA interference to downregulate ING4 expression and analyze the changes of chemo sensitivity in sensitive SPC-A1 or A549 cells. Compared with the SPC-A1/ shcontrol or A549/shcontrol cells, the level of ING4 mRNA and protein expression in SPC-A1/shING4 or A549/ shING4 cells was significantly decreased ( Figures 7A, B). Then, we determined the IC 50 value of DTX or paclitaxel using an MTT assay. Compared with SPC-A1/ shcontrol cells, the IC 50 value of DTX or paclitaxel in SPC-A1/shING4 cells was significantly increased by 39.6% or 73.7%, respectively (p < 0.05; Figure 7C). Additionally, compared with A549/ shcontrol cells, the IC 50 value of DTX or paclitaxel in A549/shING4 cells was significantly increased by 52.2% or 68.5%, respectively (p < 0.05; Figure 7C). Then, we analyzed the changes of cell-cycle distribution ( Figure 8A). Compared with SPC-A1/shcontrol or A549/shcontrol cells, the numbers of SPC-A1/shING4 or A549/shING4 cells in G 2 /M-phase accordingly decreased and the numbers of SPC-A1/shING4 or A549/shING4 cells in S-phase significantly increased (p < 0.05). However, the numbers of SPC-A1/ shING4 or A549/shING4 cells in G 0 /G 1 -phase showed no difference (p > 0.05). In addition, it was also shown that the numbers of DTX (0.08 μg/L)-treated SPC-A1 or A549 cells in G 2 /M-phase accordingly decreased and the numbers of DTX (0.08 μg/L)-treated SPC-A1 or A549 cells in S-phase significantly increased. In other previous research, it was shown that ING4 could impede the progression of the cell cycle by regulating the expression of cell cycle regulators (such as p27, cyclinD1 and SKP2), which led to cell arrest in G 2 /M phase (17). Next, flow cytometry was used to detect apoptosis. Compared with SPC-A1/shcontrol (or A549/shcontrol) cells, DTX-treated SPC-A1 (or A549) and SPC-A1/shING4 (or A549/shING4) cells showed no difference in apoptosis (p > 0.05; Figure 8B). Thus, the increased resistance of SPC-A1 or A549 cells to DTX or paclitaxel induced by ING4 downregulation was associated with a decreased number of cells in G 2 /M-phase and an increased number of cells in S-phase. Effects of ING4 Expression on the Survival Pathway of SPC-A1/DTX Cells To investigate further insight into the mechanism of apoptosis enhancement induced by overexpression of ING4, we first determined the involvement of caspase-3. Compared with SPC-A1/DTX/ control cells combined with DTX treatment (5.0 μg/L), the activation of caspase-3 protein was observed in SPC-A1/DTX/ING4 cells combined with DTX treatment (Figure 9A). Meanwhile, compared with SPC-A1/DTX/control cells combined with DTX treatment (5.0 μg/L), the activity of caspase-3 activity was significantly increased by approximately 251.3% in SPC-A1/DTX/ ING4 cells combined with DTX treatment (p < 0.01; Figure 9B). Then, we used a quantitative RT-PCR assay to detect the mRNA expression levels of some apoptosis-related genes, including survivin, Aurora-A, stathmin, XIAP, Bcl-2, Bcl-xL and Bax, which has been reported to be associated with the sensitivity of tumor Figure 9C, SPC-A1/DTX/ING4 cells combined with DTX treatment showed a significant reduction in the level of Bcl-2 mRNA expression and a significant increase in the level of Bax mRNA expression ( Figure 9C). Western blot assay confirmed the decreased expression of Bcl-2 protein and the increased expression of Bax protein ( Figure 9D). Therefore, the decreased expression of Bcl-2 and increased expression of Bax in the SPC-A1/DTX/ING4 cells leads to the downregulated Bcl-2/Bax ratio, which finally induces the activation of caspase-3. Overexpression of ING4 Increases In Vivo Chemosensitivity of SPC-A1/DTX Cells to DTX The Table S1; p < 0.05). Also, following treatment with DTX, TUNEL-staining assay showed that the rate of apoptotic tumor cells was significantly increased in tumors formed from SPC-A1/DTX/ING4 cells compared with that in tumors formed from SPC-A1/ DTX/ control cells ( Figure 10D). From these data, we concluded that overexpression of ING4 could increase the in vivo chemosensitivity of SPC-A1/DTX cells to DTX. The Expression of ING4 Is Downregulated in DTX-Resistant Lung Adenocarcinoma Tissues To investigate the association between ING4 expression in lung adenocarcinoma tissues and the clinical response to DTXbased regimens, semiquantitative immunohistochemistry on tumor biopsy specimens from 18 eligible patients with advanced lung adenocarcinoma treated with DTX combined with platinum agents was performed. As shown in Figure Fig ure 11C). Therefore, the expression of ING4 in advanced lung adenocarcinoma might be positively correlated with the response of patients to DTX. DISCUSSION Multiple factors associated with sensitivity of tumor cells to taxanes (paclitaxel and DTX) have been reported, including the MDR gene, alteration in tubulin dynamics, and differences in β-tubulin isotype expression (18,19). In particular, overexpression of β-tubulin III could induce paclitaxel or DTX resistance in association with reduced effects on microtubule dynamic instability, which suggests that upregulation of a neuronal tubulin isotype is a key contributor to taxane sensitivity in human cancers (20). Additionally, aberrant expression of some oncogenes could affect sensitivity of tumor cells to DTX (21)(22)(23). The Aurora-A gene is essential for the proper arrangement of centrosomes and microtubules, and overexpression of Aurora-A has been reported to induce increased resistance to taxanes via a decrease in spindle checkpoint activity in vitro (24). Other reports showed that suppression of Aurora-A expression could enhance chemosensitivity to DTX in human cancers, including esophageal squamous cell carcinoma, breast cancer and prostate cancer (25)(26)(27). Stathmin, also designated Op18, is a highly conserved ubiquitous cytoplasmic protein that has recently been shown to destabilize microtubules (28). Mutant p53 breast cancers exhibiting high levels of stathmin may be resistant to antimicrotubule agents and siRNA-mediated stathmin downregulation could enhance taxane chemosensitivity of tumor cells, including prostate cancer and osteosarcoma (29,30). Recently, epigenetic events, including DNA methylation and posttranscriptional regulation (microRNAs), were found to be involved in the control of gene expression, which is known to play an important role in cancer and chemotherapy drug resistance (31,32). Previously, we have identified microRNA expression profiles in DTX-resistant human NSCLC cells (SPC-A1), which provide a better understanding of mechanisms involved in drug sensitivity or resistance of NSCLC (33). To further investigate the mechanisms of DTX resistance and provide theoretical support for drug-resistant reversal induced by DTX, a DTX-resistant variant of the human lung adenocarcinoma cell line SPC-A1 (SPC-A1/DTX) was previously established. The incipient concentration of DTX was 0.008 μg/L, and the SPC-A1/DTX cell line stably grew into 5.0 μg/L DTX. An MTT assay showed that the index of drug resistance of SPC-A1/DTX was 13.20 to DTX. The establishment of a DTX-resistant lung adenocarcinoma cell model provided the foundation for further research on its chemo resistant mechanisms. In the present study, we focused specifically on improving the sensitivity to DTX and the selection of a therapeutic program for each individual patient, to identify novel genes which define the cDNA expression profiles of the parental SPC-A1 cell line in comparison with its DTX-resistant subclone SPC-A1/DTX using a cDNA microarray. Then, we attempted to provide strong molecular, biochemical and biological evidence that downregulation of the ING4 gene played an important role in SPC-A1 cell progression to DTX resistance. ING4, a novel member of the ING family, which comprises six members characterized by a highly conserved C-terminal plant home domain (PHD)like zinc-finger domain, has been reported to be involved in a variety of processes including oncogenesis, angiogenesis and DNA repair (34 (36). And ING4 plus CDDP (cisplatin) was closely associated with the cooperative regulation of extrinsic and intrinsic apoptotic pathways and synergistic inhibition of tumor angiogenesis (37). In the present study we aimed firstly to explore the roles of ING4 in DTX resistance of lung adenocarcinoma cells. We showed that the ING4 gene was significantly downregulated in DTXresistant SPC-A1 or taxol-resistant A549 cell lines when compared with nonresistant SPC-A1 or A549 cells at both transcriptional and translational levels. Ectopic expression of ING4 could significantly decrease the IC 50 value of SPC-A1/DTX or A549/Taxol cell lines, whereas siRNA-mediated ING4 downregulation could increase the IC 50 value of DTX-sensitive SPC-A1 or A549 cells. Overexpression of ING4 could inhibit in vivo growth of SPC-A1/DTX cells combined with DTX treatment. Moreover, we also found that a decreasing level of ING4 expression occurred in lung adenocarcinoma cells that responded to DTX. We then further elucidated the possible mechanisms of chemosensitivity enhancement induced by overexpression of ING4. Our results showed that overexpression of ING4 could significantly induce apoptosis enhancement and G 2 /M-phase arrest in SPCA-1/DTX cells, and the apoptosis enhancement might be associated with activation of caspase-3 induced by the decreased ratio of Bcl-2/ Bax. Next, we analyzed the effects of ING4 expression on the expression of some apoptosis-related genes such as survivin, XIAP, Bcl-xL, Bcl-2, Bax, Aurora-A and stathmin, which have been reported to affect the sensitivity of tumor cells to paclitaxel or DTX (38). We found that upregulation of ING4 could decrease Bcl-2 expression and increase Bax expression but had no effect on other genes at both transcriptional and translational levels. The decrease in the mitochondrial Bcl-2/Bax ratio is associated with caspase-3-dependent apoptosis, which is consistent with the findings of other researchers. Although Li and his colleagues showed that ING4 might play an inhibitory role in A549 cell prolifera-	2018-04-03T01:18:05.250Z	2012-07-18T00:00:00.000	{ "year": 2012, "sha1": "a1c87d8479303cd2d012532e7cbad02d3ecdbc6b", "oa_license": "CCBY", "oa_url": "https://doi.org/10.2119/molmed.2011.00230", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "bd3898f6b63d66f5e1b9d92f67a6c248c1b01c82", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
220483496	pes2o/s2orc	v3-fos-license	Baicalin regulates macrophages polarization and alleviates myocardial ischaemia/reperfusion injury via inhibiting JAK/STAT pathway Abstract Context Baicalin is an active compound which demonstrates cardioprotection effects against myocardial ischaemia/reperfusion injury (MI/RI). Objective To investigate how baicalin protects against myocardial injury and to explore its potential mechanism. We hypothesized that baicalin-modulated macrophages change from M1 (pro-inflammatory subset) to M2 (anti-inflammatory subset) under I/R stress. Materials and methods We established an ischaemia/reperfusion (I/R) model using Sprague Dawley (SD) rat, then baicalin was intragastric administration (20, 60 or 120 mg/kg) for 24 h. The rats were randomly divided into five groups (n = 10): control, I/R, I/R + baicalin (20 mg/kg), I/R + baicalin (60 mg/kg) and I/R + baicalin (120 mg/kg). Cardiac function was detected by echocardiography, HE staining and ELISA, respectively. Macrophage phenotype was examined by flow cytometry. Furthermore, IHC, qRT-PCR and WB were employed to analyse the related mechanisms. Results The study showed that baicalin (20, 60 or 120 mg/kg) significantly improved cardiac function and impeded cardiac apoptosis in rats. In addition, the repair of myocardial morphology (reduced neutrophil infiltration) further confirmed its cardiacprotective effect. Moreover, baicalin effectively decreased iNOS, IL-1β and IL-6, and up-regulated Arg-1, IL-10 and TGF-β via changing the macrophage phenotype (from M1 towards M2). Notably, treatment with baicalin also inhibited the phosphorylation levels of JAK2 and STAT3. Discussion and conclusions: It was confirmed that baicalin alleviated post-I/R myocardial injury and reduced inflammation via JAK/STAT pathway, and baicalin treatment might be recommended as a new approach for myocardial ischaemic complications. Introduction Currently, acute myocardial infarction (MI) is a common cardiovascular disease that causes high morbidity and mortality around the world. For MI patients, the most timely and effective treatment is to ameliorate myocardial ischaemia and restrict the size of myocardial infarction. To the best of our knowledge, ischaemia/reperfusion (I/R) has so far been the principal or only strategy for MI treatment, which is clinically achieved by angioplasty or thrombolytic therapy, thereby promptly restoring blood supply (Vincent et al. 2017). Yet, this sudden reperfusion should result in secondary cascade damages, known as myocardial ischaemia/reperfusion injury (MI/RI), which paradoxically deteriorated ischaemic damages and further swelled infarct size (Paelestik et al. 2017). In addition, MI/RI may trigger a large variety of pathological changes, including local acute inflammatory reactions, metabolic disorders and cell apoptosis and/or necrosis, ultimately leading to cardiac dysfunction (Kitano et al. 2014). Simultaneously, the pathogenesis of MI/RI is complex and involves multiple molecular processes such as reactive oxygen species (ROS) accumulation (Ravindran et al. 2017), calcium ion overload (Mozaffari et al. 2013), inflammatory cytokines influx (Badalzadeh et al. 2017), and mitochondrial permeability transition pore (mPTP) opening . Although there are many basic research reports on MI/RI, the clinical effects are not satisfactory, and we still need further research to find more effective clinical treatments. The pathological process of MI/RI can trigger injurious inflammatory reaction involving in the infiltration of inflammatory cells (such as neutrophils, macrophages and lymphocytes) (Epelman et al. 2015) and accumulation of pro-inflammatory cytokines, ultimately resulting in substantial cardiac dysfunction. As tissue injury occurs, inflammatory monocytes infiltrate into the surrounding area of injury and differentiate into macrophage populations, thereby regulating tissue injury and repair (Wynn and Vannella 2016). Interestingly, macrophages include two subpopulations, such as pro-inflammatory macrophages (M1-phenotype) and anti-inflammatory macrophages (M2-phenotype). Upon MI, M1 macrophages infiltrate into the injured cardiac tissues, triggering inflammation and releasing pro-inflammatory cytokines, including iNOS, TNF-a, IL-1b and IL-6 (Novak and Koh 2013). Conversely, anti-inflammatory macrophages produce anti-inflammatory cytokines (Arg1, IL-4 and IL-10), and promote the wound healing and scar formation (Nahrendorf et al. 2010). Therefore, switching the dynamic polarization of macrophages M1/M2 in time and transforming macrophages from M1 to M2, has important pathological significance for mitigating inflammation and repairing myocardial function (Mantovani et al. 2013). Traditional Chinese medicine has been used for thousands of years in China and has attracted more and more scholars' attention due to its natural, low cost and extremely low toxicity. Baicalin, a flavonoid active substance derived from the roots of Scutellaria baicalensis Georgi (Lamiaceae), has been used to treat various of diseases such as cancer (I El-Gogary et al. 2019), osteoarthritis (Wang et al. 2018), nephritis and hepatitis (Cai et al. 2008). Increasing reports demonstrate that baicalin possesses antioxidant, anti-inflammatory and anti-apoptotic activity (Luan et al. 2015;Li et al. 2017) in Ical. Previous studies found that baicalin protects rat cardiomyocytes via weakening hypoxia/ reoxygenation-induced injury (Woo et al. 2005;Lin et al. 2010). Kong et al. (2014) confirmed that baicalin protected the myocardial damage after reperfusion via the antioxidant and paracrine effects. Luan et al. (2019) revealed that baicalin attenuated myocardial I/R-induced damage, inhibited myocardial apoptosis and inflammation by Akt/NF-jB signalling. Bai et al. (2019) showed that baicalin protected cardiac microvascular endothelial cells (CMECs) in I/R rats by promoting the release of nitric oxide. However, it was still of interest to determine if baicalin could induce macrophages to regulate inflammatory response and protect myocardial injury post-I/R. The JAK/STAT pathway is involved in many important biological processes such as cell proliferation, differentiation, apoptosis and immune regulation. In recent years, several reports have proposed that JAK/STAT signalling is associated with cardiac dysfunction in MI/RI (Mascareno et al. 2001;Bolli et al. 2003). Moreover, Yang et al. (2013) reported the JAK/STAT pathway was quickly activated after I/R, Zhang et al. (2019) found propofol-alleviated inflammation and tissue injury in MI/ RI rats through inhibiting the JAK/STAT pathway. But, the role of baicalin on MI/RI in JAK/STAT signalling pathway is rarely reported. In this study, we provide the evidence that baicalin mediates cardioprotection against myocardial injury post-I/R via JAK/STAT pathway and confirm the hypothesis that baicalin stimulates macrophages change to alleviate inflammation caused by I/R. Rat I/R model All animal experiment steps were conducted in accordance with the National Institutes of Health (NIH) guide for the care and use of laboratory animals (National Academies Press 2011) and approved by the ethical committees of Wuhan Asia Heart Hospital. All SD rats were fasted for 12 h before operation. They were then anaesthetised with 2% isoflurane and fastened on the operating table, and I/R procedures were based on previous descriptions (Zhang et al. 2011). Briefly, open the left chest to expose the heart, peel off the pericardium to find the coronary artery, and ligature the left anterior descending coronary artery (LAD). After 45 min of ligation and 180 min of blood reperfusion, the same procedure was also used for sham but no ligation. Then, blood specimens were centrifuged at 400 g for 10 min. Finally, cardiac tissues and/or serum were immediately collected and stored at À80 C for further analysis. Rat administration Rats were given baicalin (20, 60 or 120 mg/kg) by intragastric administration for 24 h and were randomly grouped (n ¼ 10) as follows: control group, I/R group, I/R þ baicalin (20 mg/kg) group, and I/R þ baicalin (60 mg/kg) group, I/R þ baicalin (120 mg/kg) group. The control operation group did not have I/R but was given normal perfusion at the same time. Rats in the control group and I/R group were given 0.9% saline, yet in I/R þ baicalin treatment groups were given different doses of baicalin (once a day for two weeks), followed by I/R surgery. Echocardiography Two-hours post-I/R, SD rats were narcotised by intraperitoneal injection of 40 mg/kg pentobarbital sodium and placed in the flat position. The echocardiography measurement of cardiac function was operated by digital ultrasound equipment Vevo 2100 (VisualSonics, Ontario, Canada) and an 18 MHz transducer after reperfusion. Then, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS) and left ventricular wall thickness (LVWT) were calculated for M-mode recordings. All results were averaged over at least three consecutive cardiac cycles measuring and analysed by a blinded researcher. Measurement of pressure-volume data parameters After reperfusion, the pressure-volume tracheal catheter (SPR839, Millar Instruments Inc, USA) was inserted into the right common carotid artery, and then, the left ventricle was inserted using a pressure-volume conductance instrument (MPVS-300, Millar Instruments Inc, USA). Signals were recorded and calculated by a PVAN data analysis software (Millar Instruments Inc, USA). Haemodynamics parameters such as heart rate (HR), left ventricular systolic pressure (LVSP) and LVEF were measured. Haematoxylin-eosin (HE) staining In brief, the rat heart tissues were fixed in 10% formalin buffered. After paraffin embedding, the tissues sections were chipped to 4 mm thickness, and then stained with haematoxylin-eosin (HE) liquid for the histopathological examination. ELISA assay Serum samples were centrifuged and supernatants were collected, the serum levels of CK-MB, Mb and cTnI were detected using a ELISA kits, according to the manufacturer's protocols (Thermo Fisher, Waltham, MA, USA). Immunohistochemistry (IHC) assay Rat heart tissues were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at a thickness of 4 lm according to standard procedures. Next, the deparaffinized sections were hydrated. Furthermore, endogenous peroxidase activity was blocked by Hydrogen Peroxide Block for 15 min. After antigen retrieval in 10 mM heated citrate buffer for 10 min, the sections were incubated with primary antibody: anti-Ki67 antibody (dilution rate, 1:600) and anti-caspase-3 antibody (dilution rate, 1:600) overnight at 4 C. Subsequently, corresponding secondary antibody was incubated for 1 h at room temperature. Finally, the specimens were stained with DAB kit (Boster Biological Technology Co. Ltd, Wuhan, China). Then, the specimens were washed with PBS and images were captured under a confocal microscopy (Leica Microsystems CMS GmbH, Wetzlar, Germany). Flow cytometry screening Collected cardiac tissues post-reperfusion were quickly removed and put into the ice-filled PBS. Cardiac tissues were minced and digested (Collagenase B and Dispase II), the digestion reaction was stopped by cold flow cytometry staining buffer, the mixture was percolated, centrifuged at 400 g for 5 min at 4 C, then the liquid supernatant was cleared and the cells were resuspended in 1 mL of ice staining buffer. Cells from individual heart tissue were divided into three parts according to the cell count so that each part involved approximately 10 6 living (Trypan blue negative) cells for flow cytometry (Al-Darraji et al. 2018). Next, the isolated cells were cultivated rapidly on ice for 20-30 min with coupled primary antibodies (APC-CY5.5-coupled CD86, APC-CY7-coupled CD206 and R-PE-CY5-coupled F4/80) for the macrophage phenotype sorting. Next, after 20-30 min cultivation, the cells samples were washed two times continuity and then analysed by an Attune NxT flow cytometer equipment (ThermoFisher Scientific, Waltham, MA, USA). Macrophage phenotypes were divided into pro-inflammatory (F4-80 þ /CD86 þ ) and ant-inflammatory (F4-80 þ /CD206 þ ). Finally, manual or automatic calibration and compensation were performed for experiments using unstained and single fluorescence controls. All processes were performed at least in triplicate for each independent amplification. Statistical analysis All the experiments were conducted at least three times, and the data were expressed as mean ± SD. Statistical analysis was carried out using IBM SPSS Statistics 25.0 (IBM, Armonk, USA). Student's t-test or one-way ANOVA was used to evaluate the significance. p < 0.05 showed statistical significance. Baicalin preserved cardiac function after I/R Compared with control group, the HR, LVSP, FS% and EF% were significantly decreased in I/R (p < 0.05), the situation in Table 1 confirmed that reperfusion model was successfully constructed. On the contrary, these parameters were remarkably increased in 60 or 120 mg/kg baicalin groups compared with I/R group (p < 0.05), and there was no significant difference in the low dose (20 mg/kg) group. Interestingly, LVWT had the similar trends compared with other parameters, but no statistical difference. Baicalin repaired pathological morphology of myocardium and alleviated cardiac injury As shown in Figure 1(A), there was no apparent difference in the morphological structures of the control group, but I/R group showed obvious myocardialfibre fracture, cellular oedema, necrosis, and neutrophil infiltration. Gratifying, compared with I/R group, myocardial tissue damage was gradually repaired with the increased dose of baicalin (60 or 120 mg/kg). As showed in Figure 1(B-D), post-reperfusion, the protein expression of CK-MB, Mb and cTnI were remarkably increased (p < 0.05). In contrast, the expression of these markers was significantly decreased with different doses of baicalin treatment (p < 0.05). Baicalin inhibited cardiac apoptosis following I/R As shown in Figure 2(A-C), the expression of Ki67 was increased, while the expression of cleaved-caspase-3 was decreased, compared with control (p < 0.05). However, the levels of Ki67 and cleaved-caspase-3 were reversed after diverse doses of baicalin treatment (20, 60 or 120 mg/kg) (p < 0.05). In addition, the result from Figure 2(D,E) showed that the value of Bcl-2/Bax was increased with different doses of baicalin, which was statistically different (p < 0.05). Baicalin transformed expression of macrophage M1 and M2 biomarkers As shown in Figure 4(A), compared with control, the mRNA levels of iNOS, IL-1b and IL-6 (M1 biomarkers) were markedly increased in I/R group, and baicalin treatment (20, 60 or 120 mg/kg) effectively decreased the mRNA levels of these biomarkers (p < 0.05). Besides, the expression of M2 biomarkers (Arg-1, IL-10 and TGF-b) were down-regulated in I/R group, while baicalin reversed the down-regulation effect of I/R on biomarkers (Figure 4(B)). Furthermore, the results of iNOS and Arg-1 in proteins were consistent with that in mRNA (Figure 4(C)). Baicalin regulated the activition of JAK-STAT To elucidate the underlying mechanism of baicalin, we thus detected the activation of JAK2 and STAT3 proteins. Compared with thevcontrol group, our results revealed that the phosphorylation levels of JAK2 and STAT3 were remarkably increased in I/ R group (p < 0.05). Conversely, the phosphorylated expression of JAK2 and STAT3 proteins were decreased with baicalin treatment ( Figure 5). Baicalin attenuated MI/RI through suppressing JAK-STAT pathway We injected 2.5 lM JAK inhibitor (AG-490) into the jugular vein of rats and randomly separated the rats into groups: control group, I/R group, I/R þ AG-490 (2.5 lM) group, and I/ R þ baicalin (60 mg/kg) group, AG-490 þ baicalin group. As shown in Figure 6(A,B), the expression of cleaved-csapase3, iNOS and IL-1b were significantly increased in the I/R group, while the expression of Arg-1 and IL-10 were significantly decreased, compared with the control (p < 0.05). In addition, AG-490 or baicalin alone antagonised I/R-induced content changes of these proteins (p < 0.05). Moreover, AG-490 and baicalin co-treatment further reduced the levels of cleaved-capase3, iNOS and IL-1b and increased Arg-1 and IL-10. Discussion It is universally acknowledged that blood flow recovery in ischaemic myocardial tissue has become one of the most feasible treatments for ischaemia-induced heart disease. In this experiment, we constructed a model of myocardial I/R in rats to investigate the protective role of baicalin in myocardium post-reperfusion. This study demonstrated baicalin could improve cardiac function confirmed by echocardiography and histopathological examination. Baicalin inhibited myocardial tissue apoptosis and reduced inflammatory response through macrophage-mediated release of inflammatory factors. Moreover, baicalin down-regulated the protein expression of JAK2 and STAT3 in myocardial tissues. Numerous complex signal pathways, such as PI3K/Akt, Akt/ NF-jB, p38/MAPK, Nrf2-ARE and JAK/STAT pathways, are involved in the development of MI/RI. Among them, the JAK/ STAT signalling pathway plays an important role in the progression of MI/RI (Bolli et al. 2001). It is well known that JAKs are rapidly recruited to the receptor and activated after receiving the signal from the upstream receptor molecule, and activated JAK then catalyses tyrosine phosphorylation of this receptor, thus supplying binding sites for the SH2 domain of STATs, ultimately leading to gene transcription. In particular, JAK1 and JAK2 were activated by MI/RI, which in turn activates STAT1 and STAT3, STAT1 promotes apoptosis, whilst STAT3 protects cardiomyocyte (Stephanou 2004). Apoptosis is a crucial pathophysiological feature in the early I/R that triggers more severe heart failure (Hilbert et al. 2018). Hattori et al. (2001) found that activated JAK/STAT up-regulated Bcl-2 and down-regulated Bax, enhancing myocardial pro-survival pathways. In this study, baicalin decreased the level of cleaved-caspase-3 and increased Bcl-2/Bax ratio post-I/R (Figure 2). In order to understand whether JAK/ STAT signal is involved in baicalin-regulated myocardial apoptosis, we also detected the expression of cleaved-caspase-3 after the addition of JAK inhibitor AG-490, the result showed AG-490 or baicalin alone inhibited I/R-induced cleaved-capase3 increase, AG-490 and baicalin co-treatment further reduced the levels of cleaved-capase3 (Figure 6), indicating baicalin inhibited myocardial apoptosis induced by I/R via JAK/STAT pathway. Inflammation plays a crucial role in the recovery of MI and MI/RI (Li et al. 2015). IL-1b is an outstanding inflammatory mediator in the pre-MI/RI period (Takahashi 2011) that can increase endothelial cell permeability and stimulate the release of chemokines, which further leads to the accumulation of inflammatory cells including neutrophils and macrophages (Swirski and Nahrendorf 2013). Baicalin has been shown to have antiinflammatory effects in several diseases. Guo et al. (2019) reported that baicalin substantially decreased the levels of IL-1b, IL-6, and TNF-a in the depressive-like symptoms. Luan et al. (2019) found that baicalin inhibited myocardial inflammation through down-regulating the levels of TNF-a, IL-1b, IL-6, and IL-8 and up-regulating IL-10. Similar to these studies, we found that baicalin weakened neutrophils infiltration ( Figure 1) and decreased the release of iNOS, IL-1b and IL-6 post-I/R ( Figure 4), supporting the previous views. Macrophages are the core mediums of cardiac inflammation (Ben-Mordechai et al. 2015; Wynn and Vannella 2016) and contribute to the onset and regression of inflammation. In fact, stem cells can modify anti-inflammatory macrophages (M2) after heart damage, thereby promoting cardiac recovery (de Couto et al. 2015). In addition, the production of pro-inflammatory cytokines (such as TNF-a, IL-1b and IL-6) and their harmful effects could be reduced by regulating macrophage phenotypes (Saini et al. 2005). we present evidence for the first time in this study that baicalin transformed macrophages from pro-inflammatory/M1 to anti-inflammatory/M2 phenotype and played an immunomodulatory role after I/R. Flow cytometry data ( Figure 3) showed a significant decrease in CD86 þ (anti-inflammatory macrophages) cells and a significant increase in CD206 þ (anti-inflammatory macrophages) cells after baicalin treatment. Moreover, the expression of M1 biomarkers (iNOS, IL-1b and IL-6) were down-regulated, while that of M2 (Arg-1, IL-10 and TGF-b) were up-regulated ( Figure 4). Importantly, the transcription of JAK1/STAT3 pathway increased iNOS and contributed to the development in the late phase of I/R preconditioning , while JAK2/STAT3 signalling was critically involved in inflammation (Amani et al. 2019). Interestingly, we observed that baicalin reduced the phosphorylation of JAK2 and STAT3 ( Figure 5) and decreased the levels of iNOS and IL-1b ( Figure 6). AG-490 alone reduced the levels of iNOS and IL-1b and increased Arg-1 and IL-10, AG-490 and baicalin co-treatment further extended the results of AG-490 alone processing. Taken together, our work confirmed that baicalin-attenuated myocardial inflammatory response and switched macrophage polarisation Conclusions Our study demonstrated the protective role of baicalin on MI/RI, which is mediated via the JAK/STAT pathway. In addition, baicalin alleviated myocardial injury and inhibited apoptosis following I/R, weakened the myocardial inflammation response through switching the polarization of macrophages from M1 to M2. Hence, it could be a candidate for the treatment of MI/RI. Disclosure statement No potential conflict of interest was reported by the author(s).	2020-07-12T13:05:59.175Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "3bfdc17d8326186c513dcbead0111ea2149aa07b", "oa_license": "CCBYNC", "oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/13880209.2020.1779318?needAccess=true", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a10f044984ab47e4e161e0ac3f0d4098285f7d7f", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
28608489	pes2o/s2orc	v3-fos-license	Governance of Fracking in Africa Introduction A number of economic studies of the shale boom in the United States (US) find that shale resources are associated with economic growth (Hausman and Kellogg, 2015; Mason et al, 2015; Weber, 2012). However, there are also substantial environmental risks associated with hydraulic fracturing (fracking) (Small et al, 2014; Vidic et al, 2013), and federalism in the US has been criticized as contributing to an ineffective regulatory regime (Richardson et al, 2013; Warner and Shapiro, 2013). Despite concerns, the US has an arguably high-quality regulatory framework. US states have the majority of authority to regulate extraction and have responded to the shale boom by adapting conventional oil and gas regulations to hydraulic fracturing. Additionally, several states have enacted de facto bans on fracking (Murtazashvili, 2015). While Congress has not expanded the jurisdiction of the US Environmental Protection Agency (EPA) over fracking, the EPA coordinates scientific studies of the consequences of shale production and could theoretically impose national fracking standards. Moreover, the polycentric system of governance in the US provides access points for local level citizen participation in decisions regarding the appropriateness of shale extraction (Arnold and Holahan, 2014). US regulatory institutions governing extractive industries such as hydraulic fracturing also appear fairly competent in comparison to those of a typical developing country. Resource abundance often undermines economic wellbeing in the developing world (Boschini et al, 2007; Sachs and Warner, 2001; Van der Ploeg, 2011). Challenges for developing countries include ineffective governance, resource and revenue mismanagement, ineffective regulations that undervalue and under-tax resources, exploitation by extraction companies, and lack of transparency (Besada et al, 2015). Fortunately, the so-called ‘resource curse’ is not inevitable (Humphreys et al, 2007; James, 2015). Rather, political features of the economy, in particular the quality of governance, determine the extent resource wealth is a blessing rather than a curse (Collier and Hoeffler, 2005; Poteete, 2009b; Robinson et al, 2006). Thus, one expects that the prospects for sustainable hydraulic fracturing, which in this paper refers to extraction that balances economic growth and job creation with minimized socially costly externalities, will depend on the context within which extraction occurs. Our empirical studies consider the governance situation in Botswana and South Africa, two African countries for which widespread fracking is a very real possibility.1 To guide our empirical study, we draw upon the ‘institutionalist’ literature on governance, in particular Elinor Ostrom’s (1990, 2005) work on resource management. Institutionalist theories suggest a number of variables that should influence the prospects for sustainable fracking, including equity of distribution of the benefits and costs of resource use, information and monitoring capacity, accountability, polycentricity, democratic inclusiveness, dispute resolution mechanisms, and adaptability and flexibility. The conceptual framework and empirical analysis in this work clarify the capacity and limitations of governance as exploration for fracking begins in earnest in Botswana and South Africa. Our research finds that both countries have regulation in place that allows the national government RESEARCH Introduction A number of economic studies of the shale boom in the United States (US) find that shale resources are associated with economic growth (Hausman and Kellogg, 2015;Mason et al, 2015;Weber, 2012).However, there are also substantial environmental risks associated with hydraulic fracturing (fracking) (Small et al, 2014;Vidic et al, 2013), and federalism in the US has been criticized as contributing to an ineffective regulatory regime (Richardson et al, 2013;Warner and Shapiro, 2013). Despite concerns, the US has an arguably high-quality regulatory framework.US states have the majority of authority to regulate extraction and have responded to the shale boom by adapting conventional oil and gas regulations to hydraulic fracturing.Additionally, several states have enacted de facto bans on fracking (Murtazashvili, 2015).While Congress has not expanded the jurisdiction of the US Environmental Protection Agency (EPA) over fracking, the EPA coordinates scientific studies of the consequences of shale production and could theoretically impose national fracking standards.Moreover, the polycentric system of governance in the US provides access points for local level citizen participation in decisions regarding the appropriateness of shale extraction (Arnold and Holahan, 2014). US regulatory institutions governing extractive industries such as hydraulic fracturing also appear fairly competent in comparison to those of a typical developing country.Resource abundance often undermines economic wellbeing in the developing world (Boschini et al, 2007;Sachs and Warner, 2001;Van der Ploeg, 2011).Challenges for developing countries include ineffective governance, resource and revenue mismanagement, ineffective regulations that undervalue and under-tax resources, exploitation by extraction companies, and lack of transparency (Besada et al, 2015).Fortunately, the so-called 'resource curse' is not inevitable (Humphreys et al, 2007;James, 2015).Rather, political features of the economy, in particular the quality of governance, determine the extent resource wealth is a blessing rather than a curse (Collier and Hoeffler, 2005;Poteete, 2009b;Robinson et al, 2006).Thus, one expects that the prospects for sustainable hydraulic fracturing, which in this paper refers to extraction that balances economic growth and job creation with minimized socially costly externalities, will depend on the context within which extraction occurs. Our empirical studies consider the governance situation in Botswana and South Africa, two African countries for which widespread fracking is a very real possibility. 1To guide our empirical study, we draw upon the 'institutionalist' literature on governance, in particular Elinor Ostrom's (1990Ostrom's ( , 2005) ) work on resource management.Institutionalist theories suggest a number of variables that should influence the prospects for sustainable fracking, including equity of distribution of the benefits and costs of resource use, information and monitoring capacity, accountability, polycentricity, democratic inclusiveness, dispute resolution mechanisms, and adaptability and flexibility. The conceptual framework and empirical analysis in this work clarify the capacity and limitations of governance as exploration for fracking begins in earnest in Botswana and South Africa.Our research finds that both countries have regulation in place that allows the national government to extract rents from fracking activities.However, there are fewer institutional safeguards to ensure local government and communities benefit in the same way.Moreover, interview data from fieldwork reveals that regulatory agencies lack capacity to monitor operational compliance with regulation and provide very little information to local governments and communities, decreasing the ability for informed local level deliberation regarding shale extraction.Thus, we identify several areas where improvements on key governance dimensions may increase the chances of sustainable hydraulic fracturing. In considering fracking, which is often a highly charged political issue, it is important to discuss our perspective.While we purport to offer an institutional analysis of the governance system, all analysis, including 'positive' analysis of economics, requires making value judgements (Bromley, 2006).To that end, our perspective is that conservation of shale resources is a more realistic proposal than preservation.As Collier (2010) suggests, it is often unrealistic to ask developing countries to keep their resource as if they were in a museum, which preservation tends to ask.In contrast, conservation recognizes that current generations have an interest in using resources to enhance development prospects, while also ensuring there is enough of the resource left to benefit future generations.To that end, our paper seeks to better understand under what conditions society can capture some of the scarcity rent associated with shale production, assuming some extraction will occur, while doing so responsibly. This essay is organized as follows.Section 2 provides a conceptual framework to characterize governance of hydraulic fracturing.Section 3 and 4 consider the prospects for effective governance of fracking in South Africa and Botswana, respectively.Section 5 discusses the policy implications and concludes. A conceptual framework to analyze governance of hydraulic fracturing Natural resources have features of a common pool in that they are non-excludable and divisible.With conventional oil and gas, the institutional solution to the problem of wasteful racing to establish ownership is establishing shared common pool property rights.Under such unitization agreements, all drillers with access to a reservoir agree to share in the production profits (Libecap and Smith, 1999;Libecap and Wiggins, 1985). In contrast with conventional oil and gas, the fundamental challenge with hydraulic fracturing is dealing with environmental externalities rather than wastefulness in the race to establish property rights institutions (Holahan and Arnold, 2013).With unconventional natural gas, the shale is challenging to reach, which makes the race less of problem.However, groundwater contamination from fracking is a threat that does not affect conventional oil and gas extraction.Thus, the challenges confronting hydraulic fracturing require different institutional solutions compared to conventional oil and gas.Additionally, the property rights perspective tends to understate the politics of resource governance (Poteete, 2003). For these reasons, when considering fracking, a governance perspective is a more useful place to begin than the property rights approach.A key question is what constitutes 'governance.'A long tradition of literature conceptualizes governance mainly in terms of formal administrative capacity (Barnard, 1938;Fukuyama, 2013).Ostrom (2005Ostrom ( , 2009) ) in contrast, proposes a more encompassing notion of governance that acknowledges the complexity and diversity of governance systems, as well as the interrelationship between de jure and de facto systems of governance. Ostrom's work on resource governance, as well as the 'new institutionalism' in economics (Williamson, 2000), shares the common presumption that institutional features of political regimes determine key political, economic, and social outcomes.Since there is a vast literature on institutionalism, we discerned from the literature a number of key variables that are particularly important for understanding hydraulic fracturing.For example, the social-ecological systems (SES) approach, also pioneered by Ostrom, includes a large number of variables (dozens, in fact) (Cole et al, 2014;Ostrom, 2009).While complexity has its virtues, it can also make discerning policy implications quite challenging since there are so many moving parts in the analysis.The following list has the advantage of offering a much richer description of governance of natural resources than the property rights approach, while also offering a more parsimonious approach than the mature SES approach. The first governance variable is equity of distribution of the benefits and costs of resource use.Ostrom (1990) argues that a system of governance that attempts to more equitably distribute the benefits and costs of natural resources use may be more likely to sustain the benefits streams associated with resource extraction.With extractive industries, a challenge in the developing world is that the state is often too weak to extract its fair share of the scarcity rent associated with natural resources (Bromley and Anderson, 2012).Consequently, a key question is whether the government has in place mechanisms to ensure that government distributes the benefits of fracking widely and that communities bear the costs of fracking as a shared responsibility.This variable is important because governments may not have the ability to implement a tax upon resource extraction and there may be few mechanisms to ensure a relatively equal distribution of the environmental costs of fracking. A second governance variable is information and monitoring capacity, which includes capacity to gather information on the benefits and costs of resource use, to monitor resource use, and the presence of feedback mechanisms for decision-makers to learn about the consequences of policies and programs.Monitoring and evaluation provide insight into when it is desirable to change institutions and governance policies.The presence of such capacities are particularly important in developing countries and cannot be taken for granted (Andrews et al, 2013). Success on the dimension of information and monitoring is a question of the extent to which government organizations or institutions are able to record information regarding fracking, monitor compliance with resource use regulation, and disseminate findings to policymakers and stakeholders.Information and monitoring capacity may include the extent to which the state has ability to conduct baseline studies of groundwater and to disseminate information to concerned citizens. A third variable is accountability, which refers to the extent that users of a resource bear some of the costs of governance and are accountable for their actions.Accountability depends on establishing clear boundaries of responsibility for policies and actions and the ability of the state to enforce the rule of law (Gauri and Lieberman, 2006;Lieberman, 2011).In considering governance of fracking, accountability asks whether gas companies bear the relevant social costs associated with extraction, and the extent to which the state is capable of enforcing its rules. A fourth variable, polycentric governance, refers to overlapping systems of jurisdictions, each with shared political authority (Ostrom et al, 1961).Polycentric systems are associated with improvements in conservation of water and energy resources provided there are linkages across local jurisdictions (Blomquist and Schlager, 2005;Heikkila et al, 2011;Sovacool, 2011).This suggests that polycentric governance of fracking will improve governance outcomes, with the extent of improvement contingent on the degree to which there are mechanisms facilitating coordination across jurisdictions where fracking may occur. 2 Polycentric systems of governance vary in the extent to which they facilitate democratic inclusiveness.The fifth variable, democratic inclusiveness, refers to the extent to which political institutions facilitate access to the political process.Increasing participation should improve the quality of institutions and prospects for economic development (North et al, 2009).Local level democracy can also ensure the rights of communities are respected vis-à-vis the central government (Myerson, 2014).Citizen participation through elections or referendums, and the extent to which the political regime is democratic and encourages or allows groups to participate in the political process, are expected to improve the legitimacy of rules governing hydraulic fracturing, as well as increase the ability to implement these rules. A sixth variable is dispute resolution.Disputes are bound to arise during the process of economic development.Mechanisms for reducing disputes are important because they reduce the return to violence for resolving conflicts (Blattman et al, 2014). In the developing world, dispute resolution often occurs through both de jure (formal) and de facto (informal) legal institutions.Ostrom (1990Ostrom ( , 2005) ) emphasizes that de facto legal institutions are often effective, particularly when the state provides such forums with autonomy.Similarly, the literature on legal pluralism suggests that there are merits to increasing adjudication options (Meinzen-Dick and Pradhan, 2002), since informal legal institutions are often an efficient solution to challenges confronting local actors seeking to resolve conflicts (Leeson and Coyne, 2012;Murtazashvili and Murtazashvili, 2015).Thus, in considering dispute resolution, we consider both de jure and de facto institutions, as well as how much autonomy the state provides to informal tribunals. Finally, adaptability and flexibility are important features of governance.While adaptability of institutions is typically desirable, one expects that governance will be constrained by past choices and more prone to incremental problem solving or solving the 'wrong' problems (Pierson, 2000;Poteete, 2009b).The potential for path dependence suggests the importance of considering the extent to which the system of governance of hydraulic fracturing is able to respond to changing conditions.Table 1 summarizes the conceptual framework. Governance of hydraulic fracturing in South Africa The Karoo Basin in South Africa is estimated to hold up to 390 trillion cubic feet (tcf) of technically recoverable shale reserves, making it the eighth largest shale worldwide and the largest in sub-Saharan Africa (US Energy Information Administration, 2013) (see map -Figure 1).The energy Governance Dimension Indicators for Governance of Hydraulic Fracturing Equity of distribution of benefits and costs Extent to which the government is capable of redistributing the rents from hydraulic fracturing Information and monitoring capacity Capacity of the government to record activities of gas companies, to establish baseline environmental and health readings, citizen access to information from government, and ability to monitor the consequences of hydraulic fracturing Accountability Extent to which company officials responsible for hydraulic fracturing can be held accountable Polycentric governance Extent to which governance of hydraulic fracturing is shared with multiple jurisdictions, both vertically and horizontally Democratic inclusiveness Extent to which political processes involving hydraulic fracturing are democratic and inclusive Dispute resolution Extent to which the legal framework is capable of resolving conflicts that arise over hydraulic fracturing, including conflicts over land and land use and the environmental consequences of hydraulic fracturing Adaptability and flexibility Extent to which the regime governing hydraulic fracturing can respond to new circumstances Despite the potential promise of fracking, there are a multitude of risks associated with shale extraction, particularly at the local level.The large amounts of water required for fracking operations will likely present challenges as the Karoo Basin is an extremely water stressed region (Reig et al, 2014).Furthermore, geological characteristics of South African shale formations indicate that hydraulic fracturing has an increased risk of groundwater contamination that suggests the need for stricter regulations than in other regions (Van Tonder et al, 2013).These risks suggest that the realization of social benefits and minimization of social costs from fracking will depend on the governance situation in South Africa, considered below. Equity Distribution The fundamental mechanism governing equity distribution is the MPRDA (Mineral and Petroleum Resources Development Act, 2002), which has three key mechanisms in place to improve prospects for an equitable distribution of the benefits of hydraulic fracturing. 4First, it establishes the right of the government to levy fees and taxes on operations.Companies are required to submit their financial information to the national government for this purpose.This provides a framework for distributing the rents associated with fracking, although who receives the largest share is likely to reflect political considerations.Second, all operations must conduct an environmental impact assessment (EIA), create an environmental management plan, and submit a Social and Labor Plan (SLP) in order to obtain a license.SLPs are binding documents that stipulate how an operation will invest in the community and workforce surrounding their operations (Republic of South Africa, 2010b).Their purpose is to ensure local communities benefit from fracking and to provide them with opportunities to challenge perceived inequalities in distribution of the costs of resource extraction.Third, the broad based socio-economic empowerment charter, required by the MPRDA, stipulates that 26 per cent of operations be owned by historically disadvantaged South Africans (Republic of South Africa, 2010a).These institutions are meant to spread the wealth generated from the extractive industries domestically. 5 Despite several institutional mechanisms to improve equity of distribution of benefits and costs of fracking in South Africa, there are no mechanisms for intergovernmental transfers to different localities based on where extraction occurs.Additionally, local governments do not collect much revenue compared to total government revenue.In 2009/2010, local government collections made up only 7.5 per cent of government revenues (Republic of South Africa, 2008, 2011).The low overall proportion of revenue collected by local governments suggests they may not benefit as much as the national government from shale production even though environmental costs are likely to accrue locally.As the following informant suggests, local governments also face challenges in retaining administrative talent: If you remotely perform at the municipal level you get pulled up to the provincial or state level.So there is just a dearth of skills of the municipal level.Not because we don't have good policies in South Africa.It is on the implementation level that we fall down. 6 The institutional environment would thus suggest that local governments would receive less than their fair share of benefits from higher levels of government, while bearing a disproportionate amount of the costs of fracking.In addition, administrative capacity at the local level may be lacking, thereby undermining the quality of local governance. Information and monitoring South Africa has fairly extensive mechanisms for the national government to monitor and collect information regarding resource extraction.According to the MPRDA, the majority of the authority and responsibility for monitoring production, revenues, and compliance rests with the national government and the Department of Mineral Resources (DMR).The national DMR office approves licenses and the regional DMR offices monitor compliance with SLPs.The MPRDA stipulates that all financial information be provided to the national government. Despite a basic framework to collect information at the national level, local governments and citizens lack an effective mechanism for gathering accurate information about the potential impacts of a project.While citizens could gather information through the Public Access to Information Act, the process is arduous and places the burden of information collection on citizens.Local governments and community members may therefore know very little about the profitability of an operation or the SLP promises. Accountability The government has the ability, through the DMR, to hold companies accountable to MPRDA standards.The companies see environmental or worker-safety non-compliance with regulation as a major threat to operations as this can result in a shutdown.However, companies do not see SLP non-compliance as a threat. 7During fieldwork, informants were asked about issues related to compliance with social investments (SLPs).The following remark, from a legal/corporate social responsibility (CSR) expert, was representative of the comments on such issues: The fact of the matter is that these social and labor plans [or] mining rights are given on the basis of [social investment] promises . . .[But] I have never heard of one mining right being pulled because of not fulfilling a social and labor plan commitment. 8 Capacity constraints in monitoring compliance are especially troubling at regional DMR offices.According to a government official interviewed, companies are well aware of the DMR's constraints: With the limited capacity that we [the DMR] have . . .we are lucky in a region where we have two social plan people who can do actually the [compliance] assessment . . .So, companies are aware that we don't have the capacity, we are thin on the ground.So, some of them get away with [noncompliance], because they know . . .even if you [the DMR] find non-compliance, chances of you coming back are very slim. 9 Polycentric governance South Africa is a federal state that also includes political representation of traditional authority. 10However, local governments and traditional authorities have far less capacity than higher levels of government.The majority of agencies operate at the national level.The Ministry of Finance gathers information for royalties and taxes.The DMR has a national office with duties such as licensing, policy-making, and gathering of production statistics, while DMR regional offices have monitoring duties. Informants suggested that substantial coordination problems between levels of government or different actors also exist.For example, confusion over the respective duties of the provincial DMR and local governments can result in redundant activities or certain duties not being filled.This confusion also exists between mining companies and local governments. 11As one interviewee remarked: 'there is not a very clear line between what government's responsibilities are, in terms of service provision, and what the mines' responsibilities are.' 12 An important example of a coordination problem concerns the Kgalagadi Transfrontier Park, which stretches across the borders of South Africa and Botswana.The park is jointly managed by the two governments and the wildlife populations migrate openly between the countries (Republic of South Africa, 2015).Reports indicate that Botswana has already issued exploration licenses on their side of the park (News24, 2015: 24).Since South Africa has not granted any exploration licenses for fracking, and environmental externalities generated by exploration in Botswana have potential to affect the wildlife in South Africa, polycentricity may undermine prospects for sustainable fracking in this case. Democratic inclusiveness Although South Africa has made strides in improving democratic inclusiveness in the past decades, there are several challenges to deepening democracy as it pertains to natural resource extraction.First, while SLPs are supposed to provide communities opportunities to participate in collective resource governance, community consultation or opinion gathering in terms of issuing exploration and production licenses under the MPRDA or technical cooperation permits (which allow companies to do feasibility studies) can be inadequate (Fig, 2012). Second, even though local governments and companies may consult communities at the beginning of a project, there is often little engagement after operations begin.As a NGO official explained: I have pictures that I took last week in Limpopo, where they consulted with the community in 2004.Lots of meetings, making all kinds of promises.So I go there and I take pictures of the noticeboards of the community meeting place . . .all the notices are dated 2004, so that's the last time they did anything with the community.So I went and I asked the community, when did you last see these people?In 2004.That's when they needed a mining license.Once they got their mining license the CSR is gone, out the window. 13 Third, it is up to a company to determine who is in their ' community.'Although MPRDA and SLP guidelines stipulate that companies consult all local governments and communities about operations, community consultation standards are often poorly understood, as evidenced by several high-profile legal cases seeking to clarify what constitutes proper community consultation with respect to mining licenses.In a landmark case, Genorah Resources was granted a prospecting license under the MPRDA on Bengwenyama traditional community land.However, the community never signed the consent form, was never formally consulted, and even applied for its own prospecting license (which was refused).The case was appealed several times and finally the Constitution Court of South Africa ruled in favor of the community (Christiansen, 2013).While this decision was a win for communities, legal conflicts such as this reflect an underlying lack of clarity regarding standards for community consultation. Finally, local governments are often dependent upon mining companies for information.This information imbalance reduces community bargaining power vis-à-vis mining companies, 14 as one informant explained: So you are talking about a power imbalance, a massive power imbalance where most mine affected communities are in a rural set up . . . You have mining companies coming in with a lot of money and a lot of knowledge and talking very technical stuff, throwing a lot of information at communities, that they can't digest, and the time frames in which consultations take place just do not amount to a sufficient interrogation of the facts. And meaningful consultation means taking the facts into consideration in making up your own mind about it. There is no way that meaningful participation happens . . . it just does not happen. 15 On the other hand, civil society groups play a noticeable role in this particular debate.The Treasure the Karoo Action Group (TKAG) has led the charge against hydraulic fracturing in South Africa and created their own community voice (TKAG, 2015).They argue that under current regulations and current scientific knowledge about the impacts of fracking, 'sustainable' extraction cannot happen.The group's success in pressuring the government to put in place a moratorium on fracking in 2011 indicates that some groups have a powerful voice in the policy process, although it is important to recognize that a moratorium merely postpones key policy decisions.Furthermore, the group with the loudest voice may not represent the majority of the community.TKAG, for instance, consists predominantly of wealthier, white, land-owning farmers in the region (Cropley, 2013, Pitock, 2011). Dispute resolution As Table 2 shows, South Africa is relatively strong in terms of the rule of law compared to the rest of the region, which generally bodes well regarding institutions for resolution of disputes. While broad governance indicators can be useful, understanding prospects for sustainable shale governance requires a more nuanced perspective.Several institutional procedures provide citizens with opportunities to resolve conflicts through the legal process.The MPRDA spells out the process of dispute resolution for ' any person whose rights or legitimate expectations have been materially and adversely affected' (Republic of South Africa, 2002: 42).Individuals can appeal to the director general or minister of the designated agencies when disputes occur.If these options are exhausted, the South African courts can assert judicial review over issues.Unfortunately, the system appears to be quite backlogged.As of August 2012, there were approximately 2,000 unresolved internal disputes under the MPRDA (Mavuso, 2013). Many disputes have also gone beyond the DMR and have appealed to the High Courts, Supreme Court, or Constitutional Court to rule over disputes regarding the extractive industries.Most disputes occur around mining activities, and of these, many involve environmental issues.For example, in April 2015, a coalition of eight civil society and community organizations legally challenged a DMR decision to grant a mining right to a mining company inside the Mabola Protected Environment (Center for Environmental Rights, 2015b).At least sixteen finalized cases have been brought before higher courts since the MPRDA came into force and there are at least sixteen cases still pending judgment (Center for Environmental Rights, 2015a). The presence of institutions providing citizens with legal recourse, and the willingness of courts to exercise judicial review over issues involving extraction of natural resources, is cause for optimism regarding fracking.However, it is axiomatic in legal studies that those with resources have advantages in adversarial legal processes. 16 For this reason, one expects gas companies to have structural advantages in dispute resolution processes.In addition, the large number of pending disputes suggests the process may be inefficient. Adaptability and flexibility The process of changing regulations governing resource extraction has been fairly slow moving.It took almost a decade for the new democratic government to formally establish the new mining and petroleum laws.However, the process of adapting to shale extraction appears a bit more fluid than in the case of conventional energies. The creation of The Petroleum Agency of South Africa (PASA), the recent addition of the Gas Utilization Management Plan (GUMP), and the draft proposal on technical regulations on petroleum exploration and development demonstrate the government's commitment to specifically regulating the gas industry in the future.Additionally, the government's decision to place a moratorium on shale gas exploration from 2011 to 2012 and to only issue technical cooperation rather than exploration permits suggests at least some caution in the process of regulating fracking (Reig et al, 2014).An inter-departmental task team was also created to establish new guidelines on drilling in the Karoo and, more broadly, in South Africa (South African Government News Agency, 2013).Because fracking involves heavy water usage and a major concern is water contamination, in addition to dealing with the DMR, the new guidelines on petroleum exploration and production (yet to be formally approved) call for the involvement of the Department of Water Affairs (DMR, 2013).Each of these developments suggests at least a modicum of adaptation and flexibility in response to new challenges associated with fracking. Governance of hydraulic fracturing in Botswana Botswana is now in the early stages of granting exploration licenses for coal-bed methane (CBM).As CBM is also extracted using hydraulic fracturing, the environmental challenges confronting Botswana are similar to the issues associated with shale gas.While estimates of the extent of these reserves are unclear in Botswana, their exploitation would likely take place within valuable wildlife areas in the country, the Central Kalahari Game Reserve (CKGR), Chobe National Park, and the Kgalagadi Transfrontier Park (shared with South Africa) (Lee, 2014) (see map -Figure 1).For these reasons, it is useful to consider prospectively the governance situation in the country. Equity distribution The Botswana Government's policy is that natural resources, including mineral resources, benefit all (Collier, 2013).There are several mechanisms in place to implement this principle.Because unconventional gas in Botswana is extracted from CBM, it is covered by mineral mining rather than petroleum laws.Similar to South Africa, the Mines and Minerals Act of 1967 vests mining rights with the state.Under the 1999 Mines and Mineral Act, the government has the right to acquire a 15 per cent working interest in any licensed mining operation.Licenses are only granted to non-citizens of Botswana as an exception.Additionally, operations are required to pay a 3 per cent royalty of gross market value on mineral products to the government as an annual charge.Moreover, local governments also have the authority to levy taxes (Government of Botswana, 1965), although the ability to collect taxes depends upon the capacity of local government.The Botswana government requires EIAs, administered through the Department of Environmental Affairs.However, in contrast to South Africa, there are no specific requirements for social impact assessments or something similar to SLPs.As one interviewee explained: [CSR for companies in Botswana] is probably thirty years behind how big companies work in South Africa.It is very random.What you would get there [Botswana] is what I always call chairman's follies.Where you have the wife of the chairman or an executive's favorite pet project . . .there are no safeguards against it.Zero. 17 While most major companies in Botswana have some sort of CSR program, there are few discernible political safeguards surrounding these investments. Information and monitoring In terms of reporting and monitoring, Botswana ranks quite low among resource rich states.The Resource Governance Index ranks Botswana as the ninth worst country (out of fifty eight) for reporting practices.This is by far Botswana's worst resource governance score (Natural Resource Governance Institute, 2013). The government gathers information regarding resource production and is thus able to tax at a national level; however, it only reports these amounts in aggregate to the public.EIAs are not publically published and there is no regulation for freedom of information.Additionally, it is reportedly difficult to find experts on hydraulic fracturing in the region, and NGOs and communities have little knowledge about the consequences of shale exploitation. 18 Accountability As the government can invest up to 15 per cent in any operation, it can use this leverage to hold companies accountable.The reporting requirements at the national level allow for the national government to hold companies accountable for revenue generation.However, lack of information poses problems for both local government and citizens to hold industry accountable. There also appears to be some confusion regarding the responsibility of mining companies.As one of the informants explained: The people in those villages (near the mines) are not aggressive.They are not politically inclined.They go on with their normal lives.So sometimes you cannot really know if there is an issue . . .or they [the villages] are not sure that you're supposed to do something for them.So even (if) you ask them, what do you think of the mine, they will say, no, it is not the mine's responsibility. 19 One possibility is that citizens do not want to hold the mining companies accountable.However, it may be the case that villagers do not hold them accountable because they lack information, which makes them less politically ' aggressive.' Polycentric governance Botswana's mineral regime is fairly centralized, as rights to exploration of deposits rest with the national government in Botswana (Government of Botswana, 1999).The Department of Mines is charged with issuing licenses and permits for mining activities and producing information about revenue generation.The Botswana Unified Revenue Service and the Bank of Botswana gather information on revenue generation.The Department of Environmental Affairs is responsible for administering and controlling EIA activities.Because CBM extraction would occur inside a national park, the Ministry of Environment, Wildlife and Tourism would be involved more broadly as well (Natural Resource Governance Institute, 2013;Walmsley and Tshipala, 2007). Despite a high degree of centralization, there are a variety of lower levels of government in Botswana.Rural areas have district councils, and each area is represented at central government by a district commissioner.Local areas also have Land Boards, which hold the tribal land in trust.They are half appointed by traditional village assemblies, known as Kgotla, and half by the Minster of Land.Finally, traditional local chiefs still play a role in governance in two ways, they are chairmen of the Kgotla, connecting the community to the government, and they preside over traditional courts (Sharma, 2009). While decentralization has many benefits for natural resource governance, it is often derailed by ideological and jurisdictional conflict (Agrawal and Ostrom, 2001).In Botswana, political conflict has often undermined meaningful decentralization (Poteete and Ribot, 2011).Indeed, in the natural resource sector, political conflict has led to recentralization of natural resource governance in Botswana (Poteete, 2009a).To the extent that the experience with other natural resources is a guide, the commencement of fracking may increase political pressure to limit the role of local government in the regulation and taxation of extraction.Moreover, local governments in the country are comparatively under capacity as their entire development expenditure, and 80-97 per cent of recurrent expenditures, are met by central government. Democratic inclusiveness Botswana has long been considered an economic success story in Africa because of its strong political institutions, in particular the integration of customary governance into the political regime (Picard, 1979).The Tswana tribes that make up the majority of the population in Botswana are known for their mechanisms of local democratic participation (Acemoglu et al, 2003).This system allows for normal citizens to express their concerns in the Kgolta and to trust that these concerns are relayed to the proper authorities. 20 Despite mechanisms for local participation, one of the major complaints by foreign media has been that the government has distributed licenses for CBM exploration with little transparency and in environmentally and culturally important areas such as Chobe National Park (home to the largest elephant herds on earth) and the CKGR.For example, the government only revealed that several CMB licenses were granted after media accusations (Ramsay, 2013).Furthermore, there have been accusations that these licenses infringe on the rights of the traditional populations of the Basarwa (or San) in the CKGR with whom the government has been in a long legal battle concerning access to land.Botswana's inclusive local democratic institutions will not be effective or enhance sustainability if transparency remains an issue in CBM extraction. Dispute resolution Botswana fares relatively well in terms of rule of law (see Table 2).The success of the Basarwa (or San) in taking the government to court over their illegal removal from the CKGR suggests that, in at least some important instances, the public is able to hold the government and mining companies accountable in the courts. 21As these cases involved allegations that the government was making room for diamond mining and tourism by removing the San from their lands, but that the community successfully resisted these developments, it is implied that communities do have opportunities to use legal channels for dispute resolution in terms of CBM extraction.In addition, while the Mineral and Mines Act does not specify the process for dispute resolution, it does mention that anyone who feels dissatisfied with agreements made under the Act is entitled to arbitration under the Arbitration Act (Government of Botswana, 1999). Another aspect that potentially bodes well for Botswana is integration of de facto mechanisms of legal decisionmaking into the formal legal regime.Traditional courts, which handle about 80 per cent of criminal and 90 per cent of civil cases in the country, are an added layer of legal protection (Sharma, 2009).Although the dual court system at the local level raises potential for organizational complications or conflict -the traditional court is often preferred by citizens because it is cheaper and more approachable -additional options for legal resolution can improve prospects for dispute resolution.The caveat to this is that the extent to which de facto tribunals can operate effectively depends on the amount of autonomy extended to them by the state. Adaptability and flexibility A strong tradition of democracy with free and fair elections, both at the local and national level, combined with mechanisms for local democratic participation, indicates that the government will be able to adapt to changing perceptions and preferences as they arise.This will occur either through normal democratic channels, in particular elections and citizens expressing their preferences to district councils and commissioners, or through court action, as evidenced by the Basarwa in the CKGR. On the other hand, improving regulation requires information.The secrecy of the exploration process up to this point has left many citizens unaware of both the positive and negative effects of CBM extraction.The government has been slow to react to a call for a freedom of information act that would increase the amount of information NGOs and communities have on extractive industries in the country. 22There has been little indication that new legislation is being discussed for unconventional gas exploration.The lack of debate could be a result of little outcry from normal citizens, which may in turn reflect a lack of understanding among citizens of the nuances of CBM extraction. Conclusion From a governance perspective, several factors appear particularly important to improving prospects for sustainable hydraulic fracturing.First, additional safeguards are necessary to increase the chances that local governments profit from shale production.In both South Africa and Botswana, local governments are likely to bear the costs of hydraulic fracturing, yet it is far from clear that they will receive their fair share of the benefits. Second, there are opportunities to improve the flow of information to communities and local governments, as well as improve mechanisms for real consultation.Transparency should go beyond disclosure to include efforts to ensure the message and information is received and utilized and can be used to enhance democratic participation (Fung, 2013).While both countries have national level mechanisms in place to gather information, each government also has opportunities to package information in a way that is easier for local stakeholders to digest. Third, it is critical to address the coordination problems that arise in polycentric systems of governance.Polycentricity can be a strength provided there is good coordination among local governments and between local and national governments.Yet in both countries in this study, it is far from clear whether the coordination mechanisms that are in place are adequate. Institutional perspectives on governance, which we have applied to the cases of South Africa and Botswana, are potentially useful in comparing countries at different levels of development, or even within a country.For example, Goldberg et al have applied this approach to conclude that there is substantial variation in the quality of governance among US states, as well as variation in the extent to which states experience a resource curse with conventional extractive industries (Goldberg et al, 2008).Recent work has begun to explore the extent to which fracking is associated with a resource curse (Weber, 2014).In both developed and developing country contexts, institutional perspectives on resource governance promise to increase our understanding on the sustainability of hydraulic fracturing. Figure 1 : Figure 1: Potential hydraulic fracturing areas in Southern Africa. Table 1 : Governance variables and hydraulic fracturing. Additionally, estimates indicate that shale extraction will create an estimated 350,000 to 850,000 direct and indirect jobs (African Development Bank Group, 2013; Eberhard, 2013).Shale extraction also promises government revenue, as the Minerals and Petroleum Resources Development Act (MRPDA) (2002) establishes state ownership of sub-surface minerals. 3 Although overall access to electrification is 75 per cent, this rate is much lower in rural areas and rolling blackouts are commonly implemented to alleviate excess demand. Table 2 : Economic and governance statistics. Source: World Bank (2013).The governance indicators (except state fragility) range from −2.5 to 2.5, with 2.5 being highest-quality governance.For state fragility, higher scores indicate higher levels of fragility.	2017-10-19T20:37:23.950Z	2015-09-22T00:00:00.000	{ "year": 2015, "sha1": "e0a7547c23a6f4e2383256cad214ae749cf61813", "oa_license": "CCBY", "oa_url": "https://doi.org/10.5334/gia.aj", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "e0a7547c23a6f4e2383256cad214ae749cf61813", "s2fieldsofstudy": [ "Economics" ], "extfieldsofstudy": [ "Geography" ] }
256275744	pes2o/s2orc	v3-fos-license	RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with Ct < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses. Introduction The pandemic of COVID-19 has caused 623.8 million infections and 6.5 million deaths as of October 22th 2022 (https://covid19.who.int/ table). The pathogen of this pandemic, SARS-CoV-2, has undergone rapid and constant mutations in the process of transmissions, bringing worldwide uncertainty to the diagnostics, vaccine effectiveness and therapeutics [1]. Since November 2020, generally five rapidly expanding SARS-CoV-2 lineages have been discovered and designated as variants of concern (VOCs), i.e., the Alpha, Beta, Gamma, Delta, and Omicron [2]. Besides the current Omicron, among these lineages, the Alpha and Delta VOCs were dominant in frequency over a relatively long period of time. With gradual resumption of air travel, customs, and ports, previously isolated countries and regions are now easily exposed to the SARS-CoV-2 variants. These variants, either with higher infectivity or increased pathogenicity, could trigger outbreak waves unless they are detected timely and accurately [1,2]. Although quantitative reverse transcription PCR (RT-qPCR) performs as a gold standard for the SARS-CoV-2 detection, it is barely used to genotype the SARS-CoV-2 VOCs due to its limitations as an ampliconbased method. The current method is heavily based on in-depth wholegenome sequencing, which not only relies on bulk instruments and trained personnel but is also time-consuming and with relatively high cost [3,4]. A recent study showed that the multiplex qPCR could discriminate some of SARS-CoV-2 VOCs by targeting specific small deletions [5]. However, this method is insensitive to the point mutations and other developing variations in SARS-CoV-2. Similarly, antigen-based detection assays have lower sensitivity than RT-qPCR and have not been reported to identify SARS-CoV-2 VOCs yet [6]. CRISPR-based technology was proven to have unique advantages in molecular diagnosis over the conventional PCR methods in terms of specificity and operability [7][8][9]. Two CRISPR-based COVID-19 diagnostic methods, DETECTR and SHERLOCK, have been developed using either Cas12a or Cas13a, with the characteristics of rapid, ultrasensitive and easy-to-implement [10,11]. Moreover, CRISPR assays such as RAY and PAM-targeting methods were employed to identify the point mutations in SARS-CoV-2 due to the high specificity of Cas/guide-RNA recognition [4,12,13]. Nevertheless, the relying on well-designed ligation probes or guide RNAs somehow limited the targeting capability of these methods to diverse mutation sites. Recently, CRISPR-based point-of-care tests (POCT) of SARS-CoV-2 were reported [14,15]. The methods provided visual readout using minimal fluorescent instruments and could identify multiple SARS-CoV-2 VOCs, among the synthetic samples though. Hence, significant efforts are still needed in respect to improving the detection sensitivity and validating the methods with clinical samples. Here, we report a method combining reverse transcriptionrecombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a technology based on our HOLMES test platform [16,17], aiming to detect and discriminate SARS-CoV-2 mutations. By exchanging the crRNA sequences, the method (RRCd) achieved to discriminate the well-known single nucleotide polymorphisms (SNPs) of N501Y and T478K as well as the small deletion of ΔH69-V70, corresponding to the major VOCs such as the Alpha and Omicron. RRCd was validated in 194 clinical samples after being systematic optimization, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. The results of RRCd could be visualized within about one hour. Moreover, due to using RT-RPA-Cas12a technical route and specific sample release reagents, all procedures of RRCd could be carried out at 37 • C and with no need for RNA extraction and purification, which is very conducive to the development of POCT products. The following actions were taken to avoid the possible crosscontamination and environmental contamination: 1), the RT and RPA reactions were carried out at different biosafety cabinets, and the tubes of each step products were not opened unless cooling them off and centrifugation; 2), a nucleic acid scavenger, Sani-Cide EX3 (Cat # 42048-4, Guangzhou Smosnow Health), was used to remove the residual nucleic acid molecules in air or on the surfaces of the equipment or reagent packages before and after each reaction test. For the fluorescence-based readout, a 96-well plate containing 100 µl reaction mixture was incubated at 37 • C using the Cytation 5 Mul-Mode Reader (BioTek, VT, USA). Usually, a 60-min monitoring was used with an interval time of 30 s. The fluorescence excitation and emission wavelength were set as 485 nm and 520 nm, respectively. The relative fluorescence intensity (F/F 0 ) was calculated as the fluorescence signal relative to the starting signal. Generally, the fluorescence signals at the 30 min-reactions were quantified as F 30 /F 0. In this study, (F 30 /F 0 ) Sample / (F 30 /F 0 ) NC ≥ 1.2 indicates the positive result while (F 30 /F 0 ) Sample / (F 30 / F 0 ) NC < 1.2 indicates the negative result. For the lateral flow readout, a 100 µl-reaction mixture was incubated in an Eppendorf tube at 37 • C for 30 min. Then, the HybriDetect strip (Milenia Biotec, Giessen, Germany) was placed into the Eppendorf tube, and the C-line and/or T-line were visualized and stabilized in ~2 min. The relative greyness of the T-line (G) was quantified using ImageJ. We used the same batched strip loaded with RNase-free water as the negative control (NC). In this study, we assumed that the G Sample / G NC ≥ 1. Validation of RRCd in detection and discrimination of clinical samples To evaluate the performance of RRCd on SARS-CoV-2 clinical samples, a total of 194 swabs were collected at SITHC from March 3rd and to October 29th 2021 and were used to perform the RRCd assay. Both lateral-flow and fluorescence-based readouts were recorded. A total of 86 clinical samples were used to detect SARS-CoV-2 by targeting E gene and S gene, and a total of 108 clinical samples were used to detect and discriminate the SARS-CoV-2 VOCs by targeting S gene 501 or 478. The RRCd results were analyzed and compared with the results of RT-qPCR and genome sequencing. More experimental details can be found at Supplementary Materials. Technical route of RRCd assay The CRISPR-LbCas12a-based nucleic acid test platform HOLMES [17] was chosen as the starting core to design the technical route for RRCd ( Fig. 1 and Fig. S1). Different from the previous methods [10,11,18], RRCd combines the advantages of high sensitivity of RT-RPA [19] and high specificity of CRISPR system [7] (Table S2). More importantly, RRCd uses the Cas protein of Cas12a, which possesses a greater specificity and a lower mismatch tolerance in comparison with Cas9 and Cas13 to the nucleic acid template [7], making it more suitable for genotyping of viral variants. We conducted a two-step RT-RPA amplification on SARS-CoV-2 genes to provide adequate double-stranded DNA (dsDNA) for the following CRISPR recognition system (Fig. 1, II). By designing specific crRNA, Cas12a recognizes and cis-cleaves the dsDNA templates of SARS-CoV-2 or its variants. Meanwhile, the formation of the ternary complex of Cas12a-crRNA-dsDNA activates the trans-activity of Cas12a, which leads to the cleavage of the non-targeted single-stranded DNA (ssDNA) probe in the system (Fig. 1, III). The results of RRCd could be quantified directly by fluorescence detection of the fluorophore-quencher ssDNA (FQ-ssDNA) (Fig. 1, IIIa) or captured by lateral-flow strip using the FITC-Biotin ssDNA (FB-ssDNA) (Fig. 1, IIIb). The specificity analyses of RRCd assay Representative mutations in spike (S) protein can be used for genotyping of the SARS-CoV-2 lineages (Fig. S2). By screening optimal RT-RPA primers (Fig. S3) and crRNAs (Fig. S4) that target to the S protein encoding gene, RRCd was readily applied for the detection and discrimination of SARS-CoV-2 mutations ( Fig. 2A-F). These include the first reported Alpha and Beta mutation N501Y [20] (Figs. 2A and 2B), the recent reported T478K on Delta and Omicron VOCs [21] (Figs. 2C and 2D), and the small deletion ΔH69-V70 on Alpha and Omicron VOCs [22] (Figs. 2E and 2F). The N501Y mutation in the receptor-binding domain (RBD) of S protein was reported to cause an increased transmission and infectivity of SARS-CoV-2 [23]. The T478K in the RBD is also involved in higher transmissibility by increasing the affinity to the human ACE2 receptor [21], while the deletion of H69V70 at the N-terminal domain is shown to allow the virus to replicate more efficiently [22]. Our test results were demonstrated by both the fluorescence-based and lateral-flow readouts, representing the trans-cleavage activity of Cas12a [7,16] (Fig. 2A-F). Additionally, we confirmed this specific cleavage by leveraging polyacrylamide gel electrophoresis analyses representing the Cas12a cis-cleavage activity [7] (Fig. S5A-C). Moreover, we showed that RRCd can be used for the discrimination of SARS-CoV-2 from other phylogenetic related viruses (Fig. S6). The detection limit of RRCd To determine the limit of detection (LoD) of RRCd, we built a standard curve to dissect the correspondences between the viral copy numbers and the C t values using SARS-CoV-2 pseudovirus (Fig. S7). The C t values of each dilution were measured using RT-qPCR targeting N gene or ORF1ab gene to mimic the clinical C t ranges. Despite showing distinct kinetics, the fluorescence-based readouts revealed that the LoDs of all the targeted regions (E gene, N501, Y501, T478, and K478 of S gene) were ~10 copies per reactions, corresponding to a C t value of 35 in the RT-qPCR assay (Fig. 3). The fluorescence results from a 30-min CRISPR reaction suggested that the detection sensitivity of N501 is a slightly higher than that of Y501 (Figs. 3B and 3C), while the detection sensitivity of T478 is a slightly lower than that of K478 (Figs. 3D and 3E). It is worth noting that the detection sensitivity of the lateral-flow readouts is 2-10 folds lower than that of the fluorescence-based readouts (Fig. 3), in consistent with the previous reports [11]. The detection of Y501 and K478 was shown to be the most sensitive when using the lateral-flow readout, with the LoDs of ~10 copies per reactions (Fig. 3). To avoid false-discriminative results, we further assessed the factors that may affect the detection sensitivity in RRCd, including the sample volume and probe inputs, the usage of RNase H, T4 gene 32 protein, Bsu DNA polymerase, RPA kits, and Mg 2+ . We found the sample volume and the usage of T4 gene 32 protein [24] are the key factors affecting RRCd sensitivity mostly (Fig. S8). Validation of RRCd in detection and discrimination of clinical samples Based on the optimized procedures, we evaluated the performance of RRCd on detecting COVID-19 clinical samples isolated from nasopharyngeal and salivary swabs. In coordination with the scenario of POCT, our available samples were collected from Shanghai Customs where the susceptible patients were characterized as asymptomatic and with low virus load (Fig. S9 and Table S1). To minimize the cognitive bias, all information of the clinical samples was confidential to study staff before the detection. A validation study of SARS-CoV-2 detection was conducted on a total of 86 clinical samples (44 RT-qPCR-verified positive samples and 42 RT-qPCR-verified negative samples). Both E gene and S gene were used as the targeted sequences to acquire the double-checked detection results ( Fig. 4 and Fig. S10). RRCd identified all the positive samples with C t < 33 through both lateral-flow and fluorescence-based readouts. In addition, 42 COVID-19 negative samples were all identified without false positive results from both readouts. Samples beyond the C t value of 32 were more likely to be detected by targeting the E gene (C t ≤ 35, 100% detected) than the S gene (Fig. 4). The detection sensitivity of these results was comparable to the RT-LAMP-Cas12 based DETECTR [10] and the RT-RPA-Cas13 based SHERLOCK [11], showing a 99% positive predictive agreement (PPA) and 100% negative predictive agreement (NPA) in both readouts (C t < 33) ( Table 1). We next evaluated the performance of RRCd on discrimination of SARS-CoV-2 mutations. The 501 position of S gene was used as a target to detect SARS-CoV-2 VOCs, such as the Alpha, Beta, Gamma, and Omicron [20]. A total of 68 clinical samples, consisting of 28 genome-sequencing-verified N501 samples and 19 genome-sequencing-verified Y501 samples, were collected from March 3rd to April 10th 2021 at Shanghai Customs. RRCd discriminated all the variants from samples with a C t < 32, either using lateral-flow readouts or fluorescence-based readouts (Fig. 5A and Fig. S11). However, the discrimination ability decreased strikingly when testing the samples with C t values beyond 33, which is possibly due to the relatively high LoD of S gene 501 as shown above (Fig. 3). The 478 position of S gene was used as a target to identify Delta and Omicron VOCs [21]. A total of 40 clinical positive samples, consisting of 7 genome-sequencing-verified T478 samples and 14 genome-sequencing-verified K478 samples, were collected from May 27th and October 29th 2021. RRCd was able to discriminate all the variants targeting the S gene 478. Moreover, RRCd displayed a relatively higher sensitivity in detecting the position of 478 than that of 501, with the C t value reaching 34. The detection results were confirmed by both readouts with 0% false-discriminant rates ( Fig. 5B and S12). Taken together, these data show that a total of 89% positive predictive agreement and 100% negative predictive agreement in 194 clinical samples, demonstrating RRCd detect and discriminate the SARS-CoV-2 mutations accurately and sensitively (Table 1). Moreover, SARS-CoV-2 detection using direct lysing the clinic samples without RNA extraction and purification was demonstrated (Fig. S13). Although a relatively small sample size was used, these results indicate that the whole process of RRCd including the RT-RPA amplification and the lateral-flow detection is independent of electrical devices, which may facilitate the development of POCT products in the future. Conclusions In this study, an RRCd method aiming to detection and discrimination of SARS-CoV-2 mutations has been developed. RRCd completed in ~1 h and displayed a sensitivity down to 10 copies per reaction. By targeting and identifying the representative N501Y, T478K and ΔH69-V70 mutations, RRCd discriminated the mutations on COVID-19 samples related to SARS-CoV-2 VOCs. The method was validated in 194 clinical COVID-19 samples, with 99% PPA and 100% NPA for the samples of C t < 33 through both the fluorescence and the lateral-flow Fig. 3. The detection limit of RRCd for SARS-CoV-2 VOCs. The detection limit of the targeted regions in VOCs were determined by using 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 and 5 copies of pseudovirus RNA per reaction, respectively. The dsDNA derived from the pseudovirus RNA was incubated with Cas12a, ssDNA probe, and crRNA at 37 • C for 30 min. The fluorescence signal at the 30 min-reaction was quantified (left panel). The positive samples of lateral-flow readouts (right panel) are indicated as blue by the T-line quantification as described. Data are presented as mean ± S.D. from at least three independent experiments. For the dilutions with 10 and 5 copies per reaction, at least five independent experiments were conducted. A. LoD determination of RRCd for E gene. B-E. LoD determinations of RRCd for N501 (B), Y501 (C), T478 (D) and K478 (E) in S gene, respectively. * , P < 0.05; * , P < 0.01; ** , P < 0.001. NC, negative control using the RNase-free water. readouts (Table 1). RRCd also has the potential to be easily adapted to detect novel mutations on SARS-CoV-2 or other epidemic viruses by designing the new RT-RPA primers and crRNAs, without changing the subsequent detection procedures and ssDNA probes ( Fig. 1 and Fig. S1). Moreover, for those targeted DNAs without the proper PAM, our RRCd could introduce an exogenous 5' TTTV PAM by designing the RPA primers, which increases the adaptability of this method to target broader sites. Although displaying a high adaptability and was validated by large clinical samples, RRCd assay could only be used to detect known sequences or mutations of viruses, and it will never replace the whole genome sequencing in discovering new viral mutations. Moreover, RRCd is not able to detect all the mutations as genome sequencing since more and more mutations emerging in SARS-CoV-2. These new mutations will either increase the detection dimensions because more crRNAs are needed, or reduce the specificity of crRNA recognitions due to the undesirable mutations may happen and affect the crRNA binding sites. In addition, the LoDs of RRCd were usually higher in discriminating viral variants than detecting the wild-type sequences, and were varied between the detection of distinct mutations. These therefore require that the viral genome sequences and the optimal RT-RPA primers and crRNAs be analyzed and screened in advance. Therefore, the application scenarios of RRCd could be on site detection (e.g., self-testing), or where the power access is limited (e.g., underdeveloped areas), or when quick turnaround times are needed (e. g., hospital emergency). Further modification of this method to meet the requirements of POCT products is still needed. Table S1). In total, 47 positive samples (C t range of 13-35) and 21 negative samples were detected. The S gene 501 variants were discriminated by crRNA-N501 (green) and crRNA-Y501 (light blue) using fluorescence-based readouts. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability No data was used for the research described in the article.	2023-01-27T14:04:52.785Z	2023-01-01T00:00:00.000	{ "year": 2023, "sha1": "2e284dcd7c0c0eab0ee4fde44267ef17dd2008b2", "oa_license": null, "oa_url": null, "oa_status": "CLOSED", "pdf_src": "PubMedCentral", "pdf_hash": "f263f7af0d56188287555605d3f16ea2a771d519", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
25242222	pes2o/s2orc	v3-fos-license	Effectiveness of sound therapy in patients with tinnitus resistant to previous treatments: importance of adjustments Introduction The difficulty in choosing the appropriate therapy for chronic tinnitus relates to the variable impact on the quality of life of affected patients and, thus, requires individualization of treatment. Objective To evaluate the effectiveness of using sound generators with individual adjustments to relieve tinnitus in patients unresponsive to previous treatments. Methods A prospective study of 10 patients with chronic tinnitus who were unresponsive to previous drug treatments, five males and five females, with ages ranging from 41 to 78 years. Bilateral sound generators (Reach 62 or Mind 9 models) were used daily for at least 6 h during 18 months. The patients were evaluated at the beginning, after 1 month and at each 3 months until 18 months through acuphenometry, minimum masking level, the Tinnitus Handicap Inventory, visual analog scale, and the Hospital Anxiety and Depression Scale. The sound generators were adjusted at each visit. Results There was a reduction of Tinnitus Handicap Inventory in nine patients using a protocol with a customized approach, independent of psychoacoustic characteristics of tinnitus. The best response to treatment occurred in those with whistle-type tinnitus. A correlation among the adjustments and tinnitus loudness and minimum masking level was found. Only one patient, who had indication of depression by Hospital Anxiety and Depression Scale, did not respond to sound therapy. Conclusion There was improvement in quality of life (Tinnitus Handicap Inventory), with good response to sound therapy using customized settings in patients who did not respond to previous treatments for tinnitus. Introduction Subjective tinnitus can be defined as an auditory perception in the absence of an external sound stimulus, 1 described as a sound like a whistle or a hiss. It is estimated that over 30 million Americans have tinnitus; 2 in Brazil, it is believed that this number is about 28 million. 3 Thus, it is a public health problem. It is a common-sense conclusion among researchers that symptom severity can lead to losses in quality of life. The lack of control of tinnitus and its constant presence produces a high degree of stress; indeed, the emotional effect is variable and may range from a mild irritation associated with tinnitus, to states of anxiety, depression, and insomnia, even leading to suicide. 4 In patients with tinnitus, it is difficult to make objective measurements of emotional disorders such as anxiety and depression. However, several subjective assessment tools are available in Portuguese, and the Hospital Anxiety and Depression Scale (HADS) 5,6 is one of the most used instruments, due to its ease of application. Since tinnitus is a subjective symptom, it is difficult to analyze, measure, and treat. Thus evaluations such as acuphenometry, the use of visual analog scales (VAS), and questionnaires to determine the impact on quality of life such as the Tinnitus Handicap Inventory (THI) 7,8 are very important strategies, as are individual approaches in the treatment of these patients,. Among the therapeutic possibilities for sensorineural tinnitus, drug therapy, acupuncture, 9,10 transcranial magnetic stimulation, 11 cognitive-behavioral therapy (CBT), 12 and sound therapy (masking therapy 13 and habituation therapy) can be cited. 4 Some patients try several resources attempting to find a treatment that brings significant relief for their tinnitus. The process of habituation to tinnitus with the use of sound therapy consists of the stimulation of the ear by the presence of constant sounds, with the aim of reducing hypersensitivity in quiet surroundings. In this process, sound generators, with or without hearing amplification, are used with a neutral sound: music or white noise, at a low intensity in an attempt not to mask tinnitus, but to provide a reduction in its perception. Jastreboff 4 developed Tinnitus Retraining Therapy (TRT) as a habituation therapy that uses counseling and sound therapy. Fractal Tones Therapy 14 uses habituation therapy to reduce tinnitus, through a sound generator that produces fractal sounds (music); in this manner, the melody is maintained but does not repeat itself. To observe the effectiveness of sound enrichment in tinnitus sensation, minimum masking levels (MML) are used in order to evaluate the effect of masking the perception of tinnitus through the use of a broadband noise. 15 Currently, several protocols have been used for sound therapy; however, reports on the settings used in sound generators of hearing aids were not found in the literature. The aim of this study was to demonstrate the effectiveness of sound therapy with different types of sound generators through patients' follow-up, the relationship of sequential evaluations through previously established parameters (THI, VAS, MML), and the need for individual interventions through detailing the customized settings of these generators in patients unresponsive to previous treatments for tinnitus. Methods This study was approved by the Ethics Committee under CEP protocol No. 1090/11. Patients were instructed about all procedures of the study and signed an informed consent. A prospective study of 10 selected patients from the Tinnitus Outpatient Clinic was conducted by the otolaryngologists responsible for this department. For the research, 20 retroauricular behind the ear (BTE) hearing aids with an open fitting were donated to be used by patients bilaterally. Of these 10 patients, five used the Mind 9440 model with fractal sounds (Widex TM ) and five used the Reach 62 Model (Beltone TM ) with white noise. Patients were recruited sequentially from the beginning of the study, alternating the indication of Mind 9 and Reach 62 generators, according to inclusion and exclusion criteria. Inclusion criteria: continuous chronic tinnitus complaints for over a year without improvement with drug therapies and with no specific treatment for tinnitus for at least 3 months. Hearing loss, when present, was not the main complaint of the patient. Exclusion criteria: conductive hearing loss or changes in the external and/or middle ear. During the use of sound generators, these patients were evaluated at the beginning of therapy, and at 1, 3, 6, 9, 12, 15, and 18 months, when the generators were switched off. In all the visits, adjustments for the generators were performed when needed, and all of the following evaluations were carried out: Otolaryngology evaluation Pure tone audiometry and vocal audiometry Immitanciometry with stapedial reflex survey HADS VA S THI Acuphenometry (loudness and pitch matching) MML The HADS questionnaire was used because it is a simple, rapid assessment tool that provides data and identifies symptoms of anxiety and depression in individuals under no psychiatric treatment. To better understand the effect of tinnitus in patients, the type of tinnitus presented was considered and characterized by acuphenometry: a group with whistle-type tinnitus equivalent to pure tone, a group with hissing-type tinnitus equivalent to white noise (WN) or narrow band (NB), and a group with both types of tinnitus. In all groups, evaluations and adjustments met the same criteria. Through MML, the authors sought to ascertain the minimum intensity of tinnitus masking in ascending form by using bilateral noise: in the first instance, WN; in the second instance, NB at tinnitus frequency, and then NB at 500 Hz. Reduction in THI score ≥20 points and reduced VA S were considered as improved parameters of therapy. The five patients who used sound generators with music could choose between five types of fractal sounds, random and harmonic, known as Zen. Where necessary, the adjustments changed the rhythm (between slow and fast), frequency (between low and high), the volume in a range from 0 to 15, and, in some cases, the type of Zen sound. The other five patients using sound generators with white noise could adjust the initial volume by sound pressure level (SPL), from 0 to 100 dB SPL; use low sound cuts between 500 and 2000 Hz and high sound cuts between 2000 and 6000 Hz; insert slight, moderate, or strong modulation; and change between slow, medium, and fast speed. Patients with associated hearing loss were given a month of initial monitoring to adapt and adjust the sound amplification before being connected to the sound generator. Hearing aids with sound generator were obtained by donation from Centros Auditivos Widex and Audibel do Brasil. Results Of the total of 10 patients, five were female and five male, aged 41---78 years. Six patients presented with bilateral tinnitus showing more intensity on the left side; in three, the problem was unilateral on the left side; and one patient had unilateral tinnitus on the right side. In the analysis of tonal audiometry, it was found that two subjects had no hearing loss and eight had mild-to-moderate sensorineural hearing loss at high frequencies. Of these, only four subjects had associated hearing complaints, with the necessity for placement of amplification in their hearing aids to improve speech understanding and avoid auditory deprivation. All patients used the sound generators for at least 6 h daily throughout the treatment, regardless of the type of generator. In acuphenometry, throughout therapy, 10 patients exhibited tinnitus between 3000 and 8000 Hz; two patients started sound therapy with whistle-type tinnitus, four with hissing-type tinnitus, and four with whistle-and hissing-type tinnitus (Table 1). In the initial assessment with the HADS questionnaire, one patient (patient 8) had anxiety and depression, three patients had anxiety (patients 2, 3 and 9), and the other showed no such alterations. Of the 10 patients monitored, in nine a decrease of 20 points in THI score (with respect to basal levels, Table 2) and reduction in VA S (Table 1) were observed at the end of the sound therapy. Tw o patients, who had been consistently showing a good response to treatment, demonstrated a worse score at 15 months (patient 7) and at 18 months (patient 2). In acuphenometry, the intensity of tinnitus was reduced or remained at the same level in all patients, when comparing the beginning and the end of sound therapy. Regarding MML with WN, volume reduction or stabilization occurred in eight patients. Compared to baseline, there was a need to increase the volume of sound generators in all patients ( Table 3). The volume of MML NB at tinnitus frequency was reduced or remained at the same level in nine patients and increased only in the patient who reported no improvement with therapy. However, when MML volume with NB at 500 Hz was evaluated, this occurred in only six patients. These responses were important in setting the characteristics of the sound generators ( Table 4). The stabilization of volume and generator characteristics required 3---12 months of sound therapy. Of the six patients who used only the generator, two (patients 1 and 5) returned the devices; one continued with the therapy (patient 2), and three kept the generators for sporadic use (patients 8, 9, and 10). Of the four subjects presenting hearing loss, three remained only with amplification (patients 4, 6, and 7) and one with amplification and a generator (patient 3). Discussion Numerous proposals to determine the origin of tinnitus exist, 4,16,17 but notwithstanding the cause, the most important aspect is when this information is interpreted as something unpleasant by the limbic system. This emotional reaction can be intensified and worsened, resulting in increasing stress and exacerbation of the tinnitus sensation, with an important impact on quality of life. Very anxious individuals process emotional stimuli more rapidly than non-anxious people, 18 and even considering that anxiety has not been established as a causal factor in tinnitus worsening, HADS showed that this condition was present in three patients (2, 3 and 9) who were already using controlled 1 74 58 58 44 50 38 40 24 2 72 56 40 26 22 22 22 54 3 72 38 22 20 20 24 12 16 4 38 14 24 34 20 26 24 18 5 44 46 0 0 0 0 0 0 6 38 34 26 22 30 22 14 16 7 40 12 20 14 22 10 38 40 8 58 56 62 60 66 66 58 68 9 64 44 44 44 38 38 36 12 10 58 50 54 32 36 36 36 28 medications for their treatment. The correlation of HADS with tinnitus showed that, despite the non-occurrence of change in anxiety characteristics throughout therapy, this fact did not hinder good responses to sound therapy. In patient 8, characteristics of anxiety and depression in HADS were found from the beginning of therapy, but the patient reported that she was not in any specific treatment, because she had not thought that previous suggestions were beneficial. She was the only patient who did not achieve improvement of the impact of tinnitus on quality of life, which leads to the conclusion that depression, as reported by other authors, 19 prevented the adaptation to tinnitus. Several authors 20---23 have demonstrated the efficacy of THI in monitoring tinnitus treatments, and we, used this tool as an important measure to evaluate the impact of tinnitus on quality of life. Therefore, when considering that tinnitus is dynamic and influenced by emotional and physical changes, it was important to assess THI throughout therapy, as well as to make adjustments in the intervention when necessary. The authors observed improvement of ≥20 points in THI scores during therapy in nine patients, which is in line with the findings of other authors (Table 2). 20,24 In those subjects that worsened (patient 2 at 18 months and patient 7 at 15 months), the emotional, functional, and catastrophic aspects of tinnitus in THI were analyzed. Worsening was found in all three aspects with respect to the previous evaluation of patient 2, who had worsened emotional status due to personal factors, with no change in auditory thresholds. However, patient 7 had both worsened tinnitus and hearing thresholds after a diagnosis of cancer and the start of chemotherapy/radiotherapy. In this subject, the THI score for functional aspect was the most affected. These responses were of interest when relating the cause of worsening (emotional or functional) with the qualitative aspects of tinnitus. There are several possibilities to treat the etiology or the effect of tinnitus. Treating the cause is more difficult, given the difficulty to determine the origin of tinnitus and also because, in most cases, there is no relationship between a cure for the disease and the elimination or reduction of tinnitus. When the neurophysiological process of habituation takes place during sound therapy, the central nervous system ceases to perceive tinnitus as an unpleasant sensation and may reduce its perception or even suppress it. 4,14 Table 4 Baseline and final volume (dB) of minimum masking levels (MML) with narrow band (NB) at tinnitus frequency and at 500 Hz in sensation level, and the number of times that changes were made to generator characteristics. When measuring tinnitus with the use of VAS, we observed a difference in hissing-and whistle-type tinnitus reduction at the beginning and at the end of therapy (Table 1), without any change in pitch during the treatment. A significant reduction of whistle-type tinnitus occurred in six patients, with suppression of tinnitus in two patients at the end of treatment. However, in those with hissing-type tinnitus, the reduction was less noticeable, or the intensity remained the same, and in patient 7, who underwent chemotherapy, the authors even noted the onset of hissing in the side previously without tinnitus. The decrease of whistle-type tinnitus occurred even in patient 8, who did not achieve a good response to sound therapy, and whose HADS scores suggested anxiety and depression; this patient also noted no improvement on his THI, and an unaltered VA S for hissing. These findings suggest that the cortical representation of whistle-type tinnitus, expressed in acuphenometry by pure tone, can be more prone to modulation in the central nervous system. Nonetheless, considering the small number of subjects in this series, the authors cannot claim this as a hypothesis. According to Figueiredo et al.,25 there is a correlation between THI and VA S scores, which agrees with this study. Throughout therapy, one to four adjustments of the volume and characteristics of the sound of generators were carried out until stabilization (Tables 3 and 4). New changes occurred at 15 and 18 months in both patients who worsened (patients 2 and 7). In isolation, the intensity of tinnitus evaluated in acuphenometry did not indicate the success of the therapy. In all subjects, the baseline intensity decreased or was maintained at the same level, including in patient 8, who reported no significant improvement and did not show improvement in THI (Table 3). Despite the reduction or maintenance of tinnitus intensity after treatment of these patients, there was a need to increase the volume of the sound generators during the therapy for all subjects (Table 3). Conversely, a reduction in MML with WN in eight patients occurred, with an increase at the end in the patient with worsening of hearing thresholds (patient 7) and also in that patient who did not improve (patient 8). Thus, there was a correlation between intensity of tinnitus and MML WN intensity from the generator. With habituation, the tinnitus and the level of sound required to mask it quickly, as in MML WN, tended to diminish or stabilize, and the central nervous system tended to conform to the sound of generators, thus it became necessary to increase their volume (Table 3). Interventions throughout therapy were required to maintain the response. The literature shows that the best option for habituation is broadband WN, because it is less bothersome and covers more frequencies; 23 however, the algorithms of the hearing aids enabled changes in their characteristics. Thus, this study used other support measures to evaluate and change the characteristics of sound generators: MML NB at the tinnitus frequency, and MML NB at 500 Hz. The first had a response similar to that of MML WN, i.e., volume reduction or maintenance in nine patients. However, this relationship was not observed with MML NB 500 Hz, (Table 4). Despite the fact that low frequencies are considered more relaxing compared to high frequencies, better acceptance of changes to higher pitch was observed, but the acceptance was not good for modulation and rhythm changes. This study also observed a relationship between THI and MML WN, which was reduced in patients with good responses to treatment and without major complications during therapy. Although Figueiredo et al. 26 did not observe this effect in their study, these authors did not compare THI and MML WN throughout the treatment, but only at their initial assessment. Although several studies have demonstrated the importance of determining the mixing point for habituation of tinnitus, in the present study we did not consistently observe this, and noted it only in two patients (9 and 10) with 12 and 15 months of use of the sound generator, respectively. The findings of Tyler et al. 27 also showed that there was no need to determine the mixing point for habituation to occur. Conclusion It is possible that patients with whistle-type tinnitus have a better response to sound therapy. A correlation of MML WN with THI during therapy was observed. Apparently, there is a correlation of adjustments to sound generators with habituation of tinnitus, an aspect observed in these evaluations; this requires the customization of these settings, according to the patient's symptoms and their answers in matching pitch, loudness, and MML, which we performed on every patient's return. The patient with response to HADS for depression did not respond well to treatment. No relationship was found between the findings regarding location of tinnitus, presence of hearing loss, and type of sound generator with the other criteria evaluated in this study. Conflict of interest The authors declare no conflicts of interest.	2017-10-15T05:59:22.848Z	2015-10-16T00:00:00.000	{ "year": 2015, "sha1": "1521af2c59ebd0ffc3be086adba5cb2b582b5de8", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.bjorl.2015.05.009", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "10ed5877a5b8bc24403f3d6b33bfa6e84ca10162", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
255415426	pes2o/s2orc	v3-fos-license	Assessment of cardiotoxicity and plasma ropivacaine concentrations after serratus intercostal fascial plane block in an experimental model Serratus intercostal fascial plane block (SIFPB) has emerged as an alternative to paravertebral block in breast surgery. It involves the administration of high volumes and doses of local anesthetics (LA) that can potentially reach toxic levels. Ropivacaine is widely used in thoraco-fascial blocks; however, there is no information on the plasma concentrations attained after SIPFB and whether they are associated with cardiotoxicity. Plasma concentrations of ropivacaine and its electrophysiological effects were evaluated in eight pigs after bilateral SIFPB with ropivacaine in doses of 3 mg/kg. Plasma concentrations, electrophysiological and hemodynamic parameters were measured sequentially for the following 180 min until the end of the study. The area under the curve, the maximum plasma concentration (Cmax) and the time to reach Cmax (tmax) were calculated. The median arterial ropivacaine concentration Cmax was, 2.34 [1.40 to 3.74] µg/ml. The time to reach the highest concentration was 15 [10 to 20] min. Twenty-five percent of the animals had arterial concentrations above the lower limit concentration of ropivacaine for LA systemic toxicity (3.4 µg/ml). No alterations were observed in the electrophysiological or electrocardiographic parameters except for a prolongation of the QTc interval, from 489 ± 30 to 544 ± 44 ms (Δ11.38 ± 6%), P = 0.01. Hemodynamic parameters remained in the physiological range throughout the study. SIFPB with ropivacaine in doses of 3 mg/kg has reached potentially toxic levels, however, it has not been associated with adverse electrophysiological or hemodynamic effects. Breast cancer is one of the most common malignancies among women and surgical resection of the primary tumor with axillary dissection remains the cornerstone therapy of patients with breast cancer 1 . The anesthetic strategy usually consists of the combination of general anesthesia supplemented with local or regional anesthesia with the aim of optimizing acute pain control and patient comfort 2 . Ultrasound-guided fascial plane blocks have generated great interest as an alternative analgesic technique to the thoracic paravertebral block (PVB), considered the gold standard analgesic modality in breast surgery [2][3][4][5][6] . The interfascial plane between the serratus anterior and intercostal muscles contains the branches of the intercostal nerves and their blockade at the mid-axillary line would provide analgesia in the region of the breast and axilla. The block is termed serratus intercostal fascial plane block (SIFPB) or (BRILMA) that is, blockade of the branches of the intercostal nerves in the mid-axillary line 6,7 . Perioperative breast pain control with fascial plane blocks comprises the administration of a relatively large volume of local anesthetic (LA), usually 25-30 ml of aminoamide LA in a single dose [7][8][9] . The anatomical region of the SIFPB is widely vascularized and, like other regional techniques, potentially toxic plasma concentrations of LA can be reached 8-10 . Interventions. The animals were sedated with ketamine (20 mg/kg) 11 intramuscularly and a caudal auricular vein was subsequently cannulated. Anesthesia was induced with intravenous thiopental at a dose of 5-10 mg/ kg and was maintained with sevoflurane at 1 minimal alveolar concentration (MAC) for pigs (2.66%) 12 . Pigs were intubated and controlled ventilation was started at a tidal volume of 6-8 ml/kg, with a respiratory rate of 12 breaths per min (Heinen & Löwenstein Leon respiratory ventilator; Direx SL, Wiesbaden, Rheinland-Pfalz, Germany). The respiratory parameters were adjusted to maintain an arterial partial pressure of carbon dioxide between 35 and 40 mmHg. Throughout the experiment an infusion of 0.9% sodium chloride was administered at a rate of 5 ml kg/h. Esophageal temperature was maintained Esophageal temperature was maintained between 37.5 and 39 °C with a forced-air warming blanket (Warm Touch™ 5300A, Mallinckrodt Medical SA, Madrid, Spain). Animal instrumentation was as previously described [13][14][15] . We used a closed-chest porcine experimental model that allowed reliable cardiac electrophysiological evaluation of cardiotoxicity of local anesthetic agents (Fig. 1). The femoral vessels (veins and arteries) were cannulated percutaneously by ultrasound (Vivid S5; GE Healthcare, Wauwatosa, WI). A 5-French catheter (PiCCO, Pulsion Medical Systems AG, Munich, Germany) was inserted through the femoral artery and used to record continuous hemodynamic parameters and for cardiac output (CO) measurement by transpulmonary thermodilution. The contralateral femoral artery was cannulated for arterial blood gas sampling and analysis of plasma ropivacaine concentration. Three quadripolar catheters-one of them deflectable (Marinr, Medtronic, Minneapolis, MN), were inserted via the femoral veins under fluoroscopic guidance. The catheters were positioned in the right atrium, right ventricle, and in across the His bundle area. Catheters were used for stimulation and intracardiac electrogram recordings, which were high-pass (70 Hz) and low-pass (500 Hz) filtered. His bundle recordings were obtained with special care to avoid recording right bundle branch potentials. Stimulation was accomplished with a customized programmable stimulator (CS3 Cardio-stimulator; A.S.P. Electronic Medical, Madrid, Spain). Electrocardiographic parameters and intracavitary electrograms were continuously monitored, recorded and stored on a computer-based digital amplifier/recorder system. (LABSYSTEM PRO EP Recording System; Boston Scientific Corporation, Marlborough, MA). The animals were allowed to stabilize for 15 min before initiating the experimental protocol. Serratus intercostal fascial plane block. All blocks were performed by a single person (O.V.) with knowledge and training in ultrasound-guided regional techniques and experience in performing SIFPB on pigs 14,15 . A GE 6-12 MHz linear array probe was used, and the blocks were performed with the animals positioned in supine. A two-plane ultrasound scan was performed to identify the serratus anterior muscle, and the fifth and sixth ribs in the mid-axillary line. The SIFPB was performed with a echogenic needle (50 mm Stimuplex ® Ultra 360, B. Braun Medical S.A. Barcelona, Spain) introduced by an in-plane approach and after confirmation by hydrodissection, 15 ml of ropivacaine was administered on each side, visualizing the spread of the local anesthetic solution. Each animal received a total dose of 3 mg/kg ropivacaine. Measurements. The electrophysiological parameters measured were as follows: sinus cycle length (SCL), heart rate, PR interval, QRS duration, AH interval (the time from the earliest rapid deflection of the atrial electrogram in the His bundle recording to the onset of the His deflection), HV interval (the time from the beginning of the His bundle deflection to the earliest onset of ventricular activation), QT and corrected QTc intervals (using the Bazett formula [QTc = QT/√R − R]). Since local anesthetics slow ventricular conduction in a frequency-dependent manner as a result of blockade of cardiac sodium channels 16 , this effect was evaluated by measuring the QRS duration after ventricular pacing trains of 10-12 beat with interstimulus intervals of 400 or 500 ms (stQRS 400 or stQRS 500 ). To facilitate a precise www.nature.com/scientificreports/ QRS duration measurement during rapid ventricular stimulation, the QRS onset was timed to the pacing spike during right ventricular pacing and the QRS offset was timed to the latest lead (considering all 12 leads). The duration of the last QRS complex of each paced train was measured. Pacing was bipolar (5 mm interelectrode distance) at an intensity of 10 mA and with a pulse width of 1 ms. In all groups, the electrophysiological parameters were measured at baseline and at predetermined intervals: (2,5,10,15,20,30,40, 50, 60, 90, 120, 150 and 180 min after completion of bilateral SIFPB). Arterial blood gas measurements were performed throughout the study, but for statistical evaluation, baseline values and data at 60, 120 and 180 min after SIFP block were included. The concentration of ropivacaine was assessed by liquid chromatography coupled to tandem mass spectrometry/mass spectrometry using the method described previously for the analysis of bupivacaine, which was modified to determine the concentrations of ropivacaine 13,14 . Outcomes. The primary outcome was to quantify arterial and venous plasma ropivacaine concentrations after SIFPB and to analyze the time course of these concentrations over 180 min. The secondary outcome was to analyze whether the plasma concentrations of ropivacaine reached after SIFPB were associated with cardiac electrophysiological toxicity effects. Statistical analysis. Sample size. For sample size estimation, the reported plasma arterial concentration of ropivacaine 4.3 ± 0.6 µg/ml with potential toxicity were considered 17 , compared with transversus abdominis plane (TAP) block as a typical fascial block. For TAP blocks, the mean maximum arterial plasma concentrations of ropivacaine have been reported as 1.56 ± 0.5 µg/ml 17 . Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a bilateral contrast, 7 were required to detect a difference equal to or greater than 0.8 µg/ml, based on previous studies. A standard deviation of 0.7 µg/ml was assumed. Finally, 8 animals were included to replace possible losses. Data analysis. The analysis was conducted using the SPSS statistical package (version 20.0; SPSS Inc., Chicago, IL, USA). Results are presented as medians and and interquartile range (IQR) for non-normally distributed data www.nature.com/scientificreports/ using the Shapiro-Wilk test, and as means ± standard deviation for normally distributed variables. Ropivacaine plasma concentrations were represented by the area under the curve (AUC, trapezoid method). The maximum plasma concentration (C max ) and the time to reach C max (t max ) were calculated. Repeated measures analysis of variance was used to analyze the evolution of the electrophysiologic and hemodynamic parameters over time. Comparisons between baseline electrophysiological parameters and those obtained during peak ropivacaine concentration were performed with paired Student's t-test. Euthanasia. At the end of the experiment, the pigs were given a bolus of propofol and potassium chloride for euthanasia. Conference presentation. This study has been presented in the 2018 World Congress on Regional Anesthesia & Pain Medicine, ASRA's 43rd Annual regional Anesthesiology & Pain Medicine Meeting. Results All the animals completed the study, which lasted 299 ± 18 min. Biological parameters are shown in Table 1. They all remained stable and in physiological range throughout the study. The electrophysiological and electrocardiographic parameters are shown in Table 2. There were no electrophysiological alterations during the study period except for the QTc interval which increased from 489 ± 30 to 544 ± 44 ms (Δ11.38 ± 6%), P = 0.01. There were no significant changes in the electrophysiological parameters evaluated at baseline and during maximal ropivacaine concentration ( Table 3). The hemodynamic parameters are shown in Table 4. There was a moderate decrease of 20% in mean arterial pressure values and a 23% decrease in systemic vascular resistance index (SVRI) although the values remained in physiological ranges. Discussion The principal findings of this study were that the administration of 3 mg/kg of ropivacaine in SIFPB was associated with rapid absorption of the local anesthetic and that the concentrations achieved are similar to those described in other non-fascial chest wall blocks as PVB and intercostal nerve blocks [18][19][20][21] . We found no relevant electrophysiological toxic effects associated with the plasma concentration of ropivacaine reached after SIFP block. To the best of our knowledge, this is the first study that has evaluated plasma concentrations of local anesthetics associated with SIFP blocks and their influence on the electrophysiological parameters. We used 3 mg/kg of ropivacaine, which is the maximum dose recommended by most authors, although this is a controversial issue as the toxicity of the local anesthetic used depends on several causes 22 . The absorption of local anesthetics and the plasma concentration achieved depends, among other factors, on the block site and its vascularization, the injection speed and the total dose administered. The chest wall is a richly vascularized region, and it is potentially expected that locoregional blocks in this area will be associated with a high peak concentration of local Table 1. Biologic parameters at baseline, 60, 120 and 180 post-serratus intercostal fascial plane block. Data are showed as medians interquartile range (IQR) for non-normally distributed data using the Shapiro-Wilk test, and as means ± standard deviation for normally distributed variables. SIFPB serratus intercostal fascial plane block. www.nature.com/scientificreports/ 20 . These values are in line with ours, although we used higher doses of ropivacaine, but the time to reach the maximum concentration was shorter than in our study. This suggests that the absorption of the local anesthetic from the paravertebral space is faster compared to the serratus-intercostal interfascial space. Of note, there was significant variability in ropivacaine plasma concentrations between animals, this result is in line with the high interindividual variation in serum concentrations previously described in human's studies 23,24 . Bupivacaine plasma concentrations were recently assessed in patients undergoing breast surgery following unilateral or bilateral pectoral nerve blocks (PECS II) using 30-60 ml respectively of 0.25% bupivacaine with www.nature.com/scientificreports/ 1:400,000 epinephrine. The highest bupivacaine venous concentration after unilateral PECS was 0.038 µg/ml at 20 min 25 . In contrast in our study the highest ropivacaine venous concentration was 2.78 µg/ml attained 37 min after SIFP block. The anatomical differences of both blocks, the dose administered with and without vasoconstrictor and the study protocol in an animal vs. human model justify the discrepancies in the concentrations achieved. Arterial plasma concentrations of ropivacaine observed in our study were higher than venous plasma concentrations, showing a large arteriovenous gradient described by other authors 17,20 . Arterial concentration is closer to that which can reach target organs such as the central nervous system (CNS) and heart, thus providing more accurate information compared to venous concentrations from a toxicological point of view 17,26 . In healthy volunteers the arterial concentrations of ropivacaine associated with neurological symptoms were 3.4-5.3 µg/ml 17 . There is a controversial debate about the plasma concentration of ropivacaine that are considered toxic. Local anesthetic toxicity is considered to be a multifactorial phenomenon, and CNS stimulation produces an initial cardiovascular activation, which is followed by an intense depression if local anesthetic doses are sufficiently high 22 . Different studies have evaluated the systemic concentrations of local anesthetics in abdominal fascial plane blocks. A recent review showed that, although a limited number of patients exceeded the plasma concentration considered toxic, clinical manifestations were minimal. However, most fascial plane regional blocks were performed with the patient under general anaesthesia, which precludes assessment of neurological symptoms 27 . There are limited studies that have evaluated the effects on cardiac electrophysiology of local anesthetics at the usual plasma concentrations associated with regional blocks 28 . The administration of 200 mg of ropivacaine (~ 2.66 mg/kg) in patients undergoing interscalene brachial plexus block was associated with peak venous concentrations of ~ 2.4 µg/ml which was not followed by any electrocardiographic changes on Holter monitoring until 6 h after the block. Low doses of ropivacaine administered in healthy volunteers until the onset of neurological intoxication symptoms were associated with minimal electrocardiographic alterations, mainly an increase in QRS duration. In this study, the arterial concentrations of ropivacaine that were associated with neurological symptoms were (3.4 to 5.3 µg/ml) 17 . Our results showed that these concentrations were reached by 2 of the animals tested. However, there were no electrophysiological changes associated with peak ropivacaine concentrations compared with baseline parameters (Table 3), except for a moderate increase in the QTc interval. The QTc interval prolongation observed was significant from minute 50 to the end of the study, with a maximum increase of 55 ms at 180 min. In addition to Na + channels, ropivacaine blocks K + channels, Ca + 2 channels and HERG channels, although the action on these channels seems to be related to very high concentrations of the drug, much higher than those obtained in our study 29 . Concerning interspecies differences, it should be noted that the evaluation of potential QT-prolonging drugs is performed in animal models, and the pig is one of the useful animal species for evaluating electrocardiograms in safety pharmacology studies 30 . In this regard, it is particularly relevant to highlight the effects of sevoflurane on QTc interval prolongation shown in other studies 31,32 . Our procedure, with a duration of approximately three hours, implies a prolonged exposure to sevoflurane that may justify the increase in the QTc interval observed. Intracardiac recordings and ventricular pacing performed in our experimental model have allowed us to assess electrophysiological events in detail, which cannot be acquired with conventional electrocardiographic monitoring. The assessment of the cardiotoxic effect of sodium channel blockade induced by local anesthetics is most sensitive when studying the effects on conduction after a short period of ventricular pacing at high rates 33 . Rate-dependent conduction slowing occurs when the sodium channel blockade developed in response to the local anesthetic has insufficient time to fully dissipate during diastole because of elevated heart rates. This unmasks a phenomenon that may be hidden in sinus rhythm by observing a large increase in QRS interval duration at high pacing rates 33 . In the previously mentioned study in healthy volunteers, transesophageal atrial stimulation at 100-120 bpm was associated with a significant (8.5%) increase in QRS width 17 . In contrast, plasma concentrations of ropivacaine after SIFPB did not affect ventricular conduction even at pacing rates of 150 bpm in our study. Moderate hypotension was observed from baseline, before SIFPB, to the final post-SIFPB period. This slight decrease in mean arterial pressure does not appear to be related to apparent ropivacaine toxicity, since ropivacaine levels in the final phases were the lowest recorded during the entire study. Table 4. Hemodynamic variables on the baseline, and 10, 30, 60, 90, 150 and 180 min after serratus intercostal fascial plane block. Data are shown as medians and interquartile range (IQR). SIFPB serratus intercostal fascial plane block, SAP systolic blood pressure, DAP diastolic blood pressure, MAP mean arterial pressure, dP/dt max peak first derivative of femoral artery pressure, SVRI systemic vascular resistance index. *Arterial ropivacaine concentrations. www.nature.com/scientificreports/ The popularity of interfascial peripheral plane blocks is related to their relative ease to perform and their hypothetically low risk profile. One of the strengths of our study was the use of ropivacaine as a local anesthetic, which according to recent studies is one of the most commonly used local anesthetics in fascial blocks 34 . Limitations. Animal models allow replication of controlled clinical conditions to improve our understanding of the pharmacological and toxicological effects associated with a given regional anesthesia technique. Pigs are physiologically and anatomically close to humans and share drug metabolic pathways quite similarly to humans. Therefore, the pig is considered a very suitable model and possibly even better than many other large mammals for pharmacological research and toxicology 35 . However, interspecies differences that limit the results obtained in our study cannot be excluded. On the other hand, both the peak plasma concentration and the time to reach this concentration are comparable to results described in patients undergoing chest wall blocks. However, although the plasma concentrations associated with this fascial block can be inferred from the information obtained in this study, we must be cautious in extrapolating the results to what would happen in humans. Another limitation is that there is no information from studies that have evaluated plasma concentrations of local anesthetics associated with SIFPB in humans that would allow us to make comparisons with our findings. Conclusions In conclusion, the plasma concentrations of ropivacaine associated with a SIFPB in our porcine model using the maximum recommended dose of 3 mg/kg, have reached potentially toxic concentrations, considering the classic neurological toxicity margin described for ropivacaine in humans. However, they have not been associated with adverse electrophysiological or hemodynamic effects. The absorption pattern of local anesthetics in the SIFPB is similar to that of the intercostal and paravertebral blocks, both of which are also chest wall blocks. Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.	2023-01-05T05:09:23.764Z	2023-01-02T00:00:00.000	{ "year": 2023, "sha1": "4f4e2a8cfaa695875fa04d09f9fdbc354cfb756d", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "4f4e2a8cfaa695875fa04d09f9fdbc354cfb756d", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
1954712	pes2o/s2orc	v3-fos-license	Handover practice amongst core surgical trainees at the Oxford School of Surgery Purpose: To date no studies have specifically evaluated the use of handovers amongst core surgical trainees (CSTs) in the United Kingdom. We examined handover practice at the Oxford School of Surgery to assess and improve CSTs’ perception of handover use as well as its quality, and ultimately patient care. Methods: Based on guidelines published by the British Medical Association and Royal College of Surgeons, a 5-point Likert style questionnaire that collected data on handover practice, its educational value, and the CSTs’satisfaction with handover was given to 50 CSTs in 2010. Results: Forty CSTs (80.0%) responded to the questionnaire. The most striking findings revolved around the perceived educational value, formal training, and auditing practice of handovers throughout various units, which were all remarkably lower than expected. As a result, handover practice amongst CSTs was targeted and revised at the University Hospital’s Department of Plastic Surgery, with the implementation of targeted changes to improve handover practice. Conclusion: The execution of daily handovers was an underused educational tool amongst surveyed CSTs and may be an important modality to target, particularly in the competency-based, time-limited training CSTs receive. We recommend modifications to current practice based on our results and the literature and encourage the assessment of handover practice at other institutions. INTRODUCTION The National Patient Safety Agency in the United Kingdom (UK) defines handover as "the transfer of professional responsibility and accountability for some or all aspects of care for a patient, or groups of patients, to another person or professional group on a temporary or permanent basis" [1]. Handover is a complex process that occurs in multiple settings, at various hours, and between and within multiple health and allied health professional groups; therefore, handover practice is a recognised issue in maintaining patient safety. Handover in surgical care is topical. After initiation of the European Working Time Directive (EWTD) and New Deal in the UK, junior doctors do not normally more than 48 hours per week [2]. One of the goals in reducing working hours amongst trainees is to reduce the rates of medical and surgical errors due to potential fatigue from excessive on-call commitments [3]. To facilitate this reduction in working hours, a transition has been made from an on-call system to a shift-based rota. Appropriate handover of patient responsibility and care between staff has continued to be an integral part of good medical care. However, with the introduction of shift-based systems, several different groups of healthcare professionals might now be responsible for patient care over a 24-hour period, thus resulting in several 'handovers' of care. Thus, the role of handovers has diversified to reflect the need for compliance with working time directives as well as focus on its primary role of maintaining continuous patient care by transferring responsibilities between staff as shifts change. The Royal College of Surgeons of England identifies formal and appropriate handover as a core component for the delivery of safe patient care [4]. To preserve patient safety during handover, the British Medical Association (BMA) [1] and the Royal College of Surgeons of England [5] published national guidance on the conduct of safe clinical handovers. Briefly, these guidelines state that safe clinical handovers should be led by a senior clinician encouraging interaction between team members as a focused, structured multidisciplinary session of adequate duration. Moreover, handovers should take place in a consistent location that prevents interruptions and should be timetabled into shiftbased rotas. These guidelines also suggest that local handover practice be regularly audited as an integral part of clinical governance strategies. Overall, handover is an important professional process where clinical teams meet, communicate, interact, provide mutual support, and enable learning [6]. In the era of the EWTD, effective handover can play a substantial role in maximizing educational perspective, whilst maintaining adequate service provision. Thus, assessment of handover practice use amongst current core surgical trainees (CSTs) in the UK would be beneficial to understanding and improving its role. The first aim of our survey was to determine the status of handover practice amongst CSTs (from core training levels 1 to 3) at the Oxford School of Surgery, and the second aim was to investigate their perception of the efficacy and regional educational value of the handover process and its influence on patient safety. METHODS This study was approved by the local audit department. A questionnaire-based survey was designed in line with the guidelines of the Royal College of Surgeons of England and BMA. The survey collected data on handover practice and policy as well as the perceptions of the CSTs regarding handover efficacy, patient safety, and its educational value in surgical training. The surveyed population included all CSTs at the Oxford School of Surgery (n= 50) who worked in any hospital or surgical specialty within the region between January and April 2010. CSTs were asked to complete the questionnaire in rela-tion to their surgical specialty during the survey period. The questionnaire (Appendix) was emailed a maximum of two times to non-responders. Information regarding timing, location, duration, data transfer methods, formal policy, attendance/participation, quality, and the educational value of local handover practice was collected. Each item was assessed using a 5-point Likert scale (5, strongly agree and 1, strongly disagree). Questionnaire data was divided into the following categories: (1) handover practice and patient safety, (2) policy and training, and (3) educational value. Participants did not receive an incentive, and all information was collected anonymously. Data was transferred, verified, and stored in a security-protected database. Following analysis of this data in March 2011, major changes were implemented to improve handover practice in the Department of Plastic Surgery at the John Radcliffe Hospital (Oxford, UK). RESULTS Forty questionnaires (80.0%) were fully completed by the CSTs. These trainees represented six surgical specialties (urology, general surgery, cardiothoracic surgery, plastic surgery, trauma, orthopaedic surgery, and neurosurgery) across seven hospitals. Status of handover practice All CSTs reported their units employed a shift-based on-call rota, as opposed to a 24-hour on-call system. CSTs reported that 5 units employed a 'Surgeon of the Week' system (i.e., oncall for 7 days or at least 4 days in a row). All shifts started between 07:00 and 08:30, and the majority ( > 80%) ended between 20:00 and 21:00. Only a few CSTs reported ending their shifts earlier than 20:00. A minimum of two handovers occurred per day, one in the morning (AM) and one in the evening (PM), and 22% of CSTs reported a third intra-day handover. Seventy seven percent of CSTs reported that their handover took place in a set location and lasted between 10 and 45 minutes. For AM and PM handovers only, 70% lasted 30 minutes or longer, and 74% of all CSTs deemed their handover duration to be adequate. For AM and PM handovers, 58% and 13% reported that the on-call consultant was in attendance at the handover, respectively. Moreover, 50% of CSTs reported that a designated health team member such as a consultant, a registrar, other CST, or senior nurse usually lead their handovers. Sixty-four percent confirmed that they had the opportunity to ask questions and clarify clinical issues with their colleagues and senior members of staff. Furthermore, 77% were satisfied with the quality of information received during the handover and 58% were satisfied with the degree of prioritisation of the information/clinical http://jeehp.org J Educ Eval Health Prof 2014, 11: 3 • http://dx.doi.org/10.3352/jeehp.2014.11.3 details conveyed during handover. All CSTs reported the use of computer-assisted printouts and patient lists during the handover. Twenty-two percent of trainees reported that their handovers were designated 'bleep free' (pager free), whereas 35% reported regular delays and interruptions during their handover sessions. AM handovers tended to be a multidisciplinary session attended by allied health staff (from the Nursing, Physiotherapy, Theatre, and Bed Management Departments) according to 50% of the CSTs, but only 28% reported that their PM handover was multidisciplinary (Fig. 1). Patient safety, maintenance, and educational value of handovers Approximately a third of trainees (35%) were aware of a critical incident in the preceding twelve months related to inadequate handover practice. However, a majority (61%) of all CSTs felt their participation in the handover ensured that patients were managed safely and effectively, and 67% were satisfied with their practice of handovers (Fig. 2). Nearly half of all CSTs (48%) were aware of a formal handover policy for their designated unit, whilst only 20% reported to have received any formal training on how to conduct an effective handover. Furthermore, less than one quarter of CSTs (22%) were aware of the national guidelines on handover practice. In addition, the majority (84%) reported that handover practice was not audited in their unit (Fig. 3). Approximately half of CSTs (48%) felt that the handovers they engaged in were of educational value to their surgical training, and 38% of them strongly felt that handovers were of no educational value. Only 25% and 19% indicated they routinely provided or received feedback on each other' s performance after performing a handover, respectively (Fig. 4). DISCUSSION This is the first regional UK survey aimed at CSTs of all levels and from all surgical specialties that has targeted their practice and perception of handover in line with national recommendations. We targeted a specific category of trainees at the start of their surgical careers in the EWTD era. Only a few reports in the literature have documented the opinions of surgical trainees surrounding handover practice in the UK. These reports surveyed general surgical trainees (Northern Ireland) [7], burns trainees (UK) [8], and ENT trainees from several units (UK) [9]. A common theme amongst these reports was the shortcomings of handover practice within each specialty. In our regional study, only half of CSTs reported that an oncall consultant attended their AM handover regularly, and a lower proportion was found for the PM handovers, with a similar number reporting consultant-led handovers. The BMA guidelines state that daily involvement by senior clinicians is essential during handovers to ensure that appropriate clinical decisions are made, to demonstrate that handovers are taken seriously and are a vital part of patient safety, and to contribute to junior doctor education [1]. Differences on the perceived value of handovers amongst senior consultants could be attributed to the lack of financial or hierarchical remuneration associated with handover attendance. For example, consultants whose contracts do not typi-cally reward attendance and teaching at handover may not feel obliged to do so, particularly if a senior registrar is leading the handover process. Nevertheless, consultant attendance is also important in fostering cohesion within a unit and a teambased approach to clinical duties, therefore ensuring the sustainability of an effective handover process [10]. Consultants serve not only as leaders within their units and individual firms but also as mentors for their trainees. Moreover, they act as supervisors ensuring the competency of their trainees' actions, assuming overall responsibility for patient care, and coaching the clinical, professional, and educational develop- Despite CSTs reporting favourably on the safety of their handover practices, 35% also reported being aware of a 'critical incident' that stemmed from inadequate handover practice within the preceding 12 months. A recent study on clinical incidents amongst surgical patients at a UK hospital found that 6% of more than 300 reported clinical incidents over 3 years were likely due to inadequate handover practice [11]. Poor, incomplete, or no handovers for these patients were identified as the causes of inadequate care in three quarters of these cases. However, the reported severity and impact of these incidents were considered low in 99% of these cases, and reporting was anonymous and open to all members of staff. Thus, the frequency of reporting amongst surgical or medical trainees for these incidents of inadequate care is unknown. As a result, more critical or severe incidents may have been omitted, and this could have resulted in a reporting bias. In our study, further interpretation of incident reporting was difficult as a precise definition of 'a critical incident' was not provided in the questionnaire; therefore, our collected responses were amenable to experiential bias. In addition, some CSTs were working in the same units; therefore, the same incident could have been reported multiple times, thus reducing the accuracy of this measure. The BMA guidelines [1] state that handovers should be "interruption free"; therefore, staff should refrain from interruptions due to telephone calls and bleeps/pages. However, most surveyed units in the UK do not suggest that handovers are interruption free [11][12][13]. In our survey, only 22% of CSTs reported a bleep-free environment at handover. Because patient safety is paramount, the on-call team must be contactable at all times in case of an emergency, which could explain why these results were apparent. Handover should be a multidisciplinary event, with involvement from all healthcare professionals including appropriate clinical and non-clinical staff [1]. However, only half of CSTs reported attending AM handovers that were multidisciplinary with allied health staff also in attendance, and an even smaller percentage reported a multidisciplinary PM session. CSTs were remarkably confident in the preservation of patient safety through their participation in handovers. Perhaps the team diligence amongst the CSTs and other staff to ensure patients were appropriately managed preserved patient safety and allowed patients to receive optimal care. CSTs could have been unaware of the standards that needed to be met in their practice and of the potential 'near misses' that could have been occurring in their units due to the lack of formal training administered at handover. Hence, these observations are noteworthy and deserve further study and audit, both regionally and nationally, to determine the nature of any reported adverse incidents and to determine units where practice could be improved. The majority of CSTs reported not receiving training on how to effectively perform a handover and were not aware of any formal guidelines for safe handover conduct. This finding reiterates recent observations from a large Scottish teaching hospital (composed of medical and surgical juniors, registrars, consultants, and nurse practitioners) that identified a vital need for the formal teaching of handover skills to junior doctors [12]. A key observation in our study was the high level of dissatisfaction amongst CSTs regarding the perceived educational value of handovers. Trainees valued handover highly in terms of potential learning, but highlighted some educational deficiencies. This is likely to stem from a number of factors. A lack of formal training in handover processes, the absence of auditing of practice or outcomes, and discrepancies in regular and sustained consultant attendance (and hence leadership) may collectively serve to explain their perception of handover as being of low educational value. In the EWTD era, where opportunities for training have become concentrated, time-crucial and competency-based, all potential learning opportunities that are available for trainees should be exploited. The Royal College of Surgeons of England highlights that effective and high-quality handovers can be an "excellent training and review opportunity" that must be "maximised in a climate of shortened hours and streamlined training. " Despite acknowledgements of the potential educational benefits of formal handover sessions, there is a limited body of literature on the proposed methods of handover implementation in a trainee education programme in the UK [6]. Our results suggest the need for the introduction of several key initiatives at both the deanery and unit level. Through our experience we trialled and implemented these initiatives in one of our units (Department of Plastic & Reconstructive Surgery, John Radcliffe Hospital) (Fig. 5). Five CSTs rotated through this unit following these implementations and completed the questionnaire-based survey. All five CSTs reported improved satisfaction regarding training in handover, handover policy, in addition to a greater educational value from handover, and greater engagement within handover (data not shown). Although these are preliminary results, this is an interesting area to explore in further depth. The Oxford School of Surgery has since introduced formal handover training to CSTs at induction (at the start of their core training). At the time of commencing their surgical training, all new cohorts of CSTs will therefore be taught these formal guidelines, hopefully execute safe handovers, and utilise them as an important educational tool. Based on our experiences, we believe it should be made a priority for doctors to receive training and education on how to conduct a safe handover. This training may be most useful if conducted at the start of core training and then augmented through training with local surgical unit protocols. Trainees should be taught the reasons why safe and effective handovers are of importance, with a review of the national guidance. This training can be achieved through simple role-play scenarios, which appear to be a popular method to impart and practice requisite skills. Dedicated and consistent leadership by senior consultants during handovers is also needed to ensure trainees are supported and foster the handover experience as a potential daily learning opportunity. Efforts should also be made to encourage a multidisciplinary approach to handover, integrating with allied specialties, and encouraging attendance by all those who are most closely involved in patient care. Logistically this may be difficult to arrange with the staggered arrangements of surgical and nursing handovers, but may perhaps only require attendance by only one chosen nurse who can voice important patient issues at handover, relaying pertinent information back to nursing colleagues. Lastly, effective handovers lead to optimal patient continuity of care; therefore, further investigations to examine patient outcomes in light of handover practice, identifying potential deficiencies, and guiding best practice are needed. This survey was limited by the small number of participants, albeit they represented 80% of the CSTs in the region, were from multiple hospitals, and represented six surgical specialties. We cannot rule out the potential for a reporting bias (where CSTs reported what they felt took place at handover as opposed to what actually took place), and selection bias (where we looked at CSTs only and not registrars or consultants. Nevertheless, this UK study targeting CSTs in the EWTD era highlighted that current handover practice is compliant with national guidelines; however, its educational value should be targeted, improved, and used as a tool to maximise educational opportunities among trainees.	2018-04-03T05:02:51.164Z	0001-01-01T00:00:00.000	{ "year": 2014, "sha1": "eb93fcb0dc54c5be6ce649d764fec91c4b0feadc", "oa_license": "CCBY", "oa_url": "https://doi.org/10.3352/jeehp.2014.11.3", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "eb93fcb0dc54c5be6ce649d764fec91c4b0feadc", "s2fieldsofstudy": [ "Education", "Medicine" ], "extfieldsofstudy": [] }
266774368	pes2o/s2orc	v3-fos-license	Dietary Epigenetic Modulators: Unravelling the Still-Controversial Benefits of miRNAs in Nutrition and Disease In the context of nutrient-driven epigenetic alterations, food-derived miRNAs can be absorbed into the circulatory system and organs of recipients, especially humans, and potentially contribute to modulating health and diseases. Evidence suggests that food uptake, by carrying exogenous miRNAs (xenomiRNAs), regulates the individual miRNA profile, modifying the redox homeostasis and inflammatory conditions underlying pathological processes, such as type 2 diabetes mellitus, insulin resistance, metabolic syndrome, and cancer. The capacity of diet to control miRNA levels and the comprehension of the unique characteristics of dietary miRNAs in terms of gene expression regulation show important perspectives as a strategy to control disease susceptibility via epigenetic modifications and refine the clinical outcomes. However, the absorption, stability, availability, and epigenetic roles of dietary miRNAs are intriguing and currently the subject of intense debate; additionally, there is restricted knowledge of their physiological and potential side effects. Within this framework, we provided up-to-date and comprehensive knowledge on dietary miRNAs’ potential, discussing the latest advances and controversial issues related to the role of miRNAs in human health and disease as modulators of chronic syndromes. Introduction Among lifestyle factors, diet displays a strong impact on human health.Over decades, unhealthy dietary habits have been responsible for the spread of many severe chronic degenerative diseases such as obesity, type 2 diabetes mellitus (T2DM), cardiovascular diseases, and cancer [1].Food intake may modify gene expression as well as disease susceptibility through the regulation of several epigenetic modulators [1].MicroRNAs (miRNAs) play critical roles in gene regulation and biological processes [2,3].They are transcribed by RNA polymerase II, leading to the generation of a hairpin structure called primary microRNA or pri-miRNA, which is further processed by the RNA endonucleases located in the nucleus (Drosha) and in the cytoplasm (Dicer) to generate a duplex of 22-25 nucleotides [2].This latter is then divided into two mature sequences associated with the Argonaute protein (AGO), resulting in the formation of the RNA-induced silencing complex (RISC), which can ultimately act as a translation repressor on target genes [3].Gene expression regulation at intracellular and extracellular levels is improved by mature transcript encapsulation into extracellular vesicles, such as exosomes, secreted by multiple cell types as mediators of cell-to-cell communication [4].Because of their biocompatibility and biostability, extracellular vesicles are considered regulatory cargos in a wide range of intercellular communication and crosstalk systems [5,6].MiRNAs have been described in body fluids as blood, serum, plasma, urine, saliva, and breast milk, and changes in their circulating levels are related to a plethora of different chronic syndromes, including obesity, cardiovascular and neurodegenerative diseases, T2DM, and cancer [7,8].Preserving the human miRNA profile could contribute to the prevention of diseases and the maintenance of good health.MiRNAs have been widely reported in plants, animals, and humans [9].Plant-derived miRNAs bind to recipient target transcripts with quite perfect complementarity, acting as small interfering RNAs (siRNAs), while animal miRNAs interact with the host mRNA targets with imperfect complementarity, thus inducing their translational repression [10,11].Given this imperfect complementarity, a single exogenous miRNA (xenomiRNA) is able to recognize multiple target sites and modulate different target genes in the host [10,11].After ingestion, xenomiRNAs could modulate the gene expression by a cross-kingdom pattern or horizontal transfer of genetic information to the host [10,11].Bioactive dietary compounds, by affecting directly and indirectly gene activity, have been related to epigenetic changes, such as DNA methylation, histone acetylation, and the modulation of miRNA expression in both physiological and pathological conditions [12,13].However, the clinical relevance of food-derived xenomiRNAs in human diseases is still undefined.Several studies have demonstrated that the intricate interplay between miRNAs and nutrients could regulate health and chronic diseases, hence pointing to food modification as a pivotal tool in different diseases [14,15].However, further studies to reveal the precise mode of action of dietary active compounds and how nutrients and bioactive molecules affect miRNA expression are still required.This review aims to provide an update on the role of dietary miRNAs in health and diseases by underlining the benefits, obstacles, and controversies of the relationship between food-derived miRNAs and the pathogenesis of chronic diseases. Dietary XenomiRNAs in Health and Disease XenomiRNAs represent a family of exogenous miRNAs characterized by several dietary sources, animals and vegetables, and are able to integrate into the total miRNA profile of a recipient [16].Once in the host, these small molecules can be absorbed by the gastrointestinal tract, packaged into vesicles, released into the bloodstream, and delivered to multiple cells and tissues, thus promoting healthy state or affecting the development of chronic diseases, including cancer [17][18][19].In the following sections, the role of xenomiR-NAs from both animals and vegetables as regulators of several chronic conditions will be extensively discussed (Figure 1). XenomiRNAs from Animal Sources The following subsection is dedicated to the most studied and characterized xenomiR-NAs derived from animal sources and their involvement in chronic syndromes and cancer. XenomiRNAs from Animal Sources The following subsection is dedicated to the most studied and characterized xenomiRNAs derived from animal sources and their involvement in chronic syndromes and cancer. Meat The effects of the chronic administration of cooked pork-derived exosomal vesicles were evaluated in mouse model [22].The upregulated miR-1, miR-133a-3p, miR-206 and miR-99a plasma levels resulted in glucose and insulin metabolism impairment, as well as in lipid droplet accrual in mice liver, supporting the role of pork-derived miRNAs in the development of metabolic disorders (Figure 1) [22].The bta-miR-154c has been recently characterized by comestible parts of beef.This xenomiRNA is able to contrast the human digestion processes being absorbed from Caco-2, SW480, and SW620 colorectal cancer cells [23].However, the comprehension of the specific role of miR-154c in colorectal carcinoma requires further investigation [23]. Milk The classification of milk as an "epi-nutrient", rich in bioactive compounds and functional molecules, as well as its involvement in counteracting chronic syndromes and cancer, have been recently investigated [24][25][26][27][28].A total of 678 miRNAs were identified in bovine milk-derived exosomal vesicles [29,30].These miRNAs are involved in a wide range of cell metabolic pathways, such as miR-181 and miR-155, related to the normal function and differentiation of T and B cells, miR-let-7c, miR-17, miR-92, miR-223 implied in the regulation of immunity and inflammatory cells, and miR-30a, a regulator of autophagy in post-acute myocardial infarction (Figure 2) [31][32][33]. Meat The effects of the chronic administration of cooked pork-derived exosomal vesicles were evaluated in mouse model [22].The upregulated miR-1, miR-133a-3p, miR-206 and miR-99a plasma levels resulted in glucose and insulin metabolism impairment, as well as in lipid droplet accrual in mice liver, supporting the role of pork-derived miRNAs in the development of metabolic disorders (Figure 1) [22].The bta-miR-154c has been recently characterized by comestible parts of beef.This xenomiRNA is able to contrast the human digestion processes being absorbed from Caco-2, SW480, and SW620 colorectal cancer cells [23].However, the comprehension of the specific role of miR-154c in colorectal carcinoma requires further investigation [23]. Milk The classification of milk as an "epi-nutrient", rich in bioactive compounds and functional molecules, as well as its involvement in counteracting chronic syndromes and cancer, have been recently investigated [24][25][26][27][28].A total of 678 miRNAs were identified in bovine milk-derived exosomal vesicles [29,30].These miRNAs are involved in a wide range of cell metabolic pathways, such as miR-181 and miR-155, related to the normal function and differentiation of T and B cells, miR-let-7c, miR-17, miR-92, miR-223 implied in the regulation of immunity and inflammatory cells, and miR-30a, a regulator of autophagy in post-acute myocardial infarction (Figure 2) [31][32][33].The most abundant miRNA in milk exosomes is miR-148a, able to modulate oral cavity homeostasis as well as to inhibit 5 ′ AMP-activated protein kinase (AMPK) and phosphatase and tensin homolog (PTEN), both suppressors of mammalian target of rapamycin complex 1 (mTORC1), a pivotal regulator of multiple cell metabolic pathways [31][32][33].Moreover, milk-derived miR-148a impairs insulin secretion with diabetogenic action through its ability to promote pancreatic β-cell de-differentiation via mTORC1-high/AMPK-low phenotype [33].It has been reported that miR-148a and miR-21 alter the α-synuclein homeostasis, causing its overexpression and aggregation, with toxic effects on dopaminergic neurons and pancreatic β-cells, thus exerting a possible role in the pathogenesis of Parkinson disease and T2DM [34].MiRNAs belonging to the miR-148 family affect the immune response, suppressing calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) and the subsequent toll-like receptor (TLR)-mediated expression of major histocompatibility complex II (MHC II) in dendritic cells [31].Among miRNAs derived from milk exosomes, miR-125a is involved in the modulation of immune response to bacterial and viral aggressions, while the human homolog miR-718, still involved in immune response regulating p53, also regulates vascular endothelial growth factor (VEGF) and insulin growth factor (IGF) pathways [35], and miR-146 exerts a regulatory function in TLR signaling and in the resolution of bacterial infections [36].As bovine milk, human breast milk-derived miRNAs play a crucial role in modulating development and differentiation of immune system cells and counteracting the onset of metabolic disorders [37,38].In the first six months of lactation, miR-181a and b, miR-155, miR-125b, and the cluster miR-17-92 actively regulate the T-and B-cell maturation and the tumor necrosis factor α (TNF-α) activation, modulating the immune response of the baby [38], while miR-22-3p counteracts the development of insulin resistance and the onset of T2DM, attenuating the Wnt pathway [37].Milk-derived miRNAs are also involved in different pivotal metabolic pathways.The presence in milk exosomes of miR-181a-5p has been related to anti-atherogenic effects and reduced vascular inflammation, due to its ability to downregulate nuclear factor kappaB (NF-κB) levels [39].Another miRNA characterized from milk exosomal vesicles is miR-29, capable of targeting IL-23, a cytokine involved in intestinal damage.To this aim, treatment with milk vesicles containing miR-29 stimulated intestinal stem cell proliferation and gut recovery under several pathological conditions [40], while incubation with miR-31-5p from milk-derived exosomes improved in vitro endothelial function and promoted angiogenesis and diabetic wound healing in vivo [41].Recent studies reported that oral administration of exosomal vesicles prevented colon shortening, intestinal epithelium disruption, infiltration of inflammatory cells, and tissue fibrosis in a mouse ulcerative colitis model via inhibition of the TLR4-NF-κB signaling pathway and nucleotide-binding oligomerization domain, the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation [30].Transfection with the milk-derived miR-22 promoted cell proliferation by inhibition of CCAAT/enhancer-binding protein δ (C/EBPδ) expression and promotion of intestinal development in human intestinal epithelial cells [42].On the other hand, the milk-exosomal miR-148a and miR-30b have been correlated to adipogenic effects, supporting a correlation between milk consumption and obesity incidence [43].In vitro and in vivo studies reported that milk exosome-derived miRNAs also exert oncogenic or oncosuppressor properties (Figure 2).An association has been reported between cow milk consumption and large B-cell lymphoma development, sustained by miR-148a-3p and miR-155-5p/miR-29b-5p increase via let-7-5p/miR-125b-5p [44].As oncomir, miR-21 promotes cell growth and anabolism, encouraging cell proliferation and cancer development by activating mTORC1 [45].MiR-21 and miR-155 have been related to the most advanced stages of breast cancer progression, being involved in the development of tamoxifen resistance, metastasis formation, and worst prognosis [46].Commercial milk consumption has been correlated to an increased risk of estrogen receptor-positive breast cancer development, due to the content of multiple oncogenic factors, as miR-148a-3p and miR-21-5p [47].Milk-derived miRNAs have been associated with prostate cancer tumorigenesis promotion.In vitro studies have shown that milk-derived miR-148 increased prostate cancer proliferation by inhibiting cyclin-dependent kinase inhibitor 1B (CDKN1B) and promoted DNA methyltransferase 1 (DNMT1)-dependent epithelial-mesenchymal transition (EMT) [45].Another significant milk-derived miRNA, miR-125b, increases the development of prostate xenograft cancer targeting proapoptotic genes, as p53, p53-upregulated modulator of apoptosis (PUMA), and BCL2 Antagonist/Killer 1 (BAK1) [48], and regulates several tumorigenic pathways, including NF-κB, p53, phosphatidyl inositol 3-kinase (PI3K)/protein kinase B (AKT)/mTORC1, erb-b2 receptor tyrosine kinase 2 (ERBB2), and Wnt [45].Epidemiological studies highlighted the association between milk consumption and reduced risk of colorec-tal cancer [49].The expression of miR-148a is downregulated in colorectal cancer (CRC), where it exerts a tumor-suppressor activity interfering in NF-κB and signal transducer and activator of transcription 3 (STAT3) pathways and modulating cancer related immune response via inhibition of the programmed death ligand-1 (PD-L1) levels [45].The in vitro antineoplastic effect of miR-148a-3p overexpression also occurs through mitochondrial impairment, lipid peroxidation, and ferroptosis sustained by the Acyl CoA synthetase long-chain family member 4 (ACSL4)/transferrin receptor (TFRC)/Ferritin axis and direct solute carrier family seven-member 11 (SLC7A11) downregulation in the CRC model [50].Another buffalo milk-derived miRNA, miR-27b, exerted antineoplastic effects on HCT116 and HT-29 CRC cells by inducing mitochondrial oxidative stress, lysosome accumulation, and apoptotic cell death mediated by endoplasmic reticulum (ER) stress [51]. XenomiRNAs from Vegetable Sources Several miRNAs characterized from different vegetables and involved in chronic diseases, including cancer, have been evaluated (Figure 1).It has been described the ability of miR-156a, contained in different vegetables, such as cabbage, spinach, and lettuce, to target junctional adhesion molecule A (JAM-A), thus suppressing the development of atherosclerosis in human aortic endothelial cells through the inhibition of monocyte adhesion, occurring under inflammatory stress [52].Similarly, the miR-167e-5p, characterized by Moringa oleifera, exerted a time-and dose-dependent anticancer action in Caco-2 cells acting on the β-catenin pathway [53].The miR-159, particularly abundant in broccoli, is able to suppress breast tumor development, both in vitro and in vivo models [54,55].In silico prediction identified two miRNA sequences, bra-miR156g-5p and Myseq-330, in broccoletti Brassica rapa sylvestris, as targeting apoptosis-related human genes, although further in vitro studies on pancreatic cancer cells did not support a miR-based modulating role in cancer growth [56]. Rice An extensive study on miRNAs derived from Oryza sativa rice revealed their binding affinity for multiple human genes involved in cardiovascular and neurological diseases and cancer [57].The Oryza sativa-derived osa-miR-172d-5p exerted in vitro beneficial effects in human lung fibroblasts, suppressing transforming growth factor (TGF)-β activated kinase 1 (MAP3K7) binding protein 1 (Tab1) and TGF-β-induced fibrotic gene expression in a bleomycin-induced lung fibrosis model [58].Analyses on mice and human serum after rice consumption revealed miR-168 circulating levels associated with reduced clearance of plasmatic low-density lipoprotein (LDL) [59,60], while the rice aleurone-derived hvu-miR-168-3p increased the glucose transporter 1 (GLUT1) expression and reduced blood glucose levels by specifically inhibiting the electron transport chain complex I in both in vitro and in vivo models [61]. Ginger The action of different xenomiRNAs from vegetable sources in the modulation of oncological pathways has been reported.A report on stomach tissues of patients with different gastric conditions, such as gastritis, metaplasia, and cancer, unveiled higher plant-derived miR-168 levels in tissues of patients affected by intestinal metaplasia [62].In particular, the ginger-derived miR-1078, by regulating leptin, is related to lipopolysaccharide (LPS)-induced interleukin (IL)-6 expression, whereas miR-167a acts as a gut microbiota modulator targeting Lactobacillus rhamnosus GG SpaC pilus adhesin [63,64].Among the different miRNAs contained in the ginger-derived exosomes-like nanoparticles (GELNs), mdo-miR-7267-3p increased the production of IL-22, thus inducing mice colitis [65], while the plant-derived miR-34a, miR-143, and miR-145 act as tumor suppressors in the mouse CRC model [66]. Food-Derived Nutrients as miRNA Regulators in Chronic Diseases Nutrients affect the miRNA profile through the direct or indirect modulation of gene expression.Multiple macro-and micronutrients, such as fatty acids, carbohydrates, vitamins and phytochemicals, are able to regulate miRNA levels [31], thus rendering food epigenetics more intriguing since several pathological patterns and their related pathways are still unravelled.Herein, we report the capability of many phytochemicals, belonging to the macrocategory of polyphenols (resveratrol, curcumin, quercetin, genistein, epigallocatechin gallate), and nonpolyphenols (fatty acids and vitamins), to affect miRNA expression and the involvement of these noncoding RNAs in chronic diseases and cancer (Figure 3). Metabolic Diseases Many phytochemicals are able to counteract the pathogenesis of metabolic conditions by acting as miRNA regulators (Figure 3).Resveratrol exerted beneficial action on agerelated alterations, as senile sarcopenia, by promoting peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α) expression and myocyte differentiation by miR-21 and miR-27b upregulation and a decrease of miR-133b, miR-30b, and miR-149 levels [83].In addition, the resveratrol-induced miR-21 upregulation alleviated cognitive impairment due to insulin resistance and diabetes in mice [84].The link between saturated fatty acids (SFA) and poor health outcomes and metabolic disorders [85] is already well established; additionally, it is reported that SFA effects on human health could also depend on their influence on miRNAs [86].Rat myoblast cells treated with palmitic acid (PA) developed insulin resistance and T2DM triggered by miR-29a increase [86].In addition, PA decreased insulin-induced activation of the PI3K-AKT pathway, enhancing the expression of miR-3180-3p and miR-4632-5p, thus favoring insulin resistance development in HepG2 cells [87].Stimulation with PA treatment induced mouse cardiomyocyte injuries derived from atrial arrhythmia by targeting miR-27b [88].High fat and hypercaloric diets can modulate the levels of miRNAs involved in lipid metabolism, cell homeostasis, and fibrogenesis.Evidence demonstrates that fat-rich dietary regimens are associated with downregulation of miR-122 and upregulation of miR-200a, miR-200b, and miR-429 in liver, determining the onset of nonalcoholic fatty liver disease (NAFLD) [89,90].Similarly, treatment with Moringa oleifera prevented liver damage and nonalcoholic steatohepatitis (NASH) progression via SIRT1 upregulation and miR-21a-5p, miR-103-3p, miR-122-5p, and miR-34a-5p downregulation [91].The role of vitamins in many metabolic processes, as well as in modulating the immune system and disease prevention, has been extensively reported [92].To this aim, recent reports revealed that treatment with the carotenoid astaxanthin has been able to promote miR-382-5p expression in hepatic stellate cells, thus opposing liver dysfunctional fibrosis [93].The potential of vitamin D3 and all trans retinoic acid formulations in the prevention of diabetic cardiovascular complications via miR-126 upregulation has been revealed in diabetic mice [94].Evidence described the ability of quercetin to ameliorate diabetic nephropathy damage by miR-485-5p upregulation and yes-associated protein 1 (YAP1) suppression in human mesangial cells [95] and to attenuate testosterone secretion dysfunction in diabetic rats by reducing ER stress through miR-1306-5p/hydroxysteroid 17-β dehydrogenase 7 (HSD17B7) axis modulation [96].The combined treatment of catechin epigallocatechin gallate (EGCG) and quercetin prevented insulin resistance by increasing the expression of miR-27a-3p and miR-96-5p, which directly target Forkhead Box O1 (FOXO1), reducing the production of glucose and the transcription of gluconeogenic enzymes [97].In addition, ECGC showed therapeutic potential in obesity inhibiting white and beige 3T3-L1 and D12 preadipocyte growth by upregulated miR-let-7a and subsequent high-mobility group AT-hook 2 (HMGA2) suppression [98] and inhibiting the MAPK7 pathway by increased miR-143 levels [99].Clinical data from a cohort of women affected by overweight and insulin resistance revealed that blood orange juice consumption (500 mL per day) for four weeks resulted in upregulated miR-144-3p, miR-424-5p, miR-144-3p, and miR-130b-3p and decreased let-7f-5p and miR-126-3p levels in peripheral blood mononuclear cells, leading to attenuated IL-6 and NF-κB mRNA expression [100]. Cancer Given the beneficial effects mediated by nutrients on miRNA profile, several studies dissected their use as an oncological approach in different neoplastic contexts (Figure 3). Digestive System Cancers Resveratrol exerted anti-inflammatory effects and attenuated colitis-induced tumorigenesis by increasing miR-101b and miR-455 levels [101].In colorectal cancer, resveratrol decreased the expression levels of characterized oncomiRs, as miR-17, miR-21, miR-25, and miR-92a-2 [102], and exerted a pivotal regulation on many tumor suppressors, such as PTEN and SMAD3, as well as on the oncogenic TGF-β pathway related to progression and metastasis of colorectal cancer by targeting the expression levels of miR-1 and miR-146b-5p [103].The resveratrol-related modulation of the tumor promoter TGF-β1 was further associated with upregulated miR-663 levels as reported in the SW480 cell line [104].Genistein exhibited in vitro and in vivo anticancer effect inhibiting miR-95 and its targets AKT and serum and glucocorticoid-regulated kinase 1 (SGK1) on HCT116 cells [105,106] and enhanced miR-1275 levels thus suppressing Eukaryotic Translation Initiation Factor 5A2 (EIF5A2)/PI3K/AKT pathway in hepatocellular carcinoma [107].In mice colorectal cancer tissues, the walnut-based diet PUFAs have been associated with reduced levels of miR-1903, miR-467c, and miR-3068, along with augmented miR-297a expression levels, resulting in anti-inflammatory, antiangiogenetic, antiproliferative and pro-apoptotic effects [108,109].In vivo studies showed that vitamin D suppressed colorectal cancer cell proliferation, downregulating histone demethylase 1A (JMJD1A) by increasing miR-627 levels [110,111].The EGCG treatment was able to suppress miR-483 levels via hypermethylation of its promoter region, modulating the expression of metastatic markers as E-cadherin and vimentin in a mouse model of hepatocellular carcinoma [112], while resveratrol opposed the tumor progression via downregulation of miR-155-5p in gastric cancer cells [113]. Hormone-Dependent Cancers Resveratrol opposed the overexpressed levels of miR-17, miR-18b, miR-20a, miR-20b, miR-92b, miR-106a, and miR-106b in prostate cancer [114,115], whilst genistein was able to restore in vivo and in vitro miR-574-3p expression levels, generally downregulated in prostate cancer cells [116,117].Treatment with vitamin D positively regulated the levels of miR-100 and miR-125b in primary prostate cancer tissues, opposing tumor progression [118,119], while in breast cancer T47D and SK-BR-3, cell line treatment with retinoic acid strongly increased miR-10a expression and retinoic acid receptor β (RARβ), whose loss is associated with breast carcinogenesis [120].Vitamin D was also shown to decrease breast cancer cell capacity to elude natural killer lymphocyte attacks through the reduction of miR-302c and miR-520c expression [121,122].The effect of curcumin treatment on miR-21 was effective in inhibiting the growth of MCF-7 breast cancer cells [123].Curcumin treatment also positively regulated miR-181b, miR-34a, miR-16, miR-15a, and miR-146b-5p and down-regulated miR-19a, and miR-19b expression in estrogen receptor-positive primary cells and several breast cancer cell lines with effects on inflammatory cascade, cell cycle progression, survival, and invasiveness [124,125].The curcumin-dependent miR-146b-5p upregulation suppressed the transactivation of the breast stromal fibroblasts, responsible for the epithelial-to-mesenchymal transition in breast cancer stromal fibroblasts [126].In vitro studies proved that EGCG counteracted breast cancer progression, attenuating the expression of miR-25 [127], while genistein reduced the expression levels of onco-miR-155, the regulator of several tumor suppressors as PTEN and p27, and impaired cell mobility via p21 activated kinase 2 (PAK2) and miR-23b upregulation in breast cancer cells [128,129].Resveratrol modulated the oncosuppressor ARH-I expression and limited the "awakening" of ovarian cancer cells, reducing the oncomiR-1305 [130]. Respiratory Tract Cancers In lung cancer cells, curcumin treatment was able to inhibit cell invasion via upregulation of miR-874, which directly targets matrix metalloproteinase-2 (MMP-2), and miR-98, which suppresses MMP-2 and MMP-9 pathways by targeting LIN28A [131].In human non-small cell lung carcinoma (NSCLC) A549 cell line, curcumin-induced apoptosis and reduced migration and invasion through miR-206 upregulation and suppression of the PI3K/AKT/mTOR signaling pathway [132].In addition, treatment with curcumin suppressed tumor progression by increasing miR-192-5p expression and c-Myc reduction in NSCLC cells A427 and A549 [133].EGCG repressed c-myc expression via upregulation of tumor suppressors miR-let-7a-1 and miR-let-7g in lung cancer cells [134,135].Quercetin displayed anti-cancer properties by opposing cell survival and enhancing apoptosis in NSCLC cells by increased miR-34a-5p and downregulation of a long noncoding RNA small nucleolar RNA host gene 7 (SNHG7) [136], while in oral squamous cell carcinoma Cal-27 cells quercetin inhibited cell proliferation and metastasis via accumulation of miR-1254 and subsequent downregulation of CD36 [137], and suppressed proliferation and MMP-9 and -2 levels via a miR-16/homeobox A10 (HOXA10) axis increase [138].In vitro treatment with quercetin promoted apoptosis and repressed metastatic feature in esophageal cancer cells by upregulating miR-1-3p and suppression of Transgelin 2 (TAGNL2) expression [139], while an increase in genistein-induced mir-34a led to production of reactive oxygen species (ROS) and apoptosis in head and neck cancer [140]. Latest Dietary miRNA-Based Animal Models and Clinical Trials Most of the evidence on the effects of food-borne bioactive compounds on the regulation of human miRNAs in various diseases comes from preliminary in vitro studies, recently validated in animal models.The antidiabetic effects of quercetin and ECGC were proved in mice fed with an enriched-polyphenol diet (0.05% w/w) ad libitum for 10 weeks [97].Results revealed the capacity of quercetin and EGCG enrichment to increase miR-27a-3p and miR-96-5p and inhibit gluconeogenesis and FOXO1 pathways, with enhanced effects when combined (Table 1) [97].BALB/c mice overexpressing or not miR-483-3p were treated for two weeks with ad libitum 0%, 0.1%, or 0.5% EGCG solution, then HepG2 cells were inoculated, and the diet prolonged for two months [112].Supplementation with EGCG reduced hepatocarcinoma lung metastasis and opposed EMT via miR-483-3p inhibition (Table 1) [112].Although few clinical trials evaluating the relationship between food-borne miRNAs and human health have been conducted, the number of clinical trials is continuously increasing (www.clinicaltrials.gov)(Table 1).New insights into alterations of skeletal muscle miRNA expression when consuming 1 g/kg/h carbohydrate during the first 3 h of recovery from aerobic exercise (NCT03250234) have been recently described [146].Authors reported that carbohydrate consumption during an 80 min bout of steady-state treadmill exercise led to increased expression of miR-19b-3p, miR-99a-5p, miR-100-5p, miR-222-3p, miR-324-3p, and miR486-5p immediately following and/or within 3 h from recovery compared with a nutrient-free control, thus resulting in downregulation of breakdown protein gene expression and better muscle recovery (Table 1) [146]. Results from the RESMENA study (NCT01087086) evaluated the effect of the Mediterranean dietary pattern on miRNA levels in white blood cells of 40 patients with metabolic syndrome.Data showed that 8-week hypocaloric diet (30% caloric deficiency) based on Mediterranean diet reduced the expression of miR-155, miR-125, miR-130, miR-132, and miR-422, associated with cancer, atherogenic and adipogenic processes, and other inflammatory conditions (Table 1) [147].The effects of 60 months with a typical Mediterranean regimen (Med diet) versus a low-fat high complex carbohydrate (LFHCC) diet were correlated to T2DM development and differences in miRNA plasma levels in COR-DIOPREV study (NCT00924937) [148].Following LFHCC consumption, patients with low plasma miR-145 levels showed a higher risk of developing T2DM, as well as the subjects assuming a Med diet with low levels of miR-29a, miR-28-3p, and miR-126 and high miR-150 expression (Table 1) [148].Current therapeutic options for the treatment of antiphospholipid syndrome showed the efficacy of daily ubiquinol treatment (200 mg for 1 month) in the regulation of a profile of monocyte miRNAs and identified novel and specific miRNA-mRNA regulatory networks associated with atherothrombosis development (NCT02218476) [149].A completed clinical trial (NCT01634841) provided a systematic investigation of the role of walnuts in preventing or slowing age-related cognitive decline and macular degeneration [150].Authors screened miRNA profiles in serum samples (before and after intervention) of eight randomly selected participants from the walnut arm, showing 53 miRNAs modulated after one year assuming 15% of daily energy as walnuts, 36 of them being upregulated and 17 downregulated [150].Compared with participants in the control diet, walnuts consumed for 1 year significantly increased the serum concentration of miR-551 related to the inhibited progression of several cancers [150].The antineoplastic effects of vitamin D were evaluated in prostate cancer [118].A cohort of prostate cancer patients was treated with three different vitamin D3 doses (400, 10,000 or 40,000 IU/day) in the time between randomization and prostatectomy (approximately 3-8 weeks).Vitamin D3 assumption determined an increase in tumor suppressors miR-100 and miR-125b in both pathological and healthy prostatic tissue [118].The HYPODD study correlated the vitamin D supplementation to miR-21 circulating levels in the pathogenesis of cardiometabolic disorders [151].Hypovitaminosis D correction (50,000 UI/week for 8 weeks and then 50,000 UI/month for 10 months) ameliorated the cardiovascular risk profiles in hypertensive patients without affecting the miR-21 circulating expression (Table 1) [151].The effects of 4-week time-restricted eating (TRE), a popular form of intermittent fasting, determined downregulation of a miRNA panel (miR-4649-5p, miR-2467-3p, miR-543, miR-301a-3p, miR-3132, miR-19a-5p, miR-495-3p, and miR-4761-3p), which, in turn, could inhibit cell growth pathways while activating cell survival and promoting healthy aging (NCT03590847) [152].A study conducted on 125 subjects with hypercholesterolemia (ACTRN12619000170123) evaluated the effect of daily assumption of a nutraceutical combination containing 400 mg phytosterols, 100 mg bergamot extract, 20 mg olive extract, and 52 µM vitamin K2 up to 12 weeks of treatment [153].The evaluated nutraceutical combination did not change the serum lipid profile and inflammation-related miRNAs and biomarkers [153]. Questions Opening on the Potential Impact of Dietary miRNAs on Health and Disease Increasing evidence highlight the potential of dietary miRNAs to modulate human pathophysiology, suggesting new dietary-based intervention approaches [154].However, a preventive strategy based on xenomiRNAs denotes multiple limitations, due to their poor bioavailability, the restricted knowledge of the potential side effects related to high consumption, and the appropriate dietary intake according to their stability due to manufacturing processes and food storage [155].The bioavailability of food-targeted miRNAs after ingestion is still a controversial issue.In vitro and in vivo evidences reported upregulated miR-29b, miR-200c, miR-21-5p, and miR-30a-5p plasma levels after milk consumption, supporting the availability of these molecules [17,156].On the other hand, given the base sequence complementarity between miR-21-5p and miR-30a-5p in humans and animals, the precise assessment of xenomiRNA uptaken by diet is quite uncertain [157].Moreover, the increase in miRNA plasmatic concentration could be ascribed to endogenous responses to other milk-derived compounds [158].Analyses by real time quantitative PCR (RT-qPCR) and RNA-sequencing on human blood did not provide supporting evidences about the absorption of specific bovine miRNAs after milk consumption [159], as well as results on milk-fed mice highlighted the absence of miRNA absorption and revealed their rapid degradation in intestinal fluids [160].Analyses by RT-qPCR performed on post-prandial plasma of pigtail macaques reported absent or low-levels of nonspecific amplified miRNAs, thus supporting the scarce intestinal absorption of plant xenomiRNAs [161].Likewise, the plant-derived miR-168 has been detected in feces and gastrointestinal mucosa while being undetectable in blood, confirming that the potential availability of gastrointestinal tract is not accompanied by systemic absorption [162].On the other hand, the detection of the plant miR-172 in gastrointestinal tract, serum and feces of mice fed with plant RNA extracts, confirmed the possibility of a dietary-derived miRNA amount absorbed by intestinal tract [163].Growing evidence reported xenomiRNAs as food contaminants, describing some plant-derived miRNAs as a consequence of experimental contamination and artifacts [19,164].In addition, the biological and functional role of dietary miRNAs from different sources in human health is still controversial.Different milk-derived miRNAs have been reported to modulate oncogenic and adipogenic pathways, promoting the development of B-cell lymphoma, hormone-dependent breast and prostate cancer [43][44][45]47], as well as the ginger-related miRNA has been associated with colitis development [64] and the pork-derived miRNAs to the development of metabolic disorders in mice [22].The evaluation of food-derived xenomiRNA effects cannot refrain from considering the co-existence of a great variety of dietary bioactive compounds.In the absence of supplementary intake of dietary exogenous miRNAs, many phytochemicals or animal-based food derivatives may modify the risk of diseases, targeting the reported biological effects [29,165].Indeed, several food-derived compounds exert their regulatory effects on different diseases either by upregulating or downregulating different signaling pathways, as well as acting on many epigenetic patterns [166].Dietary constituents are able to affect epigenetic mechanisms, including DNA status modulation, histone methylation, and acetylation, by aiming key epigenetic modulators such as DNA methyltransferases and histone deacetylases, thus resulting in local or global changes in epigenetics and subsequent gene transcription and expression levels [29,165,166].Attractive strategies from a better knowledge of nutrimiromics are undeniable [167], although further studies are needed to determine the pharmacokinetics and the possible side effects of dietary miRNA-based intervention in human health.Based on current evidence here summarized, it has to take into account both the promising results of food-derived miRNAs and negative effects exerted by the dietary molecules on human health, thus their dual role in chronic diseases still represents an open door in nutrition-based strategies. Conclusions Herein, we provided an up-to-date comprehensive review of food-derived miRNA activities in human health and disease, reporting both beneficial and controversial issues of dietary miRNAs as regulators of chronic conditions.Recent reports aim to elucidate the association between altered miRNA expression and pathological processes contributing to the onset of several chronic diseases.The crucial roles of different miRNAs in the development of T2DM, insulin resistance and obesity [168][169][170][171] have been described, as well as in the progression of other chronic syndromes, such as asthma, allergy, and chronic kidney disease (CKD) [172][173][174][175][176].This evidence points out nutrient-regulated miRNAs and food-derived miRNAs as intriguing players in food-related health and disease, although the knowledge on miRNA specific role is partially known and still debatable, requiring future studies to broaden knowledge on possible molecular targets and their modulation.In this regard, lifestyle habits, affecting the expression levels of miRNAs, represent a crucial element in the perspective of preventive and personalized medicine.A healthy diet with a personalized choice of nutrients based on individual needs related to age and state of health, i.e., ranging from health to disease or disability/rehabilitative conditions, is essential for maintaining the state of health and to slow down the aging clock.To this end, dietary miRNAs could represent intervention tools in precision nutrition, although with several limitations to overcome, including bioinstability, poor availability, unknown side effects, and multiple molecular targets.Within this framework, future preclinical and clinical studies will benefit from multidisciplinary and translational design taking into consideration some host-related aspects such as microbiome, circadian rhythm and self-susceptibility, and miRNA-derived limitations, as scarce bioavailability and undefined content in body fluids, which undoubtedly add complexity in dissecting the still controversial role of dietary miRNAs in human health and diseases. Figure 2 . Figure 2. Milk-derived miRNAs and human health.Identified in exosomal vesicles, miRNAs contained in milk are involved in a wide range of metabolic pathways, as the normal function and differentiation of T and B cells, homeostasis of oral cavity, pathogenesis of Parkinson disease, antiatherogenic action, promotion of angiogenesis, and breast and prostate cancer.MiRNAs contained in milk are also able to counteract diabetes and colorectal cancer development, as well as regulate autophagy in post-acute myocardial infarction.The green arrows stand for promoting activity, while the red ones indicate an opposing role. Table 1 . Dose effect sizes, in vivo models, target miRNAs, and biological relevance to foods, constituents, and dietary regimen or supplementation.	2024-01-06T16:17:42.765Z	2024-01-01T00:00:00.000	{ "year": 2024, "sha1": "fc5dc9632afccf32d8d195060222a5356fbb09de", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2072-6643/16/1/160/pdf?version=1704272942", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a6307345cd82366769c359f55fea1f100a439760", "s2fieldsofstudy": [ "Medicine", "Biology", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
14033051	pes2o/s2orc	v3-fos-license	The Mayer-Vietoris principle for Grothendieck-Witt groups of schemes We prove localization and Zariski-Mayer-Vietoris for higher Grothendieck-Witt groups, alias hermitian $K$-groups, of schemes admitting an ample family of line-bundles. No assumption on the characteristic is needed, and our schemes can be singular. Along the way, we prove additivity, fibration and approximation theorems for the hermitian $K$-theory of exact categories with weak equivalences and duality. Introduction A classical invariant of a scheme X is its Grothendieck-Witt group GW 0 (X) of symmetric bilinear spaces over X. According to Knebusch [Kne77,§4], this is the abelian group generated by isometry classes [V, ϕ] of vector bundles V over [Mor04] and are to oriented Chow groups what algebraic Ktheory is to ordinary Chow groups, see [BM00], [FS07], [Hor08]. Using a hermitian version of Quillen's Q-construction, we have defined in [Sch08] the higher Grothendieck-Witt groups GW i (X), i ∈ N, of a scheme X, generalizing the group GW 0 (X). The purpose of this article is to prove the following Mayer-Vietoris principle for open covers. 1.1. Theorem. Let X = U ∪ V be a scheme with an ample family of line-bundles (e.g., quasi-projective over an affine scheme, or regular separated noetherian) which is covered by two open quasi-compact subschemes U, V ⊂ X. Then there is a long exact sequence, i ∈ Z, This is a special case of our theorem 10.13 which also includes versions of theorem 1.1 for skew-symmetric forms, for forms with coefficients in line-bundles other than O X and for certain non-commutative schemes. Note that we don't need the common assumption 1 2 ∈ Γ(X, O X ), and X can be singular! Theorem 1.1 is a consequence of two theorems, "Localization" and "Zariskiexcision". To explain the implication, let X be a scheme, L a line bundle on X, n ∈ Z an integer, and Z ⊂ X a closed subscheme with open complement U . With this set of data, we associate in definition 8.2 a topological space GW n (X on Z, L) which, for Z = X, n = 0 and L = O X , yields the Grothendieck-Witt space GW (X) introduced in [Sch08] whose homotopy groups are the higher Grothendieck-Witt groups GW i (X) in theorem 1.1, see corollary 8.5. The space GW n (X on Z, L) is the Grothendieck-Witt space (as defined in 2.11) of an exact category with weak equivalences and duality, namely, the exact category of bounded chain complexes of vector bundles on X which are (cohomologically) supported in Z, equipped with the set of quasi-isomorphisms as weak equivalences and duality E → Hom(E, L[n]), where L[n] denotes the complex which is L in degree −n. If Z = X, we write GW n (X, L) for GW n (X on Z, L). The non-negative part of theorem 1.1 is a consequence of the following two theorems (proved in theorems 9.2 and 9.3). They are extended to negative Grothendieck-Witt groups in §10 (theorems 10.11 and 10.12). 2.1. Categories with duality, C h and form functors. A category with duality is a triple (C, * , η) with C a category, * : C op → C a functor, η : 1 → * * a natural transformation, called double dual identification, such that 1 A * = η * A • η A * for all objects A in C. If η is a natural isomorphism, we say that the duality is strong. In case η is the identity (in which case * * = id), we call the duality strict. A symmetric form in a category with duality (C, * , η) is a pair (X, ϕ) where ϕ : X → X * is a morphism in C satisfying ϕ * η X = ϕ. A map of symmetric forms (X, ϕ) → (Y, ψ) is a map f : X → Y in C such that ϕ = f * • ψ • f . Composition of such maps is composition in C. For a category with duality (C, * , η), we denote by C h the category of symmetric forms in C. It has objects the symmetric forms in C and maps the maps between symmetric forms. A form functor from a category with duality (A, * , α) to another such category (B, * , β) is a pair (F, ϕ) with F : A → B a functor and ϕ : F * → * F a natural transformation, called duality compatibility morphism, such that ϕ * A β F A = ϕ A * F (α A ) for every object A of A. There is an evident definition of composition of form functors, see [Sch08,3.2]. The category Fun(A, B) of functors A → B is a category with duality, where the dual F ♯ of a functor F is * F * , and double dual identification To give a form functor (F, ϕ) is the same as to give a symmetric form (F,φ) in the category with duality Fun(A, B) in view of the formulas ϕ A = F (α A ) * •φ A * and ϕ A = ϕ A * • F (α A ). A natural transformation (F, ϕ) → (G, ψ) of form functors is a map (F,φ) → (G,ψ) of symmetric forms in Fun(A, B). A duality preserving functor between categories with duality (A, * , α) and (B, * , β) is a functor F : A → B which commutes with dualities and double dual identifications, that is, we have F * = * F and F (α) = β F . In this case, (F, id) is a form functor. We will consider duality preserving functors F as form functors (F, id). Note that our use of the phrase "duality preserving functor" may differ from its use by other authors! Recall that an exact category is an additive category E equipped with a family of sequences of maps in E, called conflations (or admissible short exact sequences, or simply exact sequences), satisfying a list of axioms, see [Qui73], [Kel96,§4], [Sch08, 2.1]. The map i in an exact sequence is called inflation (or admissible monomorphism) and may be depicted as , and the map p is called deflation (or admissible epimorphism) and may be depicted as ։ in diagrams. Unless otherwise stated, all exact categories in this article will be (essentially) small. 2.2. Exact categories with weak equivalences. An exact category with weak equivalences is a pair (E, w) with E an exact category and w ⊂ Mor E a set of morphisms, called weak equivalences, which contains all identity morphisms, is closed under isomorphisms, retracts, push-outs along inflations, pull-backs along deflations, composition and the 2 out of three property for composition (if 2 of the 3 maps among a, b, ab are in w then so is the third). A weak equivalence is usually depicted as ∼ → in diagrams. A functor F : A → B between exact categories with weak equivalences (A, w) and (B, w) is called exact if it sends conflations to conflations and weak equivalences to weak equivalences. For an exact category with weak equivalences (E, w), we will write wE for the subcategory of weak equivalences in E. Its objects are the objects of E and its maps the maps in w. Also, we will regard an exact category E (without specifying weak equivalences) as an exact category with weak equivalences (E, i) where i is the set of isomorphisms in E. 2.3. Exact categories with weak equivalences and duality. An exact category with weak equivalences and duality is a quadruple (E, w, * , η) with (E, w) an exact category with weak equivalences and (E, * , η) a category with duality such that * : (E op , w) → (E, w) is an exact functor (in particular, * (w) ⊂ w) and η : id → * * is a natural weak equivalence, that is, η X ∈ w for all objects X in E. We may simply say E or (E, w) is an exact category with weak equivalences and duality if the remaining data are understood. Note that if E is an exact category with weak equivalences and duality, the category wE of weak equivalences in E is a category with duality. An exact category with duality is an exact category with weak equivalences and duality where the set of weak equivalences is the set of isomorphisms. In particular, the double dual identification has to be a natural isomorphism. 2.5. Remark. If in definition 2.4, the set of weak equivalences is the set of isomorphisms, then we recover the classical Grothendieck-Witt group of an exact category with duality, see for instance [Sch08,2.9]. In this case, relation (c) says that the class [E 0 , ϕ 0 ] of a metabolic space (E 0 , ϕ 0 ) with Lagrangian i : E −1 E 0 is equivalent in the Grothendieck-Witt group to the class of the hyperbolic space H(E −1 ) of the Lagrangian E −1 . In particular, if E is the category Vect(X) of vector bundles on X, * is the duality functor E → Hom(E, O X ) and η is the usual canonical double dual identification, the group GW 0 (E, i, * , η) is Knebusch's Grothendieck-Witt group GW 0 (X) of a scheme X, denoted L(X) in [Kne77]. The hermitian S • -construction of [SY96], [HS04,1.5], which gives rise to the Grothendieck-Witt space to be defined in 2.11, is the edgewise subdivision of Waldhausen's S • -construction [Wal85]. We review the relevant definitions and start with the edgewise subdivision of a simplicial object, see [Wal85,1.9 Appendix], [Seg73, Appendix 1]. It is (uniquely) isomorphic to [2n + 1]. The assignment T : [n] → n defines a functor ∆ → ∆ where a map θ : [n] → [m] goes to the map T (θ) : n → m : p → θ(p), p ′ → θ(p) ′ . For a simplicial object X • , the edge-wise subdivision X e • of X • is the simplicial object X • • T . The inclusion [n] ֒→ n : i → i defines a map X e → X of simplicial objects. It is known [Seg73, Appendix 1] that for a simplicial set X • , the topological realization of X • and of its edge-wise subdivision X e • are homeomorphic. We need the following (well-known) variant. 2.8. Lemma. For any simplicial set X • , the map X e • → X • is a homotopy equivalence. Proof. Let X • be a simplicial set. For a small category C, write X C for the set Hom(N * C, X • ) of simplicial maps from the nerve N * C of C to X • . Note that X e is the simplicial set [n] → X n . We define bisimplicial sets X e •• and X •• by the formulas X e m,n = X m× [n] and X m,n = X [m]× [n] . Consider the following diagram of bisimplicial sets in which the horizontal maps are the canonical inclusions of horizontally respectively vertically constant bisimplicial sets, and the vertical maps are induced by the inclusions [m] ⊂ m. Once we show that all arrows labeled ∼ → are homotopy equivalences, we are done, because the right vertical map can be identified with X e • → X • . The left vertical map X e 0• → X 0• is a homotopy equivalence since it can be identified with the map X I • → X • which is evaluation at 0, where I = N * 0 ∼ = N * [1] is the standard simplicial interval. In order to see that the upper right horizontal map X e •0 → X e •• is a homotopy equivalence, it suffices to prove that for every n, the map X e •0 → X e •n is a homotopy equivalence of simplicial sets. Since the map [0] → [n] : 0 → 0 induces a retraction X e •n → X e •0 , we have to show that the composition X e •n → X e •0 → X e •n is homotopic to the identity. The (unique) natural transformation from the constant functor for which A p,p = 0 and A p,q A p,r ։ A q,r is a conflation whenever p ≤ q ≤ r, p, q, r ∈ [n]. The category S n E is an exact category with weak equivalences where a sequence A → B → C of functors Ar[n] → E in S n E is exact if A p,q B p,q ։ C p,q is exact in E, and a map A → B of functors in S n E is a weak equivalence if A p,q → B p,q is a weak equivalence in E for all p ≤ q ∈ [n]. The cosimplicial category n → Ar[n] makes the assignment n → S n E into a simplicial exact category with weak equivalences. According to [Wal85], [TT90], the K-theory space K(E, w) of an exact category with weak equivalences (E, w) is the space K(E, w) = Ω\|wS • E\|. is a simplicial exact category with weak equivalences and duality; the simplicial structure maps being duality preserving. Considering S e n E as a full subcategory of Fun(Ar(n), E), the dual A * of an object A : 2.11. Definition. Let (E, w, * , η) be an exact category with weak equivalences and duality. By 2.10, the assignment n → S e n E defines a simplicial exact category S e • E with weak equivalences and duality. The subcategories of weak equivalences define a simplicial category with duality n → wS e n E. Taking associated categories of symmetric forms (see 2.1), we obtain a simplicial category (wS e • E) h . The composition (wS e • E) h → wS e • E → wS • E of simplicial categories, in which the first arrow is the forgetful functor (X, ϕ) → X, and the second is the canonical map X e • → X • of simplicial objects (see 2.7), yields a map of classifying spaces (2) \|(wS e • E) h \| → \|wS • E\| whose homotopy fibre (with respect to a zero object of E as base point of wS • E) is defined to be the Grothendieck-Witt space of (E, w, * , η). If ( * , η) are understood, we may simply write GW (E, w) instead of GW (E, w, * , η). We define the higher Grothendieck-Witt groups of (E, w, * , η) as the homotopy groups and show in proposition 3.8 below that π 0 GW (E, w, * , η) ∼ = GW 0 (E, w, * , η), so that our definition here extends that in 2.4. 2.12. Remark. Orthogonal sum makes the spaces (wS e • E) h and GW (E, w, * , η) into commutative H-spaces. Since the commutative monoid of connected components of these spaces are groups (see proposition 3.8 and remark 3.9 below), both spaces are actually commutative H-groups. 2.13. Functoriality. A non-singular exact form functor (F, ϕ) : (A, w, * , η) → (B, w, * , η) between exact categories with weak equivalences and duality induces maps of simplicial categories compatible with composition of form functors. Taking homotopy fibres of (wS e • ) h → wS • , we obtain an induced map GW (F, ϕ) : GW (A, w, * , η) → GW (B, w, * , η) of associated Grothendieck-Witt spaces. For the next lemma, recall that a natural transformation of form functors (F, ϕ) → (G, ψ) is a map of associated symmetric forms in Fun(A, B). It is a natural weak equivalence if F A → GA is a weak equivalence for all objects A of A. Proof. The natural weak equivalence (F, ϕ) ∼ → (G, ψ) induces natural transformations of functors (wS e n A) h → (wS e n B) h and wS n A → wS n B. These natural transformations define functors [1] × (wS e n A) h → (wS e n B) h and [1] × wS n A → wS n B whose restrictions to 0, 1 ∈ [1] are the two given functors. They are compatible with the simplicial structure and induce, after topological realization, the homotopy between GW (F, ϕ) and GW (G, ψ). Next, we will associate to every exact category with weak equivalences (E, w) a category with weak equivalences and duality (HE, w) such that the Grothendieck-Witt space of (HE, w) is equivalent to the K-theory space of (E, w). In this sense, Grothendieck-Witt theory is a generalization of algebraic K-theory. 2.15. Hyperbolic categories. Let C be a category. Its hyperbolic category is the category with strict duality HC = (C × C op , * ) where (X, Y ) * = (Y, X). For any category with duality A there is a functor A h → A : (X, ϕ) → X that "forgets the forms". We define the functor (HC) h → C as the composition of the functor (HC) h → HC and the projection HC = C × C op → C onto the first factor. 2.16. Lemma. For any small category C, the functor (HC) h → C is a homotopy equivalence. Proof. The category (HC) h of symmetric forms in HC is isomorphic to the category whose objects are maps f : X → Y in C and where a map from f to f ′ : Composition is composition in C of the a's and b's. The functor (HC) h → C in the lemma sends the object f : X → Y to X and the map (a, b) to a. Write F for this functor, and let A be an object of the target category C. We will show that the comma categories (A ↓ F ) are contractible. By Quillen's theorem A [Qui73,§1], this implies the lemma. The category (A ↓ F ) has objects sequences A given by the pair of maps x : A → X and 1 : Y → Y . It follows that the inclusion C 0 ⊂ (A ↓ F ) is a homotopy equivalence. Since the category C 0 has a terminal object, namely (1 A , 1 A ), the categories C 0 and (A ↓ F ) are contractible. If (E, w) is an exact category with weak equivalences, we make HE into an exact category with weak equivalences and (strict) duality by declaring a map (a, b) : (X, Y ) → (X ′ , Y ′ ) in HE to be a weak equivalence if a : X → X ′ and b : Y ′ → Y are weak equivalences in E. Note that wHE = HwE as categories with strict duality. 2.17. Proposition. Let (E, w) be an exact category with weak equivalences, then there is a natural homotopy equivalence Proof. Consider the commutative diagram of simplicial categories where the upper vertical maps are the functors that "forget the forms". The lower vertical maps are induced by the inclusion [n] ֒→ n and are thus homotopy equivalences, by lemma 2.8. The diagonal map is a homotopy equivalence by lemma 2.16. By the "octahedron axiom" for homotopy fibres applied to the upper right triangle, it follows that the homotopy fibre of the composition of the left vertical maps is equivalent to the loop space of the fibre of p 1 : 2.18. Remark. Let (E, w, * , η) be an exact category with weak equivalences and duality. The functor F : (E, w, * , η) → (HE, w) : X → (X, X * ) together with the duality compatibility morphism (1, η X ) : (X * , X * * ) → (X * , X) is called forgetful form functor. It is a non-singular exact form functor between exact categories with weak equivalences and duality. The map (wS e • E) h → wS • E defining the Grothendieck-Witt space factors as where the second map is the homotopy equivalence in the diagram of the proof of proposition 2.17 (going right, diagonally and down). It follows that the Grothendieck-Witt space GW (E, w, * , η) is naturally homotopy equivalent to the homotopy fibre of We finish the section with a comparison result between the definition of the Grothendieck-Witt space of an exact category with duality (E, i, * , η) in terms of the hermitian S • -construction and the definition given in [Sch08,Definition 4.6] in terms of the hermitian Q-construction. We recall the relevant definitions. 2.19. The hermitian Q-construction. Recall from [Qui73] that for an exact category E its Q-construction is the category with objects the objects of E and maps X → Y equivalence classes of diagrams For an exact category with duality (E, * , η), the hermitian Q-construction is the category Q h (E, * , η) with objects the symmetric spaces (X, ϕ) in E. A map (X, ϕ) → (Y, ψ) is a map X → Y in Quillen's Q-construction, that is, an equivalence class of diagrams (3), such that the square of maps p, i, p * ϕ and i * ψ is commutative and bicartesian. Composition of maps is as in Quillen's Q-construction. For more details, we refer the reader to [Sch08,4.2] and the references in [Sch08,Remark 4.3]. In [Sch08,Definition 4.6], we defined the Grothendieck-Witt space of (E, * , η) as the homotopy fibre of the forgetful functor Q h E → QE : (X, ϕ) → X. The following proposition reconciles this definition with the one given in 2.11. This allows us to freely use the results proved in [Sch08]. Proof. For the first homotopy equivalence, the proof is the same as in [Wal85,1.9 Appendix]. In detail, let iQ h • E be the simplicial category which in degree n is the category iQ h n E whose objects are sequences X 0 → X 1 → ... → X n of maps in Q h E and a map in iQ h n E is an isomorphism of such sequences. As n varies, iQ h n E defines a simplicial category where face and degeneracy maps are defined as in the usual nerve construction. The nerve of iQ h • E as a bisimplicial set is isomorphic to the nerve of the simplicial category which in degree m are the sequences X 0 → X 1 → ... → X m of isomorphisms in Q h E and where maps are maps of sequences in Q h E (which are not necessarily isomorphisms). The latter simplicial category is degree-wise equivalent to Q h E (via the embedding of Q h E as the constant sequences). Thus the latter simplicial category (and therfore also iQ h • E) is homotopy equivalent to Q h E. Every object (A p,q ) p≤q ∈ n in (S e n E) h defines a string of maps This defines a map (iS e • E) h → iQ h • E of simplicial categories which is degree-wise an equivalence. Therefore, this map defines a homotopy equivalence on topological realizations. For the second homotopy equivalence, consider the commutative diagram of topological spaces in which the lower right two horizontal maps are defined in a similar way as their hermitian analogs above them (see [Wal85,1.9 Appendix]), the three right vertical maps are "forgetful" functors and all maps labeled ∼ → are homotopy equivalences. It follows that the homotopy fibre of the first vertical map is equivalent to the homotopy fibre of the last vertical map. Additivity theorems Additivity theorems are fundamental in algebraic K-theory. They imply, for instance, Waldhausen's fibration theorem [Wal85,1.6.4] which is the basis for the K-theory version of theorem 1.2. In this section, we prove the analogs of additivity for higher Grothendieck-Witt theory. In order to formulate them, we recall the concept of "admissible short complexes" from [Sch08, §7]. 3.1. Admissible short complexes and their homology. Let (E, w, * , η) be an exact category with weak equivalences and duality. A short complex in E is a complex in E concentrated in degrees −1, 0, 1. It is admissible if d 1 and d 0 are inflation and deflation, respectively, and the map A 1 → ker(d 0 ) (or equivalently coker( We denote by ( sCx(E), w, * , η) the exact category with weak equivalences and duality of admissible short complexes in E. If (A • , α • ) is a symmetric form in sCx(E), we write H 0 (A • , α • ) for its zeroth homology symmetric form. Its underlying object is H 0 (A • ) = ker(d 0 )/im (d 1 ), and it is equipped with the formᾱ which is the unique symmetric form such that α \| ker(d0) = α \| ker(d0) . This makes H 0 : sCx(E) → E into a non-singular exact form functor for every exact category with weak equivalences and duality E. For more details, we refer the reader to [Sch08,§7]. In the special case of an exact category with duality (where all weak equivalences are isomorphisms), the following two theorems were proved in [Sch08, Theorems 7.1, 7.4]. The K-theory version is due to Waldhausen in [Wal85, Theorem 1.4.2 and proposition 1.3.2], in view of the equivalence S e 1 E → sCx(E) : A → (A 1 ′ 0 ′ → A 1 ′ 1 → A 01 ) of exact categories with weak equivalences and duality. We will reduce the proofs of the additivity theorems 3.2 and 3.3 to the case of exact categories with dualities dealt with in [Sch08,§7]. This will be done with the help of the simplicial resolution lemma 3.7 below. For that, we need to replace an exact category with weak equivalences and duality (where the double dual identification is a natural weak equivalence) by one with a strong duality (where the double dual identification is a natural isomorphism) without changing its hermitian K-theory. This will be done in the strictification lemma 3.4 below. We introduce notation for lemma 3.4. Let ExW eDu be the category of small exact categories with weak equivalences and duality; and non-singular exact form functors as maps. Recall that a category with duality (A, * , η) has a strict duality if η = id (and in particular, * * = id). Let ExW eDu str be the category of small exact categories with weak equivalences and strict duality; and duality preserving functors as maps. Write lax : ExW eDu str ⊂ ExW eDu for the natural inclusion. The next lemma will sometimes allow us to replace an exact category with weak equivalences and duality by a simplicial exact category with duality (where weak equivalences and double dual identification are isomorphisms). 3.6. Notation for lemma 3.7. Let (E, w, * , η) be an exact category with weak equivalences and duality, and let D be an arbitrary (small) category. Recall from 2.1 that the category Fun(C, E) of functors D → E is a category with duality. It is an exact category with weak equivalences and duality if we declare maps We write Fun w (D, E) ⊂ Fun(D, E) for the full subcategory of those functors F : D → C for which the image F (d) of all maps d of D are weak equivalences in E: F (d) ∈ wE. The category Fun w (D, E) inherits from Fun(D, E) the structure of an exact category with weak equivalences and duality. In particular, for n ∈ N and (E, w, * , η) an exact category with weak equivalences and strong duality, the category Fun w (n, E) is an exact category with weak equivalences and strong duality. It has objects strings of weak equivalences and maps commutative diagrams in E. Varying n, the categories Fun w (n, E) define a simplicial exact category with weak equivalences and strong duality. Recall that the symbol i stands for the set of isomorphisms in a category. 3.7. Lemma (Simplicial resolution lemma). Let (E, w, * , η) be an exact category with weak equivalences and strong duality. Then there are homotopy equivalences which are functorial for exact form functors (F, ϕ) for which ϕ is an isomorphism. Proof. We start with some general remarks. Let C be a category, and recall that i stands for the set of isomorphisms in C. Since C = Fun i ([0], C), inclusion of degreezero simplices yields a map of simplicial categories C → (n → Fun i ([n], C)) which is degree-wise an equivalence of categories, and thus induces a homotopy equivalence after topological realization. Using the equality of bisimplicial sets where N • stands for the nerve of a category, we obtain a homotopy equivalence \|C\| The homotopy equivalence is natural in the category C. Let (C, * , η) be a category with strong duality. There is an equivalence of categories i Fun([n] op , C h ) → (i Fun(n, C)) h which sends an object (X n , ϕ n ) fn → (X n−1 , ϕ n−1 ) of the left hand category to the object equipped with the form (η Xn , ..., η X0 , 1, ..., 1). A map (g n , ..., g 0 ) (which is an isomorphism compatible with forms) is sent to the map (g n , ..., g 0 , (g * 0 ) −1 , ..., (g * n ) −1 ). The equivalence is functorial in [n] ∈ ∆ and thus induces a homotopy equivalence after topological realization. Together with (6), we obtain a homotopy equivalence of topological spaces which is natural for categories with strong duality (C, * , η) and form functors (F, ϕ) between them for which ϕ is an isomorphism. For an exact category with weak equivalences and strong duality (E, w, * , η), we apply the general homotopy equivalence (7) to the form functor wS e p E → wS e p HE induced by the forgetful form functor E → HE (see remark 2.18). Varying p, we obtain a map of homotopy equivalences after topological realization The top row gives the first homotopy equivalence of the lemma. By remark 2.18, the left vertical homotopy fibre of the diagram is GW (E, w, * , η). In view of Bousfield-Friedlander's theorem [BF78,B4], [GJ99, Theorem IV 4.9], the homotopy fibre of the right vertical map is the simplicial realization of the degree-wise homotopy fibres. By remark 2.18, this is \|n → GW (Fun w (n, E), i, * , η)\|. Before proving the additivity theorems, we give a first application of the simplicial resolution lemma and show that π 0 GW (E, w, * , η) is the Grothendieck-Witt group as defined in 2.4. 3.8. Proposition (Presentation of GW 0 ). Let (E, w, * , η) be an exact category with weak equivalences and duality. There is a natural isomorphism Proof. In view of relation 2.4 (b) and lemma 2.14, weakly equivalent non-singular exact form functors (F, ϕ) ∼ −→ (G, ψ) induce the same map on GW 0 and on π 0 GW . Therefore, we can replace (E, w) by its strictification (E str w , w) from lemma 3.7 which has a strong (in fact strict) duality. So we can assume the duality on (E, w) to be strong which will allow us to use the simplicial resolution lemma 3.7. For a bisimplicial set X •• , there is a co-equalizer diagram This is well known and follows from an examination of the usual skeletal filtration of \|X •• \| = \|n → X n,n \| = \|n → X n• \| -which the reader can find in [GJ99, Diagram IV.1 (1.6)], for instance -using the fact that the functor π 0 : ∆ op Sets → Sets preserves push-out diagrams as it is left adjoint to the inclusion functor Sets → ∆ op Sets. By the simplicial resolution lemma 3.7 and the fact (proven in [Sch08,Proposition 4.14] together with proposition 2.20) that the proposition holds when the set of weak equivalences is the set of isomorphisms, we deduce that π 0 GW (E, w) is the co-equalizer of the diagram of Grothendieck-Witt groups of exact categories with duality. It suffices therefore to display GW 0 (E, w, * , η) as the co-equalizer of the same diagram. Evaluation at the object 0 ′ of 0 defines a non-singular exact form functor F : (Fun w (0, E), i) → (E, w) which sends the object f : The form functor induces a map GW 0 (Fun w (0, E), i) → GW 0 (E, w) which equalizes d 0 and d 1 in view of the relation 2.4 (b). We therefore obtain a map from the co-equalizer of the diagram to GW 0 (E, w). To construct its inverse, consider the map from the free abelian group generated by isomorphism classes [X, ϕ] of symmetric spaces in (E, w) to GW 0 (Fun w (0, E), i) sending (X, ϕ) to ϕ : X → X * equipped with the non-singular form (η X , 1). This map is surjective and factors through relations 2.4 (a) and (c). The map induces a surjective map to the co-equalizer which factors through relation 2.4 (b) and thus induces a surjective map from GW 0 (E, w, * , η) to the co-equalizer. Since composition with the map from the co-equalizer to GW 0 (E, w, * , η) is the identity, the claim follows. Proof of theorem 3.2. In view of the strictification lemma 3.4 we can assume the duality on E to be strong. By the simplicial resolution lemma 3.7 the proof reduces further to the case of an exact category with duality (in which all weak equivalences are isomorphisms). This case was proved in [Sch08, theorems 7.4, 7.10] in view of proposition 2.20. 3.10. Proof of theorem 3.3. Theorem 3.3 is a formal consequence of theorem 3.2. Let (E, w, * , η) be an exact category with weak equivalences and duality. Consider the form functors sCx(E) → HE : . Together with the form functor H 0 : sCx(E) → E and the duality preserving functor E → sCx(E) : E → (0 → E → 0) they define non-singular exact form functors E × HE → sCx(E) → E × HE whose composition is weakly equivalent to the identity functor. By the additivity theorem for short complexes (theorem 3.2), the second form functor induces a homotopy equivalence in hermitian K-theory. It follows that the two form functors induce inverse homotopy equivalences on Grothendieck-Witt spaces and on hermitian S • constructions. Therefore, the compositions induce homotopic maps in hermitian K-theory. These compositions are (weakly equivalent to) the maps in (5). 3.11. Remark. Iterated application of theorem 3.3 implies homotopy equivalences This allows us to identify (wS e • S e • E) h with the Bar construction of the H-group wS • E acting on (wS e • E) h and leads to a homotopy fibration in which the first map is "inclusion of degree zero simplices" and the second map is the "forgetful map" (E, ϕ) → E followed by the canonical homotopy equivalence X e • → X • . In particular, the iterated hermitian S • -construction (wS e • S e • E) h is not a delooping of (wS e • E) h contrary to the K-theory situation, compare [Wal85, proposition 1.5.3 and remark thereafter]. 3.12. Remark. Define the Witt-theory space W (E, w, * , η) as the colimit of the top row in the sequence of homotopy fibrations Since the spaces in the second row get higher and higher connected, we see that If E is a Z[ 1 2 ]-linear category and "complicial", we show in [Scha] that the Grothendieck-Witt space W (E, w, * , η) is the infinite loop space associated with (the (−1)-connected cover of) Ranicki's L-theory spectrum, and its homotopy groups are Balmer's Witt groups W −i (w −1 E, * , η) of the triangulated category with duality (w −1 E, * , η). At this point, I don't know how to calculate π n W (E, i, * , η), n ≥ 2, for (complicial) (E, w, * , η) when E is not Z[ 1 2 ]-linear. Change of weak equivalences and Cofinality In this section we prove in theorems 4.2 and 4.10 the higher Grothendieck-Witt theory analogs of Waldhausen's fibration theorem [Wal85, 1.6.4] and of Thomason's cofinality theorem [TT90, 1.10.1]. Waldhausen's K-theory version of theorem 4.2 below needs a "cylinder functor". The purpose of the next definition is to define the higher Grothendieck-Witt theory analog. We first fix some notation. For an exact category with weak equivalences (E, w), write E w ⊂ E for the full subcategory of w-acyclic objects, that is, those objects E of E for which the unique map 0 → E is a weak equivalence. The category E w is closed under extensions in E, and thus inherits an exact structure from E such that the inclusion E w ⊂ E is fully exact. 4.1. Definition. Let (E, w, * , η) be an exact category with weak equivalences and duality. A symmetric cone on (E, w, * , η) is given by the following data: . In other words, the sequence P → id → C defines an exact form functor from E to the category of sequences For examples of symmetric cones, see 7.5. The proof of the next theorem will occupy most of this section. 4.2. Theorem (Change of weak equivalences). Let (E, w, * , η) be an exact category with weak equivalences and duality which has a symmetric cone. Let v be another set of weak equivalences in E containing w and which is closed under the duality. into exact categories with weak equivalences and duality such that the commutative square of duality preserving inclusions induces a homotopy cartesian square of associated Grothendieck-Witt spaces. Moreover, the upper right corner has contractible Grothendieck-Witt space. Remark. A square of homotopy commutative H-groups (such as Grothendieck-Witt spaces) is homotopy cartesian if and only if the map between, say, horizontal homotopy fibres is a homotopy equivalence, and the map of abelian groups between horizontal cokernels of π 0 's is a monomorphism. We will reduce the proof of theorem 4.2 to idempotent complete exact categories with weak equivalences and duality. We recall the relevant definitions. 4.5. Idempotent completion. Recall that the idempotent completionẼ of an exact category E has objects pairs (A, p) with p = p 2 : Composition is composition of maps in E. The idempotent completionẼ has a canonical structure of an exact category such that the inclusion E ⊂Ẽ : A → (A, 1) is fully exact (see [TT90,Theorem A.9.1], where "idempotent completion" is called "Karoubianisation"). Any duality ( * , η) on E extends to a duality (A, is an exact category with duality, call a map in the idempotent completionẼ weak equivalence if it is a retract of a weak equivalence in E. Then (Ẽ, w, * , η) is an exact category with weak equivalences and duality. Note that the natural inclusion E w ⊂ (Ẽ) w is an equivalence of categories if (E, w, * , η) has a (symmetric) cone. This is because for an object X in (Ẽ) w , the weak equivalence 0 → X inẼ is, by definition, a retract of a weak equivalence f : Y → Z in E, and, by functoriality, also a retract of 0 4.6. Lemma. Let (E, w, * , η) be an exact category with weak equivalences and strong duality which has a symmetric cone. Then the commutative diagram of exact categories with duality induces a homotopy cartesian square of Grothendieck-Witt spaces. Proof. By the cofinality theorem in [Sch08, 5.5], the horizontal homotopy fibres of associated Grothendieck-Witt spaces are contractible. Therefore, it suffices to show that the map GW 0 (Ẽ w )/GW 0 (E w ) → GW 0 (Ẽ)/GW 0 (E) between the cokernels of horizontal π 0 's is injective. For an exact category with duality A, the quotient GW 0 (Ã)/GW 0 (A) is the abelian monoid of isometry classes of symmetric spaces iñ A modulo the submonoid of symmetric spaces in A [Sch08, 5.2]. In particular, a symmetric space inÃ yields the zero class in It follows that the symmetric spaces (A, α) ⊕ H(Ā ⊕ CX) and For an exact category with weak equivalences and duality (E, w, * , η), the category Mor E = Fun([1], E) of morphisms in E is an exact category with weak equivalences and duality such that the fully exact inclusion E ⊂ Mor E : , is duality preserving. The inclusion factors through the fully exact subcategory Mor w E = Fun w ([1], E) ⊂ Mor E of weak equivalences in E and defines a duality preserving functor The following proposition is the key to proving Theorem 4.2. 4.7. Proposition. Let (E, w, * , η) be an exact category with weak equivalences and strong duality. Assume that (E, w, * , η) has a symmetric cone. Then the commutative diagram of duality preserving inclusions of exact categories with duality (all weak equivalences being isomorphisms) induces a homotopy cartesian square of associated Grothendieck-Witt spaces. The proof uses the cone category construction of [Sch08,§9]. We recall the relevant definitions and facts. 4.8. Cone exact categories. Let A ⊂ U be a duality preserving fully exact inclusion of idempotent complete exact categories with duality ( * , η). In [Sch08, §9], we constructed a duality preserving fully exact inclusion Γ : U ⊂ C(U, A) of exact categories with duality, depending functorially on the pair A ⊂ U, such that the duality preserving commutative square of exact categories with duality induces a homotopy cartesian square of associated Grothendieck-Witt spaces, and the upper right corner C(A, A) (also written as C(A)) has contractible Grothendieck-Witt space [Sch08, theorem 9.9]. We recall the definition of the cone category C(U, A), details can be found in [Sch08, §9.1-9.3]. One first constructs a category C 0 (U, A), a localization of which is C(U, A). Objects of C 0 (U, A) are commutative diagrams in U inflations with cokernel in A and deflations with kernel in A, respectively. Moreover, there has to be an integer d such that for every i ≥ j, the map U j → U i+d is an inflation with cokernel in A and the map U i+d → U j is a deflation with kernel in A. If the maps in diagram (11) are understood, we may abbreviate the diagram as ( The dual of the diagram (11) is obtained by applying the duality to the diagram: For each diagram (11), forgetting the upper left corner U 0 gives us a new object There is a fully exact duality preserving inclusion Γ : U ⊂ C(U, A) which sends an object U of U to the constant diagram Proof of proposition 4.7. By Lemma 4.6, we can (and will) assume E to be idempotent complete. Then all categories in diagram (9) are idempotent complete. We will write C(E, w) and C(E w ) instead of the categories C(E, E w ) and C(E w , E w ) of 4.8, and we will write (F, ϕ) ∼ (G, ψ) if the two non-singular exact form functors (F, ϕ), (G, ψ) induce homotopic maps on Grothendieck-Witt spaces. The strategy of proof is as follows. We will extend diagram (9) to a commutative diagram of exact categories with duality and non-singular exact form functors where the right hand square is obtained from (9) by functoriality of the cone category construction in 4.8. We will show: ( †) (GW applied to) the compositions (F, ϕ) • (I, id) and (C(I), id) • (F, ϕ) are homotopic to the constant diagram inclusions (Γ, id) of 4.8 in such a way that the homotopies restricted to E w and Mor w E w have images in the Grothendieck-Witt spaces of C(E w ) and C(Mor w E w ), respectively. Assuming ( †), the outer diagram of the left two squares and the outer diagram of the right two squares induce homotopy cartesian diagrams of Grothendieck-Witt spaces, by [Sch08, theorem 9.9]. By remark 4.3, this implies the claim of proposition 4.7. To construct diagram (12), we will define the non-singular exact form functor (F, ϕ) : Mor w E → C(E, w) as the composition of a non-singular exact form functor (F 0 , ϕ) : Mor w E → C 0 (E, w) and the localization functor C 0 (E, w) → C(E, w) such that its restriction to Mor E w has image in C 0 (E w ). Recall that (E, w, * , η) is assumed to have a symmetric cone (see definition 4.1), where i : id C and p : P ։ id denote the natural inflation and deflation which are part of the structure of a symmetric cone. The functor (F, ϕ) (or rather (F 0 , ϕ)) sends an object g : X → Y of Mor w E to the object F (g) given by the diagram In the notation (U • → U • ) of 4.8 corresponding to diagram (11), the object F (g) is given by The maps U n → U n+1 and U n+1 → U n are the canonical inclusions into the first n + 1 summands and the canonical projections onto the first n + 1 factors. The maps U n → U n are given by the matrix with u rs = 0 unless r = s = 0 or \|r − s\| = 1, and u 0,0 = g, u 0,1 = p Y , u 1,0 = i X , u r,r+1 = 1, u r+1,r = 1 for r ≥ 1. The construction of diagram (13) is functorial in g, so that F : Mor w E → C(E, w) is indeed a functor. The duality compatibility map ϕ g : F (g * ) → (F g) * for g : X → Y is the identity on X * and Y * , it is γ X on the summands P (X * ) andγ Y on C(Y * ). It is clear that F sends the subcategory Mor w E w to the full subcategory C(E w ) of C(E, w). This defines diagram (12). We are left with proving ( †). Since U 0 = X is the initial object of diagram (13), it defines a map j = j g : Varying g, the map j g defines a natural transformation j : Γ → F . Similarly, U 0 is the final object of diagram (13) and thus defines a (functorial) map q = q g : Letφ : F → F ♯ = * F * be the symmetric form on the functor F associated with the duality compatibility map ϕ (see 2.1). The formφ : Write (F I, ϕ F I ) for the composition (F, ϕ)• (I, id) of form functors. The natural transformation j above makes the canonical inclusion (Γ, id) : E → C(E, w) into an admissible subfunctor j : Γ ⊂ F I of F I. Commutativity of diagram (14) for g = id X implies that η = j ♯φ F I j, that is, j defines a map (Γ, η) → (F I,φ F I ) of symmetric spaces associated with the form functors (Γ, id) and (F I, ϕ F I ). Since the maps id and ϕ F I are isomorphisms, the symmetric space As mentioned above, the cokernel of j : . By construction, the homotopy restricted to E w has image in the Grothendieck-Witt space of C(E w ). This shows the first half of the claim ( †). For the second half, write (IF, ϕ IF ) and (IF 0 , ϕ IF0 ) for the compositions of form functors (C(I), id) • (F, ϕ) and (C(I), id) • (F 0 , ϕ), and note that (IF, ϕ IF ) is just the composition of (IF 0 , ϕ IF0 ) with the localization functor C 0 (Mor w E, w) → C(Mor w E, w). There is an obvious isomorphism of exact categories with duality in Mor w C 0 (E, w). The total complex of the square (considered as a bicomplex) is a conflation in Mor w C 0 (E, w) = C 0 (Mor w , w). It is therefore also a conflation in C(Mor w , w), hence the square is also bicartesian in C(Mor w , w). In C(Mor w , w), the horizontal maps in the square are inflations with cokernel in C(Mor w E w ) since (j, 1) is isomorphic to the C 0 (Mor w , w)-inflation (j, 1) [1] which has cokernel in C 0 (Mor w E w ). Similarly, the vertical maps in the square are deflations in C(Mor w , w) Commutativity of diagram (14) implies that the formφ IF0 on the upper right corner IF 0 of the square extends to a form on the whole bicartesian square such that its restriction to the lower left corner is the constant diagram inclusion (Γ, η) : By the additivity theorem [Sch08, theorem 7.1] (or its generalization in theorem 3.3), the form functors (Γ, id) ⊥ H ker(1, q) and (IF, ϕ IF ) induce homotopic maps on Grothendieck-Witt spaces. Since H ker(1, q) has image in C(Mor w E w ) whose Grothendieck-Witt space is contractible, we have (IF, ϕ IF ) ∼ (Γ, id) ⊥ H ker(1, q) ∼ (Γ, id). The homotopies restricted to Mor w E w have image in the Grothendieck-Witt space of C(Mor w E w ). This is clear for the second homotopy, and for the first, it follows from the proof of additivity in 3.10. Next, we prove a variant of the Change-of-weak-equivalence theorem. 4.9. Proposition. Let (E, w, * , η) be an exact category with weak equivalences and strong duality which has a symmetric cone. Then the following commutative diagram of exact categories with weak equivalences and duality induces a homotopy cartesian square of Grothendieck-Witt spaces with contractible upper right corner Consider the commutative diagram of (simplicial) exact categories with dualities (all weak equivalences being isomorphisms) in which the left hand square can be identified with the square of proposition 4.7 and induces therefore a homotopy cartesian square of Grothendieck-Witt spaces. On Grothendieck-Witt spaces, the right vertical map can be identified with the map (E w , w) → (E, w) in view of the simplicial resolution lemma 3.7. The right hand square is the inclusion of degree zero simplices. The proof of proposition 4.9 is thus reduced to showing that the right hand square of the diagram induces a homotopy cartesian square of Grothendieck-Witt spaces. Let Fun 1 w (n, E) ⊂ Fun w (n, E) be the full subcategory of those functors A : n → E for which A p → A q is an inflation, and A q ′ → A p ′ is a deflation, 0 ≤ p ≦ q ≤ n. It inherits the structure of an exact category with duality from Fun w (n, E). Further, let Fun 0 w (n, E) be the category which is equivalent to Fun 1 w (n, E) but where an object is an object A of Fun 1 w (n, E) together with a choice of subquotients is an exact category with duality such that the forgetful functor Fun 0 w (n, E) → Fun 1 w (n, E) is an equivalence of exact categories with duality. We have an exact functor Fun 0 w (n, E) → S n E w : A → (A p,q ) 0≤p≤q≤n . By the additivity theorem [Sch08, theorem 7.1] (or 3.3), this functor induces a map which is part of a split homotopy fibration The same argument applies to (E w , w) instead of (E, w) so that, varying n, we obtain a map of homotopy fibrations after topological realization which shows that the left square is homotopy cartesian. Since Fun 0 w → Fun 1 w is an equivalence of exact categories with duality, the proposition follows once we show that the inclusion I : Fun 1 w (n, A) ⊂ Fun w (n, A) induces a homotopy equivalence on Grothendieck-Witt spaces for A = E, E w . We illustrate the argument for A = E and n = 1. The general case is mutatis mutandis the same. We define two functors F, G : Both functors F and G are equipped with canonical duality compatibility morphisms, induced by γ and γ from definition 4.1, such that F and G are non-singular exact form functors. By the additivity theorem, we have IF ∼ id ⊥ IG and F I ∼ id ⊥ GI, so that GW (F ) − GW (G) defines an inverse of GW (I), up to homotopy. Proof of theorem 4.2. By lemma 2.14, GW (E w , w) is contractible. Let A = E str w be the strictification of E from lemma 3.4, and recall that it has a strong duality. Consider the commutative diagram of exact categories with weak equivalences and duality By the strictification lemma 3.4 and lemma 2.14, the square in theorem 4.2 is equivalent to the lower right square in the diagram. By proposition 4.9, the upper square and the outer diagram of the left two squares are homotopy cartesian in hermitian K-theory. Since the left vertical maps are surjective on GW 0 (because A = E str w ), it follows that the lower left square is homotopy cartesian in hermitian K-theory, by remark 4.3. Again, by proposition 4.9, the outer diagram of the two lower squares induces a homotopy cartesian square of Grothendieck-Witt spaces. Together with the facts that the lower left square is homotopy cartesian in hermitian K-theory and that the lower left vertical map is surjective on GW 0 , this implies that the lower right square induces a homotopy cartesian square of Grothendieck-Witt spaces. 4.10. Theorem (Cofinality). Let (E, w, * , η) be an exact category with weak equivalences and duality which has a symmetric cone. Let A ⊂ K 0 (E, w) be a subgroup closed under the duality action on K 0 (E, w), and let E A ⊂ E be the full subcategory of those objects whose class in K 0 (E, w) belongs to A. Then the category E A inherits the structure of an exact category with weak equivalences and duality from (E, w, * , η), and the induced map on Grothendieck Witt spaces is an isomorphism on π i , i ≥ 1, and a monomorphism on π 0 . Proof. Let U = E str w , and consider the the diagram of exact categories with weak equivalences and duality On Grothendieck-Witt spaces, the right vertical map can be identified (up to homotopy) with the map in the theorem, by lemmas 3.4 and 2.14. The rows are homotopy fibrations, by proposition 4.9, and the right horizontal maps are surjective on GW 0 (as U = E str w ). It follows that the right square induces a homotopy cartesian square of Grothendieck-Witt spaces. Since U A ⊂ U is a cofinal inclusion of exact categories with duality, the cofinality theorem of [Sch08, corollary 5.5] shows that the homotopy fibre of the Grothendieck-Witt spaces of the middle vertical map is contractible. As the right square is homotopy cartesian in hermitian K-theory, the same is true for the right vertical map. Approximation, Change of exact structure and Resolution In this section we prove in theorems 5.1 and 5.11 variants of Waldhausen's approximation theorem [Wal85, Theorem 1.6.7] which hold for higher Grothendieck-Witt groups. We explain two immediate consequences, one concerning the conditions under which a change of exact structure has no effect on Grothendieck-Witt groups (lemma 5.3, compare [TT90, Theorem 1.9.2]) and the other concerning an analog of Quillen's resolution theorem (lemma 5.6, compare [Qui73, §4 Corollary 1]). 5.1. Theorem (Approximation I). Let (F, ϕ) : A → B be a non-singular exact form functor between exact categories with weak equivalences and duality. Assume the following. (a) Every map in A can be written as the composition of an inflation followed by a weak equivalence. Then (F, ϕ) induces homotopy equivalences Proof. For the purpose of the proof, we call lattice a pair (L, λ) with L : A → A an exact functor and λ : L ∼ → id A a natural weak equivalence. Lattices form an associative monoid under composition Since λ 2 •L 2 (λ 1 ) = λ 1 •λ 2,L1 (as λ 2 is a natural transformation), lattices behave like a multiplicative set in a commutative ring. More precisely, composition of lattices allows us to generalize properties (e) and (f) to finite families of maps: (e') for any finite set of maps f i : F A i → F B i in B, i = 1, ..., n, there are a lattice (L, λ) and maps a i : LA i → B i such that F a i = f i •F λ Ai , i = 1, ..., n, and (f') for any finite set of maps a i : A i → B i in A such that F a i = 0 in B, i = 1, ..., n, there is a lattice (L, λ) such that a i λ Ai = 0 in A, i = 1, ..., n. We will refer to (e') and (f') as "clearing denominators", in analogy with the localization of a commutative ring with respect to a multiplicative subset. "Clearing denominators" together with (b) and (d) implies that we can lift non-degenerate symmetric forms from B to A in the following sense: ( †) For any non-degenerate symmetric form (F A, α) ∈ (wB) h on the image F A of an object A of A, there is a lattice (L, λ) and a non-degenerate symmetric . The proof is the same as the classical proof which shows that a non-degenerate symmetric form over the fraction field of a Dedekind domain can be lifted to a (usual) lattice in the ring. In detail, the map ϕ −1 A α : F A → F (A * ) lifts to a map a 1 : L 1 A → A * such that ϕ A F a 1 = αF λ 1,A for some lattice (L 1 , λ 1 ). The map a : λ * 1,A a 1 : L 1 A → (L 1 A) * is a weak equivalence but not necessarily symmetric. However, the difference δ = a−a * η L1A satisfies F δ = 0, so that there is a second lattice (L 2 , λ 2 ) such that δλ 2,L1A = 0. Then β = λ * 2,L1A aλ 2,L1A : L 2 L 1 A → (L 2 L 1 A) * is a non-degenerate symmetric form on LA with (L, λ) = (L 2 , λ 2 ) • (L 1 , λ 1 ), and F (λ A ) defines a map of symmetric spaces F (LA, β) = (F LA, ϕ LA F β) ∼ → (F A, α). Apart from "clearing denominators", the proof of the first homotopy equivalence in the theorem proceeds now as the proof of [Sch06, theorem 10] which was based on the proof of [Wal85, theorem 1.6.7]. We first note that under the assumptions of the theorem, the non-singular exact form functors S n (F, ϕ) : S n A → S n B also satisfy (a) -(f), n ∈ N. For (a) -(c), this is in [Wal85, lemma 1.6.6] (using the fact that in the presence of (a), the map a : A → B in (c) can be replaced by an inflation), (d) extends by functoriality, and the extension of (e), (f) to S n easily follows by induction on n by successively clearing denominators. In order to show that (wS e • A) h → (wS e • B) h is a homotopy equivalence, it suffices to prove that (wS e n A) h → (wS e n B) h is a homotopy equivalence for every n ∈ N, which, by the argument of the previous paragraph, only needs to be checked for n = 0, that is, it is sufficient to prove that is a homotopy equivalence. The last claim will follow from Quillen's theorem A once we show that for every object X = (X, ψ) of (wB) h , the comma category (F ↓ X) is non-empty and contractible. By (a) with A = 0, there is an object B of A and a weak equivalence In order to show that (F ↓ X) is contractible it suffices to show that every functor P → (F ↓ X) from a finite poset P to the comma category (F ↓ X) is homotopic to a constant map (see for instance [Sch06,Lemma 14]). Such a functor is given by a triple (A, α, f ) where A is a functor A : P → wA : i → A i , (i ≤ j) → a j,i together with a collection α of non-degenerate symmetric forms α i : A i → A * i in A, i ∈ P, such that α i = α j\|Ai whenever i ≤ j in P, and f is a collection of compatible maps of symmetric spaces f i : F (A i , α i ) → (X, ψ) in (wB) h such that f i = f j F a j,i whenever i ≤ j ∈ P. It is convenient to consider f as a map F (A, α) → (X, ψ) of functors P → (wB) h , where objects in (wB) h (or in A, B, (wA) h ) such as (X, ψ) are interpreted as constant P-diagrams. By [Sch06, Lemma 13], there is a map b : B → A of functors P → wA such that b i : B i → A i is a weak equivalence, i ∈ P, and the P-diagram B : P → A has a colimit in A such that F (B) → F (colim P B) represents the colimit of F (B) in B (the diagram B is a cofibrant replacement of A in a suitable cofibration structure on the category of functors P → A, see [Sch06, Appendix A.2], cofibrant objects have colimits, and F , being an exact functor, preserves cofibrant objects and their colimits). By (c), the natural map c → X where the first map is in the image of F and the second map is a weak equivalence in B. Let g : B → C be the composition B → colim P B → C which, by (b), is a weak equivalence since b, f , and c are. The null-homotopy to be constructed can be read off the following diagram of which we have constructed the right hand square, so far. In the diagram, a dashed arrow A X stands for an arrow F A → X in B, and solid arrows are arrows in A. By ( †), there is a lattice (L, λ) and a non-degenerate symmetric form (LC, γ) ∈ (wA) h such that F λ C : F (LC, γ) → (F C, ψ \|F C ) defines a map in (wB) h . The restrictions of γ and α i to LB i may not coincide, but their images under F coincide since in B, both are (up to composition with the isomorphism ϕ Bi ) the restriction of ψ to F LB i . Clearing denominators, we can find a lattice (L ′ , λ ′ ) such that γ \|L ′ LBi = α i\|L ′ LBi =: β i for all i ∈ P simultaneously. The outer part of the diagram involving (L ′ LB, β), (A, α), (LC, γ), (X, ψ) and the maps between them, defines a homotopy between (A, α, f ) and the constant functor (LC, γ, cF λ) : P → (F ↓ X) via the functor (L ′ LB, β, f F (bλλ ′ )) : P → (F ↓ X). This finishes the proof of the first homotopy equivalence in the theorem. 5.2. Change of exact structure. Let (A, w, * , η) be an additive category with weak equivalences and duality, and assume that A can be equipped with two exact structures 1 and 2 one smaller than the other, 1 ⊂ 2 , so that the identity functor (A, 1 ) → (A, 2 ) is exact. Assume furthermore that the duality functor * : A op → A is exact for both exact structures 2 and 2 , so that the identity defines a duality preserving exact functor The following is an immediate consequence of theorem 5.1. The purpose of the next lemma (lemma 5.5 below) is to simplify some of the hypothesis of theorem 5.1 provided the exact categories with weak equivalences in the theorem are "categories of complexes". 5.4. Definition. We call an exact category with weak equivalences (C, w) a category of complexes (with underlying additive category C 0 ) if C ⊂ Ch C 0 is a full additive subcategory of the category Ch C 0 of chain complexes C 0 such that (a) C is closed under degree-wise split extensions in Ch C 0 , (b) degree-wise split exact sequences are exact in C, (c) with a complex A in C its usual cone CA (that is, the cone on the identity map of A) and all its shifts A[i], i ∈ Z are in C, and (d) the set of weak equivalences w contains at least all usual homotopy equivalences between complexes in C. 5.1 (b), (e) and the following condition. (c') For every object E of B there is an object A of A and a weak equivalence Proof. Every map f : A → B in A can be written as the composition where i : A CA is the canonical inclusion of A into its cone CA. This shows that condition 5.1 (a) holds. To prove condition 5.1 (c) assuming conditions 5.1 (b), (e) and (c') are satisfied, Then the duality preserving inclusion A ⊂ B induces homotopy equivalences 5.7. Proof. This follows from theorem 5.1 in view of lemma 5.5 and the fact that conditions 5.1 (e) and (g) trivially hold since A ⊂ B is fully faithful, and condition (d) holds since A → B is duality preserving. We finish the section with theorem 5.11 below. It is a variant of theorem 5.1 when conditions 5.1 (e) and (f) can not be achieved in a functorial way but the form functor is a localization by a calculus of right fractions. 5.10. Context for theorem 5.11. Consider an additive functor A → B between additive categories. It induces an exact functor F : Ch b A → Ch b B between the associated exact categories of bounded chain complexes where we call a sequence of chain complexes exact if it is degree-wise split exact. We assume that F is part of an exact form functor between exact categories with weak equivalences and duality such that the duality compatibility map F * → * F is a natural isomorphism. 5.11. Theorem (Approximation II). If in the situation 5.10 above, a map in Ch b A is a weak equivalence iff its image in Ch b B is a weak equivalence, and if the functor A → B is a localization by a calculus of right fractions, then (F, ϕ) induces homotopy equivalences The proof of theorem 5.11 is a consequence of the following two lemmas. which is functorial in the pair (C, wC). In the first map, the category C is considered as a constant simplicial category, and the functor C → Fun w ([n], C) sends an object C ∈ C to the string consisting of only identity maps on C. This functor is a homotopy equivalence with inverse the functor Fun w ([n], C) → C which sends a string of maps C 0 → · · · → C n to C 0 . The composition of the two functors is the identity in one direction, and in the other, it is homotopic to the identity, where the homotopy is given by the natural transformation from C 0 1 → C 0 1 → · · · C 0 to C 0 → C 1 → · · · C n induced by the structure maps of the last string. Therefore, the first map in (19) is a homotopy equivalence. The second map in (19) is in fact a homeomorphism as it is the realization in two different orders of the same bisimplicial set. In order to prove the lemma, let σA ⊂ A be the subcategory of A whose maps are the maps which are sent to isomorphisms in B. By the natural homotopy equivalences in (19), the map \|A\| → \|B\| in the lemma is equivalent to the map \|n → σ Fun([n], A)\| → \|n → i Fun([n], B)\| which is the realization of a map of simplicial categories, so that it suffices to show that for each integer n ≥ 0, the functor σ Fun([n], A) → i Fun ([n], B) is a homotopy equivalence. By lemma 5.12 (a), this functor is a localization by a calculus of fractions, so that we are reduced to proving that G : σA → iB is a homotopy equivalence whenever A → B is a localization by a calculus of right fractions. In this case, the comma category (G ↓ B) is left filtering for every object B of B, hence contractible. By theorem A of Quillen, the functor σA → iB is a homotopy equivalence. 5.14. Proof of theorem 5.11. We only prove the first homotopy equivalence in the theorem, the second homotopy equivalence follows from this and the homotopy equivalence wS • Ch b A → wS • Ch b B which is proved in the same way (forgetting forms). A map of simplicial categories which is degree-wise a homotopy equivalence induces a homotopy equivalence after topological realization. Therefore, it suffices to show that for every n ≥ 0, the form functor (F, ϕ) induces a homotopy equivalence From exact categories to chain complexes The purpose of this section is to prove proposition 6.5 which allows us the replace the Grothendieck-Witt space of an exact category with duality by the Grothendieck-Witt space of the associated category of bounded chain complexes. 6.1. Chain complexes and dualities. Let (E, * , η) be an exact category with duality, that is, an exact category with weak equivalences and duality where all weak equivalences are isomorphisms. Let Ch b (E) be the category of bounded chain complexes Call a chain complex (E, d) in E strictly acyclic if every differential d n is the composition E n ։ im d n E n+1 of a deflation followed by an inflation, and the sequences im d n−1 E n ։ im d n are exact in E. A chain complex is called acyclic if it is homotopy equivalent to a strictly acyclic chain complex. A map of chain complexes is a quasi-isomorphism if its cone is acyclic. Write quis for the set of quasi-isomorphisms, then the triple is an exact category with weak equivalences. For n ∈ Z, the duality ( * , η) induces a (naive) duality ( * n , η n ) on Ch b E which on objects (E, d) and on chain maps f : (E, d) → (E ′ , d) is given by the formulas With these definitions, we have an exact category with weak equivalences and duality (Ch b (E), quis, * n , η n ). 6.2. Remark. The functor T : Ch b E → Ch b E given by the formula defines a duality preserving isomorphism of exact categories with duality 6.3. Remark. There is another (more natural) sign choice for defining induced dualities (♯ n , can n ) on Ch b E coming from the internal hom of chain complexes, compare 7.4. They are given by the formulas The identity functor on Ch b E together with the duality compatibility isomorphism ε n E : E * n → E ♯ n which in degree i is For the purpose of proving proposition 6.5 below, the duality ( * , η) is convenient. For most other purposes, the duality (♯, can) is more natural. It is the latter duality, which we will use from §8 on. In any case, both give rise to isomorphic exact categories with duality and thus have isomorphic Grothendieck-Witt spaces. 6.4. Exercise. Show that the exact categories with weak equivalences and duality (Ch b E, * n , η n ) and (Ch b E, ♯ n , can n ) have symmetric cones in the sense of definition 4.1 (hint: see 7.5). Proposition. For an exact category with duality (E, * , η), the functor (21) induces a homotopy equivalence of Grothendieck-Witt spaces We will reduce the proof of the proposition to "semi-idempotent complete" exact categories. This has the advantage that for such categories, every acyclic complex is strictly acyclic. Here are the relevant definitions and facts. 6.6. Semi-idempotent completions. Call an exact category E semi-idempotent complete if any map p : A → B which has a section s : B → A, pi = 1, is a deflation in E. A semi-idempotent complete exact category has the following property: any map B → C for which there is a map A → B such that the composition A → C is a deflation, is itself a deflation. This is because a semi-idempotent complete exact category satisfies Thomason's axiom [TT90, A.5.1] so that the standard embedding of E into the category of left exact functors E op → ab into the category of abelian groups is closed under kernels of surjections [TT90, Theorem A.7.1 and Proposition A.7.16 (b)]. For a semi-idempotent complete exact category, every acyclic complex is strictly acyclic. The semi-idempotent completion of an exact category E is the full subcategory E semi ⊂ E of the idempotent completion E of E of those objects which are stably in E. Clearly, E semi is semi-idempotent complete, and the map K 0 (E) → K 0 ( E semi ) is an isomorphism. If (E, * , η) is an exact category with duality, then ( E semi , * , η) is an exact category with duality such that the fully exact inclusion E ⊂ E semi is duality preserving. If (A, w, * , η) is an exact category with weak equivalences and duality, then ( A semi , w, * , η) inherits the structure of an exact category with weak equivalence and duality from ( A, w, * , η), see 4.5, so that the inclusion A ⊂ A semi is duality preserving. 6.7. Lemma. Let (A, w, * , η) be an exact category with weak equivalences and strong duality, then the inclusion A ⊂ A semi induces a homotopy equivalence of Grothendieck-Witt spaces GW (A, w, * , η) → GW ( A semi , w, * , η). Proof. For an exact category with duality (E, * , η), the map GW i (E) → GW i ( E semi ) is an isomorphism for i ≥ 1 and it is injective for i = 0, by cofinality proved in [Sch08]. The map GW 0 (E) → GW 0 ( E semi ) is also surjective, hence an isomorphism, since for every symmetric space (X, ϕ) in E semi , we can find an A in E such that X ⊕ A is in E, and thus, [X, ϕ] = [(X, ϕ) ⊥ HA] − [HA] is in the image of the map. Therefore, lemma 6.7 holds when w is the set of isomorphisms. The lemma now follows from this case and the simplicial resolution lemma 3.7 since Fun w (n, A semi ) is the semi-idempotent completion of Fun w (n, A) (for a string X n ′ → · · · → X n of weak equivalences in A semi , there is an object A of A such that X i ⊕ A is in A for all i ∈ n, so that (X n ′ → · · · → X n ) ⊕ (A 1 → · · · 1 → A) is a string of weak equivalences in A). Proof of proposition 6.5. In view of lemma 6.7, we can assume E to be semiidempotent complete, so that acyclic complexes are strictly acyclic. Moreover, by the strictification lemma 3.4, we can assume E to have a strict duality, since an exact category with duality is equivalent to its strictification. Let Ac b E ⊂ Ch b E be the full subcategory of acyclic chain complexes. It inherits the structure of an exact category with weak equivalences and strict duality from Ch b E. Consider the commutative diagram of exact categories with weak equivalences and strict duality We will show that (a) the left square induces a homotopy cartesian square of Grothendieck-Witt spaces, and that (b) the map GW 0 (E, i) → GW 0 (Ch b E, quis) is surjective. By proposition 4.9, the right hand square induces a homotopy cartesian square of Grothendieck-Witt spaces, so that by (a) the same is true for the outer square. Together with (b), this implies the proposition. We prove (a). We will show that the right-hand square induces a homotopy cartesian square of Grothendieck-Witt spaces. Then, by induction, the outer square induces a homotopy cartesian square, too. Taking the colimit over n of the Grothendieck-Witt spaces of the outer squares yields the desired homotopy cartesian square, since the Grothendieck-Witt space functor GW commutes with filtered colimits. Consider the following form functors between exact categories with duality (equipped with the obvious duality compatibility maps): These functors, together with the natural inclusions, fit into a commutative diagram of exact categories with duality The upper square induces a homotopy cartesian square of Grothendieck-Witt spaces. By additivity (theorem 3.3 or [Sch08, 7.1]), the diagonal maps s and ρ in the lower square induce homotopy equivalences of Grothendieck-Witt spaces (details below). Therefore, the outer square induces a homotopy cartesian square as well. To see that the functors s and ρ induce homotopy equivalences, consider the following functors of categories with duality We have ρσ = id. The identity functor id on Ch b [−n−1,n+1] has a totally isotropic subfunctor G ⊂ id given by By additivity [Sch08, 7.1], the identity functor id and σρ = G ⊥ /G ⊕ HG induce homotopic maps on Grothendieck-Witt spaces, thus ρ induces a homotopy equivalence. Similarly, we have rs = id, and the identity functor id on Ac b [−n−1,n+1] E has a totally isotropic subfunctor F ⊂ id given by Again, by additivity [Sch08], the identity functor id on Ac b [−n−1,n+1] E and sr = F ⊥ /F ⊕ HF induce homotopic maps on Grothendieck-Witt spaces. It follows that s induces a homotopy equivalence. This finishes the prove of (a). We are left with proving (b). We will show that The complexÃ is equipped with a non-singular symmetric formα which is α i in degree i except in degrees i = −n + 1, n − 1 where it is α i ⊕ 1. We have a symmetric space in the category of admissible short complexes in Ch b E A * n [n − 1] Ã ։ A n [−n + 1] with form (1,α, η), where for an object E of E, we denote by E[i] the complex which is E in degree −i and 0 elsewhere. The maps A * n Ã −n+1 = A −n+1 ⊕ A * n and A n−1 ⊕ A n =Ã n−1 → A n are the canonical inclusions and projections, respectively. with non-singular from (α −n ,α, α n ), where for an object E of E, we write CE[i] for the complex E 1 → E placed in degrees i and i − 1. The maps CA −n [n − 1] Ã andÃ ։ CA n [n] are the unique maps which are the identity in degree −n and n, respectively. Since CA −n [n − 1] and CA n [n] are acyclic, the form on the admissible short complex is non-singular and its zero-th homology, which is concentrated in degrees [−n + 1, n − 1], has the same class in GW 0 (Ch b E, quis) as (Ã,α). 6.8. Remark. We can equip (Ch b E, quis, * , η) with two exact structures, the one defined in 6.1, and the degree-wise split exact structure. By lemma 5.3, the two yield homotopy equivalent Grothendieck-Witt spaces. DG-Algebras on ringed spaces and dualities In this section we recall basic definitions and facts about differential graded algebras and modules over them. Besides fixing terminology, the main point here is the construction of the canonical symmetric cone in 7.5, and the interpretation of certain form functors as symmetric forms in dg bimodule categories, see 7.7. 7.1. DG κ-modules. Let κ be a commutative ring. Unless otherwise indicated, modules will always mean left module, tensor product ⊗ will be tensor product ⊗ κ over κ, and homomorphism sets Hom(M, N ) between κ-modules means set of κ-linear homomorphisms, and is itself a κ-module. Recall that a differential graded κ-module M is a graded κ-module n∈Z M n together with a κ-linear map d : M n → M n+1 , n ∈ Z, called differential of M , satisfying d • d = 0. In other words, M is a chain complex of κ-modules. A map of dg κ-modules is a map of graded κ-modules commuting with the differentials. For two dg κ-modules M , N , the tensor product dg κ-module M ⊗ N and the homomorphism dg κ-module [M, N ] are defined by the usual formulas 7.2. DGAs and modules over them. A differential graded κ-algebra (dga) is a dg κ-module A equipped with dg κ-module maps · : A ⊗ A → A and κ → A, called multiplication and unit, making the usual associativity and unit diagrams commute For dgas with involution A, B, C and M ∈ A -Mod-B, N ∈ B -Mod-C, the commutativity isomorphism of the tensor product defines an A -Mod-C-isomorphism This can be iterated to obtain for rings with involution A, B, C, D and M ∈ A -Mod-B, N ∈ B -Mod-C, P ∈ C -Mod-D an isomorphism c 3 in A -Mod-D defined by 7.4. DG-modules and dualities. Let A be a dga with involution, and let I be an A-bimodule equipped with an A bimodule isomorphism i : 7.5. The canonical symmetric cone. Let C = κ · 1 C ⊕ κ · ǫ be the dg κ-module whose underlying κ-module is free of rank 2 with basis 1 C and ǫ in degrees 0 and −1, respectively, and differential dǫ = 1 C . In fact, C is a commutative dga with unit 1 C and unique multiplication. Let where F (P ) = M ⊗ B P and the duality compatibility map is the left A-module homomorphism These properties follow directly from the definitions, and we omit the details. 7.9. Tensor product of dgas with involution. For two dgas A, V , the tensor product dg κ-module . If A, V are dgas with involution, then the tensor product dga AV is a dga with involution AV → (AV ) op : a ⊗ v → (ā ⊗v) op . Furthermore, if (I, i) and (U, u) are duality coefficients for A -Mod and V -Mod, then IU = I ⊗ U is an AV -bimodule with left multiplication (a⊗v)·(t⊗x) = (−1) \|v\|\|t\| a·t⊗v·x and right Higher Grothendieck-Witt groups of schemes Let X be a scheme, A X be a quasi-coherent sheaf of O X -algebras with involution, L a line bundle on X, Z ⊂ X a closed subscheme and n ∈ Z an integer. The purpose of this section is to introduce the Grothendieck-Witt space GW n (A X on Z, L) of symmetric spaces over A X with coefficients in the n-th shifted line bundle L[n] and support in Z. We work in this generality in order to be able to extent the localization and excision theorems of §9 to negative degrees. Recall that, unless otherwise indicated, "module" will always mean " left module". In what follows, we will denote by ⊗ the tensor product ⊗ OX of O X -modules. 8.1. Vector bundles and strictly perfect complexes. Let A X be a quasicoherent sheaf of O X -algebras with involution. The category of quasi-coherent left A X -modules (dg-modules concentrated in degree 0) is a fully exact abelian subcategory of the abelian category of left A X -modules. We denote by Vect(A X ) the full subcategory of A X vector bundles, that is, of those quasi-coherent left A X -modules F for which F (U ) is a finitely generated projective A X (U )-module for every affine U ⊂ X. As usual, the last condition only needs to be checked for those U running through a choice of an affine open cover of X. The category of A X vector bundles inherits the notion of exact sequences from the category of all (quasi-coherent) A X -modules. Note that A X vector bundles need not be locally free, since A X,x may not be commutative nor a local ring for x ∈ X. In case A X = O X , the category Vect(X) is the usual exact category of vector-bundles on X. A strictly perfect complex of A X -modules is a dg left A X -module M such that M n = 0 for all but finitely many n ∈ Z and M n is an A X vector bundle for all n ∈ Z. Denote by sPerf(A X ) the category of strictly perfect complexes of A X -modules, in oder words, the category of bounded chain complexes of A X vector bundles. Let L be a line bundle on X. Then A X L[n] = A X ⊗ L[n] is a dg A X -bimodule via the multiplication defined on sections by a(x ⊗ l)b = (−1) \|b\|\|l\| axb ⊗ l for a, b, x ∈ A X and l ∈ L[n]. We equip A X L[n] with a dg A X -bimodule isomorphism i : , εi) is also a duality coefficient for A X -Mod. In the notation of 7.9, the duality coefficient (A X L[n], εi) is the tensor product of the duality coefficient (A X , µ) for A X -Mod and the duality coefficient For a strictly perfect complex of A X -modules M , the left dg A X -module is also strictly perfect, the functor M → M ♯ n εL is exact and preserves quasi-isomorphisms. Moreover, the double dual identification can (AX L[n],εi) defined in 7.4 is an isomorphism. Therefore, the triple (sPerf(A X ), quis, ♯ n εL ) defines an exact category with weak equivalences and duality, the double dual identification being understood as can (AX L[n],εi) . If n = 0 (or ε = 1, or L = O X ), we may omit the label corresponding to n (or ε, or L, respectively), so that (A X -Mod, ♯ n ) means (A X -Mod, ♯ n 1,OX ), for instance. By restriction of structure, we have an exact category with duality (Vect(A X ), ♯ εL ). Let Z ⊂ X be a closed subscheme with open complement U = X − Z. A strictly perfect complex M of A X -modules has cohomological support in Z if the restriction M \|U of M to U is acyclic. We write sPerf(A X on Z) for the category of strictly perfect complexes of A X -modules which have cohomological support in Z. By restriction of structure, we have exact categories with weak equivalences and duality (24) ( sPerf(A X on Z), quis, ♯ n εL , ). 8.2. Definition. Let X be a scheme, A X be a quasi-coherent sheaf of O X -algebras with involution, L a line bundle on X, Z ⊂ X a closed subscheme, n ∈ Z an integer, and ε ∈ {+1, −1}. The Grothendieck-Witt space ε GW n (A X on Z, L) of ε-symmetric spaces over A X with coefficients in the n-th shifted line bundle L[n] and (cohomological) support in Z is the Grothendieck-Witt space of the exact category with weak equivalences and duality (24). If Z = X (or ε = 1, or L = O X , or n = 0), we may omit the label corresponding to Z (ε, L, n, respectively). For instance, the space GW (A X , L) denotes the Grothendieck-Witt space 8.3. Remark. By 7.5, the exact category with weak equivalences and duality (24) has a symmetric cone in the sense of 4.1. In the following proposition, we write µ : where the Grothendieck-Witt spaces on the right hand side are the ones associated with the exact categories with duality (Vect(A X ), ♯ L , ± can L ) as defined in [Sch08,4.6]. Proof. The homotopy equivalences follow from proposition 6.5, remark 6.3, and proposition 8.4. Recall that if f ∈ Γ(X, L) is a global section of a line bundle L on a scheme X, then the open inclusion X f ⊂ X is an affine map (as can be seen by choosing an open affine cover of X trivializing the line bundle L). As a special case, X f is affine whenever X is affine. Thus, for the affine cover above X = X si , all finite intersections of the X si 's are affine. In particular, a scheme with an ample family of line bundles is quasi-compact (as a finite union of affine, hence quasi-compact, subschemes) and it is quasi-separated. Recall that the latter means that the intersection of any two quasi-compact open subsets is quasi-compact (a condition which only needs to be checked for the pair-wise intersections U i ∩ U j of a cover of X = i U i by quasi-compact open subsets U i ; in our case, we can take U i = X si ). Localization and For a scheme X with an ample family of line bundles, there is a set L i , i ∈ I, of line bundles on X with global sections s i ∈ Γ(X, L i ) such that the open subsets The proofs of theorems 9.2 and 9.3 will occupy the rest of this section. First, we introduce some terminology. For an open subscheme U ⊂ X, call a map M → N of left dg A X -modules a U -isomorphism (U -quasi-isomorphism) if its restriction M \|U → N \|U to U is an isomorphism (quasi-isomorphism). A left dg A X -module M is called U -acyclic if its restriction M \|U to U is acyclic. 9.4. Notation. In 9.5, 9.6, 9.7 below, we consider the following objects: is acyclic over U , since the map s ni = (s −1 i ) ⊗\|ni\| : L ni i → O X is an X si -isomorphism, so that the Koszul complex (25) is acyclic (even contractible) over each X si . Let K(s n ) denote the bounded complex whose non-zero part (which we place in degrees −l + 1, ..., 0) is the Koszul complex (25) in degrees ≤ −1. The last differential d −1 of the Koszul complex defines a map of strictly perfect complexes of O X -modules which is a U -quasi-isomorphism since its cone, the Koszul complex, is U -acyclic. For a left dg A X -module M , we write ε M for the tensor product map Before we treat the case l > 1 (and A X = O X ; A = O X , M concentrated in degree 0), we prove the following. ( †) For every map A → G of complexes of quasi-coherent O X -modules with A strictly perfect and j * G contractible, there is an l-tuple of negative integers (n 1 , ..., n l ) and a commutative diagram of complexes of O X -modules where ι is the canonical embedding of O X (concentrated in degree 0) into the Koszul complex. It suffices to prove ( †) for l = 1, since the general case is a repeated application of the case l = 1. For the proof of ( †) with l = 1, we can assume A = O X , as above. The composition O X → G → j * j * G has contractible target and therefore factors through the cone (O X → O X ) of O X . By the case l = 1 of the lemma (proved above), there is an n < 0 such that the composition (L n → L n ) may not be the same, but their compositions with G → j * j * G are, so that, again by case l = 1 of the lemma, precomposing both maps with L n+t s t → L n makes the two maps with target G equal. Replacing n with n + t, we can assume that the two maps (0 → L n ) → G above coincide. Then we obtain a map from the push-out (L n s n → O X ) of (L n → L n ) ← (0 → L n ) → O X ) for some l-tuple of negative integers (n 1 , ..., n l ). The canonical map from the stupid truncation in degrees ≤ −1 (shifted by 1 degree) to its degree 0 part yields (a). The general case of part (b) is a repeated application of the case l = 1. For an exact category with weak equivalences (C, w), we write D(C, w) for its derived category, that is, the category C[w −1 ] obtained from C by formally inverting the arrows in w. If (C, w) is a category of complexes in the sense of definition 5.4, its derived category D(C, w) is a triangulated category. In this case, it can also be obtained as the localization by a calculus of fractions of the homotopy category K(C) of C which is the factor category of C modulo the ideal of maps which are homotopic to zero. 9.8. The derived category of quasi-coherent A X -modules. Recall that a Grothendieck abelian category is an abelian category A in which all set-indexed direct sums exist, filtered colimits are exact, and A has a set of generators. We remind the reader that a set I of objects of A generates the abelian category A if for every object E ∈ A, there is a surjection A i ։ E from a set indexed direct sum of (possibly repeated) objects A i ∈ I to E. If X is a scheme with an ample family of line-bundles, and A X a quasi-coherent O X -algebra, then the category Qcoh(A X ) of quasi-coherent A X -modules is an abelian category with generating set the set A X ⊗ L k i , i = 1, ..., n, k ≤ 0, where L 1 , ..., L n is a set of line bundles on X with global sections s i ∈ Γ(X, L i ) such that the non-vanishing loci X si are affine and cover X. For a Grothendieck abelian category A, write D(A) for the unbounded derived category of A, that is, the triangulated category D(Ch A, quis). This category has small homomorphism sets, by [Wei94,remark 10.4.5]. Coproducts of complexes are also coproducts in DA, so that the triangulated category DA has all setindexed coproducts. This applies in particular to the unbounded derived category D Qcoh(A X ) of quasi-coherent A X modules. If Z ⊂ X is a closed subset with quasicompact open complement U = X − Z, we write D Z Qcoh(A X ) ⊂ D Qcoh(A X ) for the full triangulated subcategory of those complexes E of quasi-coherent A Xmodules whose restriction E \|U to U are acyclic. 9.9. Lemma. Let A be a Grothendieck abelian category with generating set of objects I. Then, an object E of the triangulated category DA is zero iff every map A[j] → E in DA is the zero map for A ∈ I and j ∈ Z. Proof. Let E be an object of the derived category DA of A such that every map A[j] → E in DA is the zero map for A ∈ I and j ∈ Z. We can choose a surjection J A j ։ ker(d 0 ) in A with A j objects in the generating set I. The inclusion of complexes ker The same argument applied to E[k] instead of to E shows that H k E = 0 for all k ∈ Z, so that E is quasi-isomorphic to the zero complex. Next, we recall the concept of compactly generated triangulated categories due to Neeman [Nee92] in the form of [Nee96]. The following two propositions are essentially due to Thomason [TT90]. We include the proofs here because only the commutative situation is considered in [TT90], and we need the explicite versions below. For an exact category E, write D b (E) for the triangulated category D(Ch b E, quis). Recall that a fully faithful functor A → B of additive categories is called cofinal if every object of B is a direct factor of an object of A. 9.12. Proposition. Let X be a quasi-compact and quasi-separated scheme which is the union X = Proof. In the triangulated category D Qcoh(A X ), every strictly perfect complex of A X modules is a compact object. To see this, note that for an A X vector bundle A and a set M j , j ∈ J, of quasi-coherent For a quasi-coherent A U module F , consider the sheafifiedČech complexČ(U , F ) associated with this covering. In degree k it is the quasi-coherent A X -modulě where the indexing set is taken over all k + 1-tuples i = (i 0 , ..., i k ) with 0 ≤ i 0 < · · · < i k ≤ n. The differential d k :Č(U , F ) k →Č(U , F ) k+1 for the component i = (i 0 , ..., i k+1 ) is given by the formula Note that the complexČ(U , F ) is concentrated in degrees 0, ..., n. Therefore, it represents the right derived functor Rj * of j * , that is, Proof. We first check that Rj * preserves cohomological support. Denote by j U : U − Z ⊂ U , j X : X − Z ⊂ X and j Z : U − Z ⊂ X − Z the corresponding open immersions, and note that the canonical map j * X j * M → j Z * j * U M is an isomorphism for every quasi-coherent A U -module M . By the existence of an ample family of line bundles on X, we can choose a finite open cover U = {U 0 , ..., U n } of U such that all inclusions U i0 ∩ ... ∩ U i k ⊂ X are affine maps. For a complex F of quasi-coherent A U -modules, we have The units of adjunction As pull-backs of affine maps, all inclusions (U i0 − Z) ∩ ... ∩ (U i k − Z) ⊂ X − Z are also affine maps, so that the functor j Z * TotČ(U − Z) represents Rj Z * , and we obtain a natural isomorphism of functors For the proof of the lemma, note that the composition j * Rj * = j * j * TotČ(U ) = TotČ(U ) is naturally quasi-isomorphic to the identity functor via the map (27). Furthermore, the unit of adjunction F → Rj * • j * (F ), which is adjoint to the map (27) applied to j * F , is a quasi-isomorphism for F ∈ D Z Qcoh(A X ) since both complexes have cohomological support in Z, so that we can check this property by restricting the map to U , where it is the quasi-isomorphism (27). Proof. The functor in (a) is clearly conservative. It is full by the following argument. Let A and B be strictly perfect complexes of A X -modules with support in Z, Let M be the pull-back of j * E → j * j * A and the U -isomorphism A → j * j * A. The induced maps M → j * E and M → A are U -isomorphism and U -quasi-isomorphism, respectively. By lemma 9.7 with A = sPerf(A X on Z), there is a A 0 ∈ A and a U -quasi-isomorphism A 0 → M . By lemma 9.6, there is a map A 0 ⊗ K(s n ) → B such that the two maps A 0 ⊗ K(s n ) → A 0 → M → j * E → j * j * B and A 0 ⊗ K(s n ) → B → j * j * B coincide. If follows that the map f : j * A → j * B is the image of the map in D c (A X on Z, U -quis) which is represented by the diagram A ∼ ← A 0 ⊗ K(s n ) → B. Therefore, the functor in (a) is full. Any conservative and full triangle functor is faithful, hence the triangle functor in (a) is fully faithful. It follows from proposition 9.12 and Neeman's theorem 9.11 (a) that the functor in (a) is cofinal for Z = X since in this case both categories contain as a cofinal subcategory the triangulated category generated by A X ⊗ L where L runs through the line bundles on X. This shows part (d) when Z = X. In order to prove (b), write R for the compactly generated triangulated category D Qcoh(A X ) with category of compact objects R c = D sPerf(A X , quis), see 9.12. Let S ⊂ R be the full triangulated subcategory closed under the formation of coproducts in R which is generated by the set S = sPerf(A X on Z, quis) ⊂ R c of compact objects. By part (d) for Z = X proved above and proposition 9.12, we have a cofinal inclusion R c /S c → D c Qcoh(A U ). By Neeman's theorem 9.11 (b) and (c), this implies that the functor R/S → D Qcoh(A U ) is an equivalence. In particular S is the kernel category of the functor D Qcoh(A X ) → D Qcoh(A U ), so that S = D Z Qcoh(A U ) is compactly generated by D sPerf(A X on Z, quis). Since D sPerf(A X , quis) = D c Qcoh(A X ) is idempotent complete, its epaisse subcategory D sPerf(A X on Z, quis) is also idempotent complete, so that we have the identification D sPerf(A X on Z, quis) = D c Z Qcoh(A X ), by Neeman's theorem 9.11 (a). In view of (b), the map in (c) is the restriction to compact objects of the equivalence of lemma 9.14. It is therefore also an equivalence. For the proof of (d), we simplify notation by writing D c (A X on Z, w) for the triangulated category D sPerf(A X on Z, w). Let V = U ∪ (X − Z) and consider the commutative diagram of triangulated categories in which all vertical functors are fully faithful, by (a), and the middle and the right vertical functors are cofinal since all four categories have as cofinal subcategory the triangulated category generated by A X ⊗ L, where L runs through the line-bundles on X, by proposition 9.12. Therefore, the right two vertical functors are equivalences after idempotent completion. Since the two left triangulated categories are the "kernel categories" of the two right horizontal functors, and this property is preserved under idempotent completion, the left vertical functor is also an equivalence after idempotent completion. Thus, the left vertical functor is cofinal. The functor D c (A X on Z, U -quis) ֒→ D c (A U on Z ∩ U, U -quis) in (a) can be identified with the left vertical functor in the diagram since U -quasi-isomorphisms are V -quasi-isomorphisms for complexes of A X -modules cohomologically supported in Z, and since the functor Proof. We start with a standard fact about K 0 of triangulated categories. Let T 0 ⊂ T 1 be a (fully faithful and) cofinal functor between triangulated categories. Then an object T of T 1 is isomorphic to an object of T 0 if and only if its class [T ] ∈ K 0 (T 1 ) is in the image of K 0 (T 0 ) → K 0 (T 1 ). This is because the cokernel of K 0 (T 0 ) → K 0 (T 1 ) can be identified with the quotient monoid of the abelian monoid of isomorphism classes of objects in T 1 under direct sum modulo the submonoid of isomorphism classes of objects in T 0 , so that an object of T 1 defines the zero class in the cokernel if and only if it is stably in T 0 . But for triangulated categories, an object is stably in T 0 iff it is isomorphic to an object in T 0 . For the proof of the corollary, we apply this argument to the inclusion in proposition 9.15 (a) which is cofinal, by 9.15 (d). We see that j * M is isomorphic in D c (A U on Z ∩ U, U -quis) to an object j * B, where B is a perfect complex of A Xmodules with cohomological support in Z. It follows that there is a zig-zag of Let P be the pull-back of j * F → j * j * B along the U -isomorphism B → j * j * B. Then P → j * F is a U -isomorphism, and it follows that P → B is a U -quasi-isomorphism. By lemma 9.7 with A the subcategory of those strictly perfect complexes which are cohomologically supported in Z, there is a U -quasi-isomorphism B ′ → P with B ′ strictly perfect and cohomologically supported in Z. Since X has an ample family of line-bundles and U is quasi-compact, we can choose line-bundles L i and global sections s i ∈ Γ(X, L i ), i = 1, ..., l such that the set of non-vanishing loci X si , i = 1, ..., l is an affine open cover of U . By Lemma 9.6, we can find an l-tuple of negative integers n such that the composition of U -quasi-isomorphisms 9.17. Proposition. Let X be a scheme which has an ample family of line bundles, let Z ⊂ X be a closed subscheme with quasi-compact open complement, and let j : U ⊂ X be a quasi-compact open subscheme. Let A X be a quasi-coherent O Xalgebra with involution. Then for any line-bundle L on X and any integer n ∈ Z, restriction of A X vector bundles to U defines non-singular exact form functors which induce isomorphisms on higher Grothendieck-Witt groups GW i for i ≥ 1 and a monomorphism for GW 0 . If, moreover, we have Z ⊂ U , then the form functors induce isomorphisms for all higher Grothendieck-Witt groups GW i , i ≥ 0. Proof. Let sPerf K0 (A U on U ∩ Z) ⊂ sPerf(A U on U ∩ Z) be the full subcategory of those strictly perfect complexes of A U -modules with cohomological support in U ∩ Z which have class in the image of K 0 (A X on Z) → K 0 (A U on Z ∩ U ). By cofinality 4.10, the duality preserving inclusion of exact categories with weak equivalences the U -quasi-isomorphisms and duality ♯ n j * L induces maps on higher Grothendieck-Witt groups GW i which are isomorphisms for i ≥ 1 and a monomorphism for i = 0. Restriction of vector-bundles defines a non-singular exact form functor which induces a homotopy equivalence of Grothendieck-Witt spaces by theorem 5.1, where 5.1 (c) follows from corollary 9.16 and lemma 5.5; 5.1 (e) and (f) are proved in lemma 9.6; the remaining hypothesis of theorem 5.1 being trivially satisfied. If Z ⊂ U , then K 0 (A X on Z) = K 0 (A U on Z ∩ U ), by proposition 9.15 (c), so that (28) is the identity inclusion, and the map (29), which induces a homotopy equivalence of Grothendieck-Witt spaces, is the map in the proposition. Proof of theorem 9.3. The theorem is a special case of proposition 9.17. Extension to negative Grothendieck-Witt groups For an open subscheme U ⊂ X, the restriction map GW 0 (X) → GW 0 (U ) is not surjective, in general, not even if X is regular. The purpose of this section is to extend the long exact sequence associated with the homotopy fibration of theorem 9.2 to negative degrees. Theorems 9.2 and 9.3 will be extended to a fibration and a weak equivalence of non-connective spectra. 10.1. Cone and suspension of A X . The cone ring is the ring C of infinite matrices (a i,j ) i,j∈N with coefficients a i,j in Z for which each row and each column has only finitely many non-zero entries. Transposition of matrices t (a i,j ) = (a j,i ) makes C into a ring with involution. As a Z-module C is torsion free, hence flat. The suspension ring S is the factor ring of C by the two sided ideal M ∞ ⊂ C of those matrices which have only finitely many non-zero entries. Transposition also makes S into a ring with involution such that the quotient map C ։ S is a map of rings with involution. For another description of the suspension ring S, consider the matrices e n ∈ C, n ∈ N, with entries (e n ) i,j = 1 for i = j ≥ n and zero otherwise. They are symmetric idempotents, i.e., t e n = e n = e 2 n , and they form a multiplicative subset of C which satisfies the Øre condition, that is, the multiplicative subset satisfies the axioms for a calculus of fractions. One checks that the quotient map C ։ S identifies the suspension ring S with the localization of the cone ring C with respect to the elements e n ∈ C, n ∈ N. In particular, the suspension ring S is also a flat Z-module. Let X be a quasi-compact and quasi-separated scheme. For a quasi-coherent sheaf A X of O X -algebras, write CA X and SA X for the quasi-coherent sheaves of O X -algebras associated with the presheaves C ⊗ Z A X and S ⊗ Z A X . On quasicompact open subsets U ⊂ X, we have (CA X )(U ) = C ⊗ Z A X (U ) and SA X = S ⊗ Z A X (U ), by flatness of C and S. If A X is a sheaf of algebras with involutions, then the involutions on C and on S make CA X and SA X into sheaves of O Xalgebras with involution. Let ǫ = 1 − e 1 ∈ C be the symmetric idempotent with entries 1 at (0, 0) and zero otherwise. The image Cǫ of the right multiplication map ×ǫ : C → C is a finitely generated projective left C-module. It is equipped with a symmetric form ϕ : Cǫ ⊗ Z (Cǫ) op → C : x ⊗ y op → x · t y. The idempotent ǫ makes (Cǫ, ϕ) into a direct factor of the unit symmetric form (C, µ), see 7.8 7.8. Therefore, tensor product (Cǫ, ϕ)⊗ Z ? defines a non-singular exact form functor Since S is a flat C-algebra, the quotient map C → S induces an exact functor ρ : CA X -Mod → SA X -Mod : M → S ⊗ C M on categories of modules which sends vector bundles to vector bundles. The two functors ι and ρ yield a sequence of non-singular exact form functors . The functors satisfy ρ • ι = 0 because S ⊗ C Cǫ = im (×ǫ : S → S) = 0 as 0 = ǫ ∈ S. The following theorem will allow us to extend the results of §9 to negative Grothendieck-Witt groups. Proof. The image ǫC of the left multiplication map ǫ× : C → C is a right C-module. We have a Z-bimodule isomorphism η : Z → ǫC ⊗ C Cǫ : 1 → ǫ ⊗ C ǫ = 1 ⊗ ǫ = ǫ ⊗ 1 and a C-bimodule map µ : Cǫ ⊗ Z ǫC → C : Aǫ ⊗ ǫB → AǫB such that the compositions are the identity maps. It follows that η and µ define unit and counit of an adjunction between the functors A X -Mod → CA X -Mod : M → Cǫ ⊗ Z M and CA X -Mod → A X -Mod : N → ǫC ⊗ C N . Since the unit η is an isomorphism, the first functor is fully faithful. In particular, ι is fully faithful. Proposition. The sequence of triangulated categories is exact up to direct factors. Proof. The multiplication map µ : Cǫ ⊗ Z ǫC → C factors through M ∞ ⊂ C and induces an isomorphism µ : Cǫ⊗ Z ǫC → M ∞ (it is a filtered colimit of isomorphisms of finitely generated free Z-modules). is exact as κ and ρ induce right adjoint functors on derived categories, and (31) induces a functorial distinguished triangle for every object of D Qcoh(CA X ). By proposition 9.12, these triangulated categories are compactly generated. Since ι and ρ preserve compact objects, the associated sequence of compact objects -which is the sequence in proposition 10.4 -is exact up to factors 9.11. Let sPerf S (CA X ) ⊂ sPerf(CA X ) be the full subcategory of those complexes V for which S ⊗ C V is acyclic. This subcategory is closed under the involution ♯ n L , so that sPerf S (CA X ) inherits the structure of an exact category with weak equivalences and duality from sPerf(CA X ). Since ρι = 0, ι induces a non-singular exact form functor ι : sPerf(A X ) → sPerf S (CA X ). Proof. The proof is a consequence of theorem 5.1 (or of lemma 5.6). Since ι is fully faithful, conditions (e) and (f) are satisfied. Since ι induces a fully faithful functor on derived categories, by proposition 10.4, condition (b) is also satisfied. The only non-trivial condition to check is (c). By lemma 5.5, we only need to show that for every M ∈ sPerf S (CA X ) there is an A ∈ sPerf(A X ) and a quasi-isomorphism Cǫ⊗ Z A → M . Let M be a strictly perfect complex of CA X modules with S ⊗ C M acyclic. By proposition 10.4, there is a zigzag of quasi-isomorphisms Cǫ ⊗ Z B ← N → M in sPerf S (CA X ) with B ∈ sPerf(A X ). Since N ∈ sPerf S (CA X ), proposition 10.4 implies that the counit of adjunction Cǫ ⊗ Z ǫC ⊗ C N → N is a quasi-isomorphism. We apply lemma 9.7 with CA X in place of A X and U = X, A = sPerf(A X ) to the quasi-isomorphism ǫC ⊗ C N → ǫC ⊗ C Cǫ ⊗ Z B ∼ = B, and obtain a strictly perfect complex A of A X -modules and a quasi-isomorphism A → ǫC ⊗ C N . Finally, the composition Cǫ ⊗ Z A → Cǫ ⊗ Z ǫC ⊗ C N = M ∞ ⊗ C N → N → M is a quasiisomorphism. For a quasi-coherent O X -algebra A X , call an A X -module M quasi-free if it is isomorphic to a finite direct sum i A X ⊗L i of A X -modules of the form A X ⊗L i for some line bundles L i on X. Note that a quasi-free A X -module is a vector bundle. 10.6. Lemma. Let X be a quasi-compact and quasi-separated scheme, and let A X be a quasi-coherent sheaf of O X -algebras. Let A, M be quasi-coherent CA X -modules with A quasi-free. Then the following hold. This map is surjective since C → S is, proving (a). The map is also a localization by a calculus of fractions with respect to the set of elements e n ∈ C, n ∈ N, of 10.1. This shows that (b) also holds. 10.7. Lemma. Let X be a quasi-compact and quasi-separated scheme, L a line bundle on X, and let A X be a quasi-coherent sheaf of O X -algebras with involution. Then for every n ∈ Z, the Grothendieck-Witt space is contractible. Write sPerf 0 (A X ) ⊂ sPerf(A X ) for the full subcategory of those strictly perfect complexes of A X -modules which are degree-wise quasi-free. Note that this category is closed under the duality ♯ n L . We equip the category sPerf 0 (A X ) with the degreewise split exact structure. Together with the set of quasi-isomorphisms of complexes of A X vector bundles, it becomes a category of complexes in the sense of Definition 5.4. Proof. The triangle functor in the lemma is fully faithful, by lemma 9.7 with U = X and A = sPerf 0 (A X ). It is cofinal, by Neeman's theorem 9.11 (a) and proposition 9.12. Let sPerf K0 (A X ) ⊂ sPerf(A X ) be the full subcategory of those strictly perfect complexes of A X -modules M whose class [M ] is in the image of the map K 0 ( sPerf 0 (A X ), quis) → K 0 ( sPerf(A X ), quis). Then the inclusion ( sPerf 0 (A X ), quis) ⊂ (sPerf K0 (A X ), quis) of exact categories with weak equivalences induces an equivalence of derived categories, so that another application of lemma 9.7 with U = X and A = sPerf 0 (A X ) finishes the proof of the claim. Proof of theorem 10.2. By theorem 9.2, we only need to treat the case Z = X. In this case, the total spaces are contractible, by lemma 10.7. Let sPerf K0 (SA X ) ⊂ sPerf(SA X ) be the full subcategory of those strictly perfect complexes of SA X -modules E whose class [E] is zero in the Grothendieck group K 0 (sPerf(SA X ), quis) of SA X -vector bundles. Furthermore, call a map f of strictly perfect complexes of CA X -modules an S-quasi-isomorphism if ρ(f ) is a quasi-isomorphism of complexes of SA X -modules. The set of S-quasi-isomorphisms is denoted by S -quis. Consider the commutative diagram of exact categories with weak equivalences and duality ♯ n L induced by inclusions and the map of rings with involution C → S Note that K 0 of all categories with weak equivalences in the diagram is 0. For the two left hand categories, this follows from lemma 10.7 and lemma 10.8, since for cofinal triangle functors T 0 ⊂ T , the map K 0 (T 0 ) → K 0 (T ) is injective. Since the left horizontal maps are surjective on K 0 , the middle two categories with weak equivalences have trivial Grothendieck group K 0 . For the upper right corner, vanishing of K 0 follows moreover from the fact that its K 0 is generated by classes of complexes concentrated in degree 0 and the fact that every quasi-free SA X -module is the image of a quasi-free CA X -module, so that the upper horizontal map is surjective on K 0 . Therefore, the right vertical and the lower right horizontal functors, which -a priori -have images in sPerf(SA X ), have indeed image in sPerf K0 (SA X ). We will show that the upper right horizontal and middle and right vertical functors induce equivalences of Grothendieck-Witt spaces (for any duality ♯ n L ), so that the lower right horizontal functor will induce an equivalence, too. The upper right horizontal functor is a localization by a calculus of right fractions, by lemma 10.6 and 5.12 (c). Therefore, theorem 5.11 shows that it induces a homotopy equivalence of Grothendieck-Witt spaces. For the right vertical functor, the resolution lemma 5.6 (which we can apply because of lemma 10.8) shows that it induces an equivalence of Grothendieck-Witt spaces. Similarly, by lemma 10.8, for every strictly perfect complex of CA X -modules M , there is a bounded complex A of quasi-free CA X -modules and a quasi-isomorphism A → M . A quasi-isomorphism of complexes of CA X -modules is, a fortiori, an S-quasi-isomorphism, so that the resolution lemma applies to show that the middle vertical functor induces an equivalence of Grothendieck-Witt spaces. Summarizing, we have shown that the lower right horizontal functor induces an equivalence of Grothendieck-Witt spaces. By the "change of weak equivalence theorem" (theorem 4.2), the sequence of exact categories with weak equivalences and duality ♯ n L ( sPerf S (CA X ), quis ) → ( sPerf(CA X ), quis ) → ( sPerf(CA X ), S -quis ) induces a homotopy fibration of Grothendieck-Witt spaces. Using proposition 10.5 we can replace the left hand term with ( sPerf(A X ), quis ). Using the equivalence of Grothendieck-Witt spaces of the lower right horizontal functor above and cofinality (theorem 4.10) applied to the inclusion of exact categories with weak equivalences and duality ( sPerf K0 (SA X ), quis ) ⊂ ( sPerf(SA X ), quis ), we can replace the right hand term in the sequence by ( sPerf(SA X ), quis ). Since the total space in the fibration of theorem 10.2 is contractible, we obtain a homotopy equivalence of spaces Proof. This is because the sequences Proof. This is because the maps GW n (S i A X on Z, L) −→ GW n (S i A V on Z, j * L). are homotopy equivalences for i ∈ N, by theorem 9.3. Proof. The map on vertical homotopy fibres is an equivalence, by theorems 10.11 and 10.12.	2008-11-28T00:07:32.000Z	2008-11-28T00:00:00.000	{ "year": 2009, "sha1": "989f2563e74b37a33cc964c323d1adca572eae90", "oa_license": null, "oa_url": "https://link.springer.com/content/pdf/10.1007/s00222-009-0219-1.pdf", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "01806fa338a77c396f8512063b1b30114a761cd0", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
231856382	pes2o/s2orc	v3-fos-license	The Role, Function, and Mechanism of Long Intergenic Noncoding RNA1184 (linc01184) in Colorectal Cancer Background Long intergenic noncoding RNA1184 (linc01184) has been recently discovered; however, its role in human diseases is limited to date. The present study is aimed at investigating the expression pattern and mechanism of linc01184 in colorectal cancer (CRC) tumorigenesis. Methods The expression of linc01184 in CRC tissues and cell lines was compared with that in normal controls. The functions of linc01184 in CRC cells were identified by overexpression and small interfering RNA (siRNA) approaches in vitro. Meanwhile, the target gene prediction software, luciferase reporter, RNA pull-down, and western blotting assays were used to analyze the oncogenic mechanism. Results We found that linc01184 was obviously upregulated in CRC tissues and cells when compared to normal controls, and its upregulation had a positive association with the CRC progression. linc01184 knockdown significantly suppressed CRC cell proliferation and invasion and promoted apoptosis. Besides, linc01184 acted as a competitive endogenous RNA (ceRNA) by directly binding to microRNA-331 (miR-331), and its overexpression resulted in notable increases of human epidermal growth factor receptor 2 (HER2), phosphorylated Ser/Thr kinases (p-Akt), and extracellular regulated protein kinase 1/2 (p-ERK1/2) at posttranscriptional levels in CRC cells, which were antagonized by miR-331. Conclusions The findings reveal for the first time that linc01184 is an enhancer for the proliferation and invasion of CRC by functioning as a ceRNA through the linc01184-miR-331-HER2-p-Akt/ERK1/2 pathway regulatory network. The miRNAs (18~24 nt) can affect the expression of approximately 60% of protein-coding genes and act as negative regulators in cancers by binding the 3 ′ untranslated region (3 ′ UTR) of the target mRNA [22][23][24]. The lncRNAs (>200 nt), which can be divided into the sense, antisense, bidirectional, intronic lncRNAs, and large intergenic ncRNAs (lincRNAs), have been implicated to interact with DNA, RNA, and protein, exerting almost all aspects of gene regulation via the molecular mechanisms of signal, decoy, guides, scaffold, and so on [24][25][26][27]. In the oncogenesis of CRC, numerous decoy lncRNAs (such as colon cancer-associated transcript-1 (MALAT1) [19], urothelial carcinoma-associated 1 (UCA1) [28], and H19 [29]) have been identified to function as competing endogenous RNAs (ceRNAs) or RNA sponges by binding miRNA and reduce the latter action on mRNA. linc01184, which is located on chromosome 5q23.3, is a newly discovered lincRNA and associated with the racial-specific erythrocyte traits [30]; however, its potential role in CRC and other cancers is still unknown. Our previous study has indicated that miR-331 is a CRC suppressor by targeting HER2 [31]. Meanwhile, we have also observed that linc01184 contains a complementary sequence for the seed region of miR-331. Thus, we perform the study to identify whether linc01184 acts as a contributor to CRC carcinogenesis by the interaction with miR-331. In the present study, we demonstrated that linc01184 overexpression was a characteristic molecular change in CRC and had a close association with the progression of CRC. We further explored the biological roles of linc01184 in CRC cell lines in vitro and confirmed its oncogenic mech-anism that linc01184 may function as a ceRNA to sponge miR-331, subsequently upregulating the expression of its target HER2 and triggering off the activation of p-Akt/p-ERK1/2 signaling pathways at the posttranscriptional levels. Thus, this study provided the first evidence that linc01184 may be a potential biomarker and play a carcinogenic role in the progression of CRC. Materials and Methods 2.1. Tissue Samples and Cell Lines. Paired CRC and adjacent normal tissue samples were obtained from 247 patients (average age: 55:2 ± 10:4 years) who underwent radical resection of CRC but without any other therapeutic interventions at the First Affiliated Hospital of Xi'an Jiaotong University between 2010 and 2014. The study was approved by the Ethics Committee of the Hospital and was performed in accordance with the Declaration of Helsinki. All patients provided written informed consent before the study, and their information was kept anonymous in the study. 2.7. Dual-Luciferase Reporter Assay. The luci-linc01184-WT or luci-linc01184-MUT and miR-331 mimic or negative control (NC) mimic (RiBoBio) were cotransfected into HCT-116 cells by lipofectamine 3000 (Invitrogen), and the luciferase activity of the transfected cells was evaluated by a dualluciferase reporter assay kit (Promega). All experiments were conducted according to the manufacturer's protocols. 2.8. RNA Pull-Down Assay. The biotin-labeled WT and MUT miR-331 which are obtained from Invitrogen Corporation were transfected into HCT-116 cells using lipofectamine 3000 (Invitrogen). Then, the enrichment of linc01184 in transfected cells was measured by the RNA pull-down kit (Promega) in accordance with the manufacturer's protocol. 2.9. Apoptosis Assay. The HCT-116 cells which are transfected with scramble control RNA, siRNA, pCDNA-linc01184, miR-331 inhibitor (RiBoBio), and siRNA+miR-331 inhibitor, respectively, were added to a 24-well plate and cultured for 48 h. The apoptosis of harvesting cells was evaluated by the FITC-Annexin V apoptosis detection kit (BD Biosciences, Piscataway, NJ, USA) according to the manufacturer's instructions. Statistical Analysis. An independent-sample t-test, chisquared test, and Kruskal-Wallis rank test (one-way ANOVA) were used to analyze the data by SPSS 13.0 (serial number 5026743; SPSS Inc., Chicago, Illinois, USA). A p value < 0.05 (two-tailed) was considered to be statistically significant. linc01184 Was Highly Expressed in CRC Tissues and in Cell Lines. To investigate the role of linc01184 in CRC, the RT-qPCR was used to measure linc01184 expression in CRC and normal controls. We found that linc01184 expression in CRC tissues was significantly higher than that in adjacent normal tissues (2:38 ± 0:06 vs. 1:12 ± 0:05, p < 0:001) (Figure 1(a)). Using the median (2.43) as a cut-off, 137 CRC patients were defined as high linc01184 expression cases. Among them, linc01184 overexpression had a significant association with the tumor size, invasion for lymph node and depth, metastasis, differentiated type, and TNM stage (all p < 0:05) but was not significantly correlated with age, sex, and tumor location (all p > 0:05) (Table 3). Furthermore, linc01184 expression in six CRC cell lines was 2-3 times of (Figure 1(b)). The findings indicated that linc01184 upregulation may play an oncogenic role in the progression of CRC. linc01184 Knockdown Significantly Inhibited CRC Cell Proliferation and Invasion. Furthermore, loss of function assay through RNA interference was performed to elucidate the effect of linc01184 on the CRC cells in vitro. We found that compared with the treatment of scramble control, the abilities of linc01184 expression (Figure 2(a)), cell viability (Figure 2 Disease Markers by the RNAHybrid 2.2 software. TargetScan revealed that linc01184 contains complementary sites for the seed region of miR-331 (Figure 3(a)). To further investigate the interaction between linc01184 and miR-331, the luci-linc01184-WT (AUGGGAGUUCAGGGGCU) and MUT (AUGGGAGUUCCCCCCAU) were constructed (5 ′ ⟶ 3 ′ ) (Figure 3(b)). Meanwhile, the luci-linc01184-WT or MUT and miR-331 mimic or NC mimic were cotransfected into HCT-116 cells; the dual-luciferase reporter assay showed that the activity of luci-linc01184-WT could be significantly inhibited by miR-331 (Figure 3(c)). Moreover, the RNA pull-down assay was conducted in HCT-116 cells transfected with miR-331-WT and MUT probes; the results revealed that linc01184 was dramatically enriched by the miR-331-WT probe (Figure 3(d)). In addition, the RT-qPCR assay showed that the highest linc01184 expression can be obtained at ≥1 μg/mL pCDNA-linc01184 transfection in HCT-116 cells, with about three times of expression compared to empty vector (Figure 3(e)). Thus, 1 μg/mL pCDNA-linc01184 and 20 nM siRNA were used to evaluate the effect of linc01184 on the miR-331 expression. The data showed that linc01184 overexpression significantly repressed miR-331 expression, which was promoted when silencing linc01184 (Figure 3(f)). Our findings indicated that linc01184 directly targeted miR-331 and acted as a ceRNA by sponging miR-331. 3.4. linc01184 Upregulated HER2 and Inhibited Apoptosis in CRC Cell. The target relationship between miR-331 and HER2 has been identified in our previous study [31]. Thus, to explore the effect of linc01184 on HER2 expression and apoptosis, HCT-116 cells were transfected with scramble control, 1 μg/mL pCDNA-linc01184, 20 nM siRNA, 60 nM miR-331 inhibitor, and combination of siRNA and miR-331 inhibitor, respectively. We found that linc01184 overexpression or miR-331 downregulation resulted in a significant upregulation of HER2 (Figures 4(a) and 4(b)) and inhibition of apoptosis (Figure 4(c)), which were abolished by siRNA. However, the effect of siRNA on both HER2 expression and apoptosis in HCT-116 cells can be antagonized by miR-331 3.5. linc01184 Promoted the Akt/ERK1/2 Signaling Pathways in CRC Cell. To further examine whether linc01184 is involved in HER2 downstream signaling pathways, the phosphorylation levels of Ser/Thr kinases (p-Akt) and extracellular regulated protein kinase 1/2 (p-ERK1/2) under various conditions were analyzed in the present study. As presented in Figure 5(a), the protein levels of Akt and ERK1/2 are almost unchanged in HCT-116 cells transfected with 1 μg/mL pCDNA-linc01184, 20 nM siRNA, 60 nM miR-331 inhibitor, or combination of siRNA and miR-331 inhibitor. However, linc01184 overexpression or miR-331 downregulation resulted in notable increases of p-Akt and p-ERK1/2 protein levels, which was opposite to siRNA transfection. The results were also been verified in Figure 5(b) that pCDNA-linc01184 or miR-331 inhibitor transfections significantly increased the ratios of both p-Akt/Akt and p-ERK1/2/ERK1/2 with respect to scramble control transfection, which were both decreased by siRNA transfection (all p < 0:05). Furthermore, cotransfection of siRNA and miR-331 inhibitor presented no effect on the protein levels of p-Akt and p-ERK1/2 (Figures 5(a) and 5(b)). Collectively, the results suggested that linc01184 overexpression triggered off the activation of p-Akt and p-ERK1/2 signals in CRC cells, which was antagonized by miR-331. Discussion The studies of human genomes have revealed that <2% of the genomes are protein-coding genes, while many noncoding elements are transcribed into ncRNAs, yielding tens of thousands of lncRNAs that lack protein-coding capacity [32][33][34]. Until now, only a limited number of lncRNAs are well understood for their functions; however, it is very clear that lncRNAs can play abundantly regulatory and functional roles in multiple biological processes. In cancers, lncRNAs execute their molecular functions through diverse mechanisms, such as the roles of scaffolds in chromatin remodeling [35,36], enhancer RNA (eRNA) in chromatin interactions [37,38], ceRNAs to sponge miRNAs [39], and natural antisense transcript (NAT) [40]. linc01184 is a newly discovered intergenic lncRNA with 3.0 kb length, but its biological function in human diseases is limited to date. In the study, we investigated the linc01184 expression profiles in CRC, the results revealed that linc01184 was significantly upregulated in both CRC tissues and cell lines in comparison with normal controls, and its upregulation was closely associated with tumor size, invasion, metastasis, differentiation, and TNM stage of CRC. Meanwhile, siRNA-mediated linc01184 knockdown significantly inhibited CRC cell proliferation and invasion and enhanced apoptosis in vitro. These results led us to believe that linc01184 may play a carcinogenic role and is a potential biomarker in the progression of CRC. HER2 belongs to the human epidermal growth factor receptor (EGFR) family and is an oncogenic driver to be well used as the marker of prognosis and therapeutic target in ovarian [41] and breast cancers [42]; it also plays a key factor in CRC [43]. Moreover, increasing evidences have demonstrated that lncRNAs can regulate the miRNA's expression as a ceRNA in multiple cancers, including CRC [44][45][46]. Our previous study has identified that miR-331 was a CRC suppressor by targeting HER2 to inhibit p-Akt/p-ERK1/2 8 Disease Markers signals [31]. Due to the expression pattern and biological function of linc01184 being opposite to those of miR-331 in CRC, thus, we hypothesized a mechanism that linc01184 may act as ceRNA to control the expression of HER2-p-Akt/p-ERK1/2 via sponging miR-331 in the development of CRC. To validate the hypothesis of the present study, the target gene prediction software, luciferase reporter assay, and RNA pull-down assay were used to analyze the potential interactions between linc01184 and miR-331. We found that the seed sequence of miR-331 was perfectly matched with linc01184. Meanwhile, miR-331 significantly inhibited the activity of linc01184-fused luciferase and pulled down linc01184. We also found that miR-331 expression was inverse to linc01184 in CRC cells in vitro. Additionally, western blot and fold change analyses showed that linc01184 overexpression in CRC cells significantly increased protein levels of HER2, p-Akt, and p-ERK1/2, while silencing of linc01184 displayed the opposite effect. Moreover, the combined treatment of miR-331 inhibitor and siRNA-linc01184 indicated that miR-331 inhibitor could antagonize the anticancer effects of siRNA-linc01184 on cell apoptosis and deactivation of HER2/p-Akt/p-ERK1/2 signaling pathways. Therefore, the fact that linc01184 acted as ceRNA to control the expressions of HER2, p-Akt, and p-ERK1/2 at posttranscription levels via directly binding to miR-331 may be one of the potential mechanisms in CRC tumorigenesis. Conclusions In the current study, we identified linc01184 as a positive modulator and revealed its mechanism in CRC carcinogenesis for the first time. We found that linc01184 functioned as a ceRNA of miR-331 through the linc01184-miR-331-HER2-Akt/ERK1/2 pathway regulatory network, in which linc01184 suppressed the expression of miR-331, subsequently upregulated HER2, and stimulated the activation of Akt/ERK1/2 signals, thereby promoting CRC cell proliferation and invasion.	2021-02-10T05:07:05.232Z	2021-01-29T00:00:00.000	{ "year": 2021, "sha1": "ac45b81b3302fe8d70bff09fbbdb839341d08bab", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/dm/2021/8897906.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "ac45b81b3302fe8d70bff09fbbdb839341d08bab", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
234494131	pes2o/s2orc	v3-fos-license	Epidemiology of Severe Foot Injuries in US Collegiate Athletes Background: The effects of foot injuries on collegiate athletes in the United States are of interest because of the short 5-year eligibility period in the National Collegiate Athletic Association (NCAA). Purpose: To discuss the epidemiology of severe NCAA foot injuries sustained over 10 years in 25 sports. Study Design: Descriptive epidemiology study. Methods: We utilized the NCAA Injury Surveillance System, which prospectively collects deidentified injury data for collegiate athletes. Severe injuries were classified as season- or career-ending injuries, injuries with >30-day time loss, or injuries requiring operative treatment. Injury rates (IRs) were analyzed per 100,000 athlete-exposures. Results: Of 3607 total foot injuries, 18.71% (n = 675) were classified as severe, with an IR of 5.73 per 100,000 athletic-exposures. For all severe injuries, the operative rate was 24.3%, the season-ending rate 37.0%, and the career-ending rate 4.4%. The proportion of recurrent injuries was 13.9%. Men’s sports with the highest severe foot IRs were basketball (IR = 10.71), indoor track (IR = 7.16), and football (IR = 7.08). Women’s sports with the highest severe foot IRs were cross-country (IR = 17.15), gymnastics (IR = 14.76), and outdoor track (IR = 14.65). Among all severe foot injuries, the most common was a fifth metatarsal fracture. The highest contact/noncontact injury ratios were phalangeal fracture, turf toe, and Lisfranc injury. The severe injuries with the highest operative rates were Lisfranc injuries, fifth metatarsal fractures, and midfoot fractures. The severe injuries associated with the highest season-ending IRs were Lisfranc injury, midfoot fracture, and general metatarsal fractures. Severe flexor/extensor injuries had the highest career-ending IRs, followed by turf toe. Severe injuries with the highest median time loss were sesamoidal fractures, calcaneal fractures, and plantar fascial injuries. Conclusion: Of all collegiate foot injuries sustained over a 10-year period, 18.7% were characterized as severe, and 24.3% of severe injuries required surgery. Basketball was the men’s sport with the highest severe IR, and cross-country was the women’s sport with the highest severe IR. Overall, female athletes experienced slightly higher severe foot IRs as compared with male athletes. Foot injuries are common sports-related ailments that can be detrimental to individual athletes and the success of their teams. 8,10,20 Because of the different physical demands and resources available at different levels of play (high school, National Collegiate Athletic Association [NCAA], professional), it is important to understand the epidemiology of foot injuries for players at each level. The effects of foot injuries on collegiate athletes is of particular interest because of the NCAA's relatively short 5-year eligibility period. As such, it is crucial for health care professionals to maximize each athlete's career through preventative strategies, proper treatment, and rehabilitation. Despite the variety of studies in the literature examining foot athletic injuries, the majority discuss professional sports, examine individual injuries, or focus on individual sports. 7,9,[13][14][15]19 Lievers et al 14 studied foot injuries in collegiate athletes over a 5-year span. However, the authors did not distinguish the injury based on severity, and no analysis on injury mechanism or individual diagnosis was conducted. The goal of our study is to discuss the epidemiology of severe NCAA foot injuries sustained nationally over a 10-year period in 25 unique sports to better understand injury trends across all sports as well as individual sports. Understanding the epidemiology of these injuries is important in understanding the risks that NCAA athletes face and tailoring treatment to mitigate these injuries. The purpose of this study is to provide an epidemiological analysis of severe collegiate foot injuries. We hope that this leads to more informed prevention, treatment, and management of these injuries, allowing athletes to maximize playing time and performance level while minimizing time missed because of injury. METHODS An institutional review board exemption was granted for this study. The NCAA Injury Surveillance System (NCAA-ISS) prospectively collects deidentified injury data for collegiate athletes. We utilized 2 cohorts from the NCAA-ISS: 2004-2005 to 2008-2009 and 2009-2010 to 2013-2014. The complete methodology of NCAA-ISS data collection was also described by Kerr et al. 12 This retrospective study includes data on all foot injuries across 25 NCAA sports. Injury rate (IR) per 100,000 athlete-exposures (AEs), operative rate, annual IR trend, reinjury rate, mechanism of injury, in-season status (preseason, regular season, postseason), and time-loss distribution were compiled and analyzed. Data Collection The NCAA-ISS collects self-reported data across all 25 sports. Participation in the NCAA-ISS is available to all collegiate institutions but is ultimately voluntary. Therefore, the NCAA-ISS collects data from just a sample of collegiate programs. The NCAA-ISS data sets are then weighted and extrapolated to create national estimates representative of all collegiate athletes nationwide. Therefore, it is possible that not all regions or athletic divisions are equally represented. 12 Health care professionals (athletic trainer or physician) report the details of injury incidence and exposure information. The report includes the injury or condition/circumstances, body part, diagnosis, and mechanism of injury. The data are reviewed by quality control staff and updated as necessary to report treatment and time to return to play. Deidentified data are then extracted from the electronic medical record and exported through an automated verification process. These exported data are reviewed and compiled into an aggregate research database made available to researchers. Definitions A reportable injury was defined as any injury that occurred during a team-sanctioned event that required attention from an athletic trainer or physician. We identified all foot injuries by manually searching through the NCAA-ISS database by diagnosis. Injuries were grouped into the following categories: calcaneal fracture, midfoot fracture, metatarsal fractures, fifth metatarsal fracture, Lisfranc injury, phalangeal fracture, sesamoid fracture, turf toe, plantar fascia, and flexor/extensor injuries. The following injuries were classified as "other": pes cavus, fat pad syndrome, contusion, infection, spasm, bone spur, pes planus, cuboid subluxation, metatarsalgia, osteoarthritis, sinus tarsi syndrome, talus avascular necrosis, nail avulsion, and other foot injury. Ankle injuries were excluded to examine the epidemiology of severe foot injuries in this cohort. For this article, we considered talus fractures as foot injuries. Achilles and deltoid ligament injuries were considered ankle injuries and thus excluded. We subclassified severe injuries as those that were season-or career-ending, injuries with >30-day time loss, or operative injuries. A reportable AE was defined as 1 athlete participating in 1 NCAA-sanctioned practice or competition in which he or she was exposed to the possibility of an athletic injury regardless of the time associated with that participation. Operative injuries were defined as injuries in which athletes underwent surgical treatment for the reported injury. Recurrence was defined as injuries that occurred again in the same athlete after return to play. Season-ending injuries were defined as injuries in which the athletes did not return to sports during the academic year in which the injury was sustained, which included season-and career-ending injuries. Time loss was defined as the number of days missed after an injury in athletes who ultimately returned to play in the same season. Season-and career-ending injuries were excluded from timeloss calculations. Career-ending injuries were defined as those in which the player did not play the same sport at any point after the injury occurred. Injury mechanism was defined as contact or noncontact. Contact mechanisms included contact with player, surface, equipment, or out-of-bounds object, while noncontact mechanisms were defined as overuse, illness, infection, or noncontact-related force. Whether the injury occurred during preseason, regular season, or postseason was also recorded. Statistical Analysis IRs per 100,000 AEs and percentages were calculated using injury counts over AEs. The overall rate, rate by sex, individual sports rate (25 sports), and annual injury trend were reported. Rate of operative injury, recurrence, injury mechanism, season of play, season-ending injury, and time loss were calculated by total counts of foot injuries. The distributions of the injury counts and time loss were examined. The incidence rate of foot injuries was calculated by measuring the total number of injuries among the overall AEs at risk. We used a Poisson regression model in which the injuries were the outcome with the offset of AEs to estimate the incidence rate with the corresponding 95% CI for the count data. The same model was used to estimate the incidence rate ratio by including sex/contact as an independent variable. The proportion of subgroup injury was provided with the corresponding permutation 95% CI. The point estimate as well as the robust 95% CI for the ratio of the male/ contact injury proportion versus the female/noncontact injury proportion was obtained using the generalized estimating equation model with Poisson distribution to control for mild violation of the Poisson distribution assumption. Time loss was summarized as median and interquartile range because of a skewed distribution. The analyses were performed using SAS Version 9.4 (SAS Institute). RESULTS A total of 3607 foot injuries were reported in 25 NCAA sports from the 2004-2005 to 2013-2014 academic years, resulting in an overall IR of 30.62 per 100,000 AEs. Among all foot injuries, the operative rate was 4.6%, the seasonending rate 6.9%, and the career-ending rate 1.0%. For athletes who missed time for non-season-ending injuries, 55.1% returned to play within 1 week of injury, and 10.8% missed >30 days. The men's sports with the highest foot IRs were soccer (IR ¼ 56.20), cross-country (IR ¼ 38.91), basketball (IR ¼ 38.77), and football (IR ¼ 34.98). The women's sports with the highest foot IRs were gymnastics (IR ¼ 88.58), cross-country (IR ¼ 52.88), soccer (IR ¼ 46.48), and outdoor track (IR ¼ 46.28). Severe Injury Of the 3607 total foot injuries, 18.71% (675) were classified as severe, with an IR of 5.73. For athletes with severe foot injuries, the operative rate was 24.3%, the season-ending rate 37.0%, and the career-ending rate 4.4%. The proportion of recurrent injuries was 13.9%. The proportion of athletes with severe injuries who experienced recurrence was significantly higher than the recurrence proportion for all injuries. The proportion of severe contact injuries was significantly lower than the proportion of all contact injuries. The proportion of severe noncontact injuries was significantly greater than the proportion of all noncontact injuries (Table 1). Injury Type Among all 3607 injuries, the most common, outside of "other," were turf toe, fifth metatarsal fracture, and flexor/extensor injuries. Injuries with the highest contact/ noncontact ratios were phalangeal fractures, Lisfranc injury, and turf toe. Injuries with the highest severe/nonsevere injury ratios were fifth metatarsal fractures, midfoot fractures, and sesamoid fractures. The injuries with the highest operative rate were Lisfranc injury, fifth metatarsal fracture, and midfoot fracture. The injuries with the highest season-ending rates were Lisfranc injuries, midfoot fracture, and fifth metatarsal fractures. Injuries with the highest median time loss were fifth metatarsal fractures, sesamoid fractures, and midfoot fractures ( Table 2). Among all severe foot injuries, the most common injuries were fifth metatarsal fractures. The highest contact/ noncontact injury ratios were phalangeal fracture, turf toe, and Lisfranc injury. The severe injuries with the highest operative rates were Lisfranc injuries, fifth metatarsal fractures, and midfoot fractures. The following severe injuries were associated with the highest season-ending IRs: Lisfranc injury, midfoot fracture, and general metatarsal fractures. Severe flexor/extensor injuries had the highest career-ending IRs, followed by turf toe. Severe injuries with the highest median time loss were sesamoidal fractures, calcaneal fractures, and plantar fascial injuries (Table 3). Sex Across all sports, the severe IR for women (IR ¼ 5.89) was slightly higher than for men (IR ¼ 5.64). The men's sports with the highest severe foot IRs were basketball (IR ¼ 10.71), indoor track (IR ¼ 7.16), and football (IR ¼ 7.08). The women's sports with the highest severe foot IRs were cross-country (IR ¼ 17.15), gymnastics (IR ¼ 14.76), and outdoor track (IR ¼ 14.65). Male athletes had a significantly higher operation rate for severe foot injuries as compared with female athletes. Severe injuries sustained by male athletes were significantly more likely to be seasonending than severe injuries sustained by female athletes. These injuries were also more likely to be career-ending for male athletes. Finally, severe injuries sustained by male athletes were more likely to be due to contact than in female athletes (Table 4). Severe injuries that occurred via contact mechanism were significantly more likely to be season-ending, whereas severe noncontact injuries were significantly more likely to be career-ending. For men's sports, the proportion of severe contact injuries was significantly greater than noncontact, whereas in women's sports, the proportion of severe noncontact injuries was significantly greater than the proportion of severe contact injuries (Table 5). DISCUSSION The overall IR was 30.62 per 100,000 AEs, with an overall operative rate of 4.6%. In comparison with other studies, hip/groin injuries in NCAA athletes had an IR of 53.06 per 100,000 AEs, but only 1.3% of these patients required surgery. 11 Hamstring strains had an IR of 30.5 per 100,000 AEs, and quadriceps strains had an IR of 10.7 per 100,000 AEs. 4,5 Similar numbers were reported for hip flexor and hip adductor strains. 6 It is also important to compare foot IRs with knee IRs, as the knee is a common location for lower extremity injury in sports. To our knowledge, no recent study has reported on knee IRs in the NCAA; however, there are data on the incidence of knee injuries in high school athletes as well as the rates for specific ligamentous injuries in collegiate athletes. 1,21 One study examining 5 years of high school athletic data cited a knee IR of 29.8 per 100,000 AEs. 21 A study describing the epidemiology of NCAA anterior cruciate ligament (ACL) injuries in individual sports found football to be the men's sport with the highest IR (17 per 100,000 AEs) and lacrosse to be the women's sport with the highest IR (23 per 100,000 AEs). 1 The overall IRs for all sports were not indicated in this study. Hunt et al 9 reported on the epidemiology of collegiate foot injuries across 37 sports teams over 2 consecutive seasons at a single institution. This study indicated an IR of 3.80 per 1000 AEs, the equivalent of 380 per 100,000 AEs, which would be much higher than our rate of 30.62 using the NCAA-ISS database. The same study by Hunt et al cited higher foot IRs and time missed in women's sports. Consistent with their results, female athletes were more likely to miss >30 days because of injury. Conversely, we found similar IRs for men and women for all injuries as well as severe injuries. Furthermore, the following were significantly higher for male athletes when compared with female athletes: operative rates for all foot injuries, season-ending injuries, and proportion of contact injuries. Finally, Hunt et al reported an operative rate of 2.0%, whereas we found a higher operative rate of 4.6% using the NCAA-ISS database. In the present study, a significant portion of all injuries were relatively minor and did not result in prolonged functional impairment: 55.1% resulted in a time loss between 0 and 6 days, with median time loss of 4 days. Furthermore, the operative rate for all injuries was low at 4.6%. Among all foot injuries, 6.9% were season-ending, and 1.0% were career-ending. Similarly, Lievers et al 14 examined the epidemiology of all foot injuries in NCAA athletes using the same database, yet there are numerous fundamental differences between our studies. We examined 10 years of severe injuries across 25 unique teams, whereas Lievers et al looked at 5 years of data across 15 unique teams and did not differentiate by injury severity. 14 It is important to note that some of our findings may be directly influenced by the way in which we defined "severe" injury. For example, >37% of severe injuries were seasonending. This is likely influenced by the fact that our definition requires 30 days to be missed and/or the presence of operative intervention. For many collegiate sports, 30 days may represent one-third or one-fourth of an entire season; therefore, the timing in which the injury occurred in relation to the season may influence whether it was determined to be season-ending. Additionally, many injuries that require surgery may cause a player to miss >30 days, which could contribute to this finding. Within the severe injury subcategory, we examined individual diagnosis outcomes, mechanisms of injury, operative rates, sex-based discrepancies, and whether the injury was season-and/or career-ending. In terms of sex, men and women had similarly severe overall IRs. Severe foot injuries in men were most common in basketball, followed by indoor track and football. For women, severe injuries were most common in cross-country, followed by gymnastics and outdoor track. This is consistent with our findings that severe injuries in women are more likely to occur via a noncontact mechanism, whereas severe injuries in men are more likely to occur via contact (ie, football or basketball). Our findings suggest that women may be more likely to sustain significant overuse injuries, ultimately leading to a greater percentage of noncontact injuries. These findings are consistent with a study by Ristolainen et al, 18 which found female athletes to be at increased risk of overuse injuries to the ankle when compared with men. The exact cause of this difference is unclear, but 1 possible explanation is the female athlete triad. There is evidence in the literature showing that certain women's sports place pressure on women to maintain certain body weights, which can contribute to a triad of low energy, menstrual dysfunction, and decreased bone mineral density. 16 These athletes are at significantly increased risk for certain overuse injuries, such as stress fractures. 16 While the NCAA-ISS database does not provide any information regarding the health of individual athletes, this is 1 possible explanation that may contribute to our findings. Interestingly, male athletes in our study had a much higher proportion of severe injuries to nonsevere injuries than female athletes. This contrasts prior data suggesting that female athletes experience more severe injuries than male athletes in high school and college sports. 17,23 The discrepancy between male and female athletes with regard to men experiencing significantly more seasonand career-ending foot injuries may be the result of several possibilities, including biological sex or hormone differences or training behaviors as well as differences in rules, regulations, or training protocols in sports. 18 One consideration is the effect of estrogen on athletes, with studies showing that estrogen levels may affect performance and IRs, specifically placing women at increased risk for ligament injury. 3 While women may be at increased risk for ligamentous injuries as a result of higher estrogen levels, the majority of injuries classified as severe in our study were fractures and other bony injuries, which may explain the increased career-and season-ending IRs in the male cohort. That said, the literature as it pertains to sex-based differences is somewhat limited, and there is presently no consensus for why we see these discrepancies. Contact mechanisms were responsible for a greater percentage of all injuries, whereas noncontact mechanisms were responsible for a greater percentage of severe injuries. While this may seem counterintuitive, this is in line with other severe lower extremity injuries, such as ACL tears, which have been shown to occur because of a noncontact mechanism as often as 75% of the time. 2 Our findings, however, contradict a recent study examining 10 years of NCAA basketball ankle injury data, which show contact mechanisms to be responsible for a greater percentage of injuries. 22 This discrepancy may be due to the fact that the study did not differentiate between all injuries and severe injuries. Furthermore, foot injuries were not included in their study, and only basketball was evaluated. Of note, stress fractures and other overuse injuries that occur secondary to a noncontact mechanism may be more likely to occur in the foot, given that the foot bears a larger load than other areas of the body during weightbearing exercise. Because noncontact mechanisms contributed to the majority of severe injuries in our study, this suggests that noncontact mechanisms are responsible for the majority of severe foot injuries. However, it should be noted that season-and career-ending injuries were more likely to occur via a contact mechanism, suggesting that contact injuries may pose a greater threat to an athlete's career. The present study has several limitations. First, this is a database study coded by athletic trainers and team physicians with no specific central guidelines. While some data are objective measurements, there are many variables with subjective components, including the diagnosis of injury. However, all the injuries are coded per the final diagnosis released by the athletic team, and NCAA-ISS has a rigorous data-monitoring and quality control system, limiting potential bias. Second, because of the subjectivity of the coding process, we created a subclassification of severe versus nonsevere injuries. We based our cohort on time loss and/or operative intervention, which are significant factors but ultimately not the only criteria for an orthopaedist in determining whether an injury was severe. Additionally, as this is a database study, there is no standardized decisionmaking process for whether to pursue operative intervention across all athletes and providers. Because we were not able to evaluate athletes after graduation, it is possible that certain seniors characterized as having career-ending injuries may not have actually had them. As a result, we may have slightly overestimated the prevalence of severe career-ending foot injuries. This is a limitation for which we could not control within the methodology of our study. Additionally, a season-ending injury may be influenced by the time of year in which it occurred. While the NCAA-ISS collects data from the first preseason practice through the final postseason game, to our knowledge, it does not specify which phase of the season an injury occurred. Therefore, we are unable to comment on severe IR differences among the preseason, regular season, and postseason. Finally, >50% of all included injuries in this study were grouped into the "other" category. While this is but a small portion of our statistical analysis, it suggests that 1 or more common injuries were not evaluated because they were grouped into the "other" category. However, only 27.77% of severe injuries were characterized as such, suggesting that, in line with the purpose of our study, the majority of severe injuries were included. CONCLUSION Foot injuries in NCAA athletes range from minor to devastating. Severe injuries are especially of interest to orthopaedic and sports medicine providers. Of all collegiate foot injuries sustained over a 10-year period, 18.7% were characterized as severe, and 24.3% of severe injuries required surgery. Basketball was the men's sport with the highest severe IR, and cross-country was the women's sport with the highest severe IR. Overall, female athletes experienced slightly higher severe foot IRs than male athletes. Future directions include breakdown of severity by all sports as well as the specific injuries most common to individual sports.	2021-05-15T05:29:16.187Z	2021-04-01T00:00:00.000	{ "year": 2021, "sha1": "bce60516686a302d16256b7864afed2c6cbc974c", "oa_license": "CCBYNCND", "oa_url": "https://journals.sagepub.com/doi/pdf/10.1177/23259671211001131", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "bce60516686a302d16256b7864afed2c6cbc974c", "s2fieldsofstudy": [ "Medicine", "Education" ], "extfieldsofstudy": [ "Medicine" ] }
87608594	pes2o/s2orc	v3-fos-license	Raphignathus saboorii n. sp. a new species of the genus Raphignathus (Acari: Trombidiformes: Raphignathidae) from northwest Iran A new species of raphignathid mites from northwest Iran, Raphignathus saboorii n. sp. is described and illustrated. A key to all known species of Raphignathus in Iran is also given. INTRODUCTION Members of the family Raphignathidae are predacious. They can be found underneath tree bark, in lichens, in moss, in leaf litter, in pigeon nests, in soil, on a wide range of plants and in house dusts (Fan and Yin 2000;Khanjani and Ueckermann 2003). This family are easily recognized by the fused cheliceral bases, forming a stylophore; cervical peritremes not embedded in dorsal surface of stylophore and confluent coxae (Meyer and Ueckermann 1989). The genus Raphignathus Duges is the oldest genus in this family and has a worldwide distribution with more than 60 species of which seven are known from Iran, namely: R. collegiatus Atyeo, Baker and Crossley, 1961;R. gracilis (Rack 1962); R. giselae Meyer and Ueckermann, 1989;R. zaoi Hu, Jing and Liang, 1995;R. aciculatus Fan, 2000;R. hecmatanaensis Khanjani and Ueckermann, 2003;R. protaspus Khanjani andUeckermann, 2003 (Khanjani andUeckermann 2003;Ghorbani et al. 2010). In this paper a new species, R. saboorii n. sp. from northwest Iran is described and figured. MATERIALS AND METHODS Litter and soil samples were taken from apple orchards at Maragheh, East Azerbaijan Province, Iran. Measurements were taken as follows: length of idiosoma from suture between gnathosoma and idiosoma to posterior margin of idiosoma; width of idiosoma at broadest part of idiosoma; and setae from their insertion to their tips. Distances between setae were measured between their insertions. The terminology and abbreviations of dorsal and ventral setae follow Kethley (1990) and all measurements are given in micrometers (µm). Male and immature stages -Unknown. Etymology -The species is named in honor of Prof. Alireza Saboori (Professor of Entomology, University of Tehran, Iran), for his great efforts to develop of Acarology in Iran. Type material -Holotype and 1 paratype female of Raphignathus saboorii n. sp. were collected from soil in apple orchards, 20 August 2009, Maragheh, East Azerbaijan province, Iran, by Hamed Ghorbani. The holotype and paratype females will be deposited in the Arachnida Collection of Plant Protection Research Institute, Pretoria, South Africa. Remarks -The new species is very closely related to R. sceptrum Chaudhri, Akbar and Rasool (Chaudhri et al. 1979) by having: 4 pairs of genital setae, 2 pairs of setae on interscutal membrane, 2 pairs of setae on palpfemur and simple dorsal setae but can be distinguished from the later as follows: 1. R. sceptrum has one pair of small shields behind the anteromedian propodosomal shield which are absent in new species. 2. setae f1 in new species well behind the anterior margin of opisthosomal shield but in R. sceptrum f1 situated on anterior margin of opisthosomal shield. 3. tarsi I and IV with 21 and 13 setae in new species vs 19 and 12 in R. sceptrum, respectively. Raphignathus saboorii also resembles R. gracilis by having 2 pairs of interscutal setae, 2 setae on palpfemur, simple dorsal setae and absence of small shields on interscutal membrane but, can be separated by following features: 1. genital shields with 4 pairs of setae in R. saboorii vs. 3 pairs in R. gracilis 2. tarsus II with 17 setae vs. 16 in R. gracilis. 3. coxisternal III-IV is absent in new species but present in R. gracilis.	2019-03-31T13:44:14.757Z	2011-12-20T00:00:00.000	{ "year": 2011, "sha1": "7fdec770b8906107f377a04d18f2562b4ae32d2c", "oa_license": "CCBY", "oa_url": "https://www1.montpellier.inra.fr/CBGP/acarologia/export_pdf.php?id=2025&typefile=1", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "9d4c61e10431f4b2eb13bee8b72cc98513ebe83a", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology" ] }
102654638	pes2o/s2orc	v3-fos-license	Optimization of biodiesel production from castor oil by response surface methodology This work investigated the possibility of using response surface methodology based on a four factor five level central composite design to optimize biodiesel production from castor oil. The reaction variables were oil: ethanol ratio, lipozyme concentration, time and temperature. The properties of the biodiesel produced were assessed. The linear coefficient of lipozyme 2 concentration (B), temperature (D), the quadratic coefficient of lipozyme concentration (B ), time 2 2 (C ), temperature (D ), the interaction of oil/ethanol ratio and lipozyme concentration (AB), the interaction of lipozyme concentration and time (BC) had significant effect on the biodiesel yield (p<0.05).The other variableswere not significant (p>0.05).The specific gravity was (0.91), refractive index (1.41 + 0.01), viscosity (14.1), cetane number (53.9), calorific value (38.0+ o o 0.10), flash point (150 C), cloud point (7 C) iodine value (101 + 0.53), acid value (0.57 + 0.01), and saponification value (180+0.25). Maximum yield was found to be 93.0% under the conditions of oil: ethanol ratio of (1:7.14), lipozyme concentration (40 U), time (165 min) and temperature o 50 C.The properties of the biodiesel produced were largely in conformity with ASTM D6751 standards. Introduction threats to food security when used as feedstock in fuel The rapid depletion of world's petroleum industry (Koh and Mohd, 2011). reserves and increased ecological concerns have Several parameters, such as the type of prompted the demand of environment friendly catalyst, alcohol/ oil molar ratio, temperature, purity renewable energy resources (Ashish et al., 2010). The of the reactants and free fatty acid content have a development of alternative energy source to extend greater influence on the transesterification reaction the fossil fuels has received a large interest in the last (Giovanilton et al., 2011). few decades due to an increasing concern to protect This study's main objective was to develop an the environment and conserve the nonrenewable approach for better understanding the relationships natural energy resources (Vicente et al., 1998). between the variables (factors) and the response A number of processes such as chemical, ethyl ester formation to obtain the optimum enzymatic and supercritical alcohol treatment have conditionsthat will improve the biodiesel yieldusing been employed for biodiesel production (Warabi et al., response surface methodology (RSM). 2004). Transesterification reaction consists of reversible reactions between alcohol and Materials and methods triacylglycerol present in oil or fats to produce three Collection of castor seed moles of fatty acid alkyl esters and glycerol as a co- The castor seeds were collected from Sabonproduct (Abdullahi et al., 2009). The use of non-edible Gari Market Kaduna State Nigeria and were ground oils such as karanja, polanga, jatropha, and castor oil into powdered form by using a simple grinder have attracted great attention as they do not pose machine. Oil extraction Characterization of the biodiesel produced The castor oil was extracted using soxhlet The fatty acid ethyl ester was characterized for specific gravity using specific gravity bottle, extractor. About 500 ml of n-hexane was poured in a viscosity was determined using viscometer (NDJ-5A) round bottom flask and 100 g of ground castor seed model, refractive index using Abbe's refractometer packed in a filter paper was placed in the thimble (Model AR-001). The cetane number of the produced according to AOAC (2000) with modification. b i o d i e s e l w a s d e t e r m i n e d a c c o r d i n g t o Krisnangkura(1989). Calorific value was measured Transesterification reaction according to the method of Gerpen (2005), Flash Transesterification reaction was carried out in point was determined according to ASTM D 93 screw-capped vials placed inside a reciprocal shaker. (2000). The cloud points was determined according to The initial reaction mixture consisted of castor oil: ASTM D2500 (2000). Acid value, iodine and ethanol molar ratio of 1:7.14 lipozyme (40 U) saponification number were determined by titrimetric immobilized from Mucormiehei and was stirred at 200 method according to AOAC (2000). rpm along with the respective controls according to Kumari et al. (2009) Results The highest biodiesel yield of 93.0% and Design of Experiments 75.53% was obtained for experimental and predicted Central composite design integrated in responses respectively in run 18. The lowest response response surface methodology (RSM) provided in the of 5.0 % was recorded at run 23 with the predicted Design-Expert 7.1.4 (Stat-Ease, Inc) was employed to being 10.78% (Table). Figure 1 shows the 3D plot for the interaction levels of lipozymeconcentration the biodiesel yield did of oil: ethanol ratio and lipozyme concentration on not increase. However at optimum lipozyme biodiesel yield. The lower and higher levels of concentration, a maximum biodiesel yield was lipozyme did not result in higher yield of biodiesel, but obtained, but the yield decreased with increase and maximum biodiesel yield was obtained at optimum decrease in temperature. Figure 1 (f) shows the 3D lipozyme concentration. Figure 1 (b) shows that the plot for the interaction of incubation time and biodiesel yield increased with increase in oil: ethanol temperature on biodiesel yield. ratio. The yield decreased with increase in the Table 3 shows some fuel properties of incubation time. Figure 1 (c) shows the 3D plot for the biodiesel produced. The specific gravity was 0.91 and interaction of oil: ethanol ratio and temperature. The refractive index was 1.41 + 0.01. The biodiesel had yield increased with the increase in oil: ethanol ratio. the viscosity of 14.1, the calorific value of 38.0 + 0.10 Figure 1 (d) shows that at lower and higher levels of mJ/ kg and cetane number of 53.9. The flash point 0 0 lipozyme concentration the biodiesel yield did not and cloud point were 150 C and 7 C respectively. The increase. However at optimum lipozyme iodine value was 101.0 + 0.53 gI2/100?g, acid value concentration, a maximum biodiesel yield was was 0.57 + 0.01 mg KOH/g and saponification value obtained. Figure 1(e) shows that at lower and higher was 180 + 0.25 mg KOH/g respectively. Table 3. Physicochemical properties of castor oil seed biodiesel Discussion The high viscosity of the fuel reduces the In each of the three dimensional plots, the atomization but this issue can be handled with the use interaction effect of the two parameters was plotted of blends with petro-diesel (Deep et al., 2017). while the third and fourth parameters were fixed at The result shows the less likelihood of the the medium values. The decrease in biodiesel yield biodiesel produced to ignite accidentally and could be at higher oil ethanol ratio could be due to separation safely handled for storage owing to its high flash point. problem resulting from excessive ethanol, or in The low cloud point helps in keeping the biodiesel in activation of the enzyme by ethanol (Molla and liquid condition which makes transportation easier. The Nigus, 2014).The decrease in biodiesel yield at high lower calorific value could be attributed to the presence lipozyme concentration and oil ethanol ratio could of chemically bound oxygen in the fatty acid chains be that, in the presence of a high amount of lipase (Srivastava and Prasad, 2000). Aldo et al. (2003) -1 because the enzyme active site cannot be exposed reported the calorific value of 30.4 mJkg in castor oil to the substrates and many molecules of the biodiesel.The molecular weight, fatty acid chain length, enzyme aggregate together and thus results in low degree of unsaturation and degree of conjugation biodiesel yield (Liou et al., 1998). The elliptical determine the refractive index of biodiesel nature of the 3D plots shows the significance of the (Sadrolhossein et al.,2011). interactions of variables on biodiesel yield (Samukawa et al., 2000). Bello and Makanju (2011) Conclusion used ethanol; oil molar ratio of 6 to 1 and obtained The analysis of variance for the quadratic model the biodiesel yield of 92% after 3 hours of stirring revealed that lipozyme concentration, oil methanol from castor oil. ratio, temperature and incubation time are the The analysis of variance for the quadratic significant factors in biodiesel production from castor model revealed that lipozyme concentration, oil. The properties of the biodiesel produced were temperature, incubation time with their quadratic largely in conformity with ASTM D6751standards coefficients were the significant factors in biodiesel production. The value of the regression coefficient	2019-04-09T13:06:32.324Z	2017-10-30T00:00:00.000	{ "year": 2017, "sha1": "af1299267bf8098b1e33862e0cb25fc32f25a5f9", "oa_license": "CCBY", "oa_url": "https://www.ajol.info/index.php/njb/article/download/162333/151837", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "204b9561ed65b07644c55d39a2676e3647e47d03", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Chemistry" ] }
12489348	pes2o/s2orc	v3-fos-license	Impact of a CXCL12/CXCR4 Antagonist in Bleomycin (BLM) Induced Pulmonary Fibrosis and Carbon Tetrachloride (CCl4) Induced Hepatic Fibrosis in Mice Modulation of chemokine CXCL12 and its receptor CXCR4 has been implicated in attenuation of bleomycin (BLM)-induced pulmonary fibrosis and carbon tetrachloride (CCl4)-induced hepatic injury. In pulmonary fibrosis, published reports suggest that collagen production in the injured lung is derived from fibrocytes recruited from the circulation in response to release of pulmonary CXCL12. Conversely, in hepatic fibrosis, resident hepatic stellate cells (HSC), the key cell type in progression of fibrosis, upregulate CXCR4 expression in response to activation. Further, CXCL12 induces HSC proliferation and subsequent production of collagen I. In the current study, we evaluated AMD070, an orally bioavailable inhibitor of CXCL12/CXCR4 in alleviating BLM-induced pulmonary and CCl4-induced hepatic fibrosis in mice. Similar to other CXCR4 antagonists, treatment with AMD070 significantly increased leukocyte mobilization. However, in these two models of fibrosis, AMD070 had a negligible impact on extracellular matrix deposition. Interestingly, our results indicated that CXCL12/CXCR4 signaling has a role in improving mortality associated with BLM induced pulmonary injury, likely through dampening an early inflammatory response and/or vascular leakage. Together, these findings indicate that the CXCL12-CXCR4 signaling axis is not an effective target for reducing fibrosis. Hepatic fibrosis Hepatic fibrosis is a pathological wound healing response to liver damage that is characterized by excess production and deposition of extracellular matrix (ECM) components [1][2][3]. Insults that can trigger a fibrotic response include viral infection, alcohol or drug toxicity, metabolic diseases and a variety of factors that induce an inflammation response in the liver [1,4,5]. The increased deposition of ECM and its altered composition lead to progressive functional deficits [6,7]. Hepatic fibrosis and its end stage cirrhosis ranked 14 th and 10 th leading causes of death in the world and in developed countries respectively [8]. Unfortunately, this trend is expected to increase worldwide [8]. Hepatic fibrosis is reversible whereas cirrhosis, the end stage consequence of fibrosis, is generally not [2,3]. Thus, it is important to identify therapy for hepatic fibrosis, as none currently exist [4,6] and to prevent its progression to cirrhosis. It has been established that hepatic stellate cells (HSCs) are the main cells contributing to the process of liver fibrogenesis [2,4,9]. HSC are fat and vitamin A storing cells in the body [10] but following liver injury, HSC become activated and undergo a morphological transition to myofibroblast-like cells [2,11]. Activated HSCs produce an appreciable amount of ECM components [11]. Activation factors can include damaged hepatocytes, infiltrating inflammatory cells, endothelial cells, Kupffer cells (tissue marcrophages), changes in ECM composition and metabolites of toxic agents [2,11]. Pulmonary fibrosis Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible and often fatal pulmonary disorder of unknown etiology and is characterized by progressive fibrosis of the lung parenchyma leading to scarring and loss of lung function [12]. IPF primarily occurs in older adults with a median survival time of 2-4 years after diagnosis [13,14]. Prevalence in the United States has increased steadily from 202.2 cases per 100,000 people in 2001 to 494.5 cases per 100,000 people in 2011 [15]. Despite Phase 2 and 3 clinical trials indicating that pirfenidone was able to improve lung function in patients [16][17][18] there is still no current treatment for IPF as antiinflammatory, anti-fibrotic and immunosuppressive therapies have proven ineffective [12,19,20]. Hence, there is an urgent need for an effective IPF therapy. The fibrotic response in IPF appears to be driven by abnormally activated alveolar epithelial cells (AECs) which induce fibroblast proliferation, differentiation and recruitment [12]. Activated fibroblasts (myofibroblasts) secrete exaggerated amounts of ECM and destroy the architecture of the lung (reviewed in [12]). The origin of lung myofibroblasts remains a contentious issue. Recent lineage tracing studies based on a Foxd1-Cre line have shown that lung resident perivascular mesenchymal cells in addition to "lung fibroblasts" contribute to the lung myofibroblast population after bleomycin (BLM)-induced injury [21]. Circulating fibrocytes derived from bone marrow have also been reported to contribute to the myofibroblasts and type I collagen production in the lung [22][23][24][25][26][27]. However, recent studies have shown that bone marrow derived fibrocytes make a negligible contribution to type I collagen in lung fibrosis [28]. CXCR4/CXCL12 signaling and its putative role in lung and hepatic fibrosis The 7-transmembrane G-protein coupled chemokine receptor, CXCR4 and its ligand CXCL12 (SDF-1α-stromal cell derived factor-1α) [29,30] are involved in the homing of hematopoietic stem cells to the bone marrow, mobilization of stem cells from the bone marrow to the peripheral blood and injured tissues and act as a chemoattractant for different leukocyte populations [31][32][33]. CXCL12 is expressed in bile duct epithelial cells in normal human liver [34,35] and its expression is upregulated in the liver and plasma of patients with advanced hepatic fibrosis relative to control patients [35]. Both human and murine HSCs express CXCR4 and its expression increases with HSC activation [36]. In particular, CXCR4/CXCL12 signaling has been shown to induce HSC proliferation and collagen I production [36]. Furthermore, liver sinusoidal endothelial cells have also been shown to express Cxcr4 and another CXCL12 receptor, Cxcr7 after hepatic injury [37] and to participate in liver regeneration and fibrosis. In pulmonary fibrosis (PF), it was shown that circulating fibrocytes were increased in patients with stable IPF relative to controls and quantification of fibrocytes may even serve as an indicator of mortality in IPF patients [24]. Given the evidence that fibrocytes from the circulation and progenitor stem cells from the bone marrow maybe recruited to the lung during pulmonary fibrogenesis [22,23,[25][26][27], it has been argued that the mobilization of these cells to the injured lung is likely in response to CXCL12 to mediate fibrosis [23,[25][26][27]. Specifically, inhibition of CXCR4/CXCL12 signaling with anti-CXCL12 antibody reduced recruitment of CD45 + ColI + CXCR4 + fibrocytes in BLM exposed mice [25] and reduced lung fibrosis [23,[25][26][27]. CXCR4/CXCL12 antagonist There are a number of small molecule CXCR4 antagonists, initially generated as potential anti-HIV treatments or as hematopoietic stem cell mobilization agents [38]. The approved, i.v.administered CXCR4 antagonist, plerixafor (AMD3100) [39], had been evaluated in a number of BLM-induced pulmonary fibrosis models [23,26,27] including one recently-retracted article [40]. In addition, AMD3100 had been used in models of hepatic injury with reported beneficial results in a rat model of acute liver failure [41] and a report of exacerbation in a murine model of chronic liver injury [42]. Recently, an orally bioavailable CXCR4 antagonist, AMD070, had been developed and had shown safety and proof-of-concept oral efficacy in a human clinical trial for HIV treatment [43,44]. In the hope of resolving the apparently contradictory data for the effects of CXCR4/ CXCL12 modulation in models of hepatic injury and evaluating whether CXCR4/CXCL12 could be a potential therapeutic target in fibrotic diseases, we tested the efficacy of AMD070 in a BLM induced murine model of PF and in a carbon tetrachloride (CCl 4 ) induced murine model of hepatic fibrosis. Our results suggest that AMD070 was able to increase survival in BLM-induced PF but surprisingly, had no effect on lung fibrosis. Furthermore, AMD070 had no effect in a CCl 4 induced murine model of hepatic fibrosis. Similar to its predecessor AMD3100, AMD070 showed a dose response for leukocytosis attributed to CXCR4 antagonism [45,46]. In summary, our data suggest inhibition of the CXCR4/CXCL12 axis may alter the early inflammatory and vascular response to acute fibrosis but has no direct effect on the deposition of fibrotic matrix per se. Materials and Methods All animal studies described in this article had complied with the Canadian Council on Animal Care guidelines and University of British Columbia Animal Care Committee. All animal studies had also been approved by the University of British Columbia Animal Care Committee. Animals were housed in ventilated cages, maximum 5 per cage, in a 12-hour light/dark cycle and cages were changed once every 10 to 14 days. Animals received sterile food and water ad libitum and were handled aseptically. Animals were monitored at least twice daily with health monitor forms prior and post compound administration and any animal deeded to be at humane endpoint was euthanized. Criteria used for humane endpoint for the following experiments included one or more of the following: loss of >20% body weight, marked scruffed fur, hunched body, labored breathing, lack of response to stimulus and lethargic animal. To minimize animal suffering and distress, environmental enrichments such as shredded, crinkled brown paper for nest building and translucent, red polycarbonate house for shelter were provided. Isoflurane was also used as anesthetics when required as described below. Murine model of pulmonary fibrosis PF was induced in six-weeks old female CD-1 mice purchased from Harlan. Animals were randomly divided into three groups (n = 10/group): PBS plus acetate buffer control, BLM plus acetate buffer and BLM plus AMD070. Some animals in the BLM plus acetate buffer and the BLM plus AMD070 groups reached a humane endpoint prior to day 22 (hence n = 4 and n = 9 respectively) and were euthanized. On day 0, mice were anaesthetized with isoflurane and PF was induced by BLM administration. Specifically, BLM (C103610, Fresenius Kabi Canada, 2U/kg) in 40 μL PBS or vehicle control (PBS) was administrated by non-surgical endotracheal instillation with sterile disposable plastic loading pipette tips. The next day, AMD070 (Shanghai Haoyuan Chemexpress Co., 400 μg/mouse in 200 μL 30 mM acetate buffer) or 200 μL of 30 mM acetate buffer (pH 5) was administrated via oral gavage (PO) with a 20G feeding needle. AMD070 or acetate buffer vehicle was administrated daily for 21 consecutive days. Animals were euthanized with avertin overdose on day 22. Murine model of hepatic fibrosis Hepatic fibrosis was induced in eleven week old female C57BL/6 mice from JAX Labs. Animals were randomly divided into three groups: oil plus vehicle (PBS) control, CCl 4 plus vehicle (PBS) control and CCl 4 plus AMD070. As some animals were euthanized prior to study completion because they had reached a humane endpoint, final group numbers were n = 9 for oil plus vehicle (PBS) control, n = 6 for CCl 4 plus vehicle (PBS) control and n = 7 for CCl 4 plus AMD070 groups. On day 0, mice received intraperitoneal (IP) injection of CCl 4 (1 mL/kg) diluted in olive oil (1 part CCl 4 and 3 parts olive oil hence 4 mL/kg of total volume) or olive oil control (4 mL/kg) twice a week for four consecutive weeks. Starting one day prior to the initial CCl 4 treatment (day -1), dosing with either AMD070 reconstituted in PBS (50 mg/kg) or PBS vehicle control (10 mL/kg) was administered IP and continued 5 days/week for 4 weeks. Specifically, AMD070 or PBS treatments were not performed on the 2 days/week on which animals were given CCl 4 (or olive oil vehicle control). One day after the last AMD070 or PBS treatment, animals were euthanized with CO 2 . Histology and percent fibrosis analysis of the lung and liver Following perfusion with PBS, lungs were fixed in 4% paraformaldehyde at 4°C for 2 days. These were embedded in paraffin, sectioned at 1 mm apart and stained with hematoxylineosin (H&E) and Masson's Trichrome stains. For quantitation of percent PF, entire lung and fibrotic areas were outlined. Percent fibrotic area was calculated by dividing fibrotic area (mm 2 ) by total lung area (mm 2 ) and multiplying by 100. For H&E stained slides, two histology slides/animal were selected from the central region of the lungs and analyzed whereas only one histology slide/animal was analyzed with Masson's Trichrome stained slides. These analyses were performed with Image J and the analyst was blinded to sample identification. Livers were perfused with PBS then 10% formalin and the left top lobe preserved in 10% neutral buffer formalin (NBF) for a week. The fixed liver was embedded in paraffin, six cross sections at 500 μm apart generated and stained with Picrosirius Red. For quantitation of percent hepatic fibrosis, fibrotic area (Picrosirius Red) was divided by total liver area and multiplying by 100. Three histology slides/animal were analyzed. The three slides corresponded to one slide from each of the alternative six cross sections. These analyses were performed with Olympus cellSens and the analyst was blinded to sample identity. RNA isolation and quantitative real-time qPCR The top right lobe of livers were homogenized in 1 mL of Trizol (Invitrogen) and flash frozen in dry ice and stored in -80°C for RNA isolation to perform RT-qPCR. Total RNA was isolated and cDNA prepared using the High Capacity cDNA Reverse Transcription kit according to the manufacturer's instructions (Life Technologies). RT-qPCR was carried out as previously described using the standard curve method [47]. The following primer/probe sets were used to detect: Acta2 (IDT Assay-N007392.1), Probe-5'-/56-FAM/TTACAGAGC/ZEN/CCAGA GCCATTGTCG/3IABkFQ/3', Primer 1-5'-GTGAAGAGGAAGACAGCACAG-3', Primer 2: Serum aspartate aminotransferase (AST) activity Five μL of serum was used with the AST Activity Assay Kit (Sigma MAK055) as per manufacturer's instructions to determine serum AST levels. Pharmacokinetic (PK) studies and Hematology measurements AMD070 PK studies via PO. Non fasted CD-1 female mice at six weeks of age from Harlan were sacrificed at 0.5, 1, 2, 3, 6, 24 and 48 hours (n = 3/time point) post administration of AMD070 reconstituted in 30 mM acetate buffer (pH 5) (200 or 400 μg/mouse). At each endpoint, mice were euthanized with CO 2 and blood collected via cardiac puncture. An aliquot was set aside at room temperature for hematology measurements with remaining blood processed for plasma for drug concentration determination. Lungs were collected and immediately frozen on dry ice and stored at -70°C until determination of drug concentration. AMD070 PK studies via IP injections. Non fasted C57BL/6 mice at 7 to 8 weeks of age from Harlan were sacrificed at 0.5, 1, 2, 3, 6, 24 and 48 hours (n = 3/time point) post administration of AMD070 (in 30 mM acetate buffer, pH 5) (400 μg/mouse). At each endpoint, animals were euthanized with CO 2 and blood collected via cardiac puncture. An aliquot was set aside at room temperature for hematology measurements with remaining blood processed for plasma for drug concentration determination. Lungs and livers were also collected and immediately frozen on dry ice and stored at -70°C until determination of drug concentration. Bioanalysis of AMD070 in the plasma, lung and liver tissue. AMD070 concentrations in mouse plasma were determined using protein precipitation extraction followed by UPLC-MS/ MS analysis. Briefly, 100 μL aliquots of plasma were transferred to individual wells of an Isolute PPT+ (Biotage) array plate containing 300 μL of 0.1% formic acid (FA) in acetonitrile (ACN). Following a few minutes of room temperature incubation, the samples were filtered by applying N 2 (g) pressure using the Pressure+ 96 manifold (Biotage). 200 μL of sample filtrate was then transferred to a separate 96-well plate and evaporated to dryness using a Turbovap 96. The sample residue was then reconstituted into 200 μL of 0.1/5/95 FA/ACN/H 2 O (v/v/v) and analyzed using reverse phase chromatography (gradient elution) combined with multiple reaction monitoring acquisition (MRM). The mobile phases consisted of 0.1% FA in H 2 O and 0.1% FA in ACN. AMD070 was extracted from lung and liver tissue by homogenization under basic conditions followed by liquid-liquid extraction in tert-butyl methyl ether (TBME) and ethyl acetate respectively. In summary, 50 mg of lung or liver tissue pieces were cut, weighed, and transferred to homogenization tubes. 25 μL of deionized water for lung and 10 μL for liver were added to each tube, followed by the addition of zirconium (Zr) homogenization beads. The samples were homogenized for approximately 20 seconds in the BeadBeater, followed by 1 minute of centrifugation at room temperature at 13,520 rcf. 200 μL of 1 N NaOH was then transferred to each tube, followed by a second homogenization/centrifugation cycle as described above. Next, 750 μL of TBME or ethyl acetate were added to each lung and liver tubes respectively followed by a third homogenization cycle (20 sec) and 10 minutes of centrifugation (room temperature, 13,520 rcf). 500 μL of TBME or ethyl acetate extracts were then transferred to corresponding wells on 96-well plates and evaporated to dryness. The Pharmacokinetics and Pharmacodynamics of AMD070 administered PO Prior to the efficacy study of AMD070 administered by PO in the BLM-induced lung fibrosis model, we wished to confirm that AMD070 given by this route would accumulate in the lungs and in addition, had the anticipated effects on white blood cell (WBC) counts. CD-1 mice were given AMD070 by PO administration at a dose of either 200 or 400 μg/animal. Plasma and lung concentrations of AMD070, as well as complete blood cell counts were collected at various times after administration. Oral administration of AMD070 was associated with very low plasma concentrations of drug (data not shown) but a dose dependent accumulation of drug in the lungs was observed. Specifically, although mice given AMD070 at 200 μg/animal had detectable but not quantifiable drug levels, when administered at the higher dose of 400 μg/animal, reproducible drug concentrations in the lungs were observed with C max concentrations of 267 ng/g observed 6 hours after PO (Fig 1). Importantly, the IC 50 of AMD070 is 13 nM representing a concentration of approximately 4.5 ng/mL and a protein binding adjusted effective concentration (EC 90 ) to achieve 90% effectiveness of 44ng/mL [48]. The lung concentrations achieved after oral administration of AMD070 (Fig 1) were significantly above both these thresholds for at least 21 of the 24 hours after dosing as determined by WinNonlin analysis. Consistent with these findings, we observed a dose-dependent effect of orally-administered AMD070 on WBC counts in these mice. Specifically, we observed a significant, transient increase in WBCs in mice dosed with AMD070 at 200 μg/mouse, with a T max observed 3 hours following administration. At the 400 μg/mouse dose of AMD070, the WBC counts were increased and remained elevated for the full 24 hours of the study. In contrast, red blood cell (RBC) and platelet counts did not change after administration of AMD070 at either dose ( Fig 2). White blood cell differentials are provided (Fig 3) and showed that the increase in WBC counts in whole blood shown in Fig 2 were the result of increased lymphocytes; we observed no increases in neutrophils, monocytes or eosinophils in these mice. These observations are consistent with literature reports on the effects of AMD070 on WBC mobilization [45]. Efficacy of AMD070 in a murine model of pulmonary fibrosis The IC 50 and EC 90 of AMD070 against CXCR4 were 4.5 ng/mL and 44 ng/mL respectively (Fig 1) [48], indicating that AMD070 concentrations in the lung were significantly greater than the IC 50 and EC 90 for the majority of the 24 hours after PO dosing. Based on these results, it was anticipated that oral administration of AMD070 at 400 μg/animal should achieve the needed systemic and tissue exposure to elicit a biological response in the BLM-induced model of PF. In addition, inhibition of CXCR4/CXCL12 had been shown to reduce lung fibrosis in a BLM induced murine model of PF [23,26,27]. To determine whether AMD070 would have the anticipated effect, animals were randomly divided into three groups: PBS plus acetate buffer control, BLM plus acetate buffer and BLM plus AMD070. These groups were treated with BLM (or PBS vehicle control), AMD070 (or acetate buffer control) as detailed in Materials and Methods. Animals were euthanized one day after the last AMD070 treatment (Day 22). Treatment with AMD070 was not associated with reduction of lung inflammation and fibrosis in the surviving mice. H & E stained lungs showed an average of 5.4% (SEM ±2.4) lung inflammation in the BLM plus acetate buffer control; 14.2% (SEM ±3.0) in the BLM plus AMD070 and 0% (n = 10) in the PBS plus acetate buffer groups respectively (Fig 4). When lungs were stained with Masson's Trichrome stains, there was an average value of 3.7% (SEM ± 1.1) lung fibrosis in the BLM plus acetate buffer control; 10.5% (SEM ±2.5) in the BLM plus AMD070 and 0% (n = 10) in the PBS plus acetate buffer groups respectively (Fig 5). In contrast to the absence of an effect of AMD070 on lung inflammation and fibrosis in BLM-treated mice, there was a significant effect of AMD070 on BLM induced mortality. Animals receiving AMD070 and BLM, showed 90% survival over the duration of the study (Fig 6). Those animals with BLM induced PF and treated with acetate buffer exhibited significant mortality between study days 8 to 14 with 60% of these animals requiring humane euthanasia ( Fig 6). This mortality was almost entirely mitigated by daily gavage with AMD070; 90% of BLM treated animals receiving AMD070 survived to the completion of the study (Fig 6) indicating a very significant therapeutic benefit achieved by AMD070 in this disease model. Pharmacokinetics and Pharmacodynamics of AMD070 administered IP As a prelude to an efficacy study of AMD070 in a murine model of hepatic fibrosis, we wished to alter the dosing route from PO to a route less likely to cause injury during repetitive dosing for 4 weeks. Therefore, a pharmacokinetic study was conducted to compare IP and SC routes of administration. After IP administration of AMD070, plasma concentrations of AMD070 peaked 30 minutes post administration at an average of 0.77 μg/mL of plasma and descending to an average of 0.045 μg/mL six hours post administration (Fig 7). In the lung, AMD070 concentration peaked 30 minutes post administration to an average of 6.14 μg/g of lung tissue and decreasing to an average of 3.74 μg/g six hours post administration (Fig 7). In the liver, AMD070 concentration peaked 30 minutes post administration to an average of 14.3 μg/g of liver tissue and decreasing to an average of 6.47 μg/g six hours post administration (Fig 7). When compared to plasma, lung and liver concentrations after SC administration of AMD070 (data not shown), the drug concentrations achieved using IP administration were somewhat higher than those achieved with SC administration. In summary, this showed substantial drug Table 1. Consistent with these observations, there was a significant but transient increase in WBC counts observed with a T max 3 hours following IP administration (Fig 8). In contrast, both RBC and platelet levels were unaffected by AMD070 administration (Fig 8). Differential analysis showed that the increase of WBC counts was largely due to increased lymphocytes and neutrophils (Fig 8). This analysis was also performed after SC administration of AMD070 (data not shown) and the qualitative trend was identical to that achieved with IP administration but the magnitude of these changes were lower in comparison to AMD070 given IP. Based on these pharmacokinetic and pharmacodynamic observations of AMD070 given by IP and SC routes, IP administration was identified for use in the efficacy study of AMD070 in the murine model of hepatic fibrosis (below). Efficacy of AMD070 in a murine model of hepatic fibrosis Mice were randomly divided into three groups; oil plus vehicle (PBS) control, CCl 4 plus vehicle (PBS) control and CCl 4 plus AMD070 groups. After four weeks of treatment as detailed in Materials and Methods, livers were collected and stained with Picrosirius Red and analyzed for percent fibrosis. The top left lobe of the liver was analyzed for all animals. Treatment with AMD070 had no effect on percent liver fibrosis. There was a group average of 0.53%, 3.2% and 3.6% of Picrosirius red staining in the oil plus PBS, CCl 4 plus PBS and CCl 4 plus AMD070 groups respectively (Fig 9). There was also no difference in the mortality rate between these groups. To determine whether AMD070 effects transcription of genes associated with a myofibroblast phenotype, RT-qPCR was performed on liver RNA collected one day after the last AMD070 treatment. The relative transcription levels of α smooth muscle actin (αSma, Acta2) and collagen α-1(I) chain (Col1a1) relative to housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and TATA binding protein (Tbp) were not different between the CCl 4 plus PBS (n = 6) and CCl 4 plus AMD070 groups (n = 7) (Fig 10A & 10B). Serum AST levels are a marker for liver function and liver injury, and were measured prior to treatment and at the end of the experiment to both confirm CCl 4 induced liver injury and to determine if there were beneficial effects associated with AMD070 treatment that were not observed in the fibrosis measurements. Prior to CCl 4 or oil treatment, serum AST levels were similar between all three treatment groups. After treatment with CCl 4 for 4 weeks, there was a significant increase in serum AST levels, confirming that liver injury was induced by this treatment ( Fig 10C). However, those animals that received CCl 4 and AMD070 had no difference in AST serum levels compared to the mice that received CCl 4 and the vehicle control ( Fig 10C). Together, these findings indicate that inhibition of CXCR4 has limited impact on hepatic fibrosis. Discussion The overall goal of these studies was to evaluate whether CXCR4 was a potential target for therapeutic intervention in fibrotic diseases. Several studies [23,26,27] using CXCR4 antagonists have reported attenuated BLM-induced lung fibrosis in mice. Specifically, AMD3100, an antiviral compound belonging to the bicyclam derivatives that antagonize the CXCR4 receptor [49] was able to reduce lung fibrosis and fibrocyte infiltration into the lung [23,26] in a murine model of BLM induced PF. In the present study, we have used AMD070, an orally bioavailable inhibitor of CXCR4 with improved tolerability [45] and pharmacokinetics, to evaluate the CXCL12/CXCR4 signaling axis in two different murine models of fibrosis. We have taken significant care to ensure that pharmacokinetics and tissue exposure of AMD070 was sufficient to elicit biological responses and have further confirmed the pharmacodynamic activity of AMD070 in each model by monitoring hematopoietic progenitor mobilization. Pulmonary fibrosis AMD070 had no effect on the percent fibrosis in the lungs of the BLM-treated animals. As suggested earlier, this is inconsistent with studies by Makino et al. and Song et al. [23,26]. It was suggested that bone marrow derived mesenchymal stem cells (BMDMSC) migrate out of the bone marrow and there is an increase of fibrocytes in the lung between 3 and 7 days post BLM Table 1. Phoenix WinNonlin Noncompartmental analysis of AMD070 concentration in the plasma, lung and liver of C57BL/6 mice following IP or SC administration of AMD070 at 400 μg/mouse. Values for Cmax are provided as μg/mL for plasma and as μg/g for liver and lung. Values for AUC are provided as μg/mL for plasma and as μg/g for lung and liver. Plasma Lung Liver [23,26,27]. It was reported that BLM treatment significantly induced chemotactic migration of BMDMSC to the lung 3 days post treatment and that this migration was inhibited by CXCR4 antagonists [26,27]. Specifically, in vivo, AMD3100 decreased the number of fibrocytes in the lung at 3 and 7 days post BLM treatment [23,26]. Increased expression of CXCR4 and CXCL12 in the lung was also observed beginning day 3 and up to day 21 after BLM treatment [26,27]. Transplanted circulating human fibrocytes also migrated to fibrotic lungs in response to CXCR4/CXCL12 signaling in BLM induced murine PF [25]. Based on these and other observations, it has been proposed that CXCL12 chemoattracts BMDMSC and circulating fibrocytes to the lung via CXCR4/CXCL12 signaling during lung fibrosis [23,[25][26][27]. Previous reports demonstrated a role for CXCR4 in fibrosis, however, herein this was not observed. The absence of an effect of AMD070 cannot be attributed to insufficient drug accumulation in the lungs since we measured AMD070 accumulation in the lungs in significant excess of the CXCR4 IC 50 and EC 90 for 21 of the first 24 hours after dosing (Fig 1). Furthermore, AMD070 administration was associated with the expected dose-dependent increase in peripheral WBCs in these animals peaking at three hours after administration (Fig 2). This observation is consistent with AMD070's ability to induce a dose related mobilization of bone marrow hematopoietic progenitors with a peak between two to three hours post dosing in human subjects [45]. In spite of the absence of an effect of AMD070 on BLM-induced fibrosis, there was a very striking effect on mortality in this model. Specifically, AMD070 treatment was associated with an increase in survival from 40% to 90% (Fig 6). In a rat model of BLM induced fibrosis, it was suggested that a "switch" between inflammation and the fibrotic phase occurred at around day 9 [50]. Inflammatory cytokine levels (IL-1α, IL-1β, IL6 and IFN-γ) increased rapidly by 3 days post BLM treatment and remain elevated up to day 9 [50]. Concurrently, there was an increase in collagen deposition starting at day 9, which was more pronounced on day 14 and 21 with a decline of the inflammatory cytokines on day 14 [50]. Similarly, it was found that 7 days post BLM treatment, increased vascular permeability and infiltration of neutrophil and lymphocytes into the lung were observed in mice [51,52]. By 14 days post BLM administration, proliferation of fibroblasts and lung fibrosis were observed [51,52]. In our study, mortality in the BLM group occurred between days 8 to 14, a period of vascular leakage and inflammation prior to the fibrotic phase and which was almost completely mitigated by AMD070 (Fig 6). This is consistent with the report that another potent CXCR4 antagonist, AMD3100 was able to alleviate mortality associated with BLM induced PF [53]. These data lead us to conclude that a role for the CXCL12/CXCR4 signaling axis in BLM-induced fibrosis is negligible. However, there may be a very important role for signaling via CXCR4 in the earlier inflammatory and vascular leakage phase of injury in this model. Thus, this may represent an opportunity for therapeutic intervention. Hepatic fibrosis Since the effects of AMD070 on BLM induced fibrosis were uncertain, we tested the efficacy of this drug in a CCl 4 induced murine model of hepatic fibrosis and AMD070 treatment by IP injection. As was observed in the studies described above in which AMD070 was given by PO, when administered by IP injection, significant levels of AMD070 were observed in the plasma, lungs and liver of the mice for 24 hours after injection (Fig 7). In addition, IP administration of AMD070 was also associated with the expected pharmacodynamic effects on WBC mobilization (Fig 8). As expected, CCl 4 treatment induced clear evidence of liver fibrosis based on Picosirus red staining (Fig 9) and liver injury based on serum AST levels ( Fig 10C). However, treatment with AMD070 had no effect on liver function based on the AST levels ( Fig 10C) and the percent liver fibrosis was not different between the CCl 4 plus PBS vs. CCl 4 plus AMD070 groups (Fig 9). When liver RNA was evaluated for the transcript levels of Acta2 and Col1a1, AMD070 in Murine Models of Lung and Liver Fibrosis their relative abundance were not different between the CCl 4 plus PBS and CCl 4 plus AMD070 groups (Fig 10A & 10B). These data are inconsistent with the study by Saiman et al., which demonstrated that inhibiting CXCR4 with AMD3100 increased liver fibrosis and increased transcript levels of Acta2 and Col1a1 [42]. Activation of HSCs is known to play a major role in liver fibrogenesis [2,9,11]. It had been shown that CXCR4/CXCL12 signaling activates HSCs and induces their proliferation, and can lead to increased production of collagen I under fibrotic conditions [36]. Furthermore, recent studies have demonstrated that the majority of myofibroblasts following liver damage derive from "activated" HSCs [54]. AMD070 was delivered at the outset of the study and throughout the CCl 4 treatment period, with a negligible impact on multiple markers of fibrosis. These observations indicate that the CXCL12/CXCR4 signaling axis has a limited role in liver fibrosis. It is possible that other mechanisms of HSC activation independent of CXCR4/CXCL12 signaling are operating as suggested by Saiman et al. [42]. Evidence for this comes from studies in which knockdown of CXCR4 only partially suppressed the proliferative response of HSC to CXCR4/CXCL12 signaling [42]. Conclusion Taken together, our results showed little evidence for a key pathological role of signaling via the CXCL12/CXCR4 axis in either the BLM-induced PF model or the CCl 4 -induced hepatic fibrosis model. Collectively, our findings suggest that CXCR4 represents a relatively poor therapeutic target (at least in lung and liver) for modulating the fibrotic response. Instead, they suggest a significant effect of the CXCR4 antagonist on survival in the BLM-induced PF model only during the early inflammatory and vascular leakage phase. Future work should focus on exploring the biological signaling occurring during these phases of injury and recovery following BLM induced lung injury.	2016-05-12T22:15:10.714Z	2016-03-21T00:00:00.000	{ "year": 2016, "sha1": "98b79672de719d453ad25f7499df0accb5355ce7", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0151765&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "98b79672de719d453ad25f7499df0accb5355ce7", "s2fieldsofstudy": [ "Biology", "Medicine", "Chemistry" ], "extfieldsofstudy": [ "Medicine" ] }
419741	pes2o/s2orc	v3-fos-license	Extensive Basal Level Activation of Complement Mannose-Binding Lectin-Associated Serine Protease-3: Kinetic Modeling of Lectin Pathway Activation Provides Possible Mechanism Serine proteases (SPs) are typically synthesized as precursors, termed proenzymes or zymogens, and the fully active form is produced via limited proteolysis by another protease or by autoactivation. The lectin pathway of the complement system is initiated by mannose-binding lectin (MBL)-associated SPs (MASP)-1, and MASP-2, which are known to be present as proenzymes in blood. The third SP of the lectin pathway, MASP-3, was recently shown to be the major activator, and the exclusive “resting blood” activator of profactor D, producing factor D, the initiator protease of the alternative pathway. Because only activated MASP-3 is capable of carrying out this cleavage, it was presumed that a significant fraction of MASP-3 must be present in the active form in resting blood. Here, we aimed to detect active MASP-3 in the blood by a more direct technique and to quantitate the active to zymogen ratio. First, MASPs were partially purified (enriched) from human plasma samples by affinity chromatography using immobilized MBL in the presence of inhibitors. Using this MASP pool, only the zymogen form of MASP-1 was detected by Western blot, whereas over 70% MASP-3 was in an activated form in the same samples. Furthermore, the active to zymogen ratio of MASP-3 showed little individual variation. It is enigmatic how MASP-3, which is not able to autoactivate, is present mostly as an active enzyme, whereas MASP-1, which has a potent autoactivation capability, is predominantly proenzymic in resting blood. In an attempt to explain this phenomenon, we modeled the basal level fluid-phase activation of lectin pathway proteases and their subsequent inactivation by C1 inhibitor and antithrombin using available and newly determined kinetic constants. The model can explain extensive MASP-3 activation only if we assume efficient intracomplex activation of MASP-3 by zymogen MASP-1. On the other hand, the model is in good agreement with the fact that MASP-1 and -2 are predominantly proenzymic and some of them is present in the form of inactive serpin–protease complexes. As an alternative hypothesis, MASP-3 activation by proprotein convertases is also discussed. Serine proteases (SPs) are typically synthesized as precursors, termed proenzymes or zymogens, and the fully active form is produced via limited proteolysis by another protease or by autoactivation. The lectin pathway of the complement system is initiated by mannose-binding lectin (MBL)-associated SPs (MASP)-1, and MASP-2, which are known to be present as proenzymes in blood. The third SP of the lectin pathway, MASP-3, was recently shown to be the major activator, and the exclusive "resting blood" activator of profactor D, producing factor D, the initiator protease of the alternative pathway. Because only activated MASP-3 is capable of carrying out this cleavage, it was presumed that a significant fraction of MASP-3 must be present in the active form in resting blood. Here, we aimed to detect active MASP-3 in the blood by a more direct technique and to quantitate the active to zymogen ratio. First, MASPs were partially purified (enriched) from human plasma samples by affinity chromatography using immobilized MBL in the presence of inhibitors. Using this MASP pool, only the zymogen form of MASP-1 was detected by Western blot, whereas over 70% MASP-3 was in an activated form in the same samples. Furthermore, the active to zymogen ratio of MASP-3 showed little individual variation. It is enigmatic how MASP-3, which is not able to autoactivate, is present mostly as an active enzyme, whereas MASP-1, which has a potent autoactivation capability, is predominantly proenzymic in resting blood. In an attempt to explain this phenomenon, we modeled the basal level fluid-phase activation of lectin pathway proteases and their subsequent inactivation by C1 inhibitor and antithrombin using available and newly determined kinetic constants. The model can explain extensive MASP-3 activation only if we assume efficient intracomplex activation of MASP-3 by zymogen MASP-1. On the other hand, the model is in good agreement with the fact that MASP-1 and -2 are predominantly proenzymic and some of them is present in the form of inactive serpin-protease complexes. As an alternative hypothesis, MASP-3 activation by proprotein convertases is also discussed. Keywords: innate immunity, complement, lectin pathway, serine protease, proenzyme, autoactivation, reaction kinetics Extensive Basal Activation of MASP-3 Frontiers in Immunology \| www.frontiersin.org December 2017 \| Volume 8 \| Article 1821 inTrODUcTiOn The complement system, as an essential part of the innate immune response, eliminates invading microorganisms and dangerous host cells (1,2). The complement cascade is composed of more than 30 proteins. Key components of the system are serine proteases (SPs), which typically circulate in bloodstream in the zymogen form until their successive cleavage and activation (3). Complement activation can be triggered via three different, however interconnected routes: the classical, lectin, and alternative pathways, then the three routes converge into the common terminal pathway. When the classical or lectin pathways are activated, it results in the formation of the C3 convertase, C4bC2a, composed of the cleaved forms of complement factors C4 and C2 (4). The alternative pathway serves as an amplification loop, but it can also be activated on its own by the "tick-over" mechanism (5). Activation of the lectin pathway is initiated on surfaces displaying various arrays of carbohydrates or acetyl groups, which can be recognized by (at least) five different pattern recognition molecules (PRMs): mannose-binding lectin (MBL), H-ficolin (ficolin-3), L-ficolin (ficolin-2), M-ficolin (ficolin-1), and CL-LK (a heterocomplex of collectin liver 1 and collectin kidney 1) (6)(7)(8). The recognition of dangerous patterns is transformed to enzymatic signals by two proteases complexed with the PRMs, MBL-associated SP 1 and 2 (MASP-1, MASP-2). The first proteolytic step is the autoactivation of MASP-1, which then activates MASP-2. Both enzymes can cleave C2, however C4 is cleaved only by MASP-2. As a consequence both MASP-1 and MASP-2 play essential roles in the formation of the C4bC2a enzymatic complex (9)(10)(11). All MASPs have the same six-domain structure (4,7). The C-terminal SP domain is preceded by five regulatory domains in the order of CUB1-EGF-CUB2-CCP1-CCP2. CUB stands for C1r/C1s, sea urchin Uegf and bone morphogenetic protein-1 domain, EGF for epidermal growth factor domain, and CCP for complement control protein domain. Upon activation an Arg-Ile (R-I) bond is cleaved within the SP domain and the resulting two chains, termed A and B, are held together by a disulfide bridge. Mannose-binding lectin-associated SP-1 has relatively broad substrate specificity (12), as a result it is involved in coagulation (13) and certain proinflammatory reactions, e.g., cleavage of PAR receptors on endothelial cells results in their activation, and cleavage of high molecular weight kininogen produces the proinflammatory bradykinin (4,14,15). The role of two non-catalytic associated proteins (MAp19 or sMAP, MAp44, or MAP1) is less clear (16)(17)(18)(19). The third SP of the lectin pathway, MASP-3 (20), along with MAp19 and MAp44 were initially considered simply as negative lectin pathway regulators, but recent results implicated that MASP-3 has an important role in the activation of the complement system in connection with the alternative pathway (21)(22)(23)(24)(25). Factor D (FD), a single-domain SP, is a key enzyme for the alternative pathway. FD has only one natural substrate, factor B (FB) complexed with C3b (26). Until recently, it was believed that FD is activated at the site of the synthesis, maybe even before secretion (27), because only active FD could be purified from normal blood (28,29). Although in mammalian cell cultures predominantly active FD was detected (30,31), the zymogen form was also present (31). In insect cells, proenzyme FD (pro-FD) is expressed almost exclusively (22,32). Recent results by Pihl et al. (25) and personal discussions with Elod Kortvely (unpublished data) suggest that both FD and pro-FD are present in normal human blood with FD being the dominant form. A link between early complement pathways was proposed by Takahashi et al. observing the lack of alternative pathway activity in MASP1 knock-out mice. Due to the fact that MASP-1 and MASP-3 are two splice variants from the same gene such mice lacks both proteins. In the absence of both MASP-1 and MASP-3 only zymogen FD was detected. At first, MASP-1 was suggested to be responsible for pro-FD activation, although supplementing the serum from such mice with recombinant MASP-1 did not restore the alternative pathway (33). Later, MASP-3 was also implicated as an enzyme capable of converting pro-FD to active FD, and even proenzyme MASP-3 was proposed to be able to carry out such cleavage (21). Subsequent results questioned the involvement of MASP-1 and/or MASP-3 in alternative pathway activation. In the serum of a MASP-1/3-deficient patient, suffering from the Malpuech-Michels-Mingarelli-Carnevale (3MC) syndrome causing serious craniofacial defects, functional alternative pathway activity was observed (10). Nevertheless a following discussion revealed that mainly pro-FD can be detected in the blood of these 3MC patients (34). Furthermore, uncontrolled alternative pathway activity was observed in MASP-1/3 and factor H codeficient mice just like in knock-out mice deficient for factor H only (35). These data contradicted the view that MASP-1 and/or MASP-3 is involved in the function of the alternative pathway, and the exact way of pro-FD conversion remained unclear. Our previous studies clarified this controversy and the role of MASPs in pro-FD activation. First, we determined the rate constants of pro-FD cleavage for all three MASPs. We found that only the active forms of MASPs are able to convert pro-FD to FD. Next, using fluorescently labeled recombinant pro-FD, we detected the activation of this exogenous pro-FD in human blood samples. With our previously developed MASP-1-and MASP-2-specific inhibitors we ruled out MASP-1 and MASP-2 as potential pro-FD activators in resting blood (22). Then, using a MASP-3-specific inhibitor, we showed that MASP-3 is the major activator of pro-FD in general, and it is the exclusive pro-FD activator under resting conditions in human blood (23). By "resting" conditions, we mean that neither the complement nor the coagulation system is activated above the basal level. Furthermore, our results supported thrombin and/or another coagulation protease serving as a potential backup enzyme for this conversion in coagulated blood (i.e., human serum) (22,23). Our findings also imply that under resting conditions, at least a fraction of MASP-3 must be present in the active form in the bloodstream (22,23). The average serum or plasma concentration of MASP-3 was published in several studies as about 5-7 µg/ml (36)(37)(38)(39), however, the extent of MASP-3 activation has remained unknown. In this study, we aimed to determine the active to zymogen MASP-3 ratio in normal resting human blood. In order to minimize activation during sample preparation, we used EDTA plasma (pooled or individual) as starting material, then MASPs were purified on MBL-Sepharose in the presence of broad specificity inhibitors, Pefabloc and 4-nitrophenyl 4′-guanidinobenzoate (NPGB). MASPs were then detected by Western blotting. Our data imply that MASP-3 circulates mostly in the active form in human blood. Of course the question arises how MASP-3 is activated, especially in the light of the fact that MASP-3, unlike MASP-1, cannot autoactivate. In an attempt to explain the observed facts, a kinetic model of lectin pathway activation was set up, which, using reasonable assumptions, provides a solution for the phenomenon. Blood was drawn from ten healthy volunteers into S-Monovette K3E EDTA tubes (Sarstedt) to produce fresh pooled plasma. After centrifugation, samples were combined, and kept at −70°C in aliquots. Blood was drawn from seven healthy volunteers into S-Monovette K3E EDTA tubes (Sarstedt) to produce fresh individual plasma samples. After centrifugation, samples were kept at −70°C in aliquots. The study was conducted in conformity with the WMA Declaration of Helsinki. Experimental protocols were approved by the local ethics committee (permission number: TUKEB 9190-1/2017/ EKU). Informed consent was obtained for the isolation of peripheral venous blood from the donors. Two rabbits were immunized by subcutaneous injections of MASP-1-SP. The first dose was 0.4 mg MASP-1-SP with Freund's complete adjuvant. After 4 weeks, the injections were repeated four times, once in every fortnight with 0.2 mg MASP-1-SP in Freund's incomplete adjuvant. Preimmune blood was withdrawn before the treatment and test blood samples were withdrawn a week after every boost from ear vein and finally the rabbits were bled after sixteen weeks. Sera were obtained by coagulation and centrifugation of the blood samples. A two-step affinity chromatography purification method was used to obtain MASP-1-SP-specific antibodies from the pooled antiserum of two rabbits. Whole IgG was purified from the 10-fold diluted immunsera using a 10 mm × 100 mm Protein A (MabSelect SuRe, GE Healthcare) column. The sample was loaded in 10 mM Na2HPO4, 1 M NaCl, 1 mM EDTA, pH 7.0 buffer, then eluted with a linear pH gradient ending in 100 mM citrate, pH 3.0. The immunoglobulin fractions were combined and neutralized with 1 M Tris base, then further purified on MASP-1cf (R448Q)-Sepharose resin. To produce the resin, 1 ml rehydrated CNBr-Activated Sepharose 4 Fast Flow (GE Healthcare) resin was reacted with 6 mg MASP-1cf (R448Q) according to the manufacturers protocol. The suggested final washes with solutions of alternating pH were omitted. The pooled IgG fraction was twofold diluted with 20 mM Tris, 150 mM NaCl, 0.3% Tween-20, pH 7.5 buffer and applied to a 5 mm × 50 mm MASP-1cf (R448Q)-Sepharose column. Two overlapping populations of anti-MASP-1-SP antibodies, weakly and strongly binding, were eluted with linear pH gradient ending in 100 mM citrate, pH 3.0. The fractions were neutralized with 1 M Tris base and treated with 0.02% NaN3 as preservative. Part of the strongly binding population of anti-MASP-1-SP antibodies was concentrated to 1 mg/ml and conjugated with alkaline phosphatase enzyme (AP). The conjugation was performed by Alkaline Phosphatase Labeling Kit-NH2 (Abnova) using the manufacturers protocol. MasP Purification from eDTa Plasma on MBl-sepharose Mannose-binding lectin-Sepharose had been previously used by others to purify recombinant MASPs (45,46). We modified this method to purify MASPs (and MAPs) from human EDTA plasma. Recombinant human MBL was dialyzed excessively against coupling buffer (100 mM NaHCO3, 500 mM NaCl, pH 8.3), then concentrated to 4.75 mg/ml with 10 kDa cutoff concentrator. The concentration of rMBL was calculated based on the extinction coefficient of ε280 = 18,365 M -1 cm -1 and a molecular mass of a polypeptide of 24.0 kDa (not accounting for glycosylation). Half a gram of CNBr-Activated Sepharose 4 Fast Flow resin was washed with 50 ml 1 mM HCl solution, then with sterile water, and finally equilibrated with coupling buffer. The resin was mixed with 2.1 ml 4.75 mg/ml rMBL and the mixture was rotated gently for 4 h at room temperature, and subsequently the beads were washed with 10 ml coupling buffer. After coupling the resin was washed with 5 ml 100 mM Tris (pH 8.0) buffer, then incubated for overnight with 8 ml of the same buffer at 4°C, then washed with 150 mM NaCl, 50 mM Tris, 10 mM CaCl2 (pH 7.5) buffer and stored in the same buffer containing 0.02% NaN3. The coupling efficiency was about 2.5 mg rMBL per ml wet resin. Plasma, 5 ml, containing about 10 mM EDTA (pool of 10 donors or individual) was thawed at room temperature, and then was immediately mixed with 65 µl inhibitor stock solution [10 mM NPGB, 100 mM Pefabloc in anhydrous DMF (Merck)]. NaCl, 1.25 ml of 5 M, and 315 µl of 1 M Tris (pH 8.0) solutions were added to plasma containing the inhibitors, then supplemented with 100 µl of MBL-Sepharose resin. The mixture was rotated for 30 min at 0-8°C, then further 65 µl inhibitor stock solution was added. After twofold dilution with ice-cold sterile water, 85 µl of 2 M CaCl2 (12.5 mM final) was added, then it was rotated for 30 min at 4°C. Further steps were performed at room temperature however using ice-chilled buffers. The mixture containing the resin was loaded to a Poly-Prep Chromatography column (Bio-Rad, catalog number: 731-1550). The flow-through was discarded, the resin was washed with 500 µl ice-cold 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.1 mM NPGB, 1 mM Pefabloc (pH 7.5) buffer, then with 200 µl of ice-cold 50 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.1 mM NPGB, 1 mM Pefabloc, pH 7.5 buffer. After the washing steps an end cap was applied to column, and the resin was incubated with 150 µl elution buffer (50 mM Tris, 1 M NaCl, 20 mM EDTA, 0.1 mM NPGB, 1 mM Pefabloc, pH 8.0) for 5 min. Following incubation the end cap was removed and the eluate was collected, then another 150 µl of elution buffer was applied to column. The two eluted fractions were combined and after twofold dilution with ice-cold sterile water, the sample was mixed with 1/3 volume of 4× SDS-PAGE sample buffer then heated for 2 min at 95°C. In order to produce samples without inhibitors, the same experiment was performed, but the last washing and the elution buffers did not contain NPGB and Pefabloc. After elution and dilution the mixture was incubated with MASP-1cf at 215 nM final concentration at 37°C, and aliquots were removed at 1.5 and 5 h. The reaction was stopped by adding 1/3 volume of 4× SDS-PAGE sample buffer and heating for 2 min at 95°C. Western Blot analysis Proteins in the samples were separated by SDS-PAGE under both reducing and non-reducing conditions. Stacking gels (125 mM Tris, pH 6.8) contained 5%, separating gels (750 mM Tris, pH 8.8) contained 12.5% acrylamide-bisacrylamide (37.5:1) solution, separation was performed at a constant voltage of 180 V in running buffer (25 mM Tris, 192 mM glycine, 0.1% w/v SDS, pH 8.3). Proteins were transferred to nitrocellulose membranes (Bio-Rad) in 25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.4 transfer buffer. Blotting was performed at a constant current of 120 mA for 60 min. After blocking for 1 h in 20 mM Tris, 200 mM NaCl, 0.02% w/v NaN3, 5% w/v nonfat dry milk, pH 7.2 blotting buffer, membranes were incubated overnight at room temperature with MASP-1-SP-AP antibody or MASP-3 monoclonal antibody in the same buffer at 1 or 2 µg/ml, respectively. Membranes blotted with the MASP-3 antibody were washed (20 mM Tris, 200 mM NaCl, 0.02% w/v NaN3, 0.3% v/v Tween-20, pH 7.2) for 30 min, then incubated for 1 h at room temperature with alkaline phosphatase conjugated goat anti-mouse antibody at 2,000-fold dilution in blotting buffer. After washing for 45 min, membranes were visualized with NBT/BCIP solution (5 mM MgCl2, 100 mM Tris, 400 mg/l NBT, 200 mg/l BCIP, pH 9.0). Blots were scanned with an Epson Perfection 4490 scanner in 16-bit grayscale reflective mode. Densitometric analysis of the Western blots was performed using the Quantity One software (Bio-Rad). Determination of the MasP-3cf activation rate constants by Various Proteases Zymogen MASP-3cf at a final concentration of 2 µM (96 µg/ml) in 140 mM NaCl, 10 mM HEPES, pH 7.4, 0.1 mM EDTA buffer was incubated at 37°C alone (negative control), or in the presence of various proteases. The final concentrations of the tested proteases and the incubation times were as follows. Active MASP-1cf had been used previously at 100 nM (4.6 µg/ml), and samples were removed periodically for up to 5 h (11). Zymogen R448Q MASP-1cf was used at 1 µM (46 µg/ml), and samples were removed periodically for up to 13 days (the reaction was nearly complete at 6 days). Active MASP-2cf was used at 91 nM (3.9 µg/ml), and samples were removed periodically for up to 3 h, and a final overnight sample was also collected. Zymogen R444Q MASP-2cf was used at 1 µM, and samples were removed periodically for up to 6 days (no cleavage was detected). Active MASP-3cf (autocatalytic cleavage) was used at 500 nM, and samples were removed periodically for up to 5 days (no apparent increase in the amount of active MASP-3cf was detected). Zymogen MASP-3cf (self cleavage) as stated above was used at 2 µM (96 µg/ml), and samples were removed periodically for up to 6 days (no cleavage was detected). Active FD was used at 1 µM, and samples were removed periodically for up to 4 h (no cleavage was detected). Samples were analyzed by SDS-PAGE under reducing conditions, followed by Coomassie Brilliant Blue G staining. Gels were scanned with an Epson Perfection 4490 scanner in 16-bit grayscale transparent mode. After scanning the intensities of the uncleaved substrate (zymogen MASP-3cf, 48 kDa) bands were quantified by densitometry using the BioRad Quantity One software. The intensities of the product (active MASP-3cf B chain, 31 kDa) bands were quantified in a similar fashion. Substrate consumption was fitted using the IS = IB + Io × exp-(−kobs × t) equation, where IS stands for the intensity of the substrate band, Io is the intensity of the substrate band at the zero time point, and IB is the background intensity. When the substrate band overlapped with the enzyme band then the following equation was used instead: Kinetic simulations The fluid-phase activation and inhibition of lectin pathway proteases were modeled by kinetic simulations of the reaction network involving the zymogen, active, and inhibited forms of proteases and their inhibitors. The synthesis and elimination of the species were also included, resulting in a network of 25 reactions (with non-zero rate constants) between 10 species (Table S1 in Supplementary Material). The biochemical system simulator program COPASI version 4.19 (47) was used. Time course analysis was performed by solving the system of differential equations using the deterministic LSODA method (48). resUlTs isolation of a Mixture of MasPs from Blood Recombinant human MBL was coupled to CNBr-Activated Sepharose as described in the Section "Materials and Methods. " Our goal was to determine the extent of MASP-3 activation under resting conditions. Thus, we purified a pool of MASPs from EDTA plasma, and in order to prevent further activation of zymogen MASP-3 by other proteases, we used inhibitors NPGB and Pefabloc during the whole process, and the purification was carried out at 0-8°C. We mixed MBL-Sepharose with plasma in a way that the estimated immobilized rMBL to plasma PRM ratio was about 3 to 1 by mass. At the first step in the presence of 1 M NaCl and 10 mM EDTA, the PRM-MASP complexes dissociated. Then the NaCl and EDTA concentrations were reduced by dilution and excess CaCl2 was added to initiate reassociation of complexes. MASPs are expected to bind preferably to MBL-Sepharose as a result of the 3 to 1, immobilized rMBL to soluble PRM ratio. After reassociation the resin was packed into a column, and it was washed with low salt buffers to remove unbound proteins. The final elution buffer contained 1 M NaCl and 20 mM EDTA, dissociating MASPs (and MAPs) from the immobilized rMBL (Figure 1). The samples were analyzed by SDS-PAGE followed by Western blotting. For detection our polyclonal MASP-1-specific antibody, or a commercial monoclonal MASP-3-specific antibody was used. Under reducing conditions, these antibodies recognize only the B-chain of active MASPs. In order to identify the bands corresponding to the active and the zymogen variants under non-reducing conditions, we also prepared samples containing neither NPGB nor Pefabloc. Only the last washing and the elution buffers lacked the inhibitors. The eluate was incubated with MASP-1cf, and samples were withdrawn at 1.5 and 5 h. Upon this treatment under reducing conditions the bands corresponding to zymogen MASP-1 and MASP-3 disappeared, and only the B-chains are seen, as expected. Under non-reducing conditions upon addition of MASP-1cf, the faster migrating bands disappeared, and only the slower migrating bands remained. Hence, the faster migrating band corresponds to the zymogen form, and the slower migrating band corresponds to the active form using non-reduced samples under our SDS-PAGE conditions (Figure 2). MasP-3 is Mostly active, Whereas all MasP-1 is Zymogenic in the same isolates During the isolation, we used NPGB and Pefabloc SC to prevent activation of zymogen MASP-3 by any protease. Under non-reducing conditions zymogen and active MASP-3 are well separated by SDS-PAGE, on the other hand the mobilities of the two forms are only slightly different, which is favorable for the quantification. For the quantification of the two forms of plasma MASP-3, we had several reasonable assumptions as follows: (I) the MBL-Sepharose resin binds zymogen and active MASP-3 with the same affinity, and no further activation occurs during the isolation; (II) the transfer efficiencies during Western blotting are equivalent due to the small difference between the mobility of zymogen and active MASP-3 under non-reducing conditions; and (III) the commercial monoclonal MASP-3-specific antibody recognizes both forms of MASP-3 with the same sensitivity. First, we measured the activation state of plasma MASP-1 in our isolates as a control. We found that all MASP-1 remained zymogenic during the preparation, proving that the lectin pathway did not activate during the isolation (Figure 2A, lanes 1-2 and 4-5). It is in agreement with previous articles showing that MASP-1 is found in the zymogen form in the circulation (49,50). In contrast, in the same isolates we detected both forms of MASP-3 ( Figure 2B, lanes 1-2, 5-6). We performed additional Figure 1 in the presence of Pefabloc and 4-nitrophenyl 4′-guanidinobenzoate (NPGB) inhibitors. A sample was prepared in the same manner except that the inhibitors were omitted at the last elution step. Samples were analyzed by SDS-PAGE under reducing and non-reducing conditions followed by Western blotting and detected using a MASP-1-or a MASP-3-specific antibody. Both antibodies were developed against the serine protease (SP) domain of the corresponding protein, hence they can detect the whole molecule, or the B chain of the active form. three parallel isolations with pooled plasma, and each blot is shown in Figure 3A. Our results indicate that MASP-3 circulates mostly in the active form, and quantification revealed that the active-zymogen ratio was about 4 to 1 ( Table 1). low individual Variation of the active-Zymogen ratio of MasP-3 In order to assess the individual variation of the active to zymogen ratio for MASP-3, we examined EDTA plasma samples from seven healthy donors. The isolation procedure of MASPs from the individual plasma samples was the same as from the pooled samples. Each sample was analyzed three times by non-reducing SDS-PAGE followed by Western blot, then quantified. Figure 3B represents all individual samples together on the same blot membrane. In every sample MASP-3 was over 70% activated. While the total MASP-3 level can be quite different in each donor (36,37), the active to zymogen ratio showed relatively low variation ( Table 1). In Vitro activation of MasP-3 by Various complement Proteases Zymogen MASP-1 and MASP-2 are able to undergo autoactivation, and the active MASPs can activate their proenzymic counterparts (41,42), although for MASP-2 the autoactivation process is much slower (11). In contrast, Zundel et al. showed that neither the zymogen nor the active form of MASP-3 has a similar activity at all (46). We observed the same using the catalytic fragments of MASP-3 ( Table 2). It is now generally accepted that MASP-1, after autoactivation, activates zymogen MASP-2 (9)(10)(11). In vitro, active MASP-1 is able to activate MASP-3 as well (10,21), and the cleavage rate has been determined using the catalytic fragments (11). Figure 1 in the presence of Pefabloc and 4-nitrophenyl 4′-guanidinobenzoate (NPGB). Samples were analyzed by SDS-PAGE under non-reducing conditions followed by Western blotting and detection using a MASP-3specific antibody. The faint band running above the active form is probably due to non-specific binding of the antibody. Western blots were quantified as described in the Section "Materials and Methods" and the result are listed in Table 1. For this study, we determined the missing rate constants of potential reactions that can produce active MASP-3 using the catalytic fragments of MASPs (Table 2; Figure 4). Out of curiosity we checked weather FD can activate MASP-3cf, but we could not detect any cleavage (data not shown, Table 2). Similarly to active MASP-1cf, active MASP-2cf can also activate zymogen MASP-3cf at an approximately two-fold higher a rate (Figure 4A). It should be noted, however, that in the blood MASP-2 has a 20-fold lower concentration compared to MASP-1, hence its contribution to MASP-3 activation is likely to be much less than that of MASP-1 in vivo. Zymogen MASP-2cf R444Q had no MASP-3cf cleaving activity at all (data not shown, Table 2). Interestingly the stable zymogen R448Q variant of MASP-1cf was also capable to activate MASP-3cf with a 300-fold lower rate compared to the active enzyme ( Figure 4B). Although this reaction is rather slow, it might have a significance as the major variant of MASP-1 in resting blood is the zymogen form. At the same time, zymogen MASP-3cf, when incubated alone, did not show any sign of activation upon prolonged incubation (Figure 4C), demonstrating that it does not autoactivate, and it is not contaminated by any external activating proteases. Also, no significant cleavage of zymogen MASP-3cf by active MASP-3cf was observed (data not shown, Table 2). In Silico Modeling of the Fluid Phase Basal activation of lectin Pathway Proteases We have determined previously (11,12,51) and for this study ( Table 2) essentially all the possible activation rate constants between lectin pathway proteases using the catalytic fragments. These reactions include zymogen autoactivation, cross-activation between the zymogen forms, autocatalytic activation, and crossactivation of a zymogen by another active MASP. Altogether there are 3 × 3 × 2 = 18 possible reactions, however "only" 10 have non-zero rate constants and "only" 9 have a significant value (Table S1 in Supplementary Material). Activated MASP-1 and MASP-2 are constantly inactivated by C1 inhibitor and antithrombin (12,51). These reactions were also added to the reaction network (Table S1 in Supplementary Material). C1 inhibitor might reversibly inhibit zymogen MASP-1 and MASP-2 (42), however this possible effect was not included into the model, because no quantitative data are available for these interactions. Proteins are continuously synthesized and eliminated from the circulation. There are no data for human MASPs in this regard, however in mice the half life of MASP-1 and MASP-3 is about 1.5 h (52). In our model, we used 6 h (arbitrarily) for all MASPs as humans have slower metabolism. This value corresponds to a 3.31 × 10 −5 s −1 first order elimination rate constant. The same value was used for all forms of MASPs including the serpin inhibited forms. This is a valid assumption as it was shown for C1s and C1s-C1 inhibitor complex in guinea pig (53). Synthesis rates (Table S1 in Supplementary Material) were calculated from the elimination rate constants and the measured steady-state plasma concentrations of MASPs (39). Including the 10 activation, 4 inactivation, 3 synthesis, and 8 elimination reactions a network of 25 reactions was set up. Table 2. (c) Zymogen MASP-3cf alone is shown as a control demonstrating that it does not autoactivate or cleaved by any potential contaminant upon prolonged incubation. Starting with zero MASP concentrations, and assuming that MASPs are synthesized as proenzymes, simulations were run until steady-state concentrations were obtained. Simulation 1 was run using the measured activation rate constants (Figure 5 upper panels). According to simulation 1, only very little active MASP-3 is attained at steady-state (Table 3), which is not in line with our data. However, the simulation is in line with the observations that MASP-1 and MASP-2 circulate mostly as zymogens Table S1 in Supplementary Material. The reaction set is depicted in Figure 6 within the boxed area. Steady-state distributions are shown in Table 3. would correspond to a much faster reaction than that between isolated molecules. To account for this scenario the rate constant of MASP-3 activation by zymogen MASP-1 was increased arbitrarily by a factor of 200 (Simulation 2, Figure 5 lower panels). By this single modification, the active MASP-3 steady-state fraction increased to over 50% (Table 3), which is more in line with the observed >70% value. Values for MASP-1 and MASP-2 variants remained essentially the same. Of course this scenario raises several questions, which are discussed later. comparison of the activation loop sequences of MasPs, c1r, and c1s The activation loop of MASP-3 has an unusual sequence in comparison with those of MASP-1, MASP-2, C1r, and C1s ( Table 4). The activation site is a R-I bond, which is the same in all the listed proteases. However, in MASP-3 the P1 Arg residue is preceded by a Lys residue, giving rise to a paired basic (KR) motif preceding the activation site. This motif coincides with the recognition motif of basic amino acid-specific proprotein convertases, which is (K/R) − (X)n − (K/R)↓, where n = 0, 2, 4, or 6 and X is any amino acid (56). Most often the cleavage site is after consecutive basic residues. There are of course other segments in the listed proteases, where a similar motif can be found, however the activation site of MASP-3 is exposed and hence suitable for (49), and some of them circulate as serpin-inhibited complexes (54, 55). It must be noted that our simulation cannot predict the precise ratios of the different MASP forms, because several simplifications were used, however, it is suitable to predict the general trends. If we assume that a fraction of MASP-1 and MASP-3 reside on the same complexes and efficient intracomplex activation between zymogen MASP-1 and zymogen MASP-3 occurs, this Bold highlights the scissile R-I bond, whereas the potential recognition site (KR) for proprotein convertases is underlined. purified samples. For quantification, the non-reduced samples are more suitable, because the two chains of active MASP-3 are held together by a disulfide bond, hence the masses of the active and zymogen forms are virtually identical, and their mobilities in polyacrylamide gels are very similar, though still allow their separation. Mannose-binding lectin-associated SP-1 was always proenzymic, whereas MASP-3 was a mixture of active and zymogen in all tested samples with active MASP-3 being the dominant form. On average, we found that MASP-3 was about 80% active, and in individual samples it was always more than 70% active, displaying little individual variation. Using another technique, i.e., using anti-MASP-3 to pull out MASP-3, using different enzyme inhibitors and using different anti-MASP-3 antibodies on the Western blot also leads to the finding that a high proportion of the MASP-3 is found as activated MASP-3 (25). We and others have postulated before that active MASP-1 and MASP-2 is capable of activating MASP-3 (10,11,21). The rate constant for active MASP-1cf was measured previously (11), and here we also measured the activation rate constant for active MASP-2cf, which activates MASP-3cf about twice as fast as active MASP-1cf. It occurred to us that FD might reciprocally activate MASP-3, however, FD did not cleave zymogen MASP-3cf, neither did zymogen R444Q MASP-2cf. The zymogen MASP-1cf (R448Q variant) activated MASP-3cf slowly, but the rate constant was about 300-fold lower than that for active MASP-1cf. Isolated full-length recombinant human MASP-3 does not autoactivate (46), neither does the catalytic fragment as previously shown (57) and unambiguously proved here using highly purified MASP-3cf. It is notable that recombinant MASP-3 can undergo slow activation upon prolonged storage. Zundel et al. (46) observed this cleavage with the wild-type and the S645A (precursor numbering S664A) inactive variant of MASP-3 as well, and concluded that the observed activation is due to cleavage by a contaminating protease from the cell culture supernatant. Similar observations were made by Pihl et al. (25). These observations indicate that the scissile R-I (449-450 precursor numbering) bond is sensitive to proteolysis by other proteases in general. Iwaki et al. (21) detected activation of recombinant mouse MASP-3 (also produced as proenzyme) in complex with MBL-A on Staphylococcus aureus. The authors postulated it as self-activation, but it is possible that a protease from S. aureus activated MASP-3. No activation was observed on uncomplexed MASP-3, MASP-3 in complex with other PRMs, or when other activators like mannan were used (21). In all, data in the literature with full-length dimeric MASP-3 and our observations with monomeric catalytic fragments indicate that MASP-3 does not autoactivate in general, and most importantly no autoactivation of MASP-3 was observed in the fluid phase. It is enigmatic that MASP-3, which does not autoactivate, is present mostly in the active form in the circulation, whereas MASP-1, which has a potent autoactivation capacity, is proenzymic. MASP-3 must be activated obviously by another protease, or proteases. Its activator may be another lectin pathway protease, or a yet unidentified protease in the blood, or possibly it is activated before secretion within the cells (Figure 6). In order to assess the first possibility, we set up a kinetic model for the fluid-phase such cleavage. The possibility of MASP-3 activation by proprotein convertases is also discussed later. DiscUssiOn Our previous studies revealed that active MASP-3 is the exclusive pro-FD activator in resting human blood (22,23). This implied that active MASP-3 must be present in the blood, even without ongoing complement activation above the baseline level. We decided to confirm this assumption by a different approach, and to quantify the extent of MASP-3 activation, i.e., the active to zymogen ratio. Initially, we attempted to detect MASP-3 directly in plasma samples, but the available antibodies were not suitable for this purpose because of the very high background. Therefore, we decided to enrich our samples for MASP-3. A mixture of MASPs and MAps was purified from EDTA plasma using MBL-Sepharose as described in the presence of broad specificity SP inhibitors in order to avoid any activation during the isolation. We detected MASP-3, and as a control MASP-1, by Western blotting in the basal level activation of lectin pathway proteases using available and newly determined kinetic constants. Previously, we have determined the rate constants for all possible activation steps involving MASP-1 and MASP-2 using the catalytic fragments (11). In this study, we added the missing rate constant (see above) for reactions producing active MASP-3. MASP-1 and MASP-2 are known to be inhibited by C1 inhibitor and antithrombin (4,58,59), while MASP-3 is not (46,60). Rate constants for the inactivation reactions had been previously determined (12,51) and they were included into the model. Estimated elimination and synthesis rates were also included. Running the simulation with the determined rate constant does not explain the observed phenomenon, i.e., that MASP-3 is present mostly in the active form. It must be noted, however, that our kinetic model can adequately describe intermolecular reactions, but it is less suitable to describe intramolecular (intracomplex) cleavage reactions. We have attempted to modify the kinetic parameters to account for a potentially more efficient intramolecular (intracomplex) activation. By changing just one parameter-the activation reaction of MASP-3 by zymogen MASP-1 was arbitrarily increased by 200-fold-the output became similar to the observed facts that MASP-2 and MASP-1 are proenzymic, and MASP-3 is extensively activated. Moreover, the presence of serpin-inhibited MASP-1 and MASP-2 complexes was observed in blood samples by others (54, 55) even in resting blood, and our simulations are completely in line with these observations. It may occur to someone that if MASP-3 can be activated efficiently by zymogen MASP-1 intramolecularly within a complex, why would not all zymogen MASP-1 autoactivate in a similar way, which is potentially a faster reaction. This question leads to a more general problem, i.e., how the different lectin pathway complexes really look like, and how they are really activated. This is a difficult question, and several activation scenarios have been proposed (61)(62)(63). It is possible that MASP-3 is only activated on highly multimeric PRMs that can accommodate dimers of both MASP-1 and MASP-3. There are alternative explanations to account for the presence of active MASP-3 in circulation. It was proposed earlier for FD (27) that it might be activated within the secretory pathway in mammalian cells. The N-terminal sequence of pro-FD, APPRGR↓, however does not match the general recognition sequence of basic amino acid-specific proprotein convertases, which is (K/R) − (X)n − (K/R)↓, where n = 0, 2, 4, or 6 (56). In contrast, MASP-3 contains a paired basic residue motif at the activation site (SLVKR↓I), which satisfies the above-mentioned criterion. The observation that recombinant MASP-3 was always expressed as a proenzyme (21,37,46) even in mammalian cells contradicts this hypothesis. Therefore, if MASP-3 is activated intracellularly, it must be cell-type specific. Supporting this hypothesis is that furin and PACE4 can activate MASP-3 in vitro (64). In summary, we have demonstrated that MASP-3, just like FD, circulates preferably in the active form in resting human blood. It is now clear that activated MASP-3 keeps FD mostly activated in the circulation, even in the absence of any complement triggering factors. Both active MASP-1 and active MASP-2 are able to activate MASP-3 fairly well. This mechanism might be important during infections. However, both MASP-1 and -2 are present as proenzymes in resting blood. The enigma cannot be easily resolved, but the ability of zymogen MASP-1 to cleave zymogen MASP-3 may provide a solution. We set up a kinetic model for the activation of lectin pathway proteases, which, along with other activation scenarios for MASP-3, is summarized in Figure 6. Our kinetic model, which also includes inactivation by serpins, adequately explains why MASP-1 and MASP-2 are proenzymic, but it can explain extensive MASP-3 activation only if we assume efficient intracomplex activation by zymogen MASP-1. Our kinetic model provides a reasonable explanation, however, elucidating the physiological activation mechanism of MASP-3 requires further experiments. eThics sTaTeMenT The study was conducted in conformity with the WMA Declaration of Helsinki. Experimental protocols were approved by the local ethics committee (permission number: TUKEB 9190-1/2017/EKU). Informed consent was obtained for the isolation of peripheral venous blood from the donors.	2017-12-18T18:02:05.776Z	2017-12-18T00:00:00.000	{ "year": 2017, "sha1": "2987d592b67194371bec70f362129af7c32144ef", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fimmu.2017.01821/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "2987d592b67194371bec70f362129af7c32144ef", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
18506646	pes2o/s2orc	v3-fos-license	Commutants of Analytic Toeplitz Operators on the Bergman Space In this note we show that if two Toeplitz operators on a Bergman space commute and the symbol of one of them is analytic and nonconstant, then the other one is also analytic. Let Ω be a bounded open domain in the complex plane and let dA denote area measure on Ω. The Bergman space L 2 a (Ω) is the subspace of L 2 (Ω, dA) consisting of the square-integrable functions that are analytic on Ω. For a bounded measurable function ϕ on Ω, the Toeplitz operator T ϕ with symbol ϕ is the operator on L 2 a (Ω) defined by where P is the orthogonal projection of L 2 (Ω, dA) onto L 2 a (Ω). A Toeplitz operator is called analytic if its symbol is an analytic function on Ω. Note that if ϕ is a bounded analytic function on Ω, then T ϕ is simply the operator of multiplication by ϕ on L 2 a (Ω). The general problem that we are interested in is the following: When two Toeplitz operators commute, what is the relationship between their symbols? If we were working on the Hardy space of the circle instead of the Bergman space, then the following result would answer this question: • If Hardy space Toeplitz operators T ϕ and T ψ commute, then either both symbols are analytic or both symbols are conjugate analytic or aϕ + bψ is constant for some constants a, b not both 0 (Brown and Halmos [4]). More general results concerning which operators, not necessarily Toeplitz, commute with an analytic Hardy space Toeplitz operator are due to Thomson ( [10] and [11]) and Cowen [6]. On the Bergman space, the situation is more complicated. The Brown-Halmos result mentioned above fails. For example, if Ω is the unit disk, then any two Toeplitz operators whose symbols are radial functions commute (proof: an easy calculation shows that every Toeplitz operator with radial symbol has a diagonal matrix with respect to the usual orthonormal basis; any two diagonal matrices commute). Despite the difficulty of the general problem, we are encouraged by the partial results known when Ω is the unit disk. If Ω is the unit disk and T ϕ and T ψ commute, then the following hold: • If ϕ = z n , then ψ is analytic (Čučković [7]). • If ϕ and ψ are both harmonic, then either both symbols are analytic or both symbols are conjugate analytic or aϕ + bψ is constant for some constants a, b not both 0 (Axler andČučković [1]). In this note we extendČučković's first result above by replacing the disk with an arbitrary bounded domain and (more importantly) by replacing z n with an arbitrary bounded analytic function. Here is our result: Theorem: If ϕ is a nonconstant bounded analytic function on Ω and ψ is a bounded measurable function on Ω such that T ϕ and T ψ commute, then ψ is analytic. Our proof depends on the following approximation theorem: • Let ϕ be a nonconstant bounded analytic function on Ω. Then the norm closed subalgebra of L ∞ (Ω, dA) generated byφ and the bounded analytic functions on Ω contains C(Ω) (Bishop [3]). Proof of Theorem: Suppose ϕ is a nonconstant bounded analytic function on Ω and ψ is a bounded measurable function on Ω such that T ϕ T ψ = T ψ T ϕ . Our hypothesis implies that T ϕ n T ψ = T ψ T ϕ n , and thus the equations above imply that P (uϕ n ) = 0. Hence if h ∈ L 2 a (Ω) we have 0 = h, uϕ n = Ωū hϕ n dA. Because the equation above holds for every bounded analytic function h on Ω and every nonnegative integer n, Bishop's result quoted above implies that Ωū w dA = 0 for every w ∈ C(Ω). But C(Ω) is dense in L 2 (Ω, dA), and so this implies that u = 0. Thus ψ = f and hence ψ is analytic, completing the proof. Open Problems • If an operator S in the algebra generated by the Toeplitz operators commutes with a nonconstant analytic Toeplitz operator, then is S itself Toeplitz and hence (by our result) analytic? • Suppose Ω is the unit disk and ϕ is a bounded harmonic function on the disk that is neither analytic nor conjugate analytic. If ψ is a bounded measurable function on the disk such that T ϕ and T ψ commute, must ψ be of the form aϕ+b for some constants a, b? This question would have a negative answer if the disk were replaced by an annulus centered at the origin because T log \|z\| commutes with every Toeplitz operator with radial symbol.	2014-10-01T00:00:00.000Z	1998-08-14T00:00:00.000	{ "year": 1998, "sha1": "d9fe929400f77c17e786a11fd4c3e6282961ce6b", "oa_license": null, "oa_url": "https://www.ams.org/proc/2000-128-07/S0002-9939-99-05436-2/S0002-9939-99-05436-2.pdf", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "39e3436c8cf3e2bee328615633b2e2e203e21fc3", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
135450757	pes2o/s2orc	v3-fos-license	Prognostic Value of Serum Tumor Markers in Patients With Stage III NSCLC Treated With Chemoradiotherapy Background/Aim: Serum tumor markers such as carcinoembryonic antigen and cytokeratin subunit 19 fragment are generally monitored in non-small cell lung cancer (NSCLC) patients in the clinical practice. However, their clinical relevance in stage III NSCLC treated with concurrent chemoradiotherapy (CCRT) remains unclear. Herein, we examined the clinical relevance of tumor markers in those patients. Patients and Methods: We retrospectively reviewed 62 consecutive patients with stage III NSCLC who received CCRT. We examined the associations of tumor marker levels with their prognosis. Results: There was no correlation between pretreatment tumor marker levels and prognosis. Normal tumor marker levels post-CCRT were significantly associated with favorable progression-free survival (54.8 versus 14.5 months, p=0.02) and overall survival (71.7 versus 40.4 months, p=0.06) compared with high tumor marker levels post-CCRT. Conclusion: We revealed that normal tumor markers levels post-CCRT in stage III NSCLC might be a useful surrogate marker for curing those patients. the treatment of NSCLC, the prognosis of patients with stage III disease remains poor, with a median survival of approximately 20 months (6)(7)(8). Several researchers have investigated the predictive value of 18 F-fluorodeoxyglucose uptake on positron emission tomography, tumor volume, and clinical tumor response in patients with stage III NSCLC treated with CCRT (9)(10)(11)(12)(13)(14)(15). However, these studies were unable to clarify the predictive value of these factors because of various heterogeneities in the tumors and among patients with stage III NSCLC. Serum tumor biomarkers, such as carcinoembryonic antigen (CEA) and cytokeratin subunit 19 fragment (CYFRA) are generally used to evaluate NSCLC in clinical practice in Japan. However, their clinical relevance in stage III NSCLC remains unclear. Therefore, in the present study, we investigated the associations between tumor markers and clinical outcomes. Patients and Methods Patients. We retrospectively screened consecutive patients who had been diagnosed with stage III NSCLC at the Kurume University Hospital, Japan, between 2009 and 2014. Patients who had been diagnosed pathologically with NSCLC had received CCRT with platinum-containing chemotherapy and underwent tumor marker testing, before treatment or at CCRT completion were eligible for inclusion. The present study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Boards of Kurume University Hospital. Statistical analyses. We evaluated the parameters associated with the survival of patients with stage III NSCLC who had received CCRT. We assessed tumor shrinkage according to the response evaluation criteria in solid tumours (version 1.1) (16). We analyzed the associations of pretreatment levels of serum CEA and CYFRA with progression-free survival (PFS) and overall survival (OS) using Pearson's chi-squared test. PFS was defined from the date of initiation of first-line treatment to the date of disease progression or death from any cause. OS was measured from the initiation of the treatment or the initial diagnosis to the date of death or last followup. The Kaplan-Meier method was used to generate survival curves, and significant differences in the curves between the two groups were evaluated by the log-rank test. Multivariate regression analysis was performed using the Cox proportional hazards model. All variables with p-values <0.05 were included in the Cox model. All tests were two-sided, and differences were considered statistically significant at p<0.05. All statistical analyses were conducted using JMP version 11 (SAS Institute Inc., Cary, NC, USA). Results Patient characteristics. Table I lists the clinical characteristics of the 62 patients who were eligible for inclusion in the study. The median age of the patients at diagnosis was 63 (range, 43-75) years. Forty-five (73%) patients were male, and 51 (82%) had a performance status (PS) of 0. The most predominant histological type was adenocarcinoma (35 patients), followed by squamous cell carcinoma (21 patients). At the time of diagnosis, 37 (60%) and 25 (40%) patients had stage IIIA and IIIB disease, respectively. All patients received CCRT containing platinum-based agents as first-line treatment. At the time of analysis, the median follow-up duration was 45 months. Tumor shrinkage. According to tumor shrinkage, 3 (5%) patients achieved a complete response and 26 (42%) a partial response. We analyzed PFS and OS between patients who achieved a complete or partial response and those with stable disease. There were no correlations with PFS or OS in these two patient groups (median PFS: 16.5 versus 20.6 months, median OS: 58.4 months versus not reached, Figure 1). Tumor markers. Before treatment, the median serum level of CEA was 7.6 ng/ml (range, 1.0-220 ng/ml, normal <5.0 ng/ml), and that of CYFRA was 3.9 ng/ml (range, 0.8-47.4 ng/ml, normal <3.5 ng/ml). Figure 2 shows the correlations between the levels of pretreatment tumor markers and the clinical outcomes. There was no correlation between the pretreatment serum CEA level and the survival outcome (R 2 <0.01). Similarly, the pretreatment serum level of CYFRA was not associated with either PFS (R 2 =0.12) or OS (R 2 =0.17). Furthermore, we examined the associations of normal versus high tumor marker levels at CCRT completion with the clinical outcome. We stratified the patients into two groups, depending on whether they had normal or high levels of the tumor markers at the time of CCRT completion. There were no differences between the groups in terms of patient characteristics such as age (p=0.79), sex (p=0.78), smoking status (p=0.40), PS (p=0.52), histology (p=0.72), clinical stage (p=0.79), and having received chemoradiotherapy (CRT) (p=0.52) (Table II). Fourteen patients did not receive CRT because of adverse events (n=10), undergoing surgery (n=2), a decreased PS (n=1), or progressive disease (n=1). Univariate analysis revealed that having normal tumor marker levels at CCRT completion was significantly associated with a favorable PFS (median 54.8 months, Figure 3A), whereas none of the other factors examined was significantly associated with PFS (Table III). Multivariate analysis demonstrated that having normal levels of the tumor markers at CCRT completion was an independent and significant predictive factor for PFS (Table III). Univariate analysis revealed that having normal tumor marker levels at CCRT completion trended towards a favorable OS (71.7 months, Figure 3B). Multivariate analysis demonstrated that normal tumor marker levels at CCRT completion was an independent and significant predictive factor for OS (Table III). Discussion In the present study, we examined tumor markers pre-and post-CCRT in patients with stage III NSCLC and found that patients with normal levels of tumor markers post-CCRT had a better PFS and OS compared to patients with high levels. However, pretreatment tumor markers and tumor shrinkage were not associated with PFS and OS. Tumor biomarkers are useful for evaluating cancer in clinical practice regardless of the disease stage (17)(18)(19)(20)(21). outcome (17)(18)(19)(20). Only one report described that a change in tumor marker levels from before treatment to 1 month after treatment using chemotherapy was significantly correlated with clinical outcome in patients with advanced NSCLC (21). Our results also indicate that normalization of tumor marker levels at CCRT completion, regardless of the pretreatment levels, is associated with better clinical outcomes in patients with stage III NSCLC. A 5-year OS rate of 15% in stage III NSCLC patients receiving CCRT has been reported (22,23). Akamatsu et al. suggested that the 2-year PFS rate may be a reliable surrogate marker for cure, instead of 5-year OS rate, in patients with stage III NSCLC treated with CCRT (24). In the present study, the 2-year PFS rate in patients with normal tumor marker levels was 56%, and the 5-year OS rate was 67% (Figure 3). In contrast, the respective rates in the group with high tumor marker levels were 33% and 41%. Therefore, normal levels of tumor markers at completion of CCRT may be a surrogate marker indicating successful CCRT treatment of stage III NSCLC. Several investigators have demonstrated a significant association between the clinical tumor response and OS in patients with stage III NSCLC treated with CCRT (12)(13)(14). However, in the report by McAleer et al., Kaplan-Meier OS curves revealed that 90% of the responders died within 4 years (12), while the study by Kim et al. used an insufficient follow-up (median follow-up: 16 months) (13). Furthermore, only 28 patients were eligible for enrollment in the study by Lee et al. (14). Our study found no relationship between tumor shrinkage and PFS or OS after a median follow-up duration of 45 months. We consider that this may be because patients with stage III NSCLC have heterogeneous characteristics, such as tumor size, tumor invasive capacity, and number of lymph node metastases. Our retrospective study had several limitations. First, the sample size was relatively small. Second, several confounding biases were potentially introduced in this non-randomized study. Further studies in larger cohorts are warranted. In conclusion, we demonstrated that pretreatment tumor marker levels and tumor shrinkage were not associated with the prognosis of patients with stage III NSCLC treated with CCRT. Furthermore, we revealed that normal levels of tumor markers post-CCRT may be a useful surrogate marker indicating successful treatment of stage III NSCLC. Conflicts of Interest The Authors have no conflicts of interest to declare. No funding source was associated with this study.	2019-04-28T13:03:22.670Z	2019-05-01T00:00:00.000	{ "year": 2019, "sha1": "ceb7df2a11c3e2fc73398818fb319bacaa80e562", "oa_license": null, "oa_url": "http://iv.iiarjournals.org/content/33/3/889.full.pdf", "oa_status": "GOLD", "pdf_src": "Highwire", "pdf_hash": "978ca2f9d81cd7893fd80433b0d304cefe95e867", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
118838064	pes2o/s2orc	v3-fos-license	Nuclear matter properties and relativistic mean-field theory Nuclear matter properties are calculated in the relativistic mean field theory by using a number of different parameter sets. The result shows that the volume energy $a_1$ and the symmetry energy $J$ are around the acceptable values 16MeV and 30MeV respectively; the incompressibility $K_0$ is unacceptably high in the linear model, but assumes reasonable value if nonlinear terms are included; the density symmetry $L$ is around $100MeV$ for most parameter sets, and the symmetry incompressibility $K_s$ has positive sign which is opposite to expectations based on the nonrelativistic model. In almost all parameter sets there exists a critical point $(\rho_c, \delta_c)$, where the minimum and the maximum of the equation of state are coincident and the incompressibility equals zero, falling into ranges 0.014fm$^{-3}<\rho_c<0.039$fm$^{-3}$ and $0.74<\delta_c\le0.95$; for a few parameter sets there is no critical point and the pure neutron matter is predicted to be bound. The maximum mass $M_{NS}$ of neutron stars is predicted in the range 2.45M$_\odot\leq M_{NS}\leq 3.26$M$_\odot$, the corresponding neutron star radius $R_{NS}$ is in the range 12.2km$\leq R_{NS}\leq 15.1$km. Introduction Groundstate nuclear matter properties are specified by the nuclear matter equation of state e(ρ N , δ) which is simply the energy per nucleon of nuclear matter given as a function of nucleon density ρ N and relative neutron excess δ = (ρ n − ρ p )/ρ N . This equation of state is a fundamental quantity in theories of neutron stars and supernova explosions, as well as in theories of nucleus-nucleus collisions at energies where nuclear compressibility comes into play [1]. The main measured quantities which can provide information about equation of state are the binding energies and other data from finite nuclei. As the finite nuclei are in states near the nuclear matter standard state (ρ N = ρ 0 , δ = 0), which is defined as the equilibrium state of symmetric nuclear matter with minimum energy per nucleon and called also the normal state, our actual knowledge of nuclear matter is mainly about nuclear matter at state close to the point (ρ 0 , 0). In this case, the equation of state can be written approximately as [2] [3] e(ρ N , δ) = −a 1 + 1 18 which is specified by the standard density ρ 0 , volume energy a 1 , symmetry energy J, incompressibility K 0 , density symmetry L and symmetry incompressibility K s . The most interesting quantity for supernova explosion calculation is the nuclear incompressibility K 0 which dictates the balance between gravity and internal pressure of the stellar system, while the most interesting quantities for heavy-ion collision studies are the nuclear incompressibility K 0 and the symmetry incompressibility K s which influence the side-flow effects and the isotopic distributions of the collisions, respectively. There is no direct experimental measurement on these quantities. They can be determined only from data fit based on some specific nuclear model. Therefore, our actual knowledge about these quantities is essentially model dependent. Nowadays the quantities which are known with reasonable precision are a 1 , J and K 0 , being the last two still under active investigation. One of the most sophisticated data fit is given by the nonrelativistic Thomas-Fermi statistical model of nuclei with Myers-Swiatecki phenomenological nucleon-nucleon interaction [4]. It is a fit to 1654 ground-state masses of nuclei with N, Z ≥ 8, together with a constraint that ensures agreement with measured values of the nuclear surface diffuseness, giving the root-mean-square mass deviation equal to 0.655M eV . The data fits based on Skyrme nucleon-nucleon interactions give comparable results [3], whereas a model independent but approximate data fit also gives a 1 , K 0 , J and L very close to that obtained by the before mentioned data fit [5] [6]. As the σ-ω-ρ model of the relativistic mean-field theory is used widely to investigate various nuclear phenomena with success [7]- [10], it is interesting to calculate these nuclear matter quantities within this model by using the available parameter sets, to compare with those obtained by the nonrelativistic model. In addition, as these parameters are determined by nuclear ground-state properties, it is also interesting to see what the σ-ω-ρ model can predict for the nuclear system under extreme conditions of density and asymmetry. In this case, the most interesting quantities are the location e m = e(ρ m , δ) of the minimum of the equation of state for given asymmetry δ, and the generalized incompressibility K m = K(ρ m , δ) of the nuclear matter at this state [11]. Another interesting quantity is the maximum mass of neutron stars M N S calculated by the equation of state for neutron matter with δ = 1. Actually, to predict these properties of nuclear matter under extreme conditions is just one of the main goals in developing a relativistic mean-field theory [9]. The purpose of this paper is to make above mentioned calculation in comparing with results obtained by the nonrelativistic model. Section II presents the formalism and formulas used in this calculation. Section III addresses a numerical analysis on linear σ-ω-ρ model of the relativistic mean-field theory. The standard nuclear matter properties calculated from a number of parameter sets are given in Section IV, and the prediction for cold nuclear matter under extreme conditions is made in Section V. Section VI gives the summary. Appendix A displays functions F m (x) and f m (x) which are useful in the analytical expressions as well as in the numerical calculations. The Bjorken-Drell convention for four-vector [12] and the natural units withh = c = 1 are used. Formalism The σ-ω-ρ model of the relativistic mean-field theory is specified by the following Lagrangian density [9]: where , ω and b µ are the nucleon, σ, ω and ρ meson fields with masses M , m σ , m ω and m ρ , respectively, while g σ , g ω and g ρ are the respective coupling constants; b, c and c 3 are the nonlinear term coefficients, and τ are isospin matrices. The nuclear matter equation of state derived from this Lagrangian density can be expressed in terms of the nuclear energy density E as e = E/ρ N − M , and where k p and k n are the proton and neutron Fermi momenta respectively, and the function F m (x) is defined as (see Appendix A for details): The reduced effective nucleon mass ξ and thus the field φ is determined by and the field ω 0 by Knowing the equation of state, the following formulas for pressure p and generalized incompressibility K [11] can be obtained: i=p,n In the previous equation, f ′ m (x) = df m (x)/dx, and the function f m (x) is defined as (see Appendix A for details) At the standard state (ρ 0 , 0), the pressure should be zero, and In addition, the following formulas can be derived: The subscript 0 in the above formulas stands for the standard state (ρ 0 , 0), and k F is the nucleon Fermi momentum of standard nuclear matter which is related to standard density ρ 0 and nuclear radius constant r 0 as Formula (21) is well-known in the literature [7]. It is worthwhile to note that, for the linear model with b = c = c 3 = 0, the ρ-meson in the standard state is nonrelevant to Eqs.(3), (11) and (19). Thus the parameters C σ and C ω are the same both for model with or without ρ-meson, since they are determined by standard density ρ 0 and volume energy a 1 . This point will be discussed more specifically in the next Section. and Eq.(11) determining the reduced effective nucleon mass ξ becomes In addition, the equilibrium condition (19) and the expression of incompressibility (20) are reduced, respectively, to 2 3 It can be seen from Eqs.(28)-(32) that the relevant quantities are k F , C 2 σ , C 2 ω and M . Note that ξ 0 is determined by Eq. (29) and ρ 0 is related to k F by Eq. (27). Therefore, as the measured nucleon mass can be taken for M , the composite parameters C 2 σ and C 2 ω can be determined completely by the value e 0 = e(ρ 0 , 0), by using Eqs.(28)-(30) together with E 0 = (e 0 + M )ρ 0 . The procedure is as follows. At the stable equilibrium point (ρ 0 , 0), an equation involving e 0 , k F , ξ 0 and C σ can be obtained if Eqs. (28) and (30) are combined to cancel C ω . On the other hand, C σ can be solved as a function of k F and ξ 0 from Eq. (29). Substituting this function of C σ into the above-mentioned equation, the following equation involving e 0 , k F and ξ 0 can be derived: ξ 0 can be calculated from this equation, if the location (ρ 0 , e 0 ) of stable equilibrium point is chosen as input data. Having this ξ 0 together with ρ 0 and e 0 , C σ can be calculated from Eq. (29), then C ω can be determined from Eq. (28) or (30). Finally, the incompressibility K 0 can be obtained from Eq. (31). It can be shown easily that Eq.(33) is identical to Eq.(22) of Ref. [9] which is originally given in Ref. [13]. Numerically, ρ 0 and thus k F can be expressed in terms of the nuclear radius constant r 0 as Eq. (27), while e 0 can be related to the nuclear volume energy coefficient a 1 as e 0 = e(ρ 0 , 0) = −a 1 . The experimentally acceptable values are [3] The numerical calculation shows that, in the ranges 1.05fm≤ r 0 ≤ 1.25fm and 15.5MeV≤ a 1 ≤ 16.5MeV, the effective mass ξ ≈ 0.54 does not depend on the choice of r 0 and a 1 sensitively. The composite parameters C 2 σ and C 2 ω are sensitive to the choice of r 0 but not of a 1 . Fig.1 shows C 2 σ and C 2 ω as a function of r 0 for given a 1 = 16MeV. Fig.2 gives the nuclear matter incompressibility K 0 calculated by Eq.(31) as a function of a 1 for given r 0 = 1.14fm. It is not sensitive to the choice of r 0 . Furthermore, Fig.2 shows that K 0 is approximately a linear function of a 1 , in agreement with what is obtained in the macroscopic phenomenological approach to the nuclear matter [14]. In case of the Walecka model [15], C ρ = 0, other nuclear matter properties J, L and K s can be calculated also from these C σ and C ω by Eqs.(21)- (26). The calculated coefficients J, L and K s are almost constant in the range 15.5MeV≤ a 1 ≤ 16.5MeV for given r 0 = 1.14fm, On the other hand, these coefficients depend on the choice of r 0 weakly, for given a 1 . In case ρ-meson is included also in the model, the composite parameter C ρ can be determined by measured symmetry energy J through Eq. (21). The inclusion of ρ-meson contributes to the symmetry energy with an extra term J ρ (Eq.(23)) and to the density symmetry L with an extra term 3J ρ , while keeping the other coefficients a 1 , K 0 and K s unchanged. For symmetry incompressibility K s , it can be seen from Eqs. (26) and (14) that the ρ-meson contributes with a term −18J ρ to −6L and a term 18J ρ to (1/2)∂ 2 K/∂δ 2 \| 0 , and these extra terms cancel each other. Standard state nuclear matter properties There are many parameter sets for the σ-ω-ρ model of the relativistic mean field theory in the literature, some of them are listed in Table I, where L-W is taken from the pioneering Walecka linear σ-ω model [15], L-HS from the Horowitz-Serot linear σ-ω-ρ model [16], L1, L2 and L3 from Lee et al. [17], L-Z, NL-Z and NL-VT from Rufa et al. [18], NL1 from Reinhard et al. [19], NL2 from Fink et al. [20], NL3 and NL3-II from Lalazissis et al. [21], NLB, NLC and NLD from Serot [8], NL-B1 and NL-B2 from Boussy et al. [22] [23], NL-RA from Rashdan [24], NL-SH from Sharma et al. [25], TM1 and TM2 from Sugahara and Toki [26]. Most of them are collected in Reinhard's review [10]. In Table I, g 2 and g 3 are defined, respectively, as It should be noted that some of these parameter sets are given originally in values of C i instead of g i , i = σ, ω, ρ. In this case the values of g i given here are calculated from C i , m i and M by Eq. (9). It should be noted also that our g ρ is only one half of that defined in Ref. [9]. As mainly nuclear matter properties are concerned in the present calculation, the relevant parameters are only C 2 σ , C 2 ω , C 2 ρ , while the meson masses m σ , m ω and m ρ are nonrelevant ones, in case of the linear model. However, in nonlinear model the meson mass is able in some case to influence the nuclear matter property. For example, the ω meson mass m ω appears in Eq. (12) and thus has effect on nuclear matter property in the nonlinear model via the term (ω µ ω µ ) 2 . The standard nuclear matter properties related to these parameter sets are shown in Table II, where all quantities are given in MeV, except ρ 0 which is in fm −3 . In the calculation of a 1 , K 0 , J, L and K s , using formulas given in Section II and input parameters listed in Table I, Eqs. (11) and (19) should be solved simultaneously at first for ξ 0 and k F at the standard point. The calculation of ∂ 2 K/∂δ 2 \| 0 , in Eq.(26) of K s , is made numerically, as its analytical expression is too complicated to be derived. The simple numerical average among the nonlinear model sets is given as the set NL , and the Myers-Swiatecki's result [4] is shown also as the set MS for comparison. ρ 0 and a 1 give the location of nuclear matter standard state. Most values of ρ 0 given in the σ-ω-ρ model are lower than that of Myers-Swiatecki's, the later corresponds to r 0 = 1.140fm and agrees with that obtained from elastic electron scattering and muonic atom spectroscopy measurements [27] [28]. Most values of a 1 given in the σ-ω-ρ model are in the reasonable range around 16MeV, except those of L1, L2, L3, LZ and NL2 sets, which seem too large. Since a 1 is the leading term in the approximate equation of state (1), it is the main parameter in any data fit to nuclear masses. However, there is a big fluctuation around 16MeV, as can be seen from Table II. K 0 and J, the next terms to the leading a 1 in the approximate equation of state (1), are the fine tune in the data fit to nuclear masses, as shown in the droplet model of nuclei [29]. It can be seen from Table II that K 0 given in the σ-ω-ρ model is much larger than that of Myers-Swiatecki's, while J is only about 2/3 of Myers-Swiatecki's, for the linear σ-ω model; J will be increased if the ρ-meson is added also to the linear σ-ω model, but K 0 keeps the same value. This is an inherent character of linear σ-ω-ρ model, as has been shown generally in last Section. In this respect, the nonlinear terms are needed in order to reduce the nuclear incompressibility K 0 , as supported by the calculated results listed in Table II. It is worthwhile to note that, even the value of K 0 obtained from different nuclear measurements and astrophysical observations are spread over a large range from 180 to 800MeV [30], most expectations based on the nonrelativistic model are around 220MeV [11]. Being terms of order higher than K 0 and J in the approximate equation of state (1), L and K s belong to the superfine tune in the data fit to nuclear masses. Even if most values of L given in the σ-ω-ρ model seem to be larger than the acceptable one, they are still in the reasonable range around 100M eV . On the other hand, the values of K s are all positive whose sign is opposite to most expectations based on the nonrelativistic model [1]. Experimentally, K s obtained from the isoscalar giant-monopole resonance energy is between −566 ± 1350 to 34 ± 159MeV [31]. Prediction for cold nuclear matter under extreme conditions The stability condition for the state at minimum of equation of state for given asymmetry δ is [11] are also listed in the last row for comparison. It can be seen that there is no critical point for parameter sets LW, L1, L2, L3 and NL-B2. In these cases, there is a minimum for the pure neutron matter equation of state and the bound neutron matter is predicted. For other parameter sets, the neutron matter is an unbound gas system. The predicted critical point (ρ c , δ c ) is in the ranges 0.014fm −3 < ρ c ≤ 0.039fm −3 and 0.74 < δ c ≤ 0.95, with the corresponding effective nucleon mass in the range 0.87 ≤ M * /M ≤ 0.95. In addition, the predicted maximum mass M N S and the corresponding radius R N S of neutron stars, calculated by the Oppenheimer-Volkoff equation, using the σ-ω-ρ model equation of state of the relativistic mean field theory with the above mentioned parameter sets and δ = 1, are also shown in Table III. The range of the maximum mass is 2.45M ⊙ ≤ M N S ≤ 3.26M ⊙ , and the range of corresponding star radius is 12.2km≤ R N S ≤ 15.1km. Fig.3 gives some examples of ρ m (δ), where the solid curve from top to bottom in the middle range of δ corresponds to L-W, L-HS, NL-SH, TM1, NLC, and NL1; the dashed curve corresponds to Myers-Swiatecki' result. One source of deviation among these curves comes from the difference in the origin of the curves: ρ 0 = ρ m (0). In cases of L-W and L1 ρ 0 is much higher but others are close or lower than that of Myers-Swiatecki's. However, even if all the curves are rescaled to the same ρ 0 , there still exists large deviation among these curves in the middle range of δ. Fig.4 plots some examples of e m versus δ, where the solid curve from left to right on the end of the curve corresponds to L-HS, NL-SH, TM1, NL1, NLC, and L-W; the dashed curve corresponds to Myers-Swiatecki' result. All curves are close each other in the low asymmetry region, but L-W's is significantly lower than others for δ > 0.2. Fig.5 is the curve K m versus δ calculated by same parameter sets as that of Figs.3 and 4. The solid curve from top to bottom in the middle range of δ is by L-W, L-HS, NL-SH, TM1, NLC, and NL1, and the dashed curve is by Myers-Swiatecki. The diference between these curves is obvious, even if NLC and NL1's are close to each other as well as close to Myers-Swiatecki's. Summary In summary, the properties of nuclear matter at standard density ρ 0 with equal neutron and proton densities, ρ n = ρ p , are calculated at first in the relativistic mean field theory with a variety of parameter sets. The result shows that the volume energy a 1 and symmetry energy J are around the acceptable value 16MeV and 30MeV respectively, the incompressibility K 0 is reasonable only for nonlinear model while is unacceptably high for linear model, the density symmetry L is around 100MeV for most parameter sets, and the symmetry incompressibility K s has positive value whose sign is opposite to most expectations based on the nonrelativistic model. Secondly, the calculation shows that for most parameter sets there exists a critical point (ρ c , δ c ), where the minimum and the maximum of the equation of state are coincident and the incompressibility equals zero, and it falls into ranges 0.014fm −3 < ρ c < 0.039fm −3 and 0.74 < δ c ≤ 0.95; while for some parameter sets there is no critical point and the pure neutron matter is bound. The deviation among results calculated by different parameter sets is discussed. The maximum mass of neutron stars is also calculated with results in the range 2.45M ⊙ ≤ M N S ≤ 3.26M ⊙ . It is worthwhile to note that a more realistic calculation, by using a nuclear Thomas-Fermi equation of state, gives a maximum mass of neutron stars equal to 3.26M ⊙ [32]. The most of observational neutron star masses are between 1.2 − 1.8M ⊙ . As different parameter sets give results which deviate significantly from one another, in order to extract from them more reliable predictions for nuclear matter properties, more sophisticated data fit, especially the data fit to larger number of nuclear masses and other measured nuclear data is expected for the nonlinear σ-ω-ρ model of relativistic mean field theory. APPENDIX A: Functions F m (x) and f m (x) defined below are useful in the analytical expressions and numerical calculations of the relativistic mean field theory: The following formulas can be obtained: Some examples of F m (x) and f m (x) are:	2019-04-17T15:49:46.431Z	2000-12-01T00:00:00.000	{ "year": 2001, "sha1": "8d8f35fd020b84086a5bc553a57ab9cdea710cef", "oa_license": null, "oa_url": "http://arxiv.org/pdf/nucl-th/0102003", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "7bc68414127cc3dc32b0febbffcd2755906dc3b9", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
17126400	pes2o/s2orc	v3-fos-license	The role of natural killer cells and antibody-dependent cell-mediated cytotoxicity during murine cytomegalovirus infection. Definition of the functions by which the cellular immune system contributes to control of cytomegalovirus (CMV) infection should permit determination of the specific defects which result in the increased susceptibility to infection of immunosuppressed individuals. Using a murine model, we studied the cytotoxic lymphocyte response to murine CMV infection. This response was found to be biphasic. The initial phase extended from the 3rd to the 6th d after infection, was not genetically restricted, and correlated to a rise in numbers of natural killer (NK) and antibody-dependent killer (K) cells in spleens. The NK- and K-cell responses were preceded, by 24 h, by a rise in serum interferon levels, and occurred before the time when antibody could be measured in serum by neutralization. NK and K cells appear to develop the capacity for specific recognition of CMV-infected cells and the potential to contribute to control of the acute phase of CMV infection. Interferon Assays. Serum specimens from triplicate mice were collected at various times after infection and stored at -20°C. Interferon assays were performed by Biofluids, Inc., Rockville, Md. Serial 10-fold dilutions of serum were tested for interference of vesicular stomatitis virus plaque formation in L929 cells. NK-cell and ADCC Assays. A microcytotoxicity assay involving an 18-h incubation of lymphocytes with target cells similar to that which we have previously described was used throughout (1). The RBL5 cell line has been previously shown to be susceptible to lysis by NK cells and, when sensitized by allotypic antisera, to lysis by K cells (7). RBL5 target cells were labeled with [SZCr]sodium chromate (1) and resuspended either in diluted heat-inactivated BALB/c anti-C57BL/6 serum for ADCC assays, or in media for NK-cell assays. The cells were incubated for an additional 30 min at 37°C. Additional media was added to yield a final target cell concentration of 1 × 105 target cells/ml, and 0.1-ml vol were dispersed into wells of plastic round-bottomed microtiter plates (Linbro Chemical Co., Hamden Conn.). Pooled spleen cell suspensions of triplicate spleens from uninfected CBA/n mice or from BALB/c mice were prepared in RPMI-1640 (Grand Island Biological Co., Grand Island, N. Y.) with 5% fetal bovine serum at various times after infection. Lymphocyte suspensions were washed twice and then resuspended, usually at 5 × 106 cells/ml, and 0.1-ml vol were added to quadruplicate wells for test lysis. Suspensions of unlabeled RBL5 cells, 0.1 ml/well, were added for spontaneous lysis; 0.1 mi/well of 10% Brij-35 solution (Sigma Chemical Co., St. Louis, Mo.) was added for maximum lysis determination. MCMV-specific cytotoxic lymphocyte assays were performed in replicates of eight as previously described (1). The percentage of specific immune lysis (% SIL) was calculated from the equation: % SIL = test lysis -spontaneous lysis × 100. maximum lysis -spontaneous lysis Results The results of serum neutralizing antibody assays and of plaque assays for titration of MCMV infectivity in spleen cell suspensions and salivary gland homogenates are presented in Fig. 1 rise for 2-3 wk after infection, and then fell more slowly. Neutralizing antibody in serum was first detected 21 d after infection. No interferon was detected in normal mouse serum. It was first detected on the 2nd day after infection, at which time it had already reached maximum levels of 103'1 U/ml. It then declined over the next 4 d to undetectable levels. The results of cytotoxicity assays using infected and uninfected MEC as target cells are presented in Fig. 2. Specific immune lysis of MCMV-infected BALB/c MEC by syngeneic spleen cells was first detected 4 d after infection, was highest ~8 d after infection, and returned to insignificant levels by 3-4 wk. There was no significant lysis of uninfected BALB/c MEC. Specific immune lysis of MCMV-infected allogeneic C3H/HeN MEC was detected from days 3-6 after infection. The results indicated that the initial virus-specific cytotoxicity was not genetically restricted, whereas the later activity was restricted. Results of assays of spleen cells for NK-and K-cell activity indicated that the optimum incubation time was 18 h, because the % SIL was greatest at that time, and spontaneous lysis was only 20-30% of maximum lysis. Experiments were performed to determine the optimum effector:target ratio and to determine the maximum useful dilution of the BALB/c anti-C57BL/6 serum used to sensitize target cells for ADCC assays. Results of two of these experiments are presented in Table I. Similar results were obtained using spleen cells from uninfected CBA/n mice or from BALB/c mice 3 d after infection. Cytotoxic cells were not detected in spleens of uninfected BALB/c mice using these assays (results not shown). In subsequent experiments, the antiserum was routinely used at a dilution of 1:100. The optimum effector:target ratio was found to be 50:1; at higher ratios, spontaneous target cell lysis sometimes increased to unacceptable levels. Under these conditions greater % SIL was routinely measured in the ADCC assay than in the NK-cell assay. The changes in activity in response to infection of the cytotoxic lymphocytes measured by these two assays are shown in Fig. 3. CMV-infeeted BALB/c mice developed a brisk rise in splenic NK-and K-cell activity beginning 3 d after infection and persisting through day 5 or 6 after i.p. virus inoculation. The results of experiments performed to determine the cell type responsible for target cell lysis in the NK-and K-cell assays are presented in Table II. Nylon-wool nonadherent cells were equivalent in cytolytic activity to unfractionated spleen cell suspensions. Lysis ofT lymphocytes in suspensions by treatment with anti-theta serum and complement also failed to significantly affect the activity of the cytotoxic effector cells. These results indicated that the predominant cytotoxic cell type(s) detected in each of these two assays were not macrophages or B or T lymphocytes. Discussion Cell-mediated immune lysis of herpes virus-infected cells is potentially important as a host defense mechanism by which infected cells may be destroyed before virus replication has occurred. Although much attention has been given to the frequency of severe and fatal CMV infections in individuals with depressed cellular immunity (8), little is known about specific functions of different types of effector cells as mediators of resistance to these infections. We have previously defined a CTL response to MCMV infection (1). This CTL response is virus-specific, H-2-restricted, and correlates temporally with the viremic phase of infection. During this time, CTL are distributed through the peripheral blood and in cervical lymph nodes (2). These earlier findings suggest that CTL may contribute significantly to the immune control of the acute, viremic phase of infection. The studies reported here demonstrate that spleen lymphocytes develop the capacity, during days 3-6 after i.p. inoculation of MCMV, to lyse syngeneic and allogeneic MCMV-infected target cells. CTL responses to a number of virus infections have been described (1)(2)(3)(4)9). In each case, these CTL have been found to be H-2 restricted. The initial phase of the cytotoxic lymphocyte response to MCMV infection is, therefore, probably not mediated by CTL, whereas the second phase has been previously shown to be mediated by T lymphocytes (1). A biphasic response with similar characteristics has also been found during murine LCM infection (3), indicating that the pattern of response measured here is not unique to one virus. Activity of NK and K cells was found to be increased between days 3-6 after infection. In each assay system, the cytotoxic lymphocytes possessed characteristics typical of NK and K cells (10). They were nonadherent, and lacked theta antigen. Because these assays did not involve the use of MCMV-infected target cells, the cytotoxicity measured was clearly not dependent on virus-specific antibody, nor the presence of viral antigens on the surface of the target cells. However, the simultaneous occurrence of this response and the first phase of the virus-specific cytotoxic lymphocyte response suggests that the same effector cell(s) may have been involved. The mechanism by which NK or K cells could specifically recognize MCMVinfected cells is unknown. We cannot completely exclude the possibility that the cytotoxic effect of these cells was antibody dependent, but we could not detect neutralizing antibody in serum until 2 wk after this cytotoxic-cell response had abated. It is possible that MCMV infection induced the formation of nonviral antigens on infected cells which reacted specifically or by cross-reaction with antigens to which the animal was previously exposed. Regarding this possibility, it has been shown by Boyd et al. (11), that infection of cells with a variety of herpes viruses, including CMV, results in activation of endogenous type C viruses. Antigens of these viruses might be recognized by naturally occurring or preexisting antibody and thus provide specific recognition sites for NK or K cells. Alternatively, virus infection might result in differentiation of a subset of NK cells capable of specific recognition of MCMV antigens in the absence of antibody. Regardless of the mechanism by which target cell recognition occurs, NK or K cells probably serve an important role early in the course of MCMV infection. It is likely that these effector cell functions are stimulated by virus-induced interferon, and may then specifically recognize and lyse syngeneic or allogeneic MCMV-infected cells. This broadly reactive response is then replaced within 3-4 d, and as serum interferon levels fall, by the highly specific, genetically restricted CTL response. Thus, NK and K cells may contribute to the role of CTL in controlling the acute, viremic phase of MCMV infection. Summary Definition of the functions by which the cellular immune system contributes to control of cytomegalovirus (CMV) infection should permit determination of the specific defects which result in the increased susceptibility to infection of immunosuppressed individuals. Using a murine model, we studied the cytotoxic lymphocyte response to murine CMV infection. This response was found to be biphasic. The initial phase extended from the 3rd to the 6th d after infection, was not genetically restricted, and correlated to a rise in numbers of natural killer (NK) and antibodydependent killer (K) cells in spleens. The NK-and K-cell responses were preceded, by 24 h, by a rise in serum interferon levels, and occurred before the time when antibody could be measured in serum by neutralization. NK and K cells appear to develop the capacity for specific recognition of CMV-infected cells and the potential to contribute to control of the acute phase of CMV infection. Received for publication 13 August 1979.	2014-10-01T00:00:00.000Z	1979-12-01T00:00:00.000	{ "year": 1979, "sha1": "2dff1560a500c57b333b267c1e0ad198a21f2ddd", "oa_license": "CCBYNCSA", "oa_url": "http://jem.rupress.org/content/150/6/1549.full.pdf", "oa_status": "BRONZE", "pdf_src": "PubMedCentral", "pdf_hash": "2dff1560a500c57b333b267c1e0ad198a21f2ddd", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
236717155	pes2o/s2orc	v3-fos-license	A prospective comparative study of intranasal dexmedetomidine and clonidine on sedation and hemodynamic response during laryngoscopy in adult patients In general anesthesia, hemodynamic changes during endotracheal intubation are major concerns. Sedative premedications like Dexmedetomidine and Clonidine, when used intranasally are effective options for reduction of preoperative anxiety and preventing untoward hemodynamic responses at the time of induction. Thirty eight patients of ASA 1 and 2 aged 18-70 years were randomly allocated in 2 groups of 19 patients: Group C received inj. Clonidine 3 mcg/kg and Group D received inj. Dexmedetomidine 1 mcg/kg intranasally 45 min before induction. All patients were monitored after premeditation and throughout the surgery. We observed sedation, HR, SBP, DBP, MAP and SPO 2 at various intervals. Intranasal Dexmedetomidine was found to be more effective in producing perioperative sedation and more stable hemodynamics at the time of induction. Dexmedetomidine via intranasal route can be considered as a useful alternative of conventional medications to produce sedation and to blunt hemodynamic Introduction Induction of anaesthesia is a stressful and anxiety provoking experience. The fear and anxiety at the time of induction of anesthesia lead to increase in catecholamine levels. Laryngoscopy and endotracheal intubation are also associated with sympathetic stimulation which lead to tachycardia and hypertension [1] . Haemodynamic changes occurring during laryngoscopy and endotracheal intubation were first described by Reid and Brace. All these effects can be detrimental in patients with cardiovascular and cerebrovascular diseases [2] . Several techniques have been used to eliminate or suppress the stress response including deepening of anesthesia, intravenous and local lignocaine spray, intravenous nitroglycerine, hydralazine, beta blocker, calcium channel blockers, opioids, etc. The proper use of appropriate premedication may actually decrease anaesthetic and analgesic drug requirements as well as some side effects, such as postoperative emesis and untoward hemodynamic responses at the time of induction. Clonidine is a centrally acting sympatholytic drug with predominant alpha-2 agonistic action. Commonly, it has been used as an antihypertensive agent, with additional sedative, anxiolytic, and analgesic properties [3] . Dexmedetomidine is a highly selective, short-acting, alpha 2-adrenoreceptor agonist. It provides sedative, analgesic, and anxiolytic effects with minimal respiratory depression, which makes it a preferable choice among anaesthesiologist for use as an adjuvant for anaesthesia, as well as pre-medication for relieving anxiety or producing sedation before anaesthesia. The intranasal route is a convenient and effective method of administration with high rate of patient acceptance. It has been suggested that a smaller dose or routes other than rapid intravenous delivery may help to minimize the hemodynamic complications of Dexmedetomidine like hypotension, bradycardia even cardiac arrest [2] . The aim of the present study was to compare the efficacy of intranasal Dexmedetomidine and intranasal Clonidine as a premedication for producing satisfactory levels of sedation as well as blunting of hemodynamic responses due to laryngoscopy in adult patients undergoing surgery under general anaesthesia. Materials and Methods After obtaining approval from ethical committee of our institute a written and informed consent was obtained from the patients. The study was conducted in prospective randomized manner using a computer generated random number program and allocation concealment was done using serially numbered opaque sealed envelope technique where patients with even numbers were given intranasal Clonidine and patients with odd numbers were given intranasal Dexmedetomidine as premedication. This study included 38 patients of either sex between age group of 18-70 years of American Society of Anaesthesiologists (ASA) grade 1 and 2 posted for elective surgical procedure under general anaesthesia. Exclusion criteria included-patient refusal, patients with history of drug abuse, patients with pre-existing neurological or psychological disease, BMI >30, anticipated difficult airway, cardiac or respiratory system disease, hemodynamically unstable, previous history of allergy to the study drugs. All patients were explained and counselled about the procedure one day before surgery. Patients were randomly allocated to one of the two groups for administration of study drug 45 minutes prior to surgery. Baseline heart rate, blood pressure and SpO2 were recorded just before the administration of drug. Group C-received 3 mcg/kg intranasal Clonidine hydrochloride and Group D-received 1 mcg/kg intranasal Dexmedetomidine. The drugs were instilled in both nostrils using insulin syringe, with patient in recumbent position. We have used 0.5 ml per nostril as the maximum volume. The time of drug administration was noted, and the observer recorded SpO2, HR, SBP, DBP and MAP for 45 mins and sedation at 45 mins following drug administration. The sedation level was assessed by Ramsay Sedation Score. Ramsay sedation scale: 1. Anxious, agitated or restless. 2. Co-operative, oriented and tranquil. 3. Responds to command. 4. Asleep with brisk response to the stimulus. 5. Asleep with sluggish response to the stimulus. 6. Asleep with no response. In the present study, data was presented as Mean ± SD and proportion. Statistical analysis was done using t-test where P value ≥0.05 was not significant, <0.05 was significant and <0.001 was highly significant. Result and Observation The demographic profile was comparable in both groups as shown in table-1. Sedation score was comparable in both groups before premedication. After 45mins of premedication, difference in sedation score between the two groups was significant (p< 0.05). As per table-3, mean heart rate of group D is significantly less as compared to group C at all-time intervals after 20mins of premedication (p< 0.05). Mean arterial pressure of group D is significantly low as compared to group C at 40mins after premedication, at laryngoscopy and intubation and at 1 min after intubation (p< 0.05). There was no incidence of significant bradycardia, tachycardia, hyper or hypotension in either group. None of the patients experienced nausea, vomiting or respiratory depression in the study period. Discussion Attenuation of laryngoscopic stress response is a major challenge for anaesthesiologist. The satisfactory role of preoperative Clonidine and Dexmedetomidine for attenuation of laryngoscopic stress responses is well established. Nowadays besides IV route, use of Intranasal as premedication is becoming popular, specially in pediatrics population. In the present study, we compared the efficacy of intranasal Clonidine and Dexmedetomidine on the sedation and stress responses of laryngoscopy and intubation. During general anaesthesia, pre-operative anxiety and fear as well as laryngoscopy and endotracheal intubation cause sympathetic stimulation with an increase in the circulating catecholamines levels which leads to significant increase in HR and MAP. The response is initiated within 5 seconds of laryngoscopy, peaks in 1-2 min and returns to normal levels by 5-10 min. Both Clonidine and Dexmedetomidine are centrally acting α2 agonists and have sedative, hypnotic, anxiolytic, analgesic, sympatholytic and analgesic properties. They stimulate α2adrenergic inhibitory neurons in the medullary vasomotor center. Decreased central sympathetic outflow is manifested as peripheral vasodilatation and decrease in systemic blood pressure, HR and cardiac output. They have unique pharmacological property of conscious sedation and is devoid of any respiratory depression. Dexmedetomidine has a greater affinity for the α-2 receptor than α-1 receptor (1620:1) compared to Clonidine (220:1) [4] . The intranasal route is more convenient as it is relatively non-invasive, painless and easy to administer. Intranasal drug can penetrate the blood-brain barrier and reach the central nervous system directly. Due to the higher vascularity of the nasal mucosa, the drugs may access the systemic circulation rapidly, bypassing the first-pass metabolism of liver. The main disadvantages of IV premedication is that sedative action is more pronounced than analgesic effect with profound bradycardia and hypotension [2] . In present study, we observed that patients in group D achieved satisfactory sedation and better stress response to laryngoscopy as compared to group C. Souhayl Dahmani et al. (2010) studied effects of Clonidine and benzodiazepines as premedication in children and concluded that Clonidine produces better sedation, decreases post-operative pain and emergent agitation [5] . Sukanya Mitra et al. (2013) conducted a study comparing intranasal Clonidine and Midazolam as premedication in children and concluded that intranasal Clonidine produces comparable level of sedation and effective anxiolysis as intranasal Midazolam with a better recovery in children [6] . Guang Han et al. (2014) observed that intranasal Dexmedetomidine is a new, safe and effective approach for patents undergoing gastroscopy with more stable respiratory and hemodynamic parameters than intravenous Dexmedetomidine [7] . Dharmendra Kumar Yadav et al. (2018) studied effect of intranasal Dexmedetomidine and Clonidine on hemodynamic response during laryngoscopy in hypertensive adult patients. They observed that intranasal Dexmedetomidine produced a comparable perioperative anxiolysis as Clonidine with higher sedation levels, more stable hemodynamics and improved patient satisfaction [1] . Saikat Niyogi et al. (2020) noticed that both intranasal and intravenous infusion of Dexmedetomidine are equally effective for attenuation of stress response and intranasal Dexmedetomidine can be used as a safer alternative premedication [2] . Conclusion From our study we concluded that both intranasal Clonidine and Dexmedetomidine effectively produced sedation when given 45mins before the surgery and blunted hemodynamic response during laryngoscopy in adult patients requiring general anesthesia although intranasal Dexmedetomidine is found to produce better sedation and blunted stress response as compared to intranasal Clonidine.	2021-08-03T00:05:26.993Z	2021-01-01T00:00:00.000	{ "year": 2021, "sha1": "828a1d6910a6e1faf47875287c96f30ae31f3b51", "oa_license": null, "oa_url": "https://www.anesthesiologypaper.com/article/view/201/4-1-16", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "7ffdd0ab176ea792579207fcb47fe69684228b60", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
257472582	pes2o/s2orc	v3-fos-license	Water Content of Plant Tissues: So Simple That Almost Forgotten? The aim of the present review was to reconsider basic information about various functional aspects related to plant water content and provide evidence that the usefulness of measuring absolute water content in plant sciences is undervalued. First, general questions about water status in plants as well as methods for determining water content and their associated problems were discussed. After a brief overview of the structural organization of water in plant tissues, attention was paid to the water content of different parts of plants. Looking at the influence of environmental factors on plant water status, the differences caused by air humidity, mineral supply, biotic effects, salinity, and specific life forms (clonal and succulent plants) were analyzed. Finally, it was concluded that the expression of absolute water content on a dry biomass basis makes easily noticeable functional sense, but the physiological meaning and ecological significance of the drastic differences in plant water content need to be further elucidated. Introduction Water is indispensable for the functioning of all biological organisms. In plants, water has several functions in comparison to other organisms, including transport processes and transpiration. The mechanical properties of plants are highly dependent on water and its localization in tissues and cells. From a global perspective, water circulation in plants is an integral part of the natural water cycle, which is vital to the functioning of ecosystems. An analysis of the chemical and physical properties of water, as well as basic information about water uptake, transport, and transpiration in plants is beyond the scope of this review, and readers are encouraged to consult specialized reviews for this purpose [1][2][3][4]. Usually, the broad term "plant water status" is used when quantitatively describing the plant-water relationship. Plant water status has several various interrelated components or functional aspects, each describing different parts of this relationship: water potential, water movement, and water content [5]. In practice, one of the most widely used indices of the plant-water relationship is water potential, a complex parameter describing energy-related aspects of water status [6]. The water potential refers to the ability of water in the system to perform physiological functions and depends on the hydrostatic pressure in particular tissues, cells, or cellular compartments (pressure potential); the amount of dissolved solutes (solute potential); interactions with solid surfaces (matrix potential); and the effects of gravity (gravitation potential). Water movement largely depends on interactions between soil (water availability) and the atmospheric environment (air humidity, wind, etc.) through the soil-plant-atmosphere continuum, which can be particularly characterized by measuring the root pressure or transpiration rate. Within the present review, the focus will be on the third component of plant water status: water content. Saturation with water is a critical concept in understanding plant water status, as precisely formulated by N.C. Turner: "living cells need to be more or less saturated with water to function normally, but they are usually incomplete in this desirable condition" [7]. To quantify the degree of water insufficiency or unsaturation, the actual water content of tissues is expressed relative to that at full saturation or full turgor, denoted as "relative water content" (RWC). Wide use of RWC has been associated with the fact that this parameter shows a certain coherence with experimentally measured water potential. Full saturation with water is expected to occur when particular tissues reach a state of full turgor. In practice, during measurement of RWC, this is usually achieved by exposing detached tissues to water and allowing unlimited water uptake until saturation, as shown by weight stabilization. However, no special attention has been paid to the fact that different tissues of various plant species can have different values of absolute water content (in grams per mass unit) even at full saturation. However, some physiological and pathological disorders are associated with plant tissues becoming oversaturated with water, as in the case of hyperhydricity in plant tissue culture or water-soaking in plant pathogenesis. Several papers in the 1980s compared various methods for measuring plant water status [5,7,8]. These studies most commonly indicated that measurement of absolute water content either on a dry or fresh mass basis was "generally unsatisfactory because neither is stable" [5], pointing to possible diurnal and seasonal changes in both dry mass and water content. Indications of the potentially erroneous nature of absolute water content measurements found in earlier works seem to have been fully accepted, leading to the current situation in which RWC is the only water content parameter usually determined and analyzed in different functional studies with plants. There is no doubt that each analytical method has its limitations, which must be clearly stated in any case. However, the objection that "because the dry weight can change diurnally and/or seasonally, comparisons of water content on a dry weight basis are unsatisfactory" [7] loses any meaning when performing relatively short-term comparative studies where sampling is carried out at the same time of the day. Nevertheless, widely used measurements of RWC have another possible problem because of experimental manipulation with plant materials to determine water content at "full saturation" or "full turgidity". This problem has been previously discussed [5], and several more recent studies have provided experimental evidence that measurement of RWC can lead to underestimated results. Thus, it has been shown that in situations leading to internal osmotic adjustment, as in the case of both salt-affected and dehydrated plants, excess water is absorbed during the measurement procedure to obtain a "fully turgid state" [9]. As a result, measured RCW values are anomalously low. Sometimes, dry matter content, as an inverse parameter to tissue water content, has been used as indicator of functional differences between species with fast growth rates vs. species exhibiting nutrient conservation strategies [10] or to characterize yield quality, as in the case of potato production [11]. However, in order to avoid possible differences in leaf water content caused by variations in soil moisture, samples are usually rehydrated Therefore, it appears that leaf dry matter content is derived from and represents an inverse parameter for relative water content, and it is susceptible to the same technical problems as described above for the measurement of RWC. One of the problems related to absolute water content measurements and the use of the obtained results is the expression of the measurement data in a relative manner, either as a percentage of the amount of water either on a dry mass basis or on a fresh mass basis. It has been previously argued that, because of the extremely high proportion of water in fresh biomass, both types of expression are difficult to relate to any functional concept, as water content differences on a fresh mass basis tend to be extremely low (only a change in a few percentages when the actual water content changes by 30%), while differences on a dry mass basis tend to be extremely high (typical values for herbaceous plants being 500-850%) [5]. However, water content on a dry mass basis can be also expressed in absolute units, as grams of water per gram of dry mass. It can be argued that the visibility of the differences (and functional meaning of the results) increases significantly when water content is expressed in grams of water per gram of dry biomass (DM) compared to expressing it as a percentage of fresh or dry biomass. In practice, for example, changes in leaf water content from 85.22% to 80.43% were evaluated as "slight" while significant, as they corresponded only to a 5.6% decrease [12], but conversion to g H 2 O g −1 DM resulted in a decrease from 4.8 to 3.1, or 35.4%. In some studies, the same parameter (water content in grams of water per gram of dry mass) has been designated as "succulence" [13]. Alternatively, the ratio between fresh mass and dry mass, also showing the relative proportion of water, has been designated as "degree of succulence" [14]. In order to compare water content values across plant taxa, different conditions, and various experimental systems, all data were converted to absolute units (g H 2 O g −1 DM) in the present review. The aim of the present review was to reconsider basic information about various functional aspects related to plant water content and provide evidence that the usefulness of absolute water content measurements is undervalued in plant biology experiments. The main emphasis of the review is on herbaceous or semi-shrub species, with woody plants being mentioned for sake of comparison in separate places. However, an analysis of changes in plant water content due to differences in soil moisture is beyond the scope of the present review and specialized reviews should be consulted. Structural Organization of Water in Plant Tissues Water molecules in tissues of all living beings, including plants, can be part of macromolecular structures, thereby forming a network of interfaces with different properties and diverse functional roles that generally determine the structural and functional properties of macromolecules [3]. Interfacial water ("bound" water) has different properties than bulk or "free" water. Bound water has been estimated to represent approximately 30% of the total water content of plants [15]. A significant proportion of water at any particular moment in time can be attributed to the water being transported by the xylem to be transpired through the stomata, or by the phloem to ensure circulation flow through the plant. Both types of transported water enable solute transport between plant parts. In addition, water can be transported through the apoplast and symplast as well as by the transmembrane pathway [16]. While the water potential in all separate water-containing compartments (xylem, cell wall, cytoplasm, and vacuole) at equilibrium is identical, the particular components of the water potential can differ significantly. In particular, the osmotic potential is usually high in both the vacuole and apoplast, but the turgor pressure potential is extremely important in the case of the vacuole [17]. In contrast, the gravitation potential is only relevant in tall trees. It can be expected that the relative proportion of water aimed to be transpired through the leaves at any particular point is significant, given the fact that the amount of transpired water per gram of synthesized organic matter can be as high as 500 g. However, in reality, due to the high proportion of water mass in the total fresh mass of the plant, relatively fast xylem flow velocity, and high transpiration intensity, transpiration water is only a relatively small proportion of the total water content of the plant organism and can usually be ignored. The results of direct measurements are not widely available, but the amount of water transpired by individual plants of Eichhornia crassipes within an hour was calculated to be equal to 0.33-0.58% from the total amount of water in these plants [18]. Consequently, approximately 70% of water in plants can be designated as "utilizable water". The mechanical properties of plant organs are affected not only by their chemical composition and structure but also by maintenance of water-dependent turgor and rigidity. In this respect, plant tissues represent hydrostatic materials [19] and their resistance to mechanical stress is highly dependent on their water content [20]. Differences in the strength of water binding have been studied mostly from the point of view of desiccation tolerance of recalcitrant seeds [21,22]. Variations in stem water content in woody plants with respect to their drought tolerance is another relatively frequently assessed aspect of water content studies in plant biology [23]. Mostly methods based on infrared and Raman spectroscopy, isothermal sorption measurement, dielectric relaxation techniques, and nuclear magnetic resonance spectroscopy have been used to study water properties in plants. Many of these techniques are rather non-specific or require complex and bulky equipment [24]. Recent developments in the field of portable hardware for non-destructive measurement of water content by means of nuclear magnetic resonance have opened up new experimental possibilities, allowing for continuous water content measurements in growing leaves and other relatively small parts of intact plants [25]. Water in Leaves Initial assumptions that "the weight of leaves is largely water and therefore the leaf blade is composed mostly of nothing than water" [26] still seem to be valid, as only a small number of studies have addressed differences in leaf water content among different plants or their changes under the effects of variable environmental conditions. However, more information is available regarding leaf succulence with respect to drought adaptation and in response to salinity, and these aspects will be analyzed further. In different grass species, leaf water content is positively related to the proportion of the total volume occupied by mesophyll plus epidermal cells in their leaves [27]. In addition, the size of mesophyll cells can also positively affect leaf water content. Water content per unit of dry matter increases in all vegetative parts with increasing genetically determined plant growth rate, expressed as the relative growth rate [28]. In a study with 24 wild plant species cultivated under controlled conditions, plant species with the lowest relative growth rate (100-120 mg g −1 day −1 ) had whole plant water contents of 4.8-5.7 g g −1 , but the fastest growing plants (relative growth rate > 300 mg g −1 day −1 ) had whole plant water contents of 8.1-10.1 g g −1 [29]. These differences most likely resulted from higher rates of both mineral ion uptake and water absorption in fast-growing species. When two inbred lines of Plantago major with different growth rates were compared, the line with 25% higher growth rate appeared to have higher water contents in both leaves and roots (Table 1) [30]. A similar relationship has also been established for woody species. When 30 Mediterranean woody species with different post-fire regenerative strategies from a coastal shrubland were compared, the leaves of resprouting species appeared to have lower water contents, slower growth rates, and longer leaf lifespans compared to the leaves of species regenerating from seeds [45]. It is reasonable to suggest that light conditions (intensity of photosynthetically active radiation, spectral characteristics, photoperiod) can also have a pronounced impact on water content in addition to developmental and growth effects. A study with the stoloniferous plant Potentilla reptans, adapted to high light environments, showed that shading conditions resulted in decreased leaf dry mass, increased water content from 4.95 to 8.52 g H 2 O g −1 , and petioles grew taller and thinner as a result of the shade avoidance response [46]. Water in Fruits Similar to other plant products, the quality of fleshy fruits is critically dependent on their water content, affecting both storage and suitability for food processing [47]. The functional aspects of water status in fleshy fruits are largely affected by structural differences in the surface as compared to those in leaves: stomata are nonfunctional if present and the cuticle is highly differentiated but usually more water-permeable [48]. Together with an increase in solute content in developing fruits, more water accumulates, resulting in increased fruit volume [49]. It can be supposed that during growth, increased water content occurs through cellular vacuolization, but additional water is accumulated in the pectin fraction of cell walls. During the early stages of maturation, water content still increases [40]. However, the timing and intensity of changes in fruit water content are highly genotype-dependent. During the final phases of maturation and senescence, loss of cellular integrity occurs due to high activity of polysaccharide-depolymerizing enzymes, leading to fruit softening [50]. This directly results in the loss of water compartmentalization, which affects the mechanical properties of the fruit, basically changing the fruit from being crunchy to juicy. The functional aspects of water transport and accumulation during fruit development have been recently reviewed, and readers are encouraged to seek further details [47,51]. While the chemical composition of fruits is related to their relative growth rate and the climacteric/non-climacteric character of maturation [52], no comparative study involving fruit water content has been performed. Purely intuitively, one would think that the water content of mature fruits would be related to their type. Thus, berries and citrus fruits seem to be fleshier than pomes, but these organoleptic characteristics are affected mostly by chemical composition and structure instead of water content. Examples of water content values in different fruits are given in Table 2 [53][54][55][56]. It is evident that watermelons, melons, strawberries, and citrus fruits have the highest values, but bananas have among the lowest. Especially interesting with respect to water content and storage is the case of fruits of the coconut palm, Cocos nucifera, known as coconuts. Being a typical drupe, a coconut has three layers-exocarp, mesocarp, and endocarp-of which the first two layers form husk, but the hollow endocarp contains a multinucleate liquid endosperm, known as coconut water [57]. Coconut water is rich in sugars, minerals, vitamins, amino acids, etc., with an actual water content of approximately 15.2-16.0 g H 2 O g −1 DM (Table 1) [34]. DM, dry mass. In some cases, the results may be relatively inaccurate because they were read from graphs with the closest possible accuracy. If the original results were not in units of mass of water per unit of dry mass, they were converted accordingly. Water in Seeds Highly controlled changes in water content are important during plant generative reproduction. In seeds, changes in water content during development are parts of physiological changes that lead to the formation of mature seeds. Seed moisture content decreases throughout its development, mostly due to a disproportionately larger rate of assimilate accumulation in comparison to that during the seed-filling phase followed by active water loss during the maturation phase [58]. The opposite process occurs during the imbibition of quiescent seeds before germination, but this initially relies entirely on physical processes, while further changes are under tight internal control [59]. The water uptake rate of dry seeds largely depends on the structure and chemical composition of different seed parts [60]. From a practical point of view, the amount of water in seeds or "seed moisture content" is an important indicator of their expected storage life and resilience. Seeds, detached from a plant, have limited means for controlling their internal water content, which largely depends on the relative humidity of the surrounding air. When the air humidity increases (or decreases), the seed water content slowly balances accordingly, reaching so-called "equilibrium moisture content". It is important to note that seeds of a particular taxon have genotype-specific values of equilibrium moisture content for particular air humidity levels, but they also depend on temperature. Seed chemical composition has a significant effect on equilibrium moisture content, and starch-containing seeds usually have higher water sorption abilities compared to oil-containing seeds [61]. An increase in seed moisture as a result of storage in a humid atmosphere significantly reduces the seed's viability and preservation of its germination capacity, leading to a shortened expected storage period of seed material [62]. For example, even an increase in relative humidity from 20% to 30% (resulting in increase in seed moisture only from 4.4% to 5.6%) can reduce seed longevity approximately two-fold. In contrast to the majority of plant species (about 90%) for which reducing the seed moisture content and decreasing the temperature will increase seed resilience and maintain viability (aka orthodox seeds), seeds of some species do not survive dehydration or low temperatures (aka recalcitrant seeds) [62]. Due to these differences, the viability of recalcitrant seeds is best preserved when stored at high relative humidity (98-99%) and positive temperature (7-17 • C for tropical species and 3-5 • C for temperate species). Water in Vegetative Propagules Underground storage organs of geophytes, bulbs, tubers, and corms act as vegetative propagation organs, and several crop species with bulbs and tubers are essential food plants. Similar to generative propagules, i.e., seeds, the water content of vegetative propagules, such as tubers and bulbs, changes during development and maturation and has immense practical importance with respect to storage and processing. The water content of potato tubers, often expressed as an inverse parameter, dry matter content, is an important feature during potato storage as well as further for food processing [11]. For example, a dry matter content above 22% (or water content below 2.55 g g −1 DM) is necessary to gain product yield and profitability in potato chip production. There are characteristic biochemical changes during the growth of potato tubers, such as increased starch content at the expense of decreased sugar concentration, and water content decreases during potato tuber filling [41,42]. In mature tubers, water is not uniformly distributed, with lower levels in the outside than in the inside of the tuber and also lower levels at both the apical and stem ends (Table 1) [11]. Similar to genotype-dependent variability in chemical composition, water content also differs among potato cultivars [63]. In addition, agrotechnical measures significantly affect the water content of potato tubers. For example, excessive application of nitrogen fertilizers increased tuber water content, thereby reducing their quality [64]. At harvest, bulbs of onions and garlic have a relatively uniform distribution of water, including in the fleshy outer layers. To increase the shelf life, the water content of the outer layers needs to be significantly decreased, leading to the formation of several desiccated layers, i.e., the peel. As a result, the total water content decreases, for example, from 5.09 to 4.26 g g −1 DM while the water content of the outer layers is only 0.3 g g −1 DM (Table 1) [31]. During storage, bulbs of onions and garlic lose water through transpiration, which reduces their shelf life and quality. Agrotechnical measures during cultivation affect water loss from onion bulbs during storage. For example, increasing the nitrogen fertilizer rate from 100 to 150 kg ha −1 increased the water loss from 36% to 57% during 150 days of bulb storage [65]. The time of harvesting and topping as well as the duration of the drying period after harvesting also significantly affects the water content of bulbs and water loss during storage [66]. Similarly, postharvest practices have significant effects on garlic bulb quality, as indicated by changes in the water content of the peel (Table 1) [33,67]. Water in Vegetables To facilitate a comparison, the water content of different types of vegetables is given in Table 3 [68,69]. In many cases, there is no doubt that the domestication process of crops has selected for traits associated with increased water content compared to their wild ancestors. Unfortunately, more extensive comparative studies on the functional meaning of differences in water content, especially in relation to storage functions, are not available. However, these results are of key importance in the practical context of vegetable storage, food processing, etc. In general, moisture loss during storage is a critical factor that negatively affects the quality of stored vegetable products. Both high temperature and low air humidity facilitates water loss through evaporation and cuticular transpiration, which are highly genotype-dependent characteristics [70]. DM, dry mass. If the original results were not in units of mass of water per unit of dry mass, they were converted accordingly. Water Storage in Woody Plants Water storage in trees is a rather specific case, mostly due to significantly different anatomical and physiological features of woody plants in comparison to herbaceous species. Water is stored mainly in xylem conduits and extracellular spaces of living vascular tissues (aka elastic water), but capillary water can also be stored in highly lignified or dead xylem cells [71,72]. In addition, succulent trees develop fleshy tissues adjacent to sapwood-outer parenchyma layers-that act as a water storage compartment, and parenchymatous pith and cortical tissues also can act as water reservoirs [73]. The anatomical characteristics of woody stems, such as the proportion of dead and living cells, largely affect water availability during events of decreased water potential [74]. Water storage in stems tissues of woody plants acts as a buffer to compensate for variations in leaf transpiration demands [75], but capillary water mostly protects the viability of the cambium [76]. Effect of Air Humidity on Tissue Water Content "Hyperhydricity" (formerly known as "vitrification") is a term used to describe a physiological disorder that frequently occurs in plant tissue culture and often leads to a reduction in propagation and significant losses. Hyperhydricity is thought to be caused by high air humidity within cultivation vessels together with other suboptimal conditions, resulting in the glassy and translucent appearance of cultivated tissues [77]. The phenomenon has been recently reviewed in detail, including hyperhydricity-inducing conditions and possible control measures [78]. It was initially proposed that hyperhydric plants contained too much water, thus it was no surprise that the physiological basis for the phenomenon was established to be most likely associated with tissue oversaturation with water at the level of the apoplast [79,80]. Even non-hyperhydric plant tissues have higher water content under conditions of tissue culture than soil-grown plants. Thus, the leaves of Armeria maritima contained 8.0 g H 2 O g −1 DM under tissue culture conditions during the multiplication phase by shoot explants, but the water content of actively photosynthesizing leaves stabilized at 4.0-4.5 g H 2 O g −1 DM after transfer of acclimated plants to soil [81]. In general, limited gas exchange in tissue culture vessels and use of liquid and semi-liquid media lead to increased humidity in the internal environment [82]. In turn, high air humidity results in developmental abnormalities, including poor stomatal function, and leads to reduced transpiration, especially under low light conditions [83]. As unrooted explants have high rates of water uptake driven by negative osmotic potential in cells, this can lead to water oversaturation due to low transpiration rates. Initially, it was supposed that excess water accumulated in cell protoplasts due to high cell wall permeability caused by relatively low amounts of cellulose and lignin [84]. However, the application of methods for visualizing sites of water accumulation supported that extra water was accumulated in intercellular spaces [85]. As a result of cultivation on gelrite or other hyperhydricityinducing conditions, apoplastic air volumes in cultured plants were occupied by water, causing hypoxic conditions in tissues and initiating a sequence of responses characteristic of oxygen shortage, including oxidative stress [79,80]. Apart from tissue culture, the cultivation of plants in greenhouses or other closed spaces with limited ventilation can result in a buildup of high air humidity that results in different physiological alterations. Among them, high air humidity results in increased water accumulation in shoots, as shown for semi-aquatic species Ranunculus sceleratus (Table 1) [39]. Effect of Mineral Nutrition on Water Content An increase in mineral nutrient availability in several species of hydroponically cultivated ornamental indoor plants (Chlorophytum comosum, Epipremnum aureum, Plectranthus fruticosus, Tradescantia pallida) resulted in increased water content of tissues ( Figure 1) [43]. However, this effect was not evident for extremely slow-growing Anthurium spp. and Spathiphyllum spp. plants. The water content of both leaves and roots significantly increased with increasing mineral nutrient availability in coastal species Tripleurospermum maritimum [86]. The water content of both shoots and roots was increased in soil-grown Limonium sinuatum plants with increasing doses of N fertilization, but only up to the N dose that was optimal for plant growth (Table 1) [35]. The water content was increased in shoots of four organically cultivated herb species (Dracocephalum moldavica, Melissa officinalis, Nepeta cataria, Thymus vulgaris) in well-watered conditions under the effect of increasing soil amendment with compost and vermicompost, leading to increases in plant-available mineral nutrients in soil, in parallel to increased accumulation of K + and NO3 − [87]. Although the increased water content of plants by vermicompost amendment has also been noted in other studies [88,89], no clear relationship between increased water content of plant tissues and vermicompost amendment rate has been established. It is also possible that the increased water content is an indirect consequence of improved plant water status due to better water holding capacity of soil after vermicompost amendment [90]. In addition, stimulation of water uptake and accumulation in tissues are known to be the direct effects of application of humic substances, leading to an increase in the fresh mass of plants without much change in dry matter [91]. It seems that the increased water content of plants at luxury mineral nutrient availability is a result of stimulation of vacuolar development as a compartment for ion storage in a similar manner as the process that occurs in salt-adapted plant species at high salinity [92]. Effect of Biotic Interactions on Water Content Information about the effect of biotic interactions on plant water content is quite difficult to generalize, mainly due to the huge diversity of relatively specific interactions involving plants and other organisms. However, experimental evidence from soil-grown plants under controlled conditions shows that biotic interactions are important determinants of plant water status. This effect is clearly direct in some situations, such as when the organism affecting the plant directly causes changes in the water content of plant The water content was increased in shoots of four organically cultivated herb species (Dracocephalum moldavica, Melissa officinalis, Nepeta cataria, Thymus vulgaris) in well-watered conditions under the effect of increasing soil amendment with compost and vermicompost, leading to increases in plant-available mineral nutrients in soil, in parallel to increased accumulation of K + and NO 3 − [87]. Although the increased water content of plants by vermicompost amendment has also been noted in other studies [88,89], no clear relationship between increased water content of plant tissues and vermicompost amendment rate has been established. It is also possible that the increased water content is an indirect consequence of improved plant water status due to better water holding capacity of soil after vermicompost amendment [90]. In addition, stimulation of water uptake and accumulation in tissues are known to be the direct effects of application of humic substances, leading to an increase in the fresh mass of plants without much change in dry matter [91]. It seems that the increased water content of plants at luxury mineral nutrient availability is a result of stimulation of vacuolar development as a compartment for ion storage in a similar manner as the process that occurs in salt-adapted plant species at high salinity [92]. Effect of Biotic Interactions on Water Content Information about the effect of biotic interactions on plant water content is quite difficult to generalize, mainly due to the huge diversity of relatively specific interactions involving plants and other organisms. However, experimental evidence from soil-grown plants under controlled conditions shows that biotic interactions are important determinants of plant water status. This effect is clearly direct in some situations, such as when the organism affecting the plant directly causes changes in the water content of plant tissues, but this effect is indirect in other cases and may rather be related to competition for resources. One particular case of plant water oversaturation and its role in biotic interactions is associated with microbial pathogens. Many bacterial as well as several fungal pathogens induce the formation of water-soaked lesions on plant leaves during the early phase (~24 h) of infection due to local excessive water accumulation in the apoplast [93]. For Pseudomonas syringae pv. tomato interaction with Arabidopsis thaliana, water soaking is a transient phenomenon that only occurs in compatible interactions [94]. In particular, several bacterial proteins acting as effectors are necessary for the development of an aqueous apoplast environment where aggressive proliferation of bacteria occurs [93]. Plant-plant interactions in soil involve direct competition for resources, both for water and available mineral nutrients. It is highly likely that this also results in changes in plant tissue water content. In addition, plant-parasitic plant interactions represent another situation where a host plant organism acts as a source of resources for a parasitic plant, predominantly either in the form of water, carbohydrates, proteins, and amino acids from the phloem (holoparasites) or water and mineral nutrients from the xylem (hemiparasites) [95]. Rhinanthus minor, a facultative root hemiparasite, extracts as much as 20% of the water taken up by the host plant Hordeum vulgare [96]. Both water uptake and transpiration dramatically increases in R. minor plants after attachment to the host, reflected by the stomata always remaining in an open state, even at night. From the host plant side, the rate of water flow from root to shoot, into leaf sheaths, and into leaf laminae of H. vulgare plants after R. minor attachment is significantly decreased [97]. Despite a large body of evidence describing different functional aspects of the plant-parasitic plant relationship, changes in the actual water content of plant tissues have rarely been reported. One particular study explored the water regime of unattached non-parasitic vs. host-free attached R. minor plants [37]. Contrary to what was expected, the water contents of both shoots and roots of attached R. minor plants were even lower than those of unattached plants (Table 1). Thus, it appeared that the increased water flow was used only to maintain a high rate of extraction of host xylem sap for nutrient acquisition instead of increasing the amount of water in tissues. However, similar earlier studies with Rhinanthus serotinus and H. vulgare indicated that the water content of R. serotinus leaves increased after attachment to the host (Table 1) [38]. Surprisingly, usually no effect of parasite attachment on the water status of the host plant has been assessed, besides a highly host genotype-specific effect (from neutral to negative) on the growth of the host plant [98,99]. However, it was shown that water flux from root to shoot also increased in Nicotiana tabacum plants infected with the obligate holoparasite Orobanche cernua [100]. In contrast to xylem-connecting parasitic plants, where water flow is strong and unidirectional, water flow is bidirectional and weaker in the case of parasitic plants connecting to the host phloem [101]. However, the transpiration rate was increased approximately twofold in host plants parasitized by Cuscuta reflexa in comparison to control plants, together with increased photosynthesis, indicating that the parasite acted as a strong sink [102]. No data on changes in absolute water content are available for Striga hermonthica-host interactions, but it has been shown that RWC tended to increase in leaves of Sorghum bicolor plants after infection both under wet and dry soil conditions [103]. It appears that, at least for some types of interactions between plants and their parasites, the physiological status of host plants has been excited, allowing for more efficient resource acquisition. In contrast to the clearly negative effect of parasitic plants on the host plant water regime, there is reason to believe that symbiotic plant interactions, including mycorrhizal symbiosis and nitrogen-fixing bacteria, could have a positive effect in this respect. Indeed, there is a solid body of evidence that mycorrhizal symbiosis affects plant water status under conditions of water shortage [104,105]. Facilitation of water uptake by mycorrhizal hyphae is one of the proposed mechanisms in this respect [106]. A large number of studies have shown the stabilization of RWC in shoots of mycorrhizal plants under drought conditions, in contrast to a decrease in RWC in non-mycorrhizal plants [107][108][109][110][111]. Results on absolute water content are almost absent in this type of study. However, in one study, leaf water content was higher in mycorrhizal Rosa hybrida plants under severe water deficit conditions than in non-mycorrhizal plants [112]. A meta-analysis of mycorrhizal effects on plants beyond nutrient acquisition was performed to reveal if arbuscular mycorrhizal fungi had an effect of plant water content (used in a broad sense and including assimilation, leaf water, relative water content, water content, and water use efficiency) [113]. A clearly positive effect of mycorrhiza was found for water flow, mostly due to increased stomatal conductance, but no significant effect was found for water content. It seems that the increase in water potential may be related to increased osmotic potential due to synthesis in osmolytes in mycorrhizal plants [104]. Contradictory results with respect to the beneficial effects of mycorrhiza on plant water status during water shortage can be largely due to genotype-dependent differences as well as variability in experimental conditions. However, in the context of the present review, it is important to understand whether plant water content is affected in mycorrhizal plants under conditions of good water availability. Results of a single study reported that mycorrhizal Allium porrum plants had higher water content of leaves but not roots at optimum soil moisture (Table 1) [32]. In the context of improved N availability for legume plants with nitrogen-fixing bacterial symbiosis, it can be expected that water content will be increased in symbiotic plants in comparison to non-symbiotic ones, especially at low soil N concentrations. However, not many experimental results are available to support or deny this hypothesis to date. Based on one study in which both fresh and dry biomass data were available, inoculation of Trifolium pratense plants with different Rhizobium strains resulted in a decreased water content of both shoots and roots in all combinations (Table 1) [44]. On the other hand, there is evidence that inoculation of Vigna unguiculata plants with Bradyrhizobium spp. under water shortage conditions improved the drought resistance of plants, but leaf water potential and transpiration rate were not significantly affected by symbiosis [114]. Nevertheless, in a study with drought-stressed Phaseolus vulgaris, nodulation with Rhizobium spp. partially restored RWC under moderate stress conditions and delayed a decrease in RWC under severe stress conditions [115]. A similar role of nodulation with nitrogen-fixing symbiotic bacteria was also proposed in the case of salinity-affected plants, as rhizobium inoculation partially restored a decrease in RWC in Medicago truncatula plants even at high salinity [116]. Water Relationships in Clonal Plants as a Part of Physiological Integration A relatively large proportion of all known plant species can be designated as clonal or as having complex modularity in the form of potentially autonomous clonal units, i.e., ramets [117]. Clonality in plants is largely an underevaluated phenomenon with immense theoretical and practical importance [118]. Physiological integration in clonal plants involves the division of functions and sharing of resources between ramets, allowing for buffering against environmental heterogeneity. According to the principle of spatial division of functions under conditions of heterogeneous light and water availability, plants will allocate proportionally more biomass to aboveground parts, mostly leaves, of ramets in patches with high light and low water availability and proportionally more biomass in belowground parts of ramets in patches with low light and higher water availability [119]. As a result, clonal species have an advantage with respect to biomass accumulation in more spatially heterogeneous environments. However, particular types of clonal structures are associated with adaptations to habitats with different degrees of heterogeneity: in relatively homogeneous and stable habitats, clonal plants usually exhibit aggregated structure (phalanx morphology) with short spacer distances, but clonal plants with long spacers (guerrilla morphology) usually prevail in highly heterogeneous and dynamic or disturbed habitats [117,120,121]. Features of water transport between ramets within clonal genets dependent on resource availability have been studied. Typically, ramets located in wet patches take up water and transport it to ramets located in dry patches [122]. Importantly, the degree of spatial division of labor depends on the extent of spatial heterogeneity, costs of water transportation, as well as the efficiency of resource capture per unit of biomass [123]. It should be kept in mind that water uptake and transport are the principial mechanisms of mineral nutrient acquisition and distribution [124]. Moreover, water transfer occurs during establishment of new ramets in clonal trees, as in Populus tremuloides [125] and bamboo [126]. In particular, bamboo (Bambusa vulgaris and Gigantochloa apus) plants transfer water from established culms to freshly sprouted culms during their "explosive growth" phase [126]. For clonal plants, especially those belonging to the guerilla-type, ramets show ageand environment-dependent functional specialization associated with differences in their morphology. Thus, older ramets of Carex bigelowii have no aboveground structures and are specialized both for uptake of water and minerals as well as for nutrient storage [127]. Resource storage is a functionally important feature of clonal plants, but this aspect has not been particularly studied with respect to water [128]. Succulence as Drought Avoidance Succulents represent an ecological group of plants that have been studied relatively often with respect to tissue water content. In contrast to the majority of plant species without specialized tissues for water storage, plants with specialized tissues for water storage and having a swollen appearance of stems and leaves are known as "succulents." Succulence as a functional morphophysiological trait has been attributed either to an ecological strategy of drought avoidance in plants from arid environments or to an ion dilution mechanism in halophytes [92]. Succulence in halophytes will be addressed in the next chapter, with an emphasis here on plants in which water storing tissues act as a reserve water supply for photosynthetic cells during the day to buffer leaf functions against rootzone water shortage. However, even succulent plants with a clear water shortage-avoiding strategy at the cellular level do not need to have the characteristic succulent appearance at the morphological level. Several excellent reviews have summarized both the ecological significance and morphological diversity of succulent plants, functional aspects of their adaptation to the environment, as well as history of knowledge development about succulents [92,[129][130][131][132]. According to the aim of the present review, only those properties of succulent plants related to the water content of tissues will be analyzed in more detail. Many succulents have Crassulacean acid metabolism (CAM)-type photosynthesis where CO 2 assimilation and photosynthesis are temporally separated, preventing transpiration during the daytime and resulting in higher water use efficiency [133]. However, a causal relationship between the two phenomena is not clear, as CAM species are not the only plants with succulent features [134]. The relationship between leaf succulence and CAM was assessed in 10 species of the genus Sansevieria and it was found that presence of CAM was not associated with the amount of leaf hydrenchyma but rather with the manifestation of all cell succulence [135]. The most important feature associated with the morphological diversity of succulents is that water-storing (succulent) tissues can be present in any plant part, as localization in particular tissues has no functional difference with respect to the ability to temporarily ensure independence from external water sources [129]. Similarly, succulence can develop in any tissues of the plant organism, and the resulting tissues can also perform other functions in addition to water storage. Some succulent plant species rely on leaf water storage in expanded chlorenchyma cells without any specialized tissues (all-cell succulents), while other plants have developed achlorophyllous water-storing tissues, known as hydrenchyma (storage succulents) [130]. Spatial organization of storage succulents with respect to mutual arrangement of chlorenchyma and hydrenchyma is extremely variable and the physiological consequences of this variability are far from clear. Two conceptual functional problems of succulents need to be further solved: whether the amount of stored water is related to drought tolerance and whether the relative proportion of hydrenchyma is related to the amount of stored water. The concept of "utilizable water" is important part of succulence syndrome, as only a proportion of the total water that constitutes the reserve for maintaining physiological processes during the arid season is taken into account [129]. The localization of waterstoring tissues in discrete structures of living cells ensures the maintenance of the water gradient within a plant organism, thus allowing for tight physiological control of water filling and withdrawal. Both the cell size of water-storing tissues and vascular patterning are important determinants for maintaining hydraulic connectivity [134]. However, the relative amount of hydrenchyma was not a significant determinant for water content in Sanseviera species, as Sanseviera parva and Sanseviera senegambica completely lacked hydrenchyma and had relatively higher water contents than species with well-developed hydrenchyma layers (Table 4). In addition, it has been argued that the absolute amount of stored water is not the most important determinant of drought tolerance of succulent plants. During an ecophysiological study of two leaf succulent species from a semi-arid winter rainfall region in Namaqualand (South Africa), deep-rooting C 3 species Augea capensis and flat-rooting CAM species Malephora purpureo-crocea, it was established that the water content of both species was higher in winter when more water was available in the soil (Table 4) [136]. However, irrespective of the season, the water content of leaves of the CAM species M. purpureo-crocea was 33-44% higher than that of A. capensis. In epiphytic species Pyrrosia lanceolata, the relative amount of leaf hydrenchyma was positively correlated with the number of hot and rainless days only in the dry season and negatively correlated with the amount of cloud cover in the wet season [137]. However, no obvious link between the degree of succulence and macroclimatic conditions (aridity gradient in native habitats) for five Crassula species from southern Africa were found under greenhouse conditions (Table 4) [138]. Rapid uptake and recharge of water in storage tissues is supported by the development of three-dimensional venation system in leaves instead of the common two-dimensional one [139]. Decreased venation density with increased succulence leads to less efficient hydraulic function because of longer distances between veins and photosynthetic tissues. Thus, the three-dimensional venation system is an adaptation that makes it possible to resolve the contradiction between the increased leaf thickness associated with succulence and the need for efficient hydraulic function. In terms of the spatial organization of water-storing tissues, many leaf and stem succulents are characterized by the presence of specialized parenchymatous cells without chlorophyll, which are localized adjacent to photosynthetic chlorenchyma cells. These enlarged hydrenchyma cells are characterized by a less negative osmotic potential due to a lower concentration of solutes. Water shortage led to a 50% reduction in whole leaf relative water content in Peperomia magnoliaefolia, but the hydrenchyma lost 75-85% of water while the chlorenchyma lost only 15-25% of water, effectively protecting the photosynthetic function of the chlorenchyma cells [140]. Initially, under well-watered conditions, the osmotic value of the hydrenchyma was lower than that of the chlorenchyma, mostly due to the higher concentration of sugars in the latter, but the total ion concentration was similar in both cell types. However, during dehydration, osmolality in both tissue types increased to the same extent. The aerial roots of epiphytic orchids and some other epiphytes from Araceae, such as Monstera deliciosa, can absorb water from the atmosphere but the absorbed water is stored in specialized root tissues, i.e., the velamen radicum [141]. These tissues cover the root exodermis and are composed of one or several layers of dead air-filled cells, forming a sponge-like structure. When wetted, velamen cells imbibe water through capillary action. The cell layer located towards the center, the exodermis, has living passage cells within the layer of dead cells, which provide water transport from the velamen to the root cortex where photosynthesizing cells are located. Velamen cells next to the passage cells form special structures, tilosomes, which are particularly densely porous structures that are important for water transport from the velamen to passage cells and further to the cortex. Technically, these plants cannot be classified as succulents because succulence syndrome applies only to plants storing water in living cells. Other epiphytes, such as rootless species from genus Tillandsia, have a layer of water-absorptive multicellular trichomes on their leaves for efficient water transport based on capillarity [142]. Effect of Salinity on Water Content In contrast to succulence syndrome in classical succulent plants, which presumes the presence of morphological adaptations to restrict transpirational water loss, plants with watery leaves with no such adaptations are designated as "fleshy" [129]. However, xerohalophytes, as plants adapted to arid climates, will most likely have morphological adaptations to restrict water loss without having water-storing tissues, whereas hygrohalophytes will have no such characteristics. Both ionic and osmotic relationships are extremely important constituents of plant responses to soil salinity. Both sodium and potassium, the two dominant inorganic-type players, maintain their influence in aqueous medium in the form of ions. Very little substantial experimental evidence on the effect of salinity on plant water content has been obtained from studies with wild plants in native salt-affected habitats. First, it is clearly understood that any differences in actual water content between samples can be simply due to differences in soil water availability. Second, genotype-, developmentand organ-specific variability in water content are highly likely to occur. However, there is reason to believe that plant genotype likely determines salinity-induced changes in tissue water content, as shown in a study with 102 plant species from salt-affected coastal habitats [143]. For each species, water content was measured in the leaves of at least five individuals at each of several geographically distant sites on the coast of the Baltic Sea in Latvia, Estonia, Sweden, and Denmark. In Figure 2, each point represents a mean value of water content for a particular distant site, and it is evident that some species had relatively stable low or high leaf water contents below or above the middle 50% range, respectively. For example, Carex arenaria (Car are), Lathyrus japonicus (Lat jap), Filipendula almaria (Fil ulm), Phragmites australis (Phr aus), Artemisia vulgaris (Art vul), Elytrigia repens (Ely rep), and Trifolium pratense (Tri pra) had low leaf water contents, but Chenopodium rubrum (Che rub), Crambe maritima (Cra mar), Rumex maritima (Rum mar), and Spergularia marina (Spe mar) had high leaf water contents. Several species showed high variations in leaf water content above the middle 50% range, such as Honckenya paploides (Hon pep), Salsola kali (Sal kal), Plantago maritima (Pla mar), Tripolium pannonicum (Tri pan), Myosotis scorpioides (Myo sco), Chenopodium acerifolium (Che ace), and Rumex longifolius (Rum lon). Interestingly, no principle differences emerged between coastal-specific and non-specific or wetland species. Due to clear genotype-specificity in water content in salt-adapted plant species, further mostly experimental evidence from studies performed under controlled conditions will be provided. Interestingly, no principle differences emerged between coastal-specific and non-specific or wetland species. Due to clear genotype-specificity in water content in salt-adapted plant species, further mostly experimental evidence from studies performed under controlled conditions will be provided. [143]. Different color of symbols is used simply for the sake of comprehensibility. "Succulent halophytes" is a descriptive term used for plants that seem to accumulate water under increased soil salinity, presumedly serving both as a water reserve as well as for dilution of tissue salt concentration [144]. However, there are suspicions that the term "succulent" is mostly used to indicate the "fleshy" appearance of leaves and stems. Therefore, two aspects need to be solved in order to define this term from a mechanistic point of view: First, do succulent halophytes possess special water-storing structures? Second, do they accumulate more water under increasing salinity? Morphologically, a rather unique taxonomic group of halophytes is represented by species of the subfamily Salicornioideae, with Salicornieae (Amaranthaceae/Chanopodiaceae) being the only tribe [145]. The typical visual appearance of these plants includes a fleshy articulate stem with strongly reduced leaves [146]. Salicornia and Sarcocornia are the two most species-rich genera within the Salicornioideae [145]. Anatomical studies have revealed that the outer layers of the stem are composed of chlorenchymatic photosynthesizing tissues, but the inner layers with peripheral vascular bundles represent chlorophyll-less storage tissues [147]. However, a second cylinder of chlorophyll-containing [143]. Different color of symbols is used simply for the sake of comprehensibility. "Succulent halophytes" is a descriptive term used for plants that seem to accumulate water under increased soil salinity, presumedly serving both as a water reserve as well as for dilution of tissue salt concentration [144]. However, there are suspicions that the term "succulent" is mostly used to indicate the "fleshy" appearance of leaves and stems. Therefore, two aspects need to be solved in order to define this term from a mechanistic point of view: First, do succulent halophytes possess special water-storing structures? Second, do they accumulate more water under increasing salinity? Morphologically, a rather unique taxonomic group of halophytes is represented by species of the subfamily Salicornioideae, with Salicornieae (Amaranthaceae/ Chanopodiaceae) being the only tribe [145]. The typical visual appearance of these plants includes a fleshy articulate stem with strongly reduced leaves [146]. Salicornia and Sarcocornia are the two most species-rich genera within the Salicornioideae [145]. Anatomical studies have revealed that the outer layers of the stem are composed of chlorenchymatic photosynthesizing tissues, but the inner layers with peripheral vascular bundles represent chlorophyll-less storage tissues [147]. However, a second cylinder of chlorophyll-containing cells is located around the stele and separated from the water-storing parenchymatous tissues by the layer of endodermis [148]. A physiological division of functions between different layers of stem tissues of Sarcocornia quinqeflora has been described, involving the salinity-induced development of the endodermis as a barrier protecting the inner photosynthesizing tissues from salt accumulation [149,150]. For the euhalophyte Salicornia europaea, the water content of shoots tended to be higher at moderate salinity, being optimal for growth of the plant, but 800 mM NaCl treatment resulted in a significant decrease in shoot water content (Table 5) [151]. However, this effect was not evident in other studies [152]. DM, dry mass. In some cases, the results may be relatively inaccurate because they were read from graphs with the closest possible accuracy. If the original results were not in units of mass of water per unit of dry mass, they were converted accordingly. Where originally available, different letters or asterisks indicate statistically significant differences given by the authors of the respective paper. The presence of storage tissues has also been described for leaf succulent halophytes. The coastal halophyte Carpobrotus rossi (Aizoaceae) has triangular leaves with a narrow outer layer of photosynthetically active mesophyll and inner storage parenchyma cells (80% of leaf volume) surrounding a central vascular strand [173]. The storage parenchyma of salt-affected C. rossi plants contains approximately three-fold higher Na + concentrations than mesophyll cells, but the differences in water content between various parts have not been estimated. Sesuvium portulacastrum is another halophytic coastal plant species of Aizoaceae [174]. Leaves of S. portulacastrum also possess numerous layers of water-storing cells in the center surrounded by 3-7 layers of chlorophyll-containing palisade cells and an outermost layer of epidermis [175]. Tissue culture experiments showed increased water accumulation at optimal salinity but decreased water accumulation at high salinity [170], and a similar response to salinity was reported for intact plants [169]. Another succulent halophyte, Messembryanthemum crystallinum, is a C 3 species able to switch to CAM metabolism under saline, low temperature, or drought conditions [176,177]. The leaf water content of M. crystallinum plants growing at optimum salinity was extremely high, reaching 49.0 g H 2 O g −1 DM, but it decreased sharply at sea-water salinity (Table 5) [162]. Similarly, in suspension-cultured M. crystallinum cells, water content at optimum salinity was 36 g g −1 DM [164]. In addition to water storage within leaf tissues, M. crystallinum plants use water-filled epidermal bladder cells, representing modified trichomes, to accumulate Na + and other osmotically active substances [178]. In Atriplex (Halimione) portulacoides, the leaf water content increased only at an NaCl concentration stimulating plant growth (200 mM), but leaf dehydration was not evident even at extremely high salinity, causing growth inhibition (Table 5) [156]. Interestingly, at the highest salinity (800 and 1000 mM NaCl) the apoplastic water content in leaves almost doubled, which was associated with maintenance of cell turgidity. For Atriplex griffithii, the water content of leaves increased with plant age, but leaf dehydration due to salinity was evident only after 90 days of treatment, with no visible dependence on salinity level [154]. The hygrohalophyte Atriplex glabriuscula from coastal drift lines showed no pronounced changes in leaf water content with salinity, but the root water content significantly increased at an NaCl concentration optimal for shoot growth [179]. Xerohalophyte C4 species Atriplex griffithii did not show an increase in shoot water content at increased salinity, but leaf dehydration was evident at 500 mM NaCl [180]. Similar results were obtained for another C 4 xerohalophyte, Atriplex canescens [153]. Increased succulence (water content) in leaves of Suaeda salsa was associated with increased root hydraulic conductance and induction of expression of an aquaporin-encoding gene, resulting in an increased amount of aquaporin protein in the plasma membrane [14]. However, in another study, increasing salinity (up to 400 mM NaCl) did not result in changes in shoot or root water content in Suaeda salsa and Suaeda glauca, but the water content of shoots decreased at high concentration of Na 2 CO 3 (28 mM) in both species [181]. As a result of the previous analysis, it becomes clear that succulent halophytes indeed have specialized water-storing tissues and their water content usually increases with increasing salinity, but only up to a certain salinity level, followed by a decrease in water content. However, it remains to be analyzed what happens to water content under the influence of salinity in relatively salt-tolerant species that usually are not classified as "succulent halophytes". For a number of salt-secreting (Glaux maritima, Armeria maritima, Limonium vulgare, Spartina anglica) and non-salt-secreting halophyte species (Juncus maritimus, Juncus articulatus, Atriplex hastata, Atriplex littoralis) grown at moderate salinity, the shoot water content was positively correlated with the Na + accumulation rate ( Figure 3A) [182]. However, the relationship between shoot water content and transpiration rate was positive for non-saltsecreting species but negative for salt-secreting species ( Figure 3B). Species of the genus Beta have received special attention in salt tolerance studies due to relatively high salinity tolerance of both wild ancestors as well as cultivated crop forms. The water content of leaves of wild Beta macrocarpa plants tended to decrease with increasing salinity, together with a significant increase in apoplastic water content [157]. Even moderate salinity decreased the plant biomass by 40% while increasing the leaf water content in soil-grown leaf beet (Beta vulgaris var. cicla), but the water content was decreased with increasing salinity [158]. However, in a hydroponic cultivation system, increasing the NaCl concentration up to 100 mM had no significant effect on the leaf water content [183]. In another study, the salinity responses of wild beet ancestor Beta vulgaris subsp. maritima and cultivated leaf beet crop Beta vulgaris var. cicla were compared to those of soil-grown plants, and both taxa appeared to be extremely tolerant to both NaCl and KCl salinity, with no growth inhibition up to 400 mmol [184]. Most importantly, the water content of both old and young leaves of both taxa increased, with no significant changes in root water content (Figures 4 and 5). For a number of salt-secreting (Glaux maritima, Armeria maritima, Limonium vulgare, Spartina anglica) and non-salt-secreting halophyte species (Juncus maritimus, Juncus articulatus, Atriplex hastata, Atriplex littoralis) grown at moderate salinity, the shoot water content was positively correlated with the Na + accumulation rate ( Figure 3A) [182]. However, the relationship between shoot water content and transpiration rate was positive for nonsalt-secreting species but negative for salt-secreting species ( Figure 3B). Species of the genus Beta have received special attention in salt tolerance studies due to relatively high salinity tolerance of both wild ancestors as well as cultivated crop forms. The water content of leaves of wild Beta macrocarpa plants tended to decrease with increasing salinity, together with a significant increase in apoplastic water content [157]. Even moderate salinity decreased the plant biomass by 40% while increasing the leaf water content in soil-grown leaf beet (Beta vulgaris var. cicla), but the water content was decreased with increasing salinity [158]. However, in a hydroponic cultivation system, increasing the NaCl concentration up to 100 mM had no significant effect on the leaf water content [183]. In another study, the salinity responses of wild beet ancestor Beta vulgaris subsp. maritima and cultivated leaf beet crop Beta vulgaris var. cicla were compared to those of soil-grown plants, and both taxa appeared to be extremely tolerant to both NaCl and KCl salinity, with no growth inhibition up to 400 mmol [184]. Most importantly, the water content of both old and young leaves of both taxa increased, with no significant changes in root water content (Figures 4 and 5). Several species of the genus Plantago are characteristic of salt-affected habitats and are known as relatively salt tolerant. Different salinity tolerances are reported for various accessions of Plantago maritima. An accession from Tunisia was characterized as having relatively low salinity tolerance, with significant growth inhibition already at 50 mM NaCl, and salinity had no stimulative effect on water content (Table 5) [165]. However, the leaf water content was significantly decreased at 200 mM NaCl and decreased linearly with increasing salinity, but the root water content was significantly decreased at 300 mM NaCl. Comparing changes in leaf water content of three Plantago species (halophytes Plantago crassifolia and Plantago coronopus, and salt-sensitive Plantago major) differing in salinity tolerance revealed that the rate of leaf dehydration with increasing salinity was relatively similar regardless of particular tolerance level ( Figure 6) [185]. Importantly, the leaf water content was more sensitive to increasing salinity than plant development or biomass accumulation. However, for soil-grown Plantago maritima plants, the water content of both old and new leaves significantly increased under both NaCl and KCl salinity, with no dehydration evident even at 400 mM salinity, but the root water content tended to decrease with salinity ( Figure 7) [184]. Several species of the genus Plantago are characteristic of salt-affected habitats and are known as relatively salt tolerant. Different salinity tolerances are reported for various accessions of Plantago maritima. An accession from Tunisia was characterized as having relatively low salinity tolerance, with significant growth inhibition already at 50 mM NaCl, and salinity had no stimulative effect on water content (Table 5) [165]. However, the leaf water content was significantly decreased at 200 mM NaCl and decreased linearly with increasing salinity, but the root water content was significantly decreased at 300 mM NaCl. Comparing changes in leaf water content of three Plantago species (halophytes Plantago crassifolia and Plantago coronopus, and salt-sensitive Plantago major) differing in salinity tolerance revealed that the rate of leaf dehydration with increasing salinity was relatively similar regardless of particular tolerance level ( Figure 6) [185]. Importantly, the leaf water content was more sensitive to increasing salinity than plant development or biomass accumulation. However, for soil-grown Plantago maritima plants, the water content of both old and new leaves significantly increased under both NaCl and KCl salinity, with no dehydration evident even at 400 mM salinity, but the root water content tended to decrease with salinity ( Figure 7) [184]. In leaves of halophytic Lepidium latifolium plants, the water content increased by 46% at 300 mM NaCl, but it did not change in the glycophytic species Lepidium sativum (Table 5) [159]. Another halophytic species, Cochlearia officinalis, showed increased water content of leaf petioles, leaf blades, and roots under moderate NaCl concentrations, with no significant decrease at 400 mM NaCl, but the leaf water content was significantly reduced in plants treated with 400 mM KCl (Figure 8) [184]. For saline wetland species Tripolium pannonicum (syn. Aster tripolium), the leaf water content was in the range of 5.3-6.6 g g −1 DM and was not significantly affected by up to seawater salinity (Table 5) [178]. However, in another study, the water content of both the shoots and roots of T. pannonicum decreased at increasing salinity (Table 5) [172]. Similarly, coastal marsh recretohalophyte species Limonium stocksii showed decreased water contents of leaves, stems, and roots at a certain level of salinity, but not in a concentration-dependent manner [36]. In other studies with Limonium sinuatum, the shoot and root water content was either not affected by salinity [160] or decreased only at high salinity [161]. Four Mediterranean Limonium species with different geographical distribution showed extreme tolerance to salinity during early vegetative growth, with significant growth reduction only at 800 mM NaCl and characteristic leaf dehydration already at 600 mM NaCl both in leaves and roots (Figure 9) [12]. Other examples of changes in water content in salt-tolerant species are given in Table 5 [181][182][183][184][185]. In leaves of halophytic Lepidium latifolium plants, the water content increased by 46% at 300 mM NaCl, but it did not change in the glycophytic species Lepidium sativum (Table 5) [159]. Another halophytic species, Cochlearia officinalis, showed increased water content of leaf petioles, leaf blades, and roots under moderate NaCl concentrations, with no significant decrease at 400 mM NaCl, but the leaf water content was significantly reduced in plants treated with 400 mM KCl (Figure 8) [184]. For saline wetland species Tripolium pannonicum (syn. Aster tripolium), the leaf water content was in the range of 5.3-6.6 g g −1 DM and was not significantly affected by up to seawater salinity (Table 5) [178]. However, in another study, the water content of both the shoots and roots of T. pannonicum decreased at increasing salinity (Table 5) [172]. Similarly, coastal marsh recretohalophyte species Limonium stocksii showed decreased water contents of leaves, stems, and roots at a certain level of salinity, but not in a concentration-dependent manner [36]. In other studies with Limonium sinuatum, the shoot and root water content was either not affected by salinity [160] or decreased only at high salinity [161]. Four Mediterranean Limonium species with different geographical distribution showed extreme tolerance to salinity during early vegetative growth, with significant growth reduction only at 800 mM NaCl and characteristic leaf dehydration already at 600 mM NaCl both in leaves and roots (Figure 9) [12]. Other examples of changes in water content in salt-tolerant species are given in Table 5 [181][182][183][184][185]. Salinity-induced leaf dehydration has been described for several species usually not native to saline habitats, such as Silene vulgaris [186] and other species of the genus [187]; Plantago lanceolata, Plantago major, and Plantago psyllium [188]; and Mentha aquatica [184]. However, the water contents of leaf petioles, leaf blades, and stems of an extremely salttolerant accession of Ranunculus sceleratus from a sea water-affected wet beach habitat were also significantly decreased at moderate salinity [39]. Some of the examples provided above were from studies using both NaCl and KCl as salinity agents, and some differences in water content appeared between the treated plants ( Figure 8). However, not only the type of cation but also the type of anion seems to have an effect on salinity-induced changes in plant tissue water content. Thus, for four Rumex species, treatment with NaNO 3 or KNO 3 resulted in significantly higher water content of leaves in comparison to plants treated with NaCl or KCl ( Figure 10) [189]. Moreover, for the three coastal Rumex species, Rumex hydrolapathum, Rumex longifolius, and Rumex maritimus, plants treated with NaNO 2 or KNO 2 tended to have higher leaf water content in comparison to treatment with the respective chloride salts, in spite of the fact that the nitrite salts had negative effect on plant growth. At the moment, there is a lack of evidence to make any broader generalizations, but there appears to be some connection between nitrate-and nitrite-stimulated water accumulation and the increased tissue water content described above in the case of an improvement in the supply of fertilizers. To summarize, some plant taxa do not respond to increasing salinity with changes in tissue water content, at least at moderate salinity, and only mild effects on plant growth are observed, but organ specificity must be taken into account. On the other hand, if the water content changes under the influence of salinity, it can be either an increase or a decrease. As particular studies usually used only a rather limited number of salinity treatments within a particular salinity intensity range, it is difficult to determine a general dose-response pattern; however, it is reasonable to suggest that increased water content appears in species for which a particular salinity level promotes growth, but high salinity causes tissue desiccation in all species, the specific concentration of which will differ depending on the salt tolerance determined by genotype. Indeed, at least in some cases, increased shoot water content seemed to be associated with an increase in dry biomass (growth stimulation by low to moderate salinity), but a causal relationship has not yet been proven. Both types of water changes due to salinity are easily explained from a functional point of view, looking at the increase in water content as an accompanying mechanism for salt accumulation in vacuoles and the decrease in water content (tissue dehydration) as a result of salinity-induced organ senescence and/or decay. There is no doubt that, similar to variations in salinity tolerance, the response of tissue water content to salinity depends not only on genotype but also on differences in experimental conditions. For salinity tolerance studies, it is indicated that the developmental stage of plants, cultivation system (soil, hydroponics), mineral nutrient availability, light conditions (spectrum, photoperiod, intensity), temperature, mode of salt treatment, etc. are important determinants of morphological and biochemical responses to salinity [190][191][192]. Therefore, it can be expected that responses of tissue water content to salinity may also vary with changes in experimental conditions, and this could explain, at least in part, the conflicting results reported here. as salinity agents, and some differences in water content appeared between the treated plants ( Figure 8). However, not only the type of cation but also the type of anion seems to have an effect on salinity-induced changes in plant tissue water content. Thus, for four Rumex species, treatment with NaNO3 or KNO3 resulted in significantly higher water content of leaves in comparison to plants treated with NaCl or KCl ( Figure 10) [189]. Moreover, for the three coastal Rumex species, Rumex hydrolapathum, Rumex longifolius, and Rumex maritimus, plants treated with NaNO2 or KNO2 tended to have higher leaf water content in comparison to treatment with the respective chloride salts, in spite of the fact that the nitrite salts had negative effect on plant growth. At the moment, there is a lack of evidence to make any broader generalizations, but there appears to be some connection between nitrate-and nitrite-stimulated water accumulation and the increased tissue water content described above in the case of an improvement in the supply of fertilizers. Conclusions Available information on the absolute water content of plant tissues and possible functional consequences of the main regularities from both physiological and environmental aspects were analyzed in the present review. The conclusions obtained as a result of the informational analysis can be summarized as follows: (i) The phenomena of water storage and water content of tissues, apart from drought response studies, has been mostly assessed in the context of succulence syndrome either in classical succulents or succulent halophytes; (ii) High organ-and species-specific variability can be found with respect to water content in plants; (iii) The presence of "succulence" and other forms of water storage in particular plant species is not necessarily associated with relatively high water content; (iv) Salinity can have different effects on water content, even among salt-tolerant and salt-accumulating species; (v) The expression of absolute water content on a dry biomass basis makes easily noticeable functional sense. Within the present literature analysis, the highest water content values were evident in the leaves of Mesembryanthemum crystallinum, reaching 49 g H 2 O g −1 DM at 100 mM salinity, and in the stems and leaves of Tradescantia pallida, reaching 30 and 26 g H 2 O g −1 DM at high fertilizer concentration, respectively. For the lowest water content values, a number of species had leaf, shoot, or root water contents below 2 g g −1 DM under non-saline conditions. However, the fundamental structural and metabolic differences accounting for the drastic differences in plant water content remain unclear. Additionally, above all, it is necessary to clarify the functional meaning and ecological significance of these differences. Funding: This research received no external funding.	2023-03-12T15:28:23.480Z	2023-03-01T00:00:00.000	{ "year": 2023, "sha1": "36735e08111996e3ed8cadc2895b5d66b97f337e", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2223-7747/12/6/1238/pdf?version=1678415738", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "5857c0b89aebea3bfdf2ddad1facca09b78ecaf2", "s2fieldsofstudy": [ "Agricultural And Food Sciences", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
55918881	pes2o/s2orc	v3-fos-license	Three-Dimensional Elasticity Solutions for Sound Radiation of Functionally Graded Materials Plates considering State Space Method This paper presents an analytical study for sound radiation of functionally graded materials (FGM) plate based on the threedimensional theory of elasticity. The FGM plate is a mixture of metal and ceramic, and its material properties are assumed to have smooth and continuous variation in the thickness direction according to a power-law distribution in terms of volume fractions of the constituents. Based on the three-dimensional theory of elasticity and state space method, the governing equations with variable coefficients of the FGM plate are derived. The sound radiation of the vibration plate is calculated with Rayleigh integral. Comparisons of the present results with those of solutions in the available literature are made and good agreements are achieved. Finally, some parametric studies are carried out to investigate the sound radiation properties of FGM plates. Introduction Functionally graded materials (FGM) are the heterogeneous composite materials with material properties varying smoothly and continuously in one or more directions, and this characteristic properties are usually achieved through continuous change of the volume fraction of the constituent phases [1][2][3].The concept of FGM was originally proposed in 1984 by a group of material scientists in Japan as thermal barrier materials, and the superiority to conventional laminated composites of such as eliminating the stress concentration leads to a wider applications of FGM in areas such as aeronautics, astronautics, nuclear, biology, navigation [4].In practical applications, FGM structures subjected to dynamic load internal or external will generate noise and radiate sound into the surrounding medium, which may result in less comfort.On the other hand, the radiated sound carries useful information of the FGM structures that can be used for nondestructive evaluation or estimation of material properties.Therefore, the investigation of sound radiation of FGM structures is of great importance from the academic or engineering applications point of view. Plates are one of the most widely used structural components in industrial applications.Sound radiation from panel structures is a practical engineering problem that has been studied extensively.Numerical methods such as finite element method (FEM) and boundary element method (BEM) [5][6][7] are always utilized to estimate the sound radiation of structures.However, these methods are computationally expensive, especially at high frequency domain.An infinite extent of plate is always obtained in the research of sound radiation of plate.However, the plate structures are of finite size in practice.Several theories were proposed to take into account the finite size of a plate structure in sound radiation calculation, for example, the spatial windowing technique presented by Villot et al. [8].As a more general method, the vibration plates are assumed to be placed in an infinite rigid baffle, which, of course, differs from the actual situation in practice, and some authors concentrated on sound radiation from unbaffled plates [9,10].One of the authors of this paper studied the sound radiation characteristic from unbaffled rectangular plates [11].More recently, Putra and Thompson [12,13] also investigated this problem.However, the assumption of an infinite rigid baffle makes the sound radiation problem easier to solve for the fact that the velocity field equals zero everywhere except for the plate surface.In this paper, the assumption of an infinite rigid baffle is also obtained for the convenience in studying the main trends of sound radiation from FGM plate. Two-dimensional (2D) plate theories such as classic plate theory (CPT), first-order shear deformation theory (FSDT), and higher order shear deformation theories are always utilized in the research of sound radiation of plate-like structures; however, acoustic model based on three-dimensional (3D) elasticity theory may be the sole ultimate choice for more precise calculations.Due to the simplicity, the wellknown classic plate theory [14][15][16] is utilized conventionally to model the plate in the calculation of sound radiation from plate structures.The CPT is based on the Kirchhoff-Love hypothesis that straight lines perpendicular to the plane of the undeformed plate remain straight and inextensible and rotate such that they always remain perpendicular to the midplane of the plate after deformation.Nevertheless, neglecting the transverse shear effects and rotary inertia leads to overestimating the natural frequencies of the plate, especially for thick plates or structural response in high frequency range.In order to consider the transverse shear effects on isotropic plates, Reissner [17] and Mindlin [18] developed the FSDT.Hashemi et al. [19] investigated acoustic radiation of rectangular Mindlin plates in different combinations of classical boundary conditions.Cao et al. [20] investigated the sound radiation from infinite stiffened laminated plates theoretically based on the FSDT, and the comparison with Yin's method [21], which is based on CPT, shows that the model based on FSDT and CPT shows a good agreement about the estimation of sound pressure level in the low and medium frequency range, but discrepancies can be found near the coincidence frequency and in the high frequency range.Chandra et al. [22] analytically studied the vibroacoustic response and sound transmission loss characteristics of FGM plates based on a simple FSDT, which was presented by Thai and Choi [23].As is well-known to all, a shear force correction factor is required in FSDT to take in account the nonuniformity of the shear strain distribution through the thickness.The shear correction factor is typically 5/6 [17] or 2 /12 [18] for isotropic homogeneous plates; however, the constant shear correction factor is not appropriate for FGM plates due to the material properties and geometric dimension of FGM plates [24,25].In order to overcome this shortcoming of FSDT, third-order shear deformation theory (TSDT) or higher order shear deformation theory such as Reddy [26,27] was developed, and no shear correction factors are required and better precision can be achieved.Daneshjou et al. [28] studied sound transmission through relatively thick FGM cylindrical shells based on third-order shear deformation shell theory, and their work denotes that there are some discrepancies between FSDT and TSDT at high frequencies for relatively thick FGM cylindrical shells.However, studies of sound radiation from plate structures based on 3D elasticity may be the sole ultimate choice, which is, however, relatively scarce.Hwang et al. [29] presented an elasticity theory solution for acoustic radiation by a pointor line-excited fluid-loaded laminated plate.Shen et al. [30] studied acoustic radiation from multilayered anisotropic plates based on 3D elasticity model.Hasheminejad and Keshavarzpour [31] studied the active sound radiation control of thick piezolaminated smart plate, and the orthotropic laminated plate is modeled based on 3D piezoelasticity theory with the use of state space formulation.Huang and Nutt [32] developed a unique analytical formulation for sound transmission of unbounded FGM panels by employing threedimensional theory of elasticity and state space method. In the present paper, the sound radiation of FGM plates with arbitrary thickness is investigated.An analytical model of sound radiation of FGM plates based on the threedimensional theory of elasticity is proposed.By means of state space formulation and modal expansion, the threedimensional governing equations of elastodynamics are converted into a set of ordinary differential equations with variable coefficients for FGM plate.The solution of the ordinary differential equations results in the transfer matrix relating the top and bottom surface of the plate.Some similar procedures were also used in prior plates studies such as vibration [33][34][35], wave propagation [36][37][38], and sound transmission [32].The external loads including point load, line load, and distributed load are expanded into a double Fourier series form and applied on the surface.Applying the continuity of displacement and stresses at the interfaces of plate leads to global governing equations of the vibroacoustic system, and then sound radiation of the plate is calculated using Rayleigh integral with a primitive numerical scheme. Theoretical Formulation Consider a rectangular FGM plate with length , width , and an arbitrarily constant thickness ℎ, as shown in Figure 1.A Cartesian coordinate system (, , ) is located at the corner of the panel on the bottom surface of the plate.The plate is assumed to be placed in an infinite rigid baffler.The FGM plate is under the excitation of external load on the surface and the fluid structure interaction between the plate and the surrounding air is not considered. Material Properties. The FGM plate is assumed to be made from a mixture of two material phases, for example, a metal and a ceramic, as shown in Figure 1.The materials at the top surface ( = ℎ) and the bottom surface ( = 0) of the FGM panel are purely ceramic and metal, respectively.Within the top surface and bottom surface, the volume fraction of each constituent material changes smoothly through the thickness from one surface to the other, and all constituent materials are assumed in their elastic range.The material property () of the panel such as density , Young's modulus , and Poisson's ratio ] can be expressed as a function of the material properties and volume fractions of constituents [26] where the subscripts and represent the ceramic and metallic constituents, respectively.( , ) and ( , ) denote material properties and volume fraction of ceramic and metal, respectively.The volume fraction of the constituent material is assumed as [26] = ( ℎ ) where ℎ is the thickness of the panel, is the thickness coordinate, and denotes the power-law index that takes values greater than or equal to zero.Substituting (2) into (1) yields and according to (3), density , Young's modulus , and Poisson's ratio ] can be written as where is the density of the plate and u = (, V, ) T and = ( , , , , , ) T are the column vectors of the displacement and stresses, respectively.The linear relations between the strain and the displacement are where = ( , , , , , ) T is the strain components. The constitutive relations can be expressed in general Hooke's law = C, (7) where C is the stiffness matrix as follows: where is the stiffness coefficients.For the sake of generality, they are the function of ; that is, = (). Following the state space method [33,34], we select , V, , , , and as state variables.After combining ( 5)-( 7), the thickness first derivatives of the state variables can be written as Equation ( 9) can be rewritten into a compact form as where 1 = ( , , ) T and For a rectangular plate simply supported and grounded on all four sides, the boundary conditions on the edges are [34] = V = = 0, at = 0, , = = = 0, at = 0, . To satisfy the simply supported boundary conditions ( 12), the state variables are assumed as the following form [33][34][35]: where = √ −1, is the circular frequency, and (), V(), (), (), (), and () are undetermined coefficients.Substitution of ( 13) into (10) using the orthogonal property of the sinusoidal function in ( 13) and (10) yields where As is a function of the -coordinate, the system matrix K is nonconstant but -dependant.Then the problem reduces to find the solution of an ordinary differential equation system with nonconstant coefficients.The analytical solution of ( 14) can be expressed as the following form: where M( 1 , 0 ) is the matricant, which relates the value of the state vector at = 1 , that is, ( 1 ), to the value of the state vector at = 0 , that is, ( 0 ).The matricant for M( 1 , 0 ) can be defined explicitly by the so-called multiplicative integral satisfying the Peano series expansion [36][37][38]] where I is the identity matrix of dimension 6×6.If the matrix K() is bounded in the study interval, these series are always convergent [37,40].This solution to ( 14) is exact and unique. In addition, a piecewise constant method can be obtained as an alternative [41][42][43]; that is, divide the plate equally into numbers of sublayers, and the material parameters of each layer are assumed constant.It is clear that an adequate number of layers in the piecewise constant method can ensure the accuracy of the solution.In the piecewise constant method, the matricant relating the value of the state vector ( 1 ) to the value of the state vector ( 0 ) can be expressed as where is the number of sublayers and K() is the system matrix of each sublayer and takes the value at each midsurface.In addition, the matrix exponential exp((( 1 − 0 )/)K()) in ( 18) can be directly calculated by using builtin function in Mathematica or Matlab to avoid complex algebraic manipulations [32,35]. Using the matricant obtained, the relationship between the state vectors (ℎ) and (0) can be established as and the equation can be expressed in detail as where M are entries of the matrix M(ℎ, 0). Transverse Load. In this paper, only the transverse load on the surface of the plate is considered and the load (, ) is assumed to be expanded into a double Fourier series form as [44] where is the load coefficient and for a load (, ) the load coefficient is given as For a concentrated load (, ) = 0 ( − 0 )( − 0 ), where ( − 0 ) and ( − 0 ) are Delta-functions such that at = 0 , = 0 , they are of value one and vanish elsewhere, as shown in Figure 2. The load coefficient for a concentrated load can be expanded as follows according to (21) and (22): For a line load (, ) = 0 ( − 0 ), which is shown in Figure 3(a), the load coefficient can be expanded as follows according to (21) and (22): For a line load (, ) = 0 ( − 0 ), which is shown in Figure 3(b), the load coefficient can be expanded as follows according to (21) and (22): For uniformly distributed load (, ) = 0 , which is shown in Figure 4, the load coefficient can be expanded as follows according to (21) and (22): Equation ( 27) can be regrouped into the matrix form as Combining the relationship between 1 and 1 , the following equation can be obtained: Substituting ( 29) into (20), the forced response of displacement in top surface of the plate subjected transverse force on the top surface can be obtained: In a similar method, the forced response of displacement on the top surface of the plate subjected transverse force on the bottom surface can be given as where (0) = (0, 0, ) T .For free vibration, the surface boundary conditions are Equation ( 32) can be regrouped into matrix as Combining (20), a system equation can be obtained The natural frequency has an important effect on the sound radiation, and the natural frequency can be obtained by solving the equation det(M 21 ) = 0. Sound Radiation Power. When subjected to external excitation, the FGM plate will be stimulated and will radiate noise into the surrounding acoustic medium.It is convenient to characterize the sound radiated from a vibrating structure by a single global quantity such as sound radiation power.There are two different approaches generally used to determine the sound power.The first is to integrate the acoustic intensity over the vibrating surface, and the second is to integrate the squared pressure on a hemisphere in the far field [15].Only the first one is adopted in this work.The radiated sound power can be obtained by integrating the acoustic intensity over the surface of the plate [39]: where Re and superscript * denote the real part and the complex conjugate, respectively, is the surface of the plate, ẇ (r ) is the normal velocity of the surface point r , and (r) is the complex pressure amplitude at location r.For a plate placed in an infinite baffled surface, the acoustic pressure at any field point r can be expressed in terms of surface complex velocity ẇ (r ) according to Rayleigh integral [45]: where = \|r − r \| is the distance between a surface point r and a field point r, is the acoustic wavenumber and = / 0 with 0 being the speed of sound in the medium, and 0 is the density of the medium. In this paper, a primitive numerical scheme is used to solve the Rayleigh integral as [22,46].The plate is divided into × equal size rectangular elements, and the normal velocity is assumed to be constant across the rectangular elements.This approximation is sufficiently accurate if the characteristic length of the elemental radiator √ is much smaller than half of the minimum acoustic wavelength.Then the sound pressure in (36) can be written as where ẇ ( ) is the vector with normal surface velocities of the elemental radiators and Z is the radiation impedance matrix given by It is noted that the singular diagonal elements of the impedance matrix are singular when the field point is on the vibration plate surface (then = , = 0), and some approximations can be used as [22,47] The radiated sound power in (35) can be formulated as [22] = ẇ (r) R ẇ (r) , where R is the radiation resistance matrix, and for plate radiators placed in an infinite baffle the radiation resistance matrix can be written as The radiated sound power is usually written in the form of sound power level in decibel, which is defined by where 0 is the reference power and 0 = 1 × 10 −12 W. Validation Examples. In order to prove the validity of the present formulation and the developed code, the obtained results for sound radiation from the homogeneous plates as well as FGM plates have been compared with those available in the literature. The first example is sound radiation of a simply supported isotropic rectangular plate made of steel, which was analyzed by S. Li and X. H. Li [39] and Jeyaraj et al. [5].The plate is assumed to vibrate in air and with dimensions of 0.455 m × 0.379 m × 0.003 m. Figure 5 shows the comparisons of the present study with those of S. Li and X. H. Li [39] and Jeyaraj et al. [5].The finite element method based on Mindlin plate theory was used to model the plate and sound power level was calculated using the Rayleigh integral in S. Li and X. H. Li [39].The finite element method based on classical laminate plate theory was used to obtain the vibration response of the plate and the sound power level of the plate was calculated using SYSNOISE in Jeyaraj et al. [5].In the present work, the structural dynamic response of the plate is obtained by state space method based on 3D elasticity, and the Rayleigh integral is also used in calculation of sound radiation power.As is illustrated in Figure 5, an excellent agreement of the predictions of sound power level has been achieved between the present work and the work of S. Li and X. H. Li [39] and a relatively larger prediction of the sound power level is found in Jeyaraj et al. [5].This is due to the fact that the sound power level was obtained by adopting the Rayleigh integral in present work as well as in S. Li and X. H. Li [39], while that [5] and S. Li and X. H. Li [39]. was calculated using SYSNOISE in Jeyaraj et al. [5].The peaks of sound power level correspond to the natural frequencies of the plate.As depicted in the figure, an excellent agreement has been achieved among the three predictions of the first three peaks; however, there are some considerable discrepancies at high frequencies, that is, around 800 Hz.This is due to the fact that 2D plate theories were used in Jeyaraj et al. [5] and S. Li and X. H. Li [39], while the present model is based on 3D elasticity, which can give the exact solution for this system at high frequency.In conclusion, these comparisons validate the present model. The second validation example is sound radiation from FGM plates (Al/Al 2 O 3 ) under the excitation of point force which is taken from Chandra et al. [22].The plate is assumed to be simply supported on all edges and with the dimensions of 0.5 m × 0.4 m × 0.003 m.FGM plates with three distinct power-law indexes, that is, the power-law indexes = 0, = 1, and = 5, are considered.Figure 6 shows the comparison of the prediction of the present work and Chandra et al. [22], while the structural modes corresponding to peaks are listed in Table 1 and the difference between the two predictions of the structural modes is smaller than 1 Hz.The FSDT was adopted in Chandra et al. [22], and the present work is based on the three-dimensional theory of elasticity.It can be found that the two methods match well for the prediction of sound power level and the frequencies where peaks occur over a wide frequency range.It is noted that a peak is lost in the result of Chandra et al. [22] for = 5.This is for the reason that only the first 25 structural modes were considered in Chandra et al. [22] to calculate the structural response of the FGM plate, and the structural mode (, ) = (7, 1) corresponding to the lost peak, which is the 27th mode with the frequency of 1890 Hz as shown in Table 1, was not considered.There are some discrepancies in the prediction of the sound radiation power of FGM plates.As is shown in Figure 6, an excellent agreement has been achieved between the two methods for FGM plate with power-law index = 0, and some discrepancies occur at high frequencies when the power-law index changed as = 1, and a relatively bigger difference was shown for = 5.This is due to the fact that the present theoretical model is based on 3D elasticity, which can give the exact solution for this system, while the FSDT based model is capable of presenting accurate results for relatively thin plate and in low and intermediate frequency range.In addition, a shear force correction factor is required in FSDT to take in account the nonuniformity of the shear strain distribution through the thickness of the panel.A constant shear correction factor was used in Chandra et al. [22], which is usually reasonable for isotropic homogeneous plates and therefor a good agreement is achieved for = 0 due to the fact that FGM plate with power-law index = 0 is representative of monoceramic, that is, isotropic, alumina plate.However, the constant shear correction factor is not appropriate for FGM plates due to the material properties and geometric dimension of FGM plates [24,25], which results in some discrepancy between the two predictions for FGM with power-law index of = 1 and = 5.In order to illustrate this point clearly, a relatively thicker FGM plate (ℎ = 0.03 m) is considered, and the comparison of the first ten structural frequencies is shown in Table 2.The result of Chandra can be obtained following the method in references [22,23].İt can be found that a relatively bigger discrepancy exists compared with the result for FGM of thickness ℎ = 0.003 m, and a similar result is noted that the discrepancy of the two predictions for = 5 is bigger than that for = 0 and = 1. The aforementioned detail demonstrates the advantage of the present 3D elasticity based model and the necessity for the application of the present model in sound radiation of FGM plates where high accuracy is required. Sound Radiation Characteristics of FGM Plates. To investigate further sound radiation characteristics of FGM plates, several parametric studies are conducted in the previous subsection.FGM plate composed of zirconia (ZrO 2 ) at the top surface and aluminum (Al) at the bottom surface with power-law distribution of properties through the thickness with respect to volume fraction is considered.The material properties of the constituent are [22,23,28], for zirconia, = 200 GPa, = 5700 kg/m 3 , and = 0.3, while, for aluminum, they are = 70 GPa, = 2700 kg/m 3 , and = 0.3.The plate is assumed to be simply supported on all edges and placed in an infinite baffle.The air density is taken as 0 = 1.21 kg/m 3 , and the sound speed in the air is 0 = 343 m/s [22].The dimensional size of the plate is 0.4 m × 0.3 m × 0.01 m and a damping loss factor of 0.01 is taken for all the following calculations except for specially noted cases.In addition, to excite a relatively large number of modes, a unit point excitation force is applied at ( = 0.1 m, = 0.1 m) relative to the lower left corner and the frequency is varied. Investigation into the Effect of Power-Law Index 𝑁. Figure 7 shows the effects of material distribution on the sound radiation of FGM plates.Three types of power-law index are considered, that is, = 0.2, = 1, and = 5, which are representatives of FGM plates that are ceramic rich, linear, and metal rich, respectively.It can be seen that the sound radiation power of functionally graded plates is influenced significantly by the volume fraction distributions of the constituents, especially at high frequency range.As presented in (4), the power-law index has an influence on the material properties distribution, including density, Young's modulus, and Poisson's ratio simultaneously.In order to have a more accurate survey on sound radiation of FGM plates, the effects of each of graded elasticity modulus, density, and Poisson's ratio through the thickness on sound radiation of FGM plates are investigated. Figure 8 presents the effect of graded elasticity modulus on sound radiation of FGM plates.Sound radiation from assumed FGM plates of which material properties are the same as aluminum except for Young's modulus is considered.Young's modulus at the top surface and bottom surface of the plate is 200 GPa and 70 GPa, respectively, and that between the top surface and bottom surface changes gradually and continuously along the thickness direction.As is shown in the figure, the modes shift towards low frequencies with the increase of power-law index .From (4), gradually increasing the power-law index results in a proportional reduction in modulus of elasticity.Therefore, the decrease in the rigidity of the graded material through the thickness leads to a lower modal frequency of the FGM plate. Figure 9 illustrates the changes in sound radiation power from FGM plates with only graded densities from 5700 kg/m 3 at the top surface to 2700 kg/m 3 at the bottom surface.No considerable changes are seen in sound power level at very low frequencies; however, as depicted in the figure, the increase of power-law index results in a shift of the structural frequency towards high frequency range. Figure 10 shows the variation of sound radiation power with different power-law index of graded Poisson's ratio of the FGM plate.Poisson's ratio at the top surface and at the bottom surface is 0.4 and 0.2, respectively, and that between the top surface and the bottom surface changes gradually with different power-law scheme through the thickness.It is always considered that there is no need of considering the graded effect of Poisson's ratio and Poisson's ratio is always assumed to be constant in the researches of FGM structures.As depicted in the figure, the influence of graded Poisson's ratio on sound radiation of FGM plate is small compared with that of the graded elasticity modulus and the graded density.However, the effect of graded Poisson's ratio on sound radiation of FGM plate must not be overlooked, especially at high frequencies when Poisson's ratio of the constituent materials is very distinct.Some similar conclusions are also drawn for the bending and vibration of functionally graded microbeams; see [48]. Investigation into the Effect of the Plate Geometry. Figure 11 shows the variation of sound radiation power with different thickness of the FGM plate.FGM plates with constant length and width ( = 0.4 m, = 0.3 m) but different thickness, that is, ℎ = 0.004 m, ℎ = 0.01 m, ℎ = 0.016 m, and ℎ = 0.04 m, are considered, and the power-law index is taken as = 1.The plot shows that the thickness is an important geometrical parameter for the acoustic properties of the plate.With an increase of thickness, the first mode of the plate shifts towards high frequencies, and fewer peaks occur in the frequency range showed.In addition, the increase of the thickness of FGM plate leads to a reduction of sound radiation. Aspect ratio is another parameter of the plate.In order to investigate the effect of aspect ratio on the sound radiation of FGM plate, plate with distinct three aspect ratio sizes (i.e., 0.4 m × 0.3 m, 0.4 m × 0.4 m, and 0.4 m × 0.5 m) is considered and the results are presented in Figure 12.It is observed that the aspect ratio has a significant effect on sound radiation of the FGM plate. Investigation into the Effect of Damping. The result in Figure 13 shows the effects of damping loss factor on sound radiation of FGM plate.As depicted in the figure, the sound radiation power is independent of the damping loss factor at frequencies below the first mode.The damping loss factor leads to the reduction of the peaks and no considerable change of the sound power level is seen at other frequencies when the damping loss factor is relatively small.However, when the damping loss factor is relatively large, for example, 0.3, the sound power level seems to be independent of frequency.The loss factor is a function of frequency in practical applications and, in general, low values of damping are measured at high frequencies [46].Therefore, it is essential to determine the damping loss factor of the FGM plate experimentally. Investigation into the Effect of Load Type.The above observations are determined by the fact that the FGM plate is excited by a concentrated load or a point load; however, the FGM structures are subjected to different types of load in practical applications.In order to investigate the effect of force type on the sound radiation of FGM plate, three types of forces, that is, concentrated load, line load, and distributed load, are considered and the results are shown in Figure 14. The concentrated load is a 1 N force applied at the center of the plate, the line load is 10/3 N/m applied at = 0.2 m of the plate or 2.5 N/m applied at = 0.15 m of the plate, and the distributed load is 25/3 N/m 2 .It should be noted that three types of load share an equal total load acting on the plate.Only two peaks exist in the frequency range, which is due to the fact that all the modes with combination of even numbers are not well excited and only the odd-odd combination of modes (1-1, 3-1) appears.The result in the figure reveals that the force type has no effects on the frequencies where peaks appear; however, the force type has a significant effect on the sound power level.The FGM plate under the excitation of point load can radiate much larger sound power compared with the other two types of load.For example, a difference of about 10 dB is achieved between the point load and the distributed load from 500 Hz to 1000 Hz, and about 20 dB can be found around 1500 Hz.In addition, it can be found that sound radiation of the plate excited by the line load = 0 and line load = 0 shows no considerable difference from 1 Hz to about 1000 Hz and a difference of about 6 dB from 1500 Hz to 2000 Hz. Conclusions In the present paper, an analytical method for sound radiation of FGM plate based on 3D elasticity has been developed.The state space method is utilized to derive the structural model of the plate and sound radiation of the vibration plate is calculated by employing the Rayleigh integral approach.The accuracy of the present approach is validated by comparing the numerical results of sound radiation from homogeneous plates and FGM plates with those available in the literature and good agreements are achieved.Moreover, detailed comparison of the present model with FSDT based model demonstrates the advantage and necessity for the application of the 3D elasticity based model for sound radiation of FGM plates where high accuracy is required.Finally, several parametric studies on the effects of material distribution, plate geometry, damping, and force type on sound radiation of FGM plates are carried out.It is found that FGM plates have similar characteristics of sound radiation with homogeneous plates.In addition, it is found that the effect of graded Poisson's ratio on sound radiation of FGM plate must not be overlooked, especially at high frequencies when Poisson's ratio of the constituent materials is very distinct.Several practical applications are related to these analytical formulations developed in this study such as vibroacoustic sensitivity analysis, the material phase distribution identification, and parameter optimization. Figure 1 : Figure 1: Geometry and coordinates of the FGM plate. Figure 2 : Figure 2: FGM plate subjected to point load. et al. S. Li and X. H. Li Figure 5 : Figure5: Comparisons of sound power level of an isotropic plate with Jeyaraj et al.[5] and S. Li and X. H. Li[39]. Figure 7 :Figure 8 : Figure 7: Sound radiation of FGM plates with different power-law index. Figure 9 : Figure 9: The effect of graded density through thickness on sound radiation of FGM plates. Figure 10 : Figure 10: The effect of graded Poisson's ratio through thickness on sound radiation of FGM plates. 10 Figure 11 : Figure 11: The effect of thickness on sound radiation of FGM plates. Figure 12 :Figure 13 : Figure12:The effect of aspect ratio on sound radiation of FGM plates. 20 Figure 14 : Figure 14: The effect of load type on sound radiation of FGM plates. Table 1 : [22]ctural modes corresponding to the peaks in Figure6: comparison between the present work and Chandra et al.[22]. Table 2 : [22]first 10 structural frequencies of the FGM plate with thickness ℎ = 0.03 m: comparison between the present work and Chandra et al.[22].	2018-12-13T01:30:40.274Z	2016-01-01T00:00:00.000	{ "year": 2016, "sha1": "1eea355c5ea7a898761803d85b13e3b6a66d4e63", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/sv/2016/1403856.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "1eea355c5ea7a898761803d85b13e3b6a66d4e63", "s2fieldsofstudy": [ "Engineering", "Materials Science" ], "extfieldsofstudy": [ "Materials Science" ] }
199653316	pes2o/s2orc	v3-fos-license	Improving optoelectronic and charge transport properties of D–π–D type diketopyrrolopyrrole-pyrene derivatives as multifunctional materials for organic solar cell applications A series of novel diketopyrrolopyrrole-pyrene-based molecules were designed for small molecule based organic solar cell (SMOSC) applications. Their electronic and charge transfer properties were investigated by applying the PBE0/6-31G(d,p) method. The absorption spectra were simulated using the TD-PBE0/6-31G(d,p) method. The results showed that the frontier molecular orbital (FMO) energy levels, reorganization energy, the energetic driving force, and absorption spectra can be tuned by the introduction of different aromatic heterocyclic groups to the side of diketopyrrolopyrrole fragments' backbones. Additionally, the designed molecules possess suitable FMOs to match those of typical acceptors PC61BM and PC71BM. Meanwhile, the designed molecules can act as good ambipolar charge transport materials in SMOSC applications. Meanwhile, the electron and hole reorganization energies of the designed molecules are smaller than those of the typical electron and hole transport materials, respectively. Moreover, the differences between electron and hole reorganization energies do not exceed 0.046 eV. Our results suggest that the designed molecules can act as promising candidates for donor and ambipolar charge transport materials in SMOSC applications. Introduction Organic solar cells (OSCs), as promising candidates to address the global energy crisis, have attracted great attention all over the world owing to their outstanding merits, including low cost, light weight, and exibility. [1][2][3][4][5][6] In particular, the small molecule based OSCs (SMOSCs) have received tremendous interest due to their prominent advantages over polymer solar cells (PSCs), such as dened structures, relatively easy purication, and less batch to batch variation. [7][8][9][10] With continuous efforts, signicant advances have been demonstrated for OSCs. Recently, the power conversion efficiency (PCE) of PSCs and SMOSCs have exceeded 10%. [11][12][13] However, unfortunately, the overall performance of SMOSCs still lags behind that of PSCs. 14,15 Accordingly, it remains a great challenge to achieve high performance SMOSCs. To address this issue, an ideal small molecule used as the donor material should meet several requirements: (i) lower band gap and strong absorption for harvesting the solar light effectively, (ii) high hole mobility for fast charge-carrier transport to lead to high short circuit current (J sc ), (iii) suitable frontier molecular orbitals (FMO) energy levels. A deep highest occupied molecular orbital (HOMO) energy of molecules ensures a high open circuit voltage (V oc ). [16][17][18] Meanwhile, another crucial parameter is the energetic driving force (DE L-L ). The DE L-L is determined by the lowest unoccupied molecular orbital (LUMO) energy difference between the donor and acceptor. The DE L-L plays a major role in enhancing charge transfer, which should exceed the binding energy. 19,20 Generally, the fullerene derivatives PC61BM or PC 71 BM ( [6,6]-phenyl-C 61 -(or C 71 )-butyric acid methyl ester) were used as acceptors in OSCs. 21,22 With the aim to enhance the PCE of SMOSCs, one efficient method is to design electron donor-acceptor (D-A) molecular structures. 23,24 They can efficiently tune the FMOs energy levels through an intramolecular charge transfer, which leads to a narrow band gap, intense light absorption, and high PCEs. [25][26][27] Additionally, the intermolecular stacking, lm morphology, and charge transport properties can be determined by the type and length of side chains of D-A molecular structures. 28,29 It has been reported that a variety of structures, including A-D-A, D-A-D, D-p-A, D-p-D, A-p-A, and D-p-Ap-D were designed. 30,31 Among the various D-A type donor building blocks, diketopyrrolopyrrole (DPP)-based molecules have attracted great attention in terms of their outstanding features, such as good planarity, strong light absorption, high carrier mobility, good photochemical stability, and facile synthesis. 32,33 Moreover, pyrene is another promising donor building block. The pyrene derivatives can enhance the absorption due to their large aromatic hydrocarbon ring and planarity. 34,35 The planarity of molecules can improve the charge transport and interconnectivity. 36 Therefore, the planar DPP and pyrene have combined each other to develop novel donor materials. 37 Furthermore, it is worth noting that the charge mobility can been improved through introducing extended aromatic bridges to the DPP derivatives. 38 Recently, DPP-based DDP-pyrene-DDP type molecules have been reported for use as donor material for SMOSCs. It was found that these molecules exhibited high hole mobility and PCEs for SMOSCs applications. 39,40 Taking into account these merits of DPP and pyrene derivatives mentioned above, we designed eight D-A-D type molecules as the acceptor materials for improving the efficiency of SMOSCs (Scheme 1). In order to study the inuence of the DPP moiety on the optical, electronic, charge transfer properties, different aromatic heterocyclic groups have been introduced into the side of DPP in molecules. Applying density functional theory (DFT) approach, the properties of the designed molecules such as HOMO and LUMO energies (E HOMO and E LUMO ), HOMO-LUMO gap (E g ), DE L-L , the reorganization energy (l), adiabatic ionization potential (AIP) and electron affinity (AEA) were systematically investigated. The absorption spectra of the designed molecules were predicted using the time dependent DFT (TD-DFT) method. The charge transfer properties (reorganization energy, l) were also simulated. Computational methods All the electronic structure calculations were carried out performed using the Gaussian 09 program suite. 41 The molecular structures were optimized by the DFT method. Then the frequency calculations were performed at the same level of theory to conrm that all geometries are stable structures. Under the optimized structures, their absorption spectra were simulated at the TD-DFT level of theory. The all calculations were carried out with the 6-31G(d,p) basis set. The solvent effects on the optical property in chloroform solution (dielectric constant: 4.7113) were also analyzed with the polarised continuum model (PCM). 42 For the FMOs energy E HOMO and E LUMO , the optimized geometries were used to obtain E HOMO . However, it is difficult to obtain the E LUMO since the virtual orbitals can not be described denitely by theoretical method. 43 Therefore, the E LUMO were be predicted by the sum of the E HOMO and the rst vertical excited energy. 44,45 It is important to choose an appropriate method in the theoretical investigations. In order to select reasonable method, six representative functionals, B3LYP, 46 PBE0, 47 LC-uPBE, 48 uB97XD, 49 M062X, 50 and CAM-B3LYP 51 were used to optimize the geometries of molecule 1, and then using the corresponding TD-DFT method, the absorption spectra were predicted in chloroform, respectively. The calculated data are shown in Table S1 in the ESI. † As showing Table S1, † the calculated longest wavelengths of absorption (l abs ) using PBE0 (583 nm) approach compares very satisfactorily with the experimental result (589 nm). 39 Furthermore, in order to make further investigation of the validity of the selected approach, both PBE0 and B3LYP methods were also used to obtain the E HOMO and E LUMO of acceptors PC 61 BM and PC 71 BM, and the calculated results are listed in Table S2 in the ESI. † The predicted E HOMO and E LUMO of PC 61 BM (À5.98 and À3.99 eV) and PC 71 BM (À5.92 and À3.82 eV) using PBE0 approach reproduce well the experimental data (À6.00 and À3.80 eV, À6.00 and À3.95 eV, respectively). 52,53 Comprehensively, the PBE0 method arises as the best choice for characterizing the systems studied in our work. It is worth noting that the l plays an important role in determining the charge transfer. Usually, lower l value is benecial for improving the charge transfer. 54,55 Meanwhile, the external l is much small compared with its internal l in organic condensed phases. 56,57 Therefore, we only consider the internal l in this work. The electron and hole reorganization energy (l e and l h ) were be predicted by: 58 where, E + 0 (E À 0 ) is the energy of the cation (anion) structure with the optimized neutral geometry molecule. E 0 + (E 0 À ) corresponds to the energy of the neutral structure with the optimized cation (anion) geometry structure. E + + (E À À ) represents the energy of the cation (anion) calculated with the optimized cation (anion) structure, while E 0 0 is the energy of the neutral molecule at ground state. Furthermore, it is quite clear that the AIP and AEA play an important role in determining the charge transfer behavior for materials. In this work, the AIP is obtained by the energy difference between cation and neutral molecules. The AEA is determined by the energy difference between neutral and anion molecules. The l e , l h , AIP, and AEA were calculated using PBE0/6-31G(d,p) method. Scheme 1 Molecular structures of the investigated molecules. Match between the donor and acceptor It is noticeable that the electronic energy levels play important role roles to increase the PCE of SMOSCs. Small molecule donors should possess suitable energy levels with the acceptors to ensure efficient charge separation and to maximize the J sc and V oc . The deep HOMO and high LUMO levels are benecial for improving the V oc and for efficient exciton dissociation and charge separation, respectively. Moreover, small molecule donors should exhibit lower E g to guarantee high harvest sunlight efficiency and enhance the J sc . Moreover, in order to ensure the efficient electron transfer, the DE L-L should at least amount to 0.3 eV. 59, 60 We choose PC 61 BM and PC 71 BM as acceptors in our work. Additionally, the explicit molecular orbital contributions from the individual fragments to the FMOs have been investigated by partial density of states (PDOS) analysis and the results are listed in Table 1. With the aim of evaluating whether the FMOs energy of designed donors match with that of the acceptors, the DE L-L are estimated. The calculated values of DE L-L for 1-8 at the PBE0/6-31G(d,p) level are given in Table 2. The results presented in Table 2 Table S3 in the ESI †). Both the sequences of difference values are 3 < 1 < 4 < 7 < 5 < 6 < 2 < 8. It suggests that 1-8 possess suitable FMOs to match those of typical acceptors PC 61 BM and PC 71 BM for SMOSCs. Frontier molecular orbitals level and band gaps To gain insight into the inuence of the FMO energies on the optical and electronic properties of the designed molecules, the contour plots of HOMOs and LUMOs have been examined (see Fig. 1). Inspection of the distribution of FMO reveals clearly that the HOMOs and LUMOs reveal p characteristic and exhibit strong delocalization features. For molecules 1-5, the electronic cloud distributions of the FMOs spread over the whole molecules with minor contributions from p-bridge fragment (pyrene). For molecules 6 and 7, the electronic cloud distributions of HOMOs are mainly delocalized on the one side DPP fragments, whereas the corresponding the LUMOs mainly reside at another side DPP fragments of molecules. For molecules 8, the HOMO is delocalized throughout the molecule, while the electron density of LUMO is mainly localized on the one side DPP fragment of molecule. This is favorable for the charge transfer across these conjugated donor materials. It suggests that the introduction of the extended aromatic bridges to the DPP derivatives signicantly affect on the distributions of FMOs for the molecules. The evaluations of E HOMO , E LUMO , and E g for the designed molecules as well as PC 61 BM and PC 71 BM are plotted in Fig. 2. From Fig. 2 one can nd that the E HOMO values are in the order of 3 > 1 > 4 > 7 > 5 > 6 > 2 > 8, and the sequence of E LUMO values is 7 > 2 > 5 > 1 > 3 > 6 > 8 > 4. Clearly, comparing the E HOMO and E LUMO of 2-8 with those of 1, molecules 2, 5, and 7 can lower/ raise the E HOMO /E LUMO , whereas molecule 3 can raise/lower the E HOMO /E LUMO , respectively. On the other hand, molecules 4, 6, and 8 can lower both the E HOMO and E LUMO , respectively. The trend of the E g is 2 > 7 > 5 > 6 > 1 > 3 > 8 > 4. It reveals that molecules 2, 5, 6, and 7 can higher, whereas molecules 3, 4, and 8 can narrower the E g than that of 1, respectively. The above results suggest that the E HOMO , E LUMO , and E g can be tuned through the introduction of different aromatic heterocyclic groups to the molecules. Absorption and uorescence spectra Table 3 collected the longest absorption wavelengths l abs , oscillator strength f, and the main assignments of 1-8. The corresponding simulated absorption spectra are presented in Fig. 3. Inspection of Table 3 the studied molecules can be tuned effectively. With the above considerations, these molecules can act as donor material with strong absorption spectra for SMOSCs. Charge transfer properties It is commonly known that the AIP, AEA, and l are three key parameters that determine the charge transfer property. The AIP and AEA are good descriptors to estimate the energy barrier for the injection of holes and electrons, respectively. The l is the extent to estimate the ability to carry the charge in solid. Usually, lower AIP and higher AEA are favorable for better charge transport ability for hole and electron, respectively. 61,62 For l, the lower l e and l h are benecial for enhancement the electron and hole transfer rate, respectively. 54,55 The predictions of AIP, AEA, l h , and l e of the designed molecules are summarized in Table 4. It is clear that the AIP are in the order of 8 > 2 > 6 > 5 > 7 > 1 > 4 > 3. The AIP of molecules 2 and 5-8 are larger, whereas the AIP of molecules 3 and 4 are smaller than that of molecule 1, respectively. Namely, comparing the AIP of 2-8 with that of 1, molecules 2 and 5-8 can raise the AIP, while molecules 3 and 4 can lower the AIP, respectively. At the same time, the AEA is in the order of 4 > 8 > 3 > 1 > 6 > 5 > 2 > 7. Obviously, comparing the AEA of molecules 2-8 with that of molecule 1, molecules 3, 4, and 8 increase the AEA, whereas molecules 2 and 5-7 decrease the AEA, respectively. It is noticeable that the introduction of different aromatic heterocyclic groups to the molecules affects both their AIP and AEA values. A graphical representation of l h and l e has been illustrated in Fig. 4 to represent the results more clearly. Generally, Alq3 (tris(8-hydroxyquinolinato)aluminum(III), l e ¼ 0.276 eV) and TPD (N,N 0 -diphenyl-N,N 0 -bis(3-methlphenyl)-(1,1 0 -biphenyl)-4,4 0 -diamine, l h ¼ 0.290 eV) are taken as the typical electron and hole transport materials, respectively. 63,64 Apparently, the l e and l h of the designed compounds are much smaller than those of Alq3 and TPD, respectively. It indicates that both the electron and hole transfer rates of 1-8 might be higher than those of Alq3 and TPD, respectively. The decreasing sequence of l h is 4 > 6 z 7 > 2 z 5 > 8 > 1 > 3, and the values of l e are in the following order: 7 > 2 > 5 > 4 > 6 > 8 > 1 > 3. Apparently, comparing the l h and l e of molecule 2-8 with those of 1, molecules 2, 4-8 can increase both l h and l e , whereas compound 3 can decrease both l h and l e , respectively. Additionally, molecule 3 has both the smallest l h and l e values, indicating that molecule 3 has the largest hole and electron transport rates among the investigated molecules. Moreover, the differences between l e and l h values of 1-8 do not exceed 0.046 eV. It indicates that they exhibit better hole-and electron-transporting balance. Accordingly, these molecules can act as nice ambipolar charge transport materials for SMOSCs. Therefore, these molecules can be used as good candidates for ambipolar charge transport materials under the proper operating conditions. Conclusions In this article, we design a series of novel diketopyrrolopyrrolepyrene-based molecules as the acceptor materials for SMOSCs applications. The optical, electronic, and charge transporting properties were investigated by applying DFT and TD-DFT methods. The parameters such as DE L-L , l, AIP, and AEA were calculated to preliminarily evaluate the performance of the solar cell. The results show that the E HOMO , E LUMO , E g , DE L-L , AIP, AEA, and l can be tuned through the introduction of different . 3 The calculated absorption spectra of the investigated molecules (value of full width at half maximum is 3000 cm À1 ). This journal is © The Royal Society of Chemistry 2019 aromatic heterocyclic groups to the molecules. The absorption spectra were also simulated. Our results suggest that the designed molecules possess suitable FMOs to match those of typical acceptors PC 61 BM and PC 71 BM. Additionally, they can be used as ambipolar charge transport materials for SMOSCs. Conflicts of interest There are no conicts to declare.	2019-08-16T08:25:43.910Z	2019-07-17T00:00:00.000	{ "year": 2019, "sha1": "7d846b84735b404419c76b3f3efa35f29cfc329b", "oa_license": "CCBYNC", "oa_url": "https://pubs.rsc.org/en/content/articlepdf/2019/ra/c9ra04304g", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a33205f31ace7897ccb907d8696fd9bd3e80c398", "s2fieldsofstudy": [ "Materials Science", "Chemistry", "Engineering" ], "extfieldsofstudy": [ "Medicine", "Materials Science" ] }
3210116	pes2o/s2orc	v3-fos-license	Failure of Cone-Beam Computed Tomography in Detection of Fiber Post Perforation Detection of iatrogenic root perforation during post-space preparation especially in labiolingual plane can be challenging due to the two-dimensional nature of conventional radiography; this can be even more challenging if the cemented post is radiolucent. Cone-beam computed tomography (CBCT) scans were shown to be a valuable diagnostic aid in diagnosis of such cases. However, in this case, the application of CBCT did not help in diagnosis of a labial fiber post perforation in a maxillary central incisor which was finally detected through exploratory surgery. Introduction oot perforation refers to creation of an artificial and unwanted communication between the root canal system and the periodontal ligament (PDL); at the site of root perforation, an inflammatory reaction in PDL occurs and leads to the formation of a primary endodontic lesion [1]. Perforation along the root can occur as a result of root resorption, iatrogenic damages during root canal therapy (RCT) and finally during post-space preparation [2]. Iatrogenic root perforations frequently caused by inappropriate post space preparation are amongst the most common types of root perforations [3] occurring approximately between 2 to 12% of the endodontically treated teeth [4]. Accidental and unnoticed lateral root perforations during post-space preparation, show lateral bone defects on radiographies and cause formation of a sinus tract or periodontal pocket [5]. Occasionally, radiographic diagnosis of this defect in a patient with non-acute clinical symptoms is challenging as periapical radiographies show previous endodontic treatment, presence of a post and a crown which is restored without evidence of a root perforation caused during the post insertion [5]. Radiographic detection of the labial or lingual root surface perforation is even more challenging, due to superimposition of the perforation on the root [2]. In such cases, tube shift technique with at least two different horizontal angles can be helpful in identification of a misdirected post. The greatest limitation of conventional radiography, which is inability to represent the three-dimensional (3-D) anatomy of teeth and their related structures, has been overcome by introduction of cone-beam computed tomography (CBCT) [6]. However, if the cemented post is radiolucent, its detection seems almost impossible not radiographically nor in CBCT. Happening for any reason, this unwanted communication leads to destruction of the adjacent periodontal tissues [7]. The survival prognosis of perforated tooth depends foremost on prevention or control of bacterial (re)infection and epithelial migration at the perforation site [5]. Use of a biocompatible repair material to provide the best possible seal against penetration of bacteria and the ability to promote regeneration of cementum and periodontal ligament will limit periodontal inflammation [8,9]. Calcium-enriched mixture (CEM) cement is a tooth-colored cement with clinical applications similar to MTA. It offers proper sealing ability, biocompatibility, antimicrobial properties, hard tissue induction properties and shorter setting time, greater flowability and lower film thickness than MTA [10][11][12][13].The present report reviews the challenging diagnosis and surgical treatment (using CEM cement) of a radiolucent post-space root perforation on labial surface that had developed a sinus tract. In this report the inability of CBCT and efficacy of exploratory surgery in detection of the lesion, is highlighted. Case Report A young women in her early twenties was referred by a dentist, complaining of localized discomfort in the anterior maxillary vestibule subsequent to endodontic treatment and restoration of the left maxillary central incisor. Her medical history was noncontributory. Upon clinical assessment the buccal gingiva of the maxillary left central incisor had a sinus tract in the mid root area which according to the patient was occasionally formed after RCT. In the referral letter, the dentist had mentioned RCT was indicated after detection of pulp necrosis of the incisor which had a class three cavity. The tooth was not sensitive to percussion. A parallel periapical radiography was taken that indicated perfect RCT and restoration ( Figure 1A). Due to root fracture being likely, cone-beam computed tomography (CBCT) was indicated. At the first glance, normalcy was seen in different CBCT slices. However, a fenestration like lesion was detected in the three-dimensional reconstruction provided by the CBCT software ( Figure 1B, red arrows). The sinus tract was traced with a gutta-percha cone and CBCT was taken which did not show apical origin of the lesion. In other words, deviation of the tracing gutta-percha cone to the labial soft tissues in CBCT image ( Figure 1C) intensified the diagnosis. As the CBCT findings were not suggestive of the lesion origin ( Figure 1D to E), exploratory surgical flap was indicated. After buccal infiltration of 2% lidocaine containing 1:80000 epinephrine (Darupakhsh, Tehran, Iran), a submarginal Ochsenbein-Luebke flap was resected and reflected. Interestingly, after curettage of the granular tissue surrounding the fenestration, a fiber-reinforced lucent prefabricated post became visible that had perforated the root (Figures 2A and B). That segment of the post which was accessible outside the root was resected and after preparation of a small cavity inside the root, the area was sealed with CEM cement (BioniqueDent, Tehran, Iran), ( Figure 2C). The flap was replaced and sutured. After giving the post-treatment instructions the patient was dismissed. After 5 days, patient was totally asymptomatic and the sutures were removed. After two weeks the sinus tract had disappeared and the tooth was functional. The one year followup radiography was also indicative of a functional tooth with no pathology ( Figure 2D). Discussion The present report reviewed successful surgical management of a mid-root perforation during post space preparation in an upper left central incisor that was left undetected by the dentist. The post lucency prevented its emergence in radiographies and contrary to our perception, CBCT was not much helpful in detection of the lesion origin. Thus, exploratory surgery was indicated which uncovered the cause of patient's problem; labial mid-root perforation during post space preparation where the radiolucent fiber post was cemented. The perforation site was prepared/sealed using CEM cement. After one year, the tooth was symptomless and functional. Root perforation often happens iatrogenically as a result of misaligned application of burs during endodontic access preparation and search for root canal orifices, and during efforts to negotiate calcified and curved canals [2,4]. Inappropriate post space preparation for permanent restoration is yet another common iatrogenic cause of root perforation [4]. Kvinnsland et al. observed that more than half of the perforations in their cases of root perforation occurred during post-preparation procedures [3]. In some cases of endodontic practice, conventional intraoral radiography does not provide adequate information on pathologic conditions and positional relationships. Threedimensional images have become possible with introduction of CBCT technology to dentistry. The imaged area can be arbitrarily sliced and observed from three different directions [6]. Although the presence and position of a fenestration and associated bone defect were confirmed preoperatively and in three-dimensionally reconstructed CBCT image, examination with CBCT could not reveal clear evidence of a post perforation through the labial root surface of tooth 21 which could remain undetected if exploratory flap had not been indicated. In some cases when the diagnosis is doubtful and the evidence for root fracture or perforation is tenuous, it may be necessary to perform an exploratory flap [14]. In other words, it may be very reasonable to consider surgical exposure unless the nonsurgical evidence clearly indicates the presence of a fracture/perforation whose extent or position mitigates against any possibility of treatment [15]. Management of complicated endodontically treated teeth requires special consideration, particularly if the clinician is unable to elicit the proper past history, such as where, when, and how the tooth in question was treated. In such cases an approach more than just opening the pulp chamber and root canal(s) is required for a correct diagnosis [16]. Thus exploratory endodontic surgery may become necessary to help determine prognosis and plan treatment by better evaluation of Fazlyab & Asgary the damage through surgical exposure and direct visual examination of the area [15,16]. However, to the best of our knowledge almost all reports of successful exploratory surgeries explained its usefulness in detection of root fractures [15,16], resorption [17] and cemental tear [18]. Thus, this is the only report that represented indication of exploratory flap in detection of post space root perforation. In the reported case, the lesion origin was clear after raising an exploratory flap due to post material being radiolucent. However, the operator was ensured of the presence of pathogenesis in the affected area. In addition, the CBCT images made all the procedure justifiable for patient. Many important factors that are all related to infection control at the perforation site, influence the survival prognosis of the tooth [19,20]: the size of the perforation, the time elapse between creation of the perforation and its repair, and the site of perforation in relation to the level of crestal bone and epithelial attachment [21,22]. As the most critical factor determining outcome of treatment, time indicates the chance of infection of the wound site [19,20] while defect size relates to amount of trauma to the adjacent tissues and difficulty in creating seal in the affected area [9]. Perforations occurring close to the crestal zone may be complicated by formation of a periodontal pocket due to epithelial migration that forms a connection between the perforation defect and the oral cavity [20]. Down-growth of gingival epithelium to the most apical margin of perforation site can emerge, especially when accidental perforations occur in the crestal area [4,20]. Persistence of bacterial infection originating from the root canal and/or the periodontal tissues, prevents healing and brings about inflammatory sequels where exposure of the supporting tissues is inflicted. Thus, painful conditions, suppurations resulting in tender teeth, abscesses and sinus tract including bone resorptive processes may follow [3,20]. According to Patel et al. [23] perforation of the root is usually followed by the development of a sinus tract. In the presented case, the perforation was located in the mid-root area far from the crestal bone and luckily did not cause pocketing to the perforation site. Consequently, sealing of the defect was successful in controlling the intra-radicular infection. CEM cement has been recommended as an appropriate endodontic biomaterial for repair of lateral root perforations [8,9,11]. Comparison of the results of this study with the results of reports on root perforations repaired with CEM cement shows a marked improvement in the prognosis of these teeth [7,8,24,25]. In the case reported here, healing was achieved following repair of the lesion associated with a sinus tract and a bone defect with CEM cement. Conclusion In conclusion, the present case report showed that a surgical approach may be effective for treating a combined endodonticperiodontal lesion originating from a root perforation in the middle third of a maxillary central incisor with a cement lucent post, which was treated with a surgical approach using CEM cement. The presented case demonstrates that CBCT imaging technology is not always useful in detection of lesions that are not clinically and radiographically diagnosable. Endodontic surgical flaps for diagnostic purposes and management of complex endodontic problems cannot be overlooked.	2018-04-03T04:00:40.782Z	2017-07-02T00:00:00.000	{ "year": 2017, "sha1": "4511a1fa9497ed6056bc1a77200bf2747ad4f679", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "4511a1fa9497ed6056bc1a77200bf2747ad4f679", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
235302447	pes2o/s2orc	v3-fos-license	Development and Bio-Predictive Evaluation of Biopharmaceutical Properties of Sustained-Release Tablets with a Novel GPR40 Agonist for a First-in-Human Clinical Trial Sustained-release (SR) formulations may appear advantageous in first-in-human (FIH) study of innovative medicines. The newly developed SR matrix tablets require prolonged maintenance of API concentration in plasma and should be reliably assessed for the risk of uncontrolled release of the drug. In the present study, we describe the development of a robust SR matrix tablet with a novel G-protein-coupled receptor 40 (GPR40) agonist for first-in-human studies and introduce a general workflow for the successful development of SR formulations for innovative APIs. The hydrophilic matrix tablets containing the labeled API dose of 5, 30, or 120 mg were evaluated with several methods: standard USP II dissolution, bio-predictive dissolution tests, and the texture and matrix formation analysis. The standard dissolution tests allowed preselection of the prototypes with the targeted dissolution rate, while the subsequent studies in physiologically relevant conditions revealed unwanted and potentially harmful effects, such as dose dumping under an increased mechanical agitation. The developed formulations were exceptionally robust toward the mechanical and physicochemical conditions of the bio-predictive tests and assured a comparable drug delivery rate regardless of the prandial state and dose labeled. In conclusion, the introduced development strategy, when implemented into the development cycle of SR formulations with innovative APIs, may allow not only to reduce the risk of formulation-related failure of phase I clinical trial but also effectively and timely provide safe and reliable medicines for patients in the trial and their further therapy. Introduction Maintaining the drug concentration within a specific therapeutic range is a crucial factor in chronic diseases, such as diabetes mellitus type 2 (T2DM). To better control glycemia, several of the most commonly prescribed oral antidiabetic agents are available as sustained-release (SR) oral dosage forms. Such systems provide several key advantages that make them an asset in the therapeutic management of T2DM. For commonly used drugs, such as metformin or sulfonylurea derivatives, an SR formulation offers the same clinical benefits but with a reduced dosage, fewer side effects, and better patient satisfaction with the treatment compared with a standard dosage form [1][2][3][4]. Better patient compliance and lower risk of hypoglycemia make SR solid dosage forms a useful tool in the treatment of T2DM. Another benefit of SR formulations is their usefulness for drugs with a short biological half-life [5]. As a result of the reasons mentioned above, an SR formulation may appear advantageous for new antidiabetic drugs entering a clinical trial in humans, with a special case of oral dosage forms for the first-in-human (FIH) study of innovative medicines. In such cases, the determination of a simple oral solution or an immediate release capsule is not necessarily recommended. It holds true, especially if the candidate drug undergoes a rapid elimination and a long-lasting exposition is required for the clinically desired pharmacodynamic effect. A simple and robust SR product with the desired delivery rate offers a desired in vivo efficacy and reliable bioavailability estimation. In these circumstances, introducing this type of formulation should be considered and launched as early as possible. Several types of such systems are currently available on the market. SR solid dosage forms include matrix or coated tablets, multiparticulate drug delivery systems, 3D-printed tablets, or osmotically controlled drug delivery systems [6]. The last system, OROS, offers a unique zero-order kinetic release rate that is pH-independent [7]. However, the OROS technology is laborious in the development and difficult to manufacture, and owing to its non-dispersibility, it may be problematic in gastrointestinal (GI) narrowings or motility disorders [8]. Therefore, monolithic matrix tablets are still widely used as simple, robust, and easy to manufacture SR delivery systems. A thorough characterization of the newly developed SR matrix tablet should reliably assess the risk of uncontrolled drug release. Several critical factors modulate drug dissolution from hydrophilic matrices [9]. First, there are the properties of the drug, such as solubility, dissociation constant, stability, or molecular weight. Second, there are the type, properties, and particle size of the API and polymer excipients used in the matrix. Third, the formulation factors, such as the geometry of the matrix, ionic strength, or microenvironmental pH, may alter the pH-dependent solubility [10]. The SR tablet developed without considering these factors may be prone to a burst effect when a large amount of drug is released in contact with a medium or to dose dumping when the release of the drug is accelerated. Evaluation of the dissolution characteristics should reveal these unwanted and potentially threatening performances. Conventional USP compliant dissolution tests offer a quick and straightforward characterization but may lack a discriminative power to detect the impact of biorelevant media mimicking GI fluids or mechanical stress observed during GI passage [11][12][13]. In the present study, we describe the development of a robust SR matrix tablet with a novel G-protein-coupled receptor 40 (GPR40) agonist for first-in-human studies. The main aim of this paper is to propose a general workflow for the successful development of a novel SR tablet with an innovative API. The proposed step-by-step approach focuses on employing various analytical methods, including tablet swelling and water uptake analysis, through standard USP compliant dissolution tests, and sophisticated physiologydriven biorelevant dissolution tests. This novel pathway may be useful for the effective and reliable characterization of a robust SR formulation and offers a reliable prediction of in vivo pharmacokinetics. Basic Characteristics of the Formulations The active pharmaceutical ingredient (API), labeled CPL207-280-51, is an innovative BCS class I compound. This new GPR40 agonist exhibits antidiabetic properties and is intended to enter a first-in-human clinical trial. To date, there is a lack of data on the pharmacokinetics of this API in humans. The formulation process aimed to develop three strengths of SR tablets suitable for dose-escalating studies in phase I clinical trials. The tablets should resist the GI passage under both fasted and fed conditions, without the risk of dose dumping. The dosage form should release the API gradually for up to [14][15][16][17][18] h, regardless of the tablet strength, to allow a once-daily administration. The hydrophilic matrix tablets containing the labeled API dose of 5, 30, or 120 mg were manufactured by direct compression. First, the API and the excipients were mixed. Then, the mixture was compressed using an XP-1 single-punch tablet press (Korsch, Berlin, Germany) and EUD oval punch, 19 × 9 mm. The final tablets' weights and dimensions were kept fixed for all tablet strengths to assure a constant surface to volume ratio [14]. It was suspected that the overall matrix-forming ability might change upon increasing API dose because of modifying the total matrix solubility (the API is a well-soluble compound). It could result in alterations in dissolution profiles between the tablet strengths. Hence, the excipients used in the formulation were divided into two groups: matrix-forming and matrix-filling ingredients. The matrix-forming agents assured a sustained release of the API, and the fillers were responsible for tablet compression. The amount of one of the matrix formers was reduced upon increasing the API labeled dose so that the proportion between matrix formers and matrix fillers was constant regardless of the API amount. Then, total matrix solubility was established on a suitable level. All excipients were of pharmaceutical grade. In order to search for the possible interactions between the tested compound and various excipients used in pharmaceutical drug development, an incompatibility test was designed. The binary mixtures of the API and selected excipients were stored in both open and closed glass vials. The samples were stored for 4 weeks at 40 • C and 75% relative humidity. The amount of the API was analyzed using HPLC and compared with the initial value. The following excipients were evaluated: lactose monohydrate, microcrystalline cellulose (MCC), siliconized microcrystalline cellulose (SMCC), mannitol, calcium hydrogen phosphate dihydrate, dicalcium phosphate anhydrous, disodium phosphate, pregelatinised starch, Neusilin, povidone (PVP), hypromellose (HPMC), crospovidone, copovidone, crosscarmellose sodium (CCS), silicon dioxide, and magnesium stearate. Standard Dissolution Testing Test Conditions Standard dissolution tests were performed using the USP type II paddle apparatus set at 75 rpm and 37 • C. The dissolution medium was 900 mL USP phosphate buffer pH = 6.8. The samples were collected at time points of 0.5, 1, 2, 3, 5, 8, 12, 18, and 18.25 h; then, they were filtered through a 0.45 µm cellulose syringe filter. All tests were performed for at least n = 3 tablets. Determination of the API in Standard Dissolution Media Collected samples were analyzed with the high-performance liquid chromatography (HPLC) method, which was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines [15]. Five µL (for 30 mg and 120 mg tablet strengths) or 20 µL (for 5 mg tablet strength) was injected onto a Zorbax Eclipse Plus Phenyl-Hexyl column (3.0 × 150 mm, 3.5 µm, Agilent, Santa Clara, CA, USA) equipped with an inline filter. Gradient elution at a 1 mL/min flow rate was employed, and two mobile phases were used-phase A consisted of 0.1% phosphoric acid; phase B was pure acetonitrile. Initial conditions were 70% phase A: 30% phase B. After 6 min, the amount of phase A decreased to 50%; then, after 12 min, it decreased to 45%, and from 13 to 14.5 min, it decreased to 5%. After 14.5 min, the column was re-equilibrated to the initial conditions. The total run time of the method was 15 min. A UV-VIS detection was performed at λ = 275 nm. The method was linear within 1.4-140 mg/L, with the lower limits of quantitation of the calibration curve: 1.4 mg/L for the low-strength (5 mg API), 7.0 mg/L for medium-strength (30 mg), and 56 mg/L for the high-strength (120 mg) batches. Prototype Performance Evaluation Water Sorption and Swelling Kinetics Analysis The tested formulations were incubated in 0.1 M HCl, 50 mM potassium phosphate buffer pH = 3.0 with 0.1% Tween 80, and in the medium simulating intestinal fluids under fasted conditions (FaSSIF). FaSSIF comprised 0.420 g sodium hydroxide, 4.470 g sodium hydrogen phosphate dihydrate, and 6.186 g sodium chloride dissolved in 900 mL demineralized water. After the components were fully dissolved, the pH was adjusted to 6.5 with 1 M sodium hydroxide, and the volume was made up to 1000 mL with water. Into the solution, 2.240 g of FaSSIF/FeSSIF (Fed State Simulated Intestinal Fluid)/FaSSGF (Fasted State Simulated Gastric Fluid) powder (Biorelevant.com Ltd., London, UK) was added, and the medium was stirred until the powder fully dissolved. For better visualization of the hydration front, a colorant was added into both the tablet prototypes and incubation media at a concentration of approximately 0.2%. All of the reagents were of analytical grade. The tested tablets were weighed before incubation, and their dimensions were measured. They were incubated in 250 mL of preheated medium (37 • C) for 2 or 5 h, depending on the solution. Samples were withdrawn from the solution every hour, measured, and photographed (Supplementary Figure S1). After incubation, the tablets were weighed again. Then the tablets were cut with a scalpel, and their cross-sections were photographed and measured. Texture Kinetics The tablets were hydrated by incubation in beakers in 40 mL of simulated gastric fluid (SGF) pH = 1.2, in the 50 mM phosphate buffer pH = 3.0 with 0.1% Tween 80, and in FaSSIF for 2 h at 37 • C. They were also incubated for 5 h in beakers with 40 mL of phosphate buffer pH = 3.0 with 0.1% Tween 80 and in FaSSIF at 37 • C. The tablets were placed on a piece of cellulose paper to prevent adhesion to the walls of the beaker. The tablets were tested using a texture analyzer TA.XTplus Texture Analyser (Stable Microsystems, Surrey, UK) equipped with a 50 N load cell and a pushrod diameter of 35 mm. The hydrated tablets were withdrawn from the incubation medium and placed on a piece of aluminum foil on the lower plastic plate, and the test was initiated at a crosshead speed of 0.5 mm/min. The trigger force equaled 0.05 N. The load was directed perpendicularly to the long axis of the tablet. The measurement duration was 150 s at a sampling time of 0.025 s. A visual inspection of the tablets was also recorded. Biorelevant Dissolution Tests Biorelevant dissolution tests were performed in the StressTest ® device, which was first introduced by G. Garbacz and W. Weitschies [11]. The apparatus simulates the mechanical agitation of physiological intensity. In combination with the biorelevant media, the equipment mimics the physiological conditions that act on a solid dosage form in subsequent sections of the GI tract. A detailed description of the device is given elsewhere [16]. Biorelevant Test Protocols The detailed setup of all biorelevant test protocols is presented in Table 1. All of the tests were performed for n = 3-6 tablets. For simulated fasted conditions, four test programs were used, reflecting standard gastric emptying, an acidified gastric environment, increased mechanical agitation in the GI tract, and delayed gastric emptying. For simulated fed conditions, three test programs were applied: regular gastric emptying, regular gastric emptying in whole milk containing digestive enzymes, and increased gastric mechanical agitation. The detailed composition of the media is listed below in a separate section. Sampling was performed according to the chosen API determination method. Detailed information on the determination methods is given below. All samples were filtered through 1 µm poroplast filters (Dissolution Accessories, Oosterhout, The Netherlands). For UV-VIS measurements, sampling was performed in a closed-loop system. Within the first three hours, the samples were taken every 5 min, and then every 10 min until the end of the test. For HPLC measurements, 1 mL aliquots were sampled automatically at the predefined time points: 0 h, 0.5 h, every hour until 21 h, and at 22.5 and 24 h after the start of the experiment. The samples were transferred as soon as possible into an HPLC autosampler with a thermostat set at 12 • C or ambient temperature. Sample taking from whole milk containing digestive enzymes was conducted manually for up to 12 h, in which samples of 0.4 mL obtained from milk were mixed in a volumetric ratio of 1:3 with cold acetonitrile (1.2 mL), thoroughly vortexed for 5 s, and centrifuged for 10 min at 24,000× g. Then, 600 µL of the supernatant was transferred to HPLC vials, capped, and immediately transferred to a cooled HPLC autosampler. Media The composition of the media was adjusted according to the test protocols specified in Table 1. To simulate intragastric fluids under standard fasted conditions, a USP SGF sine pepsin pH = 1.8 was used. Intestinal fluids under fasted conditions were simulated using a FaSSIF/FeSSIF/FaSSGF powder concentrate dissolved in 50 mM potassium phosphate buffer (pH = 6.8) in concentration of 2.24 g/L. In the modified fasted protocols, the pH of SGF was set to 1.2 for the acidified gastric environment or to 1.8 for increased mechanical agitation protocol and delayed gastric emptying protocol. Intestinal fluids were simulated as described above. Intragastric fluids under fed conditions were simulated using a 50 mM phosphate buffer of an initial pH = 4.5. The pH of the buffer was gradually decreased to 2.0, according to the applied test protocol (Table 1). Intestinal fluids under fed conditions were simulated by adding 40 mL of a FaSSIF/FeSSIF/FaSSGF powder pre-dissolved in 50 mM potassium phosphate buffer (77.9 g dissolved in 240 mL of the buffer) to each of the test vessels. The final concentration of FaSSIF/FeSSIF/FaSSGF in the test vessels was 11.2 g/L. Immediately after the addition of the concentrate, the pH was adjusted to 6.5 with a 40% sodium hydroxide solution. In modified tests under simulated fed conditions, in which the media comprised whole milk (3.5% fat), acidification was performed similarly as in the phosphate buffer, with the gradual addition of pepsin (Merck, Darmstadt, Germany) up to the final concentration of 2.0 g/L. The intestinal transition was simulated with the addition of FaS-SIF/FeSSIF/FaSSGF powder as described above and containing pancreatin (SAFC, Hamburg, Germany) to reach a final concentration of 4.57 g/L. Determination of the API in the Media Determination of the API in the dissolution media was performed with a UV-VIS method and two HPLC methods, labeled 'A' and 'B'. All methods were validated according to ICH guidelines [14]. The UV-VIS spectroscopy method was performed with an Agilent 8543 spectrophotometric system (Agilent, Santa Clara, CA, USA). The flow-through quartz cuvettes were used, of a 10 mm path length (Hellma, Müllheim, Germany). The photometer was set to a single wavelength reference mode with a λ = 275 nm for API determination and λ = 295 nm as the reference. The method was linear within a 15-150 mg/L concentration range. HPLC method A was used to determine the API in the samples collected during tests in simulated fed conditions and determine the API in experiments involving the batches containing the lowest amount (5 mg) of the API. The resolution was obtained on a Zorbax Eclipse Plus Phenyl-Hexyl column (3.0 × 150 mm, 3.5 µm, Agilent) equipped with an inline filter and maintained at 35 • C. Gradient elution at a 1 mL/min flow rate was employed, and two mobile phases were used-A, which consisted of 0.1% phosphoric acid/acetonitrile (60:40, v:v); B, which consisted of 0.1% phosphoric acid/acetonitrile (10:90, v:v). During the first 12 min, 100% of mobile phase A was supplied. Then, the mobile phase was switched to 100% B to wash out impurities from the media. At 15.5 min post-injection, the column was re-equilibrated to the initial conditions with the mobile phase A. The total run time of the method was 22 min. The determination was performed on a fluorescence detector, with excitation wavelength λ = 220 nm, and emission λ = 309 nm and simultaneously on a UV-VIS detector set to λ = 275 nm. The quantitation limit of the fluorescence method was 0.49 mg/L, while for the UV-VIS detection, it was 2.01 mg/L. Both detection methods were linear up to 144 mg/L. The Bland-Altman analysis [17] showed that the two detection methods are in agreement for the concentrations of 5 mg/L or higher. For the lower concentrations, only the fluorescence detection method was employed. The HPLC method B was used for the determination of the API in samples collected in experiments under simulated fasted conditions. The resolution was performed on a Waters Symmetry RP18 column (3.9 × 20 mm, 3.5 µm, Waters) with an inline filter and maintained at 30 • C. Then, 10 µL sample volume was injected into the chromatographic system. Isocratic conditions were used with 0.1% phosphoric acid/acetonitrile (60:40, v:v) as the mobile phase. The total runtime of the method was 2.5 min. The detection wavelength of the UV-VIS detector was 275 nm. The quantitation limit was 2.01 mg/L, which was calculated from the ratio of the standard deviation of blank sample responses and the slope of the calibration curve, and the method was linear up to 144 mg/L. To assure complete elution of impurities that could have been retained on the column, a cleaning procedure was performed every six injections. It involved a 10-min long elution, which involved a gradual introduction of a more acetonitrile-rich mobile phase (0.1% phosphoric acid/acetonitrile, 10:90, v:v). During the cleaning procedure, the amount of the acetonitrilerich phase increased from 0 to 15% from 0.2 to 5.5 min. After this time, the column was re-equilibrated to the initial conditions. Experiment Workflow The general workflow of the study is presented in Figure 1. tation limit of the fluorescence method was 0.49 mg/L, while for the UV-VIS detection, it was 2.01 mg/L. Both detection methods were linear up to 144 mg/L. The Bland-Altman analysis [17] showed that the two detection methods are in agreement for the concentrations of 5 mg/L or higher. For the lower concentrations, only the fluorescence detection method was employed. The HPLC method B was used for the determination of the API in samples collected in experiments under simulated fasted conditions. The resolution was performed on a Waters Symmetry RP18 column (3.9 × 20 mm, 3.5 µ m, Waters) with an inline filter and maintained at 30 °C. Then, 10 µ L sample volume was injected into the chromatographic system. Isocratic conditions were used with 0.1% phosphoric acid/acetonitrile (60: 40, v:v) as the mobile phase. The total runtime of the method was 2.5 min. The detection wavelength of the UV-VIS detector was 275 nm. The quantitation limit was 2.01 mg/L, which was calculated from the ratio of the standard deviation of blank sample responses and the slope of the calibration curve, and the method was linear up to 144 mg/L. To assure complete elution of impurities that could have been retained on the column, a cleaning procedure was performed every six injections. It involved a 10-min long elution, which involved a gradual introduction of a more acetonitrile-rich mobile phase (0.1% phosphoric acid/acetonitrile, 10:90, v:v). During the cleaning procedure, the amount of the acetonitrile-rich phase increased from 0 to 15% from 0.2 to 5.5 min. After this time, the column was re-equilibrated to the initial conditions. Experiment Workflow The general workflow of the study is presented in Figure 1. First, the standard USP tests were performed to evaluate the basic dissolution profile under standard conditions rapidly. The aim was to verify that the API was released in an extended way within a 20 h time frame. The best performing formulations were selected for further in vitro performance evaluation and biorelevant dissolution tests. Second, the performance of the prototype tablets was evaluated. This stage aimed to exclude the formulations that disintegrated during incubation in media simulating gastric conditions. Leading formulations were characterized by good mechanical stability and integrity. In parallel, during the biorelevant dissolution tests in the StressTest device, the formulations were screened for the risk of potential dose dumping under various physi- First, the standard USP tests were performed to evaluate the basic dissolution profile under standard conditions rapidly. The aim was to verify that the API was released in an extended way within a 20 h time frame. The best performing formulations were selected for further in vitro performance evaluation and biorelevant dissolution tests. Second, the performance of the prototype tablets was evaluated. This stage aimed to exclude the formulations that disintegrated during incubation in media simulating gastric conditions. Leading formulations were characterized by good mechanical stability and integrity. In parallel, during the biorelevant dissolution tests in the StressTest device, the formulations were screened for the risk of potential dose dumping under various physiologyoriented conditions. The formulations that did not show an undesired drug release upon mechanical agitation and/or media change and demonstrated similar performance regardless of the API dose were selected for clinical studies. Standard USP Dissolution Tests The analysis of the API degradation in the presence of various excipients showed that none of the tested substances significantly influenced API stability. Therefore, the excipients could be safely used for manufacturing prototypes and final formulations. The compositions of all the developed batches are presented in the Supplementary Table S1. After selecting the excipients compatible with the API, the prototype formulations were tested in the USP type II paddle apparatus ( Figure 2). ology-oriented conditions. The formulations that did not show an undesired drug release upon mechanical agitation and/or media change and demonstrated similar performance regardless of the API dose were selected for clinical studies. Standard USP Dissolution Tests The analysis of the API degradation in the presence of various excipients showed that none of the tested substances significantly influenced API stability. Therefore, the excipients could be safely used for manufacturing prototypes and final formulations. The compositions of all the developed batches are presented in the Supplementary Table S1. After selecting the excipients compatible with the API, the prototype formulations were tested in the USP type II paddle apparatus (Figure 2). Figure 2A shows the dissolution profiles obtained for initially prepared formulations (A-G). The formulations varied greatly in their dissolution characteristics, ranging from an almost immediate-release (D) to a very extended one (A). The optimization of excipients' concentrations allowed obtaining the desired dissolution rate ( Figure 2B). All the manufactured prototypes (H-K) had very similar dissolution kinetics. To further characterize the prototypes and choose the leading formulation, the next steps of the development process employed varied analytical techniques involving biorelevant media and physiologically based dissolution. Table 2 presents the results of the water sorption analysis for the prototype formulation and the final tablets of different strengths and hardness 185-225 N. All of the tested batches increased their mass by approximately 100% after 2 h incubation in 0.1 M HCl. However, the transition to the phosphate buffer and FaSSIF resulted in up to a three-fold increase in the tablet mass. The water uptake did not differ greatly between tablets incubated in these two media, regardless of the tablet composition. The prototype (J) absorbed more water than the final formulations in all of the media tested. All strengths of the final formulations behaved similarly, showing robustness and good integrity. Table 2. Results of the water sorption and swelling kinetics analysis after incubation of tablets in different media. Water sorption analysis and increase of tablets' height and width are presented as comparisons with the initial measurements. The dry areas are presented as percentages of the total area of the tablet cross-section after incubation in the medium (dry area/total area × 100%). Data are presented as means with standard deviations. Table 2 shows the change in the tablet dimensions and the dry area fraction after incubation in different media. The visual observation of the tablets showed that all of the tested tablets increased mainly their relative height, whereas their relative width changed only slightly. The dimensions of the prototype increased to the greatest extent in comparison with the final formulations. Swelling Kinetics Visualization of the movement of the erosion and swelling fronts was possible due to the presence of a colorant. According to Colombo et al. [18], the erosion front is separating the matrix from the solvent, while the swelling front separates the rubbery and glassy regions in the matrix. The dissection of the tablet revealed the dry part, which remained white, while the colorant marked the colored hydrated fraction. The prototype batch (J) was the one in which the swelling front moved inwards toward the core to the greatest extent. As little as 27% of the total area of the tablet cross-section remained dry. The final batches (hardness 185-225 N) were more resistant than the prototype, especially in 0.1 M HCl. In this medium, more than 50% of the tablet remained dry. In both phosphate buffer and FeSSIF, the fraction of the tablet cross-section that remained dry was approximately 40% for all the strengths. Figure 3 presents the deformation profiles obtained after measuring the probe penetration depth into the swollen tablets, which was incubated in various media. Due to the destructive nature of the study, the texture analysis was performed only for the final batches with the hardness of 185-225 N. Texture Analysis tested tablets increased mainly their relative height, whereas their relative width changed only slightly. The dimensions of the prototype increased to the greatest extent in comparison with the final formulations. Visualization of the movement of the erosion and swelling fronts was possible due to the presence of a colorant. According to Colombo et al. [18], the erosion front is separating the matrix from the solvent, while the swelling front separates the rubbery and glassy regions in the matrix. The dissection of the tablet revealed the dry part, which remained white, while the colorant marked the colored hydrated fraction. The prototype batch (J) was the one in which the swelling front moved inwards toward the core to the greatest extent. As little as 27% of the total area of the tablet cross-section remained dry. The final batches (hardness 185-225 N) were more resistant than the prototype, especially in 0.1 M HCl. In this medium, more than 50% of the tablet remained dry. In both phosphate buffer and FeSSIF, the fraction of the tablet cross-section that remained dry was approximately 40% for all the strengths. Figure 3 presents the deformation profiles obtained after measuring the probe penetration depth into the swollen tablets, which was incubated in various media. Due to the destructive nature of the study, the texture analysis was performed only for the final batches with the hardness of 185-225 N. After incubating the tested batches for 2 h in all media, the formulations showed high resistance to deformation, regardless of the API dose. It indicates that in all tested media, the tablets contained a dry core. The high variability of deformation profiles resulted from the small differences in the water uptake and little propagation distances of the pushrod. It was observed especially in the measurements performed at 2 h due to low water penetration into the tablets (<2 mm penetration depth). Texture Analysis Fluctuations of deformation profiles decreased after a 5 h incubation in phosphate buffer pH 3.0 and FaSSIF. It suggests that the hydrogel layers surrounding the dry tablet core were stable and withheld their mechanical integrity despite higher swelling and hydration. After prolonged incubation, crossing the eroded part of the tablet required the highest force for the formulation containing 120 mg API. It indicates that the mechanical robustness of the formulation may increase with the API dose. After incubating the tested batches for 2 h in all media, the formulations showed high resistance to deformation, regardless of the API dose. It indicates that in all tested media, the tablets contained a dry core. The high variability of deformation profiles resulted from the small differences in the water uptake and little propagation distances of the pushrod. It was observed especially in the measurements performed at 2 h due to low water penetration into the tablets (<2 mm penetration depth). Fluctuations of deformation profiles decreased after a 5 h incubation in phosphate buffer pH 3.0 and FaSSIF. It suggests that the hydrogel layers surrounding the dry tablet core were stable and withheld their mechanical integrity despite higher swelling and hydration. After prolonged incubation, crossing the eroded part of the tablet required the highest force for the formulation containing 120 mg API. It indicates that the mechanical robustness of the formulation may increase with the API dose. Biorelevant Dissolution Tests According to the study protocol, the prototype batches that fulfilled the initial criteria were investigated in a biorelevant dissolution test, including physiology-driven mechanical agitation patterns. Figure 4A depicts the dissolution profiles of the prototypes (H, I, J, K) under simulated fasted conditions, as presented in Table 1. Mechanical agitation during this experiment caused an extensive release of the API into the media for almost all of the tested batches, which may cause dose-dumping episodes in vivo. The amount of the API released into the media increased to the greatest extent in the ninth hour of the experiment, when the StressTest device simulated the passage through the colon. The increase was most pronounced for batches J and K, which released almost 30% of the total amount of the API within a very short time. Batch H had two occurrences of intensified API release in the ninth and twelfth hour of the experiment. Batch I was the most durable and resistant to programmed stress events. However, the release profiles of this batch, as well as of batch H, had considerably high variability. Table 1. Under fasted conditions, the tested batches behaved similarly, and the dissolution profiles were aligned with the exception of the "Increased agitation" protocol. During this experimental setup, all tested batches, including the highest labeled API dose, were most prone to mechanical agitation. Interestingly, in the "Delayed gastric emptying" protocol, the batches showed Batches H, I, and J were tested under simulated fed conditions ( Figure 4B); dissolution from batch K was not assessed due to resemblance to batch J. Batches H and I resisted both gradually increasing acidity and mechanical agitation during the first five hours of the experiment when intragastric conditions were simulated. After adding bile salts (concentrated solution of SIF powder) in a concentration corresponding to the FeSSIF solution, pH increase, and an intensive agitation mimicking gastric emptying, formulations H and I released up to even 30% of labeled API dose within 30 min after the stress event. It indicated unwanted susceptibility to mechanical agitation in media simulating intestinal fluids. Similarly to fasted conditions, batch H was more prone to possible dose-dumping incidents. Batch J showed moderate resistance to mechanical agitation occurring during the simulated intragastric and intestinal passage. Upon obtained results, a reformulation was advised to decrease the sensitivity of the dosage form to mechanical agitation. For the characterization of the prototypes under simulated fasted conditions, an online UV-VIS method was used to determine the concentration of the API in the media. In further experiments involving final batches, the signal of a UV detector showed unwanted characteristics, such as lack of repeatability or overestimation of the API amount in the media. After investigating the possible reasons, we concluded that the online UV-VIS method lacked the necessary discriminatory power to measure the API concentrations adequately. Therefore, for the characterization of the final batches, HPLC methods were applied, as described in Section 2. Figure 4C,D show the results of these experiments. All of the tested final batches had highly similar dissolution profiles, especially under simulated fasted conditions ( Figure 4C). In addition, the variability of the profiles was lower than the prototype batches ( Figure 4A). The variability was greater under simulated fed conditions, but the dissolution kinetics and the release rate of the API from the dosage forms were comparable regardless of the dose and the applied compression force ( Figure 4D). Due to the better resistance and lower variability, the next step of the experiment involved batches compressed with a greater force. Figure 5 shows the dissolution characteristics of the final batches (hardness 185-225 N) under modified biorelevant conditions, as described in Table 1. Under fasted conditions, the tested batches behaved similarly, and the dissolution profiles were aligned with the exception of the "Increased agitation" protocol. During this experimental setup, all tested batches, including the highest labeled API dose, were most prone to mechanical agitation. Interestingly, in the "Delayed gastric emptying" protocol, the batches showed the greatest resistance to the stress events. After the transition from the acidic media to the media simulating intestinal fluids, there were little or no noticeable increases in the cumulative amount of the dissolved API; this characteristic was most evident for 120 mg formulation. However, the formulation containing the highest labeled dose of 120 mg was also more resistant to mechanical agitation in comparison with low and medium-strength tablets. Increased agitation during the experiment caused greater dissolution also under simulated fed conditions ( Figure 5). Similarly to the fasted conditions, the 120 mg dose was the least prone to the simulated stress events. The dissolution kinetics differed to the greatest extent when the media consisted of milk with digestion enzymes. At each time point, the cumulative amount of the API was lower in comparison with "Standard" and "Increased agitation" protocols. The experiment conducted in milk took twelve hours only. Therefore, the prediction of the maximum cumulative amount released within 24 h was impossible. simulated fed conditions ( Figure 5). Similarly to the fasted conditions, the 120 mg dose was the least prone to the simulated stress events. The dissolution kinetics differed to the greatest extent when the media consisted of milk with digestion enzymes. At each time point, the cumulative amount of the API was lower in comparison with "Standard" and "Increased agitation" protocols. The experiment conducted in milk took twelve hours only. Therefore, the prediction of the maximum cumulative amount released within 24 h was impossible. Discussion In the present study, we developed a robust and reliable SR dosage form of a novel GPR40 agonist for FIH pivotal clinical trial. We also introduce a framework for a comprehensive prediction of in vivo drug dissolution through physiology-oriented, biorelevant dissolution studies, standard USP dissolution, and characterization of mechanical properties of SR tablets. This novel approach has not been discussed yet in the available literature, especially in the context of the development of SR dosage forms with innovative medicines. The European Medicines Agency (EMA) published the guidelines on strategies for FIH and early clinical trials [19]. EMA advises that several doses of the drug candidate should be evaluated in healthy volunteers, besides examination of non-clinical aspects, such as determination of strength and potency, use of a relevant animal model, evaluation of pharmacodynamics, pharmacokinetics, and toxicological properties. To maintain the dissolution characteristics over up to several dozen-fold dose increases, the preclinical formulation development has to rely on a good understanding of the physicochemical properties of the API [20]. In this process, the formulation specialists are crucial, as they can aid in obtaining early relative bioavailability information by proper design of the dosage forms used in the early stages of the trials and later commercialization ones [21]. They can also propose an optimal dosage form and choose the excipients that may alter API solubility, stability, or enhance bioavailability. At the formulation development stage, critical decision points include not only the development of the prototype and stability issues but a thorough evaluation of the reliability of its galenic principles under the application conditions [22]. Later stages of FIH trials may assess food and formulation effects [21]. Therefore, the preclinical characterization of the dosage form should also estimate its performance under both fasted and fed conditions. It minimizes the risk of an undesired performance in vivo, leading to failure of the trial and consequently delays and costs due to reformulation [23]. The aim of the discussed formulation development was to achieve a dosage form with a similar dissolution rate for all tablet strengths: 5, 30, and 120 mg. Due to the required prolonged in vivo exposure to the API, reaching 14-18 h after administration, we proposed an SR monolithic matrix tablet as the dosage form. The planned clinical trial protocol involved a dose-escalation step and estimation of the possible food effects. For this reason, the second objective was to evaluate the performance of the dosage form regardless of the prandial state. The third goal was to minimize the risk of undesired characteristics, such as dose dumping. Lastly, we wanted to assure comparable dissolution kinetics under altered physiological conditions, such as decreased or elevated gastric pH and increased GI motility. In the first step, the standard dissolution tests allowed quick discrimination of the potential candidates (Figure 2A). At this stage, the matrix-filling and matrix-forming excipients were selected. As shown in Figure 2B, the USP-based standard dissolution tests allowed obtaining several candidate formulations of extended dissolution performance with a very small intra-batch variability. The dissolution from the prototypes H-K would ascertain a proper delivery within the required time frame. However, in the physiological conditions, the swollen tablet matrix is subjected to varied mechanical stress patterns and fluids of different compositions; these conditions change according to the prandial state. Therefore, we introduced more sophisticated dissolution tests that reflected the physiology of the GI tract to identify unwanted effects, such as dose dumping. The StressTest device used in the present study allows the simultaneous simulation of the mechanical agitation in the GI tract and evaluation of dissolution in biorelevant media. Initially, the most promising prototypes selected in the USP dissolution tests were evaluated under standard fasted and fed conditions, as described in Table 1. The stress patterns and pH profiles were based on the data gathered in experiments using telemetric capsules [24][25][26]. The media for the fasted state were SGF sine pepsin pH = 1.8 and 50 mM potassium phosphate buffer with the addition of FaSSIF/FeSSIF/FaSSGF in concentrations relevant to the fasted conditions; for the fed state, the medium was 50 mM potassium phosphate buffer to which FaSSIF/FeSSIF/FaSSGF was added in concentration relevant to the fed state. Usually, carbonate-based buffers, such as Hanks buffer, are preferred to simulate intestinal conditions in the fasted state. It is due to the fact that it better reflects the ionic composition and buffer capacity of small intestinal fluids [27]. In the present study, we initially used the Hanks buffer pH = 6.8 and adjusted to pH = 6.5 five hours after the beginning of the experiment. However, due to the undesired signal characteristics, the quantitative assessment of dissolution was unreliable between batches. This observation was non-formulation dependent and forced the transition from Hanks buffer to the higher capacity 50 mM phosphate buffer. For the same reason, an online UV-VIS measurement was changed to a high-throughput HPLC method, which was labeled as method 'B' in Section 2.2.3. A subsequent comparison of the dissolution characteristics in Hanks buffer versus 50 mM phosphate buffer revealed that the profiles were similar, but the tested tablets were more prone to mechanical agitation in the latter medium (data not shown). We concluded that the standard 50 mM potassium buffer was a medium of higher discriminatory power for the investigated formulations and adopted it in further experiments. The application of physiologically relevant agitation during dissolution tests in the StressTest device revealed undesired characteristics of the tested prototypes ( Figure 4A,B). The prototype tablets were prone to the applied pressure and mechanical agitation. Increased dissolution from the tablets was most pronounced under simulated fasted conditions. Although the dissolution kinetics of the prototypes were similar in the first hours of the experiment, a prolonged incubation in the medium and programmed stress events differentiated between the tested formulations. The bio-predictive dissolution tests revealed that the prototypes could release a significant amount of the API within a short time. These dose-dumping events could occur both under fasted and fed conditions. The accelerated release of API or dependability of the dosage form on mechanical stress can lead to increased pharmacokinetic variability [11,12]. Accurate estimation of pharmacokinetics is essential for innovative medicines under investigation. Therefore, a reliable dosage form with predictable dissolution kinetics and sufficient resistance to mechanical agitation in the GI tract is an asset in the planned clinical trial. This is the first study available for the public domain that shows the utilization of the StressTest device to evaluate properties of a dosage form for FIH clinical trials. Until now, the device has been successfully used to evaluate dissolution characteristics of generic SR or delayed-release products [28][29][30][31]. The composition of the prototypes was redesigned, and final batches were obtained. These batches were manufactured with two different compression forces. As a result, obtained were tablet batches with hardnesses of 140-175 N and 185-225 N, respectively. All the batches had outstanding robustness and integrity that was maintained regardless of the dose strength or the compression force used ( Figures 2C and 4C,D). Regardless of the hardness, the final batches of tablets were characterized by a similar dissolution performance under all applied test conditions. However, the batches manufactured at higher compression forces were more favorable for further technological processing such as dedusting, blistering, and even coating. Therefore, tablets of hardness 185-225 N were preferred to use in the clinical trial. The results of swelling experiments confirmed the robustness of the final formulations. The maximum incubation times in the swelling experiments (2 h for 0.1% HCl or 5 h for phosphate buffer and FaSSIF) were based on the observations from in vivo experiments with telemetric capsules [24][25][26]. The highest recorded fortitudes of the physiological stresses under both fasted (gastric emptying and ileocecal passage) and fed (gastric grinding and gastric emptying) occur within the first five hours after the intake. This time frame allowed us to estimate the performance of the formulation and the kinetics of the matrix-forming process. The test showed that the final formulations of hardness 185-225 N absorbed similar amounts of water and had comparable swelling kinetics, regardless of the API amount. It is noteworthy that a considerable area of the tablet core remained dry even after five hours of incubation in various media. It translated into an extended and regular release of the API within the desired 14-18 h time frame, as observed in the biorelevant dissolution tests. These results emphasize that simple and quick swelling experiments support and complement more complex analyses. An advanced texture analysis also confirmed observations from the StressTest device ( Figure 3). The resistance of the final batch tablets (hardness 185-225 N) to the deformation force was only slightly affected by the media. Although the tablet of the highest strength (120 mg) yielded the least to the applied force, the deformation study results were similar for all tested potencies. The dissolution tests on final batches (hardness 185-225 N) comprised various test programs aimed at mimicking altered GI conditions ( Figure 5). The tests should confirm or exclude the robustness observed under standard biorelevant protocols. For fasted conditions, we selected the conditions that emulated decreased pH of the gastric media, which could be observed in subjects with GI diseases, such as ulcers [32]. Another protocol evaluated the influence of a delayed gastric emptying time, such as in subjects with gastroparesis [33,34]. To ascertain sufficient mechanical integrity of the matrix tablets, one of the proposed protocols included prolonged mechanical agitation increased to the highest values of up to 350 mbar intensity. The increased agitation was also introduced in the test protocols under fed conditions during the intragastric stage and gastric emptying. Finally, the dissolution tests explored the possible impact of dynamically digested meals on the release of the API from the matrix tablets. Several media were suggested in the literature to simulate the composition and properties of GI fluids under the fed state. Ideally, the physicochemical properties and composition of the media should resemble a meal, such as standard breakfast in bioequivalence studies; it should also be adjustable to mirror the changes during the digestion [30]. One of the proposed is FeSSIF, which is used in the present study as a "standard" medium. Other alternatives are heat-treated whole milk and Ensure ® Plus. Another aspect is the digestion protocol. Two main approaches are dynamic digestion when enzymes such as pancreatin or lipase are added into the media [35] or the use of the "snapshot" media that reflect an early, middle, and late environment in the postprandial stomach [36,37]. In the present experiment, we used whole milk acidified with hydrochloric acid and digested it with pepsin and subsequently with pancreatin at elevated pH after simulated gastric emptying. We found that this medium is sufficiently stable and reflects well the physiological processes in the GI tract [35]. Of note, the duration of the dissolution experiments in milk was shorter than for the rest of the test setups (12 h vs. 24 h). There are several reasons that justify the selected time span. First, the test was designed to evaluate the resistance of the dosage form toward combined mechanical and physicochemical conditions that are representative of the stomach and small intestine in the postprandial state. Second, the digested milk is poorly stable during incubation at 37 • C, and the manual sampling and immediate sample processing require an extensive workload. Another aspect worth discussing is an incomplete dissolution of the API in these tests: 40-60% of the labeled amount within 12 h. The test simulates the worst-case scenario to which the dosage form can be subjected after the intake under fed conditions. Per definition, the dissolution of the tablet was not completed, and there was no plateau phase in the recorded profiles. As the API dissolved well in simulated gastric and intestinal conditions, the incomplete dissolution due to precipitation in the digested milk is unlikely. We observed that in the tests performed under increased agitation protocol, the dissolution profiles differed to the greatest extent in comparison with the standard conditions. The variability was also greatest when the most intensive stress patterns were applied. The dissolution kinetics were comparable for all tested dose levels and test protocols. The only exception was dissolution experiments conducted in milk. In this medium, the amount of the API released and the rate of this process were the lowest. Before the experiment, we evaluated whether the API could be digested by the enzymes added into the media and confirmed that the molecule is resistant to such digestion. Therefore, the observed effect did not result from the instability of the API. The decrease in the dissolution rate in milk is consistent with the results reported elsewhere [35,38,39]. Several hypotheses may explain such an observation. First, a proteinaceous film may form on the outside of the tablets from precipitates that increase tablet disintegration time [40]. This effect is most pronounced for immediate-release tablets; however, it is also likely to occur for the SR tablets. Second, due to the higher viscosity of milk than compendial media, the wetting of the tablet may be delayed [38]. Third, lipids may deposit on the outer gel layer of the matrix tablet that controls the rate of drug dissolution; these fat deposits may possess water barrier properties and prevent water from penetrating into the tablet core [39]. Milk also affects the solubility of drugs, such as dicumarol, danazol, or mefenamic acid [41,42]. It is most pronounced for BSC class II drugs. The API in the present study was classified as a BCS class I drug; therefore, its solubility should not be greatly affected by milk, and the observed dissolution variability is formulation-dependent. The outcome of this study was SR formulations of ascending strength of a novel GPR-40 agonist. They allowed a regular drug delivery within 14-18 h, according to the clinical specification. The final formulations of 185-225 N hardness, which were selected for the clinical trial, showed very consistent and similar dissolution kinetics under both fasted and fed biorelevant conditions, regardless of the dose labeled. The tablets were also resistant to non-standard conditions in the GI tract, such as delayed gastric emptying, increased mechanical agitation, or low gastric pH. The results indicate that the developed formulations bear no risk of dose dumping and allow a predictable drug delivery, which is of great importance for antidiabetic drugs. Lastly, the strength and novelty of the present study is the range and flexibility of the research protocol, connecting simple experiments with sophisticated dissolution analysis. This unique combination allows targeting specific requirements resulting from the physicochemical or pharmacokinetic properties of the API. Moreover, the biorelevant dissolution protocols specified in the StressTest device can be adopted for a wide range of GI conditions, such as simulation of the variability of the passage times, varied stress patterns, or evaluation of food effects. Customization of the conditions allows a swift reformulation and optimization of the dosage form before entering the clinical trials. Therefore, the implementation of the characterization strategy described in this paper into the development cycle of innovative medicines allows shortening the development times and reduces the risk of the FIH clinical trial failure, dose finding, and later therapy. Conclusions In this study, we showed a varied and thorough approach for the development and characterization of SR matrix tablets containing an innovative GPR40 agonist for use in the first-in-human clinical trial. The tests were performed using bio-predictive dissolution tests and a set of experiments determining the performance of the galenic principles of the formulations, such as texture and matrix formation analysis using texture analyzer and evaluation of the swelling performance. The formulations containing 5, 30, and 120 mg of the API developed according to implemented strategy were exceptionally robust toward the mechanical and physicochemical conditions of the bio-predictive tests and assured comparable drug delivery rates regardless of the prandial state and dose labeled. These characteristics were desired for the upcoming clinical trial. We conclude that the implementation of the development strategy described in this paper into the development cycle of SR formulations with innovative APIs helps not only to reduce the risk of formulation-related failure of phase I clinical trial but also to effectively and timely provide reliable medicines for patients in the trial and their further therapy. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics13060804/s1, Supplementary File containing Figure S1: A sample cross-section of a formulation after incubation in the test medium, as described in Section 2.2.2 of the manuscript. The photograph depicts the final formulation containing 120 mg API (Final-120) after 5 h incubation in FaSSIF. The addition of the colorant to the medium allowed visualization of the hydration front, and Table S1: Composition of developed formulations A-K and the final one. The values are presented as mass percentages of the total mass of the tablet. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to an ongoing development and registration process. Conflicts of Interest: This article reflects the authors' view only. Neither the Research Executive Agency nor the Polish Ministry of Science and Higher Education may be held responsible for the use which may be made of the information contained therein. 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.	2021-06-03T06:17:21.963Z	2021-05-28T00:00:00.000	{ "year": 2021, "sha1": "2c40c0f7e383b3babc18aab23aeda7e10d18c48a", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1999-4923/13/6/804/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "0537f405af7ec5a33f9da79d3bf5ab5971898723", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
234040302	pes2o/s2orc	v3-fos-license	Analysis and Treatment of Turbine’s Increasing Shaft Displacement and Thrust Bearing’s Burning Loss In this paper, the writer is to analyze the causes of axial displacement and thrust bearing temperature according to the relevant factors affecting the axial force of turbine and provides treatments. It can be used for reference for units with this phenomenon or axial force increasing. Introduction The thrust bearing of steam turbine is used to bear the axial force of steam and coupling acting on the rotor to locate the rotor position and ensure the axial clearance within the safe range. The greater the thrust, the greater the shaft displacement and the higher the thrust bearing temperature. Therefore, in addition to temperature, it is necessary to monitor the axial displacement for thrust bearing. The temperature and displacement of thrust bearing are the key monitoring objects in the safe operation of turbine. During the operation of the unit, if the temperature or axial displacement is abnormal, it should be analyzed and excluded in time to ensure the safe operation of the equipment. The 50 thousand ASU turbine of a chemical plant is EHNKS50/80/16 high temperature and high-pressure steam extraction condensing unit. About 10 months after the unit was put into operation, axial displacement and thrust bearing temperature gradually increased: the axial displacement increased by about 0.01mm/5 days; when the temperature rose from 56℃ to 72℃, the axial displacement rose faster. About 6 months later, a thrust bearing was burned out. When the tile was burned, the thrust bearing temperature did not exceed the alarm value of 105℃. At the meantime, the large shaft displacement caused to the jump. Before the axial displacement began to increase gradually, there were vibration anomalies --occasional spurts of vibration. After about 2 months of operation, the temperature and shaft displacement of the new thrust bearing replaced gradually increased. The Balance of Axial Force of Turbine Rotor and Its Rough Calculation The axial force of turbine rotor is mainly composed of four parts. The thrust caused by the pressure difference between the rotor blades and the rotor blades [1]: In the formula, ρ is the degree of reaction at average diameter D. Thrust caused by static pressure difference between front and rear guide blade [2]: Axial forces caused by differences in hub diameters between stages (including balance force) [2]: △Sg is the area difference caused by hub diameter differences. The force acting on the rotor by the coupling Fo. We can find it from the drawing of coupling. Total axial thrust: Factors Influencing Axial Force The axial force varies with the variation of the working condition. The axial force is usually proportional to the steam flow, except for the back-pressure turbine. The relationship between the pressure and flow of the back-pressure unit is nonlinear [1,2]. According to the calculation formula of rotor axial force, it can be gotten the factors that affect the axial force: throttle flow, pressure difference, reaction degree, area difference, external axial force applying to the turbine rotor. The Influence Factors of Steam Flow The intake of steam is related to the load. The greater the load, the greater the intake of steam [5]. The axial force is calculated according to the maximum power and the maximum steam intake. Therefore, the fluctuation of the load should be checked if the sudden change of the axial displacement of the unit in operation appears. The greater the load is, the greater the steam intake is and the greater the axial force is [6]. The Influence Factors of Pressure Difference When the load increases, the steam intake increases, and the pressure difference between different levels increases, the unit axial force increases. If salt of soft water exceeds the standard, it is easy to cause leaf scaling [7]. Thus, the pressure difference between the front and rear of the corresponding blade and guide impeller increases, which leads to the axial force increasing. After scaling, the friction between steam flow and blade increases, and static electricity accumulates. Wear of Inner packing gland causes to the pressure difference between stages increasing, which also reduces the balancing 3 ability of the balance face of the balance plate and increases the axial force. In general, the better the vacuum is, the greater the pressure difference is and the greater the axial force is. Only if Fg is negative, the better the vacuum is, the less the axial force is. The Influence Factors of Reaction Degree The axial force of the unit increases with the decrease of steam inlet parameters and the increase of reaction degree at all levels. The low inlet steam parameter causes to the phenomenon that the last few stages of steam contain water and the axial force increases sharply. After the steam contains water, the wet steam generates more static electricity due to the friction with the blade in the work process of the latter several stages. Since the rotor is insulated during operation, the static electricity on the rotor cannot be released, so the static electricity will accumulate more and more, and the voltage will be higher and higher [3,5]. When it reaches a certain degree, the voltage will break through the oil film in the weakest place, release the voltage, form the current, cause the electric corrosion, and then cause the burning tile. Therefore, it is very important to ensure the admission parameters complying with the design. The Influence Factors of Area Difference Hub scaling at some stages results in larger hub diameter, larger area difference, roughened blade surface, and increased axial force. Severe scaling will block the steam passage between the blades, which will not only cause the pressure difference between the stages to increase, but also cause the area difference between the hubs to change greatly. The External Influence Factors The force acting on the rotor of the coupling is related to the pretension of the coupling [8]. If the pretension of the steam inlet coupling is too small, or if the pretension of the steam outlet coupling is too large, it will increase the positive axial force of the rotor. On the contrary, the axial force of the rotor will be reduced. The turbine pulling the generator may also be subjected to axial forces caused by the generator excitation center which is not installed in place. For the bearing seat with the steam inlet end expanding displacement along the cylinder, the coaxiality of the coupling cover on its side seriously fails to meet the requirements, which causes the deflection force of the bearing seat in the expansion process. As a result, it also causes the reduction of the bearing area of the thrust bearing, and further reduces the bearing capacity of the thrust bearing. Analysis of Axial Displacement and Thrust Pad Temperature Rising Gradually According to the above influencing factors which can influence axial force, the steam turbine was inspected comprehensively on site. What can be found: the seal teeth in the steam turbine are almost completely off (the gland seal off is the cause of the occasional vibration jump of the unit), which reduces the balancing capacity of the balancing piston disc and increases the axial force. The right positioning nut of the thrust rod of the bearing house is loose, which reduces the bearing area and bearing capacity of the thrust bearing. The steam turbine pipe pulls the steam turbine to the right, which makes the cylinder move 0.55mm relative to the rotor to the right (front seal clearance 0.45mm), causing the static and static friction of the steam seal. The designed operating parameters of the steam turbine are 8.9mpa /525℃, and the actual operating parameters are 8.8mpa /470℃. The reduction of inlet steam parameter increases the reaction degree of rotor blade and increases the axial force. Moreover, due to the low steam parameters, all the steam in the last five stages of the turbine impeller included water, which caused a sharp increase in the axial force of the turbine, and the increase was related to the steam flow rate and water content. The bearing surface of the thrust bearing surface is dark gray and rough and there are also some irregular pits, which are the appearances of electrostatic corrosion. [3] Too low steam parameter may be inclined to cause static accumulation. See figure 2. The design value of pretension of the nose end coupling is less than the actual value. As a result, the coupling is under pressure when running, which increases the positive axial force of the turbine rotor. According to the above problems, it can be concluded that the axial force of the turbine has been greatly improved, resulting in the increase of axial position. With the increase of axial force, the oil film of thrust bearing is the weakest and it is also the most vulnerable to electrostatic breakdown [9]. With the constant heating of the thrust bearing current, the temperature of the thrust bearing keeps increasing and the shaft displacement becomes larger and larger. With the expansion of the electric etch surface on the thrust bearing, the bearing oil film on the thrust bearing becomes more and more unstable, and the thrust bearing burns out sooner or later. Electrostatic corrosion is shown on the right. Accident Treatment and Effect The rotor and the holding ring were returned to the factory, and the steam seal plate was embedded again. The bearing pedestal was readjusted on site to make the contact area between thrust bearing and thrust disk reach more than 90%. The minimum pass area of the balance hole was rechecked to determine the balance capacity. The stress imposed on the steam turbine inlet pipe is limited. Meanwhile, the design institute replaced the support and hanger of the steam intake pipeline. In order to ensure that static electricity is no longer harmful, the turbine rotor was installed with a brush (brush fixed on the oil seal ring --non-explosion-proof) to eliminate the corrosion of thrust bearing caused by static electricity. See the Figure 3. In addition, the steam intake parameter (8.8Mpa/505℃) was improved to reduce the ability of generating static electricity. Soften water as far as possible to avoid adding more agents or less agents, in order to reduce leaf scaling. If there are more agents, phosphate is easy to exceed the standard, resulting in phosphate adhesion on the leaf accumulation of static electricity. The steam turbine has been in operation for about 11 months since December 2019. The problem has been solved just because the turbine shaft displacement is always between 0.21--0.26mm, and the temperature is between 57--63℃. Unit load fully meets the production requirements, to ensure the benefits of customers. Preventive Measures For users and the company, that there is no problem with steam turbine is the best and most ideal state, so precautions are particularly significant to make the turbine work in normal operation. What should be done as follows: pay close attention to thrust bearing temperature and shaft displacement; Always look at long-term historical trends and be sensitive to abrupt jumps and trends in historical trend curves [10]; ensure that steam parameters are within a reasonable range; Communicate with Conclusion The case in this paper is ostensibly caused by static electricity, but it is essentially caused by a factor affecting the axial force ------unqualified steam parameters. The increase of axial force causes the oil film between the thrust plate and the thrust bearing to be weak, thus forming static electricity to be released from the thrust bearing. Therefore, to be familiar with and master the relevant factors affecting the axial force, and to understand the associated influence caused by the relevant factors can help us analyze the problem and quickly find the direction to solve the problem. This case is a typical case of the influence of comprehensive factors and it has extensive reference significance.	2021-05-10T00:03:56.476Z	2021-02-01T00:00:00.000	{ "year": 2021, "sha1": "13c94037fae23a55596cefb4a3914f6b02cba309", "oa_license": null, "oa_url": "https://doi.org/10.1088/1742-6596/1744/2/022130", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "f038c3e96760bf38e82361f62e5225c932a4a69a", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Physics", "Geology" ] }
256796983	pes2o/s2orc	v3-fos-license	Back and forth: day–night alternation between cover types reveals complementary use of habitats in a large herbivore The Complementary Habitat Hypothesis posits that animals access resources for different needs by moving between complementary habitats that can be seen as ‘resource composites’. These movements can occur over a range of temporal scales, from diurnal to seasonal, in response to multiple drivers such as access to food, weather constraints, risk avoidance and human disturbance. Within this framework, we hypothesised that large herbivores cope with human-altered landscapes through the alternate use of complementary habitats at both daily and seasonal scales. We tested the Complementary Habitat Hypothesis in European roe deer (Capreolus capreolus) by classifying 3900 habitat-annotated movement trajectories of 154 GPS-monitored individuals across contrasting landscapes. We considered day-night alternation between open food-rich and closed refuge habitats as a measure of complementary habitat use. We first identified day–night alternation using the Individual Movement - Sequence Analysis Method, then we modelled the proportion of day–night alternation over the year in relation to population and individual characteristics. We found that day-night alternation is a widespread behaviour in roe deer, even across markedly different landscapes. Day–night alternation followed seasonal trends in all populations, partly linked to vegetation phenology. Within populations, seasonal patterns of open/closed habitat alternation differed between male and female adults, but not in juveniles. Our results support the Complementary Habitat Hypothesis by showing that roe deer adjust their access to the varied resources available in complex landscapes by including different habitats within their home range, and sequentially alternating between them in response to seasonal changes and individual life history. Introduction Fitness is influenced by spatial and temporal heterogeneity in the distribution of multiple, often complementary, resources (Gaillard et al. 2010). A given habitat may not fulfil all the needs of an individual simultaneously, while the functional role of a habitat may fluctuate over time in relation to modifications in an individual's requirements, or in the value of the habitat per se (Peters et al. 2017;Couriot et al. 2018), for example, driven by vegetation phenology. In response, animals may adjust their behaviour through 'complementary habitat use', defined as the use of different habitats at different times by individuals during the course of their daily and seasonal activities (Complementary habitat hypothesis, CHH) (Dunning et al. 1992;Mandelik et al. 2012). For many ungulates, for example, accessibility to forage and protection from predation risk are two key resources that are selected (Hebblewhite and Merril 2009). Indeed, these two types of resource are both positively related to fitness, but are often spatially distinct (Benhaiem et al. 2008) and fluctuate seasonally, driving complementary use (sensu Mandelik et al. 2012) of 'open' vs. 'cover' habitats (Mysterud and Østbye 1999;Berryman and Hawkins 2006). For example, forest canopy can provide cover from adverse conditions (e.g., deep snow: Mysterud and Østbye 1995, Ewald et al. 2014, Ossi et al. 2015 and protection from predators or human disturbance (Bonnot et al. 2013), while the understorey provides seasonally rich foraging (i.e., during the vegetation green up and re-growth, Mancinelli et al. 2015). In contrast, open areas may be used by ungulates as a seasonal source of forage (Abbas et al. 2011), but mainly at night to avoid human disturbance (Godvik et al. 2009; diminish the risk of ambush predation (Lone et al. 2014;Gehr et al. 2017). Hiding cover may also be seasonally available in open habitats, for example, in summer, when row crops are mature and provide concealing (Mysterud et al. 1997;Bjørneraas et al. 2011;Bonnot et al. 2013;Dupke et al. 2017). In heterogeneous landscapes, open and closed habitats, hence, may represent composites of different resource types, and animals must alternate between them to satisfy their requirements (see Dunning et al. 1992). Large herbivores use these composites at different spatio-temporal scales, so that the open and closed habitats visited along the movement trajectories generate specific patterns of sequential habitat use (De Groeve et al. 2016). For example, at the daily scale, the day-night alternation between open and closed habitats may be linked to activity cycles e.g., foraging vs. resting and ruminating, or the forage acquisition-predation risk avoidance trade-off (Hebblewhite and Merril 2009). In turn, daily alternating use of open and closed habitats may vary in relation to seasonal changes in perceived risk (e.g., anthropogenic disturbance, hunting activity; Bonnot et al. 2013, Gehr et al. 2017, vegetation productivity (i.e., green up and senescence, or cultivation / harvesting; Peters et al. 2019), and physiological cycles (e.g., growth, reproduction, dispersal). The understanding of the complementary use of resource composites across spatio-temporal scales by ungulates could inform managers regarding the functional role of different habitat types in human-modified environments. In particular, the spatial association between open agricultural habitats and forest patches could support thriving populations of wild ungulates in matrix landscapes (Hewison et al. 2009;Linnell et al. 2020). These concepts are well-addressed within the Complementary Habitat Hypothesis framework (Dunning et al. 1992) and well established in ecological theory, but rarely tested for wild populations, especially vertebrates, as most studies concern insects (Mandelik et al. 2012). Here, we considered the patterns of alternation between open and closed habitats in European roe deer (Capreolus capreolus) to test the Complementary Habitat Hypothesis in a highly managed large herbivore (Apollonio et al. 2010) that has successfully adapted to human-modified landscapes , including open agricultural areas (Hewison et al. 2009). Roe deer is an ideal model species to test the Complementary Habitat Hypothesis because it occupies a wide range of landscapes (i.e., different arrangements of complementary habitats, sensu Dunning et al. 1992), from completely forested areas to wide open agroecosystems, exhibiting marked behavioural and ecological plasticity (Hewison et al. 1998;Morellet et al. 2013). Moreover, roe deer are known to prefer ecotonal and forest habitats, complemented by the use of open habitats (meadows and crops), typically at night (Bonnot et al. 2013;Dupke et al. 2017), in relation to their bimodal crepuscular activity pattern (Pagon et al. 2013;Krop-Benesch et al. 2013;Bonnot et al. 2020). Finally, roe deer are characterized by sex-dependent space use patterns (Malagnino et al. 2021), especially in spring and summer, when adult males display territorial behaviour (Hewison et al. 1998), and females give birth and care for their young. We applied the Individual Movement -Sequence Analysis Method (IM-SAM, De Groeve et al. 2020a) to six populations of European roe deer living in contrasting landscapes to evaluate how day-night alternation between closed and open habitats varied across the seasons and environmental contexts. We first hypothesized that day-night alternation between closed and open habitats would mirror the landscape composition (sensu Dunning et al. 1992; Table 1: Landscape Composition and Structure Hypothesis, LCSH, H1). In particular, we expected that day-night alternation would occur mainly in heterogeneous landscapes (Table 1: H1, P1). Second, we hypothesized that day-night alternation would also vary seasonally, in relation to the phenology of vegetation (Pettorelli et al. 2006), according to the Complementary Habitat Hypothesis (Mandelik et al. 2012). Specifically, we predicted frequent alternation during vegetation green-up to maximize access to high quality food in rich-open habitats, but less alternation in winter, when meadows and crops provide less food resources and are more exposed to extreme weather conditions (e.g., snow cover, wind exposure; Table 1: CHH, H2, P2.1). Also, we hypothesized that daynight alternation between open and closed habitats would vary over seasons according to key life history events that are linked to sex and age, such as births, or the rut (Table 1: H2, P2.2; Andersen et al. 2000, Bongi et al. 2008. In particular, we predicted that the day-night alternation of females and males should differ the most during the spring-summer season, when adult females should alternate less due to the constraints of provisioning for their young (Andersen et al. 2000), while adult males should alternate more to patrol and defend their mating territory, especially during rut (Johansson 1996). Therefore, we expected these sex-specific seasonal patterns to be more evident in adults than juveniles linked to reproductive status and behaviour (Sempéré et al. 1998). Materials and methods The habitat use sequences were obtained from roe deer movement trajectories of the Eurodeer database (Urbano and Cagnacci, 2021), as processed in De Groeve et al. (2020a, b), with some simplifications (see below). In the Eurodeer database, roe deer trajectories obtained from GPS collars deployed across European populations are stored and curated together with individual-based information obtained during capture (Urbano and Cagnacci, 2021). In particular, individuals sexed and aged at capture as juveniles (< 1 year old) are considered to become yearlings from 1st April (just before the birth period of roe deer over most of its range) of the year of first monitoring and for the subsequent 12 months. All other individuals are considered adults. ). The final classification of sequences into these four patterns of sequential habitat use is illustrated for two representative populations in Fig. 1 (see also Appendix S1, Figure S1.2 for the final classification of all populations). From the resulting dataset, we extracted the data for six roe deer populations with a representative number of individuals (≥ 10 individuals): Southern France (Eurodeer database study area identifier: FR8), Switzerland (CH25), Southern Germany (DE15), Southeast Germany (DE2), and Northern Italy (IT1, IT24; see Figure S1.1, Table S1.2 for a description of each population). Because we were interested in modelling seasonal variations in day-night alternation, we removed individuals which had less than 10 biweekly habitat use sequences. Our final dataset consisted of 3900 habitat use sequences from 154 animals (95 females and 59 males, made up of 44 juveniles/yearlings and 132 adults, with 22 animals switching from fawns to adults during monitoring; Table S1.2). Covariates linked to roe deer habitat use sequences To test whether day-night alternation is linked to vegetation phenology, we computed the landscape level vegetation productivity profile for each biweekly period estimated by the Normalized Difference Vegetation Index (NDVI; smoothed and pre-processed as in Vuolo et al. 2012) using the following workflow. First, we defined the study area as the Minimum Convex Polygon (MCP) of all locations from all individuals within a population. For each population's MCP, we sampled 1000 random points within that area (corresponding to a survey density of 1.06 ± 0.06 points per hectare, average ± standard deviation) and extracted the weekly NDVI values. Next, we matched each biweekly period with the corresponding averaged weekly NDVI values. Sequential habitat use through space: landscape composition and structure hypothesis (H1) First, we investigated the variation in the proportion of habitat use sequences across populations. The input for this analysis consisted of an abundance table of patterns of sequential habitat use (day-night alternation, a; homogeneous closed, c; homogeneous open, o; random, u) per population. The results were visualized using a mosaic plot, along with a chart indicating the proportion of closed habitat and closed and open edge density (as a proxy of landscape fragmentation) for each population, a circular map extract covering the population range and the number of individuals and sequences (Fig. 2). To test the Landscape Composition and Structure Hypothesis (H1), we first verified whether the probability of alternation varied across landscapes. We expressed habitat alternation as a binomial variable where day-night alternation (a) is 1 and all other patterns of sequential habitat use (c, o, u) are 0. We built Generalized Additive Mixed Models (GAMMs) with a binomial distribution of residuals, with day-night alternation as the response variable, the cyclic spline smooth of the biweek to account for temporal variation, and population as a fixed (ordered) factor, or as a factor-specific temporal effect (e.g., a population-specific spline of biweek; M1, see below and Table 1: H1; H2.1). We included individual identity as a random effect on the intercept. The most parsimonious model was selected based on minimization of the Akaike Information Criterion (Burnham and Anderson 2002). Second, we compared the landscape composition between populations, measured as proportion of To do that, for each population's MCP, we sampled 1000 random points within that area, buffered them with a 200 m radius, and measured these metrics. We built a Generalized Linear Model (GLM) with proportion of closed as the response variable with beta distribution of residuals, and population as a fixed (ordered) factor; and two GLMs with open edge and closed edge density, respectively, as response variables with gamma distribution of residuals, and population as a fixed (ordered) factor. We also applied the non-parametric Kruskal-Wallis test to each variable separately, using the measures in the buffers as repetitions, grouped by population, as a further confirmation of the robustness of the analysis. The Landscape Composition and Structure Hypothesis (H1) cannot be supported if a clear link between the probability of alternation and landscape composition and structure cannot be established, for example if the probability of alternation does not vary across populations, but the composition and structure of landscape is different. The GLM analyses were performed in R 4.0.0 (2020-04-24) with the package glmmTMB (Brooks et al. 2017). Sequential habitat use through time: Complementary Habitats Hypothesis (H2) For this set of analyses, we used habitat alternation as a binomial variable, defined as above. We built two Generalized Additive Mixed Models (GAMMs) with a binomial distribution of residuals, with day-night alternation as the response variable, the cyclic spline smooth of the biweek to account for temporal variation, and a given predictor (Model 1, M1: populationsee also H1; Model 2, M2: age x sex) for each set of models, as a fixed (ordered) factor, or as a factor-specific temporal effect (e.g., population or sex/age classspecific spline of biweek). In all models, we included individual identity as a random effect on the intercept (see full models in Table 1: H2.1 and H2.2). The most parsimonious model was selected based on minimization of the Akaike Information Criterion (Burnham and Anderson 2002). For the population model (M1), we subsequently used the ANODEV procedure (Grosbois et al. 2008) to quantify the proportion of variation in day-night alternation that was accounted for by NDVI (see Table 1, H2.1 for the formula). For visual purposes, we also modelled the annual pattern of NDVI (see Fig. 3) using a GAMM with the exact same model structure as M1 (i.e., population-specific cyclic spline smooth of the biweek, with individual identity as a random effect on the intercept), but with the population-level NDVI value of the sequence as the response variable. All statistical analyses were performed in R 4.0.0 with the packages mgcv (Wood 2017) and visreg (Breheny and Burchett 2017). Results Sequential habitat use through space: landscape composition and structure hypothesis (H1) Visually, the proportion of the four sequential habitat use patterns was not characterised by the populations. Indeed, although the homogeneous closed (c) and homogeneous open (o) sequential habitat use patterns were specific to certain populations (Fig. 2), the day-night alternation (a) was present in all populations, with approximately 40% of all habitat use sequences (38-47%) classified as such, except when closed habitats were widely prevalent (in population IT24, 14%; Fig. 2). This was statistically confirmed by the fact that the probability of alternation did not depend on population (i.e., as a fixed effect; Table 2a, Table S3.2), but only its temporal variation did (i.e., interaction between spline of biweek and population). Random sequential habitat use patterns (u) were rarely observed in any population. In contrast, landscape composition (i.e., proportion of closed habitats) differed across populations (GLMM -ref. level: FR8, IT1: 2.13, SE 0.05, p < 0.001, DE2: 1.31, SE 0.06, p < 0.001, DE15: 0.15 SE 0.05, p < 0.001, IT24 2.26, SE 0.06, p < 0.001, Table 2 Approximate significance of smoothing terms and fixed effects in the selected model (a. cyclic spline of biweek per population, AIC = 3777.7, adj R 2 = 37.1; b. cyclic spline of biweek per modality of the age and sex interaction term, added as a fixed factor, and the spline of the biweek, AIC = 3837.9, adj R 2 = 35.2) To better assess age and sex differences, we provide the general time-dependent model, followed by the age and sex differences from this general model. The latter model gives the same results as the M1 model in Table 1, which was used in the predictions of Fig. 4. Both models include individual identity as a random effect on the intercept to avoid pseudo-replication Kruskal-Wallis = 1021.7; df = 5; p < 0.0001; pairwise post-hoc t-test n.s. for: DE15/IT1). Hence, our results do not support the hypothesis that day-night alternation simply mirrors landscape composition and structure, or that alternation is limited to highly heterogeneous landscapes ( The probability of day-night alternation varied over the year with contrasting seasonal patterns among populations, as shown by the interaction between the cyclic spline smooth of the biweek being in interaction with population retained in all the best models to explain the temporal pattern of alternation ( Fig. 3 In all six populations, the probability of day-night alternation followed a bimodal pattern, with an increase in early spring (between late March and late May, from 6th to 9th biweek), followed by a drop in late spring (between mid-May and mid-July, from 9th to 12th biweek), and a second increase in Fig. 3 Predictions for the probability of habitat alternation between open and closed habitats by roe deer in the six populations over biweekly periods of the year. The smoothed averaged annual NDVI pattern is overlaid as a second y-axis. The sampling units for both curves are biweeks (dots colored by season; blue: winter; green: spring; red: summer; brown: autumn; a larger dot indicates every other fifth biweek; months indicated on x-axis for readability). The background of the panels represents the landscape (open habitat in light green vs. closed habitat in dark green) of the respective study area early autumn (between mid-August and mid-November, from 18th to 20th biweek). However, while a decrease in day-night alternation between mid-January and early-March (2nd and 4th biweek) occurred in all populations, alternation was more frequent in winter (Dec. -May), before falling in summer (May -Dec.) in South-Germany (DE15: Fig. 3, top-central plot). We found that 44% of the variation in day-night alternation across the year was explained by NDVI (Table S3.1; ANODEV). In particular, the first spring peak in day-night alternation generally corresponded to the steepest positive slope of the modelled NDVI curves (in turn varying across populations: best model including the spline of the interaction between biweek and population, Table S3.2; ΔAIC = 7754.4 with the second-best model) i.e., the spring vegetation green up in each study site (Fig. 3, overlaying NDVI curves). Our results, thus, support the prediction that day-night alternation is not a fixed property at the landscape level, but varies in time to track seasonal cycles of vegetation phenology, such as green up or harvesting (Table 1: H2.1, P2.1). We also found that the seasonal pattern in the probability of day-night alternation was significantly different between adult females and males (Fig. 4, left panel and Table 2b; AIC = 3837.9, ΔAIC > 12.0 with all other models, R 2 (adj.) = 0.35; Table S2.1). While the temporal pattern followed a similar general pattern, adult females alternated less than adult males during the summer (Fig. 4, left panel: probability of alternation for females = 0.29 (CI: 0.20-0.42) vs. males = 0.59 (CI: 0.40-0.76) at the end of June-12th biweek). On the contrary, adult females alternated more than adult males during winter i.e., from late December to early March (probability of alternation for females = 0.49 (CI: 0.34-0.63) vs. males = 0.18 (CI: 0.09-0.34) during the 3rd biweek). During other parts of the year, the confidence intervals of the predictions overlapped strongly, indicating little difference in habitat alternation between sexes. Our model also showed that the seasonal pattern in the probability of day-night alternation was not significantly different between female and male juveniles (Fig. 4, right panel; Table 2b; Table S2.1). Specifically, the temporal pattern of alternation in juveniles of both sexes followed the same trend as that of adult females. Overall, we thus found some support for the hypothesis that temporal variation in alternation between habitats is also linked to life-history constraints (Table 1: H2.2, P2.2). Finally, the two-way interaction between age and sex did not feature in the retained model as a fixed factor, indicating that the average overall amplitude in alternation across the year did not vary between factor-levels. In all models, the 'individual' random effect (i.e., including individual identity as a random effect on the Fig. 4 Predictions for the probability of day-night habitat alternation between open and closed habitats by roe deer according to sex and age (left panel: female/male adults; right panel: female/male fawns) over biweekly periods of the year. x-axis as in Fig. 3 intercept) contributed considerably towards explaining the variance (R 2 (adj.) NULL model = 0.31; see Table S2.1), indicating marked inter-individual variability in habitat alternation within a given population. Discussion In spatially heterogeneous environments, wild animals must access various resources with different spatial distributions and, often, asynchronous phenology. Our analysis shows that day-night alternation between open and closed habitats is a prevalent characteristic of roe deer movement tactics across a wide range of landscapes with contrasting composition and spatial arrangement (Dunning et al. 1992; H1 overall not supported). The most likely explanation of this behaviour is that animals use day-night alternation to access food and cover in these resource composites (Padié et al. 2015;Bonnot et al. 2018). The observed seasonal variation in day-night alternation within a given landscape supports this interpretation. Roe deer likely exploit the diversity of resources offered by those habitats at different times of the year, and according to their needs (Mandelik et al. 2012). Our results indirectly support the Complementary Habitats Hypothesis (H2) by showing marked cycles of day-night alternation over the seasons (H2.1), with clear differences between the adult sexes (H2.2). Understanding the complexity of animal resource requirements and consequent habitat use is important as it affects a range of ecosystem processes. The frequent alternation between habitats documented here likely affects transportation of seeds and nutrients between cultivated and more natural habitats (Abbas et al. 2012;Earl and Zollner 2017 for an example on roe deer), as well as spatial dynamics of parasites, including vectors of vector-borne zoonotic pathogens (Carpi et al. 2008;Mysterud et al. 2017;Chastagner et al. 2017). Further, such habitat alternation may influence human-wildlife interactions relevant for management, from harvesting efficiency and forest damage, to road-crossing and risk of deer-vehicle collisions (Hothorn et al. 2015;Passoni et al. 2021). Indeed, in highly anthropogenic European landscapes, the availability of essential resources for free-ranging large herbivores -food and cover -vary both naturally (i.e., due to phenological cycles of vegetation, Pettorelli et al. 2006), and as a consequence of human activities (e.g., agriculture and forest management practices; Lande et al. 2014). In this context, large herbivores must continually adjust their use of habitat to these changes, trading off access to resources and protection from predators and human disturbance (Godvik et al. 2009;Gehr et al. 2017). Several studies have shown that ungulates in temperate environments modulate habitat selection at different spatial (e.g., Boyce et al. 2003, DeCesare et al. 2014) and temporal scales, especially diel and seasonal scales (Dupke et al. 2017;Roberts et al. 2017;Martin et al. 2018). Yet, comparisons across contrasted environmental contexts are rare, limiting the possibility for testing hypotheses on the behavioural responses to landscape-level heterogeneity. In this work, we were able to test two alternative hypotheses on diel habitat alternation by comparing the occurrence of this behaviour spatially (across landscapes) and temporally (across seasons). According to our results, the order in which habitats were used varied over the year and across life history stages, likely in relation to different resource needs and constraints (De Groeve et al. 2020a). Temporal variation in alternation between open and closed habitats is likely due to a complementary functional use of habitats that occur within the home range (Dunning et al. 1992;Mandelik et al. 2012;Couriot et al. 2018). In our study, individuals in most populations alternated between open and closed habitats more frequently in spring, possibly due to earlier green-up in open habitats, with fresh high-quality herbaceous vegetation (Abbas et al. 2011;Dupke et al. 2017), and in the fall, perhaps due to the availability of plants with delayed leaf loss or crop stubble in open habitats. Indeed, we found that about half of the temporal variation in the probability of alternation between open and closed habitats was explained by variation in the population-level NDVI value (Fig. 3). A recent large-scale study on several boreal ungulate species showed that roe deer tended not to move across the landscape to 'surf' the green wave, instead, they maximized the 'greenness' quality of their ranges (Aikens et al. 2020). Alternating between habitats could be a tactic to achieve such maximization locally, accessing complementary resources in different habitat types (see also Peters et al. 2017). In winter, individuals in study areas with substantial forest cover (mainly IT24, DE2; Fig. 3) alternated less, and showed, instead, a consistent and more homogeneous use of closed habitats at a time when forest habitat provides thermal protection, shallower snow (Mysterud et al. 1997;Mysterud and Østbye 2006;Ratikainen et al. 2007;Ossi et al. 2015), and potentially higher food availability, either more abundant vegetation forage or supplementary food provided by hunters (Ewald et al. 2014;Ossi et al. 2017), compared to open habitats. Interestingly, the population in the agricultural landscape of Southern Germany (DE15; Fig. 3) exhibited the opposite pattern to all others, with more pronounced alternation in winter, followed by a consistent and homogeneous use of open habitats in summer. This behaviour could be due to cover-food complementation: roe deer may access open areas at night in winter to consume winter grain (no cover but valuable food resource), while using open habitat both as a food and cover source in summer, when crops are abundant in the fields and can also provide hiding cover (Bonnot et al. 2013). This pattern was not observed in Southern France (FR8), a landscape also consisting of patches of forest within an agricultural matrix. This could be due to different agricultural practices and levels of disturbance, but also because of the use by deer of small cover features in open landscapes such as tree patches and hedgerows that are not defined as forest by the forest cover layer (TCD; https:// land. coper nicus. eu/ user-corner/ techn ical-libra ry/ hrl-forest). In this paper, we used a high resolution (20 m), but static and simplified, classification of open and cover habitats. However, habitat alternation may also occur between different habitat classes (such as pastures, agricultural fields), or between forest units with different forest cover thresholds (in very dense landscapes). This suggests that roe deer compensate their need for cover within a given landscape with different types of 'functional cover' (Mysterud and Østbye 1999). To better evaluate these hypotheses, future studies should look into fine-scale variation in alternation behaviour (see Couriot et al. 2018), while explicitly accounting for the spatio-temporal availability of anthropogenic resources (Ossi et al. 2017;Ranc et al. 2020) and their revisitation patterns (Ranc et al. , 2022. As temporally dynamic remote sensing products indexing the composition and structure of habitats become increasingly available (Pettorelli et al. 2014;Neumann et al. 2015;Oeser et al. 2020), alternation between habitats could be more mechanistically linked to the resources they offer, revealing their functional role at different spatio-temporal scales (Godvik et al. 2009;Mandelik et al. 2012;Couriot et al. 2018). For instance, identification of cover vs. non-cover could be improved by quantifying habitat visibility explicitly through LiDAR surveys (Zong et al. 2022). Among-individual variation within population also seems to be a strong driver of alternation patterns that could be linked to intrinsic individual characteristics like age and sex, but also personality traits (or behavioural types) . We found a link between the temporal pattern of alternation and the sex of individual adult roe deer, with a clear contrast in the degree of day-night alternation between open and closed habitats during summer (more alternation in males) and during the winter (more alternation in females). Mammals have a timing and intensity of resource allocation to reproduction that differs by species, and sex (Williams et al. 2017). For example, the separation of sexes outside of the mating season, termed sexual segregation, is a wide-spread phenomenon among ungulates. Segregation in diet and habitat increase with higher sexual body size dimorphism, due to that body size markedly affect digestion and dietary requirements of ruminants (Mysterud 2000). Here we suggest an alternate form of adaptation to sex-specific needs, namely the diel order in the use of cover and open-habitat resources, that may not emerge as spatial separation. Despite roe deer being one of the few ungulates with a low degree of sexual size dimorphism, there are marked sex-specific differences in behaviour in spring-summer. Males are territorial from March until the rut in July-August (Sempéré et al. 1998), while females give birth in May or early June, allocating heavily to late gestation and early lactation during summer. Alternation between open and closed habitats was twice as high in males than females during the birth season and the period of intensive maternal care. Movement rate has also been shown to decrease during this period (Malagnino et al. 2021), possibly because of spatial constraints imposed by suckling newborn fawns. In contrast, males maintained a higher degree of habitat alternation throughout the summer, likely linked to territory patrolling and defence Malagnino et al. 2021). The fact that these sex-specific patterns were observed in adults, but not fawns, highlights how different habitat use of the sexes can arise from sex-specific schedules of allocation to reproduction. Sex-specific habitat alternation is inversed during winter, such that females alternate around three times more than males between December and March. This marked effect, and particularly the switch compared to the summer pattern might be related to a difference in the perception of risk between males and females linked to hunting practices (Benhaiem et al. 2008). The selective hunting pressure on male roe deer is suddenly reduced after they drop their antlers in November, to become higher for females, instead. This hypothesis should be tested in future research. The survival and reproductive performance of large herbivores depends strongly on the use of, or, as we suggest here, alternation, between habitats that are rich enough to ensure the necessary energy intake but are also safe enough to provide protection against predators . Daily habitat use in large herbivores, in particular, has been linked to circadian activity cycles (Pagon et al. 2013), to the food-cover trade-off (van Beest et al. 2013), including thermal cover (Mysterud and Østbye 1999), and to rumination cycles, where the use of cover is higher during rumination than during feeding bouts (Cederlund 1981). In this study, we showed how investigating daily alternation between habitat types may provide insights into the complementation in resource needs at different spatio-temporal scales and in relation to life-history constraints. Acknowledgments This paper was conceived and written within the EUROMAMMALS/EURODEER collaborative project (paper no. 017 of the EURODEER series; https:// www. eurod eer. org). The co-authors are grateful to all members for their support to the initiative. We thank all the field assistants that contributed to the collection of the data in the field. The EUROMAMMALS spatial database is hosted by Fondazione Edmund Mach. We are grateful to the Copernicus project for the use of free data (European Union, EEA -European Environment Agency). Here we confirm that all conditions are met for free, full and open access to this data set, established by the Copernicus data and information policy Regulation (EU) No 1159/ 2013 of 12 July 2013. Author contributions JDG conducted the research and conceived the study idea and the study design together with FC and NVdW. NM, NCB, AJMH, BG, MH, MK, and FC provided the datasets. The progress of the study was discussed in the context of Eurodeer meetings and working groups with all co-authors. JDG, FC, NR, NM conducted the data analyses. The first draft of the manuscript was written by JDG and FC and critically commented and reviewed by all co-authors. All authors read and approved the final manuscript. tions), and by the help of the Wildlife and Forest Service of the Autonomous Province of Trento and the Hunting Association of Trento Province (ACT). The GPS data collection of the CEFS-INRAE was supported by the "Move-It" ANR grant ANR-16-CE02-0010-02. The GPS data collection of SW-Germany was funded by the federal state of Baden-Wuerttemberg. BG was supported by the Federal Office for the Environment. Data availability Unfiltered classified sequences are available on Zenodo at https:// doi. org/ 10. 5281/ zenodo. 12542 30. Additional associated variables (sex, age, NDVI) were retrieved from the EUROMAMMALS spatial database hosted by the Fondazione Edmund Mach (https:// eurom ammals. org) and can be accessed upon login. The subset of the data and scripts used in the current analyses will be made available on Zenodo upon acceptance. Declarations Ethics Approval Roe deer captures and collaring were compliant to national and international welfare regulations, and approved as follows. SE-Germany (DE2): The research program in the Bavarian Forest is managed by the Administration of the Bavarian Forest National Park. Game captures were conducted in accordance with European and German animal welfare laws. The experiment was designed to minimize animal stress and handling time, and to ensure animal welfare, as defined in the guidelines for the ethical use of animals in research. Animal captures and experimental procedures were approved by the Ethics Committee of the Government of Upper Bavaria and fulfils their ethical requirements for research on wild animals (Reference number 55.2-1-54-2531-82-10); N-Italy (IT1, IT24): animal handling practice, such as captures and collar marking, complied with the Italian laws on animal welfare and has been approved by the Wildlife Committee of the Autonomous Province of Trento, 09/2004S; Switzerland (CH25): The animal capture and handling protocols were authorized by the cantonal veterinary and animal welfare services with permit number BE75/11; SW-France (FR8): prefectural order from the Toulouse Administrative Authority to capture and monitor wild roe deer and agreement no. A31113001 approved by the Departmental Authority of Population Protection; SW-Germany (DE15): The animal capture and handling protocols were authorized by the animal welfare and hunting administra-tion of the federal state of Baden-Wuerttemberg, Germany (RP Freiburg; G-09/53). Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.	2023-02-12T16:09:13.334Z	2023-02-10T00:00:00.000	{ "year": 2023, "sha1": "242eae9a52cc9be0ec973d42c439a27e1015cc93", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10980-023-01594-1.pdf", "oa_status": "HYBRID", "pdf_src": "Springer", "pdf_hash": "e85f39ee5ff36ff40070dd5ad1b5a4a51f5fc40c", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [] }
4472338	pes2o/s2orc	v3-fos-license	Proposal of a Novel Natural Biomaterial, the Scleral Ossicle, for the Development of Vascularized Bone Tissue In Vitro Recovering of significant skeletal defects could be partially abortive due to the perturbations that affect the regenerative process when defects reach a critical size, thus resulting in a non-healed bone. The current standard treatments include allografting, autografting, and other bone implant techniques. However, although they are commonly used in orthopedic surgery, these treatments have some limitations concerning their costs and their side effects such as potential infections or malunions. On this account, the need for suitable constructs to fill the gap in wide fractures is still urgent. As an innovative solution, scleral ossicles (SOs) can be put forward as natural scaffolds for bone repair. SOs are peculiar bony plates forming a ring at the scleral-corneal border of the eyeball of lower vertebrates. In the preliminary phases of the study, these ossicles were structurally and functionally characterized. The morphological characterization was performed by SEM analysis, MicroCT analysis and optical profilometry. Then, UV sterilization was carried out to obtain a clean support, without neither contaminations nor modifications of the bone architecture. Subsequently, the SO biocompatibility was tested in culture with different cell lines, focusing the attention to the differentiation capability of endothelial and osteoblastic cells on the SO surface. The results obtained by the above mentioned analysis strongly suggest that SOs can be used as bio-scaffolds for functionalization processes, useful in regenerative medicine. Introduction Bone tissue regeneration is crucial in the orthopedic field. In the past, the most useful treatments for critical-size bone defects resided in filling the gap with autologous bone graft [1]. Recently, the most advanced tissue engineering strategies are addressed to regenerate the impaired tissue using biologically-functionalized constructs. To this aim, not only the optimal construct should be a filler for the bone gap, but it should also mimic the extracellular matrix properties and provide osteogenic stimuli on native bone to trigger/guide the stimulation of the newly-forming tissue in vivo [2]. Inspired by the concept of bone tissue engineering, researchers have been proposing new constructs with osteogenic capabilities (osteoconductive and/or osteoinductive) [3,4]. An essential element for bone regeneration is also the proper vascularization, since the bone tissue is highly-vascularized and the osteogenesis is always coupled with the preliminary growth of blood vessels [5]. Thereby, in order to fill the bone gap, endothelial cells must be induced to colonize inside the newly-forming tissue, thus providing a vascular network, since osteogenic cells need an appropriate platform upon which to produce the bone matrix. In this study, the broad aim is to characterize a peculiar natural biomaterial, the scleral ossicle (SO), as a support for cell culture, and to test it as a scaffold to heal critical-size bone defects. SOs are dermal bones present in the eyeball of many lower vertebrates with bulging eyes [6,7]; in each chicken eye bulb, 13-14 ossicles form a sclerotic ring located along the scleral-corneal boundary [8]. The functions ascribed to these peculiar ossicles are to support and protect the eyeball against deformation during motion (e.g., flying or swimming) [9,10]. In this preliminary study, the SOs, extracted from the eye bulb of adult chickens, were characterized from a morphological viewpoint and tested for biocompatibility in an in vitro system. The data obtained from this preliminary research pave the way for further experimental investigations focused on the generation of 3D vascularized constructs, usable to generate new bone tissue in critical-size bone defects. The authors are aware that the SO is very small in comparison to a critical lesion (the average size of calvarial critical defects is about 8 mm in rats [11,12] and 5 mm in mice [13]) but, in the near future, our labs will be involved in the development and manufacturing of engineered 3D-matrixes that can include various SOs in order to fill lesions with critical dimensions. Extraction and Preparation of SOs Poultry processing waste, donated by a local butcher, was used as a source of SOs. Each eyeball of adult chickens (50-70 days old) was incised with a scalpel in order to extract the scleral ring. Under a stereomicroscope, the tissue membranes that cover the scleral ring were removed and each of the 13-14 SOs was disjoined. All bony plates were cleaned in PBS (phosphate buffered saline) to eliminate the residues of eye humor liquid, dried in air, and finally sterilized by exposure for 15 min under UV radiations. Size Calculation SO thickness was measured using a caliber with a sensibility of 0.01 mm. Ossicle width and length, as well as average dimensions of lacunae and canaliculi were measured by analyzing SEM images with ImageJ software (v1.8.0_101, National Institutes of Health, Bethesda, MD, USA). Profilometry Profilometric measurements were performed with a non-contact scanning confocal profilometer (scanning white light) CHR150 (STIL S.A.S, Aix en Provence, France) equipped with an optical pen of 350 µm. Each surface, measuring 300 µm × 100 µm, was scanned by 100 parallel tracings (length = 300 µm) with a point distance of 1 µm. Twelve SOs were analyzed and the corresponding R a values were calculated by the image elaboration software Gwyddion (v. 2.49 release 15 August 2017, free-software developed by Nečas et al., Brno, Czech Republic). As a term of comparison, the same approach was applied to a human stapes. . Micro Computed Tomography (µCT) Analysis Micro-CT analysis was performed on three SOs which were scanned with the high-resolution microtomography system Skyscan 1172 (Bruker Micro-CT, Kontich, Belgium) at 60 kV and 170 µA, with a rotation step of 0.3 • and a frame averaging of 3. The nominal resolution (pixel size) was set to 2.5 µm. The images obtained from the acquisition were then reconstructed by the software NRecon (Bruker Micro-CT) with corrections for alignment, depending on acquisition, beam hardening correction, and ring artifact reduction maintaining the relative pixel size = 2.5 µm. Quantitative 3D analyses were carried out with CTAn software (v.1.16.4.1, Bruker Micro-CT). After applying a global threshold, the Volume V in mm 3 , the Surface S in mm 2 and the Structure Thickness St.Th in mm were calculated. Scanning Electron Microscopy and X-ray Microanalysis Samples (four ossicles) were observed with the ESEM Quanta-200 Scanning Electron Microscope (Fei Company, Hillsboro, OR, USA). The samples were fixed for 20 min in 4% paraformaldehyde in PBS (pH 7.4) and dehydrated through a graded series of alcohol (50%, 75%, 95%, 100%) for 10 min each. The specimens were finally dried overnight and coated with gold-palladium (10 nm thickness) using an Emitech K550 sputter coater (Quorum Technologies, South Stour Avenue, Kent, UK). SEM images were collected under low and high vacuum conditions using a large field detector (LFD) and an Everhart Thornley detector (ETD), respectively. Finally, two selected samples were also analyzed via X-ray microprobe (X-ray energy dispersion spectroscopy, X-EDS) using the ESEM software INCA Suite (version 4.07, Oxford Instruments Analytical, Tubney Woods, Abingdon, Oxon, UK) acquiring five spectra for each SO. Confluent cells (80-90%) were sub-cultured routinely at a split ratio of 1:8 three times a week, 1:5 three times a week, and 1:8 two times a week, respectively, for 1G11, MAEC GFP + , and 2T3, after trypsin/EDTA digestion and cultured at 37 • C with 5% CO 2 in a humid atmosphere. Where indicated, cells were differentiated for a well-defined timing in an appropriate culture medium. Cell Differentiation In order to better monitor the endothelial differentiation over time, the positive GFP cell line MAEC was used. For endothelial tube formation assay, MAEC GFP + were stimulated using endothelial growth medium added with recombinant Vascular Endothelial Growth Factor (VEGF) at a concentration of 10 ng/mL. For osteogenic differentiation, a complete medium supplemented with 0.3 mM ascorbic acid and 4 mM β-glycerophosphate was used. The differentiation medium was changed every three days of culture. Intermediate time points were collected at different times depending on test typology. Each differentiation was performed in triplicate. Immunofluorescence Analysis For immunostaining procedure, samples were washed once with PBS followed by fixation in paraformaldehyde 4%/PBS (pH 7.4) for 20 min at room temperature (RT). Fixed specimens were rinsed twice in PBS and then permeabilized in 0.1% Triton X-100/PBS, rinsed twice in PBS, and then saturated in 3% BSA/PBS 1 h at RT. Afterward, samples were incubated with TRITC (tetramethylrhodamine)-conjugated Phalloidin (Millipore 90228, Burlington, MA, USA) diluted 1:500 in 3% BSA/PBS for 1 h at RT, washed three times in PBS, and stained with DAPI (Millipore 90229) for 10 min at RT and washed twice again with PBS. The samples were finally observed with a Nikon Eclipse 80i fluorescent microscope (Nikon Instrument Europe BV, Tripolis 100, Burgerweeshuispad 101, 1076 ER Amsterdam, Amstelveen, The Netherlands). Trypan Blue Viability Assay Cells attached to the Petri dish or to SO surfaces were washed with PBS and detached by trypsin/EDTA (Ethylenediaminetetraacetic acid) solution and then resuspended in a known volume of cell medium and counted with trypan blue in mix solution of 1:1 volume. A fraction of this solution (10 µL) was added to the Bürker chamber for the count of the trypan blue negative cells. Triplicate wells were analyzed for each condition and the standard deviation (SD) was calculated. Paraffin Inclusion and Immunohistochemistry (IHC) SOs on which 2T3 were differentiated were fixed for 1 h with 4% paraformaldehyde in 0.13 M phosphate buffer (pH 7.4), rinsed in PBS, decalcified in EDTA 5% for four days, rinsed in PBS and dehydrated through a graded series of alcohol (50%, 70%, 80%, 95%, 100%) for 20 min each. The last incubation was performed in xylene 5 min three times before the inclusion in paraffin. Samples were sectioned in 5 µm thick sections using a Leica RM2255 microtome (Leica, Wetzlar, Germany). Immunohistochemistry was carried out by deparaffinization of sections for 1 h at 60 • C, incubation of slides in xylene 10 min and a passage through a graded series of alcohol (100%, 95%, 70%, 50%) for 3 min each. Samples were incubated 10 min with a 3% H 2 O 2 solution prepared in water and rinsed twice in PBS; the antigen retrieval was performed arranging slides in a hot (95-100 • C) sodium citrate buffer (trisodium citrate dehydrate 10 mM) for 10 min; rinsed twice in PBS and treated for 1 h in a blocking buffer (10% FBS (Fetal Bovine Serum)/PBS). Slides were incubated for 16 h with anti-sclerostin primary antibody (ab30044 Abcam, Cambridge, UK) at a concentration of 1:100 in a humidified chamber at 4 • C; after rinsing in PBS, samples were incubated in horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at RT in a humidified chamber. Finally DAB (3,3 -diaminobenzidine) substrate solution (1:1 DAB-H 2 O 2 solution) was applied on the sections for 20 min at RT in a humidified chamber. Slides were counterstained by immersion in hematoxylin for 1 min, rinsed in running tap water for 10 min and mounted using DPX mounting solution (06522 Sigma Aldrich, Darmstadt, Germany) after dehydration through a graded series of alcohol (70%, 80%, 95%, 100%) for 5 min each. Statistical Analysis Data were expressed as mean ± SD. Differences between experimental conditions were evaluated by Student's t-test. In all analysis, values of p < 0.1 were considered statistically significant, while values of p > 0.1 were considered highly significant. Morphological Characterization Each SO has two sides, one convex and one concave ( Figure 1a); average measures are 3 mm in width, 4 mm in length, and 150 µm in thickness (Table 1). Statistical Analysis Data were expressed as mean ± SD. Differences between experimental conditions were evaluated by Student's t-test. In all analysis, values of p < 0.1 were considered statistically significant, while values of p > 0.1 were considered highly significant. Morphological Characterization Each SO has two sides, one convex and one concave ( Figure 1a); average measures are 3 mm in width, 4 mm in length, and 150 μm in thickness (Table 1). By means of the optical profilometry, it was possible to obtain a value of the surface roughness, expressed in terms of the R a parameter, which was 1.05 µm (Table 1). For comparative purpose, the same test was repeated on a human stapes, resulting a R a value of 1.44 µm. The reason for this comparison resides in the analogy of these two types of ossicles, since they are both continuously submitted to stereotyped stresses and strains in vivo [10]. SEM qualitative analysis allowed the identification of the ultrastructural features of the SOs by showing the characteristic presence of woven fibers interposed among numerous osteocyte empty lacunae (Figure 1b-d), where emerging canaliculi are visible (Figure 1e). On SEM images the average size of lacunae and canaliculi are also measurable ( Table 1). Volume (V), surface (S), and structure thickness (St.Th) values, calculated using µCT analysis, are reported in Table 1 and expressed as Average ± SD (Table 1). In addition to the morphological observation of the SO surface under SEM, the atoms present in the first 10 µm of its thickness were analyzed using X-ray microprobe analysis. Figure 2 shows the presence of atoms typical of bone tissue, such as calcium, chlorine, phosphorous, and sodium; the percentage of each element is indicated in Table 2. By means of the optical profilometry, it was possible to obtain a value of the surface roughness, expressed in terms of the Ra parameter, which was 1.05 μm (Table 1). For comparative purpose, the same test was repeated on a human stapes, resulting a Ra value of 1.44 μm. The reason for this comparison resides in the analogy of these two types of ossicles, since they are both continuously submitted to stereotyped stresses and strains in vivo [10]. SEM qualitative analysis allowed the identification of the ultrastructural features of the SOs by showing the characteristic presence of woven fibers interposed among numerous osteocyte empty lacunae (Figure 1b-d), where emerging canaliculi are visible (Figure 1e). On SEM images the average size of lacunae and canaliculi are also measurable ( Table 1). Volume (V), surface (S), and structure thickness (St.Th) values, calculated using μCT analysis, are reported in Table 1 and expressed as Average ± SD (Table 1). In addition to the morphological observation of the SO surface under SEM, the atoms present in the first 10 μm of its thickness were analyzed using X-ray microprobe analysis. Figure 2 shows the presence of atoms typical of bone tissue, such as calcium, chlorine, phosphorous, and sodium; the percentage of each element is indicated in Table 2. Biocompatibility Tests In order to test the SO biocompatibility in vitro, the behavior of different cell lines seeded on the SO surface were analyzed, performing the experiments in triplicate. Biocompatibility Tests In order to test the SO biocompatibility in vitro, the behavior of different cell lines seeded on the SO surface were analyzed, performing the experiments in triplicate. Firstly, in order to evaluate the absence of organic soluble compounds released in the culture media following the incubation with SOs, the pH values were measured after 24 and 48 h; the results are shown in Figure 3a. Subsequently, two viability tests were performed on 1G11 and 2T3, namely the trypan blue test and MTT assay. Cells were seeded at a density of 4 × 10 4 cells in a 24-multiwell plate for 24 ad 48 h with and without SOs. Next, detached cells were stained with trypan blue (1:1) and counted in a Bürker chamber. As shown in Figure 3b,c, the number of cells counted after 24 and 48 h on the plastic plate and SO is similar for both 1G11 and 2T3. Thus, cells were seeded in a 12-multiwell plate at a density of 9 × 10 4 cells to perform the MTT assay. Cells were grown for 24 and 48 h, either in the presence or in absence of SOs. At the end of the incubation period, MTT assay was performed (Figure 3d,e): the differences between the two experimental conditions are not statistically significant for either 1G11 or 2T3. Firstly, in order to evaluate the absence of organic soluble compounds released in the culture media following the incubation with SOs, the pH values were measured after 24 and 48 h; the results are shown in Figure 3a. Subsequently, two viability tests were performed on 1G11 and 2T3, namely the trypan blue test and MTT assay. Cells were seeded at a density of 4 × 10 4 cells in a 24-multiwell plate for 24 ad 48 h with and without SOs. Next, detached cells were stained with trypan blue (1:1) and counted in a Bürker chamber. As shown in Figure 3b-c, the number of cells counted after 24 and 48 h on the plastic plate and SO is similar for both 1G11 and 2T3. Thus, cells were seeded in a 12multiwell plate at a density of 9 × 10 4 cells to perform the MTT assay. Cells were grown for 24 and 48 h, either in the presence or in absence of SOs. At the end of the incubation period, MTT assay was performed (Figure 3d-e): the differences between the two experimental conditions are not statistically significant for either 1G11 or 2T3. Analysis of Cells Adhesion and Proliferation After assessing the biocompatibility of the support, adhesion tests were performed on three different time points in order to verify the possible occurrence of morphological and proliferative changes induced by the bone scaffold. To better understand if cells may undergo morphological changes when seeded onto the ossicle surface, immunofluorescence analyses were carried out; it emerged that the morphology of both cells, 1G11 (Figure 4a) and 2T3 (Figure 4b), is similar to that observed for the cells cultured on plastic plates (Figure 4c). Analysis of Cells Adhesion and Proliferation After assessing the biocompatibility of the support, adhesion tests were performed on three different time points in order to verify the possible occurrence of morphological and proliferative changes induced by the bone scaffold. To better understand if cells may undergo morphological changes when seeded onto the ossicle surface, immunofluorescence analyses were carried out; it emerged that the morphology of both cells, 1G11 ( Figure 4a) and 2T3 (Figure 4b), is similar to that observed for the cells cultured on plastic plates (Figure 4c). A proliferation assay was performed on 1G11 and 2T3 plated with 3 × 10 3 cells on a plastic plate and on the SO surface. For each condition, cells were counted by trypan blue staining upon 24 and 48 h of incubation. Figure 4b,d shows that the number of viable 1G11 and 2T3 cells plated on the SO surface and on the plastic plate are similar, suggesting that the cell proliferation rate is not affected by the SO surface. (c) morphology of 2T3 cultured for 24 h; and (d) negative trypan blue 1G11 counted after 24 and 48 h of culture (for the immunofluorescence images, nuclei were stained in blue by DAPI, and the cytoskeletons were stained in red by TRITC-conjugated phalloidin). Data in (b,d) were calculated by three independent experiments and presented as the mean ± SD. * p < 0.1; ** p < 0.05. A proliferation assay was performed on 1G11 and 2T3 plated with 3 × 10 3 cells on a plastic plate and on the SO surface. For each condition, cells were counted by trypan blue staining upon 24 and 48 h of incubation. Figure 4b,d shows that the number of viable 1G11 and 2T3 cells plated on the SO surface and on the plastic plate are similar, suggesting that the cell proliferation rate is not affected by the SO surface. Endothelial Differentiation Endothelial cells were tested for angiogenic potential using the VEGF-containing medium. MAEC GFP + were seeded on the SO surface for 24 h and subsequently stimulated with VEGF at a concentration of 10 ng/mL for 2, 4, and 6 h. The beginning of organization in a network-like manner was observed after 6 h of stimulation. Figure 5a shows a network-like arrangement where cells progressively organize themselves depending on the time. In order to define the cell arrangement more accurately, SEM observations were performed; as clearly visible in Figure 5c, after 6 h of VEGF stimulation, the cells (mimicking the formation process of vascular buds) showing an arrangement in circular cords that resemble the meshes of a network. Endothelial Differentiation Endothelial cells were tested for angiogenic potential using the VEGF-containing medium. MAEC GFP + were seeded on the SO surface for 24 h and subsequently stimulated with VEGF at a concentration of 10 ng/mL for 2, 4, and 6 h. The beginning of organization in a network-like manner was observed after 6 h of stimulation. Figure 5a shows a network-like arrangement where cells progressively organize themselves depending on the time. In order to define the cell arrangement more accurately, SEM observations were performed; as clearly visible in Figure 5c, after 6 h of VEGF stimulation, the cells (mimicking the formation process of vascular buds) showing an arrangement in circular cords that resemble the meshes of a network. Osteogenic Differentiation Osteoblast-like cells were tested for osteogenic potential, using medium containing ascorbic acid and β-glicerophosphate. 2T3 were cultured on the SO surface for 24 h in proliferation medium to allow adhesion. At the end of the period, the medium was changed (day 0) with differentiation medium and the conditioning was carried out for 5 days. Intermediate time points were collected at three and five days and ultrastructural and IHC analyses were performed. Osteocalcin expression increases between day 3 and day 5 of differentiation (Figure 6a), and mineralization starts to occur at day 5 (Figure 6b). Osteogenic Differentiation Osteoblast-like cells were tested for osteogenic potential, using medium containing ascorbic acid and β-glicerophosphate. 2T3 were cultured on the SO surface for 24 h in proliferation medium to allow adhesion. At the end of the period, the medium was changed (day 0) with differentiation medium and the conditioning was carried out for 5 days. Intermediate time points were collected at three and five days and ultrastructural and IHC analyses were performed. Osteocalcin expression increases between day 3 and day 5 of differentiation (Figure 6a), and mineralization starts to occur at day 5 ( Figure 6b). Discussion In the last decade, important advances in the field of bone repairing have already been achieved: from the direct replacement of defective tissues with first-and second-generation materials (such as bioinert, bioactive [16], and biodegradable materials [17]) to the application of third-generation functionalized materials (materials with the ability to signal and stimulate specific cellular activity and behavior), for the induction of bone self-healing [18]. However, challenging questions still remain unaddressed and the research of new materials is open. Scleral ossicles, that are poultry processing waste, were tested as possible innovative natural biomaterials, at no cost, to use in the generation of 3D constructs useful to improve healing of critical-size bone defects [19]. In this study, various preliminary analyses were performed in order to evaluate firstly the best set-up for the SO biocompatibility and, secondly, the adhesion, proliferation, and differentiation of endothelial and osteoblast-like cells. Discussion In the last decade, important advances in the field of bone repairing have already been achieved: from the direct replacement of defective tissues with first-and second-generation materials (such as bioinert, bioactive [16], and biodegradable materials [17]) to the application of third-generation functionalized materials (materials with the ability to signal and stimulate specific cellular activity and behavior), for the induction of bone self-healing [18]. However, challenging questions still remain unaddressed and the research of new materials is open. Scleral ossicles, that are poultry processing waste, were tested as possible innovative natural biomaterials, at no cost, to use in the generation of 3D constructs useful to improve healing of critical-size bone defects [19]. In this study, various preliminary analyses were performed in order to evaluate firstly the best set-up for the SO biocompatibility and, secondly, the adhesion, proliferation, and differentiation of endothelial and osteoblast-like cells. The first point to discuss is the suitability of the materials chosen, whose structural features make them similar to other currently used materials, such as decellularized samples from a bone bank. Many researchers, investigating the evolution, have already characterized and described the scleral ossicles [20][21][22][23][24]. Scleral ossicles, present in the eye of many lower vertebrates with bulging eyes [8,25], such as teleosts, birds, and reptiles [26,27], are devoted to restricted functions (i.e., to support and protect the eyeball against deformation during flying or diving [28]). Thus, the functional meaning of scleral ossicles differs from that of the other skeletal segments, since they are submitted to stereotyped strains for all life, and for this reason, after reaching their final size, they do not need (they even need to avoid) mechanical/metabolic adaptation. This is the reason why, in scleral ossicles (like in human auditory ossicles), the mechanism to make the bone structure stable over time, thus maintaining mechanical resistance, is to avoid bone remodeling by means of massive osteocyte programmed death [9]. Indeed, bone remodeling is a process (in response to mechanical and metabolic demands, changing over time) where firstly mature bone tissue is removed by osteoclasts and later new bone tissue is formed by osteoblasts. The osteocytes (i.e., the bone mechano-sensors) have the function to orchestrate, in response to mechanical and metabolic stimuli, the processes occurring in bone remodeling [29][30][31], which are those to be avoided in scleral ossicles [10]; therefore, scleral ossicles represent interesting acellular natural scaffolds. As already mentioned, not irrelevant is the consideration that SOs are poultry processing wastes obtained from the avian food industry and, therefore, they are available in large amounts without expensive production costs. Thus, in this preliminary study, the SOs extracted from the eye bulb of adult chicken, after characterization at the morphological level, were tested for their biocompatibility in in vitro systems. The data obtained confirm the SO suitability and encourage further investigations focused on the generation of 3D vascularized construct. To check the biocompatibility of the UV-sterilized scaffolds, the pH of the culture medium was analyzed when incubated with and without SOs after 24 and 48 h. The outcomes clearly show that SOs do not release any soluble component that can acidify the pH of the medium. Another point to discuss is that, since 1G11 [32][33][34] and 2T3 [35][36][37] are largely used to mimic, respectively, endothelium development and osteoblast-osteocyte transition in the in vitro experiments, in this study they were used to perform tests of biocompatibility and differentiation. Subsequently, the adhesion, the viability, and the proliferation of both endothelial and osteoblast cells on SOs were analyzed. Trypan blue staining and MTT demonstrated that SOs do not interfere with the normal process of in vitro culture. Finally, to investigate whether SOs affect the differentiation capability of endothelial and osteoblast cells, the tube formation by endothelial cells and the mineralization by osteoblast-like cells were monitored. As far as osteoblast-like cells are concerned, besides the specific markers of osteogenic differentiation, which are clearly expressed particularly in in vivo environments or in consolidated in vitro models, the occurrence of cell growth, osteocalcin expression increase and mineral crystal deposition are commonly accepted as early demonstrations of the differentiation process of cells seeded on the various scaffolds used in regenerative medicine [38][39][40][41][42]. As far as endothelial cells, the formation of cords arranged in network suggests the undertaken path towards endothelial differentiation. The proposal here presented agrees also with some recent suggestions by Mercado-Pagán et al. [43] according to whom "to achieve tissue regeneration and integration, cells and signals need to be incorporated into the constructs in a fashion that can establish stable graft-host tissue interfaces, and facilitate instant or rapid blood perfusion across the constructs". The present paper propounds that the "stable graft-host tissue interface" is likely properly achieved by the vascularized-SO-engineered-construct described here and already presented as a preliminary proposal [44,45]. All the observations here reported suggest that SO scaffolds show promising perspectives in terms of induced pro-angiogenic mechanism and in terms of enhanced osteoblast-like cells and activation of extracellular matrix mineralization processes. Both the capabilities to mimic vascular-like networks and to induce matrix mineralization by cell models are prerequisites to reliably reproduce the bone morphogenetic processes in vitro [46,47]. The differential analyses here performed allow an accurate prediction of the possible behavior of endothelial and osteoblast cells that could be attached to SOs and implanted as 3D-functionalized scaffolds in in vivo critical defects; as a speculative aspect, a possible expected result could be the generation of novel 3D constructs for regenerative medicine. Conclusions These findings suggest that SOs could be used as innovative natural scaffolds: after the extraction from the eye bulb of adult chickens and the functionalization with appropriate cell cultures, they could be useful in the field of the regenerative medicine for the healing of critical-size bone defects. These preliminary results are encouraging in proposing the use of these ossicles as scaffolds on which the ECs are induced to arrange themselves in a microvascular-like network, in turn, conditioning the capability of osteoblast-like cells to produce mineralizing matrix.	2018-04-03T03:02:24.830Z	2017-12-25T00:00:00.000	{ "year": 2017, "sha1": "863c0a8b18a1b35a48a1a0d5172b71e9ac36e1be", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2227-9059/6/1/3/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "863c0a8b18a1b35a48a1a0d5172b71e9ac36e1be", "s2fieldsofstudy": [ "Engineering", "Materials Science", "Medicine" ], "extfieldsofstudy": [ "Materials Science", "Medicine" ] }
262176127	pes2o/s2orc	v3-fos-license	FAKHR AL-D Ī N AL-R Ā Z Ī ’ S CRITIQUE OF AVICENNIAN PSYCHOLOGY AND THE TRANSCENDENTAL INDIVIDUATION OF HUMAN SOULS This article examines the psychological doctrines of the Ash ʿ arite theologian and polymath Fakhr al - D ī n al - R ā z ī (d. 606/1210). The starting point of the discussion is his critical reception of Avicenna ’ s (d. ca. 428/1037) psychological theories. I focus on al - R ā z ī ’ s early works on philosophy, in particular al-Mab āḥ ith al-mashriqiyya and his famous commentary on Avi cenna’s al-Ish ā r ā t wa-l-tanb ī h ā t . I show that R ā z ī affirms a theory of the human soul that is heavily influenced by his predecessor. However, I also show that offers a distinct perspective on fundamental issues. I offer a detailed discussion of two R ā zian psychological doctrines: that human souls are heterogenous, rather than constituting a single species “ rational animal ” pace Avicenna; and that they are caused by celestial entities called Perfect Natures, a theory that is adopted from the practitioners of talismanic magic. The resulting cosmological theory can be referred to as the transcendental individuation of human souls. Background The Mabāḥith is an ideal text to understand the development of al-Rāzī's psychological theory.In the psychology section of this work, al-Rāzī appropriates Avicenna's discussion and arrangement of this science as a template from which he develops his own original positions.1This appropriation is not a dialectical technique; it shows that he assents to Avicenna's general approach in classifying and outlining the science of the soul. 2 Yet the contours of a distinctive Rāzian psychology come to light once we examine the critical sections of al-Rāzī's exposition.His critique of two Avicennian theories-the differentiation of psychic faculties and univocity of human souls in species-leads to the formulation of two alternative doctrines, respectively the monadic nature of the human soul and its essential heterogeneity. 3 The third doctrine, its immateriality, is consistent with the standard Avicennian position.These are the three pillars of a systematic psychological theory that al-Rāzī would maintain and refine in later philosophical works. What is more, we learn from the psychology of the Mabāḥith that motivating al-Rāzī's critique is his skepticism regarding the epistemic procedure by which Avicenna asserts his positions.Two principles are subject to scrutiny: the epistemic claims of abstraction (tajrīd)-that it yields knowledge of a thing's noumenal reality from systematic observation of its phenomenal properties-and the related function of real definitions (alḥadd al-tāmm)-that it is an epistemologically basic insight.As an alternative, al-Razī develops a method of investigation that aims to accurately describe the human soul's nature based on its phenomenal properties, without having to rely on an Avicennian model of noetics or intellection.The resulting theory is a phenomenalist approach in psychology, whose basic components are the three doctrines mentioned above. 4In this paper we focus on al-Rāzī's critique of the univocity of the human soul and how he develops the view that human souls or classes thereof are essentially differentiated.This critique is the most important aspect of al-Rāzī's psychological doctrine, since he lays out the major points of contention with Avicenna's philosophical method.This is the key to understanding other aspects of al-Rāzī's psychology. The Investigation on the Science of the Soul A cursory look at the table of contents of the psychology section of the Mabāḥith shows how al-Rāzī thoroughly adopted the Avicennian division of this science. 5Like in Namaṭ III of al-Ishārāt wa al-Tanbīhāt, he combines the investigation on terrestrial and celestial souls as single line of investigation, which he calls the Science of the Soul (al-ʿilm al-nafs).In the eight chapters of this section al-Rāzī covers the standard Avicennian subjects: the human soul as substance, its various faculties, the external and internal faculties of perception, its immateriality, temporal generation, immanent and transcendental causes, survival after death, the saintly faculties of prophets and holy men (ʿārifīn), etc.He concludes with a relatively short discussion on the nature and movements of celestial souls.As Janssens has shown, al-Rāzī follows closely Avicenna's psychological writings, especially al-Shifāʾ. 6This appropriation was not only at a level of paraphrasing, but was at times close to verbatim restatements of the original discussions. This fidelity is tempered by the presence of a subject heading that is not originally delineated in Avicenna's psychological writings (though implied, for instance, in Chapter V.1 of the psychology of the Shifāʾ).are homogenous or whether they can be essentially differentiated. 8The issues discussed under this subject-heading mark a clear departure from the structure of the psychology section of the text so far, which not only follows the traditional order and wording of the psychology of the Shifāʾ, but also refrains from asserting positive claims.In this subchapter, al-Rāzī criticizes the Avicennian theory that human souls share a common nature as rational animals. 9He then entertains the idea that there are many essential types of human souls.His criticism is deceptively simple: he argues that Avicenna's claims are weak because they are not supported by demonstration (burhān).The following is al-Rāzī's reconstruction of three of these supposedly non-demonstrative arguments: The The first is that human souls are things that have in common (mushtaraka) their being human souls.For if some of them were differentiated from others by means of some essential constituent after their having in common their being human souls, then it would follow that they are composites.[This is] because that through which commonality [occurs] is something distinct than that through which differentiation (al-imtiyāz) [occurs].Now, if [human souls] were composite, they would be corporeal-but this is absurd.The second is that when we examined the types of human souls, we find them to be restricted to two species: perceptive and motive; and of perceptive, there are some that are universal and others particular perceptions.Now we find souls to be mutually equivalent in having these attributes apply truly to them: even though human beings differ in intelligence and ignorance, they all have in common [the ability to intellect] first principles (al-awwaliyyāt).I mean to say that when you call their attention to this, they come to realize, for instance, that which is finite in respect of ignorance.So, if you acknowledge this, the meaning of your statement, 'things that are equivalent (musāwiya) to a single thing are equal to each other (mutasāwiya),' would be inevitably understood, even if it takes some time.[Such is also the case] when the reality of the circle is mentioned to him-that it is by its nature in such a shape.For it is necessary that he conceptualizes this, even though [this conceptualization] comes after giving examples (ḍarb al-amthāl) and the exertion of thought.When this [i.e., the reality of the sphere] is understood, it is possible to understand the first figure of Euclid.The discussions regarding all the intricacies of the sciences (jamīʿ daqāʾiq al-ʿulūm) proceed in this way.Thus, it is possible that all human beings have in common the possibility of knowing objects of knowledge (mushtarikūn fī ṣiḥḥat al-ʿilm bi-maʿlūmāt) and that they have in common the possibility of being shaped by good morals (mushtarikūn fī ṣihhat al-takhalluq bi-kulli al-akhlāq).[For in the latter case], if the irascible person is forced to adopt the habits of patience, his irritability will decrease.The same must also be said about all good morals. [...] The third is that we have proven in the section on knowledge that all separate quiddities must be a thinking subject of the reality of its essence (ʿāqilat an li-ḥaqīqat dhātihā).Now our soul is a separate quiddity and is therefore a thinking subject of the reality of its essence.Moreover, we intellect of our souls nothing but the quiddity that is capable of perception and movement.Therefore, the quiddity of my soul is just this [i.e., that which is capable of perception and movement], which is something that is common between my soul and other souls, given the mentioned proofs in explaining [386] that existence is something common.Therefore, the perfection (tamām) of the quiddity of my soul is predicable of other souls.Moreover, it is impossible that [there exists] for this common thing a differentia that is constitutive of something other than myself, a differentia that [essentially] distinguishes me from something other than me.There is also no need for a distinguishing differentia in that other thing, since a single nature (al-ṭabīʿiyya al-wāhida) will not be simultaneously dependent and independent (muḥtājatan wa-ghaniyyatan maʿan).Thus, it is established that human souls are uniform in (muttafiqa fī) species.This [line of thought] is that which can be taken upon for asserting the unity of the human souls in species.But it is weak. 11 The Rejection on Avicenna Theory The common thread that links these arguments together is the capacity of the human intellect to conceptualize a thing's essential nature.The first and second argument assumes an Avicennian model of intellection, in that it involves the grasping of universals that express the essential attributes of human beings; while the third is an argument that asserts this ability, in that human beings belong to those classes of separable intellects that are capable of self-intellection.In summarizing these arguments, al-Rāzī takes his cue from Chapter V. I of the psychology of the Shifāʾ.In this Chapter, Avicenna investigates the activities and attributes that categorically distinguish human functions from those of animals. 12He then classifies these differentiating activities into two kinds of basic capacities; namely the ability to perceive universals and to act morally.When narrowed down even further, the human being's most specific attribute is the ability 'to conceptualize the universal connotational attributes belonging to the intellect that are abstracted completely of all matter.' 13 This Chapter thus showcases the process of abstraction (tajrīd), through which a philosopher can arrive at universal knowledge of particulars. Later on, in Psychology V.6, Avicenna writes that the role the intellect in abstraction is to conceptualize (taṣawwur) an object of knowledge through the method of division (tafṣīl) and combination (tarkīb). 14What he means is that the intellect structures its 11 Al-Rāzī, Al-Mabāḥith Al-Mashriqiyya Fī ʿIlm Al-ʾIlāhiyyāt Wa-Al-Ṭabīʿiyyāt, II:393-96. 12McGinnis and Reisman, "Ibn Sina," 204-8. 13McGinnis and Reisman,206. 14 McGinnis and Reisman, 241; To be sure, conceptualization, in Avicenna's scientific method, is part of the wider process of abstraction.In this thesis, however, I refer to them separately in order to emphasize the empirical aspects of abstraction and logical aspects of conceptualization, a distinction that is essential in understanding al-Rāzī's critique.For an analysis of this section of the Psychology and a discussion on the role of the cogitative faculty in conceptualization see Peter Adamson, "Non-Discursive Thought in Avicenna's Commentary on the Theology of Aristotle," in Interpreting Avicenna: Science and Philosophy in Medieval Islam: Proceedings of the Second knowledge of a thing's attributes in a logical ordering that identifies its necessary and accidental parts.On one side of this ordering, the process of division narrows down the classes of existents under which an entity can be counted as a member until one arrives at a genus that describes most accurately its basic traits.However, since this procedure looks at clusters of generalizable attributes, these basic traits identify many classes of existing things.The process of combination actualizes these universals into specific entities by distinguishing one type of existent from others of the same class.The act of combining should not be too inclusive so as to become mere abstraction, and the act of dividing should not be too exhaustive as to identify mere individuals.Finally, both must pick out essential and not accidental attributes.If one carries out this procedure properly, he will arrive at the definition of a given substance in terms of its proximate genus and differentia, whose combination (as a species) identifies a set of individuals and a corresponding universal simultaneously-the first in the external world, the second in the mind. 15For Avicenna, the notion that human beings share a common nature, viz.'rational animal,' is thus an epistemologically basic insight, which in turn serves as the first princi ple for the investigation of other aspects of the human soul. 16s a result, al-Rāzī's assessment that Avicenna does not defend the doctrine of the univocity of the human soul through demonstrative proof seems to be accurate.For the Avicennian, however, demonstration in this context is not necessary, since through abstraction the intellect is capable of gaining immediate insight into the very nature of this object. 17We see then how underlying al-Rāzī's critique is a skeptical attitude regarding the epistemic claims of abstraction (tajrīd), that they yield knowledge of a thing's ontological structure. 18Implicitly, al-Rāzī is arguing that knowledge of essences-in this case human souls-belongs to a class of knowledge that requires demonstration. Having identified the method by which Avicenna asserts his theory of the human soul, al-Rāzī focuses again on the issue of abstraction in articulating his critique.Responding to the three Avicennian proofs mentioned above, he writes, regarding the first proof, whether it is necessary to conceive the quiddity of a thing as being composed by constitutive parts (muqawwimāt), viz.genus and differentia.For al-Rāzī, it is possible to conceive the ability for rational thought, which is the differentiating attribute of human beings from animals, as a necessary concomitant (al-lāzima) rather as a constitutive: it is possible that all these things are concomitants (al-lāzima) of the substance of the soul (li-jawhar al-nafs) and are not constitutive (muqawwima) of it.To this extent, souls will differ in the perfection of their quiddity.They have in common the extrinsic necessary concomitants (al-lawāzim al-khārijiyya) in a similar way that the differentiae that are constitutive of the species of a single genus have this genus in common. 19-Rāzī's goal here is to show how Avicenna's theory that human souls are univocal in species results from the latter's commitment to a particular logical procedure he employs during conceptualization, one where human beings are constituted by the genus 'animal' and difference 'rational'. 20gainst the second, al-Rāzī interrogates whether it is possible to infer knowledge of a thing's essence through inductive reasoning.He writes that Avicenna's argument is based on […] a weak induction (istiqrāʾiyya ḍaʿīfa) from two angles.The first is that it is impossible for us to judge that all human beings are receptive to all conceptualizations (qābilan li-jamīʿ al-taṣawwurāt).The second is that it is impossible for us to judge that a soul that we know to have received an attribute (allatī qubūluhā li-ṣifatin) is receptive of all attributes.How is this the case, while it is impossible to have knowledge of all [possible] attributes? 21l-Rāzī interprets Avicenna's arguments in Chapter V.1 of the Psychology as a form of inductive reasoning.Al-Rāzī questions whether it is valid to infer from the observation that many human beings have attribute p that to be a human being is for p to be essentially predicated of human.Unless we can catalog the attributes of all members of a given species, we cannot make essential claims regarding the species itself.Without a complete sample, whatever claims we make will apply to the individuals within that incomplete sample only. 22To be sure, Avicenna would acknowledge that he has not taken 21 Al-Rāzī, Al-Mabāḥith Al-Mashriqiyya Fī ʿIlm Al-ʾIlāhiyyāt Wa-Al-Ṭabīʿiyyāt, II:396.'As for the second proof: it is a weak induction (istiqrāʾiyya ḍaʿīfa) from two angles.The first is that it is impossible for us to judge that all human beings are receptive to all conceptualizations (qābilan li-jamīʿ al-taṣawwurāt).The second is that it is impossible for us to judge that a soul that we know to have received an attribute (allatī qubūluhā li-ṣifatin) is receptive of all attributes.How is this the case, while it is impossible to have knowledge of all [possible] attributes (kayfa wa-ḍabt al-ṣifāt)?' 22 Suhrawardī in the Ḥikmat al-Ishrāq also notably used this line of reasoning when criticizing the Peripatetic theory of definition.Criticizing the idea that a correct definition must include a genus and a the most essential attribute of a given thing, viz. the differentia, he writes: 'Even if someone enumerates the essential he knows, he cannot be sure that he has not overlooked the existence of some other essential.Thus an inquirer or an opponent may challenge the person who constructs a definition, and that person will be unable to reply, 'were there another attribute, I would have known about it,' for there are more attributes that are not evident [...] Thus, if the account of all members of the human species.This is practically impossible.But he would argue that through the process of abstraction the human mind can obtain knowledge of the species' essence, even though the procedure begins with empirical observations that are not exhaustive in the way al-Rāzī envisions. Against the third, al-Rāzī antici pates a syllogistic inference that Avicenna might have constructed to avoid the second objection.This inference consists of two arguments.The first is: all separable quiddities intellect their essence 23 ; the human soul is a separable quiddity 24 ; therefore, it intellects its essence.The second is the argument that, since human souls intellect their essence and this intellection consists in the conception that human beings are capable of rational perception and movement, their essence belongs to the class of things that are capable of rational perception and movement.But al-Rāzī is skeptical that we are capable of knowing in the first place whether all separable quiddities are common in species. 25These kinds of questions belong to a class of knowledge 'for which there is no way [to knowing] (wa-dhālika mimmā lā sabīl ilayh).' 26 Though he does not elaborate further on this claim, the first and second objections above could be marshaled once again with the relevant terms, since in princi ple the epistemic claims involved in conceptualizing the univocity of separable quiddities is the same as that of human souls. These objections show how al-Rāzī's critique reveal a concern he has with fundamental aspects of Avicenna's philosophical method.In both his outline of Avicenna's arguments and his criticism against them, the issue at stake is no less than the epistemic status of abstraction as the means of gaining knowledge of a thing's ontological structure.Al-Rāzī's central concern has less to do with the empirical aspects involved in the process of abstraction, i.e. those that includes careful study of natural phenomena, as with essentialist claims inherent in the Avicennian logical system, through which conceptualization is structured. 27 The Razi's Altenative Theory on the Science of the Soul possibility exists that another essential has not been apprehended, there can be no certainty about the knowledge of the reality of the thing' Shihāb al-Dīn Suhrawardī, Ḥikmat Al-Ishrāq (The Philosophy of Illumination), trans.John Walbridge and Hossein Ziai (Brigham Young University, 1992), vols.10. 28 -11.1-5; For a discussion of this passage in the context of Suhrawardī's critique of Avicenna's epistemology, consult Hossein Ziai, Knowledge and Illumination: A Study of Suhraward's Ḥikmat Al-Ishrāq (Atlanta: Scholar's Press, 1990), vols.120-122. 26 Al-Rāzī, Al-Mabāḥith Al-Mashriqiyya Fī ʿIlm Al-ʾIlāhiyyāt Wa-Al-Ṭabīʿiyyāt, II:396. 27The ontological basis of essential predication is established, for instance, in Book V of the Metaphysics of the Healing, specifically chapters 4-9.Regarding defining actually existing things, Avicenna writes that 'inasmuch as genera and differentiae are natures that arise as [one] nature, as you have known, they are predicated of the thing defined.Indeed, we say: Definition in reality yields the meaning of one nature.For example, if you say, 'rational animal,' from this is realized the meaning of one thing which is the very animal which [in the definition] is that very animal that is rational […] [T]he consideration that necessitates that the definition itself is the thing defined does not render 'rational' and 'anima' the two parts of the definition; but rather, [it renders them] predicates of it in that it itself is [one thing], not that they are two things of a reality that are different from each other and each different from the composite.Rather, by it, in our example, we mean the thing which is itself animal, being that animal whose animality is perfected and realized through rationality'.Ibn Sīnā, The Metaphysics of the Healing, vols.7, 11-12.Having outlined his objections against the univocity of human souls in species, al-Rāzī turns to consider the possible alternative theories.He begins his discussion in his customary detached style, taking distance from the partisan positions he is about to outline.He writes that those 'who maintain the difference of souls in species' begin their arguments with the observation that [they] find in humanity persons who are knowledgeable, ignorant, strong, weak, noble, mean, good, evil, irascible, and indolent.This difference (ikhtilāf) is either 1) on account of the difference of the souls in respect of their substances or 2) because of the difference of the bodily organs, as is the case when it is said that the person whose temperament has more heat has more anger and is more intelligent in comprehension, while the person whose temperament is colder is the opposite. The second division [i.e. the difference is because the difference of the bodily organs] is false from two angles.The first is that we find two individuals who are equivalent in temperament and in external upbringing (al-taʾdībāt al-ḫāriǧiyya) but different in character (al-aḫlāq). 28Similarly, we find two individuals who are equivalent in character, but different in temperament and external upbringing.This refutes this division.Now two things that are identical in temperament and external upbringing could differ in character, since we see two individuals who are very close in temperament to the utmost degree (ġāyat al-muqāraba), but who are different to the utmost degree (ġāyat al-tabāyin) in mercy, severity, generosity, stinginess, chastity and debauchery.This is also not due to the teachings of a teacher or the supervision of parents.Therefore, it may be the case that the combination of all extrinsic factors hastens [to produce] (ittafaqa ijtimāʿ al-asbāb al-ḫāriǧiyya) chastity, yet the person through his natural disposition inclines towards debauchery.Perhaps too the opposite should come to pass.It may also be the case that the parents are vulgar and ruinous to the utmost degree, yet their child is the utmost in nobility and dignity.Such is the argument regarding other moral traits.Therefore, we know that this is due to nothing other than the substance of souls.Now two things that differ in temperament might be equivalent identical in these things [i.e. in these moral traits].The fact is, we see intelligence and cleverness from the heat, coldness, humidity and dryness of the temperament, yet the temperament of a single person could become very hot and then cool down, depending on the make-up of his soul and his primary natural disposition (ʿalá ḫilqihi al-nafsānī waġarīzatihi).If this [intelligence] were on account of the temperament, then his character would have varied. 29r the partisans of differentiation, we can account for these differences only if they are determined either by the essential difference that pertains to the individual soul, or by 28 By this latter term, al-Rāzī is thinking of wider socio-cultural influences on the individual. 29 Al-Rāzī, Al-Mabāḥith Al-Mashriqiyya Fī ʿIlm Al-ʾIlāhiyyāt Wa-Al-Ṭabīʿiyyāt, II:396-97; This passage seems to be a summary of Abū al-Barakāt's discussion Chapter 16 of Al-Baghdādī, Al-Muʿtabar Fī Al-Ḥikma, II:423-327.'On the Original and Acquired States of the Human Soul.'This Chapter begins with a similar observation: 'According to the opinion of those who have direct vision, the human soul possesses different states in their dispositions (istiʿdādihā), perfections (kamālātihā), activities (afʿālihā) and proper affinities (munāsibuhā).You will find among human beings a person who is strong, weak, noble, ignoble, knowledgeable, ignorant, chaste, vile, highminded, lowly, just, unjust, generous, stingy, patient, hot-tempered, frivolous, indolent, compassionate, severe, courageous, cowardly, keen of mind, and slow-witted.Now you will find that some of these states are the result of habits and instruction (al-ʿādāt wa-l-taʿlīm), others the result of the bodily temperaments, others the result of accidents (al-ḥawādith) that occur upon the souls with respect to that which undergoes and that with which it is afflicted (fī mā talqāhu wa-taʿāniyahu), and others the result of primary natures and essential accidents (al-gharāʾiz alawwaliyya wa-l-aʿrāḍ).' the varying determinations of the bodily organs.The preponderant cause is either internal or external to the essence. 30Yet the varying determinations of the bodily organs do not seem account for the differences in character-traits between one individual from another.There is no consistent causal link between the purported cause (i.e. the varying temperaments) and the effects (i.e. the traits of character).Individuals identical in temperament and upbringing often end up different in character.This indicates that external influences-whether bodily or in terms of pedagogy-exert no preponderating cause upon a person's behavior.Furthermore, the partisans of difference observe that the temperaments of human beings change over time and under different physical environments.If heat in the body causes intelligence in character, a person's character should also vary in accordance to the change of the temperaments.But this is not the case.A person's character-traits seem to persist through illness or other states that induce physiological changes. 31e could extend this line of thought to include socio-cultural influences.Though society determines much of our attitude and outlook in life, a great deal of our personality or idiosyncratic faults and virtues is formed and often persists in spite of common or sanctioned practices.Or, at the very least, they cannot be explained solely in terms of those sources.An individual's distinctive character-traits seem irreducibly unique to him; and the totality of these traits combined with the socio-cultural context in which he lives identifies Zayd of Marāgha, for instance, simply as Zayd of Marāgha.The partisans of difference therefore argue that if external determinations exercise no preponderant effect upon a person's behavior, then the contradictory proposition must be true; namely that a certain combination of character-traits in an individual is an irreducible and predetermined state and is therefore the result of intrinsic causes.They identify these intrinsic causes as the 'make-up of [a person's] soul and his primary natural disposition' (khilqihi al-nafsānī wa-gharīzatihi). In the next argument, al-Rāzī makes use of a line of reasoning by Avicenna in the Psychology, but for a different purpose from which it was originally intended: [I]t is known that the soul's capacity in making use (taṣarruf) of the prime matter of this world-viz.the transformation of water into fire, earth into air, and the staff into the snake-[occurs] not on account of the power of its temperament.As such this division [i.e. this second division aforementioned] is false.The Master acknowledges this when he said that the temperament that is predisposed to receiving the soul is that whose existence harmonizes [with the soul] only rarely.This division is false.Thus, it is established that the difference of the souls during these moments [occurs] on account of the difference of their substance only. 32ill reproducing the position of those who argue for the soul's difference in species, al-Rāzī deploys the argument that matter qua matter does not account for the transformations it undergoes.Separate active princi ples must account for the actuality of the material substrate.In Avicenna's cosmology this cause is the Active Intellect.Al-Rāzī in the Maṭālib assigns the same role to each Celestial Intellects, following the doctrine of the Talisman Specialists.In the psychology section Mabāḥith, however, he does not discuss the idea of transcendental causes.Admittedly, his appropriation of Avicenna's argument-attributed to Abū al-Barakāt in the Muʿtabar-is not worked out in detail. 33ut the gist of the idea is clear.The argument has already taken as a premise the idea that human beings have differentiated essences.The separate princi ple that determines this state therefore cannot be some other external cause like the Active Intellect, which in Avicennian metaphysics is responsible for the causation of universal entities, rather than particulars; the individuation (taʿayyun) of human souls occurs on account of the material substrate that receives the emanations. 34We thus see a single-minded effort to ignore the theoretical possibility of the role of the Active Intellect, because of the metaphysical commitments it entails.Al-Rāzī opts instead for what seems to be another causal explanation for substantial change; namely the existence of a human essence that is ontologically separate from its material substrate, essentially differentiated, and is an active princi ple of change.That this proposition presumes the existence of a transcendental cause is not a line of thought he follows up in the present section of the Mabāḥith. Al-Rāzī concludes his presentation of the arguments for the difference of human essences by showing a guarded preference for that position: [i]f we accept the difference of the dispositions of souls, the truth emerges that either the soul of each human being differs in species from other human souls, or it is possible that souls identical in quiddity exist.However, this is amoung that which a proof cannot be given based upon one of two contraries [viz.the two possibilities above remain indeterminate].However, it is impossible to infer (al-istidlāl) the equivalence (tasāwī) of two souls from their equivalence in the totality of actions (tasāwīhimā fī jumlat al-afʿāl).Therefore, you know that inferring the equivalence of something that possesses certain necessary concomitants, given the identity of these necessary concomitants, is false. 35e central premise that al-Rāzī is willing to accept in order to arrive at the correct view of the soul's quiddity is the difference of the soul's native dispositions, the causes of which can be either each soul's essential difference that acts as a determining princi ple, or, in the case that the soul is essentially uniform, accidental determinations, e.g., physiological factors or pedagogy.Though al-Rāzī, in outlining the views of the partisans of difference, initially set up these two propositions here as contradictory he warns us that they are in fact contraries.This means that that the possible explanations that can corroborate the truth of the initial premise-viz.that the soul has native dispositionsremain indeterminate.The third possibility seems to be that there are classes of essentially differentiated human beings rather than individually differentiated human beings. 36As a result, the problematic does not lend itself to the demonstrative procedure whereby we 36 The argument can be translated into the following propositions: 1) all human beings are essentially differentiated or 2) no human being is essentially differentiated.But since these are contraries, they can both be false due to the possibility of a third position, namely that some human beings are essentially differentiated.Abū al-Barakat's outline of these three positions in Al-Baghdādī, Al-Muʿtabar Fī Al-Ḥikma, II:381.establish the truth of one proposition by proving the falsity of its contradictory.In al-Rāzī's assessment, even the partisan of difference falls short of demonstration. What is clear is that for al-Rāzī the second proposition-that the soul is essentially uniform-is based on a weak inference.He does not think that you can infer that two souls are identical in essence simply because they appear to possess identical attributes.Rather, empirical observation seems to suggest an irreducible quality to human traits.Positing the existence of differentiated essences seems a more justifiable inference. 37Despite this assessment, al-Rāzī neither denies this proposition categorically, nor does he affirm either of the two alternatives. We can only speculate why al-Rāzī hesitates from adopting a definitive position in the Mabāḥith.It may be that he has not yet worked out the details of this inci pient theory.What is clear is that in the Mabāḥith he adheres to a consistent philosophical method in both his critique of Avicennian abstraction and defense of Abū al-Barakāt's alternative. Conclusion We have seen how he argues that Avicenna's theory that individual human souls share the same quiddity is weak because it is not supported by demonstration.He does not think that knowledge of a thing's essence can be obtained by the Avicennian procedure of abstraction, which purports to bypass syllogistic reasoning.Since al-Rāzī is skeptical of the reliability of this procedure, we should expect that claims regarding the ontological structure of things must be secured syllogistically by premises that are consist of analytic propositions. 38l-Rāzī attempts to work out this procedure in his defense of Abū al-Barakāt's position that human souls are essentially differentiated.The argument is based on inductive reasoning: systematic empirical observation shows that the character-traits of individual human beings are unchanging, despite the external circumstances of an individual's life, whether these are his physiological make-up or socio-cultural upbringing.The causal story involved in explaining a person's level of intelligence, courage, or generosity tilts in favor of positing the existence of an internal mechanism, encoded indivisibly by the various properties it expresses.True to his method, al-Rāzī does not claim that this argument is demonstrative.All he claims is that it is based on a stronger inference from the available empirical observations.One of the advantages of Avicennian theory of definitions and abstraction is that it allows for a clear distinction between a substance's necessary and accidental attributes.This distinction is crucial in scientific investigation.Al-Rāzī's alternative model collapses this distinction; Zayd's particular mannerism in speaking, writing, or laughing are no longer accidental to his being 'rational animal,' but may form the essential determinants of his being Zayd.However, for al-Rāzī, what we deem as necessary and accidental comes after the primary fact of their concrete expression in the outside world.The fact that a group of related accidents can be generalized into the universal 'rational,' is simply a mental 37 Al-Rāzī, Al-Mabāḥith Al-Mashriqiyya Fī ʿIlm Al-ʾIlāhiyyāt Wa-Al-Ṭabīʿiyyāt, II:397-98. 38Ibrahim, "Freeing Philosophy from Metaphysics: Fakhr Al-Din Al-Razi's Philosophical Approach to the Study of Natural Phenomena," 129 dan 178. ; real definitions that form the premises for Avicennian demonstrative science are non-analytic in that they identify no less the indivisible essence of a thing.On the other hand, Ibrahim identifies al-Rāzī's method as 'analytic,' in that it examines the attributes belonging to a thing's phenomenal unity without judging how the internal structure of the essence necessitates its attributes.construct.Though human beings seem to be capable of rational thought, the primary observation to which we have unmediated access remains that each individual expresses unique variations and intensities of this attribute, e.g., the intelligence of a mathematician or a physicist.The secondary, mental construction is that these variations nonetheless fall under a certain kind of conceptual category; namely the genus of rational activities. 39In other words, 'rational' is equivocally not univocally predicated of human beings. To convey how this approach works out conceptually in a way that can account for accidental and necessary attributes, al-Rāzī argues that it is possible to conceive 'rational' as an external necessary concomitant (al-lawāzim al-khārijiyya). 40This is in opposition to Avicenna's theory where 'rational' is a constitutive (muqawwima) of the quiddity. 41The observation that to be human is to be capable of rational activity does not necessarily lead to the conclusion that rational is predicated of the very essence of humanity as a constitutive part of the substance.Rather it is possible to account for the necessity, or rather the appearance of necessity, as a concomitant of the essence.Thus, he appropriates the Avicennian technical sense of 'concomitant' that is supposed to denote an attribute that is external to the constituted essence but is necessitated by it. 42This allows al-Rāzī to avoid Avicennian ontology, while making the necessary scientific observation that a human soul is capable of rational actions. 7 It deals with the question of whether human souls 4 Bilal Ibrahim developed first the idea of al-Rāzī's philosophical project as phenomenalist.This framework has allowed me to discern a distinctive Rāzian expository method and concerns despite his heavy appropriation of Avicennian categories.For a comprehensive discussion of this phenomenalist framework see Bilal Ibrahim, "Fakhr Al-Dīn Al-Rāzī's Philosophical Approach to the Study of Natural Phenomena" (McGill University, 2012); For a latest restatement, see Bilal Ibrahim, "Faḫ r Ad-Din Ar-Razi, Ibn Al-Haytham and Aristotelian Science: Essentialism versus Phenomenalism in Post-Classical Islamic Thought," Oriens 41 (2013): 379-431. 5Another source is Abū al-Barakāt al-Baghdādī (d.1164-65), whose importance for al-Rāzī cannot be understated.Much of what the latter advances as criticism against Avicenna was preceded by Abū al-Barakāt's original expository method in his famous Kitāb al-muʿtabar fī alḥikma.Comparing the psychology section this work (Muʿtabar II, 302-444) with that of the Mabāḥith shows a direct line of influence.Abū al-Barakāt raised issues that al-Rāzī also takes up against Avicennian psychology: such as the theory of faculty differentiation, the capacity of the intellect to perceive universals as well as particulars.As we shall see this is particularly true of the issue of the human soul's alleged univocity in species and the necessity for a plurality of transcendental causation.In the following we will note which arguments al-Rāzī adapts from Abū al-Barakāt, many of which are duly acknowledged.Shlomo Pines' study on the Muʿtabar remains indispensible and farsighted: Shlomo Pines, "Études Sur Awhad Al-Zamān Abū'l-Barakāt Al-Baghdādī," in The Collected Works of Shlomo Pines Vol.I (Jerusalem and Leiden, 1979); For a discussion of Abū al-Barakāt's critique of Avicennian psychology and his own original positions, consult Shlomo Pines, "La Conception de La Conscience de Soi Chez Avicenne et Chez Abu'l-Barakat Al-Baghdadi," in Archives d'Histoire Doctrinale et Littéraire Du Moyen Age (Librairie Philosophique J. Vrin, 1954). 6Jules Janssens, "Fakhr Al-Dīn Al-Rāzī' on the Soul: A Critical Approach to Avicenna," The Muslim World 102, no.3/4 (2012): 562-79. 7Unless otherwise noted, cited translations of Avicenna's Psychology of the Shifāʾ are based on the volume of translations of Arabic philosophical texts prepared by Jon McGinnis and David 19 Al-Rāzī, Al-Mabāḥith Al-Mashriqiyya Fī ʿIlm Al-ʾIlāhiyyāt Wa-Al-Ṭabīʿiyyāt, II:396. 20For full account of al-Rāzī's critique of Avicenna's notion of quiddity as constituted by these parts, see Ibrahim, "Freeing Philosophy from Metaphysics: Fakhr Al-Din Al-Razi's Philosophical Approach to the Study of Natural Phenomena," 122-69. 33 Al-Baghdādī, Al-Muʿtabar Fī Al-Ḥikma, II:388-93.under the subject heading 'On knowing the actual cause and causes of human souls.'Here he develops the same argument that matter is not a principle of change and that human souls, being incorporeal, require transcendental causes.As we shall see al-Rāzī in the Maṭālib follows Abū al-Barakāt in adopting the doctrine that human souls are caused by the Celestial Intellects. 34McGinnis and Reisman, "Ibn Sina," V. 3, 223-227. 35Al-Rāzī, Al-Mabāḥith Al-Mashriqiyya Fī ʿIlm Al-ʾIlāhiyyāt Wa-Al-Ṭabīʿiyyāt, II:388. Master [i.e.Avicenna] claims that human souls are all one in species, but said no more regarding this claim.He did not [even] provide an aporia (shubhat an ) to validate [his claim], let alone a proof.The author of the Muʿtabar [i.e.Abū al-Barakāt al-Baghdādī] denied the unity [384] [of human souls] in species and devoted a long discussion to it. 10It was acknowledged after this extended [discussion] that no one has [yet] found a demonstrative proof to validate what is sought-after [i.e. that human souls are one in species].We will mention the utmost of what is possible to say regarding this issue.Whoever maintains the unity of the souls in species argues in the following ways.	2023-09-24T16:18:57.170Z	2023-07-30T00:00:00.000	{ "year": 2023, "sha1": "49d925e095c6b7111737c38a2dbed787cc228cbb", "oa_license": "CCBYSA", "oa_url": "https://ejournal.uinbukittinggi.ac.id/index.php/islamt/article/download/6221/pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "db412ed047d7e04536072ae4c666e01c383fce31", "s2fieldsofstudy": [ "Philosophy" ], "extfieldsofstudy": [] }
204575774	pes2o/s2orc	v3-fos-license	Hall Viscosity of Composite Fermions Hall viscosity, also known as the Lorentz shear modulus, has been proposed as a topological property of a quantum Hall fluid. Using a recent formulation of the composite fermion theory on the torus, we evaluate the Hall viscosities for a large number of fractional quantum Hall states at filling factors of the form $\nu=n/(2pn\pm 1)$, where $n$ and $p$ are integers, from the explicit wave functions for these states. The calculated Hall viscosities $\eta^A$ agree with the expression $\eta^A=(\hbar/4) {\cal S}\rho$, where $\rho$ is the density and ${\cal S}=2p\pm n$ is the ``shift'' in the spherical geometry. We discuss the role of modular invariance of the wave functions, of the center-of-mass momentum, and also of the lowest-Landau-level projection. Finally, we show that the Hall viscosity for $\nu={n\over 2pn+1}$ may be derived analytically from the microscopic wave functions, provided that the overall normalization factor satisfies a certain behavior in the thermodynamic limit. This derivation should be applicable to a class of states in the parton construction, which are products of integer quantum Hall states with magnetic fields pointing in the same direction. I. INTRODUCTION The extreme precision of the quantization of the Hall resistance in integer quantum Hall effect [1] led to a topological interpretation in terms of Chern numbers [2]. The fractional quantum Hall effect [3], which emerges as a result of strong interactions, also motivated quantities that have a topological origin. These include the fractional charge for the excitations [4] and the vorticity of composite fermions that reflects through an effective magnetic field [5][6][7]. According to a general topological classification based on Chern-Simons theory [8][9][10], the fractional quantum Hall ground states are characterized not only by the electromagnetic response, e.g. the Hall conductance, but also by geometrical response, which can also be topological. An example of this is the Hall viscosity, the topic of this article. To understand the Hall viscosity as a geometrical response, let us consider applying a small deformation to a fluid. The small deformation is represented by the strain tensor u ij and strain-rate tensoru ij = duij dt , with u ij = (∂ i u j + ∂ j u i )/2 where u i (r) is the displacement at r in the ith direction. The stress tensor σ ij induced by this small deformation is given by: Of the two rank-four tensors that describe the response to the deformation, the first one λ ijkl is called the elastic modulus tensor, which measures the fluid's resistance to elastic deformation. The viscosity tensor η ijkl describes the fluid's resistance to being deformed at a given rate. Because both the stress tensor and strainrate tensor are symmetric under the exchange i ↔ j or k ↔ l, the viscosity tensor also has the symmetry property η ijkl = η jikl = η ijlk . Considering the exchange ij ↔ kl, the viscosity tensor can be further decomposed into a symmetric component η S ijkl = (η ijkl + η klij )/2 and an antisymmetric component η A ijkl = (η ijkl − η klij )/2. The symmetric component is associated with the energy dissipation of the deformation, while the antisymmetric component is nondissipative. As a result of the Onsager relation in thermal physics, the antisymmetric component survives only when the deformation is irreversible, i.e. the time-reversal symmetry is broken, such as in a quantum Hall system. In a two-dimensional isotropic system, the antisymmetric component is given by [11] It is called the Hall viscosity in a quantum Hall system, and is also referred to as the Lorentz shear modulus [12,13]. The Hall viscosity is a local bulk property of a fluid, and should therefore be present (and measurable) independent of the geometry. However, for its theoretical evaluation it has proven particularly fruitful to consider a system with periodic boundary conditions, i.e. a torus. One may see this by noting that by adiabatically changing the torus geometry, one effectively simulates a uniform infinitesimal strain rate in the fluid. Avron, Seiler and Zograf [11] (ASZ) considered the fluid on a torus defined by two sides L 1 and L 2 = L 1 τ in the complex plane. Here τ = τ 1 + iτ 2 is the modular parameter that specifies the geometry of the torus. In what follows, we choose L 1 to be real and τ 2 > 0, in which case the area of the torus is V = L 2 1 τ 2 . The number of flux quanta passing through the torus, N φ , is quantized to be an integer. (Here the flux quantum is defined as φ 0 = h/e.) The filling factor is defined as ν = N/N φ , where N is the number of electrons. It was shown in Ref. 11 that the antisymmetric Hall viscosity η A is related to the Berry curvature F τ1,τ2 in τ space (i.e. the space of torus geometries) as The wave function Ψ is a function of τ 1 , τ 2 and particle coordinates z i = x i + iy i = L 1 θ i1 + L 2 θ i2 , where θ i1 , θ i2 ∈ [0, 1) are called the reduced coordinates. The particles' physical coordinates x i and y i change with the deformation of the geometry but the reduced coordinates do not; the partial derivatives of τ 1 and τ 2 in Eq. 4 are evaluated while keeping the reduced coordinates θ i1 and θ i2 invariant. Ref. 11 further showed that in the lowest Landau level (LLL), the Berry curvature for a single particle in any orbital is 1 , implying that the Hall viscosity for a filled LLL is 4 N V . Note that 1 τ 2 2 dτ 1 ∧ dτ 2 is the volume form in τ space, implying that the Berry curvature is a constant ( 1 4 ) times the factor of volume form. ASZ further showed [11] that the integral of F τ1,τ2 in Eq. 4 in the fundamental domain of the τ plane gives the Hall viscosity, just as the integration of Berry curvature in the 1st Brillouin zone of k space returns the Hall conductivity. (Roughly speaking, the fundamental domain is the subset of all points in the τ plane which are not related by modular transformations [14]. More details on the modular transformations are given in Sec. II and Appendix A.) Lévay [15] showed that the Berry curvature for a single particle in the nth Landau level (with n = 0 for LLL) is given by The Hall viscosity for an integer number of filled Landau levels (LLs) can then be straightforwardly derived by adding the contributions of all single particles (which is valid for a Slater determinant state) to give The calculation of the Hall viscosity becomes more complicated for fractional quantum Hall (FQH) states, which do not have a single Slater determinant form. For the Laughlin state at filling 1 m [4], the Hall viscosity was shown to be η A = m 4 N V by Tokatly and Vignale [12,13], and by Read [16] by exploiting the plasma analogy. Read [16] also showed that for FQH states that can be expressed as conformal-blocks in a conformal field theory, for which a generalized plasma analogy exists, the Hall viscosity satisfies Here S is the so-called "shift", i.e. the offset of flux quanta needed to form a ground state on a sphere: The shift in the spherical geometry is a manifestation of the orbital spin, introduced by Wen and Zee [8] to describe the coupling between the orbital motion and the curvature of space. This topological quantum number is quantized and robust within a topological phase, and can thus distinguish between different topological phases. The Hall viscosity is by extension believed to be a topological quantum number for a given FQH state. In some sense, it is a manifestation of the orbital spin through a transport coefficient. Read and Rezayi [17] discussed the connection between Hall viscosity and the orbital spin i.e. Eq. 7, along with the robustness of Hall viscosity within a topological phase, by noting that the commutator of distinct shear operations is a rotation. The Hall viscosity of the Laughlin and the paired Hall states has also been extracted by Lapa et al. [18,19] from the matrix models for these states. Cho, You and Fradkin [20] showed that Eq. 7 can be derived for the Jain states from the effective Chern-Simons field theory. In this paper, our aim is to obtain the Hall viscosity of the Jain states at filling fractions directly from the explicit microscopic wave functions. According to Eq. 7, we expect An important step in this direction was taken by Fremling, Hansson and Surosa [21], who obtained the Hall viscosity for the 2/5 state using both the exact Coulomb wave functions, and a trial wave function obtained from conformal field theory (CFT) correlation functions. While approaching Eq. 7 with increasing system size, the results showed significant variation with the aspect ratio τ 2 , and also with the number of particles, presumably because the calculations were limited to small systems (10 particles or fewer). The present work has two objectives. First, a recent work [22] has demonstrated how to construct the Jain wave functions on the torus for large systems, which should allow a treatment of many fractions of the form ν = n/(2pn + 1) and also an estimation of the thermodynamic limit. Our explicit numerical evaluation of the Hall viscosity for many FQH states produces values consistent with Eq. 10 in the thermodynamic limit. The second objective for our work is to seek an analytical evaluation of the Hall viscosity for general FQH states, starting from the microscopic wave function. The unprojected wave function for the state at ν = n/(2pn + 1) is given by ZΨ n Ψ 2p 1 , where Ψ n is the normalized wave function of n filled Landau levels and Z is an overall normalization factor. We prove that the Hall viscosity of this wave function is equal to the sum of the Hall viscosities of the individual factors, producing Eq. 10, provided that we make an assumption regarding the behavior of Z in the thermodynamic limit. We further show that the Hall viscosity of the LLL projected wave function Z P LLL Ψ n Ψ 2p 1 (where P LLL is the LLL projection operator) is also given by the sum of the Hall viscosities of the individual factors, provided that we make an assumption regarding the behavior of Z in the thermodynamic limit. Numerical calculations provide support to the assumption regarding the normalization factors. We discuss the modular covariance of the Jain states, which is an important requirement from legitimate wave functions in the torus geometry as well as crucial for the evaluation of the Hall viscosity. We also find that projection of the wave function onto a well defined centerof-mass momentum sector produces a better quantized Hall viscosity for finite N , although in the thermodynamic limit the quantized value is obtained independent of center-of-mass momentum projection. In Appendices B and C we discuss this issue and show that momentum projection is a sub-leading effect in the thermodynamic limit. We briefly mention proposals for measuring the Hall viscosity experimentally. Haldane [23] suggested that it is directly related to the stress caused by an inhomogeneous electric field. Following this direction, Hoyos and Son [24] and Bradlyn, Goldstein and Read [25] have shown the Hall viscosity can be extracted from the wave vector dependent contribution to the Hall conductance. Ref. [26,27] proposed that the Hall viscosity can be extracted in a pipe-flow setup by measurement of the electroscalar potential close to the point contacts where current is injected. A similar observable effect caused by Hall viscosity has also been proposed by Ref. [28] by using hydrodynamic electronic transport in mesoscopic samples under magnetic fields. Such proposed ideas have been applied to graphene, for non-quantizing magnetic fields, and its Hall viscosity was measured by probing local electrostatic potentials in the region where the electron current is non-uniform [29]. It showed good agreement with semi-classical theory of hydrodynamic electron fluids [27,30,31]. The negative magnetoresistance and suppression of Hall resistivity were observed as a manifestation of Hall viscosity. However, these experiments were not in a quantum Hall effect regime. Similar experiment was performed on GaAs quantum wells in the classical regime, and a pronounced, negative Hall viscosity at low magnetic field was found as a signature of Hall viscosity [32]. Starting from hydrodynamics, Ref. [33] proposed another measurement protocol by taking the difference of torques acting on the Hall fluid under two opposite magnetic fields. The plan for the remainder of the article is as follows: In Sec. II we introduce the wave functions we use to evaluate Hall viscosities. These were originally constructed in Ref. 22, but here we use a slightly different (though equivalent) form, using the so-called τ -gauge, which is crucial for our analytical proof. (Both the symmetric and the τ gauges are equally good for Monte Carlo evaluations.) In Sec. III, we prove that the Hall viscosity at ν = n 2pn+1 is given by Eq. 10, provided that we assume that the overall normalization factor of the product wave functions has a certain behavior in the thermodynamic limit; this assumption is tested numerically. In Sec. IV, we numerically evaluate the Hall viscosities at fillings ν = 2/5, 3/7, 2/9, 2/3, 3/5 and 2/7 and find that the thermodynamic limit of the result is consistent with Eq. 10. We also find that both the projected and the unprojected wave functions produce the same Hall viscosity, supporting the notion that it is a topological quantity. II. COMPOSITE FERMIONS ON THE TORUS A. Modular covariance of wave functions As mentioned in the previous section, the torus is topologically equivalent to a periodic lattice spanned by a parallelogram. There are an infinite number of choices of basis vectors that span the same lattice. New basis vectors can be obtained from old basis vectors by the transformation In terms of the modular parameter τ = L 2 /L 1 , this corresponds to the transformation τ → τ = (aτ + b)/(cτ + d). Since changing the signs of all elements does not produce a new transformation, the matrices a b c d form the SL(2, Z)/Z 2 group. This group is spanned by two modular transformations T : τ → τ + 1 and S : τ → −1/τ , which, in the matrix representation, correspond to T = 1 1 0 1 and S = 0 1 −1 0 . We will define the "physical" coordinates (x, y) (also expressed as the complex number z = x + iy) with reference to the Cartesian axis. These coordinates span the whole complex plane, thus do not depend on the choice of the lattice vectors, i.e. remain unaltered upon a modular transformation. The wave functions in general depend on the modular parameter τ , and thus are not necessarily invariant under modular transformations τ → τ + 1 and τ → −1/τ . However, because a modular transformation commutes with the Hamiltonian (which is invariant under a modular transformation), all sets of degenerate states, and by extension their observables, must be closed under modular transformations. That is, a modular transformation may only mix states that are degenerate in energy. Degenerate sets of wave functions that satisfy this property are said to be modular covariant. There are 2pn ± 1 degenerate ground state wave functions at ν = n/(2pn ± 1). The set of exact ground state wave functions is of course closed under modular transformation. However, a given trial wave function does not necessarily satisfy the property of modular covariance. Explicit calculations in Ref. [21] have demonstrated that the Hall viscosity is sensitive to modular covariance of the wave function, and a well defined value is obtained only for wave functions that are modular covariant. It is therefore important to ensure that the wave function we are using to calculate the Hall viscosity is modular co-variant. The modular covariance of the Jain compositefermion (CF) wave functions can be demonstrated quite straightforwardly in the following fashion: [34] First of all, since the wave function Ψ n of n filled LLs is non-degenerate, it is modular invariant, i.e. it remains unaltered under modular transformations. (For one filled LL, this can be proved by construction of single particle coherent wave functions, using Haldane's modified sigma functions, that are modular invariant [34].) It therefore follows that the unprojected product wave function Ψ unproj n 2pn±1 ∼ Ψ n Ψ 2p 1 is also modular invariant. It further follows that LLL projected wave function Ψ n 2pn±1 = P LLL Ψ unproj n 2pn±1 is modular invariant as well. This is easiest to see when P LLL is defined as what is known as the direct projection. This P LLL is explicitly modular invariant, as evident from its definition Here a i and a † i are the LL lowering and raising operators (defined below), with a † i a i measuring the LL index of the ith particle. The situation is more subtle for a different projection, introduced by Pu, Wu and Jain (PWJ) [22] as the torus generalization of the Jain-Kamilla projection [35,36]. The PWJ projection will be used below in our calculation below, as it allows treatment of large systems in the torus geometry. The PWJ projection cannot be represented by an operator, but it is possible to show that the PWJ projected wave functions are also modular covariant; we refer to the work by Fremling [34] for a detailed proof. Interestingly the condition for modular covariance of the wave function is the same as that for the validity of the PWJ projection, namely that the states are proper states, where proper states correspond to configurations for which there are no unoccupied CF-orbitals directly beneath an occupied CF-orbital [22]. (The PWJ projection does not preserve the quasi-periodic boundary conditions for non-proper states.) Let us next address the fact that the ground state is not unique but there are 2pn±1 degenerate ground states at ν = n/(2pn ± 1), which can be chosen as eigenstates of the center of mass momentum M . As shown in the Appendix B, these can be obtained as Ψ where P M is the momentum projection operator. Under a modular transformation, we get [34] where K M,M is a unitary matrix. In other words, the set of 2pn ± 1 degenerate ground states {Ψ } is closed under modular transformation. This proves the modular covariance of the Jain CF wave functions. Finally, let us consider a translationally invariant physical operatorÔ, which must be independent of the center of mass momentum, i.e. must satisfy Ψ (M ) \|Ô\|Ψ (M ) = Oδ M,M . It follows that the expectation value O remains invariant under modular transformation provided that Ψ (M ) transforms according to Eq. 11. The Hall viscosity, given byη We note that the modular covariance of the wave functions obtained in the parton construction [37] also follows as above, because these wave functions are also products of integer quantum Hall states. B. Wave functions in the "τ gauge" A proper gauge choice can be important. The wave functions for general FQH states at ν = n/(2pn ± 1) in Ref. [22] and for the CF Fermi sea in Refs. [38][39][40][41] were first constructed in the symmetric gauge. This was crucial for implementing LLL projection using the PWJ method. Fremling [34] demonstrated that these wave functions are modular covariant, i.e. satisfy Eq. 11. Here we use the Jacobi theta functions with rational characteristics, given by [42] The special function has the periodicity properties: (13) and the only zero of ϑ a b (z\|τ ) inside the principal region lies at z 0 = (a + 1 2 )τ + b + 1 2 . Recently, Haldane has proposed [43] another building block called the "modified Weierstrass sigma function," whose advantage is that it only depends on the lattice Λ = {nL 1 + mL 2 \|n, m ∈ Z}, rather than a specific τ , and is thus explicitly modular covariant. We show in appendix D how to reformulate the Jain wave functions in terms of the modified Weierstrass sigma function. It turns out that for the analytic derivation of the Hall viscosity, it is most useful to use the "τ gauge" and to continue using the Jacobi theta functions as the building blocks. We now describe the details of the τ gauge. The vector potential for the τ -gauge is defined as in physical coordinates, corresponding to a magnetic field B = −Bẑ. The vector potential in the reduced coordinates is given by . In Eq. 4, when deforming τ , one must also move the x and y coordinates to account for the new geometry, which in turn triggers a gauge transformation relating the new coordinates to the old ones. One of the advantages of the τ gauge is that, in terms of reduced coordinates, it is τ independent, and thus no additional gauge transformation is needed when τ is varied. This feature is also shared by the symmetric gauge, but, as will be demonstrated in section Sec. III, the τ gauge is still more advantageous for analytic purposes. The quasi-periodic boundary conditions are defined as: The magnetic translation operator t(ξ) is given by where ∂ j ≡ ∂ ∂θj . The number of flux quanta through the torus is fixed to be an integer We now reformulate the wave functions of Ref. [22] in τ gauge. We write the single-particle orbital in the LLL with periodic boundary conditions according to Eq. 15 as: where the subscript "0" refers to the LLL and the nor- /L 1 is with respect to the physical coordinates. The normalization with respect to the reduced coordinates carries an extra volume factor and is N = (2τ 2 N φ ) is holomorphic in z. This form of single orbital function has been used by Lévay in Ref. [15]. Eq. 18 has its N φ zeros at z m = L 1 τ k From here on we will assume φ 1,2 = 0 unless otherwise specified. (We note that the single particle orbital in Eq. 18 is slightly different from and more compact than the form used in Ref. [22], where it was necessary to assign zeros artificially.) The single particle wave function in the nth LL is given by where we define λ ≡ τ2L1 B (θ 2 + t), and f (k) n (z,z) is given by The wave function of m filled LLs is just the slater determinant of occupied single particle orbitals: where N φ = N/m is the number of flux quanta through the torus, and χ m [f i (z j )] is the determinant of m filled LLs: Here, ni , i.e. the subscript i of f i collectively denotes the quantum numbers {n i , k i }, where n i is the LL index and k i is the momentum quantum number. (Note that the subscript of the many particle wave function Ψ ν or χ n refers to the filling factor, whereas the subscript of the single particle wave function, such as ψ n , refers to the LL index.) We use the convention that the LL index takes values n = 0, 1, · · · , with n = 0 corresponding to the LLL, while the filling factor has m = 1, 2, · · · ; we hope that the meaning is clear from the context. Specifically, the LLL wave function can also be written in a Laughlin form: where N 4 [44]. The general composite fermion wave functions before projection to LLL are written as where we omit the normalization factors. A particularly nice feature of working with the reduced coordinates θ 1 and θ 2 is that the magnetic length never enters into any expression, other than the normalization factor. This means that when wave functions are multiplied, no explicit rescaling of the magnetic length is needed to preserve the boundary conditions. Displaying explicitly the exponential factors, we have where χ n [f i (z j )] is the determinant of n filled LLs at the effective flux quanta N * φ = ±N/n defined in Eq. 24. Note that in Eq. 27, in the exponential factor the terms from different factors combine as τ N * φ + τ 2pN = τ N φ , where we have used that for ν = 1 we have N φ = N . On the other hand, in Eq. 28, which refers to the reverse flux states at ν = n/(2pn − 1), these terms combine as The LLL projection of these wave functions requires LLL projection of products of single particle wave functions of the type ψ n ψ, where ψ is some LLL wave function. Following Ref. 22 and 34, the LLL projection is given by wheref n now is an operator acting on f , which is the holomorphic part of ψ. In general this operator has the formf n =D n f 0 , wherê has two different types of derivatives:∂ z and∂ z . The first,∂ z is understood to act only on f 0 and thus has the property∂ z f 0 f = f∂ z f 0 . The second,∂ z does not act on f 0 at all and can be defined as∂ z f 0 f = f 0∂z f . Written out explicitly, for the n = 1 Landau level we havê which has exactly the same form as Eq. 54 in Ref. [22]. It is now straightforward to apply the modified PWJ projection [35,36] as shown in Ref. [22]. For the ν = n 2pn+1 states, the full LLL wave function is written as: where and ϑ(z ij ) = ϑ n (z i ) by making the replacement ∂/∂z i → 2∂/∂z i for all derivatives acting on J p i . This amounts to changing∂ z → 2∂ z in equation Eq. 30. For the LLL, g For the 1st and 2nd Landau levels, we explicitly havê and projection involving yet higher LLs can be derived analogously. We note that it is crucial for the projection that the composite fermion state be a proper state, i.e. that there are no vacant Λ-level orbitals directly underneath any occupied Λ-level orbital. If this condition is not met, periodic boundary conditions are not necessarily preserved. III. HALL VISCOSITY FROM MICROSCOPIC WAVE FUNCTIONS: ANALYTICAL APPROACH The Hall viscosity is conjectured to be related to the shift in the spherical geometry. For the Jain wave functions, the shift can be derived straightforwardly in two steps. First one can show that the shift for the unprojected wave function Ψ n Ψ 2p 1 is equal to the sum of the shifts of the individual factors. From the result that the shift for Ψ n is S = n, we obtain the result that the shift for the product is S = n + 2p. The second step is to show that the shift is preserved when the wave function is projected into the LLL. This is obviously the case, because the LLL projection in the spherical geometry keeps the system in the same Hilbert space. This suggests a possible route to deriving the Hall viscosity for the FQH states at ν = n/(2pn + 1), following the same two steps. We show in this section that we can accomplish the two steps provided that we assume a certain property for the normalization factor, which is justified by numerical calculation. While the shift on the sphere is an exact property even for finite systems, the Hall viscosity of the FQH states varies with N , becoming equal to the expression in Eq. 10 only in the thermodynamic limit. A. Hall viscosity for the unprojected wave functions The τ gauge is most favorable for an analytical proof due to the following result: Theorem: The normalized integer quantum Hall state Ψ n satisfies, in the τ gauge, the following property: where we treat, formally, τ and τ 2 as the two independent variables (which amounts to replacingτ → τ − 2iτ 2 ). Proof: The single particle orbital in the τ gauge, given by Eq. 22, may be written as where i is the momentum index, n is the LL index, , ζ k (τ ) = e iπN φ k(τ k+2(θ1+θ2τ )) and we define h n,k (τ 2 ) = H n ( τ2L1 B (θ 2 + k)). With some algebra, where we remember that τ 2 L/ B ∝ √ τ 2 and ∂ τ2 N n = 1 4τ2 N n , together with the relation H n (x) = 2nH n−1 (x), we get: To obtain this result, it is important to remember that the area V = τ 2 L 2 1 is held fixed under the variation of . Therefore, when (∂ τ2 ) τ acts on the wave function for n-filled LLs Ψ n , we have The last equality follows because the second term in the square brackets vanishes due to antisymmetrization. Q.E.D. This result can be used to derive the Hall viscosity straightforwardly, as follows. Theorem: If a normalized wave function Ψ satisfies then its Hall viscosity is given by Eq. 7. For the special case of Ψ n , Eq. 36 implies η A = N n 4V . Proof: The Hall viscosity is related to the Berry curvature in Eq. 4, which can be expressed as where the Berry connections are defined as with τ 1 and τ 2 chosen as independent variables. These can be expressed as where A τ = i Ψ\|(∂ τ )τ \|Ψ and Aτ = i Ψ\|(∂τ ) τ \|Ψ , with τ andτ chosen as independent variables. With the help of Eq. 41, it is straightforward to derive the Berry con-nections as: Eq. 44 gives A 1 = − N S 4τ2 and A 2 = 0, which implies finally producing η A = N S 4V . Q.E.D. We next prove the following theorem. provided we assume that the normalization factor Z satisfies the condition Here, we note that Ψ n and Ψ 1 in Eq. 47 are already taken as normalized, and Z is the additional factor needed for the normalization of Ψ unproj n 2pn+1 . P M projects the wave function into a definite momentum sector. Proof: First of all, it is a straightforward exercise to show (see Appendix E) that in τ -gauge we have With Eq. 36 it then follows: where in the last step we have taken the limit N → ∞ and retained the dominant term. The Hall viscosity in Eq. 48 follows according to our previous theorem. Q.E.D. The derivation depends on the assumption given by Eq. 49. The following considerations indicate that Eq. 49 is valid: • We evaluate the τ derivative of Z numerically for ν = 2/5 and show that it satisfies Eq. 49; the results are shown in Appendix F. • In the next section we evaluate the Hall viscosity directly for many unprojected Jain wave functions and find that they satisfy Eq. 48 in the thermodynamic limit. • Eq. 49 is known to be true for the Laughlin states through the use of the plasma analogy [13,16]. However, an analogous plasma analogy for Jain states is likely to be much more complicated and remains an open question. • In Appendix F we explicitly evaluate the contribution of the ∂ ∂τ2 τ ln Z term in Eq. 51 to the Hall viscosity. We find that while it provides a correction for finite N , the correction vanishes with increasing N . A comment regarding an interplay between Hall viscosity and momentum projection is in order. The commutator [(∂ τ2 ) τ , P M ] = 0 does not guarantee that the wave functions with and without momentum projection have the same Hall viscosity, since the normalization factors can be different. In fact, for finite systems, they do have different Hall viscosities, since the decomposition into the various momentum states is itself τ 2 dependent (see e.g. Appendix B). This τ 2 dependence may introduce extra berry phases and thus alter the Hall viscosity. In Appendix C we however show that this change in the viscosity is sub-leading in N and does therefore not contribute in the thermodynamic limit. We finally note that the above considerations apply to wave functions obtained from the parton construction [37], which are products of integer quantum Hall wave functions. We expect that the Hall viscosity of the wave function Ψ ν = m λ=1 Ψ n λ , with ν −1 = m λ=1 n −1 λ , is given by Eq. 7 with S = m λ=1 n λ . We note that the above proof only applies to product states where each factor has magnetic field pointing in the same direction. Generalization to reverse-flux attached states at ν = n/(2pn − 1) or to states of parton theory with negative values of n λ remains an open problem. B. Hall viscosity for the LLL projected wave functions Next we project the wave functions to the LLL. In this case we have: Theorem: The Hall viscosity of the normalized projected wave function is given by Eq. 48, provided the normalization factor Z (Z = Z) satisfies the condition Again, Ψ n and Ψ 1 are taken as normalized, and P LLL denotes LLL projection. Proof: As explained in Sec. II B, the "direct" projection is accomplished by replacement χ n ≡ χ n f in the unprojected wave function. We now prove: With this result, the Hall viscosity of the LLL-projected wave functions in Eq. 52 can be obtained in the same fashion as for the unprojected wave function. Let us first consider (∂ τ2 ) τD n f (i) 0 . We first note that [(∂ τ2 ) τ , ∂ z ] = 1 τ2 1 2 ∂ z + ∂z since z depends on τ 1 and τ 2 . We then get: (55) AsD only acts on LLL wave functions, which consequentially are analytical in z, the last term on the right-hand side of Eq. 55 may be omitted. Thereby, we effectively have: Eq. 54 then follows because: Assuming Eq. 53, we now have, in the limit N → ∞, Q.E.D. The above proof also proceeds analogously for the PWJ projected wave functions. In the PWJ projection in Eg. 32, χ 1 is decomposed into two parts: the center-of-mass part F 1 (Z) (which includes the normalization factor N 1 ) and the Jastrow factors. Noting that (∂ τ2 ) τ χ 1 = N 4τ2 χ 1 and the Jastrow factors J i s are analytical functions of τ , the center-of-mass part must be an eigenfunction of (∂ τ2 ) τ with eigenvalue N 4τ2 . The Jastrow factors can be incorporated intoχ n with the changeD → N φ∂ z − 2 N φ − N φ ∂ z . Since, mutatis mutandis, Eq. 55 still holds for the newD, we have As a final result, Eq. 58 is still valid after PWJ projection. The proof relies on the assumption in Eq. 53. For the LLL projected wave functions it is non-trivial to calculate Z numerically. However, we evaluate in the next section the Hall viscosity for the projected wave functions for several fractions and find that it converges to Eq. 48 in the thermodynamic limit, which suggests that Eq. 53 is valid. In Fig. 1 we show the Hall viscosities for various fillings calculated using Eq. 3. We use both LLL-projected and unprojected wave functions to evaluate Hall viscosities at fillings 2/5, 3/7 and 2/9 for the states with positive flux attachment. For fillings 2/3, 3/5 and 2/7, we only use the unprojected wave functions to evaluate Hall viscosities, since the PWJ projection for reverse flux sates on the torus is much more cumbersome to implement. There are many noteworthy conclusions that can be drawn from our study. • The most important conclusion from our study is that the Hall viscosities converge to the expected value in Eq. 7 for sufficiently large systems. We find that the convergence is achieved already in systems that contain on the order of ten composite fermions. Furthermore, the Hall viscosities have the expected values for both the projected and the unprojected wave functions, supporting the notion that they are a topological property of the state, independent of microscopic details. • The Hall viscosities have significant finite size corrections for small N . As shown in the previous section, a quantized Hall viscosity would be obtained for the unprojected wave functions if the overall normalization factor had satisfied Eq. 49 for arbitrary N . Our numerical results show that this equation is satisfied only in the thermodynamic limit. • The Hall viscosities of the LLL projected and unprojected wave functions are surprisingly close (though not exactly equal), indicating that the LLL projection does not significantly alter the Hall viscosity even for small N . The small deviation between them vanishes as the system size increases, as shown in the inset of Fig. 1. • The convergence of the viscosity is achieved faster for ν = 2/5 than for 3/7 and 2/9. We attribute it to a longer correlation length for the latter two, as measured, for example from the size of the quasiparticle or the quasihole excitations. As the correlation length increases, the system needs to be larger in order for a particle to "forget" that it resides on a finite geometry. Indeed, comparison with earlier works [17,44] shows that the Hall viscosity , where V is the area of torus. The length L1 is quoted in units of lB. When L1 becomes comparable to the magnetic length lB, there are obvious fluctuations in the Hall viscosity. In the thin-torus limit L1/lB → 0, the Hall viscosity approaches the value consistent with S = 1. for 1/3 converges even faster than that for 2/5. • For all of the above calculations we have assumed a square torus. We have also studied the dependence on the corner angle as well as the aspect ratio. Fig. 2 shows the Hall viscosities for different values of τ along the unit circle at τ = e iφ for N = 21 particles. The variations with φ are very small, showing again that the Hall viscosity of the projected and projected wave functions is independent of the geometry of the torus provided that the system is large enough. • To further test its robustness, we scan the Hall viscosity in the τ plane for the 16 particle system at ν = 2/5. The result is shown in Fig. 3. The Hall viscosities at different τ 1 are represented by different labels, and the horizontal axis represents ln(τ 2 ). The Hall viscosities are given by Eq. 10 for a fairly large region around τ = i, but begins to show corrections when τ deviates too far from τ = i. As expected from the modular covariance of the wave functions, the points connected by modular transformations τ → τ +1 (whose symbols have the same color) give exactly the same Hall viscosities. The curve at τ 1 = 0 is symmetric under the transformation τ 2 → 1 τ2 , as also expected from modular covariance. • In Fig. 4, we compute the Hall viscosity in the thin torus limit τ = iτ 2 as τ 2 > 1. We consider the 2/5 Jain state for two systems with N = 10 and N = 20 particles. We plot the Hall viscosity as a function of L 1 rather than τ 2 to highlight the fact that the L 1 -dependence of the viscosity is independent of the number of particles. This independence has been noted earlier in Ref. 44 for the Laughlin state, and also coincides with earlier work on the cylinder geometry [45]. One can understand this size independence by considering that the Hall viscosity should be a local characteristic of the Hall fluid. As such, if one places the fluid on an infinite cylinder or a torus makes little difference, provided that the torus is long enough. Thus the fluid will only be sensitive to the shorter circumference L 1 (the shorter edge) of the torus/cylinder, and deviations of the viscosity is expected to occur only when L 1 is comparable to or smaller than the correlation length of the fluid. It is also noteworthy that in the thin-torus limit, the Hall viscosity is seen to approach S = 1. This is expected because in the thin torus limit, the 2/5 wave function reduces to a single Slater determinant, and all LLL slater determinants have the same viscosity, η A = 4 N V . This is true of all FQH states that reduce to a single Slater determinant in the thin torus limit. • We have noted above that our results are likely also applicable to the wave functions in the parton construction, which are products of integer quantum Hall states. It is possible to further generalize the class of wave functions that produce the quantized Hall viscosity. We consider the wave function Ψ 1 \|Ψ 1 \| α as a trial wave function for ν = 1. This wave function has the same topological structure as Ψ 1 , occurs at the same shift in the spherical geometry, and may represent the physics of LL mixing due to interactions. We find that the Hall viscosity approaches the value η A = 4 N V in the thermodynamic limit independent of the value of α, as shown in Fig. 5. V. CONCLUSION In this work, we have evaluated the Hall viscosities of the Jain states at many filling factors of the form ν = n/(2pn ± 1), specifically for ν = 2/5, 3/7, 2/9, 2/3, 3/5 and 2/7. For this purpose we use LLL wave functions constructed in Ref. [22], but appropriately re-expressed in τ -gauge. The numerical results agree with Eq. 7 proposed by Read [16], which relates the Hall viscosity to the orbital spin or the shift in the spherical geometry. We also find that the Hall viscosities for the unprojected and projected wave functions are the same in the thermodynamic limit, supporting the notion that the Hall viscosity is a topological property of a FQH state. Additionally, the product form of our wave functions suggests a possible analytical derivation for the Hall viscosity of a large class of FQH states, analogous to the derivation of the shift in the spherical geometry. We show that the Hall viscosity of the unprojected and projected wave functions can be derived analytically provided we make an assumption regarding the thermodynamic behavior of an overall normalization factor Z. This assumption has been confirmed by detailed computer calculations. In this appendix, we prove that the Hall viscosity defined in Eq. 3 is modular invariant provided that Eq. 11 is satisfied. The proof for the modular transformation τ → τ + 1 is trivial, since the wave function is invariant [34]. Below we show the statement is also true for τ → − 1 τ . In Ref. [34] it is shown that under this transformation, Eq. 11 holds with K M,M = 1 √ 2pn±1 e i2π nM M 2pn±1 . We then note Hall viscosity is independent of then center-of-mass momentum, since: ACKNOWLEDGMENTS From the first line to second line, we used the factor that t CM ( L2 N φ ) is independent of τ 1 and τ 2 , as shown in Appendix E. Similarly, because t CM ( L1 N φ ) is also independent of τ 1 and τ 2 it is straightforward to see . Under the transformation τ → − 1 τ , we have τ 1 = − τ1 \|τ \| 2 and τ 2 = τ2 \|τ \| 2 . The Hall viscosity in Eq. 3 thus transforms like Given that it follows that This shows explicitly that the Hall viscosity is modular invariant for CF wave functions. Appendix B: The momentum components of the CF state It is noted in the main text that Ψ n 2pn+1 is not a momentum eigenstate, but a superposition of 2pn + 1 different momentum eigenstates, i.e. Ψ n are momentum eigenstates. It turns out that the coefficients α m have dependence on both τ and τ 2 , which, in turn, gives rise to an extra berry curvature. In this appendix and the next, using the CF formulation of the Laughlin state as an explicit example, we show how this extra Berry curvature comes about and also that it is sub-leading in the thermodynamic limit. We thus take as starting point the two formulations of the Laughlin state at ν = 1/q: The first is the momentum projected state where the center of mass is 2 . The second state version is obtained by multiplying q copies of the ν = 1 wave funcitons, Φ 1/q (z) = [Ψ 1 (z)] q . The quotient between these two functions is Thus, the only difference lies in the center of mass representations. For simplicity we will assume that N is odd such that (N − 1)/2 is an integer (which avoids carrying around cumbersome factors of one half). We may now ex- where we seek α p . We may extract α p by expanding the two wave functions mode by mode. After some algebra one finds The sums are over the k 1 , . . . , k q ∈ Z, constrained such that j k j = K. We further use the abbreviationk j = k j − K q , to show explicitly that α K = α K+q , and that α K = α −K . From Ref. [44] we may identify α K = Z is defined recursively with Z (1) t = 1. The properly normalized α s , such that s \|α s \| 2 = 1, is thus α s = The absolute value of α s in shown in Fig. 6, for τ = iτ 2 and in the entire τ -plane, for q = 2, 3. From this we see that in the thin torus limit, the CF state is almost in a momentum eigenstate, since \|α (q) s \| 2 = δ s,0 . However, in the opposite thin torus limit (τ 2 → 0), the state is in an equal superposition, with \|α In this Appendix, we compute the correction to Hall viscosity due to mixing of different momentum eigenstates, and show that it vanishes in the thermodynamic limit. We write the wave function at ν = Ψ = s α s Ψ (s) (in this section we omit the filling factor as subscript, and use s for momentum index), where s \|α s \| 2 = 1, and Ψ (s) are the momentum projected components. We further assume that Eq. 51 holds for the momentum projected states, namely that . Computing the Berry connection for the Fourier expanded states give: where the last term is simply iP 2τ2 since ∂τ preserves the momentum label. Expanding the the bra of the first term yields Similarly, directly follows. The berry curvature is thus We see here that the first term in Eq. C4 is of order one, while the second term is proportional to N . This means that in the thermodynamic limit N → ∞, the first term is sub-leading and can be dropped. This is why, for large systems, it does not really matter whether the state is a momentum eigenstate or not. The wave functions of integer filled LLs are just the slater determinants of occupied single particle orbitals: analogous to Eq. 23 in the main text. The Laughlin form for the filled LLL is: with Again, notice the τ dependence on the zeroes of the wave function. The wave functions for ν = n/(2pn ± 1) before projection to LLL are written as: where χ n [f i (z j )] has the same form as in Eq. 24. Just as in the main text there is an unusual factor in Eq. D16, originating from the relation N φ = N * φ + 2pN = −\|N * φ \| + 2pN for ν = n 2pn−1 . The LLL projection of these wave functions requires LLL projection of product of single particle wave functions of the type ψ n ψ 0 . Following Ref. [22], the LLL projection is given by where e.g. which has exactly the same form as Eq. 54 in Ref. [22]. It is now straightforward to apply the PWJ projection [35,36] as shown in Ref. [22]. For the Jain ν = n 2pn+1 states, the LLL wave function is: is defined as in Eq. 33. Theĝ and g (n) Appendix E: Proof of Eq. 50 In this appendix we prove Eq. 50. The momentum projection operator is given by [22] where q = 2pn+1 for the CF states. It projects a Ψ n 2pn+1 to eigenstates of t CM L1 N φ : P M commutes with (∂ τ2 ) τ because (∂ τ2 ) τ , t CM L1 N φ ) = 0. This can be seen by noting that in the current gauge we have These are explicitly independent of τ 1 and τ 2 , and hence commute with ∂ τ2 . Therefore, Eq. 50 does hold. Here we emphasize that P M is not modular invariant, as noted in the main text, even though there is no τ in its definition. The reason is that the reduced coordinates used in the definition of P M do change under modular transformations. For instance, under τ → − 1 τ , to keep the physical coordinates (x, y) fixed, the reduced coordinates transform as (θ 1 , θ 2 ) → (−θ 2 , θ 1 ). Appendix F: Direct evaluation of the correction term Our analytical derivation of the Hall viscosity of the unprojected Jain wave functions is based on the assumption that the overall normalization factor satisfies Eq. 49. For a numerical test, we first write (F1) We numerically evaluate the two terms on the right hand side for ν = 2/5. The results, displayed in Fig. 7 We can explicitly evaluate the contribution of this term to Hall viscosity. Without assuming the condition in Eq. 49, Eq. 51 becomes: Following the calculation in Sec. III, we get: and Finally, the Berry curvature is: In Fig. 8, we show the evaluation of ∆ for wave functions both before and after momentum projection. (They have different overall normalization factors Z.) We also show, as a sanity check, the actual difference 4η n −4 using the results from Fig. 1. As shown in Fig. 8, the correction term converges to zero quickly with increasing system sizes, especially for the normalization factor after momentum projection. The correction ∆ also agree with the difference between 4η n which is obtained in Fig. 1 and the quantized shift S = 4 for ν = 2 5 .	2019-10-15T02:48:48.000Z	2019-10-15T00:00:00.000	{ "year": 2020, "sha1": "1d5c131fc7812d1258dab2f7b46c1d4112289573", "oa_license": "CCBY", "oa_url": "http://link.aps.org/pdf/10.1103/PhysRevResearch.2.013139", "oa_status": "GOLD", "pdf_src": "Arxiv", "pdf_hash": "2bfdcd86dbf639716143e6e887a544cf0af98890", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics", "Mathematics" ] }
259239233	pes2o/s2orc	v3-fos-license	Investigation of energetic ion losses induced by long-lived saturated internal mode with energetic particle diagnostics in the HL-2A tokamak Several sets of energetic particle diagnostics, including a set of neutron flux monitoring systems, a solid-state neutral particle analyzer and a fast ion loss probe (FILP), have been used to investigate the energetic ion losses induced by the long-lived saturated internal mode (LLM) in the HL-2A tokamak. Clear experimental evidence for different levels of energetic ion losses induced by LLM, sawtooth and minor disruption has been observed. A numerical calculation for the evolution of neutron emissions was carried out with the FBURN code, and it shows that the neutron emission drop rate linearly increases with the LLM amplitude and no threshold perturbation amplitude exists, illustrating that the loss mechanism for LLM induced energetic ion loss is dominantly convective. In addition, measurement results of the FILP demonstrate that LLM tends to expel energetic ions with relatively low energy ( E<27 keV) and high pitch angle ( θ>60∘ ), and can suppress the prompt loss of energetic ions with high energy and low pitch angle to a certain degree. Furthermore, the physical process for LLM induced energetic ion loss can be explained by orbit calculations, which show that LLM induced lost energetic ions will transport from center to peripheral region first, and then get lost out of plasma. The experimental observations are successfully reproduced by calculations using the ORBIT code combined with both the NUBEAM code and the MARS-K code. The paper clearly describes the whole physical process of LLM induced energetic ion loss for the first time in the HL-2A tokamak. Introduction One of the essential preconditions for achieving self-sustained D-T burning plasma is to confine the energetic ions in fusion plasma long enough to heat the fuel ions [1]. Among the energetic fusion ions in D-T burning plasma, fusion born α particles are of particular interest because they play a vital role in plasma ignition. Actually, energetic ions via auxiliary heating are also important in D-D fusion plasma and allow us to study energetic particle interactions [2]. Hence, it is the inexorable requirement of fusion reactors to achieve excellent confinement quality for energetic ions. Energetic ion losses deteriorate the confinement quality, leading to the reduction of neutral beam injection (NBI) current drive and heating efficiency [3], which is not conducive to the enhancement of the energy gain factor. Furthermore, intense and localized energetic ion loss may damage the plasma facing components, and then cause impurity pollution to the plasma [4]. A small fraction of energetic ion loss in large magnetic fusion devices including ITER might be intolerable [5]. Understanding the behavior of energetic ion loss in fusion plasma is therefore still a crucial issue for fusion research. Numerous efforts have been made to study the energetic ion loss induced by different magnetohydrodynamic (MHD) instabilities, like toroidal Alfvén eigenmodes (TAEs) [6,7], energetic particle modes [8,9], fishbones [10,11], sawteeth [12] and tearing mode [13]. Investigations of long-lived saturated internal modes (LLMs) observed on many fusion devices [14][15][16][17] demonstrate that LLM can eject energetic ions from the plasma core region. The LLM induced energetic ion losses have been observed in MAST with a neutron emission profile monitor [18] and a fast ion loss detector [19]. However, the physical mechanism and process for energetic ion loss induced by LLM are still not sufficiently clear. It should be noted that LLM is a key MHD instability that plays an important role in the advanced scenarios for ITER operation [20], because sustained elevated central safety factor q can be achieved from the action of LLM [21]. Among the advanced scenarios, the hybrid scenario is believed to be a potentially interesting operation scenario for future fusion devices, and many devices such as MAST [14], JET [22], JT-60U [23], DIII-D [24], ASDEX-U [25], EAST [26] and HL-2A [27] have carried out experiments based on the hybrid scenario. Thus, it is of great scientific importance and practical value to investigate the behavior of energetic ion loss induced by LLM. The present work aims to investigate the physical mechanism and process of energetic ion loss induced by LLM. The experimental setups are described in section 2, in which energetic particle diagnostics including neutron flux measurement (NFM) systems, neutral particle analyzer (NPA) and fast ion loss probe (FILP) are introduced. The experimental results are shown in section 3. Physical mechanism and process analyses are depicted in sections 4 and 5, respectively. Finally, conclusions are drawn in section 6. Experimental setups The HL-2A tokamak [28] is a medium-size conventional tokamak with a closed divertor. It has a major radius of 1.65 m, and a minor radius of 0.4 m. The plasma current I p ranges from 150 to 430 kA, and its orientation is counterclockwise from the top view of HL-2A. The toroidal magnetic field along the magnetic axis B t , which is mainly created with sixteen toroidal magnetic field coils, ranges from 1.3 to 2.7 T, and is oriented to be clockwise from the top view of HL-2A. The divertor of HL-2A, which is characterized with two closed divertor chambers, enables that it can be operated in either of singlenull (SN) and double-null divertor configurations theoretically. However, only lower SN divertor configuration is considered in the present paper. The NBI system on HL-2A consists of two neutral beam lines, and each beam line is equipped with four positive ion sources. Both beam lines share the same parameters. Each beam line has a maximum injection power of 2 MW. The neutral beam particles have three energy components, i.e. full injection energy E, E/2, and E/3. Its full injection energy E has a typical value of 40 keV. The neutral beam is injected into the plasma tangentially with a tangency radius of 1.4 m and an angle of 32 • with respect to the plasma current along the magnetic axis (see figure 1(a)). Three kind of energetic particle diagnostics are used to study the behavior of energetic ion loss induced by MHD instabilities, i.e. NFM systems, NPA and FILP. The layout of the energetic particle diagnostics is shown in figure 1(a). There are two different NFM systems: the one is the ZnS detector, and the other is the 235 U fission chamber. The ZnS detector is a newly developed NFM system with an optimum temporal resolution of 1 µs [29,30]. It is ∼5 m away from the center of HL-2A. The ZnS detector is sensitive to fast neutrons (E > 0.7 MeV) rather than slow neutrons and γ/X rays. The 235 U fission chamber is a conventional NFM system with a temporal resolution of 1 ms, and it is sensitive to thermal neutrons rather than fast neutrons and γ/X rays. It is ∼11 m away from the center of HL-2A. Since the fission chamber was calibrated with a 252 Cf neutron source, it can be used as a neutron yield measurement system [31]. The NPA system in the present paper is a kind of solidstate NPA [32]. It is located at the low field side in the vacuum chamber and about 15 cm lower than the middle plane. It is equipped with a collimator facing toward the high field side through the plasma core region (see figure 1(b)). An AXUV Figure 1. Layout of the NBI system and the energetic particle diagnostics in the planform of HL-2A (a). Plasma current Ip is oriented to be counterclockwise, while toroidal magnetic field Bt to be clockwise. Poloidal view of the NPA system and its sightline, as well as the location of the FILP (b). silicon photodiode coated with a 100 nm thick tungsten film makes the NPA suitable for measurements of neutral particles with energy over 26 keV generated in the plasma core region. Its sampling rate is 1 MHz. However, the NPA system in the present paper does not have an energy resolution. The FILP [33] is located at the low field side on the middle plane in the vacuum chamber. Its detector head is ∼3 cm away from the last closed magnetic flux surface of the HL-2A plasma (see figure 1(b)). The FILP is used to detect the Larmor radius ρ i of the lost energetic ion as well as its pitch angle θ, from which the energy E i can be derived: and v are respectively the mass of the lost ion, the local magnetic field at the FILP head, the electrical charge of the lost ion, its velocity parallel to the local magnetic field, and its velocity. The FILP has a temporal resolution of 2 ms. Experimental results LLM is observed frequently in the HL-2A plasma during NBI with sufficiently high injection power. Figure 2 shows the time traces of some main parameters of an NBI plasma with strong LLMs in which some sawteeth are inserted. An NBI pulse with ∼0.5 MW in total is injected into the HL-2A plasma from 900 to 1900 ms. The typical value of the plasma current I p , the central line-averaged electron density n e and the central electron temperature T e is about 160 kA, 1.8 × 10 19 m −3 , and 2.2 keV, respectively. The sampling rates of n e and T e are respectively 1 MHz and 30 Hz. A minor disruption occurs at 978 ms, and it has a significant effect on I p , n e and T e . It can be seen from figures 2(c) and (d) that, after the minor disruption, the first LLM appears at ∼1100 ms and vanishes at ∼1220 ms, and then several LLMs appears during 1300 ms and 1900 ms together with several sawteeth. This shot makes it possible to investigate the energetic ion losses induced by LLM and sawtooth. In order to characterize the LLM, Bayesian based soft x-ray (SXR) tomography reconstruction [34] for the mode structure has been carried out. The mode structure of LLM at about 1111 ms is shown in figure 3. Both m/n = 1/1 mode and m/n = 2/2 mode exist simultaneously in the plasma core region, characterizing the existence of LLM [14]. The m/n = 1/1 mode with a frequency of ∼10 kHz and the m/n = 2/2 mode with a frequency of ∼20 kHz can also be observed in the frequency spectrogram of the SXR signal shown in figure 2(d). The normalized minor radius r/a of the q ∼1 surface is estimated to be ∼0.25. Since neutral particles exist densely in the plasma peripheral region, energetic ions lost from the plasma core region are likely to become energetic neutral particles via chargeexchange recombination interactions in the plasma peripheral region. Hence, it is possible to study the energetic ion losses in the plasma core region with the NPA system [35,36]. Figure 4 represents the flux of neutral particles with energy over 26 keV as well as its frequency spectrogram. It can be found that there exist several kinds of MHD instabilities, including mainly minor disruption, LLM and sawtooth. The neutral particle flux responds to minor disruption, LLM and sawtooth, reflecting that all the three MHD instabilities can induce the energetic ion losses. The emission rate of fusion neutrons is positively associated with the confinement property of the energetic ion population, i.e. the energetic ion loss can lead to the decrease in neutron yield. To investigate the energetic ion loss behavior, neutron diagnostics can play an important role. The LLM induced energetic ion losses have been observed in different fusion devices indeed by using neutron diagnostics [18,21]. Figure 5 shows the temporal evolution of neutron emissions measured with both the 235 U fission chamber and the ZnS detector. Both curves respectively measured with the 235 U fission chamber and the ZnS detector have the same variation trend, and the deviation between the two curves may attribute to different detection positions (see figure 1). The minor disruption at 978 ms cause a sudden drop in neutron emission. After the minor disruption, a long-lived mode appears. It can be found that the LLM during 1090 ms and 1210 ms induces a slight drop first and then a slight increase in neutron emission, and the drop gets more prominent when sawtooth occurs during 1300 ms and 1900 ms. The slight increase in neutron emission during 1150 ms and 1300 ms might benefit from the reduction in magnetic perturbation amplitude. A prominent continuous decrease in neutron emission is observed during LLM phase inserted with several sawtooth crashes, which was also observed in JET [37]. Since the decrease in neutron emissions may be caused not only by the energetic ion loss but also by the decline in main parameters of the bulk plasma, it is necessary to introduce a precise calculation for the evolution of neutron emissions, which will be described in detail in the next section. Lost energetic ions can be directly detected by FILPs which have been developed in many fusion devices [38][39][40][41]. The energetic ion losses induced by various MHD instabilities have been observed in the HL-2A tokamak by using a FILP [42]. Figure 6 presents the energetic ion losses induced by LLM and/or sawtooth in terms of distribution map measured with the FILP. An NBI pulse with ∼1 MW in total is injected into the HL-2A plasma during the entire period of the figure. Both LLM and sawtooth can be observed from the SXR signal and its frequency spectrogram. Four distribution maps in figure 6 depict the energetic ion losses related to prompt loss only (t 1 and t 4 ), both LLM induced and prompt loss (t 2 ), and both sawtooth induced and prompt loss (t 3 ), respectively. Notably, all the distribution maps during the LLM phase are similar to each other. Lost ions are mainly concentrated in three zones in each distribution map, i.e. zone α, β, and γ. Zone α is the region with relatively high energy (E ∼ 40 keV) and low pitch angle (θ ∼ 57 • ), zone β is the region with relatively low energy (E ∼ 20 keV) and high pitch angle (θ ∼ 63 • ), and zone γ is the region with relatively low energy (E ∼ 20 keV) and highest pitch angle (θ ∼ 75 • ). It can be found that the prompt loss of beam ions, which are distributed in zone α and β, dominates the energetic ion loss during NBI. In addition, energetic ion losses respectively induced by LLM and sawtooth are also observed. The spots for prompt loss at t 1 and t 4 have almost the same brightness and area, while the spots for LLM induced loss at t 2 and sawtooth induced loss at t 3 have higher brightness and larger area especially in the region of relatively low energy and high pitch angle (see zone β and γ). It illustrates that both LLM and sawtooth tend to expel energetic ions with relatively low energy and high pitch angle rather than other cases which are usually caused by other central MHD, such as TAE [43,44], NTM [45] and TM [42]. For example, TAE is likely to eject energetic ions with relatively high energy (40 keV< E < 180 keV) and low pitch angle (θ < 45 • ) in LHD [44], and NTM to eject those with comparable energy and relatively low pitch angle (θ ∼ 40 • ) in ASDEX Upgrade [45], compared with beam ions. Our result further verifies the simulation result that sawtooth crashes may provide a mechanism to expel energetic ions with relatively low energy and could be used for He ash removal [46]. Physical mechanism for energetic ion loss Mechanisms for energetic ion loss induced by MHD instabilities can be classified into three categories, characteristics of which are listed as follows [43,[47][48][49]. The dominant loss mechanism of the first category is convection. It has no threshold perturbation amplitude and does not induce thermal ion loss, and the loss quantity is proportional to the amplitude of MHD modes. The second category is dominantly diffusion. It has a threshold amplitude and the loss quantity varies quadratic with the amplitude of MHD modes. The last category is mainly stochastic process. It has higher perturbation amplitudes than the second category, and energetic ion losses as magnetic field and ion orbits become stochastic. The last category can induce thermal ion losses. Neutron emission calculation To analyze the physical mechanism for energetic ion loss induced by LLM, it is required to find out the relationship between the energetic ion loss rate and the perturbation amplitude of magnetic field. A neutron emission analysis code called FBURN [50] is used to perform a precise calculation for the evolution of neutron emissions. The required input parameters are listed below. Electron density profile: It is measured with the far-infrared laser interferometer [51]. Electron temperature profile: It is measured with the multipoint vertical-Thomson scattering diagnostic [52]. Ion temperature profile: It is measured with the charge exchange recombination spectroscopy [53]. Ion density profile: The profile of deuteron density is obtained from the equation n D = ( Z eff −ZC 1−ZC )n e , where Z eff is the effective plasma charge, Z C the atomic number of carbon and n e the electron density. Effective plasma charge: The effective plasma charge measured with the visible bremsstrahlung diagnostic [54] show that Z eff ∼ 2.5 before the minor disruption and Z eff ∼ 4 after the plasma recovered from the minor disruption. Neutral beam deposition profile: The neutral beam deposition profile is calculated using the same method as the neutral beam model of the Simulation of Spectra code [55]. Figure 7 shows the calculated neutral beam deposition profile for shot #39020 with a total NBI power of 0.5 MW. The proportion of the three energy components of neutral beam particles, i.e. E, E/2, and E/3, is 0.44, 0.39, and 0.17, respectively. The neutral beam deposition profile is peaked at the plasma center, which implies that most neutral beam particles are deposited in the plasma core region. With the input parameters described above, the FBURN code can calculate the neutron emission profiles, as shown in figure 8. Neutron emission profiles are peaked at the center as expected, illustrating that fusion reactions mainly occur in the plasma core region. Both LLM and sawtooth occur in the region around q ∼ 1 surface (ρ ∼ 0.25). It can be found that the neutron emission profiles are much distinct from each other in the plasma core region with normalized radius ρ ⩽ 0.4, while they are almost indistinguishable in the peripheral region with normalized radius ρ > 0.4. It indicates that the energetic ion loss in the MHD active phase mainly occurs within the normalized radius of 0.4. Analysis for loss mechanism The neutron emission evolution curves both measured with neutron diagnostics and calculated with the FBURN code are compared in figure 9. The calculated neutron yield agrees well with the measurement before 1100 ms, which illustrates that the calculation results are sufficiently accurate. The agreement between calculation and measurement for the minor disruption is due to that FBURN has considered the prominent thermal ion losses induced by the minor disruption at 978 ms. In addition, the deviations between the calculation curve and the experimental curves during LLM and/or sawtooth can be observed after 1100 ms. The FBURN calculation has considered the prompt loss of beam ions without any MHD activities by setting the diffusion coefficient of energetic ions to be 0.2 m 2 s −1 , which is estimated from neutron emission measurements during MHD quiescent phase. Among its input parameters described above, only the calculated neutral beam deposition profile has a correlation with energetic ions, while other input parameters are associated with the bulk plasma. It should be noted that the anomalous energetic ion loss induced by MHD instabilities is not included. Hence, the deviation between the experimental curves and the calculation curve during LLM and/or sawtooth illustrates that energetic ion loss happens in the period. Comparison between the experimental and calculated neutron emission evolution curves shows that, the LLM during 1090 ms and 1210 ms induces a ∼15% reduction in neutron emission, and both the LLMs and sawteeth during 1300 ms and 1900 ms induces more than ∼30% reduction in neutron emission. Figure 10 shows the dependence of the drop rate of total neutron emission rate on LLM amplitude measured by a magnetic probe located on the internal surface of the vacuum vessel. The drop rate of total neutron emission rate linearly increases with the perturbation amplitude, i.e. the energetic ion loss rate has a positive linear relation on the perturbation amplitude. In addition, it can be found from the positive intercept of the fitting line (the red solid line) that no threshold perturbation amplitude exists in the relation. Both characteristics indicate that the dominant loss mechanism for LLM induced energetic ion loss is convection, which is also the dominant loss mechanism of TAE induced energetic ion loss [43,47,56]. The relation between energetic ion loss rate and perturbation amplitude is verified by calculations, which will be discussed in section 5.2. Analysis for energetic ion loss process Since the energetic ion loss rate is proportional to the light output of the scintillator of the FILP head, the energetic ion loss rate induced by LLM and sawtooth can be estimated from the statistical analyses of the distribution maps in figure 6. Energetic ion loss distribution functions of light output vs. energy and pitch angle are shown in figure 11. Prompt loss is the average of energetic ion loss at t 1 and t 4 . LLM induced loss and sawtooth induced loss are respectively evaluated as the increment of energetic ion loss at t 2 and t 3 from the prompt loss. The LLM induced energetic ion loss is estimated to be 13% of the prompt loss, and the sawtooth induced energetic ion loss is estimated to be 75% of the prompt loss. However, the estimated sawtooth induced loss herein may contain partial or total LLM induced loss. Hence, the estimation value of sawtooth induced energetic ion loss is revised to be 62%-75% of the prompt loss. Since the time scale of LLM is much larger than sawtooth, the energetic ion loss induced by LLM is a mild process that has a much smaller loss rate than prompt loss, while the energetic ion loss induced by sawtooth is a drastic process compared with that induced by LLM. According to the neutron signal, less than 10% neutron yield reduction is observed during the sawtooth crash. Figure 11(a) shows that LLM induced energetic ion losses concentrate in zone β and γ, and those induced by sawtooth cover a much wider energy range. It indicates that energetic ion losses induced by LLM have relatively low energy (E < 27 keV) compared with the prompt loss. Figure 11(b) shows that both LLM induced and sawtooth induced energetic ion losses concentrate in zone β and γ, i.e. energetic ion losses induced by LLM have relatively high pitch angles (θ > 60 • ) compared with the prompt loss. Negative light output for the LLM induced loss curve appears during an energy range 37 keV < E < 55 keV and a pitch angle range 56 • < θ < 60 • (in zone α). It means that the energetic ion losses in zone α during LLM phase are less than the prompt loss, i.e. part of the prompt loss with high energy and low pitch angle is suppressed by LLM, so that the redistribution of energetic ions occurs with the presence of LLM. It is reported that Alfven instability induced redistribution of energetic ions can reduce the central magnetic shear leading to a reversed shear in a tokamak [57]. It indicates that the redistribution of energetic ions may produce a certain influence to the current redistribution, and even the formation and sustainment of flat or weakly reversed q profiles which are worth studying in the advanced scenarios for ITER operation [21]. Further, it can be speculated that LLM induced energetic ion loss might be related to some observation of internal transport barrier formation during central MHD such as LLM or fishbone, in which poloidal flow is enhanced [58]. The orbits of energetic ions in a magnetic field can be calculated with the following equation, m i where m i is the mass of energetic ion, e i the charge of energetic ion, ⃗ v the velocity of energetic ion, ⃗ B the magnetic field strength which can be obtained from the equilibrium reconstructed by the EFIT codes [59] combined with the Faraday rotation angle from polarimeter, as well as the constraint of central qvalue during sawtooth [60]. The calculated poloidal projections of the lost energetic deuteron ion orbits, which are calculated backward in time from the FILP head, are drawn in figure 12. Three main components in the distribution maps in figure 6 have been considered in the calculation. For the first component (zone α: E = 40 keV, θ = 57 • ), both trapped and passing particle orbits are observed, and the trapped particle orbits pass through the plasma core region. For the second component (zone β: E = 20 keV, θ = 63 • ) and the third component (zone γ: E = 20 keV, θ = 75 • ), only trapped particle orbits are observed, and all the trapped particle orbits pass through the peripheral region. The first component has a brighter spot because it corresponds to the maximum energy and therefore dominates according to figure 7. Additional magnetic perturbation is introduced to induce anomalous energetic ion losses when MHD instabilities are exited. Since LLM is excited around q ∼ 1 surface [37] which is shown by the yellow dash circles in figure 12, the energetic ions in the plasma core region are expected to be expelled to the peripheral region via convection as shown by the white arrows in figure 12. For the first component (see figure 12(a)), the lost energetic ions expelled from the plasma core to the peripheral region on the low field side (see the white arrows) have no chance to encounter lost particle orbits. On the contrary, the lost energetic ions expelled from the plasma core to the peripheral region on the low field side (see the white arrows) for the second component (see figure 12(b)) are likely to encounter lost particle orbits and be recorded by the FILP system. It explains why LLM tends to expel energetic ions with relatively low energy and high pitch angle. Please note that the loss orbits of the third component are located at the plasma edge. It illustrates that the energetic ions during LLM instabilities transport from the plasma core region to the peripheral region first, and then being scattered into the loss orbit, which is the same as the energetic ion transport behavior during TAE instabilities [61]. Notably, there exists another possible explanation for the reduced brightness of the main spot during LLM activity. In figure 12(a), both trapped orbits have banana tips located on the high-field side, i.e. their pitch angles are close to precession reversal ω D = 0. Banana guiding centers of such particles can be trapped by LLM and experience wide (few cm) super-banana oscillations [62]. Such super-banana particles will cross the last closed flux surface well outside the midplane, i.e. these loss ions cannot be detected by FILP. In this instance, the conclusion that LLM tends to expel energetic ions with relatively low energy should be relaxed. In order to confirm which explanation is more reasonable, theoretical calculations for LLM induced energetic ion loss is therefore necessary. Theoretical calculations for LLM induced energetic ion loss To reproduce the experimental observations for LLM induced energetic ion losses, theoretical calculations are carried out. figure 13(b)). It can be interpreted as due to the slowing down process of energetic ions in plasma. In order to calculate the distribution map of lost energetic ions with the ORBIT code [64], the perturbation displacement structure of LLM mode should be input the calculation as a necessary parameter. Figure 14(a) presents the perturbation displacement structure of the LLM mode calculated with the MARS-K code [65]. It can be found that m = 1 mode has a prominent perturbation displacement, while m = 2 and 3 modes have rather smaller displacements, which can also be seen in figure 2. The perturbation displacement shown by the green curve in figure 14(a) corresponds to the 2/1 mode, while the second harmonic in figure 2 is the 2/2 mode. However, since the ratio of the 2/2 and 1/1 components is sufficiently small which is estimated to be ∼1% according to figure 3, the discrepancy between calculation and experiment has very limited impact on subsequent ORBIT calculations. Since magnetic probes are located on the vacuum vessel, they cannot measure the absolute mode amplitude of core-localized mode. Herein, we just set the mode amplitude of LLM to be 65 Gs, which is about two times the measured value in figure 10. The factor of 2 is used in the ORBIT simulation here because the mode amplitude of core-localized mode is much higher than the measured value with the edge magnetic probes. Beside the perturbation displacement structure of LLM mode calculated with MARS-K, the ORBIT calculations take the energetic ion distribution calculated with NUBEAM as an input parameter. It should be noted that, unlike the distribution maps of lost ions measured with the FILP, the lost energetic ion in the ORBIT calculations is defined as the energetic ion whose guiding-center can cross the last closed flux surface in 5 ms. The distribution map of lost energetic ions in the pitch-angle vs. energy plane calculated with the ORBIT code is depicted in figure 14(b). The ORBIT calculation here has simulated 500 thousand particle markers. Figure 14(b) shows that lost energetic ions with relatively low energy (E < 20 keV) and high pitch angle (θ ∼ 62 • ) are more likely to be expelled during LLM mode. Conversely, lost energetic ions with relatively high energy (E > 20 keV) and low pitch angle (θ < 60 • ) are negligible. Hence, the calculation results are in qualitative agreement with the measurement results, i.e. the experimental observations that LLM tends to expel energetic ions with relatively low energy and high pitch angle are reproduced by the theoretical calculations. In addition, the dependence of the energetic ion loss rate on the magnetic perturbation amplitude caused by LLM can be obtained from calculations with the ORBIT code, as shown in figure 15. The ORBIT calculations here have simulated 70 thousand particle markers for each magnetic perturbation amplitude. Figure 15 shows that the energetic ion loss rate increases with the magnetic perturbation amplitude linearly, and the intercept of the fitting line (the red solid line) is about Figure 15. Dependence of energetic ion loss rate on magnetic perturbation amplitude caused by LLM. The energetic ion loss rate increases with the magnetic perturbation amplitude linearly, and no threshold perturbation amplitude for energetic ion loss exists. 1.5%, indicating that no threshold perturbation amplitude exists for LLM induced energetic ion losses. The calculation result has the same linear relation as the experimental result shown in figure 10 which shows that the mechanism for LLM induced energetic ion loss is dominantly convective. However, there is a quite large difference (∼5 times) between the measured drop of the neutron rate during LLM and the fast ion loss rate calculated by the ORBIT code. There are mainly two aspects of factors for causing the difference. One is that the redistribution of energetic ions is also a significant effect in LLM active plasma in addition to ion loss, which has also been observed in the spherical-torus device [21]. Hence, the measured drop of the neutron rate includes the contribution of the redistribution of energetic ions. The other is that the ORBIT calculations take the NUBEAM calculation results, which considered the moderated energetic ions, as an input parameter. Notably, the initial beam-born ions suffering prompt loss was not considered in the NUBEAM calculation results. Thus, the fast ion loss rate calculated by the ORBIT code is underestimated, since the ORBIT calculations did not consider the prompt loss of initial beam-born ions. Summary Energetic ion losses induced by LLM have been measured and investigated in the HL-2A tokamak by means of energetic particle diagnostics, including the NFM systems, the solidstate NPA and the FILP. Measurements show that LLM, sawtooth and minor disruption can lead to varying degrees of energetic ion losses. Neutron emission calculation has been performed by using the FBURN code with input parameters without considering MHD induced energetic ion losses. Calculated neutron emission profiles show that the energetic ion losses in the MHD active phase mainly occur within the normalized radius of 0.4. The deviation between the neutron emission evolution curves obtained from calculation and experiment illustrates that energetic ion losses are induced by LLM and/or sawtooth. Dependence of the drop rate of total neutron emission rate on LLM amplitude shows that the energetic ion loss rate linearly increases with perturbation amplitude and no threshold amplitude exists in the relation which is verified by the later calculation with the ORBIT code. Hence, the loss mechanism for LLM induced energetic ion loss is dominantly convective. It is observed with the FILP system that the energetic ion loss induced by LLM is a mild process that has an ion loss rate of 13% of that of the prompt loss. On the one hand, LLM tends to expel energetic ions with relatively low energy (E < 27 keV) and high pitch angle (θ > 60 • ) rather than other cases. On the other hand, LLM can partially suppress the prompt loss of energetic ions with high energy and low pitch angle, indicating that the redistribution of energetic ions and even plasma current may occur with the presence of LLM. The orbit calculations for energetic ions in a magnetic field not only satisfactorily explain the physical process of energetic ion loss induced by LLM, but also show that the lost energetic ions will transport from the plasma center to the peripheral region first, and then get lost out of plasma. In addition, the experimental observations for LLM induced energetic ion losses are also reproduced by theoretical calculations.	2023-06-24T20:01:52.485Z	2023-06-23T00:00:00.000	{ "year": 2023, "sha1": "a9d202df4cc21afe6634f8b975816899a425b7d8", "oa_license": "CCBY", "oa_url": "https://iopscience.iop.org/article/10.1088/1741-4326/acdca5/pdf", "oa_status": "HYBRID", "pdf_src": "IOP", "pdf_hash": "a9d202df4cc21afe6634f8b975816899a425b7d8", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
932769	pes2o/s2orc	v3-fos-license	Dynamic Learning Agents and Enhanced Presence on the Grid Human Learning on the Grid will be based on the synergies between advanced software and Human agents. These synergies will be possible to the extent that conversational protocols among Agents, human and/or artiﬁcial ones, can be adapted to the ambitious goal of dynamically generating services for human learning. In the paper we highlight how conversations may procure learning both in human and in artiﬁcial Agents. The STROBE model for communicating Agents and its current evolutions shows how an artiﬁcial Agent may ”learn” dynamically (at run time) at the Data , Control and Interpreter level, in particular exemplifying the ”learning by being told” modality. The enhanced telepresence research, exempliﬁed by Buddyspace, in parallel, puts human Agents in a rich communicative context where learning effects may occur also as a ”serendipitous” side effect of communication. The integration of the two streams of research will be the result of a workpackage within the E-LeGI EU Integrated Project, currently under negotiation. INTRODUCTION The concept of Learning Agent is seducing as much as confusing.There is no clear definition of what a software Agent is, and often, in the best AI tradition, one calls Agents both software and human Agents communicating in a network for performing jointly a task.The "learning" specification for an Agent may refer to learning as it occurs in human Agents within a scenario such as the one of the E-LeGI project, or else "machine learning" as it may be introduced in software.In this paper we wish to address the issue of Learning both in artificial and in human Agents [3].In order to define our research directions, we need to identify a minimal lexicon, and its associated choices as a precondition for the comprehension of further statements.As we wish our model to be the simplest possible one, we will perhaps risk to oversimplify more complex situations; eventually extensions will be treated later as a result of critical remarks.We assume first that artificial Agents are just software programs -and their associated processes -that at least are autonomous, distributed and able to communicate asynchronously with the environment (consisting only of other Agents) by means of a communication language that is independent on the content of the communication.Objects, for instance, are neither autonomous nor properly communicating, as the communication language consists just of selectors for methods (interfaces) thus is not independent of the objects themselves.Looking at the literature, in spite of the high popularity of the Agent literature, one may seldom find Agents that really respect all the minimal requirements for agentship.In previous papers, in order to give a provocative definition for Agents, we proposed to consider them as "crazy" Operating Systems (the adjective denoting their autonomy in reacting to messages). In the E-LeGI ambitions, perhaps the most challenging one is the personalization of learning services for humans.That will not be achieved unless a minimal formal, computational model of human Agents will be exploited during the generation of the corresponding services.We take this challenge by trying to develop a computable model for Agents that can serve also the purpose of modelling humans in a deliberately well-circumscribed context.Therefore, the issue of learning for human Agents is put into correspondence with the same for artificial Agents.Our model for artificial Agents has to be capable of modelling artificial learning; its corresponding software Agents similarly have to show learning properties that are sufficient for modelling human learning and therefore personalizing the services. Again with the risk of oversimplifying, we will initially consider for human as well as for artificial Agents just three types of learning: 1. Learning by being told, the classical "instructional effect".2. Learning by abstracting and generalizing (or by classifying examples, extracting rules and forming "theories").3. Learning as a side effect of communication, what we like to call "serendipitous"1 learning. We know that in a parallel paper [7] type 2 learning is extensively addressed.Perhaps what they call induction and abduction in theory construction (or Ontology negotiation) may be reconducted to our abstraction and generalization.At any rate, most of the machine learning work has been performed in this direction, while type 1 and 3 of learning described above -for artificial Agents -did not really get much attention in the literature. The STROBE model and its evolution Humans learn facts, rules (or procedures), and languages necessary to understand messages stating facts or procedures, as well as necessary for generating behaviour when applying a particular procedure to parameters.Although we are strong believers in the cognitive constructivist learning paradigm, we nevertheless focus on this highly restricted area of learning, which contains important elements that are so naturally inherited from the computational metaphor.Indeed facts, rules and languages are such as Data, Control and Interpreter levels in computing.These three abstractions levels may be found in all programming languages.One may distinguish Data (information) and Control (procedures) levels which corresponds to define new simple data and new procedures abstracting on the existing ones, from the Interpreter level which means to identify the way of evaluating an expression, or defining a special form which cannot be defined at the Control level.Currently, the two first levels could be reached during execution but the challenge is to allow Interpreter level modifications at run time, in order to generate processes. In order for conversational processes in E-LeGI to be effective, they have to generate services that help humans to learn facts, rules and... languages.That will be possible insofar we model human Agents by means of artificial Agents able to learn dynamically facts, rules and languages.As a resulting side effect, we will have the opportunity to use artificial Agents that learn (by being told) during conversations with other artificial Agents, thus that show a dynamic behaviour that adapts to the context.The STROBE model proposes an architecture to support this Agent behaviour. The key initial idea is to give to Agents an environment as a representation of any type of knowledge and consider them as REPL (Read, Eval, Print, Listen) interpreters. In the STROBE (STReams, OBjects, Environments) [2] model, that inherits most of its features from classical lambda calculus [8], denotational semantics [9] and the Scheme language we have identified a few basic properties for Agents: 1. First class2 Environments to model memory: linking variable names to values, under the commitment that types are on the values (dynamic typing) and that procedural abstractions are first class.2. First class OBjects to model the control: the classical loop (message, reaction with another message).In fact this option, very practical as a first approximation, is due to be abandoned for an Actor version, including a dynamic scheduler.3. First class STReams, modelling the evolving conversational processes by using the delayed (lazy) evaluation of values associated to expressions.4. First class continuations (K), in order to model non determinism and multiple conversational threads, i.e: a formal model of the rest of the process consuming the results from the current one once it will be terminated or suspended.5. First class interpreters (I), modelling how to generate processes from procedures.They are included in the above described environments. For the representation of the interlocutor in a conversation STROBE uses the concept of Cognitive Environments [1] which give to the Agent a "partner model" represented by an environment dedicated to this Agent.In this environment a dedicated interpreter is stored and used to interpret messages (and their content) sent by this Agent.Actually, messages' interpretation is done in a given environment and with a given interpreter.Learning at the Data and Control level consist in modifying the dedicated environment; learning at the Interpreter level (meta-level) consist in modifying the dedicated interpreter.Figure 1 illustrates an Agent representation.Within this model, it is not difficult to envison "learning-by-being-told" of variables (Data) or procedural abstractions (Control) insofar both are acceptable when declared bound to names by an external Agent that has the right to "teach me" about facts or rules.That means that an Agent can learn from another one simple information (Data) and procedures (Control) using an interpreter to evaluate "assertion" typed messages (such as definition or assignment). What is not straightforward is how to learn at run time special forms or any other modification that affects the interpreters.In the human Agent scenario, it is not difficult to teach a fact or a rule; what is less simple is to teach a piece of "language", i.e. teach people how to modify dynamically their interpretation behaviour for new facts or rules. The STROBE model makes it possible by allowing Agents to modify dynamically their interpreters. Agents, human and artificial ones, may be considered, in a first approximation, quite similar.Our challenge will be to show that both may be represented by combinations of primitives (environments, objects (procedures, schedulers), streams, continuations, interpreters) within the same model. The Scheme architecture Scheme classic REPL loop interpretation consists in waiting for a user expression, read and interpret this expression, send back the result and wait for the next expression.This represents eventually a typical Data and Control level modification.The higher level, Interpreter level, is not directly accessible.Our model uses another architecture.Instead of interpreting user expressions with the current interpreter, this one calls (via apply-procedure) another interpreter (stored in the dedicated cognitive environment), that interpret the user expressions.Thus, user expressions may modify the interpreter which evaluates them.Our idea is to use this architecture with our Agents.If one considers the three following interpreters each characterized by two procedures: • Scheme: eval and apply • Meta-eval: evaluate and apply-procedure • Meta-ambeval: ambevaluate and ambapply-procedure Then the classic REPL loop is equivalent to (eval e r k).where e is the expression to evaluate, r the environment and k the continuation3 .Our is: (eval (apply-procedure 'ambevaluate e r ks kf)).We need eval because Scheme is the programming language used; apply-procedure is used as a dedicated interpreter selector, and provides the first class environments.Finally, the last interpreter, the dedicated one (ambevaluate), is used to evaluate the message and its content4 . Example of meta-level learning: "teacher-student" dialogue We consider that the goal of education is to change the interlocutor's state.This change is done after evaluating new elements brought by the communication.The example in figure 2 (see also [5]) shows that a STROBE Agent can modify its way of seeing things (i.e. of evaluating messages) by "changing" its dedicated interpreter while communicating.It is a standard "teacher-student" dialogue, though of course highly simplified in light of our earlier comments.An Agent teacher asks to another Agent student to broadcast a message to all its correspondents.However, student does not initially know the performative5 used by teacher.So, teacher transmits two messages (assertion and order) clarifying to the student the way of processing this performative.Finally, teacher formulates again its request to student and obtains, this time, satisfaction.Figure 2 describes the exact dialogue emerging from the experimentation.After the last message process, the student function dedicated to the evaluation of message (ambevaluate-kqmlmsg) is modified.Thus a part of its interpreter was dynamically changed.The corresponding code in its environment dedicated to this conversation is changed.Then student Agent can process broadcast messages. TEACHER STUDENT Here is the definition of the square procedure: Ok, I know now this procedure: (kqmlmsg 'assertion teacher student '(define (square x) (* x x))) (kqmlmsg 'ack student teacher '(.)) Broadcast to all your current correspondents: Sorry, I don't know this performative: (kqmlmsg 'broadcast teacher student '(order (square 3))) (kqmlmsg 'answer student teacher ''(no-such-performative broadcast)) Ok, here is the method to add this performative to those you know: Here is the code you have to generate and add to your ambevaluate-kqmlmsg function: Ok, I have added this code in a binding of my environment: (kqmlmsg 'assertion teacher student learn-broadcast-code-msg) (kqmlmsg 'ack student teacher '(.)) Run this procedure: Ok, I have just modified my interpreter: (kqmlmsg 'order teacher student '(set!ambevaluate-kqmlmsg learn-broadcast-code))) (kqmlmsg 'executed student teacher '(.)) Broadcast to all your current correspondents: Ok, I broadcast (kqmlmsg 'broadcast teacher student '(order (square 3))) (kqmlmsg 'order student ... '(square 3)) Notice that learn-broadcast-code-msg message indicates how to generate the new function code taking into account the previous student code definition, and to store it in the learn-broadcast-code variable.This is a constructivist method. This toy-example is very simple but interesting because it shows the potentiality of such a model.We consider that it is a meta-level learning because a part of the Agent interpreter is dynamically changed.Another paper [6] describes also how using a nondeterministic interpreter in the model can enable the dynamic specification of a problem, in order to fit with dynamic service generation scenarios, such as necessary on Grids.The paper gives a typical e-commerce scenario example. LEARNING AS A SIDE EFFECT OF COMMUNICATION The examples provided in the previous section are of course characteristic of a very narrow spectrum of learning activities, namely those that occur during a particular kind of synchronous (i.e.real time communicative) interaction.Although the overwhelming majority of distance learning and e-learning literature emphasizes either the asynchronous space (particularly via discussion forums) or the one-to-many large-scale synchronous activities afforded by streaming media, there are nevertheless important and indeed profound opportunities that arise during very small-scale synchronous interactions (i.e.one-to-one or among very small study groups up to say, three or four participants).We note in particular the opportunistic learning dialogues that can occur in real time in such intensive tutorial situations, and which are precisely suited for the examples presented earlier.Although seemingly small and specialised in nature, it is nevertheless the case that if tutorial dialogues eventually occur between human and artificial agents, then there are in fact no practical limits to scalability, because every one-to-one interaction that involves an artificial agent can be replicated hundreds, thousands, or even millions of times. For the time being, our research progresses by studying the nature of synchronous interactions that occur between two, three, or four human agents.The ideal paradigm for this is to investigate and facilitate the learning interactions that take place via the world's fastest-growing software phenomenon: Instant Messaging.In the context of the E-LeGI project, we provide a custom environment for learners, called 'BuddySpace' [4] which can be summed up as "Instant Messaging + Smart Maps = Enhanced Presence".It provides continual 'background presence awareness' of peers, by deploying significant extensions to the open-source XML technology from the Jabber Software Foundation.As argued in Smart Mobs [10], tools like BuddySpace leverage the overwhelming power of social cohesiveness that can be brought about by knowledge of the presence and location of others in both real and virtual spaces.We know also from the work of Reffell and Eklund (2001) that this kind of presence awareness is used by students to locate resources, for quick exchange of information and to organize meetings either online or face-to-face. In reality, Enhanced Presence is much more than just 'messaging' and 'maps'.In particular, we aim to provide tools that enable us to express the entire situated context of the learner, which is clearly a lot more than just 'location X' and 'online' or 'offline'.The learner's current state of mind, including goals, plans and intentions, must be understood, as well as the way this connects with ongoing activities and devices accessible to the learner.When all these are modelled, plausible inferences can be drawn about what the leaner wants and needs to know, and this gives us an important 'foot in the door' for addressing the problem of delivering the right knowledge to the right people in the right place at the right time.So far, this notion of 'right knowledge' has been nothing more than a Knowledge Management 'slogan', but our belief is that Enhanced Presence capabilities, linked to the STROBE model, can make this dream a reality. CONCLUSION In this paper we presented the current progress of our work in two domains: Agent Communication Languages and Enhanced Presence.These are, at a first sight, very different one another nevertheless we have shown that they may become highly synergic and perhaps also mutually dependent within a rich experimental context such as the one of E-LeGI.The scenarios for integration of these two streams of research are currently under discussion.We perceive their properties as consisting of the identification of models and tools for generating learning services that enable and facilitate co-learning effects, i.e. humans AND machines construct their own representations -learn -by exploiting the representation of the partner through conversations. This social constructivist approach extends to machines the full right membership of societies of learning Agents both as "destinations" of knowledge emerging from these societies, and as a continuous source of knowledge digitally represented and stored and potentially consumed by other members through conversations.In these societies, it will be important to partition the responsibilities among members according to their best capacities: exploiting therefore Human Agents for what they are best in, and, at the same time, Artificial Agents for their optimal performance.The consequence is to adopt an approach in Distributed Artificial Intelligence where Humans are privileged in their best roles (e.g.motivation, trust, depth of conceptual analysis ...) and Machines in other roles (computing fast and reliably, instantaneous transmission of information through the net, storing and retrieving ...). FIGURE 1 : FIGURE 1: Agent attributes for representing others	2014-10-01T00:00:00.000Z	2003-12-03T00:00:00.000	{ "year": 2003, "sha1": "d9c6c7a424e7be073c35b9b1af813041681eaf61", "oa_license": "CCBY", "oa_url": "https://www.scienceopen.com/document_file/a84b3c5f-d344-4ecb-9f04-75cb96a8d07c/ScienceOpen/001_Eisenstadt.pdf", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "4d9228743563610e8733d83eec171359c7e93761", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
244554067	pes2o/s2orc	v3-fos-license	ENERGY-SAVING PAINTS AND VARNISHES AND COATINGS FOR AGRICULTURAL MACHINERY The choice of paints and varnishes and painting technologies for agricultural machinery is made taking into account its purpose and operating conditions. Therefore, when choosing a painting technology, it is necessary to take into account the weather resistance of the resulting paintwork, oil resistance, petrol resistance and heat resistance, as well as chemical resistance and non-toxicity, depending on the operating conditions. According to the authors, the standards of applied paintwork materials are outdated, since in recent years better and more durable materials have appeared on the market. SPA Lakokraspokrytie offers as an alternative fast-drying energy-saving alkyd-urethane, urethane and alkyd-acrylic paintwork materials and also offers a two-layer energy-saving dyeing technology. INTRODUCTION The choice of paints and varnishes and painting technologies for agricultural machinery is made taking into account its purpose and operating conditions. By purpose, agricultural machinery can be divided into three classes [1]. The first includes tractors, engines and self-propelled chassis. The second class includes tillage machines, machines for sowing and planting, for preparing and applying fertilizers, for protecting plants, for irrigation, various types of combines, headers and trailers for transporting goods, machines for cleaning grain and seeds, harvesting and processing vegetables, as well as machines used in gardening. The third class of agricultural machinery includes equipment for animal husbandry and forage production. Agricultural machinery of the first and second class is operated in atmospheric conditions. Therefore, when choosing a painting technology, it is necessary to take into account the weather resistance of the resulting paintwork. For parts and assemblies exposed to gasoline, mineral oils and high temperatures, oil, petrol and heat resistant materials are selected. Machines and equipment for applying fertilizers, as well as for livestock complexes, must have chemically resistant paintwork. In addition, paint products for the protection of the livestock complex should be non-toxic, should not affect the reproductive functions of animals and reduce the sanitary characteristics of the received food. PRACTICAL PART Currently, the choice of paint and varnish materials and the technology of their use is regulated by GOST 6591-91 "Coatings for paint and varnish tractors and agricultural machines. General technical requirements "and GOST 23852-79" Paint coatings. General requirements for the choice of decorative properties ". So, for example, facing parts of agricultural machines must have a coating class of at least IV-V, on the remaining surfaces a VII coating class is allowed according to GOST 9.032-74. Coatings should be smooth, glossy or semi-glossy. According to the color scheme for agricultural machinery, red, orange, yellow, blue and blue colors are selected. For painting agricultural machinery in accordance with GOST 6591-91, weather-resistant enamels such as AC-182, PF-115, PF-188, ML-152, XV-518, XC-758 and others are recommended. Priming, as a rule, is made with alkyd primers GF-0119, GF-021, GF-017. These are energy-intensive LMK with high-temperature (80-110 ºС) and long-term drying (from 35 to 90 minutes). It should be noted that until now, many manufacturers of agricultural machinery use PF-115 enamel for coloring their products, despite the fact that, according to GOST, the period of application of this enamel is limited to 01.01.1994. The above paintwork has a service life in temperate and cold climates up to 5 years. Higher service life (up to 7 years) is provided by coatings primed with water-borne materials applied by electrodeposition, dipping and spraying (primers V-KF-093, V-KCh-0207, V-ML-0143), as well as powder coatings. In our opinion, this standard is outdated from the point of view of the applied paints and varnishes, since in recent years better and more durable materials have appeared on the market. In foreign practice, trends in the development and application of paintwork for agricultural machinery are: • replacement of conventional paintwork materials with paintwork materials with a high solids content and with water-based materials; • application of formulations without harmful air pollutants; • minimization of the amount of VOCs; • optimal compatibility of application methods; • obtaining high-quality paintwork at lower costs. For example, on a new painting line for potato diggers at the Grimme Landmaschinenfabrik plant, since 1995, products have been painted by dipping followed by powder coating. Surface preparation was carried out by iron phosphating. In 2003, iron phosphating was replaced by zinc phosphating, and dip priming was replaced by a thick layer (up to 45 microns) cataphoretic priming. The German company Eisenmann for the Czech Republic develops and installs a painting line for agricultural machines with 11 zones for surface preparation by cataphoresis followed by a coating layer. At the John Deere Werke plant (USA), a fully automated line for painting agricultural equipment with the quality of painting a car was launched. The painting line includes a 13-stage surface preparation, cataphoresis priming followed by painting with acrylic powder coatings. The rise in energy prices both abroad and in Russia leads to the need to save electricity. The problem of energy saving abroad is solved through the rational loading of the painting line, the use of diagonal ventilation systems in the painting and drying chambers, the use of heat exchangers to use the recovered heat, for example, for heating surface preparation baths, heat recovery from drying chambers, the use of special sliding gates for maximum heat preservation [2][3][4]. In Russia, energy savings are mainly solved through the use of energy-saving paintwork materials [5]. Such energy-saving materials include alkyd-acrylic, urethane, acrylic-urethane materials, which are currently widely represented on the Russian market [6]. The assortment of paints and varnishes, produced by SPA Lakokraspokrytie, also includes fast-drying energysaving alkyd-urethane, urethane and alkyd-acrylic paintwork materials. The paint systems for painting agricultural machinery based on these materials are shown in Table 1. These materials are distinguished by accelerated drying not only at elevated (up to 80 ºС) temperatures, but also under natural conditions (3-5 hours), high decorative properties, high durability (8-10 years in temperate and cold climates and 4-5 years in tropical climate). The use of quick-drying energy-saving materials is very important, especially in Russia. This is due to the fact that most of the agricultural machinery enterprises were built in the middle of the last century. The equipment on the painting lines has exhausted its resource and requires reengineering [3]. When reengineering paint lines, there is a simultaneous reduction in the area allocated for the paint area and an increase in the range of products to be painted. So, for example, in JSC "Sibselmash" reengineering of paint production was carried out. For painting mediumsized parts, SPA Lakokraspokrytie proposed a two-layer energy-saving painting technology based on AU-1-201 soil-enamel and AU-1518 "Universal-Lux" enamel for medium-sized and large parts, which makes it possible to place a line of automatic and manual painting by the method spraying. Replacing the existing technology with an energy-saving one allows not only to reduce the production area for painting, but also to improve the quality and durability of the resulting paintwork. Energy-saving technology was also used in the development and implementation of a mechanized line for painting soil-cultivating equipment in Saransk (customer JV Gaspardo-RM LLC), in the development of technical proposals for the reconstruction of the painting area of Morozovskselmash OJSC (Rostov Region) and Voronezhselmash OJSC ". However, it should be noted that the most advanced technologies for painting agricultural machinery are technologies with cataphoretic priming and powder painting. The use of these technologies will increase the competitiveness of the manufactured agricultural machinery in the world market. The equipment for the painting line of tractor cabs using cataphoretic priming followed by painting with melamine-alkyd enamels of accelerated drying was developed for the Kharkov Tractor Plant and the Automobile Plant (Elabuga). CONCLUSION The use of powder technology for painting agricultural machinery meets the requirements of environmental safety of production, can significantly reduce production areas for painting due to a single-layer application, provides a high-quality durable paintwork with high wear resistance and resistance to aggressive environments [7][8][9][10]. This technology is especially suitable for the coloring of tillage and livestock equipment, as well as equipment for the production of feed. The powder coating line was introduced by SPA Lakokraspokrytie at JSC BelAgromash-Service for painting large frame structures. A unique mobile complex of equipment for the application of powder coatings was created, allowing in tact production to paint large-sized (5350 * 800 * 2500) frame structures. All powder coating equipment, including the spray booth, is located on a movable platform that fits on rails laid in the concrete floor pit. The platform moves sequentially along the product and allows painting the product along its entire length. Thus, SPA Lakokraspokrytie has extensive experience not only in the development of modern technologies for painting agricultural machinery, but also in the development of modern painting lines, the manufacture of complex equipment for surface preparation and painting of this technique. At present, SPA Lakokraspokrytie is the only specialized research and production association in Russia for the development of equipment and technology for applying liquid paint and varnish materials and powder paints. SPA carries out the whole range of works related to the preparation of the surface of products for painting, design and manufacture of painting equipment, flow painting lines and their implementation in various fields of industry.	2021-11-25T16:17:16.174Z	2021-01-01T00:00:00.000	{ "year": 2021, "sha1": "1c2a8555d623283e07835af08a3c2bd59692b243", "oa_license": "CCBY", "oa_url": "https://www.confer.cz/metal/2021/download/4165-energy-saving-paints-and-varnishes-and-coatings-for-agricultural-machinery.pdf", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "eaf58f9e1b9f4ccffbd0fdb942b96b3edb66fe62", "s2fieldsofstudy": [], "extfieldsofstudy": [] }
15604588	pes2o/s2orc	v3-fos-license	Rigorous Asymptotics for the Lam\'e and Mathieu Functions and their Respective Eigenvalues with a Large Parameter By application of the theory for second-order linear differential equations with two turning points developed in [Olver F.W.J., Philos. Trans. Roy. Soc. London Ser. A 278 (1975), 137-174], uniform asymptotic approximations are obtained in the first part of this paper for the Lam\'e and Mathieu functions with a large real parameter. These approximations are expressed in terms of parabolic cylinder functions, and are uniformly valid in their respective real open intervals. In all cases explicit bounds are supplied for the error terms associated with the approximations. Approximations are also obtained for the large order behaviour for the respective eigenvalues. We restrict ourselves to a two term uniform approximation. Theoretically more terms in these approximations could be computed, but the coefficients would be very complicated. In the second part of this paper we use a simplified method to obtain uniform asymptotic expansions for these functions. The coefficients are just polynomials and satisfy simple recurrence relations. The price to pay is that these asymptotic expansions hold only in a shrinking interval as their respective parameters become large; this interval however encapsulates all the interesting oscillatory behaviour of the functions. This simplified method also gives many terms in asymptotic expansions for these eigenvalues, derived simultaneously with the coefficients in the function expansions. We provide rigorous realistic error bounds for the function expansions when truncated and order estimates for the error when the eigenvalue expansions are truncated. With this paper we confirm that many of the formal results in the literature are correct. Introduction The Lamé equation is Lamé's equation first appeared in a paper by Gabriel Lamé in 1837 [14]. It appears in the method of separation of variables applied to the Laplace equation in elliptic coordinates. Lamé functions have applications in antenna research, occur when studying bifurcations in chaotic Hamiltonian systems, and in the theory of Bose-Einstein condensates, to name a few (see [24, § 29.19]). Mathieu's equation is where λ and h are real parameters. We consider the interval z ∈ (0, π). When λ assumes the special values a m or b m+1 for m = 0, 1, 2, . . . , Mathieu's equation admits even or odd periodic solutions denoted ce m (h, z) or se m+1 (h, z) respectively. These functions first arose in physical applications in 1868 inÉmile Mathieu's study of vibrations in an elliptic drum [15]. Since they have appeared in problems pertaining to vibrational systems, electrical and thermal diffusion, electromagnetic wave guides, elliptical cylinders in viscous fluids, and diffraction of sound and electromagnetic waves, to name a few. In general, they appear when studying solutions of differential equations that are separable in elliptic cylindrical coordinates. For an insight to see how Mathieu functions appear in physical applications see [16]. We wish to obtain uniform asymptotic approximations for the Lamé and Mathieu functions, and asymptotic expansions for their respective eigenvalues, as the parameters ν in Lamé's equation and h in Mathieu's equation become large. We denote for the moment the parameter h in Lamé's equation to be h L , to avoid confusion with the parameter h in Mathieu's equation. We have in the limit k → 0 + from [24, § 22.5(ii)] that lim k→0 + sn(z, k) = sin z, and lim k→0 + thus if ν → ∞ in such a way that as k → 0 + , ν(ν + 1)k = 2h for some constant h, then we can rewrite the limit of Lamé's equation in the form Thus for ν = − 1 2 + 1 4 + 2h k 2 we have lim k→0 + Ec m ν z, k 2 = ce m h, and With this in mind, we derive rigorous results for the Lamé functions and their eigenvalues, and deduce analogous results for Mathieu's equation using this limiting relation. For a general overview of the Lamé and Mathieu equations, see [2,5,29]. For a more detailed study of Mathieu's equation see also [17]. Whilst the results for asymptotic expansions of the Lamé functions and their respective eigenvalues for parameter ν large are not so abundant, analogous problems for the Mathieu functions and their eigenvalues for parameter h large have been studied extensively. The main results in the Lamé case can be found in [11,12,18,19,20]. These results are all formal, meaning they are not accompanied with any error analysis. None of the results about the functions have been published in [24, § 29.7], and only limited formal results about the corresponding eigenvalues can be found there. In [24, § 29.7(ii)] it is stated that one could derive from the results of [28] asymptotic approximations for the Lamé functions. However in that paper the results are given without much justification and the error bounds given for the approximations do not make sense in the intervals where the approximant is exponentially small. Here the results we give for the Lamé functions are new; we give a two term uniform asymptotic approximation for the Lamé functions in terms of parabolic cylinder functions for ν large complete with error bounds, and we show this holds uniformly in the interval z ∈ [0, K]. We also make a rigorous statement about the corresponding eigenvalues. We also give asymptotic expansions for the functions and eigenvalues in a shrinking neighbourhood of the origin, which correspond with the few formal results in the literature. The main results in the Mathieu case can be found in [3,4,6,7,8,9,10,13,17,25,26,27,28]. In [17,28], error estimates are written down for a one term asymptotic approximation of the Mathieu functions but these are given without any justification, and the error does not make sense in the intervals where the approximant is exponentially small. In [13], similar results are given for the functions with similar issues for the error estimates; the results about the eigenvalues however seem reasonable, but methods of obtaining terms in the eigenvalue expansions seem cumbersome. The most satisfactory work thus far is contained in [4]. Here Dunster derives uniform asymptotic approximations for all complex values z when −2h 2 ≤ λ ≤ (2 − d)h 2 , d > 0, with error bounds either included or available for all approximations. These approximations involve both elementary functions and Whittaker functions. He also includes rigorous statements related to the eigenvalues a m and b m+1 . The remaining papers stated include only formal results, without any satisfactory error analysis. Here we consider only the real interval z ∈ (0, π) as many physical applications are restricted to real variables. The results we give are uniform asymptotic approximations complete with error bounds in the interval z ∈ 0, π 2 , given in the most natural form for the case we consider. Since we restrict ourselves to real variable analysis we can make stronger statements about the error bounds for the functions and their respective eigenvalues than given in [4]. We also give asymptotic expansions for the functions and eigenvalues in a shrinking neighbourhood of π 2 , which correspond with the formal results in the literature. Overview This paper is written in two parts: In Part I we derive two term uniform asymptotic approximations for the Lamé functions Ec m ν z, k 2 and Es m+1 ν z, k 2 which hold for z ∈ [0, K], and rigorous approximations for the large order behaviour of their respective eigenvalues a m ν and b m+1 ν , m = 0, 1, . . . , as κ → ∞, where κ = ν(ν + 1)k. Treating the Mathieu functions and their respective eigenvalues as a special case of those in the Lamé case, we obtain simply uniform asymptotic approximations for the Mathieu functions ce m (h, z) and se m+1 (h, z) which hold for z ∈ 0, π 2 , and rigorous approximations for the large order behaviour of their respective eigenvalues a m and b m+1 , as h → ∞. Part II uses a simplified method to derive asymptotic expansions for both the Lamé and Mathieu functions and their respective eigenvalues. We can compute as many terms as we like in these expansions. The price to pay is that these function expansions only hold for z = O(κ −1/2 ) and z = π 2 + O(h −1/2 ), as κ → ∞ and h → ∞ respectively. These intervals at least encapsulate all of the interesting oscillatory behaviour of the functions. We give rigorous and realistic error bounds for the function expansions once truncated, along with order estimates for the error when the eigenvalue expansions are truncated. First, we will summarise the relevant properties of the Lamé and Mathieu functions and their respective eigenvalues in the upcoming section. and interlace such that The normalisations in [24, § 28.2] can be rewritten as and to complete their definitions, the signs are determined by continuity from We summarise their properties and give boundary conditions in Table 2. From formal asymptotic expansions given in [24, § 29.7] we deduce that s → 0 as κ → ∞, hence in this limit (4.2) has two coalescing turning points. The turning points of our equation are at x = ±s and thus we apply the theory of Case I in [21]. In this case uniform asymptotic approximations are in terms of the parabolic cylinder functions U − 1 2 κσ 2 , √ 2κζ and U − 1 2 κσ 2 , √ 2κζ . For the standard notation see [24, § 12.2]. Following Olver [21], new variables relating {x, w} to {ζ, W } are introduced by the appropriate Liouville transformation given by the dot signifying differentiation with respect to ζ, where σ is defined by From this we denote that 0 < s < 1 corresponds to 0 < σ < σ * , where σ * = 2 arcsin(k) πk . Since ζ = ±σ corresponds to x = ±s, integration of the second of (4.4) yields −s These equations define ζ as a real analytic function of x. There is a one-to-one correspondence between the variables x and ζ, where ζ is an increasing function of x, and we denote ζ = −ζ * , 0, ζ * to correspond to x = −1, 0, 1. It follows that x(ζ, σ) is analytic both in ζ and σ for ζ ∈ (−ζ * , ζ * ) and σ ∈ (−σ * , σ * ). Alsoẋ is non-zero in these intervals. Performing the substitution t = sτ in (4.5) we expand the integral and obtain and by reversion In the critical case s = σ = 0 we have from the third of (4.6) which gives Thus we deduce that as s, σ → 0 ζ * → 2 arctanh(k) k . The transformed differential equation is now of the form where ψ ζ, k 2 =ẋ 2 φ x, k 2 +ẋ 1/2 d 2 By construction, the apparent singularities in the above function at ζ = ±σ, corresponding to x = ±s, cancel each other out so that ψ ζ, k 2 is well-behaved there. To check this one could expand ψ ζ, k 2 around this point. One could also note that in (4.2), φ x, k 2 has singularities at x = ±1, where as ψ ζ, k 2 does not blow up there. However ζ and therefore ψ x, k 2 will have branch point singularities there. For our advantage in the error analysis, we write our differential equation in the form where ψ ζ, k 2 = ψ ζ, k 2 − ψ 0, k 2 = ψ ζ, k 2 + σ 4 − s 4 2σ 2 s 4 , and correspondingly where This gives ψ 0, k 2 = 0. (4.12) On inspection it follows that since s, σ and the variables x and ζ are all bounded as κ → ∞, and the apparent singularities at x = ±s and ζ = ±σ cancel each other, we have for ζ ∈ [−ζ * , ζ * ] ψ ζ, k 2 = O(1), uniformly in this limit. On applying Theorem I of [21, § 6] with u = κ, α = σ and ζ 2 = ζ * , we obtain the following solutions of (4.9) valid when ζ ∈ [0, ζ * ). Choosing as a consequence of (4.12) and thus defining the variational operator as the bounds obtained are . The functions E, M and later N are defined in [21, § 5.8]. It follows from (4.12) and the evenness of ψ ζ, k 2 that thus clearly l 1 − 1 2 κ σ 2 is bounded as κ → ∞. Hence Applying similar analysis to the above, again from Theorem 1 in [21, § 6], we obtain We can extend this analysis to include the point ζ * since ζ and ψ ζ, k 2 are bounded there. where C m ν , S m+1 ν , η m ν,c and η m+1 ν,s are constants, and s m ν , σ m ν , W m ν,1 and W m ν,2 are defined with respect to either h = a m ν or h = b m+1 ν in the previous section. Let's first consider m odd. In correspondence with the boundary conditions we require The requirement at z = K in (5.1) gives thus from (4.16), (4.15) and (4.13), and since as κ → ∞ (see [24, § 12.9]) necessarily η m ν,c is exponentially small in this limit. Note that here we have assumed that κ σ 2 = O(1) as κ → ∞, and we show later, in (5.3), that this assumption is justified. Similarly the requirement at z = K in (5.2) gives is exponentially small as κ → ∞. Hence for both these cases by considering the requirements at z = 0 we have the condition and to satisfy this we require where j is an odd integer. Let's now consider m even. In correspondence with the boundary conditions we require By similar reason to the m odd case, when considering the boundary condition at z = K both η m ν,c and η m+1 ν,s are necessarily exponentially small as κ → ∞. Hence by using the boundary condition at z = 0 we have the condition To satisfy this we require that where j is an even integer. √ 2κζ tend to the zeros of D j √ 2κζ , the parabolic cylinder functions with exactly j zeros in it's oscillatory interval (see [24, § 12.11]). Thus in correspondence with the number of zeros of the Lamé functions and those of D j √ 2κζ , we deduce that j = m and as such from (4.7) and (4.10) we have that We deduce from (4.7) and (4.3) that 6 Approximations in terms of parabolic cylinder D functions The Lamé functions decay exponentially on either side of the oscillatory interval in (−K, K). Our approximant in Section 4, U − 1 2 κ σ 2 , √ 2κζ , displays the appropriate exponentially decreasing behaviour when ζ is large and positive, but when the variable is large and negative it becomes exponentially large. If our argument − 1 2 κ σ 2 had been exactly a negative half-integer, which it is not, then the approximant would have exhibited this wanted exponentially decaying behaviour when the variable is both large and positive and large and negative. With respect to (5.3) we define thus it makes sense to split up (4.8) so that From (6.1) and since s m ν , σ m ν and the variables x and ζ are all bounded as κ → ∞, we have for in this limit uniformly. Now our approximant will have the desired property of decaying exponentially on either side of the oscillatory interval for large positive and large negative ζ. We obtain the solutions for (6.2) We have that In Part II, Section 9, we will show that We combine this with (4.11) and obtain This time in the error analysis we choose it so that and, hence, take as the variational operator The bounds for the errors are . We define for convenience From (4.14) clearly l m,1 is a bounded constant as κ → ∞. Hence we have Applying similar analysis to the above, again from Theorem 1 in [21, § 6], we obtain Thus from (6.3), (4.4) and (4.1) we obtain are O e −κζ 2 * κ m+1/2 as κ → ∞. Only when ζ nears the endpoint the contribution from the D m √ 2κζ + m ν,2 ζ, k 2 term is comparable to D m √ 2κζ + m ν,1 ζ, k 2 . These functions will be made unique by their normalisation. A second term in the approximation in terms of parabolic cylinder D functions In [21, § 11] uniform asymptotic expansions are included. The two term version is of the form in which we have taken A 0 = 1 and for B 0 (ζ, κ) we have dt, whereσ = (2m + 1)/κ. This coefficient depends on κ and its dominant part is To simplify the integrand we take the final equation in (4.6) and let κ → ∞. The result is This relation can be inverted We use this in and are able to evaluate the integral in (6.6) and obtain 32ζB 0 (ζ) = k 2 + 1 ln 1 where C(ζ, k) = 2k coth kζ 2 /2 − k 2 − 1. Thus our two term approximation is as κ → ∞ and similarly for Es m+1 ν z, k 2 . Normalisation constant We now want an approximation for C m ν and S m+1 ν . Due to the complicated nature of the mapping between x and ζ, it is not simple to express one in terms of the other. With respect to (3.1) we consider the integral Using only this first term in an expansion for the solution, we can only obtain coefficients for up to 1/h terms. Any further terms in an expansion for the solution would contribute to further terms in the normalisation constant expansion, which we will not consider here. Since the first terms in their respective function uniform approximations are the same for large κ, it will be the same for both C m ν and S m+1 ν . First we perform the transformation x = sn(z, k) to obtain then another transformation to the ζ variable to obtain As the oscillatory behaviour of D m √ 2κζ happens in a shrinking region of the origin as κ → ∞, we seek to approximate the integral around this point to get an approximation for C m ν . Thus we consider an expansion of the form as ζ → 0, and substituting this into the relation From this we can determine, by matching (6.7) and (6.8), the c k terms, and obtain as ζ → 0. From this we obtain as ζ → 0. With respect to this, we consider as a first approximation for C m ν the integral and letting t = √ 2κζ, we have the approximation In correspondence with (3.2) and that for large z, both η c m and η s m+1 are O e −2hζ 2 * h m+1/2 as h → ∞, the relationship between z and x is defined by x = cos z and by evaluating the integrals (4.6), the relationship between x and ζ is defined by and λ m corresponds to either a m or b m+1 depending on the solution we are considering, and We have that Two term approximations are given by where for B 0 (ζ) we have the relation Uniform asymptotic expansions In Part I, uniform asymptotic approximations were given for the Lamé and Mathieu functions when a parameter became large. A third term in an asymptotic expansion could not be computed due to the complicated nature of the transformation of the independent variable. In Section 8 of this part we employ a simpler transformation than in Part I such that we can construct formal asymptotic expansions for the Lamé functions and their corresponding eigenvalues in the forms where t = √ 2κ sn (z, k), the coefficients A s (t) and B s (t) are just polynomials, and the µ s are constants in terms of m and k. The function expansions will clearly only make sense when t = O(1) as κ → ∞. In Section 9, we analyse the asymptotic expansions for the eigenvalues and give an order estimate for the error corresponding to the truncated eigenvalue expansion. Then in Section 10 we use these results to give rigorous and realistic error bounds for the function expansions upon truncation. In Sections 11 and 12 we identify our expansions with the Lamé functions and then summarise our results. Finally in Section 13 we give analogous results for the Mathieu functions and their corresponding eigenvalues as a special limiting case of those given for the Lamé functions and their corresponding eigenvalues. Uniform asymptotic expansions of the Lamé functions In this section we construct formal asymptotic expansions for solutions of Lamé's equation and corresponding eigenvalues. We do this by performing a simpler transformation than employed in Part I. The oscillatory behaviour of the Lamé functions happens in a shrinking neighbourhood of the origin as κ → ∞, and it can be shown that around the origin ζ behaves approximately like sn(z, k). Thus the variable in the parabolic cylinder function around this point behaves approximately like √ 2κ sn(z, k). This motivates the next simpler transformation. t-plane Letting t = √ 2κ sn(z, k) in (1.1) we have where z ∈ (−K, K) corresponds to t ∈ − √ 2κ, √ 2κ . Supposing in accordance with (5.4) that where n is a positive integer and µ n can be re-expanded in a sensible manner. We can then write Lamé's equation in the form This equation is split in such a way that constructing a formal asymptotic expansion in terms of parabolic cylinder functions D m (t) in the form seems sensible. However one should observe that this splitting only makes sense when t = O(1) as h → ∞, hence this formal expansion is only sensible for this range of t. We denote this range as (−t * , t * ). One should note that whilst this appears to be a new ansatz, there are similar expansions given in the literature for the Mathieu functions. These expansions are given in terms of D m−4j (t) for j ∈ Z instead of in terms of D m (t) and D m (t), but using the recurrence relations for the parabolic cylinder functions it is obvious these expansions are equivalent up to normalisation. An expansion in the form we have given allows us to differentiate easily and hence seems the most natural in this case, also allowing us to perform rigorous error analysis. We seek solutions which are either even or odd respective to the parity of m. Since D m (t) is either even or odd respective to when m is either even or odd, we deduce that A s (t) and B s (t) must be even and odd respectively. Substituting (8.3) into (8.2) and equating powers of κ, we have the recurrence relations for A s (t) and B s (t) +t(m) tA s−2 (t) + 2tB s−2 (t) + 2B s−2 (t) = 0, (8.5) wheret(m) = 1 4 t 2 − m − 1 2 . Neither of these relations separately determine solutions for A s (t) or B s (t) from previous coefficients, thus we differentiate (8.4) to obtain an expression for A s (t) and substitute it into (8.5); this gives the third order inhomogeneous differential equation for B s (t) Once B s (t) is determined, we can use (8.4) to determine A s (t). There will be freedom in choosing the integration constants in the A s (t) terms, with identification of our solutions made unique by their normalisation. General coef f icients B s (t) and A s (t) Using variation of parameters we obtain the general solution for B s (t) By expanding the Wronskians we derive the relations Then without loss of generality we can rewrite the indefinite integrals in b i s (t) 3 i=1 as definite integrals from 0 to t, and since B s (t) is supposed to be an odd function we have to take c 1 = c 2 = 0. Thus Although we consider t to be O(1) as κ → ∞, we still want an expansion which exhibits the correct behaviour at the endpoints of the interval. Expanding the squared term in (8.8) and splitting it into three separate integrals, large variable asymptotics for the parabolic cylinder functions (see [24, § 12.9]) tells us that the terms involving D 2 m (t) or D m (t)D m (t) grow no faster than polynomials when t becomes large. Since to ensure the boundedness of our formal expansion as t becomes large, µ s is determined uniquely by the condition that This condition will also ensure boundedness as t → −∞. Note that in the next section we will derive an alternative method to compute the µ s terms which we use to compute the coefficients as it is simpler. Consider first s = 0. From (8.7) we see that b 0 (t) = 0, thus ensuring that B 0 (t) is odd we obtain the general solution and then Rearranging, on the κ 0 level of the asymptotic expansion (8.3) we have Thus having this term in B 0 (t) equates to B 0 (t) = 0 and an extra constant term in A 0 (t), and since we have freedom in the arbitrary constant terms in A s (t), we can take c 3 = 0 without loss of generality. For simplicity we take A 0 (t) = 1 and adopt the convention A s (t) = 0 for s ≥ 1. With these choices we have and expressing the D m (t) in (8.9) via [24, formula (12.7.2)] in terms of Hermite polynomials we can evaluate the integral in (8.9) and obtain In the case s = 1, it can be shown using integration by parts that for µ 1 which satisfies (8.9) Thus clearly in this case, if the correct c 3 is chosen in (8.8) then B 1 (t) is exactly an odd polynomial. Using similar observations as in the s = 0 case, we also note that if this multiple of D m D m is included in the B 1 (t) term, the expansion can be rearranged so that this term is instead represented in the constant term of A 1 (t). Taking B 1 (t) to be exactly an odd polynomial, we get that A 1 (t) is an even polynomial. If one would go through the details to compute the representation for B 1 (t) as a polynomial plus this multiple of D m D m , one would see that it would appear for all s ≥ 1 that if the previous A s (t) and B s (t) terms are all polynomials, then the A s (t) and B s (t) terms can be represented as polynomials. Polynomial coef f icients B s (t) and A s (t) To obtain explicit expressions for A s (t) and B s (t) we try substituting in polynomial expansions with undetermined coefficients. Take A 0 (t) = 1 and B 0 (t) = 0, then for s ≥ 1 we consider A s (t) and B s (t) in the form Substituting these into (8.6) and (8.4) we obtain the recurrence relations for coefficients (2i + 3)(2i + 2)(2i + 1)b s,i+1 + (4m + 2)(2i + 1)b s,i − 2ib s,i−1 From these recurrence relations it is observed that only a finite number of the a s,i and b s,i are non-zero. The orders are displayed in Table 3. In this manner the coefficients are determined recursively. We deduce by considering the difference of (8.10) and (8.11) in the case i = 0 that µ s = (2m + 1)b s,0 − 2a s,1 . (8.12) In the case that s is even then we start with i = 2s − 1 in (8.10) to determine b s,2s−2 . Then we take i = 2s − 2 and determine b s,2s−3 , and so on. Once every power of t is eliminated, we are left with a constant equation which we must make zero by specifying µ s ; in this manner the eigenvalue terms are determined uniquely. These µ s terms are the same as those specified by the condition (8.9), since it is required for solutions B s (t) which grow no faster than polynomials. Once the coefficients in the polynomial expression for B s (t) are determined, and the corresponding µ s , then the coefficients in the polynomial expression for A s (t) can be determined from (8.11). In the case that s is odd then we have to start with i = 2s in (8.10) to determine b s,2s−1 . Returning to the z-plane: odd solutions In [24, § 28.8] expansions for the odd Mathieu functions are given which exhibit the correct odd behaviour, which an expansion in the form (8.3) would not when transformed back into the z-plane. We want something similar for the Lamé functions. Once we transform our formal expansion back into the z-plane, over the whole real line they behave like the even Lamé functions Ec m ν z, k 2 . We want to construct a similar expansions which when transformed back into the z-plane behave like the odd Lamé functions Es m+1 ν z, k 2 . We need our expansion to have the property The Jacobi elliptic function cn(z, k) is even around the origin and odd around z = −K and z = K. Hence again letting t = √ 2κ sn(z, k) we consider a formal expansion for a solution of (8.2) of the form This has the correct behaviour at z = −K and z = K. Again we will require that P s (t) is even and Q s (t) is odd. By writing we can express the formal solution in the form Expanding this square root, we can rewrite this formal expansion as where the connection between P s (t) and A s (t), and Q s (t) and B s (t) is given by where a b is the generalised binomial coefficient. Thus we determine the P s (t) and Q s (t) terms by connection with the A s (t) and B s (t) derived previously. By connection with the previous case we get the same eigenvalue expansion to all orders for both a m ν and b m+1 ν . An interlude: error analysis for the eigenvalue expansions In this section we match our eigenvalue expansions with the eigenvalues a m ν and b m+1 ν , and give order estimates for the expansions on truncation. The theory of this subsection is included in [17], where it is employed for Mathieu's equation. For this we consider Sturm-Liouville theory; for a fuller treatment on Sturm-Liouville theory see [1]. We have the differential equation The regular SL theory states that these eigenvalues are real and can be ordered such that λ 0 < λ 1 < · · · < λ ν < · · · → ∞ and these eigenvalues λ ν correspond to unique eigenfunctions y ν (x) with exactly ν zeros in (a, b). Normalised eigenfunctions form an orthonormal basis such that The following theorem is Theorem 1 in [17, § 1.52] Theorem 9.1. Consider SL to be a self-adjoint differential operator defined on a subspace A of L 2 [[a, b], w(x)dx] containing all the twice differentiable functions which satisfy the boundary conditions (9.1) and corresponding λ ν such that Let y(x) ∈ A be such that ( y, y) = 1. Now define a remainder function R(x) such that Proof . We have that (R, y k ) = ((SL + λ) y, y k ) = ( y, SLy k ) + λ( y, y k ) = (λ − λ k )( y, y k ), and using this in and these eigenvalues are real and can be ordered such that Eigenvalues of Lamé's equation The functions Ec m ν z, k 2 have exactly m zeros in (−K, K). The normalised eigenfunctions w m ν z, k 2 form an orthonormal basis such that Let L ν be the operator Letting t = √ 2κ sn(z, k), we define the truncated expansions corresponding to w m ν z, k 2 as where the A s (t) and B s (t) terms were derived previously, and c m ν,n is defined to be a function of κ so that where the dash represents differentiation with respect to t. Thus clearly w m ν,n z, k 2 ∈ A since cn(−K, k) = cn(K, k) = 0. We also define the truncated eigenvalue expansions a m ν,n = (2m + 1)κ + 2 where the µ s were derived previously and define R m ν,n z, k 2 such that L ν + a m ν,n w m ν,n z, k 2 = R m ν,n z, k 2 . We consider the operator L ν acting on D m (t) and derive L ν (D m (t)) = (2m + 1) 1 + k 2 2 t 2 − κ − k 2 (2m + 1) + κ 1 + k 2 t 4 4κ + k 2 t 6 8κ D m (t) Using the recurrence relations tD m (t) = D m+1 (t) + mD m−1 (t), D m (t) = mD m−1 (t) − 1 2 tD m (t), (9.5) we can rewrite w m ν,n z, k 2 given in (9.2) such that for s ∈ {0, . . . , n}, A s (t)D m (t) and B s (t)D m (t) are sums of varying orders of parabolic cylinder functions with constant coefficients dependent only on m and k; it then follows from (9.4) that with the solution rewritten in this form, we have R m ν,n z, k 2 = c m ν,n κ −n (· · · ) + κ −1 (· · · ) + · · · , where the terms inside the brackets are sums of varying orders of parabolic cylinder functions with constant coefficients dependent only on k and m. Note that this remainder term will be finite sum and clearly R m ν,n z, k 2 ∈ L 2 [[−K, K], dz]. As w m ν,n z, k 2 ∈ A we have that min k a m ν,n − a k ν 2 < R m ν,n , R m ν,n . In Part I, Section 5, we proved that We need an order estimate for R m ν,n , R m ν,n . The integrals we must consider then are of the form where i, j ∈ N 0 . Performing the substitution t = √ 2κ sn(z, k) we obtain Since when t is large D m is exponentially small it follows that Considering the first term of the expansion for w m ν,n z, k 2 in (9.2) we have as κ → ∞. Hence it follows from (9.3) that c m,n = O(κ 1/4 ) as κ → ∞. Similar observations would give us that and so as κ → ∞. The error analysis for b m ν,n would in the same manner give the order estimate Hence we have from (8.1) that a m ν = (2m + 1)κ + 2 In the same manner we would deduce that b m+1 ν = (2m + 1)κ + 2 This result also follows from the difference between a m ν and b m+1 ν being exponentially small as κ → ∞ (see [24, § 28.8]). Error analysis for the uniform asymptotic expansions of the Lamé functions We now use the results of Section 9 to obtain strict and realistic error bounds for the functions expansions derived in Section 8. Define the differential operator and consider t ∈ (−t * , t * ). We have the truncated expansion corresponding to an even solution such that we have the exact solution w m ν t, k 2 = w m ν,n t, k 2 + m ν,n t, k 2 . (10.1) We define the remainder term R m ν,n t, k 2 such that L ν w m ν,n t, k 2 = R m ν,n t, k 2 and split the eigenvalue such that h 2κ = m + 1 2 + n s=1 µ s κ s + µ n+1 κ n+1 , and note we just proved that µ n+1 = O(1) as κ → ∞. Since the coefficients A s (t) and B s (t) satisfy (8.4) and (8.5) it follows that R m ν,n t, k 2 = O κ −n−1 , as κ → ∞. Applying L ν to (10.1) we obtain m ν,n and denoting the right hand side of this equation Ω m ν,n t, k 2 , by use of variation of parameters we have In accordance with [22, § 6.10.2] we define J(τ ) = 1, H(τ ) = 1 + k 2 − k 2 τ 2 2κ and Since we consider t ∈ (−t * , t * ) where t * = O(1) as κ → ∞, the error analysis is much simpler than the analysis Olver uses in [21] as we have \|K (t, τ ) \| ≤ k 0 , and \|∂K (t, τ ) /∂t\| ≤ k 1 , where k 0 and k 1 are O(1) as κ → ∞. Thus again in accordance with [22, § 6.10.2] we define P 0 (t) = k 0 , Q(τ ) = 1, P 1 (t) = k 1 (10.2) (we do not define P 2 (t) as we do not need to bound \|∂ 2 K(t, τ )/∂t 2 \| to carry out our analysis), and finally the constants κ = 1, κ 0 = k 0 , κ 1 = k 1 . These eigenvalue coefficients match the formal results given in [24, § 29.7].	2015-11-24T05:17:28.000Z	2015-07-17T00:00:00.000	{ "year": 2015, "sha1": "8e80a27d8ad9c27d32d9a66e9fd5ac70cb78291f", "oa_license": "CCBYSA", "oa_url": "http://www.emis.de/journals/SIGMA/2015/095/sigma15-095.pdf", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "5cc3606b89cdcc48df26e2b6ff384e9a64e9cd41", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
153397999	pes2o/s2orc	v3-fos-license	Model-Free Discretisation-Invariant Swaps and S&P 500 Higher-Moment Risk Premia We derive a general multivariate theory for realised characteristics of `model-free discretisation-invariant swaps', so-called because the standard no-arbitrage assumption of martingale forward prices is sufficient to derive fair-value swap rates for such characteristics which have no jump or discretisation errors. This theory underpins specific examples for swaps based on higher moments of a single log return distribution where exact replication is possible via option-implied `fundamental contracts' like the log contact. The common factors determining the S&P 500 risk premia associated with these higher-moment characteristics are investigated empirically at the daily, weekly and monthly frequencies. surrounding the Lehman Brothers collapse in September 2008 market rates were frequently more than 5% greater than their fair-values -see Ait-Sahalia et al. [2012]. The fair-value swap rate is set so that the swap's expected pay-off at inception is zero. Since the seminal paper of Demeterfi et al. [1999], the computation of a theoretical fair value for a variance swap rate, i.e. the expected realised variance, has been the subject of extensive academic research. The standard fair-value variance swap rate calculation supposes three things, namely: (1) monitoring of the realised leg happens continuously; (2) the discounted underlying price follows a martingale diffusion process; and (3) vanilla options on the underlying with the same maturity as the swap are traded at a continuum of strikes. As long as these assumptions hold the fair value of realised variance -which, under assumption (1), becomes the quadratic variation of the log price -can be derived precisely from the market prices of these vanilla options by applying the replication theorem of Carr and Madan [2001]. See Jiang and Tian [2005] for further details. However, in the real world none of these assumptions hold. Consequently, a considerable body of literature has developed on quantifying the errors associated with these assumptions. In practice the realised leg of the swap must be monitored discretely and the theoretical fair-value derived under assumption (1) is subject to a bias, which decreases with the term of the swap. As shown by Carr and Lee [2009] this discretisation bias is dominated by the third moment of returns, so it is largest during excessively volatile periods. Moreover, the continuous fair-value, i.e. the quadratic variation of the log price, under (over) estimates the correct (discretely-monitored) fair value when the third moment is negative (positive). Jarrow et al. [2013] show that, for some otherwise reasonable price processes, a discretelymonitored swap rate need not exist. They derive discretisation error bounds that get tighter as the monitoring frequency increases and prove several results on the convergence of the discretely-monitored swap rate to its continuously-monitored counterpart, assuming some specific stochastic volatility diffusion processes. Bernard and Cui [2014] generalize some of the results in Jarrow et al. [2013] and give conditions for the discretisation bias to be positive or negative, and Hobson and Klimmek [2012] derive model-free discretisation error bounds and super-and sub-replication strategies for hedging variance swaps. Relaxing assumption (1), Broadie and Jain [2008] derive analytic fair-value variance (and volatility) swap rates based on the discrete-monitoring assumption. However, these swap rates are not model-free. They consider several stochastic volatility diffusion and jump models, claiming that for most realistic contract specifications the discrete monitoring error is actually smaller than the error due to the violation of assumption (2). Bernard and Cui [2014] extend their analysis to include a much wider variety of processes, but their results are still not entirely model free. Ignoring the jump component in an underlying process induces another bias to the fair-value swap rate, whose sign and size depend on the direction and magnitude of the jumps. Rompolis and Tzavalis [2013] derive bounds for this jump bias and demonstrate, via simulations and an empirical study, that price jumps induce a systematic negative bias which is particularly apparent during excessively volatile periods. Thus, the jump bias and the discretisation bias work in the same direction, to substantially under-estimate the fair-value swap rate when the term of the swap includes a particularly volatile period. This is potentially good news for the investor. If the issuer omits to add on to his premium to cover these two known biases then the investor could pay a much lower fixed rate than he would if the swap were priced under more realistic assumptions. By contrast, the issuer of the swap could find himself under-hedged during exactly those excessive volatile periods that drive the variance swap market. Hence, there are good reasons for issuer's premiums to contain substantial add-ons to cover the uncertainty in the size and sign of jump and discretisation biases. Indeed, Ait-Sahalia et al. [2012] demonstrate that traded swap rates can deviate significantly from fair-value quoted rates, especially for longer-term variance swaps, during periods where there are large jumps in the price of the underlying. Jiang and Tian [2005] also address the problems attendant to assumption (3), i.e. impossibility of exact replication due to the availability of only a finite set of strikes for traded vanilla options. They derive upper bounds for the truncation error (i.e. the error due to the use of a finite range of strikes) and use a model-dependent simulation to illustrate that truncation errors are negligible if the strike range is more than two standard deviations from the separation strike. Model-free error bounds are also provided, but they are not as tight as the model dependent ones. Based on a finite number of traded strikes Davis et al. [forthcoming] derive much tighter model-free arbitrage bounds for continuously-monitored variance swap rates. They also find that market rates are surprisingly close to their lower bound yet remain consistent with the absence of arbitrage. The terms and conditions of a standard variance swap define the realised variance as the average squared daily log return on some underlying, commonly an equity index. However, a different definition for the realised characteristic could result in fair values that are easier to price and hedge. Martin [2013] advocates the use of a sum of squared 'simple' returns, rather than log returns, to define the realised variance. With this modification both jump and discretisation biases are minimised. Likewise, the gamma swaps described by Lee [2010] weight the realised variance characteristic in such a way that replication and valuation are relatively straightforward under the continuous martingale assumption. The idea to change the definition of the realised characteristic leads us to the pathbreaking work of Neuberger [2012], which lays the foundation for our research. Neuberger demonstrates that a different modification of the definition of realised variance results in a swap which is completely free from any errors arising from assumptions (1) and (2) above. In other words, an exact theoretical fair-value swap rate can be derived for any price process without assuming continuous monitoring. Indeed, the expected realised variance does not depend on the frequency of monitoring and the same fair-value swap rate applies whether the floating leg is based on intra-day, daily, weekly, or monthly returns. In fact, the monitoring of the floating leg does not even have to be regular; any time-partition of the term of the swap can be defined without affecting the fair-value swap rate. The only assumption made about the underlying is that the market is free from arbitrage opportunities. The crucial condition is that the function used for defining the floating leg (the realised characteristic) must satisfy an aggregation property. Using this property Neuberger introduces a particular third-moment swap where the floating leg is based on a new realised skewness characteristic. The starting point of our research is an extension of Neuberger's ideas to other characteristics of a price (or a log return) distribution. We introduce a vector space (over R) of time-discretisation invariant (TDI) swaps which contains an infinite variety of second, third and higher-moment swaps, and swaps that are not even associated with moments of the underlying distribution. The realised leg of a TDI swap is defined in such a way that the discrete monitoring error is zero, which implies that the jump error is also zero, and that the aggregation property is satisfied. We show that, provided some technical conditions, the absence of a discrete monitoring error as well as the aggregation property are equivalent to a second order system of partial differential equations and provide elegant analytic solutions for the characteristics. The practical importance of our work is that, based only on the noarbitrage assumption and without requiring any further model specifications, the theoretical fair-value rate for a TDI swap can be derived exactly from vanilla option prices, i.e. both the discretisation bias and the jump bias are zero. An infinite variety of TDI swaps can be defined. For brevity we focus on just a few interesting examples, including third-moment and fourth-moment swaps for which the associated risk premiums have relatively low correlation. In all these examples the theoretical fair-value swap rate may be expressed in terms of prices of certain synthetic fundamental contracts, of similar ilk to the log and entropy contracts introduced by Neuberger [1994] and Neuberger [2012]. Since the same fair-value rate applies to a realised characteristic irrespective of the monitoring frequency, the fair value of a floating-floating swap which exchanges a daily-monitored for a weekly-monitored realised characteristic is zero. However, we demonstrate both theoretically and empirically that the risk premium on a TDI swap is non-zero, in general, and that it will depend on the monitoring frequency. This leads to the definition of frequency swaps, which exchange one monitoring frequency for another. Similarly, the TDI risk premium depends on the maturity of the swap. This observation motivates the introduction of calendar swaps, which exchange two TDI characteristics that refer to different maturities, and which provide direct access to the forward variance, third moment or fourth moment risk premiums. While it is reasonable in practice to assume an arbitrage-free market, so that the underlying is a martingale, the supposition that options on a continuum of strikes are traded is certainly not valid. Indeed, as previously discussed, when this assumption is needed an approximation error appears in the actual calculation of the theoretical fair-value swap rate. This error can be relatively large, especially during volatile markets when the volatility skew is pronounced -see Alexander and Leontsinis [2011]. Motivated by this observation we introduce a subspace of swaps whose fair value may be computed exactly, without the need for numerical integration, as a discrete weighted sum over vanilla option prices at the available, traded strikes. These 'strike-discretisation invariant' swaps also preserve the TDI property, so we refer to them using the more general term discretisation invariant (DI) swaps. A particular subset of DI swaps is called straddle swaps because their theoretical swap rates are simply the product of the market prices of a call and a put options with the same strike. The returns on these swaps have very low correlation with returns on variance swaps. In the following: Section 2 sets the background by briefly describing the errors that enter the calculation of theoretical rates for standard variance swaps and relating them to the aggregation property introduced by Neuberger [2012]; Section 3 presents the aforementioned second order system of partial differential equations, derives the vector space of realised characteristics for TDI swaps, develops examples of variance and higher-moment swaps for which the fair-value swap rates and replication portfolios may be expressed in terms of fundamental contracts, and focuses on a particular subset of DI swaps called 'straddle swaps' for which replication and valuation are particularly simple; Section 4 analyses the risk premiums on TDI swaps under the geometric Brownian motion assumption and introduces 'frequency swaps' and 'calendar swaps' based on TDI characteristics; The empirical results are presented in Section 5, and Section 6 concludes. All proofs are in the Appendix. 2 Background and Motivation The distinction between martingale and non-martingale processes will be central to our arguments so it helps to distinguish them in our notation. Univariate martingale processes are here denoted using upper-case letters; and non-martingales with lower-case. We can simplify notation because we need to consider only one maturity date, T , but various partitions of the interval Π := [0, T ] are also needed, in particular the regular partition Π D := {0, 1, . . . , T } which we term the 'daily' partition for brevity. The increments along a partition are denoted using a 'carat' (.), e.g. for some process y we setŷ t := y t − y t−1 for increments under the daily and let x := {x t } t∈Π be the log futures price process, i.e. x t := ln F t . Using our notation the standard, daily, realised variance may be written: wherex t := x t − x t−1 denotes the daily log returns. In practice, the floating leg of a variance swap is set equal to the average realised varianceT −1 Π Dx 2 t , whereT denotes the number of trading days during the lifespan Π, rather than the total variance as in (1). However, including this level of detail would only add an unnecessary level of complexity to our analysis. Carr and Wu [2009] discuss the idealised case where continuous monitoring is possible. In other words, the realised variance (1) becomes the quadratic variation of x, denoted x T . Then, under the assumption of a generic decomposition of the underlying process into a pure jump and a pure geometric diffusion component, they apply the replication theorem of Carr where q(k) denotes the forward price of a vanilla out-of-the-money (OTM) option with strike k and maturity T . 2 The jump error, η, is zero when F follows a pure diffusion. The second main source of error in the theoretical fair-value swap rate stems from the fact that the realised leg of the swap is monitored only at discrete points in time. For instance, when monitoring is based on the daily partition the discrete monitoring error may be written Both these errors affect the theoretical price of the fixed leg. For instance, with the realised variance (1) the fair-value variance swap rate may be written By ignoring these errors the risk-neutral expectation of the pay-off becomes η + ε rather than zero and therefore the estimator for the variance risk premium is biased under the standard variance swap pricing formula. In practice, the integral in (3) that is used to approximate the fair-value swap rate must be estimated using the prices of vanilla options that are actually traded. So there is a third, estimation bias affecting the actual computation of the fair-value rate approximation. Typically we use a fairly restricted range of quoted strikes, because deep-OTM options lack 1 In an arbitrage-free market, as introduced by Harrison and Kreps [1979], the bank issuing a variance swap to a representative investor will compute this expected pay-off under a risk-neutral measure Q. In a complete market the risk-neutral measure for a representative investor corresponds to the market implied measure M (see Breeden and Litzenberger [1978]). In this case a unique fair value for the variance swap rate may be derived as the expectation of realised variance under M. sufficient liquidity to have reliable prices. Indeed the estimation error can be quite significant, especially during volatile periods, as shown by Alexander and Leontsinis [2011]. The second aim of our research is to define characteristics that are also free from this estimation error because their fair-value can be derived without using the replication theorem of Carr and Madan [2001]. This second property is written Π N → Π for brevity. Clearly, the daily partition Π D is a special case of Π N where t i = i and T = N . To be completely general we allow the realised characteristics to describe distributional properties of an n-dimensional stochastic process z := {z t } t∈Π ∈ R n . Then, given a continuous function f : R n → R, the realised whereẑ i := z t i − z t i−1 denote the increments in z along Π N . If it exists (and this depends on the choice of f and z) we define the f -variation of z as the continuously monitored limit of the realised characteristic, i.e. In the following we only consider characteristics f with f (0) = 0, otherwise the limit in (5) would not be finite. 3 The f -variation is a purely theoretical construct that, if it exists, can be used to derive a theoretical fair-value swap rate by taking its expected value based on some assumed process for the underlying. This is the approach taken by Jarrow et al. (2013) and many other papers that analyse the discrete monitoring error for variance swaps. As long as the f -variation exists and is finite the discrete monitoring error for an f -swap under the partition Π N may be written For instance, with z = x and f (x) =x 2 the definition (5) corresponds to the quadratic variation of the log price and the discrete monitoring error is given by (2). For a given f and z there may be zero, one or more partitions Π N of Π for which ε N (f, z) = 0. Our focus is on those combinations of f and z for which If (7) holds ∀ Π N then it holds for the trivial partition Π 1 = [0, T ], for which the above Note that the lack of path-dependence of this expectation also implies that the jump error η must be zero. In other words, when the discrete monitoring error is zero under all partitions then, even if investors differ in their views about jump risk in an incomplete market, they would still agree on the fair-value f -swap rate as long as they agree on the measure for E[.]. The aggregation property (8) was introduced by Neuberger [2012], albeit with different notation and motivation. He also noted that exact pricing of a discretely monitored swap is possible when the aggregation property (AP) holds because the replication theorem of Carr and Madan [2001] is applicable to the right hand side of (8), which he called the 4 Although the absence of a discrete monitoring error (7) is a stronger assumption than the aggregation property (8), we show in Corollary 1 that the two properties are equivalent as long as f is twice continuously differentiable and the f -variation of z is finite. In other words, if the f -variation does exist (and we do not need to assume this for the main results in our paper) then (8) holds for a pair (f, z) iff ε N (f, z) = 0. implied characteristic. 5 The AP does not hold for (f, x) when f (x) =x 2 is the standard variance characteristic, but Neuberger finds two alternative variance characteristics: The log characteristic l (x) := 2 ex − 1 −x for which the AP holds when x is the log of any martingale; and the entropy characteristic h (x) := 2 xex − ex + 1 which satisfies the AP under the additional assumption of independent incrementsx i , i ∈ {1, . . . , N }. Using Taylor expansion about the origin, one can see that both g ≡ l and g ≡ h may be associated with the second moment of the distribution ofx, because they satisfy limx →0 g (x) /x 2 = 1. Neuberger also classifies the functions f which satisfy the AP when: (i) z = {F, ψ} and Two particular subsets of this second class of AP-characteristics correspond to v gt : . These are called the log and entropy variance processes because they are closely related to the log contract, which pays x T , and the entropy contract, which pays F T x T at maturity, respectively. Because the log characteristic is an APcharacteristic w.r.t. the log of any martingale, x, one can change the definition of the floating leg of a variance swap from (1) to Π D l (x t ), and the result will be a log variance swap that has no discrete monitoring error and no jump error. In this case (3) becomes Neuberger also values and replicates a third-moment AP characteristic whose risk premium is strongly correlated with the variance risk premium (see Kozhan et al. [2013]). This further motivates our search for a more general class of (f, z) for which the AP holds and through which more diverse risk premiums can be accessed. Discretisation Invariant Swaps Consider a multivariate stochastic process z ∈ R n which contains only deterministic functions of futures prices F := {F t } t∈Π ∈ R d of d tradable assets, or derivatives on these assets, in an arbitrage-free market. The process z may e.g. contain futures prices or the logs of these prices and we make the minimal, arbitrage-free assumption only to ensure that the futures prices follow a multivariate Q-martingale. Let ∆ ∈ R n×d and Γ ∈ R n×d×d denote the first and second partial derivatives of z w.r.t. F. Now let f : R n → R be some twice-differentiable deterministic function and denote by J (ẑ) ∈ R n the Jacobian vector and H (ẑ) ∈ R n×n the Hessian matrix of first and second partial derivatives of f w.r.t.ẑ. A time-discretisation invariant (TDI) swap is any f -swap on z for which the discrete monitoring error (7) is zero and consequently the aggregation property (8) holds. Two trivial TDI swaps follow immediately from the definition: (a) if f is linear, say f (ẑ) = a ẑ for some a ∈ R n , then (8) holds for any process z because so (8) holds for any function with f (0) = 0. Note that (7) also holds in both cases: in (a) because z f T = z T − z 0 and in case (b) because z f T = 0, provided f (0) = 0. Therefore, in order to find the most general set of characteristics that define TDI swaps we shall assume that z can be stochastic and we consider only those characteristics f ∈ C 2 for which f (0) = 0. Theorem 1: Equivalence of the Aggregation Property If (f, z) is such that either the AP (8) holds, or the f -variation of z exists and (7) holds, then the following second-order system of partial differential equations holds: Moreover, if F follows a diffusion with finite f -variation, then (7), (8) and (9) are equivalent. The above system can be solved numerically to yield, for given z, the characteristics that define a TDI swap on z. However, we are only interested in the analytic solutions of (9), for which we can value and replicate realised TDI characteristics, as in the following: Theorem 2: Time-Discretisation Invariant Characteristics Let F follow any d-dimensional martingale process and set z = (F, x) with x = ln F. 6 Then the solutions to (9) form vector space over R, defined by: The idea of the proof is to find candidates for V by solving (9) for this particular z and then show, by straight forward evaluation of (7), that the necessary condition is sufficient. The generality of the conditions for this theorem allows TDI swaps to be defined for a wide variety of underlying variables. For instance, we can include the log contract Λ t : Note that with z = (F, Λ, x) we can relate the characteristics introduced by Neuberger [2012] to specific characteristics in V. 7 Before examining some specific examples of TDI swaps on z = (F, x) we prove one further result which provides a characterisation of the value process for a general TDI swap in a form that clarifies how to replicate them. From henceforth we assume that replication is under the risk-neutral measure Q. Also, for ease of exposition we use the daily partition Π D in the text, although all proofs in the Appendix are for general Π N . Since the fair-value f -swap rate equals the risk-neutral expectation of the realised f -characteristic, the value process V := {V t } t∈Π of the f -swap is given by Theorem 3: Replicating TDI Swaps The increments along Π D of the value process for a TDI f -swap may be written Theorem 3 characterises the unrealised profit and loss (P&L) which accrues to the issuer of a TDI swap who pays the fixed swap rate E Π D f (ẑ t ) and receives the floating leg defined by the realised characteristic. When the elements of F and the contract U := {U t } t∈Π are both tradable the swap can not only be priced exactly, but is also replicable in discrete time by means of a dynamic trading strategy. For instance, the value increments of the swap on the log characteristic l (x) : 0 and the value increments of this swap may be However, it is more convenient to follow Theorem 3 and use the square contract to writeV Σt =Σ t − 2F t−1Ft . Hence, this swap can be hedged by selling a square contract and dynamically holding 2F t−1 futures from time t−1 to t. Example 2: Covariance Swap. Let z = (F, Σ) and f (ẑ) =FΣ. This TDI characteristic describes the covariance of the value increments in the futures and square contract. The fair value swap rate can be derived using the replication theorem of Carr and Madan [2001] as The P&L on this swap is obtained from setting α = 0 in Theorem 3, with Ω 12 + Ω 21 = 1, the other elements of Ω being zero, yieldinĝ Hence, this covariance swap can be hedged by selling a cube contract and holding F t−1 squared contracts plus Σ t−1 futures from time t − 1 to t. the fair-value swap rate is a third moment. The unrealised P&L for this swap may be written: Hence, the swap can be hedged exactly by selling a cube contract and dynamically holding F t−1 + 2F 0 square contracts as well as Σ t−1 − 4F 0 F t−1 futures. The nice association of this swap with the third moment comes at the price that the hedge ratios depend on the initial price level F 0 . Example 4: Fourth Moment Swap. Let z = (F, Σ, Q) and consider the characteristic Following Theorem 3 we havê Hence, this swap can be hedged exactly by selling a quartic contract and dynamically holding cubed, squared and forward contracts according to the above hedging ratios. , so the swap can be hedged exactly by selling an entropy contract and dynamically holding F t−1 log contracts as well as Λ t−1 futures contracts. For another example set z = Λ and f (ẑ) =Λ 2 . Then we have a squared-log swap, so-called because the fair-value swap rate is E (Λ T − Λ 0 ) 2 , which may be expressed in the form Υ 0 −Λ 2 0 . This swap rate corresponds to the variance of the log price since Υ 0 − Λ 2 Following Theorem 3 we haveV Υt :=Υ t − 2Λ t−1Λt . Hence, this swap can be hedged exactly by selling a squared log contract and dynamically holding Λ t−1 log contracts. The pricing and hedging of all swaps presented in the above examples relies on the replication of certain fundamental contracts. Table 1 summarises the pricing formulas and Table 2 the hedge portfolios for these contracts, all obtained by applying the theorem of Carr and Madan [2001] to the implied characteristic. The difference between the two replication methods is that the pricing formula is based on OTM options which are more liquidly traded -but not investable due to the stochastic separation strike -and the hedge portfolio involves options that are OTM only at inception but remain investable. In particular, the hedge portfolios describe buy-and-hold strategies that require no dynamic rebalancing. However, for an ex-post analysis the two representations are exchangeable and we choose to implement the pricing formula for liquidity sake. Contract Variable Fair Value Pricing Formula Log One of the challenges faced by issuers of standard variance swaps is to hedge the realised variance through dynamic rebalancing of an options portfolio which is tilted towards the low Hedge Portfolio Cash Futures Puts Calls strike options via the weight k −2 in the replication formula for the log contract. This factor is also present, though moderated by the factor (1 − ln k), in the squared-log contract. In the entropy contract, which is another generalized variance swap contract, the weight is only k −1 , but still this causes problems because it places most weight on very low strike options. These are the illiquid and expensive deep-OTM put options for which demand much exceeds supply during market crashes, because they provide insurance for risk-averse investors. The illiquid market in such options on single-name equities during the financial crisis of 2008-9 is the main reason why equity variance swaps are now focussed mainly on indices, rather than individual stocks. An additional advantage of the new cube and quartic fundamental contracts is that they are tilted more toward high-strike options, the OTM calls where transactions costs are lower and the market is more liquid. In all cases integration over a continuum of option strikes is necessary, but in practice vanilla options are traded on a relatively small number of discrete strikes. We now introduce a class of strike-discretisation invariant swaps that can be priced and replicated exactly based only on the available vanilla option prices, while preserving the time-discretisation invariance property. We refer to these swaps as discretisation-invariant (DI) swaps because, like TDI swaps they have the same fair value, independent of the partition Π N , which is free from both discrete monitoring and model-specific (e.g. jump) error. But also, their fair-value can be computed exactly without requiring an integral approximation based on a continuum of traded option strikes. By the same token, these swaps can be hedged exactly, without having to replicate (imperfectly) the log, or entropy, or other fundamental contract. Let P := {P t } t∈Π and C := {C t } t∈Π describe the forward price processes of d vanilla put options and d vanilla call options, with identical, traded strikes k, on an underlying futures F with maturity T . That is, where 1 := (1, . . . , 1) ∈ R d . Assume w.l.o.g. that the traded strikes k := (k 1 , . . . , k d ) ∈ R n are ordered such that k 1 < k 2 < . . . < k d , and denote byP andĈ the increments in P and C, respectively, along some partition of [0, T ]. By Theorem 2, the vector space contains all TDI characteristics f for z = (P, C). The fixed leg of the corresponding swap can be derived as However, in the case that Ω is a lower triangular matrix, we have since the strikes are in ascending order and hence either the put pay-off or the call pay-off is zero, for each component. Therefore an exact swap rate can be derived only based on the prices P 0 and C 0 of traded vanilla options with strikes k, without using the replication theorem of Carr and Madan [2001]. Now, by Theorem 3, the value increments of this swap arê Hence, the swap can be hedged exactly by dynamically holding α − C t−1 Ω j puts and γ − P t−1 Ω j calls with strike k j for j = 1, 2, . . . , d. Example 6: Straddle and Strangle Swaps. Let P := {P t } t∈Π and C := {C t } t∈Π describe the forward price processes of a vanilla put option with strike k p and a vanilla call option with strike k c , i.e. P t := E t (k p − F T ) + and C t := E t (F T − k c ) + . Since z = (P, C) follows a Q-martingale, f (ẑ) =PĈ is a TDI characteristic and the corresponding fair-value swap rate is E Under the additional restriction k p ≤ k c , E [P T C T ] = 0 so the fair value swap rate is simply the negative of the product of the call price and put price. Following Theorem 3,V St = −P t−1Ĉt − C t−1Pt and the swap can be hedged exactly by dynamically holding P t−1 calls and C t−1 puts from time t − 1 to t. Risk Premiums By definition, the expected realised characteristic for a TDI swap is independent of the monitoring frequency under Q. However, its expectation under the physical measure P, and therefore the risk premium that it captures, is not. The relationship between model assumption and risk premiums for TDI swaps is an interesting area for future research which goes beyond the scope of this paper. In this section we are content to derive an expression relating the risk premium to the monitoring frequency for a simple TDI variance swap, based on a geometric Brownian motion for the underlying price. To this end, consider the variance swap of Example 1, under the assumption with µ = 0 so that the variance risk premium may also be non-zero. Assume further that the swap is monitored under the regular partition Π N = {iτ } i=0,...,N for some N ∈ N + , with τ = T /N so that the monitoring frequency increases as τ decreases. We compute the expectation of the realised characteristic under P and examine how the deviation of this expectation from the fair-value swap rate (i.e. the risk premium) depends on τ . In Example 1 we have z = F and f (ẑ) =ẑ 2 so the fair-value swap rate is E Q (F T − F 0 ) 2 = F 2 0 e σ 2 T − 1 > 0. In the Appendix we prove that the expected realised characteristic under P may be expressed in the form: where κ := 2µ + σ 2 , so that κ = 0 corresponds to the case where ln F follows a martingale under the physical measure. Now using Taylor expansion of the exponential terms above and letting τ → 0 we obtain the following expression for the expected realised characteristic of a continuously monitored variance swap: The risk premium for a continuously monitored swap may thus be written We show in the Appendix that the sign of the risk premium coincides with the sign of κ − σ 2 . Now, to determine the relationship between τ and the risk premium, consider d dτ The sign of the derivative coincides with the sign of \|κ\| − σ 2 , as follows from some tedious algebra. That is, for small negative drifts (−σ 2 < µ < 0) the risk premium on the discretely monitored variance swap increases with the monitoring frequency, converging to a negative limit. For both µ = 0 and µ = −σ 2 the risk premium does not depend on the monitoring frequency, being equal to zero in the first and negative in the second case. For positive or strongly negative drifts (µ < −σ 2 ) the risk premium decreases as the monitoring frequency increases. Empirical Analysis Our empirical study is based on the daily closing prices P t and C t of all traded European put and call options on the S&P 500 between January 1997 and December 2013. We eliminate quotes that fulfil any of the following criteria: less than seven calendar days to maturity, more than 365 calendar days to maturity, zero trading volume, mid-price ≤ 0.5 or an implied Black Scholes volatility ≤ 1% or ≥ 1. For each trading day, we further delete all quotes that refer to the same maturity if less than three different strikes are traded. The forward price is backed out via put-call-parity for each maturity from the pair of quotes whose strike minimises \|P t − C t \|. This forward price is also used as the separation strike between OTM put and call options, i.e. we use the put price for k < F t and the call price for k ≥ F t . We then apply a cubic spline smoothing algorithm according to Fengler [2009] in order to preclude static arbitrage in both the strike and maturity dimension (calendar arbitrage). This yields OTM option prices q t (k) that cover an equally distributed grid of 2000 strikes k j on a six-σ-range around the forward price, σ being the average implied volatility of the sample. Outside the domain of the spline we assume the implied volatility to be constant and equal to the implied volatility at the closest strike. These data are integrated numerically w.r.t. k to derive time series of daily prices for the contracts with fixed maturity dates in Table 1. 9 We then calculate trading day, weekly (five trading days) and monthly (20 trading days) value increments (i.e. profit and loss, P&L) for these contracts and the corresponding increments for the log variance, the variance, covariance, third moment, fourth moment, entropy, squared-log and straddle swaps based on Theorem 3 and the example applications. 10 We aim to provide a large-sample time-series analysis of the properties of TDI and DI swaps, examining risk premiums over a period of 16 years. To this end we convert the contracts with fixed expiry dates into synthetic, constant-maturity contracts that are still investable, i.e. replicable by holding a portfolio of futures and options. For this purpose we follow Galai [1979], applying linear interpolation of the option-price value increments, not the prices themselves, between the two adjacent maturities. We apply a similar constant-maturity transformation to the swap value increments, i.e. the value increment between time t − 1 and time t of a contract Φ with constant time-to-maturity t c iŝ Φ ut whereΦ lt andΦ ut denote the increments in the prices of the contracts with fixed maturity dates T l and T u . 11 Now, by definition, and the same holds for our TDI swaps, since they are portfolios of these contracts. However, under the physical probability measure the expected increments in these contracts and swaps need not be zero, in the presence of a risk premium. Table 3 shows the average risk premiums for 30-day swaps that are monitored daily, 9 For example, the entropy contract is approximated by Ψ t ≈ F t x t − 2000 j=2 k −1 j q t (k j ) (k j − k j−1 ) and similar approximations apply for Λ, Υ, Σ, Q and H. 10 Note that it is only possible to determine a value increment if at least three options with different strikes and the same fixed maturity are quoted on both trading days. 11 From now on there is a slight of notation here in that the incrementsF t ,Λ t , etc. refer now to increments in the constant-maturity time series, rather than the fixed maturity series that we have used for developing the theory. Table 3: Average risk premiums over 16 years, for 30-day constant-maturity contracts based on daily, weekly and monthly monitoring, for: futures (F ), covariance swap (V P ), third moment swap (V Q ), fourth moment swap (V H ), log variance swap (V Λ ), variance swap (V Σ ), squared-log swap (V Υ ), entropy swap (V Ψ ) and straddle swaps with strikes 1000, 1100 and 1200. weekly and monthly over the entire 16-year sample period. Besides the daily partition Π D we include results for the weekly and monthly partitions, denoted Π W and Π M . Each premium is computed as the average value increment divided by its standard deviation, and annualised to enable comparison between daily, weekly and monthly increments. The premium for the variance contracts are negative, those for the third moment swaps and straddle swaps are positive, and the fourth-moment risk premium is small and negative, on average. By contrast, the P&L on the third-moment swap decreases only during these crisis periods, when the negative skew in realised returns on equity indices becomes pronounced. During crisis periods it is also more negative for daily-monitored third-moment swaps than it is for weekly or monthly monitored swaps. Otherwise, during stable trending markets it is small and generally positive, particularly since the crash in September 2008, and does not depend much on the monitoring frequency. During the last 5 years of the sample, except during August 2011, the skew in realised equity index has been more positive than the skew in the implied characteristic, which is inferred from option price distributions. The risk premium on the fourth-moment swap is more variable over time. The returns on this swap are sensitive to downward jumps in the index and the P&L is strongly positive during the crisis periods. It also tends to exhibit a highly variable but generally negative P&L immediately prior to a market crash. For instance, this is evident during the period leading up to the technology crash in year 2000 and during 2007, the period preceding the banking crisis. At these times realised returns on S&P500 futures have significantly less kurtosis than the kurtosis in the distribution implied by option prices, with the implied distribution reflecting a high degree of uncertainty about forward prices. Notice that the fourth-moment swap has a particularly variable, negative P&L during 2013, the last year of the sample, a period when S&P500 futures prices exhibited a very strong upward trend amid much uncertainty in the global economy. and 1200 when monitored at different frequencies. 12 The risk premium on these swaps can be large and negative during a crisis, e.g. in September 2008 and August 2011. Otherwise, the risk premium is small and positive and greater for straddle swaps that are monitored weekly or monthly than for straddle swaps that are monitored daily. Table 4 presents correlations between the daily (and weekly and monthly) unrealised P&Ls on the moment and straddle swaps that we have previously examined. In each case moments variances straddles For instance, at the daily frequency V P has a correlation of 0.98 with V Σ , 0.78 with V Λ and 0.90 with V Ψ . Hence, the risk premium on the covariance swap is already accessible through trading variance swaps. As is expected from several previous empirical studies the correlation between the daily P&L on the futures and the variance swaps is around −0.6; this decreases (in magnitude) marginally when measured (and monitored) at the weekly and monthly frequencies. The P&L on all variance swaps are very highly correlated at every frequency. Thus, as is also evident from Figure 1, these swaps compensate the investor for downward shocks in the futures by a strongly positive realised variance. The third-and fourth-moment swaps are more interesting. As expected there is a strong positive correlation between the futures P&L and the P&L on the third-moment swap, but this decreases with frequency so that the monthly P&Ls have a correlation of 0.7. The empirical features of this third-moment swaps and the one considered by Kozhan et al. [2013] are very similar. In particular, the third-moment swap may be attractive to variance-swap issuers as a hedge given its strong positive performance during crisis periods, when large losses are made on short variance swaps positions. However, given the very high negative correlation between the P&Ls on third-moment and variance swaps no attractive new diversification characteristics have been identified here. By contrast, the fourth-moment swap could be an excellent diversifier of both equity and variance. It has an unusually significant negative correlation with the futures which also is largely independent at frequency. The correlation between the futures P&L and V H is −0.88 at both daily and weekly frequency and −0.89 at the monthly frequency. Clearly, fourth-moment swaps have the potential to offer equity diversification, but their risk premium is clearly distinct from the variance premium. Until now we have considered swaps with a synthetic, constant maturity of 30 days. At each monitoring period (daily, weekly or monthly) we have re-balanced our position using the investable methodology of Galai [1979] to roll over into another swap with 30-days to maturity. To investigate how the characteristics of these synthetic contracts compare with longer maturities, Table 5 presents the standardized risk premiums on the S&P500 futures contract and the TDI moment swaps of Examples 1, 3 and 4, with realised legs monitored under the daily, weekly and monthly partitions. The risk premium decreases in magnitude with the maturity of each swap. For instance, the risk premium on the 30-day variance swap Σ is −0.72 compared with −0.46 for the 180- 30 90 180 30 90 180 30 90 180 30 90 (a) Frequency Swaps: These exchange two realised legs of the same maturity that are monitored at different frequencies. Conveniently, the AP implies that the fair-value rate on this type of swap is zero, by definition, but the risk premium may be positive or negative depending on the sample period; and (b) Calendar Swaps: These exchange two realised legs of different maturities that are monitored at the same frequency. Since all payments up to the lower maturity cancel out, these swaps give access to forward variance, skewness and kurtosis and therefore the corresponding risk premium term structure. From Table 5 the risk premium is positive for variance calendar swaps, negative for third moment calendar swaps and close to zero (no more than 0.02) for fourth moment calendar swaps when receiving the longer and paying the shorter maturity. The largest absolute premium (-0.39) is paid on a weekly monitored 180-for-30-day third moment swap, indicating strong backwardation of the skewness term structure. Another interesting feature about the fourth-moment frequency swap is that the P&Ls from these swaps are very highly correlated as we move along the term structure, as is evident from the fact that the black, blue and red lines in this chart from Figure 3 almost coincide. A possible explanation for this is that the risk premium on the fourth moment swap is dominated by expectations about jumps in the index, and that such expectations are only held over the short-term. In other words, all the jump risk premium is contained already in the 30-day fourth moment swap. Conclusion The advantages to a prospective issuer of TDI swaps (and DI swaps in particular) are evident: the residual hedging risks are much smaller than they are for standard variance swaps; the theoretical fair-value swap rate is independent of the monitoring frequency (it is the same for swaps that are monitored daily as for those that are monitored weekly or monthly based on the same realised characteristic); and not only is the fair-value swap rate free of a discrete monitoring error, it is model independent, indeed we can derive it assuming only that the futures price follows a martingale. For DI swaps exact fair values can be computed from traded option prices even without requiring the approximation of numerical integration. Theorem 1 allows us to find all characteristics which have this property, by solving a second order system of partial differential equations, for any set of deterministic functions of a multivariate martingale process. Theorem 2 focusses on a particular sub-class of these swaps, i.e. those for which the characteristic depends only on a multivariate martingale itself, and its logarithm. In this case, the non-trivial TDI characteristics are quadratic forms of the martingale processes plus exponentials for the log-martingale processes. The rich variety of TDI characteristics that capture third, fourth and higher-moment risk premiums is accessed by letting the underlying multivariate martingale processes contain these conditional moments. Within this variety of time-discretisation invariant (TDI) swaps we have focussed on some special swaps corresponding to second, third and fourth-order moments of a single futures price or log return distribution. These swaps have fair-values that can be computed from those of so-called 'fundamental contracts', each of which can be derived from vanilla option prices using the standard replication theorem. The fair-value computation still has an error, though relatively small, because we must use numerical integration over the option prices at traded strikes to approximate the optionprice integral formula for the fair-value of the fundamental contracts. However, a second sub-class of TDI swaps can be defined for which this strike-discretisation error is zero. These 'discretisation invariant' swaps have characteristics defined by bilinear forms of traded call and put prices. Again, an infinite variety of such DI swaps exists and we have only examined 'straddle' swaps in depth, which are based on a single put and call at the same strike. Our empirical analysis, spanning a 16-year sample period, demonstrates that a diverse variety of risk premiums are available to trade via these swaps. By contrast with the variance and third-moment swaps introduced by Neuberger [2012], and later analysed empirically by Kozhan et al. [2013], the fourth-moment and straddle-swap risk premiums that we introduce are not highly correlated with the variance risk premium. Indeed, some novel sources of risk become tradable via the creative use of these new swaps and they should be attractive to investors seeking new sources of diversification as equities, commodities and bonds become more highly correlated. Furthermore, the lack of error in the pricing formulas for discretisation-invariant swaps, plus the exact dynamic hedging portfolios that can be used to replicate them, considerably reduce the uncertainties faced by their issuers. Applying (15) to each summand in (4) yields where m(t) := max{t i ∈ Π N \|t i ≤ t}. Taking the limit as Π N → Π yields the f -variation where J := J (0) and H := H (0). With (15) and (17), the condition (7) is equivalent to Substituting (13) and (14) in the above, and considering that E Q [dF t ] = 0, shows that (7) is equivalent to Applying a spectral decomposition to the symmetric matrix in curly brackets above yields where Λ t = diag λ 1 t , . . . , λ d t is a diagonal matrix of eigenvalues and E t is an orthogonal matrix of eigenvectors. In order to derive a necessary condition for (7), following the same logic of refinement as before, we can select a specific local volatility process. Let us now assume that where ξ ∈ R is an arbitrary constant. Because exp {YXY −1 } = Y exp {X} Y −1 for X, Y ∈ R d×d as long as Y is invertible, we have Inserting (19) and (20) into (18) which implies that all eigenvalues in Λ t must be equal to zero. Hence we know that both sides in (19) are zero and, given that this must hold for all F t and z 0 , we can write where F andẑ are independent variables. We have derived this d × d system of partial differential equation based on the assumption that F follows a particular local volatility process, so it represents a necessary condition for the more general case where F can be any martingale diffusion. However, since (21) is also sufficient for (18) to hold, the two conditions are equivalent. A.2 Proof of Corollary 1 The proof of Theorem 1 can be performed analogously for the AP by substituting (15) and (16) into condition (8) and yields the same solution (21). This version does not require the existence of the f -variation. Swaps associated with α are TDI since lim Π N →Π Π N α ẑ i = α (z T − z 0 ) even without expectation for any process. For the swaps associated with Ω we can apply where the only requirement is that F follows a martingale (not necessarily an Itô process). Finally, for all swaps associated with β we have Therefore, if z = (F, x) , the necessary condition (21) is sufficient for all martingales. A.5 Risk Premiums Let the forward price follow the dynamics dF t = F t σdW Q t = F t µdt + σdW P t , F 0 > 0, for some σ > 0 and consider the regular partition Π N = {iτ } i=1,...,N with τ = T /N for some N ∈ N + . Recall that E P r [F t ] = F r e µ(t−r) for r ≤ t and E P 0 [F 2 t ] = F 2 0 e κt with κ = 2µ + σ 2 , where setting µ = 0 yields the corresponding expectations under the risk-neutral measure. The fair-value swap rate of the TDI variance swap of Example 1 is The expectation under P of the discretely monitored realised characteristic is e κiτ − (2e µτ − 1) e κ(i−1)τ = F 2 0 (e κτ − 2e µτ + 1) The risk premium on a continuously monitored swap has the same sign as κ − σ 2 . For κ = 0 we can apply the inequality ln (1 + σ 2 T ) < σ 2 T to show that the risk premium is negative. If κ = 0 we observe that the risk premium is zero for κ = σ 2 (i.e. µ = 0). Due to the dominance of exponential growth (decay) the risk premium is positive (negative) for κ < σ 2 (κ > σ 2 ). In order to determine the asymptotic of the risk premium as τ → 0 we look at which has the same sign as \|κ\| − σ 2 . It is easy to verify that the derivative is zero for the case κ = σ 2 , which corresponds to µ = 0. For κ = 0 we apply the inequality ln (1 − µτ ) < −µτ to show that the derivative is negative. For all other cases we consider the monotonic function (1 − e xτ ) /x, with a removable discontinuity at the origin, to argue that (1 − e µτ ) κ = (1 − e κτ ) µ if and only if κ = µ, which corresponds to the case κ = −σ 2 where the derivative again equals zero. The conclusion about the sign of the derivative follows from continuity.	2016-02-04T03:01:19.000Z	2014-04-04T00:00:00.000	{ "year": 2014, "sha1": "811c500e2ab1d92d7797b0dc95e859bf50d71fa1", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "08b98ab7f2d997f0a5e277f212d838a788c15854", "s2fieldsofstudy": [ "Mathematics", "Business", "Economics" ], "extfieldsofstudy": [ "Mathematics", "Economics" ] }
263648889	pes2o/s2orc	v3-fos-license	Place-Based Strategies Addressing Neighborhood Environments to Improve Perinatal and Preterm Infant Outcomes Preterm birth (defined as birth <37 weeks of gestation) is a significant health concern globally, with lasting implications for individuals, families, and society. In the United States, high preterm birth rates among Black and low-income populations likely result from differences in environmental exposures. Structural racism and economic disadvantage have led to unequal distribution of polluting industrial sites and roadways across society as well as differential access to health-promoting resources which contribute to preterm birth risk. Once born, preterm infants remain at risk for numerous environmentally responsive adverse health outcomes that affect growth and development throughout childhood and adulthood. In this commentary, we describe associations of neighborhood environments with pregnancy and preterm infant health outcomes and propose strategies to address harmful exposures that affect families across the lifespan. Introduction In the United States, the preterm birth rate rose 4% in 2021 to 10.49%, after an overall decline from 2007 to 2020 [1], with persistent significant racial disparities.The preterm birth rate is 50% higher for Black compared to White individuals [1][2][3].Black infants are also three times more likely to be born <28 weeks' gestation, which represents the most vulnerable neonatal population for multiple medical morbidities and mortality [4].Racism, not race, contributes to racial disparities in preterm birth rates in the United States through unequal exposures to environmental factors, such as air pollution, violence, crowded housing, low levels of greenspace, and discriminatory policies [2,5,6].Many of the same environmental exposures that are associated with preterm birth are also associated with adverse health outcomes after birth, particularly among preterm infants.Understanding the relationship of the environment with perinatal health is a vital step in identifying modifiable factors and developing place-based strategies to reduce inequities in preterm birth risk and long-term adverse health outcomes of preterm infants after discharge from the neonatal intensive care unit (NICU). Preterm infants are at risk for numerous adverse outcomes, including, but not limited to, neurodevelopmental impairment, bronchopulmonary dysplasia (BPD), long hospital lengths of stay, and increased healthcare utilization throughout childhood [7][8][9][10].BPD represents the most common chronic morbidity of preterm birth and is a lifelong disease with extensive healthcare burden and cost for patients and families [11][12][13].Significant racial disparities exist in early life outcomes of infants born very preterm; Black preterm infants have a higher mortality rate (12.9% vs. 11.7%) and higher readmission rate (23.2% vs. 18.9%) compared to White preterm infants [14].It is important to note that racism, not race itself, contributes to the racial disparities present among preterm infants [15].The environment, dynamically shaped over time through policies, migration, and external factors, is a compilation of multiple exposures that may modify the risks of adverse perinatal health outcomes.Examining environmental contexts is crucial to identify at-risk families, address underlying drivers of adverse outcomes, and develop place-based interventions to optimize perinatal health and equity. Environmental exposures may be broken down into micro-environmental and macroenvironmental factors [2].Micro-environmental exposures are specific to the individual's non-genetic exposures and may include diet, physical activity, smoking, and social relationships.Macro-environmental exposures are those that are shared by a community and over which individuals may have less control, such as ambient air pollution, extreme heat waves, neighborhood violence, and other community characteristics.Importantly, these exposures do not happen in isolation as the macro-environment may influence the micro-environment.For instance, an individual's residence in a community that is a 'food desert' may affect dietary intake.We will focus on measured exposures of the macro-environment, specifically the physical environment including neighborhood characteristics and air pollution. A growing body of literature demonstrates associations of neighborhood environments with adverse health outcomes [16,17].Numerous indices have been developed to characterize neighborhood environmental characteristics that may affect health including, but not limited to, the Centers for Disease Control and Prevention (CDC) Social Vulnerability Index (SVI), the Area Deprivation Index (ADI) developed at the University of Wisconsin, the Child Opportunity Index, and the Community Material Deprivation Index developed at the University of Cincinnati [18][19][20][21].Each of the metrics incorporates various factors such as household size and composition, income, poverty levels, education, and insurance status to describe neighborhood deprivation and vulnerability.Understanding the relationship of the neighborhood environment with pregnancy-related outcomes and preterm infant health after discharge from the NICU is central to the ability to develop interventions to improve perinatal health outcomes. In this commentary, we will highlight known associations of the macro-environment with both preterm birth risk and preterm infant health outcomes and discuss potential interventions to improve health and address inequities. Neighborhood Deprivation Numerous studies demonstrate associations of neighborhood disadvantage with preterm birth.A 2016 systematic review and meta-analysis of cross-sectional studies investigating poverty, deprivation, racial residential segregation, and crime in the United States found a 27% higher risk of preterm birth among the most disadvantaged neighborhoods when compared to the least disadvantaged neighborhoods [22].In a 2023 retrospective cohort study using the US Vital Statistics data, Salazar et al. reported an association of the Maternal Vulnerability Index (MVI)-comprised of 43 area-level indicators reflecting the physical, social, and health care environments of mothers and birth parents-with preterm birth [23].The MVI ranged from 0 to 100 and residing in a county with the highest MVI level (80-100) was associated with 18% elevated adjusted odds of extreme preterm birth compared to the lowest MVI (0-20), even after adjusting for individual level confounding variables such as race and ethnicity, insurance, maternal education, and maternal health characteristics. Residential Segregation Longstanding structural residential segregation contributes to today's racial disparities in neighborhood conditions and thus in perinatal health.A cohort study in New York state investigated the effects of redlining during the Great Depression-a racially discriminatory home loan practice-and demonstrated higher preterm birth rates in ZIP codes historically defined as "Hazardous" by the Home Owners' Loan Corporation map [24].In a study of 376 counties in the United States, Mehra et al. found a 6.9 increase in the odds of preterm birth of Black women compared to White women residing in racially isolated counties relative to non-racially isolated counties [25].Similarly, a cross-sectional study of birthing parents in Chicago, Illinois, found an association of 12% higher adjusted-odds of preterm births among Black women residing in redlined neighborhoods relative to non-redlined neighborhoods [26].These findings support a causal pathway connecting profound structural inequities to racial disparities in perinatal outcomes. Air Pollution A key environmental exposure that varies by neighborhood is air pollution.Air pollution is a heterogeneous mixture of harmful particles and gases that cause adverse health outcomes through localized airway irritation as well as systemic inflammation [27,28].The Environmental Protection Agency (EPA) has identified and regulates six "criteria" air pollutants that are associated with poor human health outcomes: particulate matter, carbon monoxide, lead, nitrogen oxides, ozone, and sulfur dioxide [29]. Herein, we focus on fine particulate matter smaller than 2.5 µm in diameter (PM 2.5 ) which is the product of vehicle emissions, power plants, burning of fuels, tobacco smoke, and cooking.PM 2.5 exposure is not equally distributed across society.Air quality can vary drastically neighborhood to neighborhood, city to city, state to state, and country to country [30][31][32][33][34].In the United States, communities of color are disproportionately exposed to PM 2.5 [6]. A wealth of animal and human studies on small and large scales link air pollution exposure during pregnancy to adverse birth outcomes, including preterm birth.A 2012 systematic review and meta-analysis by Stieb et al. reported increased odds for preterm birth attributable to increased PM 2.5 exposure, with predominant effects from third trimester exposures [35].A more recent systematic review and meta-analysis from 2017 by Li et al. also demonstrated increased odds of preterm birth with interquartile increases in PM 2.5 exposure throughout pregnancy [36].Lastly, Bekkar et al. investigated increases in air pollution exposure and extreme heat exposure related to climate change in a 2020 systematic review and meta-analysis [37].Among over 30 million live births, PM 2.5 was significantly associated with increased risk of preterm birth.Importantly, the authors reported that the populations most affected included Black birthing people and birthing people with medical comorbidities such as asthma.Given the concurrent racial disparities in preterm birth and air pollution exposure, reducing PM 2.5 levels represents a critical target to reduce preterm birth and improve equity. Neighborhood Environment and Outcomes of Infants with BPD While there is a paucity of studies focused on neighborhoods and outcomes of preterm infants overall, several studies have examined associations of neighborhoods and outcomes among infants with BPD.Banwell et al. performed a retrospective cohort study in the metropolitan Philadelphia and Baltimore regions among infants with BPD to investigate the association of ADI with respiratory health outcomes in these infants.The authors identified an association of ADI with readmission, emergency department visits, and activity limitations among infants with BPD [38].Additionally, the authors found that infants with discharge addresses in the highest tertile of ADI were more likely to be Black, publicly insured, and born with lower gestational ages and birth weights [38].This study emphasizes two important points: (1) families that live in more deprived neighborhoods are more likely to have preterm infants and (2) preterm infants are more likely to be discharged home to deprived neighborhoods.Furthermore, the strong associations of neighborhood deprivation with race and insurance status support that publicly insured and Black families likely have higher rates of preterm birth and that their preterm children have worse outcomes than their White counterparts partly due to differences in environmental exposures. Others have also found that environments are associated with post-discharge outcomes of infants with BPD.In 2021, Deschamps et al. investigated neighborhood disadvantage and respiratory outcomes in the first year after NICU discharge in France.Among infants with BPD, infants living in disadvantaged areas had higher risk for hospital readmission [39].After adjusting for perinatal and discharge-related clinical characteristics and season of birth, the authors found that respiratory-related readmissions were nearly 3-fold higher among infants living in disadvantaged areas [39].Members of our team also investigated the association of SVI with medically attended respiratory illness in the first year after NICU discharge among 378 infants with BPD in the Philadelphia area [40].After adjusting for gestational age, infant sex, birth year, BPD severity, and insurance status, higher SVI was associated with elevated odds of medically attended acute respiratory illness in the first year after NICU discharge.Furthermore, while adjustment for race and ethnicity attenuated this relationship, SVI mediated (potentially explained) 31% of the Black-White disparity in emergency department visits [40].These studies suggest that neighborhoods matter for respiratory health outcomes among preterm infants with BPD. Neighborhood Environment, Infant Mortality, and Development Associations of the neighborhood environment with neonatal mortality, neurodevelopmental outcomes, and follow-up clinic attendance also exist [41][42][43].Janevic et al. found that racial and economic neighborhood segregation was associated with neonatal mortality, specifically finding that infants residing in neighborhoods with the highest relative concentration of Black residents in New York City had higher neonatal mortality risk compared to infants in neighborhoods with the highest relative concentration of White residents [42].Nwanne and colleagues found that infants from high-risk neighborhoods, defined as a maternal residential census block group with a score ≥75th percentile on a neighborhood risk index constructed using eight measures from the 2012-2016 American Community Survey (ACS), were more likely to have neurodevelopmental impairment, lower markers of cognitive function, and lower reading scores compared to infants from low-risk neighborhoods [43].Similarly, Fraiman et al. quantified disparities in infant follow-up participation and attendance between Black infants and White infants, infants of non-English speaking compared to infants of English-speaking families, and infants with a home address in an area of very-low Child Opportunity Index compared to a home address in an area of very-high Child Opportunity Index [41].Taken together, there are numerous associations of neighborhood disadvantage with adverse health outcomes among preterm infants, raising the question as to which neighborhood interventions could improve the health of preterm infants most substantially. Air Pollution Exposure and Infant Outcomes Air pollution not only increases the risk of preterm birth, but also adversely affects the health of infants after birth.Mechanistically, this may be due to the direct and indirect effects of air pollution exposure and its ability to cause local inflammation and damage in the lung, but also to translocate across the pulmonary vasculature and precipitate systemic inflammatory responses [27,44].Given the role of climate change and human factors in the increase in occurrence of natural air polluting phenomena such as wildfires, the racial disparities inherent in air pollution exposure, and the fact that the majority of individuals in the world are exposed to higher levels of air pollution than deemed "safe" by the World Health Organization (WHO) and EPA, it is increasingly important to understand how these exposures contribute to preterm infant health outcomes [6,[45][46][47]. Building on the well-established line of evidence linking PM 2.5 exposure with adverse cardiovascular and respiratory complications in adults and children [48][49][50][51], several recent studies have investigated the association of PM 2.5 exposure with respiratory outcomes in preterm infants [52][53][54][55][56]. Teyton et al. performed the largest retrospective cohort study, to date, of 1,983,700 term and preterm infants in California.The authors found an association of each 5 µg/m 3 increase in PM 2.5 exposure with increased odds of infection-related and respiratory-related emergency department visits among term and preterm infants.The stronger association was among preterm infants, suggesting that preterm infants may be particularly sensitive to worse air quality than their term counterparts [52]. These findings are consistent with less granular studies that have investigated proxies of air pollution exposure, namely residential proximity to a major roadway, environmental tobacco smoke exposure, and indoor combustion exposure and their relationship with respiratory health among preterm infants.Collaco et al. found that proximity to a major roadway and traffic-related air pollution exposure was associated with activity limitations and nighttime cough among infants with BPD [53].In a separate study, Collaco et al. reported an association of higher hair nicotine levels among infants with BPD residing in households with caregiver reported smoking and an association of higher hair nicotine levels with increased hospitalizations and decreased activity levels [56].Rice et al. demonstrated an association of exposure to combustible sources of indoor air pollution (i.e., gas or propane heat, gas or wood stove, gas or wood burning fireplace) with increased risk of hospitalization and chronic respiratory symptoms including activity limitation and cough among infants and children with BPD [54]. Air Pollution and Infant Neurodevelopment Numerous studies suggest that prenatal exposure to air pollution is associated with adverse neurodevelopmental outcomes in preterm infants [57,58].Similarly, evidence exists highlighting an association of postnatal air pollution exposure and adverse neurodevelopmental outcomes, including autism, in term infants and children [59,60].The relationship between postnatal air pollution and preterm infant neurodevelopmental outcomes has not been fully explored but represents an important area for further study and a potential avenue to intervene to improve outcomes for this particularly vulnerable population. Neighborhood Strategies to Improve Outcomes; a Focus on Greenspace and PM 2.5 Given the clear associations of neighborhood deprivation and air pollution with perinatal and preterm infant health outcomes, it is of utmost importance to develop strategies to mitigate these exposures.Furthermore, given residential segregation and disparities in exposures and outcomes, addressing neighborhood health will be required to improve health equity.There are numerous opportunities to address these outcomes at an individual and policy level.Urban planning efforts, tax incentives for positive local businesses, and building and supporting community centers to enhance community engagement may all improve perinatal health. Greenspace Greenspace, which has been shown to confer multiple health benefits, is another neighborhood factor that can be shaped and influenced by public policy to improve health.A 2020 systematic review and meta-analysis found an association of increased residential greenspace with higher birth weight, lower odds of small for gestational age birth, and a non-significant association with lower odds of preterm birth [61].A retrospective cohort study of nearly four million births in California found an association of increased greenspace with decreased risk of overall preterm birth, with the strongest association for very preterm birth (28-<32 weeks gestation) [62]. On an individual level, numerous studies have investigated greenspace and health outcomes in the perinatal and postpartum periods.Tiako et al. found that residential tree canopy coverage was associated with reduced stress among pregnant people in urban settings [63].South et al. performed a pilot randomized trial to increase greenspace among postpartum individuals and found that among trial participants, those in the intervention arm had more and longer visits in nature and a non-significant trend towards lower postpartum depression scores [64].Given the association of greenspace with reduced risk of preterm birth [65,66] and the reported relationships of increasing access to greenspace with improved maternal mental health outcomes, efforts to increase community greenspace represents a promising intervention.Greenspace may also serve to improve overall community health and reduce exposure to other factors-such as stress and crime-that have been associated with an increased risk of preterm birth [67][68][69]. Greenspace may also encourage physical activity [70].There is evidence that specific elements of neighborhood health contribute to maternal health and risk of preterm birth.Kash et al. investigated neighborhood walkability as a risk factor for preterm birth phenotypes in Philadelphia and found that walkability-as defined by publicly available Walk Score ranking-was associated with decreased odds of medically indicated preterm birth (i.e., delivery due to maternal or neonatal complications) [71].However, walkability is not evenly distributed throughout cities and may not represent the same set of positive influences in largely Black or White neighborhoods.Indeed, in this study, the association of walkability was only protective for White individuals, suggesting that walkability alone, without safety, is insufficient to reduce inequities; in Philadelphia, neighborhood violence levels are higher in neighborhoods with a higher proportion of Black residents [72]. Addressing Air Pollution Given the relationships among PM 2.5 exposure, preterm birth, and adverse health outcomes of preterm infants, identifying, investigating, and implementing interventions to reduce PM 2.5 exposure is critical to improving health.Policy interventions may represent the most effective intervention in reducing PM 2.5 exposure.Importantly, the United States EPA sets and reviews air quality standards under the Clean Air Act, which was passed in 1963, amended in 1990, and most recently amended in 2022 as part of the Inflation Reduction Act.From 2000 to 2022, the EPA has reported a 42% decrease in PM 2.5 nationally [73].Given the success in the Clean Air Act to decrease in PM2.5 nationally over the past 22 years, policies to improve air quality work.Continued advocacy for policies such as the Clean Air Act, emissions standards requirements, and renewable energy is an important opportunity to improve environmental health for birthing people and preterm infants [74].Furthermore, thoughtful design of childcare facilities and their geographic relationship to major roadways and sources of air pollution may represent an additional opportunity to limit potentially harmful exposures to PM 2.5 . Interestingly, community-level interventions, such as increasing greenspace and urban vegetation, may also serve to reduce air pollution in these settings.Greenspace is a unique intervention in that it provides green havens in otherwise urban environments that can be used for leisure and recreation.Trees and urban vegetation also play an independent role in removing air pollution from the environment [75][76][77][78].Furthermore, studies demonstrate an association of lower overall and nighttime temperatures with increased tree canopy and urban vegetation [79,80].These findings demonstrate the dual roles of greenspace to directly improve health while also decreasing levels of air pollution and extreme heat which can increase risks of preterm birth and adverse infant outcomes.Future research is needed to study the effects of greening interventions on preterm birth risk and preterm infant respiratory and developmental health to determine whether such interventions could improve perinatal health. In addition to policies such as the Clean Air Act and greening interventions, at an individual family level, air purifiers represent an option that may be a feasible intervention to improve health outcomes.Air purifiers can reduce indoor air pollution exposure and have been shown to effectively reduce indoor PM 2.5 concentrations [81,82].Furthermore, the use of air purifiers has been shown to significantly improve allergic symptoms in children with allergic rhinitis [83].A pilot study in Baltimore also demonstrated feasibility of randomizing women with children less than one year of age to receive air purifiers and secondhand smoke education, finding that the families receiving the air purifiers had significantly decreased PM 2.5 levels and high participant satisfaction [84].Similarly, interventions aimed at decreasing indoor air pollution sources, such as installation of electric or clean-burning stoves in the place of biomass or gas combustion as well as improved ventilation, have demonstrated improvements in respiratory health in children in developing countries and rural areas in the United States [85,86].Interventions to reduce indoor air pollution through purification, ventilation, and decreased combustion may represent feasible interventions for families with preterm infants to optimize their respiratory and overall health. Healthcare Interventions to Overcome Structural Inequities Neighborhood improvements take time.As a healthcare community, we can intervene to overcome structural inequities and address some unmet needs and barriers to self-care and health care.These strategies may buffer some of the adverse neighborhood exposures by supporting families through stressors that are common in under-resourced communities. Partnering with birthing people to improve access to and interaction with perinatal care providers may represent a strategy that can mitigate preterm birth risk in at-risk populations.Doulas are trained professionals who provide continuous social support to their patients before, during, and after the childbirth process [87].Community doulas often come from the same communities as birthing people and thus can often provide information and resources that are accessible to families in addition to advocating for birthing people during healthcare visits [88].Evidence suggests that doula care is linked with improvements in numerous perinatal outcomes [89,90].Given potential disparities in access to doula care, two recent studies highlighted the results of implementing doula supports in populations of low-income women and women of color.Thomas et al. reported lower rates of preterm birth (6.3 vs. 12.4%, p < 0.001) among women receiving doula support in the New York City Department of Health and Mental Hygiene's Healthy Start Brooklyn initiative compared to women not receiving doula support [91].Kozhimannil et al. compared birth-related outcomes for Medicaid recipients who received doula care with Medicaid recipients receiving no doula care and found lower cesarean and preterm birth rates [92].Improving access to doula care in at-risk populations may help bridge the gap between an individual and the healthcare environment, providing emotional, physical, and informational support, and represents an exciting strategy to improve birth outcomes for birthing people from neighborhoods with fewer resources. There are other opportunities for partnership with families at the healthcare level to disrupt pathways by which infants-often preterm infants-from disadvantaged areas suffer worse health outcomes.Interventions to fulfill the unmet needs of caregivers represent an opportunity to improve health outcomes by enabling holistic and family-centered infant care.For example, partnering with families to provide transportation to follow-up clinics may improve attendance and participation in follow-up care.However, simply providing rides may be insufficient.A systematic review from 2020 showed mixed results of subsidizing transportation on healthcare outcomes but noted that transportation assistance is more likely to be effective when offered in tandem with other interventions aimed at reducing social and economic barriers to health [93]. Interventions such as the use of Community Health Workers (CHWs) and housing improvements have also been shown to be beneficial in reducing disparities in outcomes related to pediatric diseases such as asthma [94,95].A similar approach to preterm infants with BPD may be an avenue to improve respiratory health outcomes.Integrating CHWs into the care of infants with BPD and systematically targeting the home environment of infants with BPD to decrease exposure to respiratory irritants could improve the respiratory health outcomes of infants with BPD and would take significant investment from both the community and the healthcare system. On a policy level, interventions such as cash assistance, in the form of the expansion of pre-existing benefits such as Earned Income Tax Credits and Child Tax Credits, as well as unconditional cash transfers to caregivers of term infants have been associated with decreased food insufficiency, reduced child poverty, and improved markers of brain activity [96][97][98][99].However, not all studies demonstrate benefit as Mergerison et al. recently found an association of increased odds of low birth weight with exposure to Child Tax Credit advance payments administered from July to December 2021 [100].However, studying the impacts of the expanded Child Tax Credit and Earned Income Tax Credit for families with preterm infants is needed to understand their impacts on the outcomes of this vulnerable population after birth. Similarly, the benefits of unconditional cash transfers have not been sufficiently investigated among preterm infants, though an initial study found that the provision of unconditional cash transfers resulted in higher parental visitation, more skin-to-skin time, and higher rates of breastfeeding during NICU admission [101].These policy-level interventions are poised to reduce disparities in health outcomes of preterm infants and should be studied rigorously in the future. Conclusions Both pregnancy-related and preterm infant health outcomes are influenced by the neighborhood environment.Neighborhood deprivation, air pollution, greenspace, and other community factors are intertwined exposures that affect health.Environmental disadvantage confers a risk of preterm birth and, importantly, those same preterm infants are often discharged to a high-risk neighborhood environment.Addressing structural inequities in environmental exposures will be required to reduce perinatal and preterm infant health inequities.Only with the implementation of numerous state-sponsored and healthcare-initiated, place-based strategies at the societal and individual level will meaningful improvements in perinatal health and health equity be achievable.	2023-10-05T15:24:04.158Z	2023-10-01T00:00:00.000	{ "year": 2023, "sha1": "7a8dd5f5fc362b50be66ab15b48769ab10c9c375", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2227-9067/10/10/1646/pdf?version=1696256112", "oa_status": "CLOSED", "pdf_src": "PubMedCentral", "pdf_hash": "b8fd9afe6ff75ee60c321a6175319dd3c45fa659", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
15795468	pes2o/s2orc	v3-fos-license	Anomalous Effects of"Guest"Charges Immersed in Electrolyte: Exact 2D Results We study physical situations when one or two"guest"arbitrarily-charged particles are immersed in the bulk of a classical electrolyte modelled by a Coulomb gas of positive/negative unit point-like charges, the whole system being in thermal equilibrium. The models are treated as two-dimensional with logarithmic pairwise interactions among charged constituents; the (dimensionless) inverse temperature $\beta$ is considered to be smaller than 2 in order to ensure the stability of the electrolyte against the collapse of positive-negative pairs of charges. Based on recent progress in the integrable (1+1)-dimensional sine-Gordon theory, exact formulas are derived for the chemical potential of one guest charge and for the asymptotic large-distance behavior of the effective interaction between two guest charges. The exact results imply, under certain circumstances, anomalous effects such as an effective attraction (repulsion) between like-charged (oppositely-charged) guest particles and the charge inversion in the electrolyte vicinity of a highly-charged guest particle. The adequacy of the concept of renormalized charge is confirmed in the whole stability region of inverse temperatures and the related saturation phenomenon is revised. Introduction This paper deals with physical situations when one or two "guest" charges, say arbitrarily charged colloidal particles with a hard core of radius σ, are immersed in a classical electrolyte modelled by an infinite Coulomb gas of positive/negative unit charges. In order to obtain explicit results we consider the point-like limit of the guest charges, i.e. σ/λ → 0 where λ is a characteristic correlation length of electrolyte species; the obtained results are not expected to be applicable to large-sized colloids. If the charges of the guest particles are sufficiently large, anomalous counterintuitive phenomena emerge in the system [1]. One of such phenomena is the appearance, under some circumstances, of an effective (i.e., mediated by the electrolyte) attraction between likecharged colloids. While the traditional DLVO theory [2,3] always predicts an effective repulsion between two like-charged colloids [4,5], experimental measurements [6,7] and numerical simulations [8,9] provide evidence for attraction, especially within confined geometries (close to a dielectric wall or between two glass plates) but also in the bulk of the electrolyte [10]. It was argued that the effective attraction of two like-charged colloids in the presence of a single wall can arise also from a non-equilibrium hydrodynamic effect [11,12]. Another interesting effect is the overcharging, or the charge inversion, of a highly charged colloid [13]. This effect occurs when the number of electrolyte counterions in the vicinity of the colloidal surface becomes so high that the colloidal bare charge is locally overcompensated. The charge inversion has been observed experimentally by electrophoresis [14] and in simulations [15]. Its theoretical explanation is based on Wigner-crystal theories [16,17]. The latter effect is related to the concept of renormalized charge [18,19,20,21,22,23]. The true electric potential far from the colloid immersed in a weakly-coupled electrolyte is supposed to exhibit the Debye-Hückel form, but with a renormalized-charge prefactor which is different from the bare charge of the colloid. An important feature, which occurs in the framework of the nonlinear Poisson-Boltzmann equation, is that the renormalized charge saturates monotonically at some finite value when the colloidal bare charge goes to infinity [22,23]. Monte-Carlo simulations of a salt-free colloidal cell model [24] indicate the existence of a maximum in the plot of the renormalized charge versus the bare colloidal charge. Téllez and Trizac [25] considered the possibility of a more general phenomenon of potential saturation. A theoretical elucidation of anomalous phenomena requires to go beyond mean-field approximations by incorporating electrostatic correlations among electrolyte particles. Heuristic phenomenological approaches applied so far are based on plausible, but not rigorously justified, arguments. Some exactly solvable models are needed. The best candidates are two-dimensional (2D) Coulomb systems with logarithmic pairwise interactions among the charged constituents. The 2D Coulomb gas of ± unit point-like charges is stable against the collapse of positive-negative pairs of charges at high enough temperatures, namely for β < 2 where β is the (dimensionless) inverse temperature or coupling constant. The collapse point β = 2, at which the collapse starts to occur, is equivalent to the free-fermion point of the Thirring representation of the 2D Coulomb gas [26,27]; although the free energy and the particle density diverge, the truncated Ursell correlation functions are finite at this point. In a recent work [28], we have solved exactly the 2D problem of one colloid immersed in the Coulomb gas just at the free-fermion point. An explicit form of the induced electric potential as a function of the bare colloidal charge was derived at every point of the space. Based on this exact result, the concept of renormalized charge was shown to fail in this strong-coupling regime. On the other hand, the anticipated phenomenon of the electric potential saturation was confirmed at the free-fermion point. Our present aim is to extend the exact treatment of the guest-charge(s) problem to the whole Coulomb-gas stability region of inverse temperatures 0 ≤ β < 2. A first important step towards this aim has already been done by solving exactly the equilibrium statistical mechanics of the 2D Coulomb gas in the stability regime (the bulk thermodynamics, special cases of the surface thermodynamics and the large-distance behavior of the two-body correlation functions) via an equivalence with the integrable 2D Euclidean sine-Gordon theory; for a short review, see ref. [29]. As is shown in this paper, the problem of one (two) guest charge(s) immersed in the Coulomb plasma is related to the evaluation of one-point (two-point) expectation values of the exponential field in the sine-Gordon theory. Based on recent progress in the latter topic, we derive explicit formulas for the chemical potential of one guest charge and for the asymptotic large-distance behavior of the effective interaction between two guest charges. The exact results imply, under some circumstances, an effective attraction (repulsion) between like-charged (oppositely-charged) guest particles and the charge inversion in the electrolyte around a highly-charged guest particle. The adequacy of the concept of renormalized charge is confirmed in the whole stability region 0 ≤ β < 2. The related saturation phenomenon is revised. The paper is organized as follows. Basic facts about the bulk properties of the 2D symmetric Coulomb gas are summarized in Section 2. Section 3 deals with the problem of one guest charge in the electrolyte. The effective interaction between two guest charges immersed in the electrolyte is studied within a form-factor method for the equivalent sine-Gordon model in Section 4. Based on the exact results of Section 4, the concept of renormalized charge and the related saturation phenomenon are tested in Section 5. A brief recapitulation and some concluding remarks are given in Section 6. Basic facts about the 2D Coulomb gas We consider an infinite 2D plane Λ of points r ∈ R 2 , filled with a homogeneous medium of dielectric constant = 1. The electrostatic potential v at a point r, induced by a unit charge at the origin 0, is given by the 2D Poisson equation The solution of this equation, subject to the boundary condition ∇v(r) → 0 as \|r\| → ∞, reads The free length constant r 0 will be set for simplicity to unity. This definition of the Coulomb potential in 2D maintains many generic properties (e.g., sum rules [30]) of "real" 3D Coulomb fluids with the interaction potential v(r) = 1/\|r\|, r ∈ R 3 . The symmetric Coulomb gas consists of two species of point-like particles with opposite unit charges q j ∈ {+1, −1}; to simplify the notation, the elementary charge e is set to unity. The bulk properties of the system in thermodynamic equilibrium are usually treated within the grand canonical ensemble. The ensemble is characterized by the (dimensionless) inverse temperature β, which plays the role of the coupling constant, and by the couple of equivalent particle fugacities z + = z − = z. Since the length scale r 0 in (2.2) was set to unity, the true dimension of z is [length] −2+(β/2) . The grand partition function of the plasma is defined by is the number of positively (negatively) charged particles and N = N + + N − . The system is stable against the collapse of positivenegative pairs of unit point-like charges provided that the corresponding Boltzmann factor r −β is integrable at short distances in 2D, i.e. for β < 2. In what follows, we shall restrict ourselves to this stability region of coupling constants. To introduce the averaged many-particle densities, we denote by · · · β the standard thermal average. At the one-particle level, one considers the number density of particles of one sign Due to the charge symmetry and space homogeneity, n + = n − = n/2 where n is the total density of particles. At the two-particle level, one considers the two-body number densities which are translationally invariant, n qq ′ (r, r ′ ) ≡ n qq ′ (\|r − r ′ \|). The two-body densities decouple at asymptotically large distance onto the product of the corresponding one-body densities, lim r→∞ n qq ′ (r) = n q n q ′ . It is therefore natural to introduce the Ursell functions, U qq ′ (r) = n qq ′ (r) − n q n q ′ , which go to 0 as r → ∞. It is also useful to consider the pair distribution functions g qq ′ (r) = n qq ′ (r)/(n q n q ′ ). The short-distance behavior of the two-body densities is dominated by the Boltzmann factor of the corresponding pair Coulomb potential [31,32]. In particular, the pair distribution functions behave like Here, the excess (i.e. over ideal) chemical potential of the Coulomb-gas species is defined by and µ ex Q with arbitrarily-valued Q represents an extended definition of the excess chemical potential: µ ex Q is the reversible work which has to be done in order to bring a particle of charge Q (in units of the elementary charge e) from infinity into the bulk interior of the considered Coulomb gas. In the case of oppositely-charged particles, formula (2.6) reduces to According to Eq. (2.1), −∆/(2π) is the inverse operator of the Coulomb potential v(r). The grand partition function of the 2D Coulomb gas (2.3) can be thus turned via the Hubbard-Stratonovich transformation (see e.g. ref. [33] being the 2D Euclidean action of the sine-Gordon model. Here, φ(r) is a real scalar field and Dφ denotes the functional integration over this field. The fugacity z is renormalized by the (diverging) self-energy term exp[βv(0)/2], without changing the z-notation. The one-and two-body densities of the charged particles in the plasma are expressible as averages over the sine-Gordon action as follows The short-distance behavior (2.9) is equivalent to Under this conformal normalization of the exponential field, the divergent self-energy factor (which renormalizes z) disappears from statistical relations calculated within the sine-Gordon representation. The short-distance formula (2.14) is the special case of a more general relation valid in a restricted region of the parameters a and a ′ such that the one-point average e i(a+a ′ )φ be finite. The sine-Gordon model (2.11) is integrable [34]. Its particle spectrum consists of one soliton-antisoliton pair (S,S) with equal masses M and of S −S bound states ("breathers") {B j ; j = 1, 2, . . . < 1/ξ} whose number at a given b 2 depends on the inverse of the parameter The mass of the B j -breather is given by and this breather disappears from the spectrum just when m j = 2M. The breathers exist only in a subinterval of the stability region 0 ≤ b 2 < 1/2 (0 ≤ β < 2) of the point-like Coulomb gas. The lightest B 1 -breather, usually called the elementary one, has the mass m 1 = 2M sin πξ 2 (2.18) and disappears from the particle spectrum just at the free-fermion point b 2 = 1/2 (β = 2). The soliton-antisoliton pair is present in the spectrum up to the Kosterlitz-Thouless transition point b 2 = 1 (β = 4) at which the sine-Gordon theory ceases to be massive. The (dimensionless) specific grand potential ω of the 2D Euclidean sine-Gordon model, defined by was found in ref. [35] by using the Thermodynamic Bethe ansatz: Under the conformal normalization of the exponential fields (2.14), the relationship between the fugacity z and the soliton/antisoliton mass M was established in ref. [36], where Γ stands for the Gamma function. Note that the mass M has dimension of an inverse length. As a consequence of Eqs. (2.20) and (2.21), one has Relations (2.21) and (2.22), together with the equality n 2z = e ibφ (2.23) determine explicitly the density-fugacity relationship and consequently the complete thermodynamics of the 2D Coulomb gas in the stability region [37]. One guest charge in the electrolyte Let us consider a point-like particle of charge Q with Q being an arbitrarily valued real number; when Q is interpreted as the valence it has to be an integer. The charge is put into the bulk interior of the 2D electrolyte, say at the origin 0. The electrostatic potential induced by the guest charge at a point r ∈ R 2 is equal to −Qln\|r\|. Its effect on the constant species fugacities z ± is the following: z q → z (1) q (r) = z\|r\| βQq . The excess chemical potential of the guest particle is given by represents an obvious functional generalization of the definition (2.3) of the grand partition function with position-dependent particle fugacities. Performing the Hubbard-Stratonovich transformation, the sine-Gordon representation of Ξ[z (1) q (r)] takes the standard form of Eq. (2.10) with the action We first shift the scalar field φ(r) → φ ′ (r) = φ(r) − i4Qb ln\|r\|. Using subsequently the Poisson equation ∆ln\|r\| = 2πδ(r) and integrating by parts with vanishing boundary contributions at \|r\| → ∞, one gets where the average is taken with the usual sine-Gordon action S(z) given by Eq. (2.11). When Q = ±1, one recovers the previous result (2.13) derived for the plasma constituents. Note the obvious symmetry µ ex Q = µ ex −Q . An exact formula for the expectation value of the exponential field e iaφ , where the sine-Gordon parameter b 2 lies inside the stability region 0 ≤ b 2 < 1/2 and a is a free real parameter, was conjectured by Lukyanov and Zamolodchikov in ref. [38]. In terms of our notation a = Qb [see Eq. (3. 3)], their formula reads The formula was "guessed" on the base of three exactly solvable cases of the sine-Gordon theory: the semi-classical limit b 2 → 0, the free-fermion point b 2 = 1/2 [39] and the special value of a = b, see Eq. (2.22). The validity of the formula was supported later by a "reflection" relationship with the imaginary Liouville theory [40], a numerical study of the sine-Gordon model in finite volume [41] and a variational perturbation theory [42]. Other checks, provided by the Coulomb-gas representation, are presented in the next two paragraphs. The integral (3.5) is finite provided that \|Q\| < 1/(2b 2 ); at \|Q\| = 1/(2b 2 ), the integrated function behaves like 1/t for t → ∞ what causes the logarithmic divergence of the integral. In the Coulomb-gas picture, the interaction Boltzmann factor of the Q-charge with an opposite unit plasma charge (counterion) at distance r, r −β\|Q\| , is integrable at small r in 2D if and only if β\|Q\| < 2. The stability region for µ ex Q therefore is expected to be \|Q\| < 2/β; there is a collapse at \|Q\| = 2/β characterized by µ ex Q → −∞. With regard to the relationship β = 4b 2 we conclude that the pair of Eqs. (3.4) and (3.5) passes the guest-charge collapse test. The way in which e iQbφ diverges as \|Q\| approaches the collapse value 1/(2b 2 ) is another check provided by the Coulomb-gas representation. To show this fact, let us attach a hard core of radius σ around the guest Qcharge. The effect of the Q-charge on the species fugacities is the following: z q → z q (r) = z q \|r\| βQq θ(\|r\| − σ), where θ is the Heaviside step function. The procedure analogous to that outlined between Eqs. where the average is taken with the action We expand e iQbφ(0) σ in the lowest σ-order around the sine-Gordon action S(z): Close to the collapse value of the guest charge, i.e. when Q = 1/(2b 2 ) − ǫ with ǫ → 0 + , one expands Since the regularized e iQbφ(0) σ has to be finite at the collapse Q = 1/(2b 2 ), it follows from Eqs. (3.9) and (3.10) that the singular behavior must take place. It is shown in the Appendix that the singular behavior Two guest charges in the electrolyte Let us put two point-like particles into the bulk of the Coulomb plasma, the one with the charge Q 1 at the point r 1 and the other with the charge Q 2 at the point r 2 . The electrostatic potential induced by these two charges at a point r ∈ R 2 is equal to −Q 1 ln\|r−r 1 \|−Q 2 ln\|r−r 2 \|. The constant species fugacities z ± are thus modified as follows: z q → z (2) q (r) = z\|r − r 1 \| βQ 1 q \|r − r 2 \| βQ 2 q . The excess chemical potential of the guest 1,2-charges as a whole is given by Under the Hubbard-Stratonovich transformation, the sine-Gordon representation of Ξ[z (2) q (r)] takes the standard functional form of Eq. (2.10) with the action Shifting the scalar field φ(r) → φ ′ (r) = φ(r)−i4Q 1 bln\|r−r 1 \|−i4Q 2 bln\|r−r 2 \|, applying the Poisson equations ∆ln\|r − r j \| = 2πδ(r − r j ) (j = 1, 2) and integrating by parts, one arrives at exp −βµ ex Q 1 Q 2 (\|r 1 − r 2 )\| = e iQ 1 bφ(r 1 ) e iQ 2 bφ(r 2 ) (4.3) where the average is taken with the usual sine-Gordon action (2.11). The effective interaction energy between the guest 1,2-charges is defined by With respect to the relation (3.3), it holds exp [−βE Q 1 Q 2 (\|r 1 − r 2 \|)] = e iQ 1 bφ(r 1 ) e iQ 2 bφ(r 2 ) e iQ 1 bφ e iQ 2 bφ (4.5) At asymptotically large distance \|r 1 − r 2 \| → ∞, the two-point correlator e iQ 1 bφ(r 1 ) e iQ 2 bφ(r 2 ) decouples onto the product e iQ 1 bφ e iQ 2 bφ and so the interaction energy goes to zero as it should be. From Eq. (4.5) one then gets − βE Q 1 Q 2 (\|r 1 − r 2 \|) ∼ e iQ 1 bφ(r 1 ) e iQ 2 bφ(r 2 ) e iQ 1 bφ e iQ 2 bφ − 1, This means that the asymptotic large-distance behavior of the effective interaction energy between the guest particles is related to the large-distance behavior of the corresponding two-point correlation function of exponential fields associated with the 2D sine-Gordon theory. For the 2D Euclidean sine-Gordon model, like for any integrable 2D theory, the two-point correlation function of local operators O a (a is a free parameter) can be formally expressed as an infinite convergent series over multi-particle intermediate states [43], where ǫ indexes the particles [say ǫ = +(−) for a soliton (antisoliton) and ǫ = j for a B j -breather] and the rapidity θ ∈ (−∞, ∞) parametrizes the energy and the momentum of the corresponding particle. The form factors are the matrix elements of the operator at the origin, between an N-particle in-state (being a linear superposition of free one-particle states \|Z ǫ (θ) ) and the vacuum. The first N = 0 term of the series expansion (4.7) corresponds to the decoupling O a O a ′ . The form-factor representation (4.7) is particularly useful in the limit \|r − r ′ \| → ∞ where the dominant contribution to the truncated correlation function O a (r)O a ′ (r ′ ) − O a O a ′ comes from a multi-particle state with the minimum value of the total particle mass N j=1 m ǫ j , at the point of vanishing rapidities. As was already mentioned, the lightest particle in the stability region 0 ≤ b 2 < 1/2 is the elementary breather B 1 . For this particle, the one-particle form factors F a (θ) 1 and 1 F a ′ (θ) = F a ′ (θ) 1 of the exponential operator O a (r) = exp [iaφ(r)] were calculated in refs. [44,45]: and ξ is defined in Eq. (2.16). Since the form factor (4.10) does not depend on the rapidity, the integration over θ in (4.7) can be done explicitly by using the relation where K 0 is the modified Bessel function of second kind [46]. On the base of the asymptotic equivalence (4.6), the large-distance behavior of the effective interaction energy is finally found in the form Here, the symbol [Q] stands for the ratio [Q] = sin (πβQ/(4 − β)) sin (πβ/(4 − β)) (4.14) Using the thermodynamic formulas derived at the end of Section 2, the mass m 1 (which plays the role of the inverse charge-charge correlation length of the Coulomb-gas particles) is expressible as where κ = √ 2πβn denotes the inverse Debye length. The β-dependence of the parameter λ, defined by Eq. (4.11), reads An interesting feature of the result (4.13) is that the effective interaction energy factorizes into the product of separate charge contributions [Q] from each of the guest particles. We would like to emphasize that the asymptotic formula (4.13) was derived for the guest particles of point-like nature. Its rigorous validity is therefore restricted to such guest charges which do not collapse with an opposite unit counterion from the electrolyte, i.e. to the values \|Q 1 \|, \|Q 2 \| < 2/β. Since the function [Q] is analytic at every real Q, the stability border \|Q\| = 2/β does not represent an exceptional point at which a singularity emerges [like it was in the case of the excess chemical potential determined by Eqs. (3.3)-(3.5)]. The explanation of this important fact follows from the definition (4.4) of the effective interaction energy: if one of the guest charges passes or is beyond its collapse value, say \|Q 1 \| ≥ 2/β, both excess chemical potentials µ ex Q 1 Q 2 (\|r 1 − r 2 \|) and µ ex Q 1 tend to −∞ in such a way that their difference is expected to keep a finite value. We therefore suggest that the formula (4.13) remains valid for arbitrary real values of Q 1 and Q 2 , and corresponds to the limit of a small hard-core radius σ (such that m 1 σ << 1) around the guest charges. In what follows, we shall refer to this conjecture as "the regularization hypothesis". The interaction energy E Q 1 Q 2 (r) is repulsive (attractive) at asymptotically large distance r when it goes to zero from above (below). When the amplitudes of the guest charges are the same, i.e. \|Q 1 \| = \|Q 2 \|, the interaction energy (4.13) exhibits the vacuum-type behavior: it is repulsive for Q 1 = Q 2 and, since [−Q] = −[Q], attractive for Q 1 = −Q 2 . The situation is more complex when the amplitudes of the guest charges differ from one another. Let us analyze the plot of [Q] as the function of (say positive) bare charge Q for a fixed value of β from the stability range 0, 2). In the interval Q ∈ 0, (2/β) − (1/2) , [Q] increases monotonically from 0 to its maximum at the end-point of this interval. In the subsequent interval Q ∈ (2/β) − (1/2), (4/β) − 1 , which contains the stability border Q = 2/β, [Q] is the decreasing function of Q but still keeps the positive sign of Q. Such behavior means physically that by increasing the bare charge of one of the guest particles the effective interaction energy weakens which is a counterintuitive phenomenon. The function [Q] changes the sign when Q passes the value (4/β) − 1; under the assumption of validity of the regularization hypothesis, this indicates an effective change of the sign of the bare charge Q. Appropriate combinations of the Q 1 , Q 2 -charges in the factorized relation (4.13) can therefore lead to an effective attraction (repulsion) between likecharged (oppositely-charged) guest particles. The described scenario repeats itself when increasing Q due to the periodicity relation [Q] = [Q + (8/β) − 2]. The above discussed plot of [Q] versus the (positive) bare charge Q is presented graphically for various values of the electrolyte coupling constant β in Fig. 1. The solid-line parts of the plots correspond to the interval 0 < Q < 2/β for which the formula (4.13) is valid rigorously, the dashedline parts of the plots correspond to Q ≥ 2/β when the formula (4.13) is applicable under the assumption of the regularization hypothesis. Renormalized charge Let us put one point-like particle of charge Q at the origin 0 and look for the evoked density profiles n q (r) of the electrolyte species q = ±1. One can formally follow the procedure for two guest particles (one of which has the charge q = ±1 of the electrolyte species) outlined at the beginning of Section 4, to obtain Note that in the special case Q = 0 the spatially-homogeneous relation (2.23) is reproduced. At asymptotically large distance r from the guest Qcharge, the straightforward application of the form-factor method explained in Section 4 provides the result where the symbols [q], [Q] are defined by Eq. (4.14) and the parameters m 1 , λ by the respective relations (4.15) and (4.16). The charge density ρ of electrolyte unit charges is defined as ρ(r) = n + (r) − n − (r). Since [q] = q for q = ±1, one finds that The average electrostatic potential ψ induced by the guest Q-charge is related to the charge-density profile through the 2D Poisson equation, ∆ψ(r) = −2πρ(r) (5.4) Inserting here the asymptotic formula (5.3) and considering the circularly symmetric Laplacian ∆ = r −1 d r (rd r ), the dimensionless electric potential βψ can be shown to behave at large distance r from the Q-charge as follows In the Debye-Hückel limit β → 0 and for finite Q, it holds [Q] ∼ Q, m 1 ∼ κ and λ ∼ β. Eq. (5.5) thus reduces to the well-known result (see e.g. refs. [1,28]) The asymptotic behaviors (5.5) and (5.6), considered respectively in terms of the dimensionless combinations m 1 r and κr, exhibit the same type of the falloff. This fact confirms the adequacy of the concept of renormalized charge [18]- [25] in the stability weak-coupling regime of the Coulomb gas. Eq. (5.5) is consistent with Eq. (5.6) provided that one introduces the renormalized charge Q ren as follows This formula can be simplified to where the positive amplitude A(β) is given by dt π t sin t (5.9) As was mentioned above, because of the point-like nature of the guest Q-charge the rigorous validity of the result (5.8) is restricted to \|Q\| < 2/β. Changing Q from 0 towards positive real values, the renormalized charge (5.8) increases monotonically up to its maximum A(β) at the point Q = (2/β) − (1/2). Increasing then Q from (2/β) − (1/2) up to the stability border 2/β, Q ren paradoxically decreases while still keeping the positive sign of the bare charge Q. This scenario resembles the one observed in the Groot's Monte-Carlo simulations of the salt-free (only counterions are present) colloidal cell model [24]. Under the assumption of validity of the regularization hypothesis, one can further increase the value of Q beyond 2/β in the relation (5.8). Q ren changes its positive sign to the negative one at Q = (4/β) − 1. This change of the sign is closely related to the effect of charge inversion [13]- [17]: since the total screening cloud of electrolyte particles must compensate exactly the bare charge Q of the guest particle (the electroneutrality sum rule), the fact that the charge density (5.3) goes to 0 at asymptotically large distance r from above is the evidence of the charge inversion starting at some distance from the guest Q-charge. The renormalized charge Q ren is a periodic function of Q. This is why going with Q → ∞ does not imply the saturation of Q ren at some finite value. Instead, Q ren oscillates between the two finite ±A(β) extremes. With regard to Eq. (5.5), the induced electrostatic potential exhibits the same type of the oscillatory behavior as Q → ∞ what contradicts the idea of the monotonic electric potential saturation [25]. Conclusion Let us summarize briefly the crucial results of the present work. Section 3 deals with the case of one point-like particle of charge Q (with \|Q\| < 2/β) immersed in the bulk of the stable 2D Coulomb gas. Passing to the sine-Gordon representation, we were able to relate in Eq. (3.3) the excess chemical potential µ ex Q of the guest charge to the expectation value of the exponential field. The explicit form of the latter quantity was conjectured by Lukyanov and Zamolodchikov [38], see Eqs. (3.4) and (3.5), and subsequently verified by various methods. Our Coulomb-gas formulation provides two other checks of this conjecture: the guest-charge collapse test at \|Q\| = 2/β and the predicted singular behavior (3.11) of the exponential-field expectation close to the collapse value of Q = (2/β) − ǫ (ǫ → 0 + ). The problem of two guest point-like particles, charged by Q 1 and Q 2 and being at distance r from one another, is studied in Section 4. Using the form-factor method for the two-point correlation functions in the sine-Gordon formulation of the problem, we have derived the explicit formula (4.13) for the effective interaction energy of the two guest charges E Q 1 Q 2 (r) at asymptotically large distance r → ∞. This formula is valid rigorously for \|Q 1 \|, \|Q 2 \| < 2/β; in a subspace of this region of charge values we have noticed an anomalous weakening of the effective interaction when one of the guest charges is increasing. Since in the definition (4.4) of E Q 1 Q 2 (r) the divergences of chemical potentials (caused by the collapse of guest charge with electrolyte counterions) cancel with each other, we have suggested an extended validity of the formula (4.13) for arbitrary values of Q 1 and Q 2 (the regularization hypothesis). Then, under certain circumstances (especially, the inequality \|Q 1 \| = \|Q 2 \| must hold and one of the guest charges has to be large enough), the result (4.13) implies an effective attraction (repulsion) between like-charged (oppositely-charged) guest particles. The adequacy of the concept of renormalized charge was confirmed in Section 5. This is not a surprise: in the whole stability interval of inverse temperatures 0 ≤ β < 2, the large-distance behavior of two-point correlators is determined by the form-factor of the same particle from the sine-Gordon spectrum, namely the B 1 -breather with the lightest mass m 1 playing the role of the inverse correlation length of electrolyte species. The large-distance behavior of the induced electric potential (5.5), considered in terms of the dimensionless combination m 1 r, is therefore basically the same in the Debye-Hückel limit β → 0 as well as at every point β which belongs to the stability interval, up to the renormalized-charge prefactor. The renormalized charge Q ren , considered as a function of the (positive) bare charge Q, exhibits a maximum at Q = (2/β) − (1/2) which is in the range \|Q\| < 2/β of the rigorous validity of the formula (5.8). Under the assumption of validity of the regularization hypothesis, increasing Q can produce the effect of charge inversion. Going with Q → ∞ in Eq. (5.8) does not imply the saturation of Q ren at some finite value. Instead, Q ren oscillates between two finite extremes. The same property holds for the electric potential in the electrolyte region which contradicts the idea of the monotonic potential saturation [25]. The previous results obtained at the free-fermion (collapse) point β = 2 [28] are different from the present ones concerning the stability interval 0 ≤ β < 2 in the following aspects. Firstly, the concept of renormalized charge fails at β = 2. Secondly, at β = 2, when the bare charge Q → ∞ the induced electrostatic potential saturates monotonically at a finite value in each point of the electrolyte region. The reason for the fundamental differences is obvious. The lightest B 1 -breather disappears from the particle spectrum of the sine-Gordon model just at β = 2, and the asymptotic behavior of two-point correlation functions at this coupling constant is governed by the soliton-antisoliton pair. Since m 1 → 2M as β → 2, the particle mass in the exponential decay is a continuous function of β at β = 2. On the other side, the inverse-power-law asymptotic behavior, which is determined by the formfactor of the dominant particle(s) in the sine-Gordon spectrum, undertakes an abrupt modification when passing through the β = 2 point. The basic qualitative features of the results obtained at the free-fermion point β = 2 are expected to be present also for such β > 2 where the soliton-antisoliton pair exists. It is known [34] that the soliton-antisoliton pair disappears from the sine-Gordon particle spectrum (and the sine-Gordon theory ceases to be massive) at the point b 2 = 1 (β = 4) which corresponds to the Kosterlitz-Thouless transition of infinite order from the conducting (fluid) phase to the insulating phase. We conclude that the 2D results obtained in the weakcoupling regime of the Coulomb gas 0 ≤ β < 2 differ substantially from those in the strong-coupling regime 2 ≤ β < 4. where the argument of the exponential on the rhs of Eq. (A.5) can be written as Finally, considering in Eq. (A.5) the integral representation of the logarithm of the Gamma function [46] lnΓ Re(x) > 0 (A.11) the proof of the desired formula (3.11) becomes accomplished.	2014-10-01T00:00:00.000Z	2005-03-09T00:00:00.000	{ "year": 2005, "sha1": "762beeb54dd55a27cf2fed1d82d50c807eb92313", "oa_license": null, "oa_url": "http://arxiv.org/pdf/cond-mat/0503201", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "639d2c51d33eee28617f6cb88527cf0291e29322", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
258685148	pes2o/s2orc	v3-fos-license	The clinicopathological characteristics of gastric cancer and precancerous conditions in gastric DLBCL and MALT lymphoma patients: a multi-center retrospective study Abstract Objective The objective of this study is to explore the clinicopathological characteristics of gastric cancer and precancerous conditions in patients with primary gastric lymphoma. Methods We analyzed 474 cases of primary gastric lymphoma, mainly DLBCL and MALT, from three clinical centres retrospectively, and compared the clinicopathological parameters of primary gastric lymphoma patients complicated with gastric cancer, precancerous conditions, or with no complications. Results A total of 5.1% of the patients with primary gastric lymphoma were diagnosed with gastric cancer, including metachronous gastric adenocarcinoma (3.2%) and synchronous gastric adenocarcinoma (1.9%). Of the patients with gastric lymphoma, 14.6% had precancerous conditions including atrophy (14.6%), intestinal metaplasia (8.9%), and low-grade intraepithelial neoplasia (1.9%). Primary gastric lymphoma patients with an ulcerative type (p = 0.009) and Lugano classification stage IIE + IV (p < 0.001) lymphoma had a higher risk of complicating with gastric cancers or precancerous conditions. The rate of infection of Helicobacter pylori (Hp) was 68.4% in patients with primary gastric lymphoma, which was higher in patients with MALT lymphoma (p < 0.001), Lugano classification stage I + II (p < 0.001), and patients complicated with precancerous conditions and gastric cancer (p < 0.001), especially gastric cancer of the intestinal type (p = 0.04). Gastric cancer (95.8%) and precancerous conditions (91.3%) occurred mostly in Hp-infected primary gastric lymphoma patients, with a minor subset of Hp-eradicated patients. Primary gastric lymphoma patients had a higher detection rate of early gastric cancer (25.0%) and a five-year survival rate (40.0%) than the general Chinese population. Conclusions Patients with primary gastric lymphoma have a high risk of developing gastric cancer and precancerous conditions, and this risk may be related to Helicobacter pylori infection. Follow-up of primary gastric lymphoma provides an opportunity for the detection of early gastric cancer. Key messages 5.1% of the patients with primary gastric lymphoma were diagnosed with gastric cancer. 14.6% of the patients with gastric lymphoma had premalignant lesions including atrophy (14.6%), intestinal metaplasia (8.9%), and low-grade intraepithelial neoplasia (1.9%). Primary gastric lymphoma patients complicating with gastric cancer had a higher infection rate of Helicobacter pylori (100.0%), a detection rate of early gastric cancer (25.0%) and a five-year survival rate (40.0%) than the general Chinese population. Introduction The incidence and mortality of gastric cancer rank first among malignant tumours of the stomach, with about 1.09 million new cases and 768,000 deaths each year globally [1]. Approximately 90% of gastric cancer is gastric adenocarcinoma [2], and the next primary gastric lymphoma is the second most common gastric malignant tumour [3]. In treating these cancers, clinicians need to differentiate between gastric cancer and primary gastric lymphoma if masses, ulcers, or other lesions are found in the stomach, and current identification methods include pathology biopsy and endoscopy, as well as other imaging examinations [4]. However, rare cases of primary gastric lymphoma complicated with synchronous gastric cancer and metachronous gastric cancer have attracted the attention of clinical researchers in particular. The occurrence of primary gastric lymphoma is closely related to Helicobacter pylori ( H. pylori), especially mucosa-associated lymphoid tissue (MALT) lymphomas and some diffuse large B-cell lymphomas (DLBCL) [5]. H. pylori infection can lead to chronic inflammation, atrophy, intestinal metaplasia, low-grade intraepithelial neoplasia, and the intestinal type of gastric adenocarcinoma [6][7][8]. However, few studies have shown that patients with primary gastric lymphoma have a significantly increased risk of developing malignant solid tumours, such as gastric cancer [9,10], and the question of whether patients with primary gastric lymphoma have an increased risk for gastric cancer and precancerous conditions remains controversial [11,12]. This study retrospectively analyzes the clinicopathological characteristics of gastric cancer and precancerous conditions in patients with primary gastric lymphoma from three clinical cancer centres in China and aims to provide clinical evidence for the correlation between primary gastric lymphoma and gastric cancer. Study objects and groups Our study sample consisted of 474 primary gastric lymphoma patients diagnosed at three Chinese cancer centers between January 2010 to August 2020, including 268 patients from the First Affiliated Hospital of Xi'an Jiao Tong University, 119 patients from Shaanxi Provincial People'ss Hospital, and 87 patients from Beijing Tsinghua Changgung Hospital. Our inclusion criteria were the presence of the following: clinical and pathological diagnosis of primary gastric lymphoma and regular endoscopic follow-up, and our exclusion criteria were the presence of complications with malignant tumours other than the stomach, previous upper digestive tract surgery, failure to cooperate with gastroscopy, serious cardiopulmonary disease, and serious liver, kidney, or mental disease. A diagnosis of primary gastric lymphoma was made based on the Dawson criteria [13]. We broke down the sample into two experimental groups and a control group. First, the gastric cancer group included cases diagnosed with synchronous gastric adenocarcinoma within 6 months or metachronous gastric adenocarcinoma beyond 6 months from primary gastric lymphoma diagnosis. Second, the gastric precancerous conditions group included cases with atrophy, intestinal metaplasia, or low-grade intraepithelial neoplasia at initial endoscopy and during follow-up, excluding patients who progressed to gastric cancer. The cases that didn't develop gastric cancer or precancerous conditions were placed in the control group. Gastroscopy Gastroscopy follow-up of patients diagnosed with primary gastric lymphoma was carried out regularly in accordance with the guidelines presented in Matysiak-Budnik et al. [14]. A standard gastroscopy requires adequate preparation before the examination, where the endoscopist should carefully observe a patient's H. pylori infection status, background mucosa, changes in the original lesions, and suspicious malignant lesions by white light endoscopy, chromoendoscopy, linked colour imaging, narrow band imaging, and magnifying endoscopy. After careful observation, we took a biopsy of suspicious lesions for histopathological examination as well. Diagnosis of gastric cancer and precancerous conditions Atrophy and intestinal metaplasia can be diagnosed by gastroscopy or pathology alone. Diagnosis of gastric cancer and low-grade intraepithelial neoplasia must be confirmed by histopathological examination of a biopsy or surgical specimens. Endoscopic manifestations of atrophic gastritis include the pale appearance of gastric mucosa, thinning of the gastric mucosa, increased visibility of vasculature, and loss of gastric folds [15,16]. The degree of atrophic gastritis is assessed using the Kimura-Takemoto classification system or Operative Link for Gastritis Assessment (OLGA) classification [15][16][17]. The pathological diagnosis of atrophic gastritis is based on the loss of normal glandular epithelium gastric glands [15,16]. Intestinal metaplasia is manifested as a gray-white flat bulge on white light endoscopy, and regular vessels with ridge/tubular or tubulovillous glands, particularly with a light blue crest on magnified narrow band imaging, mostly on an atrophic background [18]. The degree of intestinal metaplasia is assessed using the Operative Link on Gastric Intestinal Metaplasia Assessment (OLGIM) system [15,16]. The pathological manifestations of intestinal metaplasia are divided into complete intestinal metaplasia and incomplete intestinal metaplasia. Complete intestinal metaplasia resembles the small intestinal epithelium, while incomplete intestinal metaplasia resembles the colonic epithelium [19]. The classification of gastric cancer and intraepithelial neoplasia was defined by World Health Organization [20]. Low-grade intraepithelial neoplasia has limited architectural abnormalities and only mild to moderate cytological atypia, with hyperchromatic, elongated, pseudostratified nuclei [21]. H. pylori detection and eradication H. pylori infection status was determined by H. pylori detection and patients' histories of eradication therapy. For detection, we used a 13 C-urea breathing test, a 14 C-urea breath test, or a rapid urease test to detect H. pylori. A positive result for any of the above tests confirmed the presence of an infection with H. pylori, which we denote as 'Hp-infected' , and negative results without previous H. pylori eradication therapy were judged to be 'Hp-uninfected' . Negative results after H. pylori eradication therapy was denoted as 'Hp-eradicated' . In order to avoid false negative test results, we required an interval of at least 4 weeks from antibiotics and at least 2 weeks from proton pump inhibitor and bismuth before H. pylori detection could take place. Patients were also required to fast for at least 2 h before the examination. Clinicopathological data collection Clinicopathological and demographic data of all the study subjects were also collected and analyzed. Demographic data included age and sex, and the clinicopathological characteristics of primary gastric lymphoma included diagnosis time, pathological type, lesion location, endoscopy manifestation, Lugano Classification, B symptoms, H. pylori infection status, treatment and prognosis. The clinicopathological characteristics of gastric cancer included symptoms, diagnosis time, lesion location, largest tumour size, TNM staging, pathological type, Lauren classification, H. pylori infection status, treatment and prognosis. Finally, the clinicopathological characteristics of gastric precancerous lesions included diagnosis time, lesion location, pathological type, H. pylori infection status, treatment and prognosis as well. We successfully contacted 391 patients by telephone on 22 May 2022 to 124 months (median: 49 months) after their initial diagnosis of primary gastric lymphoma. The remaining patients were lost. Statistical analysis We used SPSS 24.0 (SPSS Inc., Chicago, IL, USA) for the statistical analysis of all of our data. Categorical variables were expressed as the number of cases (percentage) and analyzed by Pearson χ 2 tests or Fisher's exact test between groups, and continuous variables that conformed to normal distribution were represented by mean ± standard deviation and analyzed by Student's t-tests and ANOVA tests. Continuous variables that didn't conform to normal distribution were presented as median (interquartile range) and analyzed by Mann-Whitney U-tests. The overall survival rate and its risk factors were evaluated by the Kaplan-Meier survival analysis. Our threshold for a statistically significant test result was p < 0.05 for all tests. Demographic and clinicopathological characteristics of patients with primary gastric lymphoma In total 474 patients with primary gastric lymphoma were enrolled in our study. As shown in Table 1, the patients' average age was 52.6 ± 5.5 years (27 to 73 years), and 63.5% of the patients were male. The most common involved locations were the gastric body (55.9%) and gastric antrum (45.1%), and ulcerative type (32.5%) was the most common endoscopic manifestation of primary gastric lymphoma, and the pathological types of primary gastric lymphoma were mainly DLBCL (47.5%) and MALT lymphoma (45.6%). The risk of gastric cancer in patients with primary gastric lymphoma A total of 5.1% (24/474) of primary gastric lymphoma patients had been diagnosed with gastric cancer, all of which were gastric adenocarcinomas. As shown in Among the patients with gastric adenocarcinoma, 1.9% (9/474) and 3.2% (15/474) had synchronous gastric cancer and metachronous gastric cancer, respectively. Only one patient who had synchronous gastric cancer was diagnosed at the same time with gastric lymphoma. The median diagnosis interval between metachronous gastric cancer and primary gastric lymphoma was about 3 years (-5 to 11 years). Only one patient who had metachronous gastric cancer was diagnosed before gastric lymphoma. We found no significant differences in sex, age, lesion location, largest tumour size, pathological type, Lauren classification, or TNM stage between the synchronous gastric adenocarcinoma group and the metachronous gastric adenocarcinoma group (Table 3). The risk of H. pylori infection in patients with primary gastric lymphoma, gastric cancer, or precancerous conditions The H. pylori infection rate of primary gastric lymphoma patients was 68.4% (324/474), and as defined above the remaining patients were Hp-uninfected (20.0%, 95/474), and Hp-eradicated (11.6%, 55/474) ( Table 5). Gastric antrum (p < 0.001) and pylorus (p = 0.014) involvement were more common in primary gastric lymphoma patients who were HP-infected, and patients with MALT lymphoma accounted for 56.2% of Hp-infected primary gastric lymphoma patients. MALT lymphoma was more common in Hp-infected primary gastric lymphoma patients compared to Hp-uninfected and Hp-eradicated patients (p < 0.001). The H. pylori infection rate of MALT lymphoma patients was significantly higher than that of DLBCL patients as well (MALT lymphoma, 182/216, 84.3% vs. DLBCL 123/225, 54.7%; p < 0.001). In addition, primary gastric Age of patients when diagnosed with gastric cancer. b diagnosis interval between gastric cancer and primary gastric lymphoma of each case is presented as an estimated whole year. The diagnosis of gastric cancer earlier than gastric lymphoma is indicated by a negative number, and the diagnosis of gastric cancer later than gastric lymphoma is indicated by a positive number. lymphoma patients with Lugano classification stage I + II also had a higher H. pylori infection rate than patients with Lugano classification stage IIE + IV (p < 0.001). The H. pylori infection rate of primary gastric lymphoma with Lugano Classification stage I + II was as high as 89.5% (Table 5). For primary gastric lymphoma patients split into groups with gastric cancer, precancerous conditions, or neither, the H. pylori infection rate was significantly higher in the patients with gastric cancer and precancerous conditions (p < 0.001). Actually, gastric cancer (95.8%, 23/24) occurred mostly in Hp-infected primary gastric lymphoma patients and rarely occurred in patients after H. pylori eradication (4.2%, 1/24 Similarly, gastric precancerous conditions also mostly occurred in Hp-infected primary gastric lymphoma patients (91.3%, 63/69), a few patients after H. pylori eradication (5.8%, 4/69), and rare Hp-uninfected patients (3.4%, 2/69). Additionally, gastric cancer of the intestinal type was more common in Hp-infected primary gastric lymphoma patients compared to Hp-uninfected and Hp-eradicated patients (p = 0.04) ( Table 5). Gastric cancer of the intestinal type was only found in Hp-infected primary gastric lymphoma patients. Treatment and prognosis of patients with primary gastric lymphoma, gastric cancer, or precancerous conditions All Hp-infected primary gastric lymphoma patients received H. pylori eradication therapy, and the overall success rate of H. pylori eradication was 87.1%, with no difference in MALT lymphoma (89.9%), DLBCL (88.3%), and other pathological types (84.6%). H. pylori eradication therapy was effective in 40.9% of primary gastric lymphoma patients, 55.8% of MALT lymphomas patients, and 28.6% of DLBCL patients. Among them,17.7% of primary gastric lymphoma patients, 28.1% of MALT lymphoma patients, and 7.1% of DLBCL Primary gastric lymphoma patients with Lugano classification stage IIE + IV, and those who were Hp-uninfected, failed to eradicate H. pylori, or didn't respond to H. pylori eradication therapy received chemotherapy (75.9%), targeted therapy (53.2%), surgery (12.4%), radiotherapy (6.8%), and immunotherapy (5.9%). Most patients (67.1%) received a combination of therapies. The prognosis of primary gastric lymphoma was generally well. The five-year overall survival rate and three-year overall survival rate of primary gastric lymphoma patients were 62.6% and 73.1%, respectively. Discussion We found a high risk of gastric cancer and precancerous conditions for primary gastric lymphoma patients from our multi-centre retrospective cohort study. A total of 5.1% and 14.6% of primary gastric lymphoma patients had gastric cancer and precancerous conditions, respectively. This is of particular concern in China, where the number of new cases and deaths of gastric cancer each year is close to 50% of all the world's cases [22,23]. Chronic inflammation and the progressing cascade of precancerous conditions caused by H. pylori are high-risk factors for gastric cancer [24], and primary gastric lymphoma is a gastric malignant tumour second only to gastric adenocarcinoma in prevalence [3]. Pathological examination of gastroscopic biopsy is the gold standard for the diagnosis of primary gastric lymphoma. The majority of primary gastric lymphoma are of B-cell lineage pathologically, of which DLBCL accounts for 45% to 59%, and MALT lymphoma accounts for 38% to 48% of primary gastric lymphoma cases worldwide [25,26]. Cases of primary gastric lymphoma that are complicated with gastric adenocarcinoma are relatively rare in clinical practice, and therefore there are few studies on this topic, most of which are case reports and small-sample or single-centre studies. However, these previous studies and our research have shown that the presence of gastric malignant lymphoma increases the incidence of gastric cancer. Previous studies have shown that patients with primary gastric lymphoma face a significantly higher risk of developing other malignant tumours, including gastric cancer [27,28]. Amiot et al. [29] showed that patients with gastric MALT lymphoma had a 16-fold increased risk of gastric cancer compared with the French general population [29]. Our research also found that 5.1% of primary gastric lymphoma patients had gastric cancer, much higher than for the general Chinese population with a 5-year prevalence of 0.0276%. Inaba et al. [9] found that 7.2% (10/139) of Japanese patients with primary gastric lymphoma treated with radiotherapy developed metachronous gastric adenocarcinoma, which was higher than the prevalence of gastric adenocarcinoma in the general Japanese population. Old age, H. pylori infection, gastric mucosal change of chronic gastritis, and intestinal metaplasia were also found to be possible risk factors for metachronous gastric adenocarcinoma [9]. Capelle et al. [10] also showed that Dutch patients with gastric MALT lymphoma were six times (2.4%, 34/1419) more likely to have metachronous gastric adenocarcinoma than the general Dutch population, and Ishihama et al. [28] reported that 3.3% (4/121) of primary gastric malignant lymphoma patients develop synchronous gastric adenocarcinoma in Japan, a much higher figure than for the Japanese population in general (about 0.05%). The diagnosis interval between metachronous gastric cancer and primary gastric lymphoma varied widely. As previously reported, 91.2% to 100% of gastric cancer in primary gastric lymphoma patients were metachronous gastric adenocarcinoma developed after lymphoma [9,10]. Inaba et al. [9] reported that the mean latent period between primary gastric lymphoma and metachronous gastric adenocarcinoma was 43.1 months (range: 7.9 to 90.8 months). Our research reported 1.9% and 3.2% of primary gastric lymphoma patients had synchronous gastric cancer and metachronous gastric cancer, respectively, which were similar to those reported in previous literature, and higher than that in the general Chinese population. There are even fewer studies on primary gastric lymphoma complicated with gastric precancerous conditions. However, the studies that have been conducted have found that atrophy and intestinal metaplasia appear in 54% to 91% and 51% to 70% of the surrounding tissue of gastric MALT, respectively [12,30]. In addition, Capelle et al. [10] found that 31% (440/1419) of Dutch gastric MALT patients had gastric precancerous conditions, with 4.6% having atrophic gastritis, 21.3% having intestinal metaplasia, and 5.1% having dysplasia. Later, they also found that precancerous conditions existed in 67.5% (27/40) of gastric MALT patients simultaneously and that the prevalence of atrophy, intestinal metaplasia, and dysplasia was 20%, 35%, and 12.5%, respectively [11]. No investigations of primary gastric lymphoma patients complicated with gastric cancer and precancerous conditions in China have previously been conducted. Despite this, our observations for the prevalence of gastric cancer in primary gastric lymphoma patients are consistent with most earlier studies. However, we must pay attention to the endoscopic diagnosis of gastric adenocarcinoma both for the initial endoscopy and the endoscopic review during the follow-up of patients with primary gastric lymphoma. The prevalence of gastric precancerous conditions in primary gastric lymphoma is lower in our study than in earlier ones, and possible reasons for this include a lack of high-definition endoscopy, and insufficient knowledge and limited diagnostic ability of endoscopists on gastric precancerous conditions in the earlier years. Moreover, the sample size and time span of the study subjects are also factors that cannot be ignored. Insufficient amounts taken for biopsy might lead to insufficient bases for pathological diagnosis. In addition, the follow-up of gastric precancerous conditions in our study was consistent with Correa's cascade [24]. This study also analyzed the clinicopathological characteristics of primary gastric lymphoma, gastric cancer, and precancerous conditions, including H. pylori infection. We found that most primary gastric lymphomas were ulcerative type and Lugano Classification stage I + II, and were mostly confirmed to be DLBCL and MALT lymphomas, and we also found that primary gastric lymphoma patients with ulcerative type and Lugano classification stage IIE + IV had a higher risk of developing gastric cancer and precancerous conditions as well. Furthermore, the H. pylori infection rate was higher in patients with MALT lymphoma, Lugano classification stage I + II, and patients with precancerous conditions and gastric cancer, especially gastric cancer of the intestinal type. Our findings on clinicopathological features of primary gastric lymphoma are consistent with those of other studies, which we discuss below. One earlier study found that about two-thirds of primary gastric MALT lymphoma patients were diagnosed at stage I, and that stage IV accounted for only 4.2% [31]. Primary gastric lymphoma with stage IV had disseminated extranodal involvement or concomitant supradiaphragmatic nodal involvement according to the modified Lugano staging of primary gastrointestinal lymphoma [32]. We also had less than 10% of stage IV patients in our study. The ulcerative type was the most frequent presentation at endoscopy [31]. The appearance of an ulcer generally indicates invasion of the muscularis mucosae and deeper layers, which is related to severe Lugano classification stage and high-grade lymphoma [31]. It has also been shown that high-grade lymphomas presented more commonly as ulcerative type, being more frequently diagnosed in stage > I when compared with low-grade lymphomas [33]. We also found that the ulcerative type was an important risk factor for developing gastric cancer and precancerous conditions in primary gastric lymphoma, as was having a severe Lugano classification stage. A possible reason for this is that patients with ulcerative types and severe stages may have had a longer course of the underlying disease. For patients under follow-up care for primary gastric lymphoma, we need to distinguish metachronous gastric cancer and precancerous conditions from the recurrence of gastric lymphoma. We noticed that the median largest tumour size of 4 cm in metachronous gastric cancer was too large considering the median diagnosis interval of 3 years between primary gastric lymphoma and metachronous gastric cancer. A study of minute gastric cancer also showed that the mean growth rate was 0.0071 mm/day, and it took an average of 3.42 years to grow to 5 mm, with 95% of cases being well-differentiated tubular adenocarcinoma [34]. One possible reason for the larger tumour size of the metachronous gastric cancers in our study is that some gastric cancers may have been present before or at the time of diagnosis of primary gastric lymphoma but were missed. The small sample size of 15 metachronous gastric cancer patients is also a very important factor. In addition, 20% of metachronous gastric cancers were undifferentiated adenocarcinomas, which grow fast. All of these factors may exist, but no matter what the cause, gastric cancer should not be missed. Therefore, careful endoscopic and pathological evaluation is required to make the diagnosis. Rentien et al. [35] found that precancerous conditions were more frequent in gastric MALT lymphoma than DLBCL and H. pylori-associated gastritis, and more frequent in the MALT lymphoma area than other areas in the stomach. However, our study didn't find differences in the prevalence of precancerous conditions in gastric MALT lymphoma and DLBCL. Therefore, the prevalences of precancerous conditions among different pathological types of gastric lymphoma need to be further studied with large samples. For our observed high H. pylori infection rate of primary gastric lymphoma, studies have even shown that H. pylori infection is related to adenocarcinoma of the distal stomach and gastric lymphoma [5,6,8]. Some studies have even shown that H. pylori infection was present in nearly 90% of primary gastric lymphoma patients [12,31]. H. pylori can cause chronic inflammation, gastric precancerous conditions, and intestinal-type gastric adenocarcinoma [6,8]. Therefore, we indeed observed that the H. pylori infection rate was higher in patients with primary gastric lymphoma complicated with gastric cancer and with precancerous conditions, and the underlying biological relationship between H. pylori, primary gastric lymphoma and gastric cancer, and precancerous conditions warrants further study. We also found that H. pylori eradication was effective in 40.9% of patients with primary gastric lymphoma, including 55% in MALT lymphoma and 28% in DLBCL, suggesting the necessity of eradicating H. pylori therapy in patients with primary gastric lymphoma. H. pylori eradication is the first choice for the treatment of gastric MALT lymphoma, and it has been reported so far that eradication of H. pylori can achieve a complete pathological response in 47% to 100% of H. pylori-positive gastric MALT lymphoma [5]. However, the role of H. pylori eradication in gastric DLBCL is still controversial. Gastric DLBCL with evidence of MALT is classified as DLBCL (MALT), which is not high-grade transformed MALT lymphoma or de novo DLBCL [5]. Several studies have found that some gastric DLBCL patients respond to H. pylori eradication therapy. A multicenter prospective study has reported that 80% of patients with early-stage gastric DLBCL (MALT) achieve complete remission at a median of 4.0 months after H. pylori eradication [36]. Several other studies also showed that some H. pylori-positive gastric DLBCL(MALT) patients respond to H. pylori eradication [37][38][39]. There were also cases of gastric de novo DLBCL patients who regressed after H. pylori eradication [40,41]. A study in Japan showed 27% of gastric de novo DLBCL patients achieved complete remission with eradication [42]. The efficacy of H. pylori eradication in DLBCL patients might be related to histological type and depth of invasion [42]. BCL10, NF-κB (p65), and CagA expression were also found to help predict the efficacy of H. pylori eradication in patients with early-stage gastric DLBCL(MALT) [36]. The results of our study and previous studies suggest that prospective studies on the efficacy of H. pylori eradication in gastric DLBCL patients are promising. In addition, a previous study suggested an increased risk of metachronous gastric cancer in gastric DLBCL patients treated with chemotherapy, which can be explained by the fact that patients with advanced stages needed chemotherapy [9]. Amiot et al. [29] also found an increased risk of metachronous gastric cancer in gastric MALT Lymphoma patients who received immunotherapy. Our study didn't find the negative effects of chemotherapy, radiotherapy, immunotherapy, and targeted therapy on the development of metachronous gastric cancer in primary gastric lymphoma patients. The association and mechanism of various therapies for lymphoma and the risk of solid tumours, including gastric cancer, remain to be further investigated. Furthermore, whether gastric cancer or gastric malignant lymphoma was treated first depended on the time of diagnosis. For patients with synchronous gastric cancer, the pathological types, stages and expected prognosis of gastric cancer and lymphoma should be taken into consideration to determine the treatment plan. Extending survival and improving quality of life is the primary goal at all times. It's also worth noting that we found no significant difference in prognosis between primary gastric lymphoma patients and complicated with gastric cancer. The main possible reason that gastric cancer didn't significantly affect survival in primary gastric lymphoma patients was that the sample size was too small to represent the population. A previous study reported that the 5-year overall survival rates for gastric DLBCL and MALT lymphomas were 89.6% and 97.7%, respectively, while the 5-year survival rates for patients complicated with gastric cancer were 75.0% [10]. Differences in survival were not statistically analyzed in the study because only 139 patients with primary gastric lymphoma were included, 10 of whom had gastric cancer [10]. A few other studies had similar limitations. In addition, early gastric cancer accounted for 25% of patients with gastric cancer in our study, with a high survival rate and little impact on the prognosis. Regular follow-up of gastric lymphomas provided opportunities for the detection of early gastric cancer, which is also related to the detection and monitoring of precancerous conditions. One earlier study showed that up to 76% of gastric cancer in primary gastric lymphoma patients were of early type [28]. Other studies have shown increased prevalences and progression of intestinal metaplasia and dysplasia during follow-up for gastric lymphoma, which associated with the occurrence of carcinoma [32,43]. We also found a higher detection rate of early gastric cancer (25.0%) in primary gastric lymphoma patients than in the general Chinese population (11%) [44], which may explain the better prognosis and higher 5-year survival rate of gastric cancer complicated in primary gastric lymphoma patients (40.0%) than the gastric cancer patients (27.4%) in China [23]. Our study also found that the development of gastric cancer and precancerous conditions complicated in primary gastric lymphoma conformed to Correa's cascade [24]. Therefore, the follow-up of patients with primary gastric lymphoma may provide an opportunity for early detection of gastric cancer, thereby improving the prognosis of gastric cancer. The primary advantage of our study is that it is the first Chinese study of primary gastric lymphoma complicated with gastric cancer and precancerous conditions. Moreover, our study is a multi-centre study and has the largest sample size among the current studies on gastric lymphoma complicated with gastric cancer and precancerous conditions, followed by the Dutch study [10]. There are also some limitations to the study. The three centres included in this study are located in urban areas in China, two of which are located in the same city. Therefore, there may be bias among the study subjects. In addition, endoscopists could diagnose atrophic gastritis and intestinal metaplasia based on endoscopic findings, and biopsy pathology was not performed on all these patients. Although pathology is the 'gold standard' , pathological evaluation of gastric atrophy and intestinal metaplasia is also based on endoscopic observation by endoscopists and is related to the sampling location and number. Endoscopic evaluation of gastric atrophy and intestinal metaplasia also relies on the endoscopic equipment and the experience of the endoscopists. Conventional white-light endoscopy showed a poor correlation between histological and endoscopic findings in the diagnosis of gastric atrophy and intestinal metaplasia [16]. High-definition white-light endoscopy can identify atrophy and intestinal metaplasia better than conventional white-light endoscopy [45]. Narrow-band imaging and white-light endoscopy nontargeted biopsies may present more promising results for the diagnosis of atrophy and intestinal metaplasia [46,47]. Narrow-band imaging-guided biopsies further improved the diagnostic yield of atrophy and intestinal metaplasia [48]. We included patients with atrophy and intestinal metaplasia both endoscopically and pathologically diagnosed in the gastric precancerous conditions group. While high definition white-light endoscopy and/or narrow-band imaging were used in most cases, except in some early cases. Furthermore, because of our lack of knowledge about the grading of atrophy and intestinal metaplasia, many patients were not graded, especially before 5 years ago. We cannot accurately judge the severity of atrophy or intestinal metaplasia according to the endoscopic pictures at present. However, if we discard cases that are not graded for precancerous conditions, we will lose a lot of cases and information, and we will underestimate the probability of precancerous conditions in patients with primary gastric lymphoma. Therefore, we included these patients with precancerous conditions that were not graded for severity in the precancerous conditions group. It does cause bias. Conclusion In summary, we find that patients with primary gastric lymphoma have a high risk of developing gastric cancer and precancerous conditions and that this risk may be related to H. pylori infection. Follow-up of primary gastric lymphoma may provide an opportunity for early detection of gastric cancer. Endoscopists should pay attention to the endoscopic findings of metachronous gastric cancer, synchronous gastric adenocarcinoma and precancerous conditions in patients with primary gastric lymphoma. Data availability Statement The data used to support the findings of this study are available from the corresponding author upon request.	2023-05-16T06:17:07.383Z	2023-05-15T00:00:00.000	{ "year": 2023, "sha1": "9d928ebb3c4ac1fe024299edfd1a8a1eae7da6e2", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1080/07853890.2023.2193423", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "b386f20066456e5d06a1f440ab377721dd2e1444", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
206440270	pes2o/s2orc	v3-fos-license	Skin optical clearing potential of disaccharides Abstract. Skin optical clearing can significantly enhance the ability of biomedical optical imaging. Some alcohols and sugars have been selected to be optical clearing agents (OCAs). In this work, we paid attention to the optical clearing potential of disaccharides. Sucrose and maltose were chosen as typical disaccharides to compare with fructose, an excellent monosaccharide-OCA, by using molecular dynamics simulation and an ex vivo experiment. The experimental results indicated that the optical clearing efficacy of skin increases linearly with the concentration for each OCA. Both the theoretical predication and experimental results revealed that the two disaccharides exerted a better optical clearing potential than fructose at the same concentration, and sucrose is optimal. Since maltose has an extremely low saturation concentration, the other two OCAs with saturation concentrations were treated topically on rat skin in vivo, and optical coherence tomography imaging was applied to monitor the optical clearing process. The results demonstrated that sucrose could cause a more significant increase in imaging depth and signal intensity than fructose. Introduction Biomedical optical imaging techniques provide a powerful tool for accessing structural and functional information of tissues. 1 However, the strong scattering of tissues induced by the mismatch in refractive indices (RIs) of different tissue components limits the penetration depth of light in tissue and reduces the contrast and depth of biomedical optical imaging.][15][16][17][18] Skin is a protective layer of the body for preventing invasion of light or others into internal tissue, and is also a barrier layer for accessing physiological characteristics via noninvasive optical imaging. 19Undoubtedly, topical treatment of skin OCAs should be an available way for increasing the penetration of light into tissue and enhancing the optical imaging contrast, resolution, and depth. 15,16,20][26] Khan et al. 27 proposed an optical clearing potential (OCP), the ratio of reduced scattering coefficient before and after treatment of OCAs, to evaluate the optical clearing efficacy of skin.Some positive results have been obtained, e.g., hydroxyl-terminated organic compounds demonstrated higher OCP than others. Thereafter, Hirshburg et al. further found that OCAs' OCP correlated with its ability for destabilizing the collagen structure because collagen located in the dermis is the main scattering source in skin. 26,28,29Then they employed molecular dynamics (MD) simulation to study the interaction between collagen and alcohols, and they predicted the OCP by means of analyzing the propensity to form hydrogen bonds and bridges. 28,29The MD theoretical simulation was further verified by the experiments, 28,29 and the MD simulation would effectively avoid the blindness and time-consuming of experiments.Meanwhile, they found that increase of OCA molecular size and the number of hydroxyl groups could further increase the efficacy of TOC. 28oting that sugars have hydroxyl groups, Wang et al. investigated sugars-induced skin optical clearing efficacy and found that fructose had the best OCP among monosaccharide-OCAs through MD simulation and experimental demonstration. 16Actually, disaccharides have a larger molecular size and more hydroxyl groups, but the OCP of disaccharides received little attention, and there is no comparative study between monosaccharides and disaccharides.Recently, some innovative TOC methods for ex vivo brain and other organs were developed, such as Scale, 30 ScaleS, 11 SeeDB, 12 CUBIC, 31 and so on; alcohol (glycerol and sorbitol) and sugar (fructose and sucrose) were also used, respectively. In addition, all of these methods usually include tedious steps or complex components, which make them unsuitable for in vivo skin because of their time-consumption and toxicity. In this study, the OCP of sucrose and maltose, as the representatives of disaccharides, was investigated systematically by theoretical simulation, ex vivo and in vivo experiments, and compared with fructose.MD simulation was used to predict the OCP of the agents according to its propensity to form hydrogen bonds.Due to the three sugars having different saturation concentrations, various concentrations were prepared.After ex vivo skin samples were immersed in each solution, the reduced scattering coefficients were measured by a commercially available spectrophotometer with an integrating sphere to evaluate the optical clearing efficacy.Further in vivo studies were performed by using OCT to evaluate the improvements in OCT imaging depth and signal intensity after topical treatment of saturated agents on in vivo rat skin. Chemical Agents Considering the solubility of disaccharides and theoretical investigation, sucrose and maltose were selected as the representatives to compare with fructose, which has been reported to have a greater OCP among the three monosaccharides. 16igure 1 shows the chemical structures, respectively. In this study, sucrose, maltose, and fructose were purchased from the Sinopharm Chemical Reagent Co., LTD (Shanghai, China).To evaluate the OPC of the OCAs, various agents with some concentration gradient were prepared for the ex vivo experiments.Their RIs were measured using an Abbe digital refractometer (WAY-2S, Shanghai YiCe Apparatus & Equipment Co., LTD, Shanghai, China).Since skin optical clearing methods have universality for various optical imaging techniques and the pH value of OCAs may produce influences on the fluorescence proteins or dyes, the pH values of each OCA with different concentrations were measured using pH-Indicator strips (Sigma-Aldrich Co., Shanghai, China).The RIs and the pH values of each agent at different concentrations (Con.) were listed in Table 1. Molecular Dynamics Simulation MD simulation was employed to study the interactions between chemical agents and collagen.Here, the OCP of each agent was quantitatively evaluated by analyzing the number of hydrogen bonds formed between OCAs and collagen mimetic peptides.MD simulation software CHARMM (version-36, Chemistry at Harvard Macromolecular Mechanics, Harvard University, United States) with CHARMM22 force field and the carbohydrate force field was used.1BKV (Protein Data Bank ID) and a regular GPO peptide ½ðGPOÞ 10 3 (G, glycine; P, proline; and O, hydroxylproline) are representative collagen mimetic peptides for MD simulation. 29The information of the computational hardware is as follows: Intel(R) Core(TM) i5 CPU at 2.80 GHZ, 6 GB of physical RAM, Linux.The MD simulation of all agents proceeded in parallel, and the runtime was about 17 h. Each agent was applied to simulate with both 1BKV and GPO, respectively.On account of disaccharide molecular size limits, 10 disaccharide molecules (sucrose or maltose) were placed randomly around the collagen mimetic peptide at a radial distance of 12.0 Å away from the cylindrical axis of the peptides. 29By contrast, 12 monosaccharide molecules (fructose) were placed in a similar way according to the previous study. 16Each production run lasted 600 picosecond (ps) and the coordinates were recorded every 1 ps.To prevent diffusion of the agent molecules away from the collagen, a cylindrical potential shell was applied, and the harmonic constraints were used to anchor the peptide.The detailed simulation process was reported by Hirshburg et al. 29 The average numbers of hydrogen bonds between each agent and mimetic peptides during 600 ps were analyzed after the entire simulation.The cutoff radius of hydrogen bonds was set to 2.4 Å. 32 The number of hydrogen bonds formed between every molecule and collagen every 1 ps was counted to evaluate the skin OCP of sucrose, maltose, and fructose, respectively. Animal Preparation Male Sprague-Dawley rats (60 g, n ¼ 40) were bought from Wuhan University Center for Animal Experiments (Wuhan, China).All animals were fed under a specific pathogen free level of feeding condition.The animal care and experimental procedures were approved by the Experimental Animal Management Ordinance of Hubei Province, China.Rats were anesthetized by an intraperitoneal injection of a mixture of 10% urethane and 2%-α-chloralose with a dosage of 0.8 mL∕100 g.After rats were thoroughly unhaired with depilatory cream, they were placed on the experimental platform. Ex Vivo Experiments Thereafter, we removed the dorsal skin and cleaned the subcutaneous fat, fascia, and blood completely.Then the fresh skin samples (5 cm × 5 cm, N ¼ 95) were immersed in phosphate buffer solution and kept under 4°C until experiments were performed in <24 h.The fresh dorsal skin samples were randomly divided into 19 groups according to Table 1. A micrometer (Chengdu ChengLiang Tools Group Co., Ltd., Chengdu, China) was used to measure the thickness of skin sandwiched between two glass slides at four different directions.To reduce artificial measurement error, the measurements were done by the same person.The spectrophotometer (Lambda 950, PerkinElmer, Shelton, Connecticut) with an integrating sphere (150MM RSA ASSY, Labsphere, North Sutton), a dual-beam system, was applied to measure the reflectance and transmittance of the samples.The diameter of the integrating sphere is 150 mm with a circle reflectance-entrance (r ¼ 12.70 mm) and a transmittance-entrance.The transmittance-entrance combines two semicircles (r ¼ 8.11 mm) and one rectangle (w × h ¼ 16.22 mm × 7.78 mm). 33The spectrophotometer's software WinLab was used to calibrate.Each measurement needed to scan a baseline, and 100% transmittance was performed without samples, 100% reflectance was performed by a reflectance standard (Labsphere), 0% transmittance and reflectance were in the absence of light to reflect the dark noises.After the skin samples were immersed into each solution for 45 min, the thickness of the skin samples and the reflectance and transmittance spectra were measured again. Based on the reflectance, transmittance, and thickness of each sample, the inverse adding-doubling method was applied to calculate the reduced scattering coefficient without introducing correction factors.5][36] Further, the reduced scattering ratio (RSR) was calculated as follows: 28 where μ 0 before and μ 0 after are the reduced scattering coefficient (λ ¼ 635 nm) before and after OCA treatment. Optical Coherence Tomography for Assessing Optical Clearing Potential In Vivo The imaging principle of OCT indicates that it has the capacity to image the tissue within 1 to 3 mm depth in vivo, [37][38][39] which makes it a powerful tool to assess the optical clearing process in tissues. 22,23,40In this study, we used a commercial OCT system (OCP930SR, Thorlab Inc., Newton, New Jersey) working at ð930 AE 5Þ nm with ð100 AE 5Þ nm full-width-half-maximum, with an optical power of 2 mW, a maximum image depth of 1.6 mm, an axial resolution of 6.2 μm (in air), and a lateral resolution about 10 μm.The saturated sucrose (78.9%) or saturated fructose (67.1%) induced skin optical clearing efficacy was assessed quantitatively by analyzing the improvements in imaging depth and signal intensity at the given depth. Here, 12 rats were used for in vivo experiments.Initially, the prepared rats were placed on the experimental platform.The OCT B-scan images were recorded through the intact dorsal skin, tape stripped skin, and 6, 12, 18, 24, 30, and 36 min after topically applied saturated sucrose or saturated fructose on the skin, respectively.In order to reduce the mirror reflection caused by OCA, the OCA was quickly wiped away prior to acquiring the images and then was reapplied.It took about 30 s to wipe away and reapply the OCA, and 30 s acquire the images. To facilitate analysis, the acquired images were flattened to enable averaging of the signal intensity profiles over the whole image so as to reduce the effects of the heterogenetic distribution of cutaneous absorbers and scatters.The flattening procedure was carried out with two steps: finding the z position of the skin surface at each lateral x position in the image and then translating the column of pixels at that x to bring the skin surface to a common axial position. 5Figure 2 shows an example of flattening the skin by the image analysis.What is worth noting is that the flattening procedure may distort the tissue structure somewhat because it shifts pixels so that the skin surface is aligned at one constant z-axis position.However, such spatial distortion has minimal effect on the deduced light penetration since this study only paid attention the one-dimensional penetration of light into the skin. 5Results Hydrogen Bonds Formed Between Collagen and Agents Previous studies indicated that the higher-order structures of collagen could be stabilized by the hydration shell and water-mediated hydrogen bonds. 28,32Nevertheless, the formation of hydrogen bonds as well as hydrogen bond bridges between collagen and chemical agents could disrupt the hydration shell and water-mediated hydrogen bonds, 29,32 which would destabilize the higher-order structure of collagen.Thus, the propensity of chemical agents to form hydrogen bonds could be used to predict the OCP of agents.Figure 3 shows the hydrogen bonds formed between the three agents and collagen mimetic peptide, it is expected that the more hydrogen bonds and hydrogen bond bridges an agent forms, the more effectively the hydration shell and water-mediated hydrogen bonds are disrupted. The average numbers of hydrogen bonds formed between each agent and two collagen mimetic peptides per ps are calculated and listed in Table 2.We find that sucrose forms the most hydrogen bonds among the three agents.That is, sucrose has the strongest ability to disrupt the hydration shell and watermediated hydrogen bonds.Previous research has shown that the RSR increases as collagen solubility increases. 41Hence, it predicts that sucrose has the best skin OCP, while maltose is the second best and fructose is the worst, respectively, among the three agents. Ex Vivo Experiment for Reduced Scattering Ratio Further, to verify the prediction of MD simulation, the reduced scattering coefficient before and after skin samples were immersed into each agent was calculated, and then the RSR was deduced.The slope of the RSR was used to define the OCP of each agent. 28Figure 4 shows the RSR of skin samples treated with each agent at different concentrations for 45 min, and it can be seen that the RSR increases with the concentration of an agent for the same OCA.Considering three concentration regions (A: 1-2 M; B: 1-3 M; C: 1-6 M) for each agent, different slopes were calculated separately.The results show that there is an excellent correlation (R 2 ) between the experimental data and the fitted data for each agent.The slope in concentration region A is in a descending order of sucrose, fructose, and maltose.Likewise, in concentration region B, the slope of sucrose is larger than that of fructose.According to Ref. 28, the slope is defined as the OCP of a chemical according to all the fitting data points, and the OCPs of sucrose, maltose, and fructose are 1.05, 0.65, and 0.48, respectively.Thus, sucrose and fructose have the best and worst skin OCP among the three agents, which is consistent with the MD simulation results. It should be noted that different chemicals have different saturation concentrations, and OCAs-induced optical clearing efficacy increases with its concentration.Furthermore, statistical analysis at common concentrations and the highest concentrations for fructose and sucrose were made, respectively.The results show that the RSR values of sucrose at the same concentration are obviously larger than those of other two agents (p < 0.05).However, there is no significant difference between the RSR of sucrose (3 M) and fructose (6 M) (p > 0.05). Agent-Induced Improvements in OCT Imaging Depth and Signal Intensity Although MD simulation and ex vivo experimental results demonstrated that sucrose has a better OCP than that of fructose, the skin optical clearing efficacy caused by saturated sucrose is almost the same as that by saturated fructose.Moreover, both are on the basis of the direct interactions between the dermal collagen and the OCAs, which neglects the barrier of the stratum corneum that limits the penetration of OCAs into the dermis. In this work, the in vivo rat skin optical clearing performances of saturated fructose and saturated sucrose were evaluated using OCT. Figure 5 shows the typical OCT results recorded through the intact rat skin in vivo, tape stripped skin, and 6, 12, 18, 36 min after the topical application of fructose (top row) or sucrose (bottom row) on the skin, respectively.By comparing the initial and the tape stripping, we find no significant changes, whereas after topical application of fructose or sucrose, the OCT imaging depth and signal intensity in the deeper region are enhanced gradually as optical clearing develops. Figures 6(a)-6(b) show the typical normalized OCT signal intensity profiles after treatment of saturated fructose or sucrose for different times, respectively.It is found that OCAs' treatment can evidently increase the signal intensity.To quantitatively evaluate OCAs-induced changes in imaging depth, the depth at which the signal intensity attenuates to 1∕e of the normalized OCT intensity was calculated, respectively.The imaging depth can reach up to 450 or 550 μm after treatment of saturated fructose or saturated sucrose for 36 min, respectively, but the initial imaging depth is only 200 μm. Figure 6(c) demonstrates the changes in image depth versus treatment of OCA, and the slope of linear regression analysis was also used.The results show that the slope of saturated sucrose (0.33) is higher than that of saturated fructose (0.23), respectively.Further statistical analysis using one-way analysis of variance (ANOVA) with SPSS (version 16.0, IBM) shows that there exists an extremely significant difference (p < 0.01) between the two slopes. Furthermore, we calculated the changes in signal intensity at 300 and 400 μm before and after OCA treatment for different times [see Figs.7(a)-7(b)].It demonstrates that both OCAs can significantly enhance the signal intensity at 300 and 400 μm.The linear regression slopes of sucrose and fructose are 0.49 and 0.44 for the changes in signal intensity at 300 μm, and 0.38 and 0.27 for that at 400 μm, respectively.Further statistical analysis using one-way ANOVA shows that there is an extremely significant difference for that at 400 μm (p < 0.01) between the linear regression slopes of fructose and sucrose, but no significant difference for that at 300 μm (p > 0.05). Discussion In this work, the optical clearing potential of disaccharides was investigated through the MD simulation, ex vivo, and in vivo experiments.Sucrose and maltose were chosen as typical disaccharides due to the limited availability of the force field files of disaccharides.Previous investigations showed that sugar-alcohols were common OCAs of skin optical clearing.For instance, fructose had better a OCP than glycerol, sorbitol, DMSO, and glycerol by the ex vivo experiments; 26,41,42 and it was the best one among the three monosaccharides, such as glucose, fructose, and ribose from the MD simulation to experiments. 16hus, we used fructose to compare with the disaccharides in this study. The MD simulation revealed that the OCP is in the order of sucrose, maltose, and fructose.This is the reason that sucrose could form the most hydrogen bonds among the three agents, so molecular size and the numbers of hydroxyl groups play an important role in skin optical clearing.The occupation of the water-mediated hydrogen bonds sites by the OCAs-agent molecules would disrupt the hydration shell because of the formation of hydrogen bonds between OCAs-agents and collagen. 26Since disaccharides have a larger molecular size than monosaccharides, it has a bigger coverage on the collagen surface and observably shields water surrounding the collagen peptides.Thus, the OCP of disaccharides is stronger than that of monosaccharides, which implies that a higher OCP may be realized by extending the chain length for sugar-alcohols. The theoretical prediction of the OCP of OCAs was demonstrated by ex vivo experiments.The results indicate that sucrose has a larger OCP than fructose, which is inconsistent with that reported in previous work. 41The correlations (R 2 ) between the experimental data and the fitted data in Ref. 41 were only 0.73 and 0.78, for sucrose and fructose, respectively, whereas the R 2 reach up to 0.993 and 0.974 in this work. Undoubtedly, the OCP is an important parameter to evaluate the OCA-induced skin optical clearing efficacy.However, each chemical has a different solubility or saturation concentration.It is well known that a high concentration of OCA can induce better optical clearing efficacy for the same chemical.Therefore, OCA-induced optimal optical clearing efficacy not only depends on the OCP, but also on its saturation concentration.Thus, it is easy to understand why fructose with a lower OCP and higher concentration (6 M) could cause almost the same ex vivo skin optical clearing efficacy as sucrose with a higher OCP and lower concentration (3 M). In addition, both the theoretical prediction and ex vivo experiments neglect the barrier function of the epidermis.Therefore, OCAs-induced in vivo skin optical clearing efficacy may be relative to the penetrability of OCAs.Since the saturation concentration of maltose is extremely low, for the in vivo skin optical clearing experiments, only the saturated sucrose was compared with the saturated fructose.Results indicated that both OCAs would increase the signal intensity in the deeper region.Fructose treatment decreases the signal intensity in the shallower region, whereas this situation was less obvious for sucrose treatment.This inconsistent phenomenon should be the result of the different influences of the OCAs on surface reflectivity, which was also observed by other investigators when they used different OCAs.For instance, Ghosn et al., 43 and Wen et al. 5 reported similar changes with our results of the fructose treatment; and Zhong et al. reported that optical clearing of human skin enhanced the OCT signal at all depths; 44 Xu et al. found that optical clearing of porcine skin in vitro increased the reflectance from deeper dermis although the OCT signal from superficial skin did not change. 45Meanwhile, the deeper penetration depth promoted the enhancement of image contrast, which was demonstrated by Wen et al., 5 Larin et al., 6 Proskurin et al., 46 and Bonesi et al. 47,48 OCT imaging results showed 1.5 to 2.5 times enhancements in penetration depth or signal intensity after topical application of saturated fructose and sucrose, respectively.The imaging depth for the intact rat skin was only about 200 μm, which was insufficient for obtaining the dermal information, whereas after OCAs treatment, the imaging depth could reach 450 to 550 μm, which can be almost enough to penetrate the whole dermis.4][15][16][17][18] Especially, the pH of fructose decreases with its concentration, which shows a meta-acid with a pH of 5.5 at a concentration of 6 M, whereas the pH of the sucrose increases with its concentration, which is neutral with a pH of 7 at a concentration 3 M.The acid materials may quench fluorescent protein, 50 which indicates that sucrose will be more befitting for fluorescence imaging. In addition, in vivo skin optical clearing efficacy also depends on the penetration of OCAs.If some chemical penetration enhancers, such as zone, thiazone, DMSO and so on, are introduced, 13,42,51 the imaging depth will be further enhanced.This information is expected to play a much more significant role in clinical application 21 and basic research. Conclusion In this work, the OCP of sucrose, maltose, and fructose was predicted by MD simulation, which was verified by the ex vivo experiments.The results indicated that sucrose had the best skin OCP, but there is no significant difference between sucrose at 3 M and fructose at 6 M for induced skin optical clearing efficacy.The in vivo OCT imaging experiment indicated that the imaging depth and signal intensity were obviously improved after topical treatment with the saturated fructose and sucrose, and that sucrose is better than fructose.This study combined MD simulation with ex vivo and in vivo experiments to hunt for a novel preferable skin OCA from common disaccharides, which expands the reagent library of skin OCAs.It is hoped that sucrose will play an important role in optical clearing-based application and research. Fig. 2 Fig. 2 (a) Original OCT image and (b) computer-flattened image.Bright color indicates the strong OCT signal intensity. Fig. 4 Fig. 4 RSR of ex vivo rat skin as a function of agent concentration.(a-c) The slope of each agent is obtained by linear regression analysis of RSR data in different saturated concentration regions (inset).The data are shown as mean AE standard deviation (p < 0.05). Fig. 5 Fig. 5 Computer-flattened OCT images of rat dorsal skin treated with saturated fructose or saturated sucrose, respectively. Fig. 6 Fig. 6 Typical temporal dynamical profiles of normalized OCT signal intensity after treatment with (a) saturated fructose and (b) saturated sucrose, respectively.(c) The relative changes in imaging depth (ΔD∕D 0 ) at different treatment time with the two saturated solutions.Data are mean AE standard deviation, N.S. refers to no significant difference, means significant difference (p < 0.05), and ** means extremely significant difference (p < 0.01). Fig. 7 Fig. 7 Relative changes in signal intensity (ΔI∕I 0 ) at the depth of (a) 300 μm and (b) 400 μm through the in vivo rat skin treated by saturated fructose and saturated sucrose, respectively.Data are mean AE standard deviation, N.S. refers to no significant difference, * means significant difference (p < 0.05), and ** means extremely significant difference (p < 0.01). Table 1 The RIs and the pH value of each agent at different Con.(M refers to mol/L). Table 2 Average number of hydrogen bonds formed.	2018-04-03T04:41:19.195Z	2016-08-01T00:00:00.000	{ "year": 2016, "sha1": "c768692ca3fb02ca1621d8aedfb151d01bbed40c", "oa_license": "CCBY", "oa_url": "https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics/volume-21/issue-8/081207/Skin-optical-clearing-potential-of-disaccharides/10.1117/1.JBO.21.8.081207.pdf", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "130be7b1e03fd516cbc9a15b65177a3fe93cbd65", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Medicine", "Chemistry", "Engineering" ] }
55293083	pes2o/s2orc	v3-fos-license	Tarkovsky ’ s Philosophy of Love : Agape in Stalker and Sacrifice This article explores how the main characters in Tarkovsky’s movies Stalker and Sacrifice exhibit the Christian ideals of agape. With the New Testament, agape acquired metaphysical primacy and distinctive meaning as it came to be identified as originating from God and portraying a sacrificial act exemplified by the crucifixion of Jesus. Hence, agape necessarily entailed strong altruistic ethos and passionate commitment for the well being of others. Besides demonstrating the way in which Tarkovsky’s main heroes in Stalker and Sacrifice exhibit the ideals of agape while each going through their own unique “hero’s journey”, the article also seeks to unravel how Tarkovsky’s philosophy of love goes beyond mere “religious obstructionism”. Although Zizek argues that Tarkovsky’s ultimate message and solution to the ills of human existence is “religious obstructionism; that of self-sacrifice”, Tarkovsky’s philosophy of love provides much more complex and profound picture by entailing mystical characteristic and spiritual depth. Indeed, if one removes its metaphysical dimension, Tarkovsky’s philosophy of love may even align with the secular tempered existentialist and humanist tradition. Introduction This article seeks to unravel Tarkovsky's philosophy of love, which is complementarily embodied in the two movies that he had directed: namely; Stalker and Sacrifice.According to Zizek (2006), "religious obstructionism; that of self-sacrifice" is the ultimate message or solution that Tarkovsky provides to the ills of human existence through his movies of Stalker and Sacrifice.However, statements and analysis given by Tarkovsky in his own interviews (Gianvito, 2006) and in his classical book, Sculpting in Time (1987), provide us a much more complex and profound picture.After all, in his own book, Tarkovsky (1987:200) declares that his main function, as a director, "is to make whoever sees my films aware of his need to love and to give his love, and aware that beauty is summoning him."According to the central thesis of this article, transformation through love (not the "religious obstructionism; that of self-sacrifice") is the ultimate message or solution that Tarkovsky provides to the ills of human existence through these two movies. But then what kind of love does Tarkovsky refer to while stating about humans' "need to love and give love"?In order to support the central thesis, the article will first demonstrate how and why Tarkovsky's ideal of love is deeply rooted in the the Christian notion of Agape as formulated by the spiritual leaders of that tradition such as John and Saint Paul (Wirzba and Benson, 2008).Secondly, the article will argue how Tarkovsky's philosophy of love goes far beyond mere "religious obstructionism" by entailing mystical characteristic and spiritual depth akin to perennial philosophy (Huxley, 1945).Even so, if one removes its metaphysical dimension, Tarkovsky's philosophy of love is not in contradiction but rather in conjunction with the secular tempered existentialist or humanist tradition espoused by Erich Fromm (1956 and1999), Viktor Frankl (1992), Rollo May (1969).All these, in turn, constitute one of the main reasons why Tarkovsky's movies are still appealing to the non-religious spectators in our "secular age" (Taylor, 2007). To this end, the article will be divided into four sections.First section will provide key analysis of Tarkovsky's philosophical reflections on Sacrifice and Stalker while providing a key narrative for both movies.The second section will expose how Tarkovsky's ideal of love derives from the Christian ideal of Agape, which particularly stands for the "love of God for men" irrespective of humans' sin or love in God (Nygren, 1953;Lewis, 1960;May 1969;and Selig, 2015).Third section will scrutinize how Tarkovsky projects his ideal of Agape on the main heroes in both Stalker and Sacrifice.Utilizing Joseph Campbell's notion of Hero's Journey, one may even call them spiritual heroes who go through a spiritual crisis to create a better version of themselves (Bray, 2017 andCampbell, 2004).After briefly revealing the mystical and spiritual depths of Tarkovsky's cinematic language, the fourth section will highlight that if one leaves aside his metaphysical dimension, Tarkovsky's philosophy of love can be reconciled with secular tempered humanist tradition espoused by scholars such as Erich Fromm and Viktor Frankl (Robbins, 2015). Tarkovsky's Philosophical Reflections on Stalker and Sacrifice Tarkovsky's "Stalker" is one of the greatest landmarks in cinema history.The movie's sensational landscapes, ethereal music and imageries, along with the script's psycho-philosophical depths, renders it an exceptionally enigmatic piece of visual poetry (Turovskaya, 1989.The plot of the movie is loosely based on the science fiction novel, Roadside Picnic, written by Arkady and Boris Strugatsky.The plot revolves around the journey that three people make to a place called "the zone" (zona).The zone is a deserted and forbidden area closely protected by the government authorities in which there is a room where it is believed that people's most innermost wishes are fulfilled (Pourtova, 2017).Travelling into the zone is not only challenging because of the forbidden entry but also due to its dangerous nature.The zone is a place full of complicated and deadly traps (McLenachan, 2014). Given the dangers and difficulties of reaching the zone, people, who would like to take the risk of traveling into the zone, have to hire a professional guide or a "stalker" -hence the name of the movie.Accordingly, the plot of the movie unfolds as the "Writer" and the "Professor" embark upon an existential Odyssey with the "Stalker" into the zone.Towards the end of the movie, the "stalker" announces that they have finally reached to the room in the zone.However, both the writer and the scientist refuse to enter the room and the trio return without entering the room. In his untimely book Sculpting in Time, Tarkovsky (1987:199) unequivocally articulates the central message that he intended to communicate through his movie by asserting that; "In Stalker I make some sort of complete statement: namely that human love alone is-miraculously-proof against the blunt assertion that there is no hope for the world.This is our common, and incontrovertibly positive possession.Although we no longer quite know how to love…" Furthermore, Tarkovsky (1987:200) maintains: In the end, everything can be reduced to the one simple element, which is all a person can count upon in his existence: the capacity to love.That element can grow within the soul to become the supreme factor, which determines the meaning of a person's life.My function is to make whoever sees my films aware of his need to love and to give his love, and aware that beauty is summoning him. At first, Tarkovsky's assertion is that his movie Stalker is fundamentally about the miraculous nature of human love, which may occur as quite puzzling.Nevertheless, in the third section it will be demonstrated how the two heroes in Stalker and Sacrifice (stalker himself and Alexander) embody the ideals of love according to Tarkovsky. Sacrifice is the last masterpiece of Tarkovsky, which he produced while living in exile from the Soviet Union (The Sacrifice, 1987).The film was shot in Sweden just before Tarkovsky's untimely death due to lung cancer.In the movie, Tarkovsky utilizes the allegory of tree of life in both the first and the last scene of the Sacrifice in order to powerfully evoke his notion of transformation through love. Since Plato, various thinkers have resorted to the use of allegories to render complex ideas visible and intelligible (Tarnas, 2011).The Sacrifice also opens and closes with the allegory of 'tree of life'.In the first scene, Alexander asks his son (Little Man) to give him a hand as he tries to plant a dead barren tree near the lake.While planting the tree together, Alexander tells the Little Man a story about how once upon a time an old Orthodox monk named Pamve planted a tall barren tree on a mountainside just like he is doing.Then, Pamve entrusted his young disciple Ioann Kolov to water the dead tree every day until it came to life.Every morning, Ioann would fill the bucket with water and climb up the mountain to water the dead tree.Ioann has done the same thing for three years and one day, as he climbed up the mountain, he saw that the whole tree was covered in blossoms! The narrative of Tarkovsky's Sacrifice unfolds as radio announces the outbreak of a total nuclear war, which will bring the annihilation of life on earth.Confronted with the almost certain future of nuclear holocaust on the planet Earth and the "deadly, sickening, animal fear" that comes along with it, Alexander turns inward and prays to God to spare the human kind.In return, he promises to sacrifice and "relinquish everything that binds" him to life; his house and family including his most beloved son, the Little Man.Alexander wakes up the next day experiencing that his prayer to God has been heard and that everything is normal. To keep his promise of sacrifice, Alexander takes his family for a walk in the countryside and then secretly comes back home to put his house on fire.When the family arrives home, Alexander confesses that he has burnt the house while running around in frenzy.Ambulance comes and two paramedics chase and seize Alexander who appears to have gone mentally ill. While discussing on the role of an artist in society, Tarkovsky (1987:40) emphasizes that the idea of love necessarily entails an act of sacrifice.Indeed, this is one of the reasons why Tarkovksy chooses to direct the movie Sacrifice.While reflecting on his movie, he elaborates further on the indissoluble link between the idea of love and sacrifice by stating: What nobody seems to understand is that love can only be one-sided, that no other love exists, that in any other form it is not love.If it involves less than total giving, it is not love…I am interested above all in the character who is capable of sacrificing himself and his way of life-regardless of whether that sacrifice is made in the name of spiritual values, or for the sake of someone else, or of his own salvation, or of all these things together (Tarkovksy, 1987:217). Certainly, Tarkovsky's (1987:200) emphasis on the ontological primacy of the "capacity to love", becoming aware of the "need to love and to give love" in Stalker intersects and complements the remarks made above on the altruistic nature of sacrificial love.Indeed, this is also how the two movies, Stalker and Sacrifice, complement each other in displaying Tarkovsky's philosophy of love.As the next section will show, Tarkovks's points directly reflects the disintictive meaning that Agape came to identified with the New Testament where Agape's metaphysical and ontological primacy is tied with its sacrificial characteristic (Brady, 2003).The next section will explore the ontological primacy of love and its sacrificial characteristics as the two most distinguishing components of Agape embodied by the Christian heritage. Early Christian Heritage of Agape in New Testament: John and Saint Paul The Christian notion of Agape stands for the unconditional love of God for humankind regardless of his or her sins.In his famous book The Four Loves, C.S Lewis (1960) distinguished and described Charity (Agape) as "the highest level of love known to humanity: a selfless love that is passionately committed to the wellbeing of others".Following Lewis, a pioneer in existential psychology, Rollo May (1969: 37-38), expanded on these four types of love and enumerated them as: 1-Libido, sexually charged lust. 2-Eros, the drive of love to create and the ascending urge towards higher forms of being and relationship. Since the beginning of the ancient Greek civilization, Eros, as the "drive of love to create and pro-create" (May 1969: 78), played a significant role in shaping the mental imaginary of the Western mind (Tarnas, 2011).With Plato, the concept of Eros has established itself as that unfettering force which uplifts men towards heavenly ideas and thus serving as a vehicle for the particular to ascend to the universal (Wagoner, 1997).Certainly, for Plato it was through the reason that one starts to seek the particular beauty in this world of appearance and then charge the wings of Eros to ascend to the world of ideas. Nevertheless, the Christian notion of Agape is cherished as the highest form of love available to humankind since Agape originates from the God (Nygren, 1953).In contrast to Eros, Agape is the pure form of divine love and it is devoid of any egocentric acquisitiveness towards the object of the beauty and love (Abrahamov, 2011: 12-13).Although Nygen clear cut anthitehtical typology between eros and agape is not without its critics (Armstrong. 1961;Streiker, 1964), his dichatomous typology of two loves is valuable to the exent that it captures the most distinguishing features of Agape embodied by the New Testament [See Table 1]. Eros versus Agape. Nygen's Two Types of Love Eros is primarily man's love; God is the object of Eros. Even when it is attributed to God, Eros is patterned on human love. Agape is primarily God's love; "God is Agape." Even when it is attributed to man, Agape is patterned on Divine love. Eros is acquisitive desire and longing. Eros is an upward movement. Eros is man's way to God. Agape is sacrificial giving. Agape comes down. Agape is God's way to man Eros is egocentric love, a form of self-assertion of the highest, noblest, sublimest kind. Agape is unselfish love, it "seeketh not its own," it gives itself away. Eros is the will to get and possess which depends on want and need. Agape is freedom in giving, which depends on wealth and plenty. Source: Adopted from Anders Nygren (1953. 210) Although the term Agape appeared numerous times in various meanings since Homer (Brady, 2003:54), it acquired a new metaphysical context and distinctive meaning through the New Testament.With the New Testament, agape came to be identified with unconditional and self-sacrificing love of God for humans which is best examplified through the crucifixion of Jesus.Indeed, the suffering of Jesus at the cross as the son of God for all the human sins plays the most central role in the imagination of the Christian psyche.Thus agape is not just an abstract unconditional divine love but love demonstrated by the sacrifice of God's most beloved son!According to this article, the most distinguishing features of Agape is composed of two elements.First, Agape retains utmost ontological, metaphysical and epistemological primacy due to the very fact that it "originates in God and God is love" (Wirzba, 2008;Brady, 2003) Second, Agape is quintessentially sacrificial due to the atonement of Jesus and thus necessarily contains the altruistic and other-centred love ethos (Hill, 2002). Indeed, the first First Epistle of John establishes how agape originates in God by declaring that: "Beloved, let us love one another, because love is from God; everyone who loves is born of God and knows God.Whoever does not love does not know God, for God is love."(Brady, 2003:75).By equating love with God, agape is then granted ontological, epistemological and metaphysical priority.But then, the Christian notion of agape invevitably contains sacrificial ethos as John continues: "God's love was revealed among us in this way: God sent his only Son into the world so that we might live through him.In this is love, not that we loved God but that he loved us and sent his Son to be the atoning sacrifice for our sins."(John 4:7-12) Hence, Agape's metaphysical and ontological primacy is indisolloubly linked with its sacrificial characteristic. One can observe the same sacrificial emphasis in the letters of Saint Paul.In the Chapter 13 of his letter to the Corinthians, for instance, Saint Paul establishes "faith, hope and love" to be the most fundamental Christian virtues.Among the three virtues "faith, hope and love", however, Saint Paul considers love as being the greatest.According to Saint Paul (Wagoner, 1997: 43) love has the utmost ontological and epistemological value among the three since "Love never fails… It always protects, always trusts, always hopes, always preserves." As stated previously, agape necessarily contains other centred altruistic characteristic since it is devoid of egocentric acquisitiveness.This characteristic is certainly expressed by Saint Paul (Wagoner, 1997: 42) when he declares: "Love is patient, love is kind.It does not envy, it does not boast, it is not proud… It does not dishonor others, it is not self-seeking, it is not easily angered, it keeps no record of wrongs."In his letter to Epistalians, Saint Paul makes sure to provide the sacrificial act of Christ as a model for Agape to be emulated by all Christians.As Paul declares: "God in Christ has forgiven you.Therefore be imitators of God, as beloved children, and live in love, as Christ loved us and gave himself up for us, a fragrant offering and sacrifice to God" (Eph.5:1-3).Indeed, Agape as an unselfish love which gives itself away through the act of sacrifice is an ideal which every Christian must aspire for.As Paul urges upon, "If the love of God was manifested in the self-giving of Christ, how could the love of Christ be shown to others except in the same way?" (Brady, 2003:69).Given the basic tenents of Agape particularly outlined by Saint John and Paul, the next section will demonstrate how Tarkovksy's heroes reflect the ideals of Agape. Tarkovsky's Spiritual Heroes Reflecting the Ideals of Agape While Alexander's sacrificial act for the sake of saving the World from nuclear catastrophe in the movie Sacrifice is quite evident, Tarkovsky asserts that his movie Stalker is fundamentally about the fact that miraculous nature of human love may occur as less obvious.Yet, when one focuses on the main character of the movie, it is not difficult to see why Tarkovsky makes that statement.In his interviews on Stalker, Tarkovsky (Gianvito, 2006: 61) emphasizes that out of the three main characters who embark on the journey to the zone, the stalker is "the one who pleases me the most" and that he reflects "the best part of me". Indeed, in the eyes of Tarkovsky, the main character stalker's selfless devotion to serve people by taking them to the zone makes him a spiritual hero.For stalker, the belief in the zone and belief in the hope that zone may bring to humanity is what really matters.Whether the zone's acclaimed miracles are truly real or not is not the ultimate concern.As Tarkovsky (Gianvito, 2006: 57) comments in his interviews, "What is important to Stalker is to light a spark, a belief in the heart of people." Furthermore, Tarkovsky appraises the selfless love and irrational devotion that the stalker's wife has towards her husband despite all the miseries caused by her husband's declared mission."Her love and her devotion" Tarkovsky (1987:198) remarks, "are that final miracle which can be set against the unbelief, cynicism, moral vacuum poisoning the modern world, of which both the Writer and the Scientist are victims."Indeed, both stalker and his wife have a strong commitment to a higher purpose, which requires a great deal of sacrifice, just like the main character Alexander in the movie Sacrifice!Hence, it is quite evident how these three characters (stalker along with his wife and Alexander) exhibit the ideals of Agape, "a selfless love that is passionately committed to the well-being of others". To reframe it in terms of the Jung inspired comparative mythologist Joseph Campbell's notion of "hero's journey" or monomyth (Campbell, 2004), Stalker is in the quest and in service for people to find hope, love and happiness for themselves.Just like in every hero's journey, the stalker is confronted with a challenging task.Yet, it is through this very challenge that the hero also succeeds at creating a better and higher version of himself or herself.Hence, although both the heros in Stalker and Sacrifice are anti-hero in regard to the criteria of Aristotelian tragic hero, they are still heros with specific journeys and challenges formulated by the monomyth of Campbell (2004). In the case of Stalker, the hero goes through a spiritual crisis while fighting against the chronic cynicism and resignation, "poisoning the modern world, of which both the Writer and the Scientist are victims."Achallenge or crisis is necessary for any hero's journey.In the case of the character stalker, the crisis is spiritual as Tarkovsky (1987:93) states: "I believe that it is always through spiritual crisis that healing occurs.A spiritual crisis is an attempt to find oneself, to acquire new faith."Accordingly, the hero stalker (Tarkovsky, 1987:93) goes through a challenge, "moments of despair when his faith is shaken; but every time he comes to a renewed sense of his vocation to serve people who have lost their hopes."Certainly, stalker's journey as a hero mirrors the journey of the movie's director.After all, just like the hero stalker who is commissioned to "light a spark, a belief in the heart of people", Tarkovsky (1987:200), as a director, declares his function "to make whoever sees my films aware of his need to love and to give his love, and aware that beauty is summoning him."Indeed, it become evident how the central messages of both Stalker and Sacrifce add up to what Saint Paul wrote in Corinthians 13:13 "And now these three remain: faith, hope and love.But the greatest of these is love." In the case of Sacrifice's main character Alexander, the challenge of Hero's Journey is even more daunting than the challenge posed to stalker and his wife.Tarkovsky (1987:222) describes Alexander as "a character who is perpetually crushed by depression" and who "has grown to hate the emptiness of human speech." In a world where "word has lost all mystery and magic and speech has become mere chatter empty of meaning" as Alexander bitterly comments in the movie, it is no coincidence that the Sacrifice starts with Alexander telling the story of Pamdev and young disciple Ioann Kolov.According to Alexander Pamdev, the story embodies a deeper meaning which he shares with the Little Man: Say what you will, but a method, a system, has its virtues.You know, sometimes I say to myself, if every single day, at exactly the same stroke of the clock, one were to perform the same single activity…the world would be a very different place.Yes, something would change!It would have to!Indeed, young disciple Ioann Kolov's full integrity with his word and sheer devotion in climbing the hill every morning to water the dead dry tree for three years has the utmost significance and meaning in a world where "word become mere chatter empty of meaning."This is also the reason why it is essential that Alexander keeps his word to God by burning his house.Hence, by keeping his word to God, Alexander does not only save the World from nuclear catastrophe but also from chronic cynicism where the word hast lost its all meaning. For Tarkovsky (1987:209), Alexander is not a hero in the classic sense but "a thinker and an honest man, who turns out to be capable of sacrifice in the name of a higher ideal."Given Alexander's vow to God, his action of burning the house may appear as mentally ill to an outside observer.As Tarkovsky (1987:209) observes: "He nevertheless takes the crucial step, thereby infringing the rules of normal behaviour and laying himself open to the charge of folly...It may well be that through individual exertions such as his, which nobody notices or understands, world harmony is preserved."Indeed, this is where Alexander gets to be classified as one of Tarkovsky's spiritual heroes and exhibits the Christian ideal of Agape; an unselfish love which gives itself away through the act of sacrifice. Then, the final scene of the Sacrifice unfolds.Camera shows the Little Man carrying the bucket to water the dead tree planted by his father in the first scene of the movie.In the meantime, the ambulance drives Alexander away from home.After watering the dead tree, the Little Man lies underneath the barren tree. Wondering about the mysteries of the world and the creation, he utters; "In the beginning was the Word.Why is that, Papa?"Although Alexander is gone and cannot provide the Little Man with any answers, the world underneath that tree is filled with mystery and magic.There is only silence accompanied with Bach's "Erbarme Dich, mein Gott". Then, the camera slowly but steadily moves upwards.The camera movement stops once it reaches to the top of the barren tree where the rising sunlight flickers through its branches.The time is suspended and we are invited to witness the subtle movements of something that is becoming alive.It is in this very moment that the tree of life allegory reaches its full potential.The possibility of dead barren tree becoming alive with blossoming flowers, as in the story of Pamdev, is an image and allegory that powerfully stands for Tarkovsky's philosophy of transformation through love which prerequisites absence of egoistic acquisitiveness and strong altruistic self-devotion and sacrificial act.Yet, perhaps, it is not really the tree that we see out there in the world that is dead.The tree of life inside us had already been dead for a long time and we were not aware of it!Given his terminal cancer and being in exile, Tarkovsky completes the final scene of the film by dedicating it to his son Andriosha who is apart from him living in the Soviet Union "with hope and confidence." Mystical Depths of Tarkovsky in our "Secular Age" The unique cinematic language and the visual poetry of Tarkovsky critically rest upon his innovative and masterly utilization of the time (Bello, 2014) and visual texture (Johnson and Graham, 1994).In Stalker, for instance, Tarkovsky accentuates the very density of time and the rotten material texture of the physical setting (the very face of stalker himself, cars, and indoors buildings) to provide the effect of strong spiritual reality behind the disintegrating physical."It is this disintegration of the very material texture of reality" as Zizek (2006) perceptively remarks, "which provides the spiritual depths".Hence, through the innovative use of time itself and material texture, "Tarkovsky affects us at a level which is much deeper, much more crucial for our experience than all the standard spiritual motive of elevating ourselves above material reality and so on."(Zizek, 2006). Indeed, Tarkovsky is able to deliver the spiritual motive of elevating ourselves above material reality through not only the script and the plot of the movie but also the spectator's personal experience of the density of time and the visual poetry, which permeate throughout his movie (Baglivo, 1984).In addition to these, the haunting Zen-like music of Stalker and the uncontaminated natural beauty of the landscapes in the Zone (in stark contrast with the rotten physical world surrounding that beauty) are also crucial elements in creating that unique mystical effect in Tarkovsky's cinema.In the very last scene of Sacrifice, our notion of time gets suspended when the camera movement stops once it reaches to the top of the barren tree where the rising sunlight flickers through its branches.Spectator is invited to witness the transcendent reality while gazing at the subtle movements of tree branches. I consciously use the term "unique mystical effect" in Tarkovsky cinema simply because the experience of the density of time and the presence of hidden but unified spiritual reality behind the multitude of the material world embody the key aspect of mystical thought.Despite their variety of expressions among different religious or philosophical traditions throughout the centuries and across different continents, Bertrand Russell (1997: 179) sums up the three cores features of the mystical thought: (1) That all division and separateness is unreal, and that the universe is a single indivisible unity; (2) That evil is illusory, and that the illusion arises through falsely regarding a part as self-subsistent; (3) That time is unreal, and that reality is eternal, not in the sense of being everlasting, but in the sense of being wholly outside time. In this sense, watching Tarkovsky's movies is a reminiscent of having a mystical visionary experience.This is not something surprising given the fact that being in touch or in connection with Agape (as a distinct notion of divine love, which stands for the God's unconditional love for human kind) would necessarily entail some sort of mystical sensation or "personal religious experience" as famously examined by William James (2017) in his "Varieties of Religious Experience".Although Tarkovsky mystical roots on love and metaphysics is located in the cultural color of Orthodox Christianity, mysticism appear in almost every religious tradition.Furthermore, it is an integral part of not only Eastern but also Western Philosophy since its very inception by Pythagoras, Parmenides and Plato (Russell, 1974). If one removes its metaphysical dimension from his philosophy of love, Tarkovksy's (1987:200) emphasis on the primacy of love "which is all a person can count upon in his existence", the necessity to develop one's "capacity to love" or becoming aware of the "need to love and to give love" are in conjunction with the themes adhered by secular tempered existentialist or humanist tradition.(Erich Fromm, 1956 and1999;Viktor Frankl 1992). Indeed, in classical book Art of Loving, Erich Fromm achieved a Copernican revolution by reversing the order of question from "Am I loved?" to "How much can I love?" which involves a shift of perspective.Indeed, people are more commonly interested in finding and receiving love from external sources rather than generating love and the capacity to love from within.As Fromm (1956: 36) observes: "Infantile love follows the principle: "I love because I am loved."Mature love follows the principle: "I am loved because I love." For many of us, the problem with love is primarily about whether we are loved enough or are we certain that we are loved.However, we never seriously confront the question 'How much we can love?' Usually, people chase all their life to be loved by others in vain and get disillusioned or destructive in their search for love since they never seriously confront themselves about the fact that their inner or spiritual tank is empty of love, empty of love for themselves and for all the existence around them. Indeed, it was in the concentration camps of Nazi Germany that Viktor Frankl came to realize the ontological supremacy of love over everything.While clinging to the image of his beloved wife in the concentration camps, Frankl (1992: 48-49) realized that: For the first time in my life I saw the truth as it is set into song by so many poets, proclaimed as the final wisdom by so many thinkers.The truth-that love is the ultimate and the highest goal to which man can aspire.Then I grasped the meaning of the greatest secret that human poetry and human thought and belief have to impart: The salvation of man is through love and in love. Hence, it is critical to observe how Tarkovksy's (1987:200) emphasis on the primacy of love "which is all a person can count upon in his existence", the necessity to develop one's "capacity to love" or becoming aware of the "need to love and to give love" are in conjunction with the themes exposed above.	2018-12-07T11:45:15.074Z	2018-07-02T00:00:00.000	{ "year": 2018, "sha1": "c0d62a13b677fca48996b13e22cacc808be4563a", "oa_license": "CCBY", "oa_url": "http://kutaksam.karabuk.edu.tr/index.php/ilk/article/download/1490/1164", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "c0d62a13b677fca48996b13e22cacc808be4563a", "s2fieldsofstudy": [ "Philosophy" ], "extfieldsofstudy": [ "Philosophy" ] }
226036363	pes2o/s2orc	v3-fos-license	Oxidation of Sulphites by Electrodes Made of Novel Materials for Use in Microbial Fuel Cells Catalysts based on novel carbon forms (Fullerenes С60/С70, Higher Fullerenes and Double Wall Carbon Nanotubes) were applied to facilitate the process of oxidation of sulfites to sulfates. The electrodes with incorporated new catalysts are able to desulphurize and remove toxic pollutants. The electrochemical characterization included steady state polarization curve analysis. The electrodes that incorporate fullerene structures were found to yield the highest current densities. A short overview considers the existing sulfur oxidizing microbes that should facilitate the creation of a workable microbial fuel cell. Introduction The removal of pollutants while simultaneously generating energy will help safeguard the environment and enable the development of innovative fuel cells. In this regard a principle objective is to build new electrodes and catalysts able to remove toxic pollutants where sulfide is oxidized to sulfite through sulfite reduction by Dsr. The sulfite generated by rDsr is then oxidized to sulfate by other enzymes [2]; (ii) oxidation of sulfite to sulfate by a mononuclear molybdenum enzyme known as sulfite oxidoreductase [3]. In bac-terial sulfate reduction, bacteria respire sulfate and yield sulfide. The process consists of four paths [4,5]. It has been shown that organotrophic bacteria are capable of the oxidation of thiosulfate to tetrathionate (these bacteria are referred to as T-HSOB) and are found in the redox layer of the Black Sea [6]. Microbial Fuel Cells (MFCs) with graphite anodes and graphite cathodes were built in an aerobic seawater environment and in anoxic marine sediment [7]. The anode electrode was embedded in anoxic marine sediments while connected through electronic circuits to a similar electrode in the overlying aerobic seawater (the cathode), thus building a MFC. The MFC had a power yield of 0.01 W/m 2 and can supply electronic instrumentation [8]. The enrichment with microorganisms from the family Geobacteraceae on graphite anodes allowed these microorganisms to conserve energy, supporting their growth by oxidation of organic compounds with an electrode that served as the electron acceptor [9]. Advanced catalyst supports including carbon nanotubes, aerogels and graphene have been tested in the past [10]. Applied also in catalyst synthesis is a method to fabricate the electro-catalyst for the electrodes using a lyophilization process [11]. Key sources when describing the electrochemical oxidation of sulphites in alkaline solutions are [12,13]. These researchers showed that the anodic oxidation of sulphite irreversibly yields sulphate and dithionate. The oxidation of SO 3 2at the anode was found to initiate at 1.2 V vs SHE in alkaline solutions. An OHradical is added to the sulphite ion at relatively high potentials. The anodic oxidation of sulphite ions under alkaline conditions has been studied comprehensively by J Lu et al. [14]. The adsorbed species suffer deprotonation at pH < 7 and are then subject to oxidation. In solution, sulphite exists in the form HSO 3 and SO 3 2with the following equilibria between these species [15]: . Novel electro-catalytic materials such as higher fullerenes and carbon nanotubes are studied in our current research. Higher fullerenes are fabricated by applying the carbon arc method in a quartz reactor followed by sublimation. These include the fairly stable species C 74 , C 78 , C 80 , C 82 , C 84 , C 86 , C 88 , C 90 , C 92 , C 94 , C 96 , C 98 , C 100 . Characteristic of higher fullerenes is that the bonding sites between the pentagon atom groups are usually found to be the most reactive. Materials and Methods In this study, we use "higher fullerenes", made by the method of Deener and Alford, also called "narrow gap fullerenes" [16]. Sodium sulfite (Na 2 SO 3 ), sodium chloride NaCl and higher order fullerenes were purchased together with manganese acetate and polypyrrole from Sigma Aldrich. Fullerenes C 60 /C 70 and DWCNTs were pur-chased from CEC Research, Houston, Texas. The Vulkan XC-72 particles with a particle size of 50 nm were purchased from the Cabot Corporation and prepared in accordance with [17]. The catalysts studied were lyophilized fullerenes C 60 /C 70 , higher fullerenes and DWCNTs (2-11 mg) dispersed in 6 ml of distilled water in an ultrasonic bath for 15 minutes. Subsequently, 40 mg of manganese acetate are slowly added to the aqueous suspension together with 60 mg of polypyrrole. These ternary mixtures were then baked at 180 °C for 12 hours in a Teflon autoclave. Thus, manganese oxides cover the fullerene and nanotube structures with polypyrrole binding. The electrodes under investigation have a geometric area of 1and 10 cm 2 . The electrodes were prepared from a catalyst mixture and Teflonized carbon black (60 mg/cm 2 Vulcan XC-72 (35% Teflon)) as a binder [18]. The mixture is compressed on both sides of a stainless-steel collector at 150 °C and pressed at 300 kg/cm 2 ( Table 1). XRD Analysis Shown in Figure 1 is an XRD of the catalyst incorporating higher fullerenes and manganese oxides. . Electrolyte 1М Na 2 SO 3 -132 g/l + 18 g/l NaCl.	2020-10-30T13:10:04.374Z	2020-03-16T00:00:00.000	{ "year": 2020, "sha1": "e244d2fa41e6b7c988a7dfbfbec784b9f0b55c75", "oa_license": "CCBY", "oa_url": "https://biomedres.us/pdfs/BJSTR.MS.ID.004381.pdf", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "797acbce0ad75add05ce83ed8789de0eb3e9bcd4", "s2fieldsofstudy": [ "Engineering", "Environmental Science", "Materials Science", "Chemistry" ], "extfieldsofstudy": [ "Chemistry" ] }
225574437	pes2o/s2orc	v3-fos-license	Wits Appraisal Among Qatari Females : A Comparative Study DOI: http://dx.doi.org/10.24018/ejmed.2020.2.4.365 Vol 2 \| Issue 4 \| July 2020 1 Abstract — Introduction: The goal of cephalometric analysis is to determine the skeletal and dental relationships that exist in an individual patient. The objectives of the present study were to establish the Wits appraisal for Qatari females and to compare the results with previous reported results in different racial groups. Materials and Methods: The sample consisted of 34 lateral cephalometric radiographs of Qatari female patients with age range from 18 to 25 years old presented with well-balanced face and acceptable profile. Results: The mean value of the Wits appraisal for Qatari females in the present study was -0.62 ± 3.8. The Wits appraisal mean value of the present study revealed that no statistically significant difference between the Qatari females and the original data of Jacobson. On the other hand, significant differences were found when compared to other populations from several reports. Conclusion: The Wits appraisal among Qatari females was -0.62 with standard deviation of 3.8. Care should be taken in growing and developing children where variation in dental height exists and may confuse the Wits value. The Wits appraisal could be used as a moderator to ANB angle in boarder line cases. It is recommended to incorporate angular measurements using 3D imaging software which increases diagnostic accuracy for the least amount of existed discrepancy. I. INTRODUCTION The goal of cephalometric analysis is to determine the skeletal and dental relationships that exist in an individual patient [1], [2]. This is by comparing the individual's measurements with a normal reference group, so that differences between the patient's actual dentofacial relationships and those expected for his or her racial or ethnic group are revealed [3]- [10]. The most common angle used was the ANB angle suggested by Rediel [11] and used by Steiner [12]. It indicates the antero-posterior relationship between the maxilla represented by (SNA) angle and the mandible represented by (SNB) angle [13], [14]. However, it is reported that ANB angle is not without limitations with respect to its accuracy due to several factors. Among these factors include age, the spatial position of Nasion point, the upward or downward rotation of the jaw or the anterior cranial base change in relation to the occlusal plane, and the degree of facial prognathism [15]- [18]. To effect of these factors, a proposed diagnostic tool was introduced by Jenkins [19] and later was modified by Jacobson as "Wits" appraisal [9] Jacobson introduced the Wits appraisal as a reliable indicator of the sagittal relationship between the maxilla and the mandible. He drew a perpendicular line from Point A and Point B to the occlusal plane [9]. The horizontal linear measurement between the perpendicular lines indicate the skeletal relationship between the maxilla and the mandible. His sample consisted of 21 adult subjects with excellent occlusion and noticed that point BO was approximately 1 mm ahead of point AO. With a range of -2 to 4 mm and mean reading of 1.17 ± 1.9 mm. Since then, several studies were conducted in different races to establish the Wits value. Robertson and Pearson found in sample from South wales that the Wits appraisal was 0.1±1.9 in males and 0.3±1.7mm in females. [20] On the other hand, the Wits appraisal in a South Chinese sample was -4.88±3.61 in males and -4.47±4.19 mm in female [21]. Whereas in Nigerians it was -4.15±3.65mm for males and in females was -3.06±2.96 [22]. In addition to the aforementioned studies, few reports from the Arab populations were published such as the study of Al-Barakati who established the Wits value in a group of Saudi dental students and concluded that there was no significant difference between her sample and the original sample of Jacobson [23]. However, she found significant differences when she compared her sample to other samples such as, the Chinese and British Caucasians. On the contrary, Zawawi in another group of Saudi samples found that there was significant difference between males and females (-0.73±2.48 and 1.79±2.06 respectively). He stated that Wits appraisal appears to be more gender-specific and less ethnicityspecific. In the State of Qatar, there is no data on the Wits values. Therefore, the aims of this study are to establish the Wits values in a sample of Qatari female individuals and to compare the results with other populations from different racial and ethnic backgrounds reported in the literature. A. Subjects The sample consisted of 34 lateral cephalometric radiographs of Qatari female patients with age range of 18 to 25 years old (Mean 20.69±4.9 years). There were no enough cephalometric radiographs for male patients to conduct an appropriate analysis and compare to female patients. Therefore, measurements were made on female patients. They were selected according to the following criteria: well-N. ALSayed, Hamad dental Centre, Qatar. F.A. AL-Jawad, Hamad Dental Center, Qatar. A. AL-Qaisi, Hamad Dental Centre, Qatar. Wits Appraisal Among Qatari Females: A Comparative Study Hayder Abdalla Hashim, Najah AL-Sayed, Feras Abed AL Jawad, Ayah AL-Qaisi balanced face and acceptable profile, skeletal class I and Class I occlusion with normal overjet and overbite, minor or no crowding or spacing, and no history of orthodontic treatment. Ethical approval and consent form were obtained. B. Method A good quality digital lateral skull radiograph was taken by Planmeca ProMax (84 kV-16mA. Total filtration 2.5 mm AI) manufactured in Finland. The radiograph was taken by well-trained technician at 5-feet from each patient looking straight to her own eyes on the mirror. Each radiograph was hand traced by one operator. The Wits appraisal was recorded by drawing straight perpendicular lines from Point A (Subnasale) and Point B (Supramentale) respectively to the functional occlusal plane at 90° and measuring the horizontal distance from point AO to BO. (Fig 1). C. Statistical Analysis Microsoft Excel Program was used for the data analysis. Descriptive statistics, including the mean and SD were computed for each measurement. For analytical analysis, Student's t-test was used to assess whether there was significant difference between Wits appraisal in Qatari females and Jacobson data as well as with previously published data from other racial and ethnic groups. The level of significance was set at p<0.05. III. RESULTS The error of the method was assessed by double measurements taken at least one-week interval on five randomly selected cephalograms. The t-test result indicated that no statistically significant differences between the two readings at 5 % level. (P>0.05). According to Table I, the mean value of the Wits appraisal for Qatari females in the present study was -0.62 ±3.8. Table II revealed that there was no statistically significant difference between the Qatari females and Jacobson's data. No statistical significant differences were noted when comparing the present data with data from Jacobson, central region of Saudi Arabia, Kuwaiti and Black American. On the other hand, significant differences were noted when compared to data from Chinese, Nigerians and Western region of Saudi Arabia (Table III). IV. DISCUSSION Cephalometric analysis is a key component for diagnosis, assessing growth changes and treatment planning for orthodontists by measuring not the relationship of teeth with the basal bone, but also the inter-relationship of jaw elements and cranial structures [25]. Therefore, Careful planning and analyzing the craniofacial structure is imperative in order to achieve favorable outcomes. Although ANB angle is the most common measurement to evaluate the sagittal relationship between jaws, it is not without limitations. As a result, other analyses were introduced to overcome the shortcomings of the ANB angle. Among these, is the Wits appraisal which was introduced by Jacobson in an attempt to give a better and adequate sagittal measurements for skeletal discrepancies of the maxilla and the madible by using the occlusal plane as a referrence instead of the cranial plane [9]. The mean value of the Wits appraisal in the present study revealed no statistical significant differences with the data of Jacobson. This finding was also consistent with other reports from other ethnic backgrounds such as, the South Walesh, Saudis, Kuwaitis [20], [23], [26], [27]. On the other hand, significant differences were revealed when compared to data from Chines, Nigerians and Western region of Saudi Arabia [21], [22]. Such differences could be attributed to the diversity of the ethnic background of the population investigated in these studies. Also could be related to the great systematic errors between the different studies, specifically when defining the occlusal plane. More over, Zawawi reported that, such variation in the definition between the observers; could lead to errors and those errors could be overcomed by well trained and calibrated reseachers to reduce the inter-and intra-examiner errors [24]. One draw back of Wits appraisal analyses is the vertical posterior inclination of the mandible in high angle cases. This high posterior vertical incilnation could affect the position of B point as well as the perpendicular line from B point to the occlusal plane by moving more posteriorly. One possible soultion to this; is cancelling this effect by establishing a formula of a figure as the same method proposed in case of Nasion point movement, which affect the inclination of the anterior cranial base line by substraction or addion of 7 degrees to all measured angles related to Nasion point or anterior cranial base. Applying cone beam computed tomography (CBCT) could be of great help which give three dimensional view [28]- [29]. The outcome of such studies will supply the orthodontist with valuable information applicable to diagnosis and treatment planning. In support to this suggestion, Abou Kheir and Kau stated "Mathematical analysis is a valid method to measure mandibular asymmetry with respect to the third dimension which is usually absent in clinical photos and panoramic radiographs as well as cephalogram. Incorporation of angular measurements using 3D imaging software increases diagnostic accuracy for the least amount of asymmetry present" [30]. The Wits appraisal is a simple linear measurement used as an adjunctive diagnostic tool which overcome the draw back of other analyses when invetisgating the anteroposterior skeletal relationship. Furthermore, it is very logical to consider that we should not treat patient according to numbers. It is noteworthy to remember that cephalometric interpretation not only depends on a number of different values and also not to only one single absolute value [24].This was confirmed by Jacobson, who stated that no single parameter in cephalometric should be depend upon as the only absolute value [9]. Wits appraisal could be more reliable than the ANB angle when assessing the sagittal dental base discrepancy. This is in line to findings from other previous published studies [9], [31], [32]. Accordingly it is recommended to use the Wits appraisal as a moderator of the ANB angle in treatment of orthodontic patients in boader line cases [22]. V. CONCLUSION The Wits appraisal among Qatari females was -0.62±3.8 which is in accordance to Jacobson data. Care should be taken in growing children where the effect of dental height variations may confused the Wits value.The Wits appraisal could be used as a moderator to ANB angle in boader line cases. Incorporating angular measurements using 3D imaging software which increases diagnostic accuracy for the least amount of descrepancy present is highly recommended. ACKNOWLEDGEMENT We would like to thank Hamad Medical Corporation/ Hamad Medical Research Center for sponsoring this research (Sub-Research Proposal Number 15041/150). The authors heartily acknowledge Dr. Hashim AL-Hussain for his help in statistical analysis procedure of this study. Thanks, extended to Dr. Mutaz Ahmed for his invaluable comments and support and to professor Anil Sukumaran for preparing the final temple.	2020-07-30T02:02:36.463Z	2020-07-11T00:00:00.000	{ "year": 2020, "sha1": "d52e57c0cafe5f83e297012b63431282f8f2b90b", "oa_license": null, "oa_url": "https://ejmed.org/index.php/ejmed/article/download/365/213", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "4f3838ac2296cb737dd9bb2c4774acb7452f4dfb", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
216071419	pes2o/s2orc	v3-fos-license	Double-targeted knockdown of miR-21 and CXCR4 inhibits malignant glioma progression by suppression of the PI3K/AKT and Raf/MEK/ERK pathways Background Currently, miR-21 and CXCR4 are being extensively investigated as two unrelated key regulators in glioma malignancy. In this study, we investigated the combined effect of these two factors on glioma progression. Methods We confirmed the expression of miR-21 and CXCR4 in malignant glioma tissue and glioma cells with qRT-PCR and western blotting. Single-targeted knockdown of miR-21 and CXCR4, as well as double-targeted knockdown of miR-21 and CXCR4 lentiviral vectors were constructed and they were transfected to U87 and U251 cells. Cell proliferation, apoptosis, invasion, and migration from different treatment groups were assessed by MTT assay, Flow Cytometry analysis, Transwell analysis, and Scratch assay, respectively. U87 xenograft mice were constructed to detect roles and potential mechanisms of miR-21 and CXCR4 in malignant glioma tumor growth. Results The expression of miR-21 and CXCR4 was increased in tumor tissues and cell lines. Inhibition of miR-21, CXCR4, and miR-21 and CXCR4 together all reduced the migration, invasiveness, proliferation and enhanced apoptosis in glioma cells, as well as reduced tumor volume and mass in xenograft model. The inhibition effect was strongest in double-targeted knockdown of miR-21 and CXCR4 group, whose downstream pathways involved in AKT axis and ERK axis activation. Conclusions Our findings reported that double-targeted knockdown of miR-21 and CXCR4 could more effectively inhibit the proliferation, migration, invasion and growth of transplanted tumor and promote cell apoptosis, which were involved in the PI3K/AKT and Raf/MEK/ERK signaling pathways. Introduction Glioma is the most common and feared type of tumor, with an annual incidence of 1 per 10,000 population (Yang et al., 2018). Currently, treatments for malignant glioma are mainly surgical resection, followed by chemotherapy or radiotherapy (Andrea et al., 2013). However, these comprehensive treatments are not very effective and the median survival of patients afflicted with malignant glioma is less than 1 year, due to the aggressive proliferation and insidious invasion of cells (Skalsky andCullen 2011, Põlajeva et al., 2012). Thus, recent studies are focusing on the factors that can effectively control the invasive and proliferative capacity of malignant glioma cells to develop targeted salvage treatments to improve patients' life quality. miR-21 is an oncomiR which attracts many attentions, because it takes part in almost all steps of tumor growth and metastasis [6]. Evidence shows that miR-21 is overexpressed in gliomas and glioma cells compared to normal tissues, and its expression level is positively correlated with glioma grade (Tao et al., 2013, Lei et al., 2014. Elevated miR-21 expression leads to increased glioma cell proliferation invasion, and also chemo-resistance, which indicates poor prognosis and tumor recurrence of glioma patients (Gabriely et al., 2008, Kwak et al., 2011, Melnik 2015, Hermansen et al., 2016, On the other hand, downregulation of miR-21 leads to repression of anti-apoptotic capacity, reduction of migratory and invasion, as well as increase of chemical-induced death in glioma cells (Gabriely et al. 2008, Loges et al., 2012, Luo et al., 2017, Shao et al., 2017. All of these make miR-21 not only a potential glioma marker for diagnosis and prognosis, but a target for novel therapeutic intervention. Another factor that draws great attention for playing a crucial role in malignant glioma biological regulation is C-X-C Chemokine Receptor 4 (CXCR4), a transmembrane G-protein-coupled receptor. Statistics shows that CXCR4 level is elevated in malignant glioma when compared to normal cells derived from the same tumor (Zhang et al., 2007). Particularly, its overexpression is concentrated in invading regions, and associated with increased glioma tumor grade and malignancy (Chatterjee et al., 2014, Yang et al. 2018. Numerous studies suggest that the overexpression of CXCR4 facilitates proliferation, angiogenesis, invasion, metastasis, as well as chemotherapy and radiotherapy resistance of glioma in several glioma cell lines and mouse models (Rubin et al., 2003, Bajetto et al., 2006, Redjal et al., 2006, Mercurio et al., 2016, Gagner et al., 2017, Eckert et al., 2018. Administration of either CXCR4 neutralizing antibody or CXCR4 siRNA impairs the enhanced glioma malignancy (proliferation, angiogenesis, invasion, metastasis, and post-chemotherapy and post-radiotherapy recurrence) and increases median survival in in vivo glioma models (Rubin et al. 2003, Redjal et al. 2006, Esencay et al., 2010, Choi et al., 2014, Mercurio et al. 2016, Wang et al., 2016, Yadav et al., 2016, Bathen et al., 2017, Gagner et al. 2017, Yang et al., 2017. Thus, the potential of CXCR4 as an anti-tumor target has been a popular research topic. The two moleculars miR-21 and CXCR4 as a single target respectively have drawn wide attention of researches. However, several studies have shown that in gene therapy of cancers, combination of multigene therapy tends to be more effective than single-gene therapy strategy, which may provide a promising treatment in cancer. Herein, we investigated the combined effects of miR-21 and CXCR4 by lentiviral mediated gene recombinant technology on glioma malignancy. Double-targeted knockdown of miR-21 and CXCR4 was performed in glioma cell lines (U87 and U251) and its effects on glioma malignant progression were evaluated in cells and xenograft mouse models. Knockdown of either miR-21 or CXCR4 decreased glioma proliferation, invasion, and migration and enhanced apoptosis. Double-targeted knockdown generated a significantly enhanced inhibition effect on tumorigenicity even compared to single-targeted knockdown of miR-21 or CXCR4 alone. Also, we found that decrease of tumor progression was related to PI3K/AKT and Raf/MEK/ERK pathways which are responsible for glioma growth, invasiveness, chemo-and radio-therapy resistance, and recurrence (Loges et al. 2012, Shao et al. 2017). Thus, this study might also pave ways for further research on glioma pathology and physiology. miR-21 and CXCR4 expressions were increased in malignant glioma tissues and cells In order to determine the effect of double-targeted knockdown of miR-21 and CXCR4 on malignant glioma tumorigenicity, we first confirmed the dysregulation of these two factors in malignant glioma tissues. qRT-PCR was employed to assess the expression of miR-21 and CXCR4 at genetic level. Both miR-21 and CXCR4 average expressions were significantly up-regulated in glioma tissues compared to paracancerous tissues ( Fig. 1A-1C). Further, we examined the expression of miR-21 and CXCR4 in two invasive-prone glioma cell lines (U87 and U251) in comparison to a normal glial cell line (HAC). The expression levels of miR-21 and CXCR4 were both significantly increased in U251 and 6-fold higher in U87 ( Fig. 1D-1F). The results suggested that miR-21 and CXCR4 might play important roles in mediating malignant glioma aggressiveness, as well as the tumorigenicity. The construction of stable anti-miR-21, sh-CXCR4, and anti-miR-21 + sh-CXCR4 glioma cells We constructed miR-21 knockdown (anti-miR-21), CXCR4 knockdown (sh-CXCR4), and miR-21 and CXCR4 double-targeted knockdown (anti-miR-21 + sh-CXCR4) U87 and U251 cells to investigate the anti-miR-21 + sh-CXCR4 generated effect on glioma tumorgenicity. qRT-PCR was used to detect the efficiency of miR-21 knockdown at genetic level and data showed that the knockdown efficiency reached more than 70% in both U87 and U251 cells ( Fig. 2A). The efficiency of sh-CXCR4 was measured by qRT-PCR and western blot and the results showed that CXCR4 expression level in U251 and U87 cells was significantly repressed compared to negative control and mock group ( Fig. 2B-2D). Moreover, the construction of anti-miR-21 + sh-CXCR4 was successful with the efficiency of miR-21 and CXCR4 inhibition in U87 and U251 being comparable to anti-miR-21 or sh-CXCR4 treatment alone generated effects ( Fig. 2E-2G). Double-targeted knockdown of miR-21 and CXCR4 inhibited proliferation and enhanced apoptosis of glioma cells To explore the roles of miR-21 and CXCR4 in sustaining aggressive proliferation and anti-apoptosis in glioma cells, functional experiments were performed. MTT assay was performed on transfected U87 and U251 cells for the evaluation of cell proliferation at different time points (0, 24, 48, 72 hr). In comparison to negative control group, anti-miR-21 or sh-CXCR4 alone was able to significantly decrease cell proliferation of U87 and U251 ( Fig. 3A and Fig. 3B). Interestingly, in anti-miR-21 + sh-CXCR4 group, the inhibition efficiency was significantly higher than that in anti-miR-21 or sh-CXCR4 alone group. Next, Flow Cytometry analysis was used to assess the apoptosis in these transfected cells. Statistically more apoptotic cells were found in both anti-miR-21 and sh-CXCR4 groups, and as we expected, anti-miR-21 + sh-CXCR4 group generated the most apoptotic cells ( Fig. 3C and Fig. 3D). Thus, our results demonstrated that anti-miR-21 + sh-CXCR4 had an enhanced effect on both suppressing glioma cell proliferation and pro-apoptosis. Double targeted knockdown of miR-21 and CXCR4 inhibited invasion and migration of glioma cells Transwell assay was performed to evaluate the regulation of miR-21 and CXCR4 on glioma cell invasiveness. Anti-miR-21 or sh-CXCR4 alone was able to significantly limit the invasiveness of glioma cells ( Fig. 4A and Fig. 4B). Here our data presented that anti-miR-21 + sh-CXCR4 significantly inhibited the cell invasiveness compared to single-targeted knockdowns (anti-miR-21, and sh-CXCR4) ( Fig. 4A and Fig. 4B). Anti-miR-21 or sh-CXCR4 alone significantly reduced migration in both U87 and U251 cells; while, anti-miR-21 + sh-CXCR4 suppressed inhibited the cell migration compared to single-targeted knockdowns in both cell lines ( Fig. 4C and Fig. 4D). This revealed the potent repressive effects of anti-miR-21 + sh-CXCR4 on capability invasion and migration of glioma cells. Double targeted knockdown of miR-21 and CXCR4 slowed tumor growth in glioma xenograft mouse model To better illustrate the roles of miR-21 and CXCR4 together in tumor growth of glioma, we employed a U87 xenograft mouse model. No obvious difference between the average body weight of mice from different treatment groups (Lv-NC, anti-miR-21, sh-CXCR4, and anti-miR-21 + sh-CXCR4, Fig. 5A). We measured tumor volume every 5 days. The growth of glioma tumor was significantly suppressed in anti-miR-21 or sh-CXCR4 treatment alone groups compared to negative control, while the anti-miR-21 + sh-CXCR4 treatment obviously reduced the tumor growth compared to the anti-miR-21 or sh-CXCR4 group ( Fig. 5B and 5C). Furthermore, the miR-21 and CXCR4 level in xenografts were detected. And further, the results had confirmed that the remarkable effect on tumor growth suppression was contributed by inhibition of miR-21 and CXCR4 ( Fig. 5D-5F). Thus, our data reported that anti-miR-21 + sh-CXCR4 could diminish growth of glioma xenograft in vivo. Double-targeted knockdown of miR-21 and CXCR4 inhibited malignant glioma progression by suppressing of the PI3K/AKT and Raf/ MEK/ ERK pathway We further investigated the potential downstream pathways that might be responsible for the repressive effect of anti-miR-21 + sh-CXCR4 on tumor progression. In this study, we focused on two pathways 1) PI3K/AKT and 2) Raf/ MEK/ ERK, which both played a vital role in glioma cell fate, and were aberrant in the miR-21 and/or CXCR4 overexpression environment (Ching and Hansel 2010, Guo et al., 2013, Dongfeng et al., 2014, Du et al., 2015, Han et al., 2016, Shao et al. 2017). The results from western blot analysis presented no statistically difference in AKT, ERK1/2 among different treatment groups both in glioma cells and xenografts ( Fig. 6A-6C). However, anti-miR-21, sh-CXCR4, and anti-miR-21 + sh-CXCR4 all showed a significantly suppressive effect on p-AKT, and p-ERK1/2, with anti-miR-21 + sh-CXCR4 group presenting the strongest inhibition effect in both cell lines and xenografts. These results demonstrated a potent suppressive effect of anti-miR-21 + sh-CXCR4 on activation of AKT axis and ERK axis. These results suggested that double-targeted knockdown of miR-21 and CXCR4 inhibited malignant glioma progression by suppressing of the PI3K/AKT and Raf/ MEK/ ERK pathway. Discussion Glioma is the most common primary malignant brain tumor. Several novel therapies have been developed for the treatment of malignant glioma, including gene therapy, targeted molecular therapy, immunotherapy, and stem therapy. Among them, gene therapy presents great efficiency in suppressing glioma progression (Han et al. 2016). Particularly, the combination of multi-gene therapy tends to be more effective than the single-gene one (Jin et al., 2010). miR-21 is the first miRNA reported involved in human malignant glioma regulation (Melnik 2015). Its expression has been reported positively correlated with clinical grade of glioma malignancy (Lei et al. 2014, Hermansen et al. 2016. Also, the vital roles of miR-21 in glioma initiation, maintenance, and survival have been proved by numerous studies (Tao et al. 2013, Lei et al. 2014, Yeh et al., 2015, Hermansen et al. 2016, Luo et al. 2017, Wang et al., 2017. Studies show that miR-21 downregulation represses cell proliferation and invasiveness, enhances apoptosis, and sensitizes resistant to chemo-and radio-therapy (Loges et al. 2012, Lei et al. 2014, Wang et al., 2015, Yeh et al. 2015, Luo et al. 2017, Wang et al. 2017. In our study, we confirmed the increased miR-21 in malignant glioma tissues and glioma cells as well as the regulation of it in glioma progression. CXCR4 is another factor that plays a vital role in glioma pathology (i.e. neoplastic transformation, malignant tumor progression, infiltration, metastasis, angiogenesis, and vasculogeneis) (Dutt et al., 1998, Ono and Freed 1999, Eckert et al. 2018. Our data confirmed that CXCR4 expression was up-regulated in malignant glioma tissues and glioma cells in comparison to normal tissue or glial cells. The potential of CXCR4 as a promising therapeutic target for malignant glioma treatment has been extensively investigated and several targeting drugs have already been undergoing clinical trials (Eckert et al. 2018). However, most research topics are focused on employing CXCR4 antagonist as a single treatment or in complement with radio-or chemo-therapy (Eckert et al. 2018). In our present study, we reported that double-targeted knockdown of miR-21 and CXCR4 presented a better suppressive effect on tumor progression when compared to single-targeted knockdown of miR-21 or CXCR4 alone. Both PI3K/AKT and Raf/MEK/ERK are common and crucial signaling for cancer development (Serena et al., 2011). Activated ATK and ERK phosphorylate and activate downstream proteins involved in regulating cell survival, proliferation and metabolic pathways (Redman et al., 2013, Dueppers et al., 2015. Studies demonstrate that dysregulation of both pathways are observed in various cancers and the inhibition of both pathways would be promising strategy for cancer therapy (Dueppers et al. 2015). However, direct inhibition of these pathways is unpractical given the complexity of their crosstalk regulatory mechanisms (Ghayad and Cohen 2010). Interestingly, our results presented that double-targeted knockdown of miR-21 and CXCR4 powerfully suppressed PI3K/AKT and Raf/MEK/ERK activation, which might have significant therapeutic values and offer some insights for further investigation of the molecular mechanism in glioma pathology. Since the overexpression of miR-21 and/ or CXCR4 is also observed in many other cancers, such as, ovarian, gastric, colonic, pancreatic, breast and prostate cancers (Melnik 2015, J Richardson 2016), our study could also shed some light on the efficacious treatment development for them. Conclusion In summary, the knockdown of miR-21 or CXCR4, double-targeted knockdown of miR-21 and CXCR4 could more effectively inhibit the proliferation, migration, invasion and growth of transplanted tumor and promote cell apoptosis, which were involved in the PI3K/AKT and Raf/MEK/ERK signaling pathways. Therefore, double-targeted miR-21 and CXCR4 may be potential and promising therapeutic targets for malignant glioma salvage treatment. Samples A total of 25 patients were recruited in this study. All patients were diagnosed with malignant glioma, and diagnosis was confirmed by both clinical symptoms and the histology consensus from at least two neuropathologists at the Department of Oncology in the First Affiliated Hospital of Zhengzhou University. The surgical removed tumor tissues and adjacent peritumoral brain tissues were immediately snap frozen using liquid nitrogen and then stored at -80°C fridge for further qRT-PCR and western blot analysis. Written informed consents for the pathological analyses and experiments were obtained from all of the donors before their surgery. All samples were handled and processed in compliance to the institutional ethical and legal standards, which was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Cell lines and culture Human glioma cell lines U87 and U251, normal glial cell HAC and embryonic kidney cell HEK-293T were obtained from the American Type Culture Collection (ATCC, USA). All cells were cultured in Dulbecco's modified Eagle medium (DMEM) /high glucose (Hyclone, Logan, UT) supplemented with 10% FBS (Invitrogen, CarIsbad, CA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) and maintained in humidified incubator at 37 °C with 5% CO 2 and 95% of air. The relative CXCR4 and miR-21 levels were obtained from comparing to the expression level of GAPDH and U6 snRNA, respectively. The relative expression levels were calculated using 2 -ΔΔCt method. Western blot assay Cultured cell and collected tissue samples were first washed twice with ice-cold PBS and then lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails. 30 μg of protein was separated by 10% SDS-PAGE and then electrophoretically transferred onto a PVDF membrane (Milipore, Bedford, MA). The blots were incubated in 1 × PBS containing 5% skimmed milk powder for 1 hr at room temperature to block the nonspecific binding. Following washing (TBST, 3×, 10 min each) , blots were incubated with primary antibodies overnight at 4°C. After another wash, blots were exposed to horseradish peroxidase-conjugated secondary antibody for 2 hr at room temperature, and visualized using the enhanced chemiluminescence detection kit (Sigma). The following primary antibodies were used in this study: CXCR4 ( and phospho-ERK1/2 (Cell Signaling Technology). The protein levels were digitalized using ImageJ 1.48 version (NIH, Bethesda, Maryland) (Java 1.8.9_66) and relative protein levels were obtained by comparing the protein level to the level of β -actin. Lentiviral infection of glioma cells (U87 and U251) Before the infection, glioma cells (U87 and U251) were routinely cultured and seeded on 6-well plates at a density of 5 x 10 4 cells/ well. When cell density reached 50% confluence, cells were infected with lentivirus particles (MOI=20) in presence of 8 μg/ml polybrene (GeneCopoeia, Guangzhou, China) for 24 hr. Then the culture were replaced by fresh culture medium containing 2% FBS and cultured for another 72 hr. The effectiveness of lentiviral particles that expressing anti-miR-21, sh-CXCR4, or anti-miR-21 + sh-CXCR4 was examined by qRT-PCR and western blot as described above. MTT assay Logarithmically growing glioma cells (U87 and U251) with different treatments were plated into 96 wells culture clusters (Costar, Cambridge, MA, USA), at a density of 2000 cells/well and cultured for different time points (0, 24, 48, 72 hr). Cell viability was assessed by adding 10 μl MTT (0.5 mg/ml) to each well and incubated for 4 hr. After removing the cell medium, 100 μl DMSO was added and cell proliferation was measured at 490 nm wavelength. Measurements of cell viability for each treatment were done in triplicates from three independent experiments. Treatments included: 1) anti-miR-21, 2) sh-CXCR4, 3) anti-miR-21 + sh-CXCR4, 4) Lv-NC. Transwell assay The invasion of glioma cells (U87 and U251) was assayed using BioCoat Matrigel invasion chambers (BD Bioscience) with poly carbonic membrane (6.5 mm in diameter, 8 μM pore size). Before the start of the assay, the chambers were inserted into 24-well culture plates and pre-treated with serum-free DMEM medium at 37 °C for 30 min. In the Transwell assay, the transfected cells were resuspended in 300 μl serum-free DMEM at a density of 5 × 10 4 cells/ml and added to the upper chamber. 500 μl DMEM supplemented with 20% FBS was added into the lower chamber of each well. The plates were incubated in humidified incubator (37°C, 5% CO 2 ) for 20-24 hr. After the incubation, non-migrated cells in the upper chamber were removed mechanically with a cotton swap, while the invaded cells in the lower side of the membranes were fixed with 5% glutaraldehyde for 15 min at 4 °C, washed with PBS twice, and stained with 1% crystal violet for 15 min. Pictures were photographed at 200× magnification, and cell numbers were counted from at least five randomly selected fields. Scratch assay Transfected glioma cells (U87 and U251) were suspended and plated in 6-well plates until they reach 70-80% confluence. At the initial time, a scratch was made on the cell monolayer using a sterile 200 μl micropipette tip. Pictures were photographed at different time points (0, 24 hr), with 40 × magnification. Generation of tumor xenografts and in vivo treatments All the animal procedures were approved by Ethics of Animal Experiments of the First Affiliate Hospital of Zhengzhou University, and all the experiments were performed in strict accordance with the institutional guidelines. BALB/C nude mice weighting 21.2±3 g and being 5 weeks old were housed in sterile cages under standard conditions (23 ± 2°C, 45-55% humidity, 12 hr light duration). The mice were allowed for free access to water and acclimatized for one week before the experiments. The mice were randomly divided into 4 groups. Immediately prior to the xenograft experiment, the transfected (Lv-NC, anti-miR-21, sh-CXCR4, and anti-miR-21+sh-CXCR4) cells were suspended to single-cell in sterile saline. The transfected U87 cells were injected into the left axillary subcutaneous location of BALB/C nude mice. The xenograft procedure was designed according to the published ones (Qin et al., 2017, Li et al., 2018. In brief, immediately prior to the xenograft experiment, the transfected cells were suspended to single-cell in sterile saline. Following the surgery, mice were monitored for tumor growth every 5 days. The formula V (volume) = (long diameter × short diameter ∧ 2)/2 was used to calculate the tumor volume. Mice were weighted and sacrificed at 4 weeks post-xenograft; then tumors were collected and measured. Immediately after the measuring, xenografts were stored at -80°C for further qRT-PCR and western blot analysis. Statistical analysis Statistical analysis was performed with SPSS 19.0 with an overall significance level of P < 0.05. Differences between the groups were computed by two-tailed unpaired Student t-Test. protein levels in transfected U251 and U87 cells were measured using western blot. (E) miR-21 and CXCR4 levels in transfected U251 and U87 cells were measured using qRT-PCR. (F and G) CXCR4 protein levels in transfected U251 and U87 cells were measured using western blot. *P < 0.05.	2020-04-16T09:14:57.424Z	2020-04-14T00:00:00.000	{ "year": 2020, "sha1": "73e23030c03c27170d174f9dc7b1df0aee62a3dd", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/bmri/2020/7930160.pdf", "oa_status": "GREEN", "pdf_src": "BioRxiv", "pdf_hash": "84bb5b0e8301110bfc029919558539e5803b471b", "s2fieldsofstudy": [ "Biology", "Medicine", "Chemistry" ], "extfieldsofstudy": [ "Biology", "Chemistry" ] }
25630307	pes2o/s2orc	v3-fos-license	Correlations in interference and diffraction Quantum formalism of Fraunhofer diffraction is obtained. The state of the diffraction optical field is connected with the state of the incident optical field by a diffraction factor. Based on this formalism, correlations of the diffraction modes are calculated with different kinds of incident optical fields. Influence of correlations of the incident modes on the diffraction pattern is analyzed and an explanation of the ''ghost'' diffraction is proposed. Introduction Correlations of states play an important role in quantum cryptography [1,2] , teleportation [3,4] , and computation [5−11] theory. Correlated states are generated usually by nonlinear optical processes [12] or by the beam splitter [13] . In this paper, we consider correlations in interference and diffraction. On the one hand, the diffraction or interference modes have some interesting correlation properties. On the other hand, correlations of the incident modes has a notable influence on the interference or diffraction pattern, in particular, it is the key to the explanation of the "ghost" diffraction [14] , an interesting quantum effect. Interference can be regarded as a special case of diffraction. To analyze correlations in interference and diffraction, we need a quantum formalism of diffraction. In the early days of quantum electrodynamics(QED), it had been proved that the Maxwell equations which underpin diffraction remain true when the fields are quantized [15−17] . In quantum optics the entire mode structure of the diffraction field is still determined by the Helmholtz part of the wave equation. The role played by quantum mechanics is in determining the states of the diffraction modes from the states of the incident modes. However, no systematic approach in determining the states of the diffraction modes has been proposed. In this paper, we first solve this problem. By introducing the quantum Kirchhoff boundary condition, we connect the states of the diffraction modes with the states of the incident modes by a diffraction factor. Then correlations of the diffraction modes with different kinds of incident optical fields are calculated. Influence of correlations of the incident modes on the diffraction pattern is analyzed. The "ghost' diffraction is also explained based on this formalism. We consider Fraunhofer diffraction. This kind of diffraction is most important. In Section 2, we introduce the equivalent scalar optical field and the quantum Kirchhoff boundary condition. The equivalent scalar optical field simplifies the problem of scalar diffraction, in which the variation of polarization through diffraction is not considered. The quantum Kirchhoff boundary condition is equivalent in physics to the Kirchhoff boundary condition in classical scalar diffraction yet overcomes the difficulty that the classical Kirchhoff boundary condition destroys the commutation relations of the field operators. In section 3, we obtain quantum formalism of Fraunhofer diffraction. The normal characteristic functions of the diffraction modes are connected with those of the incident modes by a diffraction factor. From the characteristic functions, correlation properties of the diffraction modes are analyzed in Sec. 4. In this section the diffraction pattern is also calculated with entangled incident states. An explanation of the "ghost" diffraction is proposed. 2 The equivalent optical field and the quantum Kirchhoff boundary condition In the diffraction problem the incident and diffraction optical fields are free. The free quantized electromagnetic field can be expanded into plane wave modes: where µ is polarization index and − → k · − → e − → k µ = 0. The annihilation and creation The frequency of the optical field remains unchanged through diffraction. So we only need consider fields with a definite frequency ω. That is, in the expansion (1) only the terms with − → k = ω c need be considered. Let In scalar diffraction theory, the boundary condition at the diffraction plane is independent of the orientation of the optical field, and the variation of polarization of the optical field through diffraction need not be considered. So we can introduce the following equivalent scalar optical field by neglecting the polarization index. where the box-normality of space has been used and S is the cross-section area of the box. The commutator (2) yields the following commutation relation of the equivalent optical field at the diffraction plane z = 0 In scalar diffraction the equivalent scalar optical field can be in place of the real optical field. The diffraction problem is much simplified by introducing the equivalent scalar optical field. In classical scalar diffraction theory the Kirchhoff boundary condition states: the optical field remains unchanged through the diffraction aperture Σ and decays to zero through the diffraction screen [18] . This boundary condition can not be used directly in the quantum case because the postulate that the optical field decays to zero through the diffraction screen destroys the commutation relations of the field operators. To keep consistent with quantum theory, we introduce the following quantum Kirchhoff boundary condition. The equivalent optical field ε (x, y, z = 0) before diffraction is generally in a complicated entangled state and we use ρ (z = 0 − ) to represent its whole density operator. The quantum Kirchhoff boundary condition says: When passing the diffraction screen all modes of the field ε (x, y, z = 0) (x, y ∈ S − Σ) at the screen undergo such a strong dissipation that after the screen they are all in the vacuum state. At the same time, the modes of the field ε (x, y, z = 0) (x, y ∈ Σ) at the aperture undergo no dissipation at all. From quantum dissipation theory [19,20] , the total density operator ρ (z = 0 + ) after diffraction is expressed as where the notation tr S−Σ indicates trace of all modes at the screen. This boundary condition for scalar diffraction is equivalent in physics to the classical Kirchhoff boundary condition. Yet it is consistent with quantum mechanics as it results from the quantum dissipation theory. In next section we use this boundary condition to derive quantum formalism of diffraction. Quantum formalism of Fraunhofer diffraction In Fraunhofer diffraction the incident and diffraction optical fields are expanded into the plane wave modes and the role played by quantum mechanics is in determining the states of the diffraction modes from the states of the incident is the density operator of the incident mode − → k ′ 0 and other incident modes are supposed in the vacuum state. First we derive the reduced normal characteristic function Using Eq. (5) and the inverse trans- we get where Σ and S represent area of the diffraction aperture and the whole diffraction plane, respectively, and the notation T r indicates trace of all modes. We define the energy transmissivity λ as λ = Σ S . Its physical meaning is the ratio of the energy of the diffraction optical field to the energy of the incident optical field. The Fraunhofer diffraction factor f − → k is defined as Eq. (7) is therefore simplified to Eq.(10) connects the reduced normal characteristic function of the diffraction mode b − → k with that of the incident mode a − → k ′ 0 by a simple diffraction factor. Similar to the derivation of Eq.(10), the total normal characteristic function of all diffraction modes b − → k has the form The above results are obtained with the supposition that only the incident mode − → k ′ 0 is not in the vacuum state. If all the incident modes are in an entangled state, and we use χ to indicate its whole normal characteristic function. Eq. (11) can thus be generalized to Eq. (12) The final result (12) is similar to the quantum description of the beam splitter. For the beam splitter, the input and output modes are linked by a canonical transformation [21] b where a 1 , a 2 are input operators and b 1 , b 2 are output operators. The parameters r and t should satisfy r 2 + t 2 = 1. From Eq. (13), we obtain the relation of the normal characteristic function between the input and output modes Eqs. (14) and (12) are very alike in the form. However, some important differences lie in their derivation. In diffraction the input and output modes cannot be put in a canonical transformation, which may be seen from the relation Only when the energy transmissivity λ = Σ S = 1 , i.e., when there is no diffraction screen, the input and output modes can be linked by a trivial canonical transformation. So unlike Eq. (14), Eq. (12) is not a direct result of the inputoutput theory [22,19] . In the derivation of Eq. (12), the quantum Kirchhoff boundary condition plays an essential role. S . When Σ 1 , Σ 2 tend to zero, Eq. (12) ; ξ has the following form i.e., the incident mode is in a coherent state. Under this condition, the diffraction modes are not correlated and all in coherent states. With any other kinds of incident optical fields the diffraction modes are in an entangled state. The above discussion shows that the diffraction modes are generally correlated. In experiments correlation of the photon number is widely used, so we first calculate the correlation coefficient of the photon number of two diffraction modes .The correlation coefficient is defined by where n − → k i (i = 1, 2) denotes the number operator of the mode − → k i . After some calculation, from Eq. (11) we obtain where F n is the Fano factor of the incident mode − → k ′ 0 , i.e., and h i in Eq. (19) is defined by The relation between η and F n is illustrated in Fig. 1 Fig . 1 If the incident mode is in a thermal state, F n = n − → k ′ 0 + 1 and η tends to its maximum value 1 with n − → k ′ 0 >> 1 . The correlation coefficient η gets its with the incident mode in a Fock state. Though η ≈ 1 if the incident mode is in a thermal state with n − → k ′ 0 >> 1, the diffraction modes are not correlated perfectly in this case. That can be seen from residual variance of the variables n − → k 1 and n − → k 2 in the linear regression. The residual variance of the variable n − → k 1 has the form [23] V ar where β 1 and β 2 are linear regression coefficients. Suppose h 1 = h 2 and So in this case the residual variance is very large. In fact, the equation η → 1 under the condition n − → k ′ 0 → ∞ results from the infinite variance of n − → k 1 and n − → k 2 .We can not conclude from η → 1 that the diffraction modes are correlated perfectly. For the beam splitter, the correlation of the number operator of the output modes has the same form as Eq. (19). However, there are still some differences. First, the equation 1 h 1 + 1 h 2 = 1 holds for the beam splitter whereas in diffraction we have 1 h 1 + 1 h 2 < 1 . So for the beam splitter, the correlation coefficient of the output number operators can attain its minimum value -1 with the input mode in a Fock state. Second, in diffraction or interference correlations of many modes can be generated whereas the beam splitter is only used to prepare twomode entangled states. Correlation coefficients describe correlation properties of a pair of specialized operators. Several approaches to the description of quantum entanglement have been proposed. In particular, Schlienz and Mahler interpreted the difference between the entangled state and the product state as the entanglement [24] . Suppose ρ is the density operator of the whole system and ρ a = tr b ρ , ρ b = tr a ρ , where the subscripts a and b represent two subsystems. The Schlienz-Mahler measure is defined by [24] where N (ρ) indicates the dimension of the density operator ρ and γ defined above satisfies 0 ≤ γ ≤ 1. However, the more recent papers distinguish quantum entanglement from classical correlations [25−30] . The entanglement is interpreted as the degree of inseparability. The entangled state is said to be inseparable if it can not be expressed as a mixture of product states of two subsystems. In this interpretation, the Schlienz-Mahler quantity γ measures the total correlations rather than pure quantum entanglement. It is now believed that pure quantum entanglement can not be fully described by a single quantity [29] . Bennett et. al. defined two quantities: [26,29] "entanglement of formation" defined as the least number of shared singlets asymptotically required to prepare ρ by local operations and classical communication, and "distillable entanglement" defined as the greatest number of pure singlets that can asymptotically be prepared from ρ by local operations and classical communication. And recently, Vedral et. al. introduced a new measure of entanglement [30] , which interprets the entangle-ment as the minimum distance to all separable states. These measures have the desirable feature that their expectations can not be increased by local operations, but the disadvantage of being hard to evaluate because of the implied optimization. The question is still open in this direction. Though the Schlienz-Mahler quantity γ in fact measures the total correlations, it is superior to the correlation coefficients, since it is not limited to specialized observables. In the following we use the Schlienz-Mahler quantity to analyze correlation properties of the diffraction modes. Before doing this, we first introduce the following lemma. Lemma. Suppose ρ 1, ρ 2 are two density operators of boson fields, and 2 (ξ) are normal characteristic functions of ρ 1 and ρ 2 , respectively, then we have Proof. If generalized functions (such as derivatives of delta functions) are permitted, the existence proof of P-functions of Boson fields has been given by Klauder and Sudarshan [31,19] . So tr (ρ 1 ρ 2 ) can be expressed as where P 1 (α) and Q 2 (α) are P,Q-functions of the density operators ρ 1, ρ 2 respectively. The P,Q-functions are Fourier transformations of the normal and anti-normal characteristic functions. So Eq. (26) can be rewritten as tr (ρ 1 ρ 2 ) = d 2 ξ π χ (n) where χ (a) (ξ) indicates the anti-normal characteristic function. Eq. (27) is equivalent to Eq. (25) . This completes the proof. We calculate the Schlienz-Mahler quantity γ with a thermal incident optical field. From Eq. (11) the normal characteristic function of the diffraction modes − → k 1 and − → k 2 has the form where N is the mean photon number of the incident mode. For thermal states, the dimension of the density operator N (ρ) → ∞. Eq. (24) together with Eq. (25) yields where From Eq. (29) it is obvious that γ tends to zero if N → ∞ or N → 0.	2017-09-14T21:10:31.125Z	1997-06-18T00:00:00.000	{ "year": 1997, "sha1": "cf7c126bf8bde6c83a4ed17c619dbc5ee17dfc2a", "oa_license": null, "oa_url": "http://arxiv.org/pdf/quant-ph/9706042", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "cf7c126bf8bde6c83a4ed17c619dbc5ee17dfc2a", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
6747857	pes2o/s2orc	v3-fos-license	The role of protein kinase C δ activation and STAT3 Ser727 phosphorylation in insulin-induced keratinocyte proliferation Activation of the STAT family of transcription factors is regulated by cytokines and growth factors. STAT tyrosine and serine phosphorylation are linked to the transcriptional activation and function of STAT. We have previously described a unique pathway inducing keratinocyte proliferation, which is mediated by insulin stimulation and depends on protein kinase C δ (PKCδ). In this study, we assessed STAT3 activation downstream of this pathway and characterized the role of PKCδ activation in STAT3 tyrosine and serine phosphorylation and keratinocyte proliferation. Following insulin stimulation, STAT3 interacted with PKCδ but not with any other PKC isoform expressed in skin. Activated forms of PKCδ and STAT3 were essential for insulin-induced PKCδ-STAT3 activation in keratinocyte proliferation. Abrogation of PKCδ activity inhibited insulin-induced STAT3 phosphorylation, PKCδ-STAT3 association and nuclear translocation. In addition, overexpression of STAT3 tyrosine mutant eliminated insulin-induced PKCδ activation and keratinocyte proliferation. Finally, overexpression of a STAT3 serine mutant abrogated insulin-induced STAT3 serine phosphorylation and STAT3-induced keratinocyte proliferation, whereas STAT3 tyrosine phosphorylation was induced and nuclear localization remained intact. This study indicates that PKCδ activation is a primary regulator of STAT3 serine phosphorylation and that PKCδ is essential in directing insulin-induced signaling in keratinocyte proliferation. Introduction STATs (signal transducers and activators of transcription) are cytoplasmic proteins that function as transcriptional activators. Seven STAT family members have now been identified including: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 (Akira, 1999;Akira, 2000;Horvath, 2000;Levy and Darnell, 2002). The use of isoform-specific transgenic knockout mouse models defined discreet physiological roles for each of the STAT family members. Among the seven known STAT proteins, STAT3 is unique. Only the STAT3-null phenotype in mice results in an embryonic-lethal phenotype at day 6-7 of fetal development, which cannot be compensated by any of the other STAT species expressed within the visceral endoderm (Akira, 1999). Furthermore, the importance of STAT3 for specific organ development was confirmed by experiments in which STAT3 expression was conditionally ablated in defined organs (Bromberg and Darnell, 2000;Sano et al., 2000;Takeda et al., 1997;Wen and Darnell, 1997). STAT3 has a specialized role in skin development. In the absence of STAT3 expression, both skin remodeling and hair cycle progression are severely disrupted (Sano et al., 1999;Sano et al., 2000). The activation of STAT proteins involves tyrosine phosphorylation, dimerization, nuclear translocation and activation of transcription by binding to DNA-response elements of target genes (Bromberg and Darnell, 2000;Levy and Darnell, 2002). The STAT proteins are distinctive among transcription factors in containing an SH2 (Src-homology 2), phosphotyrosine-binding domain. The SH2 domain interacts with sites of tyrosine phosphorylation to recruit the STATs to receptor complexes. Each of the STATs is differentially activated by various extracellular ligands including growth factors, cytokines and hormones, allowing differential intracellular processing of transcriptional signals (Bromberg and Darnell, 2000). During activation, STAT proteins can be specifically phosphorylated on both tyrosine and serine residues. Tyrosine phosphorylation of STAT3 at a single tyrosine residue (Tyr705) located at the Src-homology domain is essential for the activation of STAT3 (Bromberg et al., 1998). Similarly, a single serine site (Ser727) located in a conserved Pro-X-Ser-Pro sequence was shown to regulate STAT3mediated transcriptional activation (Bromberg et al., 1998;Decker and Kovarik, 2000;Lim and Cao, 1999;Turkson et al., 1999). However, although STAT serine phosphorylation was shown to be induced by several factors including interferon, epidermal growth factor and IL-6, its exact role in regulation Activation of the STAT family of transcription factors is regulated by cytokines and growth factors. STAT tyrosine and serine phosphorylation are linked to the transcriptional activation and function of STAT. We have previously described a unique pathway inducing keratinocyte proliferation, which is mediated by insulin stimulation and depends on protein kinase C ␦ ␦ (PKC␦ ␦). In this study, we assessed STAT3 activation downstream of this pathway and characterized the role of PKC␦ ␦ activation in STAT3 tyrosine and serine phosphorylation and keratinocyte proliferation. Following insulin stimulation, STAT3 interacted with PKC␦ ␦ but not with any other PKC isoform expressed in skin. Activated forms of PKC␦ ␦ and STAT3 were essential for insulin-induced PKC␦ ␦-STAT3 activation in keratinocyte proliferation. Abrogation of PKC␦ ␦ activity inhibited insulin-induced STAT3 phosphorylation, PKC␦ ␦-STAT3 association and nuclear translocation. In addition, overexpression of STAT3 tyrosine mutant eliminated insulin-induced PKC␦ ␦ activation and keratinocyte proliferation. Finally, overexpression of a STAT3 serine mutant abrogated insulin-induced STAT3 serine phosphorylation and STAT3induced keratinocyte proliferation, whereas STAT3 tyrosine phosphorylation was induced and nuclear localization remained intact. This study indicates that PKC␦ ␦ activation is a primary regulator of STAT3 serine phosphorylation and that PKC␦ ␦ is essential in directing insulin-induced signaling in keratinocyte proliferation. The protein kinase C (PKC) family of serine-threonine kinases plays an important regulatory role in a variety of biological phenomena (Dekker and Parker, 1994;Toker, 1998). The family is composed of at least 11 individual isoforms. In skin, protein kinase C (PKC) signaling was shown to be a major intracellular mediator of proliferation and differentiation pathways (Denning et al., 1996;Dotto, 1998;Matsui et al., 1992;Ohba et al., 1998). Utilizing both pharmacological activators of PKC as well as genetic approaches, we and others identified distinct roles for specific PKC isoforms in the induction of keratinocyte proliferation and differentiation in vivo and in vitro Dlugosz et al., 1992;Dotto, 1998;Verma, 1988). Specifically, we previously showed that growth factors such as insulin-like growth factor-1 (IGF-1) and insulin, although similar in their structure, diverge in their downstream signaling to mediate skin keratinocyte proliferation. Furthermore, insulin-induced proliferation is uniquely mediated by activation of PKC␦ (Jain et al., 1999;Novotny-Diermayr et al., 2002;Shen et al., 2001). In this study we demonstrate STAT3 activation and serine phosphorylation downstream of insulin-induced PKC␦ activation and keratinocyte proliferation. However, although PKC␦ activation is indispensable for insulin action in keratinocytes proliferation, eliminating STAT3 serine phosphorylation does not abrogate insulin-mediated PKC␦ activation and keratinocyte proliferation. Results The role of STAT3 in signaling of keratinocyte proliferation In previous studies, we identified a unique pathway involved in mediating insulin action in keratinocytes. This pathway uses the activation of PKC␦ as a crucial signaling factor downstream of insulin to induce keratinocyte proliferation. One of the factors involved downstream of PKC␦ signaling and which is affected by various cytokines and growth factors, is STAT3 (Boulton et al., 1995;Jain et al., 1999). To determine whether insulin signaling in keratinocytes may mediate its effects via STAT3 activation, we first examined the ability of insulin to regulate the phosphorylation state of STAT3 in these cells. As early as 5 minutes following insulin stimulation, tyrosine phosphorylation of STAT3 was induced; phosphorylation peaked by 15 minutes following stimulation (Fig. 1A). The phosphorylation of STAT3 was accompanied by its nuclear translocation, as observed by immunofluorescence (Fig. 1B). Specificity of STAT3 activation to the insulin-signaling pathway was further corroborated by following STAT3 association with the insulin receptor. As early as 2 minutes following insulin stimulation, activated STAT3 was physically associated with the insulin receptor (Fig. 1C). Moreover, insulin did not induce association of STAT3 with the insulinlike growth factor-1 (IGF-1) receptor, suggesting that the specificity of insulin-induced STAT3 activation to insulin receptor mediated signaling (Fig. 1C). Insulin induces STAT3 activation and nuclear translocation in mouse primary keratinocytes. (A) Keratinocytes following 5 days in culture were stimulated with 10 -7 M insulin for the times indicated. STAT3 immunoprecipitates were probed with anti-p-tyr-STAT3 (top panel). Equal loading of gels was confirmed by reblotting with STAT3 antibody (bottom panel). Relative optical density of four representative blots is presented in arbitrary units (mean ± s.d.). (B) Keratinocytes were plated on glass slides. Cultures (5 days old) were stimulated with insulin for 5 minutes, fixed in ethanol and analyzed by immunofluorescence, using anti-p-tyr-STAT3, followed by FITC-conjugated secondary antibody. Cells were viewed by confocal microscopy. (C) Keratinocytes were stimulated with 10 -7 M insulin for the times indicated. STAT3 immunoprecipitates were probed with anti-IR␤ antibody (top panel) and with anti-IGF 1R␤ antibody (middle panel). Equal loading of gels was confirmed by reblotting with STAT3 antibody (bottom panel). The experiment was repeated twice. Bar, 20 m. Effects of insulin on the PKC␦-STAT3 complex Our previous studies identified PKC␦ as a primary mediator of insulin signaling in keratinocyte proliferation. Since PKC␦ activation was previously linked to STAT3 signaling, we next investigated whether the PKC␦ isoform could physically bind to STAT3. Using recombinant PKC adenoviruses, keratinocytes were transduced with recombinant PKC adenoviruses where ␤-gal-infected keratinocytes were used as Journal of Cell Science 119 (3) controls for the viral infection (Miyake et al., 1996;Ohba et al., 1998). Lysates were subjected to immunoprecipitation with antibodies to PKC isoforms expressed in skin including: PKCs ␣, ␦, and , and immunoprecipitates were then subjected to western blot analysis using a STAT3-specific antibody. The overexpression of various PKC isoforms effectively induced protein expression five-to tenfold above basal levels associated with constitutive activation of the PKCs isoforms ( Fig. 2) (Alt et al., 2004). Under unstimulated basal conditions, STAT3 was associated with PKC␦ but not with PKC isoforms ␣, or , expressed in skin (Fig. 2). Furthermore, as can be seen in Fig. 3A, following stimulation by insulin, a dramatic increase of STAT3 serine phosphorylation was observed within the complex. Tyrosine phosphorylation was also increased, indicating STAT3 activation. However, no induction in STAT3 tyrosine or serine phosphorylation was observed in PKC immunoprecipitates of insulin-stimulated keratinocytes and keratinocytes overexpressing PKC isoforms ␣, or ( Fig. 3B and results not shown). Although PKC-overexpressing keratinocytes expressed similar levels of each of the PKC isoforms, constitutive association and phosphorylation of STAT3 was evident only with PKC␦ (Fig. 2). A direct link between PKC␦ activity and complex formation and activation was next confirmed by abrogation of PKC␦ activity. Overexpression of a kinase inactive PKC␦ form (dominant-negative PKC) reduced STAT3-PKC␦ complex formation (Fig. 4A,B) and decreased both tyrosine Fig. 2. Protein kinase C ␦ (PKC␦) specifically associates with STAT3. Keratinocytes (5 day cultures) were infected with isoform-specific PKC ␣, ␦, and recombinant adenoviruses, or with adenovirus encoding ␤-galactosidase (␤-Gal) as a control, for 1 hour. OE, overexpression. Following infection, cells were incubated for 24 hours, then extracted and immunoprecipitated (IP) with antibodies against PKC␣, PKC␦, PKC and PKC. Immunoprecipitates were subjected to western blot analysis using isoform-specific anti-PKCs or anti-STAT3 antibodies. Results presented are representative of at least three experiments. Fig. 3. Insulin-induced PKC␦ activation regulates STAT3 association and phosphorylation. (A) Keratinocytes were untreated (-) or treated with insulin for 5 minutes (+). PKC␦ immunoprecipitates were subjected to western blot analysis using antibodies against STAT3, antiphosphotyrosine-705-STAT3 (p-tyr), anti-phosphoserine-727-STAT3 (p-ser) and anti-PKC␦. Relative optical densities of the blots are presented in arbitrary units. Relative optical density of four blots is presented in arbitrary units (mean ± s.d.). (B) Primary keratinocytes were either untreated or infected for 1 hour with isoformspecific PKC recombinant adenovirus, or with ␤galactosidase (␤-Gal) adenovirus as control. Control cells were either untreated (C) or stimulated with insulin for 5 minutes (Ins 5Ј). Cells were extracted and immunoprecipitated (IP) with isoform-specific PKC antibodies. The immunoprecipitates were subjected to western blot analysis using anti-PKCs, anti-STAT3 or anti-phosphoserine-727-STAT3 (STAT p-ser) antibodies. Experiments were repeated three times. and serine phosphorylation of STAT3 following insulin stimulation (Fig. 4B,C). Similar results were obtained when we inhibited PKC␦ activity using the PKC␦ inhibitor, rottlerin (results not shown). Collectively, these results confirm the link between insulin-induced PKC␦ activation and the STAT3 phosphorylation state. PKC␦-STAT3 association also depends on the activation of STAT3 Using a STAT3 wild-type construct and a dominantnegative STAT3 mutant (STAT3-705-tyrosine mutant), efficient expression of STAT3 could be achieved in keratinocytes, at levels 5-to 20-fold above basal expression (Fig. 5A). As expected, overexpressed wild-type STAT3 was constitutively phosphorylated on tyrosine residues and was found complexed with PKC␦. Similarly to the endogenous protein, insulin stimulation for 5 minutes further increased tyrosine phosphorylation of the overexpressed STAT3 (Fig. 5B). The importance of tyrosine residue 705 to the insulin-induced activation of STAT3 was further verified using a STAT3 tyrosine mutant in which Tyr705 was exchanged with phenylalanine. Following overexpression of the dominant-negative STAT3 tyrosine (705) mutant, insulin could no longer induce STAT3 tyrosine phosphorylation (Fig. 5B). This confirmed STAT3 Tyr705 to be essential for STAT3 activation by insulin (Fig. 5B). PKC␦ and wild-type STAT3 were physically associated, and stimulation with insulin further augmented this association (Fig. 5C). Interestingly, overexpression of the tyrosine-inactive STAT3 mutant did not abrogate constitutive PKC␦-STAT3 complex formation. However, insulin-induced association was abolished, suggesting that STAT3 activation is not a prerequisite for complex formation with PKC␦. Similarly to the endogenous STAT3 protein, in keratinocytes overexpressing wild-type STAT3, inhibition of PKC␦ activity by overexpression of a dominant-negative PKC␦ decreased STAT3 tyrosine phosphorylation as well as its association with PKC␦ ( Fig. 5D). Similar results to those obtained by overexpressing a dominant-negative form of PKC␦ were obtained using rottlerin, a specific inhibitor of PKC␦ activation (results not shown). However, the STAT3 activation state also contributed to insulininduced activation of PKC␦, as demonstrated in PKC activity assays. As early as 5 minutes following insulin stimulation, PKC␦ activity was increased in PKC␦ immunoprecipitates ( Fig. 6A) and PKC activity was found to reside within STAT3 immunoprecipitates (Fig. 6B). Furthermore, dominant-negative STAT3-inhibited insulin-induced activation of endogenous and overexpressed PKC␦ (Fig. 6A,B). Overall, these results suggest that the active form of PKC␦ is required for the formation of the insulin-induced STAT3-PKC␦ complex, and emphasize the crosstalk between PKC␦ and STAT3 in regulating the activation state of the PKC␦-STAT3 complex. Nuclear translocation of STAT3 STAT3 activation is characterized by induction of tyrosine phosphorylation and dimerization of STAT3 followed by (B) Keratinocytes were either infected for 1 hour with ␤-Gal or WT STAT3 adenovirus constructs, or double infected with DN PKC␦ followed by WT STAT3 recombinant adenovirus infection. Following infection, cells were incubated for 24 hours and overexpressing cells were untreated (-) or stimulated with insulin for 5 and 30 minutes. PKC␦ immunoprecipitates were subjected to western blot analysis and probed with anti-phosphoserine-727-STAT3 (STAT3-p-ser), anti-PKC␦ and anti-STAT3 antibodies. (C) Keratinocytes were either uninfected (-) or infected (OE) for 1 hour with recombinant dominant-negative PKC␦ (DN PKC␦) adenovirus. After 24 hours, cells were left untreated (-) or treated with insulin for 5 minutes. PKC␦ immunoprecipitates were subjected to western blot analysis and probed with anti-phosphotyrosine-705-STAT3 (STAT3-p-tyr) and anti-PKC␦ antibodies. Relative optical density of three blots are presented in arbitrary units (mean ± s.d.). Experiments were repeated at least three times. nuclear translocation of the STAT3 complex. Therefore, we next investigated the link between insulin-induced PKC␦ activation and STAT3 nuclear translocation. In non-stimulated keratinocytes, no STAT3 was detected in the nucleus (Fig. 7A). However, as early as 5 minutes following insulin stimulation, tyrosine-phosphorylated STAT3 was observed in the nucleus, where the signal was retained for 15 minutes, and faded 30 minutes following stimulation. Pretreatment with rottlerin before insulin stimulation, completely inhibited translocation of STAT3 to the nucleus, but some tyrosine-phosphorylated Journal of Cell Science 119 (3) (A) Total cell lysates were subjected to western blot analysis using anti-STAT3 antibody. (B) Overexpressing cells were either untreated (-) or treated with insulin for 5, 15 and 30 minutes. Lysates were immunoprecipitated with anti-HA antibody and immunoprecipitates analyzed by western blotting, using a anti-p-tyr-STAT3 antibody. (C) Overexpressing cells were treated with insulin for 5 and 30 minutes. PKC␦ immunoprecipitates were analyzed by western blotting, using anti-HA and anti-STAT3 antibodies. Equal loading of gels was confirmed by reblotting with PKC␦ antibody. Relative optical density of three representative blots is presented in arbitrary units (mean ± s.d.). (D) Keratinocytes were either infected (OE) for 1 hour with WT STAT3 adenovirus constructs or double infected with DN PKC␦ followed by WT STAT3 recombinant adenovirus infection. 24 hours following infection, cells were untreated (-) or treated with insulin for 5 minutes. HA immunoprecipitates were subjected to western blot analysis and probed with antiphosphotyrosine and anti-HA antibodies. Experiments were repeated at least three times. Keratinocytes were either uninfected (-), or infected with DN STAT3 adenovirus. Following infection (24 hours), cells were untreated, or stimulated with 10 -7 M insulin (Ins) for the designated times (5 or 30 minutes). (A) PKC␦ or (B) STAT3 were immunoprecipitated from protein extracts using specific anti-PKC␦ and anti-STAT3 antibodies. PKC␦ and STAT3 immunoprecipitates were analyzed for PKC activity using an in vitro kinase assay as described in Materials and Methods. Each bar represents the mean ± s.d. of three determinations in three separate experiments. Values are expressed as picomoles of ATP per dish per minute. Highly significant differences were found (P<0.0001) for both IP PKC␦ and STAT3 compared with levels in the respective controls at 5 minutes. Insulin-induced PKC␦ and STAT3 activation STAT3 could be detected in the cytoplasm of insulinstimulated cells (Fig. 7A, lower panel). Interestingly, following insulin stimulation, the nuclear translocation of STAT3 was not accompanied by nuclear translocation of PKC␦. Under basal conditions, PKC␦ expression was detected mostly in the cytoplasm, whereas in some cells, a perinuclear distribution was noticed. Following insulin stimulation, PKC␦ distribution was shifted to the perinuclear area including the nuclear membrane but no protein was seen within the nucleus (Fig. 7B). Inhibition of PKC␦ activity by pretreatment with rottlerin prior to insulin stimulation, completely abolished redistribution of PKC␦ expression following insulin stimulation (data not shown). Role of STAT3 serine phosphorylation To directly study the functional contribution of STAT3 serine phosphorylation to insulin-induced PKC␦ activation in keratinocyte proliferation, we next constructed a HA-tagged STAT3 Ser727 to Ala mutant. Ser727 located at the Pro-X-Ser-Pro motif was previously shown to contribute to transcriptional activation of STAT3 (Chung et al., 1997;Jain et al., 1999;Novotny-Diermayr et al., 2002). This mutant (SmS) was introduced into an adenovirus construct and used to follow the STAT3 activation state in response to insulin stimulation. STAT3 SmS was efficiently expressed in keratinocytes (Fig. 8). No serine phosphorylation was observed in the overexpressing keratinocytes, in either the basal state (Fig. 9B) nor in response to insulin stimulation (Fig. 8C), confirming that the Ser727 residue is uniquely phosphorylated in response to insulin. However, surprisingly, in the basal state, constitutive tyrosine phosphorylation of the serine mutant STAT3 was increased at least five-to tenfold above levels observed with wild-type STAT3 (Fig. 8B), and the STAT3 serine mutant was localized to the nucleus of unstimulated, overexpressing cells (results not shown). Furthermore, the STAT3 serine mutant was constitutively associated with PKC␦, but no further increase in STAT3 tyrosine phosphorylation and association with PKC␦ was seen in response to insulin stimulation (Fig. 8C). The role of STAT3 in keratinocyte proliferation In previous studies, we identified a pathway which links PKC␦ activation by insulin to keratinocyte proliferation (Shen et al., 2001). Therefore, we next studied the involvement of STAT3 in this pathway. Overexpression of PKC␦ and overexpression of STAT3 were able to induce keratinocyte proliferation to a similar degree (Fig. 9A). No proliferation was induced when cells were infected with dominant-negative constructs of STAT3 and PKC␦ as well as by overexpression of the STAT3 serine mutant (Fig. 9A, SmS). These results further confirmed that STAT3 serine phosphorylation is required for induction of keratinocyte proliferation. Moreover, abrogation of PKC␦ activity by infection with a DN PKC␦ construct inhibited proliferation induced by STAT3 overexpression (Fig. 9A). We next investigated the role of STAT3 serine and tyrosine Journal of Cell Science 119 (3) phosphorylation and the activation state of PKC␦, in the regulation of insulin-induced keratinocyte proliferation. Following overexpression of WT STAT3, DN STAT3, SmS and kinase active (WT) and kinase inactive (DN) PKC␦, only inactivation of PKC␦ significantly inhibited insulin-induced keratinocyte proliferation (Fig. 9B). These results suggest an imperative regulatory role for PKC␦, in mediating insulin signaling via the STAT3-PKC␦ pathway in insulin-induced keratinocyte proliferation. However, STAT3 mutants were unable to abrogate insulin-induced keratinocyte proliferation suggesting a lesser role for STAT3 compared with PKC␦ in the regulation of insulin action in keratinocytes, The role of PKC␦ and STAT3 in inducing keratinocyte proliferation was also confirmed by FACS analysis (Fig. 9C and results not shown). Increase in the percentage of actively proliferating S-phase cells was detected following overexpression with WT PKC␦ and WT STAT3 recombinant adenoviruses whereas infection with DN PKC␦, DN STAT3 and SmS did not induce cell proliferation (Fig. 9C). Interestingly, by following the Sub-G1 population we identified that in comparison to ␤-Gal overexpressing cells, only overexpression of WT PKC␦ but not overexpression of DN PKC␦, WT STAT3 or STAT3 mutants induced keratinocyte apoptosis (Fig. 9C, A-apoptotic cells). Collectively, the results suggest that PKC␦ plays a specific role in regulation of STAT3 727 serine phosphorylation and mediation of insulin-induced keratinocyte proliferation. Discussion The STAT family of proteins are transcriptional activators, which can be activated in response to a variety of stimuli including cytokines, growth factors and hormones (Calo et al., 2003;Heim, 1999). However, the mechanism of STAT activation is unique, compared with that of other transcriptional activators. The presence of an SH2 domain and a C-terminal tyrosine residue, which becomes phosphorylated upon stimulation, provides the basis for the interaction of STATs with several signaling complexes. Such signaling pathways can directly regulate subsequent homo-and Relative optical density of three representative blots is presented in arbitrary units (mean ± s.d.). (D) Keratinocytes were infected for 1 hour using recombinant adenoviruses encoding WT STAT3 and STAT3 serine mutant (SmS). Cells were stimulated with insulin for 5 and 30 minutes. Lysates were immunoprecipitated with PKC␦ antibody and analyzed by western blotting, using anti-HA. Equal loading of gels was confirmed by reblotting with PKC␦ antibody. Relative optical density of blots is presented in arbitrary units (mean ± s.d.). Experiments were repeated at least three times. heterodimerization of the protein to direct its localization to the nucleus. In the nucleus, STATs bind DNA to induce transcription of specific genes (Chatterjee- Kishore et al., 2000;Imada and Leonard, 2000;Levy and Darnell, 2002). In this study, we describe a unique pathway in skin keratinocyte proliferation, which involves STAT3 activation downstream of insulin-mediated PKC␦ activation. The association between STAT3 serine phosphorylation and PKC␦ was previously established in other STAT3-mediated pathways including stimulation with IL6, LIF and EGF (Jain et al., 1999;Novotny-Diermayr et al., 2002;Schuringa et al., 2001). In addition to PKC␦, several other kinases including Erk, p38, and JNK have been shown to directly or indirectly induce phosphorylation of STAT3-Ser727 (Abe et al., 2001b;Chung et al., 1997;Jain et al., 1998;Jain et al., 1999;Kuroki and O'Flaherty, 1999;Lim and Cao, 1999;Sengupta et al., 1998;Xu et al., 2003). However, the specific physiological role for this interaction was not determined. Our study establishes a direct link between STAT3 serine phosphorylation and the activation and regulation of keratinocyte proliferation in response to insulin signaling in the skin. Our previous studies suggested that the activation of PKC␦ by insulin is unique and is not induced by other factors such as EGF, PDGF, IGF or KGF (Shen et al., 2001). This study extends these results to identify STAT3 activation and serine phosphorylation as downstream regulators of insulin-mediated signaling in keratinocyte proliferation. Moreover, the physical linkage between PKC␦ and STAT3 was shown to be specific and does not involve other PKC isoforms expressed in the skin. Upon insulin-induced activation of PKC␦, STAT3 becomes phosphorylated on both serine and tyrosine residues, leading to subsequent STAT3 nuclear localization and activation. Abrogation of PKC␦ activity was able to inhibit the physical association of STAT3 with PKC␦ as well as to inhibit STAT3 phosphorylation, nuclear translocation and activation. Furthermore, abrogation of either PKC␦ activity or serine phosphorylation of STAT3-blocked PKC␦ and STAT3mediated keratinocyte proliferation. However, although abrogation of PKC␦ activity was able to inhibit STAT3induced keratinocyte proliferation, a dominant-negative form of STAT3 was not able to efficiently inhibit PKC␦-induced proliferation. These results suggest that serine phosphorylation of STAT3 by PKC␦ is indispensable for the downstream STAT3 signaling in mediation of keratinocyte proliferation. Furthermore, STAT3 mutants were not able to inhibit insulin-induced proliferation. These results could be associated with the inefficient physical link between PKC␦ and STAT3 mutant forms (SmS and DN STAT3) allowing the activation of endogenous STAT3 by insulin-induced PKC␦ activation. However, other possible explanations could be related to the STAT3 mechanism of action. STAT3 as a transcription activator acts downstream of various signaling pathways including signaling by IGF-1 and Leptin. The convergence of IGF-1 and Leptin pathways with the insulin signaling pathway can lead to indirect effects of insulin via alternative pathways following the blockade of insulin-STAT3 S -11.9 Apo-2.9 S -37.6 Apo-24. activation (Kim et al., 2000;Xu et al., 2005). Therefore, chronic effects of insulin on cell proliferation measured 18-24 hours following stimulation does not reflect directly just acute activation of PKC␦ and STAT3 but rather is the consequence of the overall compensatory mechanisms. Finally, although our results clearly show that insulin does not phosphorylate STAT3 mutants, constitutive complex formation of STAT3 with PKC␦ and nuclear localization of the STAT3 mutant protein suggest alternative mechanisms distinct from protein phosphorylation to transmit insulin signaling in cell proliferation. Although the role of tyrosine phosphorylation as an essential step for activation of STAT3 is well established (Darnell et al., 1994;Levy and Darnell, 2002), the precise role of a single serine phosphorylation event, such as the one associated with the activation of Ser727 in STAT3, was not known. Indeed, in several studies, serine phosphorylation was shown to coincide with and contribute to transcriptional activation following stimulation in vitro (Ceresa and Pessin, 1996;Decker and Kovarik, 2000;Gotoh et al., 1996). However, several recent studies suggested that STAT3 serine phosphorylation following stimulation with EGF, NGF or IL-6 has a regulatory role distinct from that of STAT tyrosine phosphorylation and activation (Abe et al., 2001a;Kovarik et al., 1999;Lim and Cao, 1999). In keratinocytes, tyrosine and serine phosphorylation of STAT3 seem to coincide following insulin stimulation. However, in cells expressing a STAT3 Ser727 mutation, serine phosphorylation was completely abrogated whereas STAT3 tyrosine phosphorylation was significantly increased. These results are in agreement with recent results obtained in transgenic mice expressing a form of the STAT3 Ser727 mutant (Shen et al., 2004). Although fibroblasts carrying the serine mutation are defective in their transcriptional response in vitro, fibroblasts induced by oncostatin M exhibit intact tyrosine phosphorylation of STAT3. Furthermore, in the serine-mutant mouse, many of the downstream signaling pathways associated with STAT3 remain intact (Shen et al., 2004). Interestingly, in the serinemutant mouse, STAT3 serine phosphorylation was found to specifically affect levels of members of the insulin like growth factor (IGF1) family and to influence growth-hormonemediated signaling. Furthermore, the phenotype observed with this mutant was associated with marked growth retardation, reduced thymic epithelial cell number and induction of apoptotic cell death (Shen et al., 2004). These results are in agreement with our study supporting a role for STAT3 activation and serine phosphorylation in the physiological proliferation of keratinocytes under the regulation of another member of the insulin family of growth factors, insulin. Overall, these results suggest that rather than transcription per se, serine phosphorylation is involved in directing the specificity of the signaling pathway. Indeed, serine phosphorylation could be associated with a variety of mechanisms regulating cell signaling leading to calibration of the signal and a specific physiological outcome. These mechanisms include the induction of conformational changes, induction of specific complex formation, and phosphorylation and regulation of other downstream effectors by PKC␦ and STAT3. The unique ability of a transcription factor such as STAT3 to physically interact via an SH2 domain with various signaling protein complexes would also provide a mechanism whereby a downstream element such as STAT3 could affect Journal of Cell Science 119 (3) PKC␦ activation via phosphorylation or direct interaction with various components of the signaling complex. The association of STAT3 with cell proliferation was established in several model systems. STAT3 was found to be activated and constitutively phosphorylated in many tumor cells in vitro and in vivo (Bromberg et al., 1999;Frank, 1999;Takeda et al., 1998;Turkson and Jove, 2000) including the induction of the oncogenic phenotype in Src-transformed fibroblasts (Bromberg et al., 1998;Silva, 2004;Turkson et al., 1998). In addition, specifically in skin, targeted deletion of STAT3 abrogates skin remodeling and is associated with a blockage of hair-cycle progression and impaired wound healing (Akira, 2000;Sano et al., 1999;Sano et al., 2000). Interestingly, similarly to STAT3, PKC␦ activation in several model systems was shown to mediate signaling by growth factors such as EGF, NGF, PDGF, insulin, TNF␣ and IL-6 (Gliki et al., 2001;Jackson et al., 2001;Jain et al., 1999;Kontny et al., 1999;Novotny-Diermayr et al., 2002;Robin et al., 2004;Shen et al., 2001). However, although STAT3 was shown to be involved in mediating cell growth and oncogenic potential, further studies suggest PKC␦ to be primarily involved in cell differentiation and apoptosis; thus inhibition of PKC␦ activity and loss of PKC␦ during transformation are associated with cell growth and oncogenesis (Fujii et al., 2000;Li et al., 1999;Li et al., 1996b;Lucas and Sanchez-Margalet, 1995). Our results suggest possible explanations for the role of PKC␦ in directing the specificity of signal activation in keratinocytes. Under physiological settings in normal primary keratinocytes, PKC␦ activation is associated with STAT3 and linked to insulin signaling and keratinocyte proliferation. However, in cell transformation and during tumor progression, PKC␦ expression is downregulated or inactivated (Geiges et al., 1995;Joseloff et al., 2002;Langzam et al., 2001). The resulting loss of STAT3-PKC␦ complexes could contribute to the transformed phenotype by enabling STAT3 phosphorylation and activation to come under the regulation of a variety of other growth factors distinct from insulin (Bromberg et al., 1999;Calo et al., 2003;Chan et al., 2004). Alternatively, the fact that only PKC␦ but not STAT3 overexpression could induce cell apoptosis suggests that PKC␦-mediated apoptosis is distinct from the proliferative pathway which is regulated by both PKC␦ and STAT3 activation. Another possible explanation for the differences between PKC␦ and STAT3 regulation could be based on intracellular localization. For example, our results in primary murine keratinocytes demonstrated PKC␦ translocation to the perinuclear region following insulin stimulation to be independent of the nuclear translocation of STAT3. Furthermore, our study has confirmed that whereas serine phosphorylation is crucial for functional outcome, the serine mutation does not abrogate nuclear translocation. These results are in agreement with recent studies suggesting that the Cterminus trans-activating domain and not the N-terminal domain regulates STAT3 nuclear localization (Ma et al., 2003;Pranada et al., 2004). Therefore, although STAT3 serine phosphorylation is coupled to PKC␦ translocation to the nucleus in a variety of transformed cell lines, the perinuclear localization of PKC␦ in primary keratinocytes could result in a different physiological outcome in response to STAT3 phosphorylation and transactivation (Buchner, 2000;Goodnight et al., 1995;Jain et al., 1999). Overall, these results suggest a specific role for PKC␦ as a serine kinase of STAT3 and as a regulator of insulin-mediated STAT3 signaling in normal skin physiology. Furthermore, the interaction of STAT3 with PKC␦ facilitates PKC␦ function and is essential for insulin-induced proliferation of skin keratinocytes. Future studies will be aimed at understanding the mechanisms that underlie the specificity of the signal induced by STAT3 serine phosphorylation versus tyrosine phosphorylation in keratinocyte physiology and the pathology of skin cancer. Isolation and culture of murine keratinocytes Primary keratinocytes were isolated as described from newborn BALB/C mice (Dlugosz et al., 1995). Keratinocytes were cultured in Eagle's minimal essential medium (EMEM) containing 8% fetal bovine serum pretreated with Chelex-100 (BioRad). To maintain a proliferative basal cell phenotype, the final Ca 2+ concentration was adjusted to 0.05 mM. Serum starvation was induced by maintaining keratinocytes in medium containing 0.05% serum for 24 hours. Experiments were performed 5-7 days after plating. Total cell extracts and western blots Whole-cell lysates were prepared by scraping cells on ice into 300 l lysis buffer containing 5% SDS, 20% 2-␤-mercaptoethanol and 50% western upper gel buffer (0.5 M Tris-HCl, pH 6.8). Lysates were homogenized, boiled for 5 minutes, and centrifuged at 28,000 g for 20 minutes. Supernatant containing the total cell protein extracts was transferred to a fresh tube. Protein loading buffer was added to an equal volume of the total cell extracts, samples were boiled for 5 minutes and centrifuged. Supernatants were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). Specific protein bands were detected by immunoblotting using specific antibodies and visualized by enhanced chemiluminescence. Preparation of cell extracts in RIPA buffer For crude membrane fractions, cells were washed with cold PBS three times, resuspended in RIPA buffer without SDS containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% deoxycholic acid, 1% Triton X-100, 0.25 mM EDTA (pH 8.0), and protease and phosphatase inhibitors (10 g/ml aprotinin, 10 g/ml leupeptin, 2 g/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 200 M NaVO 4 and 5 mM NaF). Lysates were incubated for 30 minutes at 4°C on ice and centrifuged at 4°C for 30 minutes at 28,000 g. The supernatant containing the soluble protein fraction (cytoplasmic and membrane) was transferred to another tube and used for immunoprecipitation. Protein concentrations were measured using a modified Lowry assay (Bio-Rad DC Protein Assay Kit). Western blot analysis of cellular protein fractions was carried out as described (Li et al., 1996a). Immunoprecipitation and western blot analysis Protein cell lysates, prepared in RIPA buffer, were precleared by mixing 0.3 ml cell lysate with 25 l of Protein A/G Sepharose (Santa Cruz Biotechnology), and the suspension was rotated continuously for 30 minutes at 4°C. The preparation was then centrifuged at maximal speed at 4°C for 10 minutes. Supernatants were incubated with specific polyclonal or monoclonal antibodies to the individual PKC isoforms (diluted 1:100), anti-STAT3, anti-phosphotyrosine-STAT3, or antiphosphoserine-STAT3 antibodies overnight at 4°C followed by incubation with protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) for 2 hours. The suspension was then centrifuged at maximal speed for 10 minutes at 4°C, the pellet was washed twice with RIPA buffer, twice with cold PBS and subjected to western blot analysis. Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membrane (0.2 m) (BIO-RAD) and blotted with specific polyclonal or monoclonal antibodies. Experiments were repeated several times, as indicated in the figure legends, and blots of repeated experiments were analyzed by densitometry. Arbitrary units were normalized to a similar scale for all original figures maintaining the differential quantitative responses. Statistical analysis (mean ± s.d.) was performed accordingly. Recombinant adenovirus constructs The recombinant adenovirus vectors were constructed as described (Miyake et al., 1996). A dominant-negative mutant of mouse PKC␦ was generated by substitution of the lysine residue at the ATP-binding site with alanine (Ohba et al., 1998). Adenoviral vectors containing the genes for HA-tagged STAT3 and HA-tagged DN STAT3 (AxCASTAT3 and AxCAdnSTAT3), which contained a CAG promoter (chicken ␤actin promoter with cytomegalovirus enhancer), were prepared by homologous recombination in HEK293 cells by using pAxCA 1w cosmid cassette, as described previously (Miyake et al., 1996). The dominant-negative activity of STAT3F gene was demonstrated by the abrogation of its tyrosine phosphorylation. A serine phosphorylation STAT3 mutant adenovirus was generated by the COS-TPC method using cosmids and recombinant adenoviruses containing pEFHA-Stat3-S727A expression vector (Miyake et al., 1996). PEFHA-Stat3S-727A was prepared by introducing a point mutation at Ser727 to Ala (Abe et al., 2001a). Viral titer was adjusted by the TCID 50 method according to the manufacturer's protocol (Takara Shuzo, Japan) (Miyake et al., 1996). Briefly, a virus solution from HEK293 cell lysates was serially diluted into 96-well collagen-coated plates. A row of uninfected cells was used as controls. Complete cytopathic effect was judged by microscopic evaluation after 11-13 days of infection. Wells in which the virus induced a full cytopathic effect were counted and the 50% tissue culture infectious dose was calculated (TCID50) in correlation with the number of plaque-forming units. More than 95% of keratinocytes in the culture dish expressed the transduced protein following recombinant adenovirus infection as analyzed by ␤-galactosidase adenovirus infection. Overexpression of specific proteins by using of the recombinant adenovirus vectors The culture medium was aspirated and keratinocyte cultures were infected with the viral supernatant containing PKC␦, DN PKC␦, WT STAT3 or DN STAT3 recombinant adenoviruses at an multiplicity of infection (MOI) of 12 for 1 hour. The cultures were then washed twice with PBS and re-fed low-Ca 2+ MEM (0.05 mM) for 24 hours. ␤-Galactosidase adenovirus was used as a negative control to exclude possible deleterious effects of the vector itself. Following infection, cells were incubated for 24 hours, and then were left untreated or treated with insulin, and used for proliferation assays, activity assays, immunocytochemistry assays or for immunoprecipitation and western blotting. PKC activity Specific PKC activity was determined in freshly prepared immunoprecipitates from keratinocyte cultures following appropriate treatments. These lysates were prepared in RIPA buffer without NaF. Activity was measured with use of the SignaTECT Protein Kinase C Assay System (Promega, Madison, WI) according to the manufacturer's instructions. Neurogranin (AAKIQAS*FRGHMARKK) was used as the substrate for PKC activity in these studies. Cell proliferation Cell proliferation was measured by [ 3 H]thymidine incorporation in six-well plates. Cells were pulsed with [ 3 H]thymidine (1 Ci/ml) for 1 hour. After incubation, cells were washed five times with PBS, and 5% TCA was then added to each well for 1 hour. The solution was removed and cells were solubilized in 1 M NaOH. The labeled thymidine incorporated into cells was counted in the 3 H window of a Tricarb liquid scintillation counter. Immunofluorescence Primary keratinocytes were plated on ProbeOn Plus glass slides. Keratinocytes after 5 days of culture were left untreated or treated with insulin for 5 minutes and 30 minutes with or without pre-treatment with rottlerin for 7 minutes, infected either with PKC␦, DN PKC␦, or DN STAT3 adenoviruses for 1 hour, washed twice with Ca 2+ -and Mg 2+ -free PBS and maintained in culture in 0.05 mM Ca 2+ EMEM. After infection, keratinocytes were incubated for 24 hours, then fixed in methanol for 10 minutes followed by permeabilization with 0.2% Triton X-100 for 5 minutes, rinsed with PBS and incubated for 18 hours at 4°C, with specific anti-PKC, antiphosphotyrosine STAT3 or anti-STAT3-phosphoserine antibodies diluted in 1% BSA in PBS. After incubation, slides were washed twice for 10 minutes with PBS and incubated with biotinylated secondary anti-rabbit antibody for 40 minutes, washed twice in PBS and incubated with streptavidin-FITC for 40 minutes. Following two washes in PBS and one wash with double-distilled water, slides were air dried and mounted with Vectashield mounting medium (Vector). Fluorescence was examined by laser scanning confocal imaging microscopy (MRC1024, Bio-Rad, UK). This study was supported by the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum (DKFZ) and Israel's Ministry of Science (MOS). Research was supported in part by the Israel Science Foundation (grant No. 4801).	2017-08-28T04:39:28.926Z	2006-02-01T00:00:00.000	{ "year": 2006, "sha1": "d370b9a140ad359452de1d176cfbfaafd931b73a", "oa_license": "CCBY", "oa_url": "http://jcs.biologists.org/content/119/3/470.full.pdf", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "d370b9a140ad359452de1d176cfbfaafd931b73a", "s2fieldsofstudy": [ "Biology", "Computer Science" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
53420970	pes2o/s2orc	v3-fos-license	A Three-Dimensional Quantum Simulation of Silicon Nanowire Transistors with the Effective-Mass Approximation The silicon nanowire transistor (SNWT) is a promising device structure for future integrated circuits, and simulations will be important for understanding its device physics and assessing its ultimate performance limits. In this work, we present a three-dimensional quantum mechanical simulation approach to treat various SNWTs within the effective-mass approximation. We begin by assuming ballistic transport, which gives the upper performance limit of the devices. The use of a mode space approach (either coupled or uncoupled) produces high computational efficiency that makes our 3D quantum simulator practical for extensive device simulation and design. Scattering in SNWTs is then treated by a simple model that uses so-called Buttiker probes, which was previously used in metal-oxide-semiconductor field effect transistor (MOSFET) simulations. Using this simple approach, the effects of scattering on both internal device characteristics and terminal currents can be examined, which enables our simulator to be used for the exploration of realistic performance limits of SNWTs. I. INTRODUCTION As the channel lengths of metal-oxide-semiconductor field effect transistors (MOSFETs) scale into the nanometer regime, short channel effects 1 become more and more significant. Consequently, effective gate control is required for a nanoscale MOSFET to achieve good device performance. For this reason, silicon nanowires, which allow multi-gate or gate-all-around transistors, are being explored. [2][3][4][5][6][7] In Ref. [2], the authors reported a parallel wire channel transistor, whose channel can be viewed as a wire with a triangular cross-section. In Refs. [3][4][5][6], wires with rectangular cross-sections were used to fabricate different types of tri-gate/gate-allaround FETs. At the same time, cylindrical Si nanowires with diameters as small as 5nm have also been synthesized by the chemical vapor deposition technology. 7 These recent experiments have shed light on the potential applications of silicon nanowire transistors in future electronics. To deeply understand device physics of silicon nanowire transistors (SNWTs) and to assess their ultimate performance limits, simulation work is necessary and important. In contrast to a planar MOSFET, which has a uniform charge and potential profile in the transverse direction (normal to both the gate and the source-to-drain direction), an SNWT has a three-dimensional (3D) distribution of electron density and electrostatic potential. As a result, a 3D simulator is required for the simulation of SNWTs. In this paper, we propose a 3D self-consistent quantum simulation of SNWTs based on the effective-mass approximation (whose validity in the nanoscale device simulation has been established in Ref. [8]). The calculation involves a selfconsistent solution of a 3D Poisson equation and a 3D Schrödinger equation with open boundary conditions. Using the finite element method (FEM), we solve the 3D Poisson equation to obtain the electrostatic potential. At the same time, we solve the 3D Schrödinger by a (coupled/uncoupled) mode space approach. [9][10][11][12] which provides both computational efficiency and high accuracy as compared with direct real space calculations. Since the (coupled/uncoupled) mode space approach treats quantum confinement and transport separately, the procedure of the calculation is as follows: Step 1: Solve the 3D Poisson equation for the electrostatic potential; Step 2: Solve a 2D Schrödinger equation with a closed boundary condition at each slice (cross-section) of the nanowire transistor (see FIG. 1) to obtain the electron subbands (along the nanowire) and eigenfunctions; Step 3: Solve (coupled/uncoupled) 1D transport equations by the nonequilibrium Green's function (NEGF) approach [13][14][15] for the electron charge density; Step 4: Go back to Step 1 to calculate the electrostatic potential. If it converges, then calculate the electron current by the NEGF approach (as in Step 3) and output the results. Otherwise continue Step 2 and 3. Different transport models (in Step 3) are implemented into our simulator. In this paper, we will discuss both ballistic NEGF model, which gives the upper performance limit of SNWTs, and a dissipative NEGF model with a simple treatment of scattering with the Büttiker probes, 14,16,17 which offers an efficient way to capture scattering in the quantum mechanical framework. A rigorous treatment of scattering and a detailed calculation of bandstructures are very important to understand physics in Si nanowires in detail. However, the huge computational cost involved in such a rigorous model can prevent it from being used for extensive device simulation and design. As we will show later, the use of the effective-mass approximation and the simple treatment of scattering with the Büttiker probes greatly reduces the computational complexity while still capturing the essential device physics of SNWTs (i.e., 3D electrostatics, quantum confinement, source-to-tunneling and scattering, etc), so the method we discuss in this paper can be used as a practical 3D quantum approach for device study and design of SNWTs. 18 This paper is divided into the following sections: Sec. II describes our methodology for ballistic SNWTs and provides the basic equations, Sec. III discusses the simulation results for ballistic SNWTs with arbitrary cross-sections (e.g., triangular, rectangular and cylindrical), Sec. IV introduces the Büttiker probes for the treatment of scattering and shows relevant results, and Sec. V summarizes key findings. Figure 1 shows a schematic structure of the Si nanowire transistors simulated in this work. II. THEORY FOR BALLISTIC SILICON NANOWIRE TRANSISTORS This intrinsic device structure is connected to two infinite reservoirs, the source and drain (not shown), so the source/drain (S/D) extension regions are terminated using open boundary conditions. As shown in FIG. 1 (b), a uniform grid with a grid spacing of a is used along the channel (x) direction. In the y-z plane (the cross-section of the SNWT), a 2D finite element mesh with triangular elements is generated by Easymesh-1.4, 19 which allows us to treat nanowires with arbitrary cross-sections (e.g., triangular, rectangular and cylindrical, as shown in FIG. 1 (c)). By doing this, a 3D finite element mesh with prism elements is constructed. When solving the Poisson equation, the 3D Laplacian is directly discretized by the FEM approach. The obtained linear system is solved using a Preconditioned Conjugate Gradient method with Incomplete Cholesky factorization. More details about the numerical techniques can be found in Ref. [9]. As mentioned earlier, we solve the 3D Schrödinger equation by the mode space approach, [9][10][11] which is based on an expansion of the active device Hamiltonian in the subband eigenfunction space. As a result, we need to solve a 2D Schrödinger equation by the FEM at each slice of the SNWT to obtain the subband eigenenergy levels and eigenfunctions (modes). After that, the original 3D device Hamiltonian is transformed into a 1D Hamiltonian in the x direction, which can be used to calculate electron density and current within the NEGF formalism. In this section, we will first give an overview of the coupled mode space (CMS) approach for the SNWT simulation (part A), which is mathematically equivalent to a direct real space solution if adequate modes are included (to be discussed later). 9,10 Then we will introduce the uncoupled mode space (UMS) approach (part B) and a fast uncoupled mode space (FUMS) approach (part C), which are a simplification of the CMS approach to provide high computational efficiency. The simulation results (in Sec. III) illustrate that the UMS and FUMS approaches show excellent agreement with the CMS approach for the SNWT simulation. A. The coupled mode space (CMS) approach In this part of the work, we will briefly review the Coupled Mode Space (CMS) approach and list basic equations for our particular case of interest. In the 3D domain, the full stationery Schrödinger equation is given by where 3D H is the 3D device Hamiltonian. Assuming an ellipsoidal parabolic energy band with a diagonal effective-mass tensor (for the case that the effective-mass tensor includes non-zero offdiagonal elements, please refer to Ref. [12]), 3D H is defined as here * x m , * y m and * z m are the electron effective mass in the x, y, and z directions, respectively, and ( ) , , U x y z is the electron conduction band-edge profile in the active device. We note that the effective mass varies in the y and z directions due to the transition between the Si body and the SiO 2 layer. Now let us expand the 3D electron wavefunction in the subband eigenfunction space, Now we multiply by ( ) , ; m y z x ξ on both sides and do an integral within the y-z plane. According to Eq. (5), we obtain the following 1D coupled Schrödinger equation From the derivation above, it is clear that the CMS formalism (Eqs. (7) and (8) After the device Hamiltonian H is obtained, we can calculate the electron density and current using the NEGF approach. The NEGF approach, a widely used method for the simulation of nanoscale electronic devices, has been discussed in Refs. [13,14]. Here we list the relevant equations for our particular case. The retarded Green's function of the active device is defined as 14 where the device Hamiltonian, H, is defined by Eq. (10), S Σ is the self-energy that accounts for the scattering inside the device (in the ballistic limit, it is equal to zero), 1 Σ ( 2 Σ ) is the selfenergy caused by the coupling between the device and the source (drain) reservoir. If we discretize the equations by the 1D (in the x direction) Finite Element Method where S 0 is a X X N N × The self-energies, 1 Σ and 2 Σ , are defined as 10,15 [ ] [ ] where Then the 1D electron density (in m -1 ) for mode m can be calculated by where f is the Fermi-Dirac statistics function, and S which is determined by the applied bias. The electron density obtained by Eq. (22) is a 1D distribution (along the x direction). To obtain a 3D electron density, we need couple Eq. (22) with the quantum confinement wavefunction for mode m, The total 3D electron density needs to be evaluated by summing the contributions from all the subbands in each conduction band valley. Then this 3D electron density is fed back to the Poisson solver for the self-consistent calculations. Once self-consistency is achieved, the electron current is computed by where the transmission coefficient, T(E), can be evaluated as 14 To obtain the total electron current, we also need to add up current components in all the conduction band valleys. B. The uncoupled mode space (UMS) approach In the simulation of SNWTs, we assume that the shape of the Si body is uniform along the x direction. As a result, the confinement potential profile (in the y-z plane) varies very slowly along the channel direction. For instance, the conduction band-edge ( ) Hamiltonian H becomes a block-diagonal matrix 11 22 Since all the input matrices at the RHS of Eq. (12) are either diagonal or block-diagonal, the where ( ) m G E (m=1,2,…,M) is the Green's function for mode m and is obtained as and Knowing the retarded Green's function, the spectral density functions due to the source/drain contacts for each mode m can be obtained as 14 , (p=1,2,…,N X ). After that, the electron charge density is computed by Eqs. (22) and (23). For the calculation of electron current, the total transmission coefficient can be written as a summation of the Finally, Eq. (38) is inserted into Eq. (24) to compute the electron current for the SNWT. As we will show in Sec. III, for SNWTs, this uncoupled mode space approach shows excellent agreement with the CMS approach while maintaining higher computational efficiency. (The validity of the UMS approach for planar MOSFET simulation has been established by Venugopal et al 11 by doing a careful study of the UMS approach vs. 2D real space approach.) C. A fast uncoupled mode space (FUMS) approach As described earlier, for both CMS and UMS approaches, we need to solve N X 2D Schrödinger equations (see Eq. (4)) in a self-consistent loop to obtain the electron subbands and eigenfunctions. For the device structures simulated in this work, this part of simulation usually takes more than 90% of the computational complexity, which makes parallel programming necessary. To increase the efficiency of our simulator and to make it executable on a single processor, we introduce a fast uncoupled mode space (FUMS) approach, 9.10 Here the average conduction band-edge ( ) , U y z is obtained as where L X is the total length of the simulated SNWT (including the S/D extensions). After III. RESULTS FOR BALLISTIC SILICON NANOWIRE TRANSISTORS In this section, we first verify the validity of the FUMS approach by comparing its results with those obtained by the UMS and CMS approaches. Then we adopt the FUMS as a simulation tool to explore device physics (i.e., both internal characteristics and terminal currents) of ballistic Si nanowire transistors with different types of cross-sections (e.g., triangular, rectangular and cylindrical). A. Benchmarking of the FUMS approach As mentioned in Sec. II, for both CMS and UMS approaches, we need to solve a 2D Schrödinger equation (shown in Eq. (4)) at each slice of the SNWT to obtain the electron subbands and the corresponding eigenfunctions (modes). Figure 2 shows the electron wavefunctions at a slice of the SNWTs with a triangular, rectangular or cylindrical cross-section, respectively. After solving all the N X 2D Schrödinger equations, the electron subband levels are obtained (see FIG. 3, circles). For the FUMS approach, however, only one 2D Schrödinger equation needs to be solved, and the subband profile can then be calculated by Eq. (42). Figure 3 clearly illustrates that this approximation method (solid lines) provides excellent agreement with the rigorous calculation (circles), which shows that the FUMS approach correctly computes the electron subbands in SNWTs. It is clear that all the three approaches are in excellent agreement (<0.5% error), thus indicating that the FUMS approach, which has much higher computational efficiency than CMS and UMS, is an attractive simulation tool for modeling Si nanowire transistors. Although the sample device structure we use in FIG. 3 and FIG. 4 is a cylindrical SNWT, our conclusion is also applicable for SNWTs with arbitrary cross-sections. In the following parts of this work, we will use the FUMS approach to investigate the device physics in various SNWTs. B. Device physics and characteristics The NEGF transport model we use in this work provides an opportunity to illustrate the local density of states (LDOS) of the simulated SNWTs. Figure 5 shows the LDOS together with the electron subbands for a ballistic cylindrical SNWT with 10nm gate length and 3nm Si body thickness. Strong oscillations in the LDOS plot are clearly observed, which is due to the quantum mechanical reflection. To be specific, the states injected from the drain are reflected off the drainto-source barrier at the high drain bias and these reflected states strongly interfere with the injected ones. At the source end, the states injected at energies around the source barrier are also reflected and interfere. It should be noted that the occurrence of quantum inference in ballistic SNWTs relies on the quantum coherence (complete preservation of electron phase information) inside the devices. If scattering (dephasing mechanism) is included, as we will see in Sec. IV, the quantum interference and the oscillations in the LDOS are smeared out. In addition, the presence of states below the first electron subband is also visible in the LDOS plot, which is caused by source-to-drain tunneling. 20 Figure 6 plots the 1D electron density (in m -1 ) profile along the channel of the simulated cylindrical SNWT. It is clearly observed that the oscillations in the LDOS of the device result in an oscillation in the 1D electron density, even at the room temperature and more apparent at low temperature (77K). In general, such an oscillation in the electron density profile occurs in all kinds of transistors with 1D channels (e.g., the carbon nanotube transistor 21 ). It is interesting to mention that there is no evident oscillation in the electron density profile in a planar MOSFET (see FIG. 8 on p. 3736 in Ref. [11]) although its LDOS also bears strong oscillations (see FIG. 4 on p. 3735 in Ref. [17]). The reason is that in a planar MOSFET there is a transverse direction (normal to both the Si/SiO 2 interfaces and the channel direction), in which the electron wavefunction is assumed to be a plane wave, thus resulting in numerous transverse modes in the device. These transverse modes wash out the oscillations in the LDOS and cause a smooth electron density profile. So the oscillation in the electron density profile is a special property of SNWT as compared with planar MOSFETs. Figure 7 illustrates the calculated transmission coefficient (from Eqs. (38) and (39)) for the simulated cylindrical SNWT. When the total electron energy increases above the source end of the first subband, the electrons start to be injected into the channel, so the transmission coefficient begins to increase from zero. As the electron energy continues to go up, the second and third subbands (modes) become conductive successively, which results the step-like shape of the transmission coefficient curve. We also observe that the transmission coefficient is above zero even when the total electron energy is below the top of barrier of the first subband, which is the evidence of source-to-drain tunneling. In FIG. 8, we compare the I DS vs. V GS characteristics for SNWTs with triangular, rectangular and cylindrical cross-sections. Two interesting phenomena are evidently visible: 1) the cylindrical wire (CW) and triangular wire (TW) transistors have higher threshold voltages, V TH (that is defined as I DS (V GS =V TH )=10 -8 A when V DS =0.4V), due to stronger quantum confinement (the cross-section areas of the CW and TW are smaller than that of the RW for the same Si body thickness), and 2) the CW SNWT offers the best subthreshold swing and the highest on-off current ratio (under the same gate overdrive, V GS -V TH ) due to its good gate control. These results clearly show that our simulator correctly treats the 3D electrostatics, quantum confinement and transport in SNWTs with arbitrary cross-sections. IV. TREATMENT OF SCATTERING WITH BÜTTIKER PROBES In this section, we apply a simple quantum treatment of scattering based on the Büttiker probes 14,16,17 to our SNWT simulation. The simulation results show that this simple model captures the essential effects of scattering on both internal device parameters (e.g., charge distribution and electrostatic potential) and current-voltage characteristics. (A detailed treatment of scattering within the NEGF formalism is important to deeply understand physics in Si nanowires, and it will be discussed in future work.) A. Theory The simple treatment of scattering with the Büttiker probes has been adopted by Venugopal and coworkers 17 for the simulation of nanoscale MOSFETs. Due to the similarity between the transport calculations of a MOSFET and an SNWT, here we will follow the basic concepts and formalism of the method described in Ref. [17] while making necessary modifications and corrections for the case of SNWT simulation. In the ballistic regime, as we know, electrons move through the device coherently, with their energies and phase information conserved. When scattering is present, however, electrons' momenta and energies could be altered and their phase information may be lost. Based on this observation, virtual probes (Büttiker probes) are attached to the device lattice (in the channel direction), which serve as reservoirs that absorb electrons from the active device, modulate their momenta and/or energies, and then reinject them back to the device. The difference between the probes and the S/D contacts is that the probes can only change the electron momentum/energy and not the number of electrons within the active device. 17 Figure 9 shows the 1D device lattice (in the channel direction) for an SNWT with the Büttiker probes attached. Each probe is treated as a virtual 1D lattice (in the ' x direction) that is which implies that the scatting in the device is strong. As we will show later, the Büttiker probe strength can be analytically related to the electron mean-free-path, which allows us to calibrate the parameters in our simulation to mimic a low field mobility that can be measured experimentally. 17 It should also be noted that since we treat each probe as a reservoir, a Fermi level ( i µ , 2,..., 1 − ) needs to be assigned to the probe, and the values of these probe Fermi levels have to be adjusted to achieve current continuity (i.e., the net cuurent at each probe is zero). The mathematical formalism used to treatment this physical structure is described in the following paragraphs. As we show in Sec. II, the retarded Green's function for mode m is obtained as where the coupling energy between adjacent lattice nodes (in the x direction) is ( ) and the total self-energy matrix turns to Inserting Eqs. (43) and (47) into Eq. (31), the retarded Green's function m G can be evaluated. Knowing m G , the state spectral function due to injection from the S/D and all probes for mode m is obtained as 17 The local density of states due to injection from reservoir i is then obtained as and the 1D electron density (in m -1 ) for mode m can be calculated by The transmission coefficient between any two reservoirs i and r can be evaluated as The net current density (at energy E) at reservoir i including contributions from all reservoirs (labeled by r), modes (labeled by m) and valleys is and the net current at reservoir i is As mentioned in Ref. [17], while the S/D Fermi levels are determined by the applied voltages, the Fermi levels of the probes have to be adjusted to ensure current continuity, which implies that the net current at each probe must be zero, Inserting Eq. (53) into (55), we obtain Solving this nonlinear equation group (56) by Newton's method, 17 the Fermi levels where λ is the electron mean-free-path, which relates to the low field electron mobility by the following equation for a 1D conductor (the λ~0 µ relation for a 2D conductor is described in Ref. [23]), V. SUMMARY In this paper, we present a computationally efficient three-dimensional quantum simulation of various silicon nanowire transistors based on the effective-mass approximation. The coupled/uncoupled mode space approaches are adopted to decompose the 3D device Hamiltonian, which greatly reduces the simulation time while keeping excellent computational accuracy. The use of a fast uncoupled mode space further scales down the computational complexity and makes our simulator executable on a single processor. This enables our approach to be used as a practical 3D quantum model for extensive device simulation and design. Although we mainly focus on ballistic simulations in this work, a simple treatment of scattering with the Büttiker probes, previously applied to MOSFET simulations, is also implemented in our SNWT simulator. This model is a one input parameter model and the parameter we use can be related to the experimentally measurable low field mobility of electrons. As a result, the implementation of this simple scattering model endows our SNWT simulator with the ability to explore the realistic performance limits of SNWTs. The cross-sections of the simulated triangular wire (TW), rectangular wire (RW) and cylindrical wire (CW) FETs. T Si is the silicon body thickness, W Si is the silicon body width and W Wire is the wire width. For the TW, the direction normal to each gate is <111>, so the channel is <101> oriented. In contrast, for the channel of the RW, both <101> and <100> orientations are possible. For the CW, we assume the channel to be <100> oriented. . 1 (c)), is 3nm, and the oxide thickness is 1nm. The source/drain (S/D) doping concentration is 2×10 20 cm -3 and the channel is undoped. The solid lines are for the approximation method (solving a 2D Schrödinger equation only once) used in the FUMS approach, while the circles are for the rigorous calculation (solving 2D Schrödinger equations N X times) adopted in the UMS and CMS approaches.	2018-10-31T23:30:47.513Z	2004-03-30T00:00:00.000	{ "year": 2004, "sha1": "75028935242ad23f5d5fff41f043a7c9dea22a8b", "oa_license": null, "oa_url": "https://docs.lib.purdue.edu/cgi/viewcontent.cgi?article=1113&context=ecepubs", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "c5bcc28d3a083b4704694f9db5c7d896555a5fe4", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
216294163	pes2o/s2orc	v3-fos-license	Postmarketing surveillance on the clinical use of edoxaban in patients with nonvalvular atrial fibrillation (ETNA‐AF‐Japan): One‐year safety and effectiveness analyses Abstract Background The safety and effectiveness of edoxaban in real‐world clinical settings have not yet been elucidated thoroughly among Japanese patients with nonvalvular atrial fibrillation (NVAF). We report the one‐year interim results of ETNA‐AF‐Japan (Edoxaban Treatment in routiNe clinical prActice in patients with nonvalvular Atrial Fibrillation: UMIN000017011), an ongoing two‐year study. Methods ETNA‐AF‐Japan is a prospective, real‐world multicenter observational study that analyzes the long‐term safety and effectiveness of edoxaban. Physicians recorded clinical characteristics, bleeding events, and clinical events of ischemic stroke and systemic embolism, among others. Results In total, 11 569 patients with NVAF were enrolled. The demographic and clinical characteristics of the patients in the safety analysis set (n = 11 107) were a mean age of 74.2 ± 10.0 years; female sex, 40.6%; age ≥75 years, 52.4%; body weight ≤60 kg, 54.3%; creatinine clearance ≤50 mL/min, 31.2%; mean CHADS2 score of 2.2 ± 1.3. The mean treatment period was 311.2 days (median; 366.0 days), and ~80% of patients continued edoxaban treatment. In the safety analysis, the incidence of all bleeding events was 6.32% [95% CI: 5.87, 6.79] (n = 702), and for major bleeding, it was 1.08% [0.90, 1.29] (n = 120). In the effectiveness analysis set (n = 11 059), the incidence of ischemic stroke (excluding TIA) or systemic embolism was 1.10% [0.92, 1.32] (n = 122). Conclusions At one‐year follow‐up, the results showed no major concerns about the safety and effectiveness of edoxaban in Japanese patients with NVAF in a real‐world clinical setting. \| INTRODUC TI ON In patients with nonvalvular atrial fibrillation (NVAF), treatment with a direct oral anticoagulant (DOAC) [1][2][3][4] for ischemic stroke prevention is now preferred over warfarin and especially considered for use in patients who are planning to start anticoagulant therapy as described in the "2013 Guidelines for Pharmacotherapy of Atrial Fibrillation" (JCS 2013) of the Japanese Circulation Society 5,6 and the 2018 European Heart Rhythm Association Practical Guide. 7 However, the guides have pointed out that patients with atrial fibrillation (AF) typically have concomitant diseases and other risk factors, and their long-term, day-to-day experience with the treatment warrants further investigation. A study of the long-term use of a DOAC in a large sample-size (≥10 000 patients) that analyzes the reduction in risk of ischemic stroke and systemic embolism and the occurrence of bleeding episodes would help physicians understand the most appropriate way to treat patients, particularly elderly patients. This is critical in Japan where ≥25% of the population is ≥65 years old (2015 census), and those with AF are a little over three-quarters of a million. 8,9 Edoxaban is a once-daily DOAC that directly and reversibly inhibits factor Xa and is indicated for long-term use in patients with NVAF to prevent ischemic stroke and systemic embolism. [10][11][12][13] Edoxaban is available in two formulations: tablet and orally disintegrating (OD). Swallowing is a concern for elderly patients, and so the OD formulation is particularly useful because it helps them take the drug daily and consistently in the long-term. Edoxaban has two more indications: treatment and prevention of recurrence of venous thromboembolism, and prevention of postoperative venous thromboembolism after lower extremity orthopedic surgery. The efficacy and safety of edoxaban were confirmed in phase-3 ENGAGE AF-TIMI-48; 1 because it was a pivotal confirmatory study and designed as a randomized controlled trial (RCT), the patient population had strict inclusion and exclusion criteria. Since these studies do not include all patients who would potentially benefit from taking the drug, it is necessary to investigate the safety and effectiveness of edoxaban in a real-world clinical setting. ETNA-AF-Japan (UMIN000017011) was initiated to collect such data over a two-year period. We have published three-month interim analysis results in September 2018 14 that reported on patient demographics, clinical characteristics, and dosing status. Here, we report on the one-year interim analysis of data that also includes safety and effectiveness analyses of edoxaban. Furthermore, we analyzed the safety and effectiveness of treatment in patients with a specific background, such as those ≥75 years old, including patients whose body weight is ≤60 kg and who have other factors. \| Study design ETNA-AF-Japan is a real-world, prospective, multicenter observational study that aims to collect the baseline and clinical characteristics of Japanese patients with NVAF and analyze the safety and effectiveness of edoxaban in these patients. This postmarketing surveillance (PMS) was conducted according to the "Good Post-marketing Study Practice of the Ministry of Health, Labor, and Welfare of Japan." Detailed methods of this study were published in the three-month report. 14 \| Patient population Eligible patients were adults with NVAF who were to receive edoxaban for the first time to prevent ischemic stroke and systemic embolism. Further requirements for enrollment were the ability to start treatment during the enrollment period, availability for long-term follow-up, and agreement to provide written informed consent to participate at the time of registration. \| Starting daily dose and administration The recommended daily oral dose, as described in the package insert (PI) in Japan, was 60 mg for adult patients weighing >60 kg and 30 mg for those weighing ≤60. 11 The 30 mg dose was also recommended for patients with one or more of the following: renal dysfunction (creatinine clearance, CLcr ≤50 mL/min), and concomitant use of the following P-glycoprotein (P-gp) inhibitors: quinidine, verapamil, erythromycin, or cyclosporine. \| Statistical analysis For categorical variables, proportions were calculated. For continuous variables, summary statistics (mean, SD) were calculated. To determine the one-year outcomes, we analyzed data up to the 390day follow-up period for each patient. The Kaplan-Meier Plots were presented for bleeding events and clinical events, respectively. All statistical analyses were performed using SAS System Release 9.2 (SAS Institute Inc). \| Disposition of patients In total, 11 569 patients with NVAF were enrolled. At one-year, we had collected case report forms for 11 469 patients and ex- \| Starting daily dose and adjustment factors The starting daily dose of edoxaban prescribed to patients in the safety analysis set was 60 mg in 27.6% and 30 mg in 72.4% ( Note: Data are presented as number (%) unless otherwise indicated. Abbreviation: SD, standard deviation. a Neither labile international normalized ratio nor alcohol use were counted; thus, the highest total score was seven. for dose adjustment factors were excluded. Thus, background information for 10 877 patients was used to determine how edoxaban was used, as described in the PI (Figure 1 \| Edoxaban treatment status The mean and median duration of edoxaban treatment during the and plan to receive an invasive procedure (n = 25, 0.2%). \| Safety analyses The cumulative incidence rate of bleeding events has increased over the year ( Figure 2). Figure 3A shows the incidence of bleeding events during the edoxaban treatment period (details in Supplement 3). In the safety analysis set (n = 11 107), the incidence of all bleeding events was 6.32% (n = 702); major bleeding was 1.08% (n = 120), and CRNMB was 2.81% (n = 312). Gastrointestinal bleeding was the P-gp inhibitors c) Others most common type (Supplement 3). The incidence of all bleeding events was 6.26% for the 60 mg dose group and 6.34% for 30 mg; for major bleeding, these were 0.72% and 1.22%. Figure 3B shows the incidence of major bleeding events categorized by HAS-BLED scores. Patients with scores of 2, 3, or 4 had an incidence of 1.30%, and those with scores of 0 or 1 had an incidence of 0.47%. Patients with a score of ≥5 had an incidence of 7.55% for major bleeding. \| Effectiveness analyses The cumulative incidence rate of clinical events has increased over the year ( Figure 2). Figure 4A summarizes the incidence of clinical events during the edoxaban treatment period. In the effectiveness analysis set (n = 11 059), the incidence of thromboembolic events (ischemic stroke [excluding TIA] or systemic embolism) was 1.10% (n = 122): 1.05% (n = 32) at the 60 mg dose and 1.12% (n = 90) at the 30 mg dose. The incidence of thromboembolic events categorized by CHADS 2 and CHA 2 DS 2 -VASc risk scores are shown in Figure 4B,C, respectively. For CHADS 2 , the incidence of thromboembolic events in patients with lower risk scores (0 and 1) was 0.64%, and was 1.35% for those with higher risk scores (≥2). For CHA 2 DS 2 -VASc, the incidence of events was 0.72% for patients with lower risk scores (0-3) and was 1.53% for patients with higher risk scores (≥4). Table 4 shows the safety and effectiveness of recommended doses of edoxaban treatment in patients with a specific background. Patient groups whose incidence of major bleeding was more than double that of all patients (1.13%) were the dual antiplatelet therapy (DAPT) use group (4.35%) and severely low body weight (<40 kg) group (3.00%). Moreover, patient groups whose incidence of thromboembolic events was more than double that of all patients (1.19%) were the DAPT use group (4.97%), cerebrovascular disease group (2.74%), and antiplatelet agent use group (2.56%). \| D ISCUSS I ON ETNA-AF-Japan is an observational study of over 10 000 NVAF patients treated with edoxaban. ETNA-AF-Japan, a real-world study in contrast to an RCT such as ENGAGE AF-TIMI 48, includes various types of patients with multiple risk factors. Therefore, the study could provide information to aid in the physicians' understanding of bleeding risks and the clinical benefits of anticoagulation with edoxaban. 19 We report here the one-year follow-up on the safety and effectiveness of edoxaban. \| Baseline demographics and clinical characteristics As reported previously, 14 patient backgrounds in this study were somewhat different from those in the phase-3 study (ENGAGE AF-TIMI 48). 1 ETNA-AF-Japan included patients with CHADS 2 score ≤1, severe renal dysfunction (CLcr <30 mL/min) and DAPT use, which were excluded from ENGAGE AF-TIMI 48. 1 Patient backgrounds such as age, body weight, and risk scores in terms of ETNA-AF-Japan were similar to those in the real-world SAKURA AF Registry, 20,21 Fushimi AF Registry, 22 and other DOAC observational studies, [23][24][25][26][27] suggesting that ETNA-AF-Japan reflected a real-world population of Japanese patients with NVAF. \| Dose levels and adjustment factors The lower dose of 30 mg edoxaban was administered to approxi- \| Clinical outcomes (safety and effectiveness) At one-year follow-up, the incidence of all bleeding events was 6.32%, and was 1.08% for major bleeding. Although a direct comparison is inappropriate, the incidence of major bleeding was lower in the present study than in ENGAGE AF-TIMI 48 overall data (2.75% of patients/year) 1 and in ENGAGE AF-TIMI 48 Japanese patient data (3.38% of patients/year). 31 Major bleeding events were mostly gastrointestinal bleeding, which was similar to that of ENGAGE AF-TIMI 48. In the SAKURA AF Registry 20 and Fushimi AF Registry, 22 the incidence rate of major bleeding was 1.21% and 1.8% of patients/ year, respectively. Although these observational studies differ from ETNA-AF-Japan in terms of the follow-up period and drugs prescribed, the incidence of major bleeding in ETNA-AF-Japan (1.08%) was not higher than those in other registries. At one-year followup, the incidence of clinical events was 1. \| Safety and effectiveness in the 60 mg and 30 mg groups The mean HAS-BLED, CHADS 2 , and CHA 2 DS 2 -VASc score in the 30 mg group (2.1 ± 0.9, 2.3 ± 1.4 and 3.8 ± 1.6) were higher than those of the 60 mg group (1.7 ± 1.0, 1.8 ± 1.3 and 2.7 ± 1.5). These results suggest that the lower dose (30 mg) was given not only to patients at high risk of bleeding but also to patients at high risk of stroke. Thus, the safety and effectiveness results for the 30 mg group are particularly important. The incidence of all bleeding in the 30 mg (6.34%) and 60 mg (6.26%) groups were similar, and the incidence of major bleeding in the 30 mg (1.22%) was higher than 60 mg (0.72%) group. However, the incidence of major bleeding in the 30 mg group (1.22%) was not higher than that in other real-world studies (SAKURA AF Registry, 1.21% of patients/ year; Fushimi AF Registry, 1.8% of patients/year). The incidence of thromboembolic events was similar in the 30 mg (1.12%) and 60 mg (1.05%) groups. Therefore, these preliminary, one-year results suggested that there are no major concerns about the safety and effectiveness of edoxaban at the lower dose in a real-world clinical setting. \| Safety and effectiveness outcomes in patients with a specific background Patients with a specific background (eg, low body weight, patients provide information about the safety and effectiveness profile of these patients, which may be helpful for physicians in clinical practice. The incidence of major bleeding was more than double in patients who had factors of DAPT use and severely low body weight than in all patients in the study. In the phase 3 study, patients using DAPT were excluded, and information on low-weight patients weighing <40 kg was limited. However, the results of this study point to a higher incidence of major bleeding in these patient groups. The factors for which the incidence of thromboembolic events was more than double that of all patients in the study were history of cerebrovascular disease, antiplatelet agent use, and DAPT use. Cerebrovascular disease includes a history of ischemic stroke, which had a high probability of recurrence. Moreover since an antiplatelet agent or DAPT is administered to patients who are at a high risk of a thromboembolic event, it is expected that incidence rates for these patients would be high. Therefore, it becomes necessary to monitor patients carefully for bleeding or thromboembolic events in the presence of the following factors: severely low body weight, cerebrovascular disease, antiplatelet agent use, or DAPT use. \| Limitations First, the present study was an open-label observational study with no comparative arm. Therefore, it is not possible to compare edoxaban ACK N OWLED G EM ENTS The authors express their deepest gratitude to the investigators who conducted this survey and provided valuable data. This study was funded by Daiichi Sankyo Co. Ltd. Writing and editorial assistance was provided by Kazuhiro Kanmuri PhD, Inter-Professional Inc.	2020-03-26T10:33:02.245Z	2020-03-24T00:00:00.000	{ "year": 2020, "sha1": "0368c5e69dce8b8ecebb9c10be6434314325bad5", "oa_license": "CCBY", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/joa3.12332", "oa_status": "GOLD", "pdf_src": "Wiley", "pdf_hash": "35cd0f379858b1c9f022f14a7c6b4175b10e232e", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
62836278	pes2o/s2orc	v3-fos-license	Demographic Characteristics and Etiology of Hydrocephalus Patients attended at Tertiary Care Hospital in Bangladesh Background: Hydrocephalous can occur at any age. Objectives: The purpose of the present study was to see the Demographic Characteristics and Etiology of Hydrocephalus Patients. Methodology: This cross sectional study was conducted in the Department of Neurosurgery at Bangabandhu Sheikh Mujib Medical University, Dhaka from April 2009 to September 2010 for a period of one and half year. Patients presented with obstructive hydrocephalus at any age with both sexes were included as study population. Detailed socio-demographic history as well as the associated etiological factors was recorded in pre designed data collection sheet. Result: A total number of 60 obstructive hydrocephalous patients were recruited for this study after fulfilling the inclusion and exclusion criteria. The mean age with SD was 17.95±19.15. Acquedctal stenosis was the most common etiology of hydrocephalous which was 31(51.7%) cases followed by posterior fossa midline tumour, CPA tumour and pineal region tumour which were 14 (23.3%) cases, 9(15.0%) cases and 6(10.0%) cases respectively. Conclusion: Obstructive hydrocephalous is most commonly found in younger age group which is caused by acquedctal stenosis. [Journal of National Institute of Neurosciences Bangladesh, 2015;1(2): 47-49] Introduction Hydrocephalus is an abnormal enlargement of the ventricles due to an excessive accumulation of CSF resulting from a disturbance of its flow, absorption or, uncommonly, secretion 1 .The incidence of hydrocephalus in the new born varies from 0.2-0.5 per 1000 birth 2 .Little is known about the factors influencing the development of hydrocephalus.Stoll et al 3 studied environmental and genetic factors in congenital hydrocephalus (CH) in 96 children.It has been reported 33 teratogenic agents including physical injury, irradiation, nutritional deficiency, nutrition excess and various chemicals and drugs which produce hydrocephalus 4 .It was found that there was a positive correlation between the higher dose of radiation and production of hydrocephalus in children conceived during the period of radiation fall out 5 .Experimental observations have shown that intrauterine viral infection may also cause hydrocephalus 2 .Obstructive hydrocephalus is one of the common conditions in neurology hospital which requires surgery.Therefore it is important to know the etiological factors as well as the demographics characteristics of obstructive hydrocephalous patients. Methodology This was a cross sectional study which was conducted the Department of Neurosurgery at Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh from April 2009 to September 2010 for a period of one and half year.All patients presenting with obstructive hydrocephalus and age more than 6 months were included as study population.Communicating hydrocephalus, previously shunted patient, abnormal ventricular anatomy in imaging were excluded from this study.Detailed information regarding severe systemic diseases like DM, HTN, age less <6 months, post menifgngitic hydrocephalus were recorded.On admission, a detailed history was taken from the patient or from patient's attendants.Thorough general and neurological examinations were carried out by the researcher himself.Images findings (CT scan and MRI) were used to confirm hydrocephalus.Prior to commencement of this study, the research protocol was approved by the ethical review committee of BSMMU.All the data were checked and edited after collection.Then the collected data were analyzed by SPSS 16 th version (statistical package for social science) computer software program.Percentage was calculated to determine the proportion of the findings.Results were presented in tabulated form.Statistical significance was set at p<0.05 Results A total number of 60 patients were recruited for this study after fulfilling the inclusion and exclusion criteria.Majority were in the age group of 0.6 to 10 years of age group which was 32(53.3%)cases followed by 11 to 20 years, 41 to 50 years and 31 to 40 years which were 9(15.0%)cases, 6(10.0%) and 5(8.3%) cases respectively.Minimum age of hydrocephalous was 6 months.Maximum age of hydrocephalous was 70 years.The mean age with SD was 17.95±19.15(Table 1). Out of the sixty patients male was 35(58.3%)cases and female was 25(41.7%)cases.The ratio of male and female was 1.4:1(Table 2). Discussion Hydrocephalus can occur at any age, but it is most common in infants and young children or in adults over the age of 60.The present study consists of 60 patients.Age range was 6 months to 70 years.According to the National Institute of Neurological Disorders and Stroke, hydrocephalus affects approximately one in every 500 children.The study of Koch and Wegmer 4 shows that obstructive hydrocephalous occur more frequently in the first decade of life which is consistent with the present Conclusion In conclusion obstructive hydrocephalous is most commonly found in younger age group.In the majority of cases acquedctal stenosis is the most frequently detected etiology of obstructive hydrocephalous.Posterior fossa midline tumour is the 2nd most common cause of obstructive hydrocephalous. Table 1 : Distribution of the Patients according to Age Table 2 : Distribution of the Patients according to Sex Table 3 : 9istribution of the Patients according to the Etiology of Hydrocephalus (n=60)Demographic Characteristics and Etiology of Hydrocephalus Patientsstudy result.There is a clear impact of age on obstructive hydrocephalous occurrence rate.In this study, out of 60 patients 35 (58.36%) were male and 25 (41.7%) were female.In a study it has reported that out of 19 patients' male predominance found6.The causes of hydrocephalus are still not well understood.Hydrocephalus may result from inherited genetic abnormalities such as the genetic defect that causes aqueductal stenosis or developmental disorders such as those associated with neural tube defects including spina bifida and encephalocele7.Other possible causes include complications of premature birth such as intraventricular hemorrhage, diseases such as meningitis, tumors, traumatic head injury, or subarachnoid hemorrhage, which block the exit of CSF from the ventricles to the cisterns or eliminate the passageway for CSF within the cisterns8.Obstructive hydrocephalus can be a congenital condition and this is in similar findings of the present study where acquiductal stenosis is the most common cause of obstructive hydrocephalous.In these cases, it typically results from a genetic disorder such as spina bifida or as a complication of premature birth with brain hemorrhage.In other cases, the hydrocephalus is an acquired condition that develops later in life due to a brain tumor or cyst, head injury, or an infection such as meningitis.In another study Gangemis et al9presented 140 patients with obstructive hydrocephalus and have reported that the common etiology of hydrocephalus was acquiductal stenosis in 95 cases and compression by tumour in 45 cases.In present study, the etiology of hydrocephalus is acquiductal stenosis in 31 cases and compression by tumour in 29 cases which is consistent with the previous study.	2018-12-22T21:23:53.946Z	2017-04-06T00:00:00.000	{ "year": 2017, "sha1": "6e684460493499cf72cc2de17af1929d73c3bf7b", "oa_license": "CCBYNC", "oa_url": "https://www.banglajol.info/index.php/JNINB/article/download/29850/20013", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "6e684460493499cf72cc2de17af1929d73c3bf7b", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
8621156	pes2o/s2orc	v3-fos-license	A Novel Technique for Post-Prostatectomy Catheter Traction Background Prostate traction is one way to control post-prostatectomy bleeding. The most popular method involves traction with a catheter fixed to the thigh with adhesive bands. However, this method has its own drawbacks. Objectives We aimed to simplify this traction procedure and to overcome its disadvantages. Patients and Methods From 2007 - 2015, a new method was used to control post-prostatectomy bleeding in 152 patients. This technique involved inducing pressure on the prostate neck with an indwelling catheter attached to a partially filled urine bag to control bleeding after a prostatectomy. Results The new method effectively controlled post-prostatectomy bleeding. A few patients required surgical intervention. Conclusions Post-prostatectomy catheter traction using a semi-filled urine bag was an acceptable alternative to the standard method to control post-operative bleeding. Background Although several minimally invasive methods of prostatectomy have been introduced (1,2), open prostatectomy and trans-urethral resection of the prostate (TURP) have not been completely abandoned (3,4). However, these two old but highly effective methods have an important disadvantage. Incomplete hemostasis during the operation makes post-surgical bleeding one of the major drawbacks of these older methods. In comparison, the new methods like laser prostatectomy or bipolar-TURP involve great care to prevent this complication (5,6). Inducing pressure on the prostate neck using an indwelling catheter for traction is one of the most important techniques to control postprostatectomy bleeding (7). Placing an adhesive band between the catheter and the thigh to affix the catheter to the leg ( Figure 1) is the most popular method for prostate traction (7). This paper presents a new and simple procedure for prostate traction after TURP or open prostatectomy. Objectives Catheter traction using adhesive bands attached to the thigh is associated with drawbacks, which we hoped to improve by describing this new method. hours postoperatively, 163 patients required prostate traction based on the severity of the bleeding. All these patients (open prostatectomy or TURP) received a three-way 22 or 24 French prostate irrigation catheter. A closed urine bag was hung from the end of the bed, which permitted the irrigation fluid to collect in the bag ( Figure 2). The weight of the irrigation fluid in the urine bag produces direct pressure on the bladder neck, and this pressure is gradually increased. Whenever the irrigation color changes favorably to clear, the urine bag is incised at the fluid level in the urine bag. After the incision, additional irrigation is permitted to drain from the incision line, and the semi-filled urine bag maintains adequate pressure on the bladder neck to control the bleeding. Results The mean age of patients was 65 years old. The average weight of the prostate (in both TURP and open prostatectomy procedures as measured by pathology) was 38 ± 12 grams. Patients experienced a mean hemoglobin decrease (hemoglobin on the day prior to surgery minus the hemoglobin on the first post-operative day) of 4 ± 3 gram per dl. With this method, only 11 patients undergoing TURP needed second look coagulation procedures, which were all carried out during the same session as the primary intervention using the same spinal anesthesia. Only one open prostatectomy case had to be reopened the next morning, and this patient was found to have high-grade prostate cancer upon permanent pathology. The other 151 patients who received this type of traction soon transitioned to a favorable irrigation color. A blood transfusion was administered to 92 patients to compensate for chronic blood loss after the traction was removed near the time of discharge. Discussion Because of the nature of open prostatectomy and TURP, complete hemostasis intraoperatively is impossible (6), which has increased the value of nursing after prostatectomy to an even higher level than that for the operation itself (4). In order to prevent intravesical clot retention resulting from incomplete hemostasis, continuous bladder irrigation for one or two days after surgery is recommended (2). However, irrigation prevents clot production and therefore has minimal effects on controlling bleeding. For a long time, urethral catheter traction has been accepted as the most effective way to control this postoperative bleeding (7). The application of pressure over the bladder neck caused by the traction of the overinflated catheter balloon occludes the veins and arteries that enter the prostate capsules through the vesicoprostatic junction. In addition to this mechanism, the obstructed vesicoprostatic junction also does not permit blood to enter the bladder and instead compresses it within the prostate capsule space, which aids in achieving hemostasis. Today, the most effective way to elicit pressure on the prostate capsule is catheter traction with the fixation of a urethral catheter to the thigh or, less commonly, to the abdomen (7) (Figure 1). However, there are some disadvantages of fixing it to the thigh: 1) Patients must keep their lower extremities immobile because the efficacy of the traction decreases with any type of leg mobility and knee bending. 2) Immobility of the lower extremities, especially in aged patients and those who have previously undergone pelvic surgery, is a risk factor for deep vein thrombosis (DVT), a lethal complication (8). 3) Any knee movements alter the amount of pressure elicited by this method. 4) Sweating and leakage around the urethral catheter from the urethral meatus allows the adhesive bands to become wet, and they gradually loosen over time, making the traction less effective. In contrast, with this new and simple method, the patient can readily move his legs, including lower extremity physiotherapy to prevent DVT, without interfering with traction (9). The pressure is also completely adjustable from patient to patient according to the amount of fluid Two potential limitations of this method are the possibility of kinking and occlusion of the line connected to the urine bag at the point where the line hangs over the bed and separation of the catheter from the urine bag due to the pressure. These two factors are dependent on the quality of the medical products. In our experience, a volume of up to 500 cc in the urine bag is not likely to cause these problems. In short, traction using the weight of a semi-filled urine bag to control bleeding after open prostatectomy or TURP is effective, easy to set up, and time saving.	2018-04-03T01:09:49.815Z	2016-06-07T00:00:00.000	{ "year": 2016, "sha1": "3d9dcfae862892b61dd71275805f3a665002ed9f", "oa_license": "CCBYNC", "oa_url": "https://europepmc.org/articles/pmc5039959?pdf=render", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "3d9dcfae862892b61dd71275805f3a665002ed9f", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
261243495	pes2o/s2orc	v3-fos-license	Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema Chronic inhalation of cigarette smoke is the major cause of sterile inflammation and pulmonary emphysema. The effect of carbon black (CB), a universal constituent of smoke derived from the incomplete combustion of organic material, in smokers and non-smokers is less known. In this study, we show that insoluble nanoparticulate carbon black (nCB) accumulates in human myeloid dendritic cells (mDCs) from emphysematous lung and in CD11c+ lung antigen presenting cells (APC) of mice exposed to smoke. Likewise, nCB intranasal administration induced emphysema in mouse lungs. Delivered by smoking or intranasally, nCB persisted indefinitely in mouse lung, activated lung APCs, and promoted T helper 17 cell differentiation through double-stranded DNA break (DSB) and ASC-mediated inflammasome assembly in phagocytes. Increasing the polarity or size of CB mitigated many adverse effects. Thus, nCB causes sterile inflammation, DSB, and emphysema and explains adverse health outcomes seen in smokers while implicating the dangers of nCB exposure in non-smokers. DOI: http://dx.doi.org/10.7554/eLife.09623.001 Introduction Tobacco smoking is linked to a long and growing (Barnes, 2014;Carter et al., 2015) list of fatal illnesses (e.g., emphysema, cancer, and stroke) and is the major preventable cause of human death. Despite public awareness of the harmful effects of smoking, in many large developing countries the prevalence of smoking is growing (Eriksen et al., 2014). Compounding this risk is particulate air pollution due to the combustion of organic materials including biomass fuels, slash and burn agriculture, and coal (Furlaneto et al., 1969;Arif et al., 1993;Dadvand et al., 2014). While our understanding of the immune basis of smoke-induced sterile inflammation has increased, the molecular mechanism underlying emphysema and its persistence despite smoking cessation remains unclear (Cosio et al., 2009;Kheradmand et al., 2012). Even less is known regarding the healthrelated inhalation effects of atmospheric and workplace airborne carbon particulates. Innate immune cells such as alveolar macrophages and neutrophils are recruited to the lungs in response to cigarette smoke (Salvi, 2014). Several human studies and pre-clinical models of smoke-induced emphysema have also confirmed that lymphocytes (T and B cells) and lung antigen presenting cells (APCs) play pathogenic roles in chronic lung inflammation in smokers (Shan et al., , 2014Churg et al., 2012b). Prior work has shown that increased concentrations of pro-inflammatory cytokines such as IL-6, IL-1β, and IL-17A are among the most important hallmarks of smoke-mediated lung inflammation (Shan et al., , 2014Chang et al., 2014). We and others have previously shown that IL-17A plays a critical role in smoke-induced emphysema in humans and in mouse models of disease Kurimoto et al., 2013;Zhang et al., 2013;Chang et al., 2014). Further, adoptive transfer of lineage-negative CD11c + myeloid dendritic cells (mDCs) isolated from the lungs of smoke-exposed mice to naive mice recapitulates smoke-induced lung disease, indicating a direct causal relationship between mDC and emphysema . Despite these advances, the mechanism by which smoke induces the inflammatory mDC phenotype remains completely unknown. Tobacco smoke contains many noxious chemicals (e.g., carbon monoxide, sulfur, nitrogen dioxide, nitric oxide, and methane), aromatics (e.g., benzene, toluene, and xylene) and chlorinated (e.g., methyl chloride, chloroethene, and chloroform) volatile organic compounds, as well as particulate matter (Wang et al., 2012;Perfetti and Rodgman, 2013;Salvi, 2014). One or more of these agents is thought to underlie the carcinogenic potential of smoke, involving at least eight different cancers; accordingly, the role of volatile carcinogens found in smoke has been studied extensively (Pope et al., 2011;Carter et al., 2015). Far less is known about the pathogenic effects of particulate matter that is suspended in smoke and which includes nanoparticulate carbon, metal oxides, and inorganic salts. Histopathological analysis of the lungs of heavy smokers invariably reveals dark-staining anthracotic pigment often attributed to poorly soluble material found in tobacco smoke (Mitchev et al., 2002). Anthracotic pigment is also found in the lymph nodes of smokers (Churg et al., 2005), but its chemical composition and potential contribution to smokingrelated diseases have not been explored. In this study, we show that the anthracotic material found in the lung of human smokers is nanoparticulate carbon black (nCB), a hydrophobic material that accumulates specifically in highly activated CD1a + lung APCs. Raman spectroscopy, hyperspectral imaging, and high-resolution transmission electron microscopy (HR-TEM) were used to confirm this observation and show that nCB further accumulates in the lung and APC of mice exposed to smoke. Moreover, we show that nCB eLife digest Smoking for many years damages the lungs and leads to a disease called emphysema that makes it difficult to breathe and is often deadly. There are thousands of chemicals in cigarette smoke and many of them have been linked to the development of lung cancer, although it has been difficult to pinpoint those that are responsible for smoking-related emphysema. Moreover, cigarette smoke also contains large numbers of small particles and relatively little is known about the role played by these particles in smoking-related disease. One of the hallmarks of long-term smoking is a blackening of the lung tissue that persists even if someone stops smoking. Previously, little was known about the composition of the substance that causes this blackening, or its significance in the development of emphysema. Now, by studying lung tissue taken from smokers with emphysema, You et al. have shown that this black substance is made of nano-sized particles of a material called carbon black (which is also known as elemental carbon). These nanoparticles are produced by the incomplete combustion of the cigarettes. You et al. also confirmed that nanoparticles of carbon black can cause emphysema in mice. Closer examination of the lung damage caused by the nanoparticles revealed that they trigger breakages in DNA, which leads to inflammation of the lung. And because the nanoparticles cannot be cleared, they are released into the lung when cells die, which perpetuates lung inflammation and damage. You et al. then went on to show that nanoparticles of carbon black can be modified in a way that allows them to be cleared from the lungs. Such modifications could potentially protect people who are exposed to carbon black nanoparticles in the environment or in workplaces where carbon black is used, such as factories that produce automobile tires and other rubber products. given by inhalation to mice in amounts that are comparable to human exposures is alone sufficient to cause sterile inflammation and induce robust T helper 17 cell (Th17) responses and emphysema, implicating the potential health risks of airborne nCB that contaminates a wide range of workplace and domestic environments (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2010). Detection of nCB in lungs of smokers In contrast to the white or pink appearance of normal lungs, the lungs of heavy smokers are typically dark brown or black (Churg et al., 2005). During the routine preparation of lung phagocytic cells including CD1a + mDCs and APCs, we observed the same anthracotic pigmentation in lung cells from smokers ( Figure 1A,B), whereas the same cells isolated from non-smokers lacked the pigment. We previously showed that activated mDCs from smokers and smoke-exposed mice promote Th1 and Th17 responses . To determine the nature of the anthracotic pigment from mDCs, we first performed HR-TEM of the residual black material after complete proteolytic digestion of human emphysematous lung and observed the aggregates that were composed of 20-50 nm spheroids ( Figure 1C). To further extend these observations, we examined CD1a + mDCs from human emphysematous lung tissue using Raman spectroscopy and hyperspectral mapping which showed the signature for pure nCB and not inorganic salts or metal oxides ( Figure 1D-H). Thus, nCB is extensively deposited as solid material in the lungs of smokers and specifically accumulates in lung APC. Next, using our established mouse model of cigarette smoke-induced emphysema (Shan et al., , 2014), we exposed mice to 4 cigarettes daily or air for 4 months to examine whether nCB accumulates in lung APCs. When compared to air-exposed mice, we confirmed that isolated CD11c + APCs and cells present in bronchoalveolar (BAL) fluid (macrophages >90%) contain particulate matter with the Raman spectral signature of nCB within each cell ( Figure 1I). These findings indicate that both humans and mice chronically exposed to cigarette smoke accumulate nCB within phagocytic lung APCs. nCB induces emphysema in mice We have previously recapitulated smoke-induced lung sterile inflammation and emphysema by adoptively transferring lineage-negative CD11c + mDCs isolated from the lungs of smoke-exposed mice to naive mice, which revealed the direct, causal role of mDCs in emphysema . As we found that these mDCs contained nCB, we sought to determine if nCB was alone sufficient to induce emphysema. We first determined that the commercial nCB does not desorb polycyclic aromatic hydrocarbons (PAHs), as determined by Soxhlet extraction followed by gas chromatography mass spectroscopy (GCMS) (Harwood and Moody, 1989). Mice were then exposed twice weekly for 6 weeks to hydrocarbon free, hydrophobic, neutral surface charged nCB (average particle size 15 nm), to achieve a total lung dose of ∼1% of wet lung weight (mg/g), which approximates human lung nCB burdens (Figure 2-figure supplement 1). 4 weeks after the last intranasal instillation of nCB, harvested lungs were extensively anthracotic, similar in appearance to lungs of long-term smokers ( Figure 2A). Physiologically, the nCB challenge-induced enlargement of the alveolar spaces ( Figure 2B) concomitant with significant increases in total lung volume quantified by micro-CT imaging and unbiased lung morphometry measurement (mean linear intercept; MLI) ( Figure 2C,D), both hallmarks of emphysema. Mice exposed to nCB showed significantly increased numbers of macrophages, neutrophils, and lymphocytes in BAL fluid as compared to vehicle (PBS)-challenged control animals ( Figure 2E). Consistently, increased lung inflammation was accompanied by higher concentrations of inflammatory cytokines and chemokines (Figure 2-figure supplement 2) as well as elastolytic matrix metalloproteinases (MMPs) 9 and 12 ( Figure 2F), all of which are characteristic features of cigarette smoke-induced emphysema in human patients and animal models of this disease Churg et al., 2012a). Similarly, both lung parenchymal CD11c + mDCs and BAL fluid macrophages showed an accumulation of nCB as detected by hyperspectral imaging (Figure 2G-I). Gross and microscopic examination of the lungs at 7 and 18 months following the last nCB exposure showed persistence of nCB, lung inflammation, and hyperinflation (Figure 2-figure supplement 3-5). Thus, nCB-as an insoluble byproduct of tobacco combustion as shown above and also delivered through a non-smoking model-accumulates in lung and airway APCs, is poorly cleared from the lungs, and is alone sufficient to cause lung inflammation and emphysema in mice. Representative hyperspectral image of lung CD1a + cells from a patient with emphysema (E-H): a reference sample of nanoparticulate carbon black (nCB) was used to generate a signature spectral library (E) using CytoViva Hyperspectral Imaging System. Each colored spectra represents the spectral profile of a distinct area of the nCB sample, which were used in combination to map nCB present in cells. (F) Bright field (BF), (G) dark field (DF), and (H) overlay CB signature spectrum of lung CD1a + cells. Positive signals were pseudo-colored red to aid visualization. Scale bar: 20 μm. (I) Raman spectrum yielded in lung CD11c + and macrophages isolated from lungs of mice exposed to smoke for 4 months; CB reference (CB Ref) signal indicates solid CB sample. SMK: 4 months of cigarette smoke. Inset images for cell type correspond to Raman spectra indicating the subcellular localization of CB. The brightness of each 2 μm × 2 μm pixel, representing one spectrum, indicates the height of the graphitic band of CB at 1600 cm −1 compared to the background, such that brighter pixels indicate more CB. nCB activates APCs to secrete pro-Th17 cytokines and inhibits regulatory T cell differentiation in vitro We previously determined that cigarette smoke induces lung APC activation in human patients and mice, which then induces Th17 cell differentiation in naive T cells . To determine whether nCB specifically induces Th17 responses in vivo, we first examined lung mDCs from nCB intranasal-challenged mice. Lung CD11c + CD11b hi mDCs were significantly increased in the lungs of nCB-challenged mice when compared with controls ( Figure 3A,B). nCB also selectively induced lung Th17 but not Th1 responses relative to control animals ( Figure 3C,D and Figure 3-figure supplement 1). Lung CD11c + APCs isolated from nCB-challenged mice secreted significantly more of the Th17 cell growth factors IL-6 and IL-1β, along with other pro-inflammatory cytokines and chemokines, but not IL-12 or IL-4 (IL-4 was undetectable in both PBS and nCB groups), which promote Th1 and Th2 cell differentiation, respectively (Figure 3-figure supplement 2). To determine if lung APCs from nCB-challenged mice induce specific T cell differentiation programs in vitro, we cocultured naive splenic CD4 + T cells with CD11c + cells isolated from lungs of nCB-or PBS-challenged mice. Lung APCs from nCB-challenged mice induced significantly more IL-17A, but neither IFN-γ nor IL-4 production, when compared to controls ( Figure 3-figure supplement 3). Lung Th17 responses persisted for at least 7 months following the last nCB challenge (Figure 3-figure supplement 4). Further, Il-17a −/− mice were resistant to nCB challenge as assessed by their attenuated increases in lung volume, lung immune cell infiltration, and the reduced destruction of alveoli ( Figure 3E-H) when compared to identically treated WT mice. Thus, in vivo nCB selectively induces chronic lung Th17 responses, which are crucial for CB-induced emphysema in mice. We next explored whether nCB plays a direct role (i.e., independent of APCs) on T helper cell differentiation. To address this question, we polarized T cells toward Th1, Th17, and regulatory (Treg) phenotypes in the presence or absence of nCB in vitro. We found that nCB did not affect Th1 or Th17 cell differentiation directly ( Hydrophobicity of nCB correlates with pathogenicity The previous findings demonstrated that when deposited in the lungs, nCB activates mDCs and induces durable Th17-dependent inflammation and emphysema in mice. To determine the mechanism of nCB-mediated lung pathology, we next investigated whether its physicochemical properties could account for its immunostimulatory function. Whether manufactured or found in the lungs of smokers with emphysema, nCB is very hydrophobic and completely insoluble in aqueous media. Conjugating polyethylene glycol to nCB (PEG-nCB) renders the material hydrophilic and miscible with aqueous solutions (Hwang et al., 2014). Mice challenged with intranasal PEG-nCB using the same protocol (Figure 2-figure supplement 1) failed to develop emphysema as assessed by quantitative CT-based lung volume measurements, MLI and microscopic evaluation of the lungs ( Figure 4A,B,C). Further, we detected less anthracotic pigment in the lung parenchyma, suggesting that in contrast to hydrophobic nCB, PEG-nCB could be cleared from the lungs ( Figure 4C). Microscopic inspection of isolated BAL fluid cells from PEG-nCB-challenged mice showed intact phagocytic cells compared to that of hydrophobic nCB, suggesting that the latter may induce less cytotoxic effects on phagocytic cells (Figure 4-figure supplement 1). In support of this, we found that the release of lactate dehydrogenase (LDH), an indicator of cytotoxicity, was enhanced in macrophage-like RAW 264.7 cells exposed to nCB as compared to PEG-nCB (Figure 4-figure supplement 2). Consistent with the failure to induce emphysema and cell death, exposure to PEG-nCB also resulted in attenuated recruitment of macrophages, neutrophils, and lymphocytes to the lung when compared with hydrophobic nCB ( Figure 4D). This reduction in inflammation was accompanied by reduced expression of Mmp9 and Mmp12 transcripts in BAL fluid cells as compared with nCB-challenged mice ( Figure 4E,F). The markedly reduced inflammatory nature of PEG-nCB was further underscored by the reduced concentrations of pro-inflammatory cytokines and chemokines detected from freshly collected lung homogenates of PEG-nCB-challenged mice (Figure 4-figure supplement 3), including decreased IL-6 and IL-1β levels ( Figure 4G,H). Critically, PEG-nCB failed to induce lung Th17 cells when compared to nCB-exposed animals ( Figure 4I,J). Thus, the proinflammatory potential of nCB is intimately tied to its hydrophobic surface and ability to induce cytotoxicity of phagocytic cells. nCB-mediated induction of DNA damage and Erk signaling activates APCs We conducted additional studies to determine how nCB activates APCs to secrete pro-inflammatory cytokines (e.g., IL-6 and IL-1β) and chemokines. In response to nCB, but not PEG-nCB, reverse phase protein array (RPPA) identified the activation of several DNA damage (e.g., PARP, p-Chk2, p-ATM) and MAPK/Erk (p-ERK, p-MEK1/2)-response proteins ( Figure 5A and Figure 5-figure supplement 1). Consistent with these data, we found that nCB, but not PEG-nCB, induced DNA double strand breaks Mmp12 (F) gene expression in BAL cells isolated from the above group of mice. Lung homogenate collected from indicated groups of mice were measured for IL-6 (G) and IL-1β (H) by ELISA. Representative intracellular staining (I) or cumulative analysis (J) of Th17 cells in the lungs. *p < 0.001, p < 0.01, p < 0.05 as determined by the one-way ANOVA and Bonferroni's multiple comparison test. n = 4 to 6 per group, and data are mean ± SEM and representative of two independent studies. DOI: 10.7554/eLife.09623.016 The following figure supplements are available for figure 4: (DSB) as determined by phosphorylation of Histone 2AX (H2AX) on serine 129 (γH2AX) (Figure 5B,C). Further, the induction of DSB was inversely dependent on the size of nCB as we observed progressively fewer DSB with increasing nCB size ( Figure 5-figure supplement 2). We next examined whether CB-induced DSB could account for the pro-inflammatory responses seen in APC. Human monocyte-derived dendritic cells (MDDCs) treated with Nu7026, an inhibitor of the DNAdependent protein kinase catalytic subunit (Wilmore et al., 2004;Zhou et al., 2014), exhibited reduced IL-6 production in a dose-dependent manner in response to nCB but not LPS ( Figure 5D). Moreover, in nCB-exposed RAW 264.7 cells, transfection of a specific siRNA against ataxia telangiectasia mutated (ATM)-a serine-threonine kinase that coordinates repair of doublestranded DNA breaks (Guo et al., 2010)-significantly reduced expression of IL-6 and TNFα, two inflammatory cytokines that are induced through ATM ( Figure 5-figure supplement 3). To further examine whether induction of Th17 responses is dependent on nCB-mediated DNA damage, CD11c + lung mDCs isolated from nCB-challenged mice were co-cultured with splenic CD4 T cells in the presence of either Nu7026 or Ku55933, an inhibitor of ATM (Li and Yang, 2010), for 3 days. As expected, mDCs isolated from nCB-challenged mice promoted Th17 cell differentiation, which was significantly reduced in response to Nu7026 or Ku55933 ( Figure 5E) while Th1 and Th2 cell differentiation remained unchanged ( Figure 5-figure supplement 4). Together, these findings suggest that nCB-mediated DNA damage is required for the induction of pro-inflammatory cytokines in mDCs and Th17 cell differentiation. Moreover, nCB exposure in a dose-and time-dependent way increased phosphorylation of Erk ( Figure 5F), and similar inhibition of MEK1/2 with U0126, an inhibitor of MAP kinases (Newton et al., 2000), reduced IL-6 production in response to nCB exposure ( Figure 5G). Together, these findings indicate that hydrophobic nCB activates DNA damage responses and induces MAPK/Erk signaling coincident with the induction of Th17 responses. ASC-mediated assembly of the inflammasome complex is required for nCB-induced Th17 responses and emphysema The inflammasome detects danger signals released in response to cell injury and sterile inflammation and the adaptor protein ASC (apoptosis-associated speck-like protein containing CARD) was shown to be required for inflammasome-dependent caspase-1-mediated conversion of pro-IL-1β to mature IL-1β (Kono et al., 2012). In response to nCB exposure, lung CD11c + mDCs increased IL-6 and IL-1β expression and RAW 264.7 cells released more LDH, consistent with the concept that nCB induces both sterile inflammation and necrotic cell death. To determine if ASC is also required for nCBinduced Th17 responses and emphysema, Pycard −/− mice were challenged intranasally with nCB. When compared to WT mice treated identically, Pycard −/− mice showed attenuated emphysema ( Figure 6A-C) and reduced macrophage, neutrophil, lymphocyte, and mDC infiltration into the lungs ( Figure 6D,E). Consistently, lung mDCs of Pycard −/− mice produced less IL-6 and IL-1β and poorly activated splenic T cells to differentiate into Th17 cells when compared with WT mDC ( Figure 6F-H). Freshly collected lung homogenates from Pycard −/− mice challenged with nCB also showed reduced inflammatory chemokine production compared with WT mice (Figure 6-figure supplement 1). Thus, the earliest immunological events induced by nCB include ASC activation and inflammasome assembly, which are in turn required for nCB-mediated Th17 responses and emphysema. Discussion Evidence from experimental systems and human translational studies strongly support a role for chronic inflammation-and Th17 cells in particular-in the initiation and progression of emphysema in smokers Eppert et al., 2013;Kurimoto et al., 2013). A characteristic feature of the anthracotic pigment of smokers' lungs is that such discoloration persists even long after smoking has ceased (Churg et al., 2005). In this study, we addressed the role of insoluble anthracotic pigment that is universally found in the lungs of smokers with emphysema in driving this pathological response. Although several chemical identities have been proposed, our study is the first to clearly identify the anthracotic pigment as nCB and show that it accumulates specifically in human lung phagocytic cells. (E) Relative abundance of lung mDCs (CD11c + CD11b high ) isolated from whole lung tissue in the same group of mice. IL-6 (F) and IL-1β (G) concentrations detected by ELISA in the supernatant of lung CD11c + cells isolated from indicated group of mice after overnight culture. (H) IL-17A concentration detected by ELISA in the supernatant of splenic CD4 + T cells co-cultured with lung CD11c + cells isolated from indicated group of mice for 3 days in the presence of anti-CD3 (1 μg/ml). p < 0.001, p < 0.01, *p < 0.05 as determined by the one-way ANOVA and Bonferroni's multiple comparison test; n = 3 to 7 per group, and data are mean ± SEM and representative of two independent studies. DOI: 10.7554/eLife.09623.025 The following figure supplement is available for figure 6: Our functional studies are also the first to show that nCB administered to the lungs in pathophysiologically relevant amounts can induce sterile inflammation and emphysema that is indistinguishable from disease induced by exposure to cigarette smoke in mice. Thus, nCB is likely the major component of smoke that causes long-term lung toxicity. Furthermore, our findings have major implications regarding the safety of activities involving the chronic inhalation of smoke and the need to control the particulate composition of air. Since nCB is used extensively in the rubber, plastics, and composites industries, the exposure levels should also be controlled in the workplace. Our findings also elucidate both the nature of and the mechanism by which inflammation in the lungs of heavy cigarette smokers is perpetuated even long after cessation of cigarette smoking. Both chronic exposure to cigarette smoke and inhalation of nCB mediate similar inflammatory responses that are characterized by the activation of lung mDCs, differentiation and accumulation of Th17 cells, and lung parenchymal destruction (emphysema) (Shan et al., 2014). In part, this sterile inflammatory response to nCB is due to activation of the inflammasome pathway. Specifically, we found that inhaled nCB induces the production of IL-1β and IL-6, two pro-inflammatory cytokines that are required for mDC-mediated differentiation of Th17 cells and emphysema development. This inflammation persists and lung damage continues to accumulate even after smoking cessation due to the insoluble nature of nCB. Although readily taken up by phagocytic lung cells that could theoretically be expectorated or migrate out of the lungs via the lymphatics (Corry et al., 1984), these cells most likely undergo cell death too rapidly in response to nCB ingestion for any of these potential clearance mechanisms to operate efficiently. The nCB is then released in the lung by the cells it kills, only to be taken up again and kill subsequent phagocytes. nCB thus establishes an unending cycle of cell death that, if sufficiently pronounced, will trigger activation of the inflammasome pathway in response to the release of danger-associated molecular patterns (DAMPs) from dying cells (Piccinini and Midwood, 2010). Immune responses both rapidly kill invading pathogens and solubilize antigenic and adjuvantlike pathogen-derived substances to facilitate their removal and thus terminate the potentially deleterious inflammation. Both of these fundamental immune functions are thwarted in the context of nCB accumulation, leading to a perpetual cycle of lung inflammation and damage. Our findings, therefore, raise concerns that other insoluble environmental nanoparticles may, if inhaled, accumulate in lung phagocytic cells and induce similar pathology. In support of this, inflammasome-activated IL-1β has been shown to play a major role in lung sterile inflammation induced by other nanoparticles associated with lung diseases (Merget et al., 2002). ASC is required for the assembly of pro-caspase 1 in order to yield caspase 1 for the activation of pro-IL-1β to IL-1β (Franchi and Nunez, 2012). We show that inhaled nCB can activate the inflammasome pathway that results in production of mature IL-1β. In addition, inflammasome sensors activated by nCB-damaged cells require ASC activation because lung mDCs isolated from Pycard −/− mice failed to increase IL-1β and showed attenuated Th17 responses. A critically important physical feature of nCB, accounting in large part for its pro-inflammatory potential, is its hydrophobic character. Exposure to large quantities of hydrophobic nCB has been shown to induce cell injury, pyroptosis and generate reactive oxygen species (ROS) in cultured cells (Reisetter et al., 2011). One particular characteristic of nCB that correlates with its toxicity is its large surface area; larger forms of elemental carbon have much less potential to induce cell injury (Oberdörster et al., 2005) and, as we have shown here, damage DNA. A single burning cigarette can generate approximately 10 12 particles that vary in size from 1 micron to a few nanometers in diameter (Sahu et al., 2013). The deposition site of particulate matter in the lungs of smokers is governed largely by size, with larger particles depositing in the mouth and upper airway while smaller particles are deposited in progressively smaller and more distal airways (Adam et al., 2006;Baker and Dixon, 2006). For our studies, we used nCB spheroids with a nominal size of 15 nm that aggregate in clusters of 3-4, forming 50-75 nm per particle. However, in aqueous solution, this material forms macro-aggregates that fail to distribute evenly in the lung after intranasal challenge as does nCB delivered by smoke inhalation. We were partially successful in alleviating this confounding factor by adding sucrose to the nCB in aqueous solution. Nonetheless, although we endeavored to deliver nCB to mice in amounts that matched actual burdens found in human lung, it is likely that we did not fully recapitulate the in vivo particle size and distribution of nCB acquired through smoke inhalation. Further studies are required to define how nCB size affects in vivo toxicity as defined in these studies. Thermal and chemical analyses have shown that a combustion heat of 350-550˚C yields black carbon (BC) that contains PAHs that are linked to inflammation (Bleck et al., 2006), but combustion at much higher temperatures (650-1100˚C) produces PAH-free CB (Watson et al., 2005); we used this material heated to higher temperatures for intranasal administration in mice. A critical question related to our studies is, therefore, which form of carbon, BC or CB, is deposited in the lung during smoking, and how much do PAHs contribute to smoking-related lung inflammation. The nCB used in our studies lacked detectable PAHs by gas chromatography mass spectrometry analysis, which has a sensitivity limit of 1 part in 10 10 by mass. While PAHs are present in soot and diesel exhaust particles (Garza et al., 2008), neither Raman spectroscopy nor hyperspectral imaging can distinguish CB from BC as could be found in human lungs. However, X-ray analysis of the melting of small metal particles has revealed the temperature distribution inside a burning cigarette as 850-920˚C during active inhalation, decreasing to 700˚C during the smoldering phase; this temperature regime is consistent with the creation of predominant CB during smoking (Baker, 1974). Moreover, as PAH-free CB recapitulates almost entirely the pathology induced by smoke exposure, we conclude that sterile inflammation and pulmonary emphysema are primarily the result of CB accumulation in the lung and not that of BC or PAHs therein during smoking. Among signaling pathways that are important for APC activation and inflammation, we found that phosphorylation of Erk was significantly upregulated in cells exposed to CB treatment in a dose-and time-dependent manner while phosphorylation of p38 or JNK were not changed. The most notable, and unexpected, result of our RPPA analysis was the upregulation of several DNA damage enzymes by nCB exposure. This led us to confirm that nano-sized CB induces DSB in DNA as detected by the expression of γH2AX, an ATM-regulated pathway. We show that larger forms of CB result in attenuation of DSB, indicating that the size of CB is an important factor in its genotoxicity. Activation of this DNA repair pathway is in turn linked to the production of pro-inflammatory cytokines such as IL-6 and IL-1β that are required for Th17 differentiation. In addition, microRNA-22 (miR-22) has been shown to be upregulated in lung APC of mice exposed to smoke or nCB and is critical in Th17 responses through activation of AP-1 complexes and histone deacetylase (HDAC) 4 (Lu et al., 2015). Thus, together with DAMPs released by cells killed by nCB, nCB-induced DNA damage accounts for much of the inflammatory nature of nCB. The mechanism by which nCB cleaves DNA and the additional biological consequences of this adverse property remain active areas of investigation in our laboratory. In summary, our findings show that inhalation of cigarette smoke leads to the accumulation of nCB in airway APCs (macrophages and mDCs). This insoluble material promotes perpetual Th17 cellmediated lung inflammation in part through the double-stranded cleavage of nuclear DNA. These findings largely explain the persistent and incurable nature of smoking-related lung disease. Because no medical means of removing accumulated lung nCB exists, our findings underscore the need for all individuals and societies to minimize the production of and exposure to smoke-related particulate air pollution and industrial nCB. Materials and methods Mice C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Pycard −/− mice (C57BL/6 background; Pycard encodes for Asc protein [Mariathasan et al., 2004]) were obtained from Dr Vishva Dixit (Genentech, South San Francisco, CA). Il-17a −/− mice (C57BL/6 background) were obtained from Dr Chen Dong (The University of Texas MD Anderson Cancer Center, Houston, TX). All mice were bred in the transgenic animal facility at Baylor College of Medicine. All experimental protocols (AN-4589) used in this study were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and followed the National Research Council Guide for the Care and Use of Laboratory Animals. Reagents MEK1/2 inhibitor U0126 was purchased from Cell Signaling (Danvers, MA). DNA-PKc inhibitor Nu7026 was purchased from Tocris (Bristol, UK). ATM inhibitor Ku55933 was purchased from Millipore (Billerica, MA). Pierce LDH Cytotoxicity Assay Kit was purchased from Life Technologies (Grand Island, NY), and LDH release was measured according to the manufacturer's instructions. Nanoparticle characterization and preparation Various sizes of carbon black nanoparticles (nCB) were obtained from Cabot Corporation (15 nm, Monarch 1100, Lot 1278105;35 nm, Vulcan 9A32, Lot: CS-5822;70 nm diameter, Sterling NS1, Lot 761510, CAS# for CB: 1333-86-4;Alpharetta, GA). The nCB, although listed to be 15 nm, is more precisely described by the manufacturer to have 15-nm CB particles that are arranged in clusters of 3-5 particles, much as grape clusters, so the actual size is ∼50-75 nm diameter clusters. Conjugation of polyethylene glycol to nCB (PEG-nCB) was performed as described (Zhou et al., 2014). Briefly, 15-nm nCB (250 mmol) was dispersed in tetrahydrofuran (THF) using bath sonication for 3 hr. Then, 4,4′-azobis(4-cyanopentanoic acid) (ACPA) was added to the nCB dispersion in a three-step process. The first portion of ACPA (7 mmol) was added and stirred continuously for 24 hr at 70˚C. This addition was repeated at 24 hr and 48 hr. The mixture was cooled to room temperature, filtered through a 45-μm pore Teflon membrane, washed with THF, ethanol and acetone for three times, and vacuum dried (60˚C, 100 Torr), producing carboxyl-functionalized nCB. Carboxyl-functionalized nCB (2 mmol) was dispersed in dimethylformamide for 30 min, then mixed with N,N′-dicyclohexylcarbodiimide (0.8 mmol), mPEG-NH 2 (0.04 mmol), and dimethylaminopyridine (2 flakes). The reaction was stirred for 24 hr, transferred to a dialysis bag (molecular weight cut-off, 5.0 kDa), dialyzed in running deionized water for 1 week, and filtered through a 0.22-μm pore Teflon membrane. X-ray photoelectron spectroscopy performed on an a PHI Quantera SXM scanning X-ray microprobe with 26 eV passing energy, 45˚takeoff angle, and a 100-μm beam size. Thermogravimetric analysis performed on a TA Instruments Q-600 Simultaneous TGA/DSC. FTIR spectra were recorded using a Nicolet FTIR with an ATR attachment. By TGA, PEG-nCB contains 42-50% PEG by mass. By X-ray photoelectron spectroscopy, the PEG-nCB surface is 15% carbon, 56% oxidized carbon, 1.2% nitrogen, and 28% oxygen. Animal model of nCB-induced emphysema We suspended 20 mg of endotoxin-free nCB or 47 mg PEG-nCB (containing 20 mg of nCB by weight) in pre-warmed 1 ml tert-butyl alcohol with 1% sucrose. The mixture was then frozen at −80˚C for 24 hr and was placed on a vacuum pump and lyophilized until dry for 24 hr. nCB or PEG-nCB mixed with 1% sucrose were rehydrated in sterile PBS to achieve the concentration of 0.5 mg/50 μl, vortexed and sonicated in a water bath for 10 min before administration. Mice were deeply anesthetized with isoflurane, and 50 μl droplets containing 1% sucrose in PBS (vehicle) or nCB or PEG-nCB (mixed with 1% sucrose in PBS) were applied to the nares. Mice were challenged twice per week for 6 weeks and were sacrificed 1 month after the last challenge. Quantification of experimental model of emphysema The severity of lung parenchymal destruction (emphysema) was determined by computed tomography (CT) methods . Briefly, mice were anesthetized with etomidate (30 mg/kg) and placed in an animal CT scanner (Gamma Medica, Salem, NH), and completed images of the chest were obtained by the Animal Phenotyping Core in Baylor College of Medicine. Amira 3.1.1 software (FEI, Hillsboro, OR) was used to process the images and quantification of emphysema in three dimensions. The MLI used for measurement of mouse lung destruction (e.g., lung morphometry) was calculated as previously described (Shan et al., 2014). Briefly, an unbiased observer randomly selected ten fields from the left lobe (large airways and vessels were excluded). Paralleled lines were placed on serial lung sections and MLI was calculated by multiplying the length and the number of lines per field, divided by the number of intercepts. Analysis of experimental model of emphysema BALF and lung tissue were collected as previously described (Goswami et al., 2009). Briefly, mice were anesthetized with etomidate and BALF was collected by instilling and withdrawing 0.8 ml of sterile PBS twice through the trachea. Total and differential cell counts in the BALF were determined with the standard hemocytometer and HEMA3 staining (Biochemical Sciences Inc, Swedesboro, NJ) using 200 μl of BALF for cytospin slide preparation. Mouse lungs were dissected to prepare single-cell suspensions; alternatively, lungs were fixed with instillation of 4% paraformaldehyde solution via a tracheal cannula at 25-cm H 2 O pressure followed by paraffin embedding and were sectioned for histopathological studies. Hematoxylin and eosin (H&E) staining was performed as described (Goswami et al., 2009). Mouse immune cell isolation from lung, spleen, and bone marrow-derived dendritic cell (BMDC) culture siRNA transfection Mouse ATM siRNA and scramble siRNA were purchased from Sigma-Aldrich. Mouse RAW 264.7 cells were transfected with Nucleofection kit (Lonza, Basel, Switzerland) according to the manufacturer's instructions. RAW cells treated with siRNA were incubated for 6 hr before CB treatment for overnight. Western blot RAW 264.7 cells or BMDCs were harvested, pelleted, washed with PBS and lysed in RIPA (Radioimmunoprecipitaiton Assay) buffer (Sigma-Aldrich) with a cocktail of proteinase and phosphatase inhibitor (Thermo Scientific, Waltham, MA). The protein concentration of whole cell lysate was detected by BCA kit (Thermo Scientific). Equivalent amounts of protein in each sample were resolved by SDS-PAGE and transferred into nitrocellulose membranes. Membranes were blocked in 5% nonfat-dried milk in PBS with 0.05% Tween 20. Rabbit anti-mouse phospho-Erk (Cell Signaling, Danvers, MA) was used for protein detection. Immunostaining Cytospins of single cell suspensions were fixed with 4% formaldehyde, permeabilized with 0.5% saponin, and blocked with 3% BSA and Fc receptor Blocker (BD BioSciences). Then cells were stained with anti-γH2AX (Millipore) for overnight and detected by antibodies labeled with DAPI (4′,6diamidino-2-phenylindole) and Alexa Fluor 488. Images were detected with Nikon ECLIPSE TE2000 and NIS-Elements software version 2.30 and Leica DFC300 FX. RPPA analysis of RAW 264.7 cells treated with nanoparticles RPPA analysis was performed at the University of Texas MD Anderson Proteomic Core facility. Control RAW cells (untreated) and nanoparticle treated (100 μg/ml nCB and 100 μg/ml PEG-nCB) for 24 hr in triplicates were washed, pelleted, and subjected to RPPA analysis. A detailed description of sample processing and data analysis is available on the website of the core facility. Heatmaps were generated by the softwares Cluster and Treeview. VA Merit Award David B Corry, Farrah Kheradmand The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.	2017-06-19T08:59:45.717Z	2015-10-05T00:00:00.000	{ "year": 2015, "sha1": "b46d0f288a47e9407b6d15a8da4316101e69bde9", "oa_license": "CCBY", "oa_url": "https://doi.org/10.7554/elife.09623", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "6300d534ddc1f794e5ea5c2d87c3388055659252", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
13762334	pes2o/s2orc	v3-fos-license	Coronary endarterectomy to facilitate bypass surgery for patients with extensive stenting of the left anterior descending artery Introduction Procedural full metal jacket (FMJ) results in an increasing number of high-risk ‘stent-loaded’ patients who are referred to cardiac surgeon and complete revascularization cannot be achieved with the conventional technique [1,2]. The following report describes the technique used to allow enblock long-segment stent removal by coronary endarterectomy (CE) and left anterior descending (LAD) repair with left internal thoracic artery (LITA) graft. Introduction Procedural full metal jacket (FMJ) results in an increasing number of high-risk 'stent-loaded' patients who are referred to cardiac surgeon and complete revascularization cannot be achieved with the conventional technique [1,2]. The following report describes the technique used to allow enblock long-segment stent removal by coronary endarterectomy (CE) and left anterior descending (LAD) repair with left internal thoracic artery (LITA) graft. CE consists in opening LAD in its middle portion. After that a proper plane has been found, a long arteriotomy is performed. Under direct vision and avoiding traction, the whole atherosclerotic core and the stents are dissected and removed en-block. The distal intima is tacked using 7/0 polypropylene suture. Case 1 A 52-year-old male. Admitted for acute coronary syndrome because of three-vessel disease with a FMJ-LAD proximally occluded, with 50% of ejection fraction. Percutaneous coronary intervention allowed only a partial LAD reperfusion and patient was referred to surgery. The other three target vessels were grafted and the CE was performed (Fig. 1a). The LAD opening, 11 cm long, was repaired with a tailored on-lay vein patch and a pedicle LITA was anastomosed onto it. Postoperative course was uneventful and the patient was discharged on oral anticoagulation therapy. Patency of the LAD bypass graft was showed on coronary computed tomography angiography control, 6 months later (Fig. 1b). Case 2 A 54-year-old male. Admitted for acute coronary syndrome because of three-vessel disease with a FMJ-LAD proximally occluded and ejection fraction of 45%. The patient was referred to surgery after ineffective several attempts of percutaneous coronary intervention. The other two target vessels were grafted and the CE on LAD was performed. The LAD opening, 4.5 cm long, was repaired using a skeletonized LITA as on-lay patch. Postoperative course was uneventful and the patient was discharged on dual antiplatelet, after coronary computed tomography angiography control that showed patency of the LAD bypass graft (Fig. 2a). Six months later, due to onset of acute chest pain a coronary angiography was performed showing subocclusion of right coronary artery graft, percutaneously treated, whereas the LITA-LAD graft was patent (Fig. 2b). Discussion LAD is the major revascularization site in coronary surgery and incomplete revascularization of this vessel has been proven to be a predictor of higher long-term mortality after coronary artery bypass grafting [3]. The presence of long stented segment LAD may result in having difficulties in finding an appropriate site for bypass grafting when conventional techniques are used. Therefore that could force bypass grafts to be anastomosed to a more distal site and the compromise runoff 'per se', decreasing graft patency [4]. Once FMJ procedures have been performed, the only effective way to adequately treat restenosis is by CE with stents extraction, followed by surgical revascularization. Because stents cling firmly to the intima, the most suitable technique, in our experience, is performing CE under direct vision (open technique), rather than traction technique that could be associated either to the risk of 'snowplow' effect or to the risk of destruction of the artery [2,3,5]. In conclusion, CE with open technique represents a feasible and effective method for treating FMJ-LAD.	2018-04-03T02:06:32.377Z	2017-10-01T00:00:00.000	{ "year": 2018, "sha1": "1031bfe10373fe8f8dc40d912d47c30715787486", "oa_license": "CCBYNCND", "oa_url": "https://journals.lww.com/coronary-artery/fulltext/2018/06000/Coronary_endarterectomy_to_facilitate_bypass.14.aspx", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "1031bfe10373fe8f8dc40d912d47c30715787486", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
252438677	pes2o/s2orc	v3-fos-license	Scope of Pre-trained Language Models for Detecting Conflicting Health Information An increasing number of people now rely on online platforms to meet their health information needs. Thus identifying inconsistent or conflicting textual health information has become a safety-critical task. Health advice data poses a unique challenge where information that is accurate in the context of one diagnosis can be conflicting in the context of another. For example, people suffering from diabetes and hypertension often receive conflicting health advice on diet. This motivates the need for technologies which can provide contextualized, user-specific health advice. A crucial step towards contextualized advice is the ability to compare health advice statements and detect if and how they are conflicting. This is the task of health conflict detection (HCD). Given two pieces of health advice, the goal of HCD is to detect and categorize the type of conflict. It is a challenging task, as (i) automatically identifying and categorizing conflicts requires a deeper understanding of the semantics of the text, and (ii) the amount of available data is quite limited. In this study, we are the first to explore HCD in the context of pre-trained language models. We find that DeBERTa-v3 performs best with a mean F1 score of 0.68 across all experiments. We additionally investigate the challenges posed by different conflict types and how synthetic data improves a model's understanding of conflict-specific semantics. Finally, we highlight the difficulty in collecting real health conflicts and propose a human-in-the-loop synthetic data augmentation approach to expand existing HCD datasets. Our HCD training dataset is over 2x bigger than the existing HCD dataset and is made publicly available on Github. Introduction In recent years, quick and easy access to online health information has changed the way people eat, exercise, and interact with medical professionals (Bujnowska-Fedak and Wegierek 2020). As we continue to live through a global pandemic, people are increasingly relying on online resources for health advice (Loomba et al. 2021). However, a significant limitation of online health platforms is their inability to provide consistent, contextual health advice. For instance, while many information sources promote taking daily aspirin after a heart attack, this advice is dangerous to those on warfarin, as combining the medicines can have lifethreatening side-effects (See example 5 in Table 1). Moreover, as new clinical evidence emerges, some health advice will inevitably become outdated, generating conflicts with recent findings. Such situations motivate the need for automatic detection of conflicting health information across multiple relevant sources to facilitate safer interactions with online health platforms. This task is referred to as health conflict detection (HCD). Given two pieces of health advice statements, the goal of HCD is to both identify the presence of a conflict and to recognize what type of conflict occurs 1 . Different types of conflicts are presented in Table 1. Recognizing the conflict type will aid the user in interpreting the reason for a conflict correctly. For example, consider sample 5 in Table 1. Simply expressing that a conflict is present does not guide the user on how to react to the conflicting information on aspirin consumption. Explicitly stating the conflict type communicates how one should be careful regarding when (temporal) aspirin should be taken as well as if (conditional) aspirin should be consumed. With more people relying on online health websites and the surge of a global infodemic (Buchanan 2020), detecting inconsistent/conflicting health information at large is even more critical now, as widespread health misinformation is a risk to public health and extremely expensive to combat (Cornish 2020). Conflicting health information appears in many forms across the internet. HCD was first introduced in (Preum et al. 2017a,b) where the authors introduce a novel HCD dataset using data extracted from several health apps and medical information websites. More recently, the need for conflict detection on other sources, including social media, has presented itself. Consider the sub-reddit r/AskDocs, a forum with 380k+ members where anyone can ask questions to a verified medical professional for free. In Table 9 we highlight conflicting pairs of advice statements from registered nurses and licensed physicians identified on r/AskDocs to 1 HCD does not aim at discounting a source of information as health conflicts can occur between a pair of advice even when both advice statements are correct (Carpenter et al. 2016). Also, conflict resolution is beyond the scope of HCD as it requires the expertise of a professional healthcare provider and is often subjective to an individual. Advice 1 Advice 2 Conflict Type 1 Limit intake of sweetened drinks, snacks and desserts by eating them less often and in smaller amounts. Many fruits need little preparation to become a healthy part of a meal or snack. Direct 2 Limit liquids before bed. To prevent dehydration, drink plenty of fluids unless your doctor directs you otherwise. Temporal 3 Pasteurized dairy products, milk, cheese, yogurt, spinach are rich in calcium Dilute full-fat milk in your cereal with water. This reduces the absorption of sugar and decreases fat intake. Sub-typical 4 Eat dark green vegetables such as spinach, collard greens, kale and broccoli Avoid sudden increase of cruciferous vegetables if you are on Coumadin. They may affect your treatment and dosage Conditional 5 Aspirin helps prevent future blood clots and decreases the risk of death after a heart attack. Aspirin, aspirin-like drugs (salicylates), and nonsteroidal anti-inflammatory drugs ... may have effects similar to warfarin. These drugs may increase the risk of bleeding problems if taken during treatment with warfarin. Conditional, Temporal Table 1: Example health conflict pairs from the HCD dataset. Each sample contains a pair of health advice regarding a common topic. The conflict label is annotated with respect to the common topic between pieces of advice. Conflict topics are bold in the examples above. illustrate the need for more reliable and consistent social interaction on health. In this study, we build on top of the initial HCD work proposed in (Preum et al. 2017a,b) by focusing on a scaleable approach to HCD data expansion. (Preum et al. 2017a,b) use a rule based solution to detect health conflicts. Although the proposed solution results in high recall the following issues limit its applicability to detect conflicting health information at large. First, rule base solutions are brittle and harder to generalize. Secondly, their solution requires a prohibitively expensive amount of annotation for each piece of advice and is hard to scale to other data. So, the next step in the advancement of HCD is to develop an end-to-end system which does not require granular levels of annotation and can work in a production environment on free-text inputs. The recent advances in transfer learning inspire us to investigate the scope of pre-trained language models to detect health conflicts automatically. Successful implementation of a transfer learning based solution can effectively address this critical challenge of automatically detecting inconsistent/conflicting health information at large. One of the main challenges with using pre-trained language models for HCD is that they require sufficient labeled data for fine-tuning and the HCD task is inherently resource-constrained. For example, given a set of 500 health advice statements, there exists (500) 2 = 250000 candidate advice pairs from which a very small set will be conflicting -making data collection extremely difficult. Given the limited number of health advice pairs presented in (Preum et al. 2017a,b), this study explores methods of synthetic conflict augmentation towards making HCD suitable for use in a transfer learning framework. After expanding the original HCD training set by 2x, we explore the task in the context of pre-trained language models. Finally, to verify prediction reliability, we test if transformer-based predictions value the same information as human annotators when asked to identify the content most important to the prediction of a conflict. In summary, we considering the following research questions: RQ1: How effective pre-trained language models are in detecting conflicting health information? We provide thorough analysis of five different language models on the HCD task, investigating the challenges associated with understanding different conflict types. We additionally contrast our work with a related task, Natural Language Inference, which aims to classify pairwise inputs as entailed, contradictory, or neutral (Williams, Nangia, and Bowman 2018). We investigate the relationship between notions of conflict and contradiction by applying a pre-trained NLI classifier to the HCD task. RQ2: Are synthetic health conflicts able to aid in the prediction of real health conflicts? While synthetic conflicts are shown to be linguistically and semantically valid, they are often factually incorrect and contain distributional drift in terms of style. So, we explore the capability of synthetic conflicts to aid in the prediction of real conflicts. RQ3: Do language models agree with humans when identifying features most influential to conflict detection? The interpretability of AI systems is crucial to integration of machine learning in healthcare systems (Tjoa and Guan 2019). We use a popular model interpretability tool, Captum (Kokhlikyan et al. 2020), to identify the input tokens our top performing language model attributes as being most important to the prediction of a given output. We wish to explore if language models pay attention to known conflict indicators such as the conflict topic and label-specific semantic phrases (LSSPs) (e.g. temporal or conditional clauses that result in a temporal or conditional conflict). Problem Formulation Inspired by the definition presented in (Preum et al. 2017a), we define the input to a health conflict detection (HCD) task as a pair of health advice, (W 1 , W 2 ) each with a common topic or object of an advice, t. HCD is a multiclass, multilabel classification problem. Each advice pair contains one or more of the following labels. Direct Conflict: Occurs when the topic of a health advice pair has opposite polarity. If a piece of advice W i suggests the user take action a, a direct conflict occurs when another piece of advice W j suggests the user against taking action ¬a. In Table 1, our direct conflict example has topic snack. We see how advising fruit as a healthy snack directly conflicts with the suggestion to limit sweet snacks. Temporal Conflict: Occurs when a health advice pair disagrees about when to take some action a. In Table 1, our temporal example agrees one should drink liquids, but disagrees as to when consumption should occur. Sub-Typical Conflict: Occurs when the topic in reference to the suggested action disagree in type. In Table 1, we see that Advice 1 supports milk consumption while Advice 2 speaks negatively only in regards to full-fat milks. Conditional Conflict: Occurs when a conflict's occurrence depends on some condition. In Table 1, our conditional example displays how Advice 1 and Advice 2 are only conflicting if the user is taking Coumadin. Non-Conflict: Occurs when two pieces of advice are non-conflicting. We note that this list of conflict types is non-exhaustive, as we do not explore the quantitative and cumulative conflicts defined in (Preum et al. 2017b), due to limited available annotation. Such conflicts may be explored in future works. Dataset The following section is organized as follows. First, we provide an overview of existing HCD data. We then discuss our attempts at performing data augmentation on existing HCD samples. Next, we discuss how new data was collected. Finally, we detail our human-in-the-loop augmentation approach, which alleviates problems faced during our attempts at data generation and augmentation. Additional dataset details are included at the end of the section. Real Health Advice Dataset from PreCluDe The Pre-CluDe dataset contains 3285 pieces of advice statements from authentic health websites, health apps, and drug usage guidelines. The data covers general health topics, 34 Table 3: The F1 score of DeBERTa-v3 when trained using additional data using various popular augmentation strategies. These results highlight the complexity involved in augmentation of pair-wise multi-sentence inputs and the need for more advanced augmentation algorithms. chronic diseases, and their relevant prescription medications collected from authentic and reliable health websites and popular health apps. The list of diseases and medications are selected based on a set of real prescriptions for multimorbidity. These advice statements can be single sentence or multi-sentence. Each of these advice statements were annotated for topics of advice (e.g., advice on a food item, drugdrug interaction, drug-food interaction, exercise). In addition each advice was annotated based on the polarity of the advice with respect to each topic. Then potential pairs of conflicting advice statements were generated based on common topics. Each of these potential pairs of conflicting advice statements were then annotated by at least three reviewers to determine whether they are conflicting and the type of potential conflicts (if any). The details of this annotation process are described in multiple published literature (Preum et al. 2017c(Preum et al. , 2018. The PreCluDe dataset is the source of the 2006 real conflicting advice pairs utilized in this study. Synthetic Dataset Generation using Human-in-the-loop Data Augmentation In attempt to expand the size of existing HCD data, we first explored various automated methods of textual augmentation. The result of these experiments can be found in Table 3. Our first experiment employed PEGASUS (Zhang et al. 2019) for paraphrase generation. Given some Advice 1, we paraphrase it's corresponding Advice 2 to generate a new sample. Unfortunately, this method was prone to the removal or alteration of the conflict topic, producing incorrect samples. We additionally tried a similar approach using Back Translation between German and English, but little textual diversity was observed. We also explored a text generation approach using GPT-Neo (Black et al. 2021), where we employed in-context learning to generate new samples, with prompt structure inspired by GPT3-MIX (Yoo et al. 2021). Specifically, given a prompt and a small set of example advice pairs, GPT was asked to then generate the Advice 2 for a provided Advice 1. Unfortunately, this method failed to produce consistent coherent outputs. In each experiment we double the size of the dataset (thus generating n = 1398 samples) using the described augmentation methods. Note that we additionally report a result denoted Aggregated Set, which is an evenly distributed mix of all three augmentation approaches. New Online Health Advice Dataset To expand upon the existing HCD dataset with additional real-world samples, we scraped 4 popular health information websites including WebMD 2 , MedLine 3 , Covid protocols 4 , and CDC 5 . We collected a total of 47,600 candidate advice statements from a wide variety of health topics. Three annotators were then tasked with annotating a random sample of 5393 advice statements, each answering the following questions: 1) Does this statement contain health advice? 2) What are the topics of the advice? 3) What is the polarity of each topic in a piece of health advice? This allowed us to generate potential conflicting advice pairs by considering samples with common topics containing opposite polarities. The annotators found 2715 samples which were in fact health advice, and thus eligible for inclusion in a conflicting advice pair. Unfortunately, very few useful organic conflicts were found when pairing together samples with opposite topic polarities. This occurred because 1) there is a wide variety of topic entities annotated, leaving few opportunities for sample pairing, 2) many topics have inherent static polarity (e.g. cancer is always negative), which makes finding a conflict for such samples difficult. We thus concluded that automatic conflict generation using randomly-sampled, real-world text was not a feasible strategy to significantly increase the dataset size. Human-in-the-loop Data Augmentation The failure of the two aforementioned approaches to data generation inspired us to take a human-in-the-loop approach for data augmentation using mTurk. This approach allows us to generate training samples which encode the relevant linguistic phenomena associated with each conflict type, even if the style and prose of human-written samples does not perfectly reflect real-world data. Although the synthetic data generated in this process is most often not clinically accurate, they are helpful to capture the linguistic phenomena of conflicting textual information. In this study, we recruit 88 master-qualified mTurk workers. Additionally, we only recruited US-based annotators with a minimum 90% acceptance rating. To generate a synthetic advice conflict, an mTurk worker (Turker) is provided with a real health advice statement, the topic of the advice, and instructions on how to write samples for each conflict type. For example, a Turker may be prompted with the advice statement "Drink less alcohol." If asked to write a conditional conflict, a valid synthetic sample would be "Drink less alcohol if you suffer from heart disease". Full details of the data collection process are depicted in Figure 2. Of the 2715 newly collected health advice statements deemed useful, an additional round of human review extracted 649 advice statements most suitable for augmentation. Each sample was annotated for each conflict type, and then verified by at least one research assistant at the authors institution. The resulting dataset after sample verification contains 1897 unique advice statements from Amazon's mTurk platform 6 . We display the distribution of health domains included in the synthetic dataset in Table 4. Dataset Details In total, there are 2006 real annotated advice pairs, and 1897 synthetic pairs. Our training set contains 2825 samples, comprised of a mix of real and synthetic advice pairs. We evaluate our model on two test sets: One with entirely real-world data, and another which contains only synthetic advice pairs. The full data distribution is shown in Table 2. The ratio of single-sentence advice to multi-sentence advice is 3.07. The length of a multi-sentence advice can vary from (2, 11) sentences. On average, one health advice is 1.4 sentences long, containing close to 25 words. Given that HCD is a pairwise task, each input thus requires modeling of about 2.8 sentences, demanding longrange dependency modeling. Methods RQ1: How effective pre-trained language models are in detecting conflicting health information? To investigate the strengths and weaknesses of pre-trained language models on the HCD task, we aim to first quantify performance when fine-tuning transformer-based models for HCD. We fine-tune 5 different models for each architecture in our experiments. Four out of these five models are 1-vsall classifiers for each conflict type, with the fifth being a multi-label classifier. We then report results on two different test sets, each containing exclusively real or synthetic data. These experiments will identify (i) which conflict-types are most and least challenging to detect and (ii) allow us to quantify the difficulty level in predicting real vs synthetic test samples. We additionally explore the relationship between conflict detection and the relevant NLP task of Natural Language Inference, which includes contradiction detection. To do so, we apply a DeBERTa-v3 model fine-tuned on the MNLI dataset (Williams, Nangia, and Bowman 2018) and consider contradictory predictions to be conflicts in our 1-vs-all HCD setting. This experiment aims to highlight the differences between conflicts and contradictions and motivate the need for HCD-specific algorithms. We additionally experiment with intermediate fine-tuning (Chang and Lu 2021) on MNLI, before fine-tuning on HCD. This experiment aims to explore if leveraging the subtle relationships between MNLI and HCD provides any boost in predictive performance. RQ2: Are synthetic health conflicts able to aid in the prediction of real health conflicts? We have thus far motivated the need for better health conflict detection solutions, which will require more data to train reliable end-to-end models. Thus, RQ2 measures the effectiveness of the human-in-the-loop synthetic data augmentation technique. This experiment evaluates the predictive power of synthetic training samples on real-world health conflicts. To evaluate, we train 3 separate classifiers on each conflict type, using real-only, synthetic-only, and real+synthetic training data respectively. RQ3: Do language models agree with humans when identifying features most influential to conflict detection? With the long-term goal of deploying a scaleable HCD system to alert health information consumers of potential conflicts, we aim to explore the reliability of transformer-based model's by taking a low-level look at their predictions on the HCD task. To do so, we use the Layer Integrated Gradients algorithm (Sundararajan, Taly, and Yan 2017) provided by Captum (Kokhlikyan et al. 2020), to quantify the attribution of each input token to the prediction of the conflict label. Specifically, Captum allows us to produce a numeric attribution score for each input token. This enables visualization of token importance by toggling the opacity of highlighted tokens based on the magnitude of their attributions. An example Captum output for each conflict type is shown in the prediction of the positive class. Tokens highlighted in red contribute positively towards the prediction of the negative class. In this experiment, we employ 3 human annotators experienced in health conflict detection to answer the following two questions about the tokens Captum identifies as most important to the prediction of the positive class. 1. Does the model identify the topic as important to classification? 2. Does the model identify the correct Label-Specific Semantic Phrase (LSSP) as important to classification? Where LSSPs are defined as the topic-altering tokens which generate a specific conflict. In other words, the annotator is asked to look at a given sample, gather their understanding of the correct Topic and LSSP, and confirm their intuitions align with the transformer model by observing which tokens DeBERTa-v3 highlights in green. Given the importance of model interpretability in making health-related decisions (Vellido 2020;Dai, Sun, and Wang 2020), this experiment will verify if transformers are making judgements which align with human intuitions. In this experiment, each annotator looks at 10 samples from each class, from each test set (80 samples in total). All samples were selected randomly from the set of correct predictions made by the DeBERTa-v3 model. Each annotator provides a binary response (Yes/No) for each question, and the average score is reported. Evaluation Setting To ensure results are robust to random weight initializations, we run each experiment 3 times, each with a different random seed. Each reported result is the macro average of all experiments. For each individual experiment, we compute the F1 score of the positive class in a 1-vs-All setting or the weighted F1 in the multi-label setting. All metrics are computed using the scikit-learn package (Pedregosa et al. 2011). We note that we are unable to perform cross-validation due to the fact that a given advice statement may be paired with multiple other advice statements. Thus, performing cross validation runs the risk of input memorization as has been observed in similar pairwise inference tasks (Gururangan (Reimers and Gurevych 2019) and GloVE (Pennington, Socher, and Manning 2014) each function by first computing a single embedding vector for each piece of advice. Next, the embeddings for Advice 1 and Advice 2 are concatenated and fed into a linear classification head using scikit-learn. In these experiments, only the classification head undergoes training. Each dynamic contextual embedding model (BERT, Bio+Clinical BERT, RoBERTa, DeBERTa-v3) is fine-tuned useing the pair-wise clasification paradigm outlined in (Devlin et al. 2018) for a fixed 5-epochs with the AdamW (Loshchilov and Hutter 2017) optimizer set with learning rate = 2e − 5 and a weight decay = 0.01 to combat overfitting. We use the standard Cross Entropy loss for each task. In the case of 1-vs-All experiments, the loss is weighted using the distribution of the training labels to account for class imbalance. Results RQ1: How effective pre-trained language models are in detecting conflicting health information? Model Analysis We report the performance of 5 different transformer-based classifiers in Table 6 as well as a random guess, lexical feature, and word embedding-based baseline. The lexical model, which uses TFIDF (Manning and Prabhakar 2010) for feature generation with a class-weighted Random Forest classifier (Breiman 2001), under-performs random guess on 90% of experiments across all conflict types. This is expected as TFIDF generates features based on word count and frequency, ignoring the fact that the input text is comprised of two disjoint advice statements which need to be compared. Additionally, we note that the Random Forest model was unable to overcome the severe class im- balance with TFIDF features, often over-predicting the nonconflicting class for all labels. The static embedding methods, SBERT and GloVE, provide an improvement over our lexical baseline while outperforming random guess in most experiments. However, static embedding methods are not constructed to perform pair-wise comparison of longer texts. Thus, all textual comparison must happen in the classification head, which is shown to be inferior to the dynamic embedding approaches shown in Table 6. Transformer-based models such as BERT provide significant improvement over both TFIDF and static embeddings, as their contextual embedding approach permits direct comparison of two texts. The BERT baseline, however, is shown to be inferior to more modern transformer models such as RoBERTa and DeBERTa-v3, both of which outperform BERT on 80% of experiments. From our experiments using Bio+Clinical BERT (B+C BERT), we show that BERT's performance issues are not due to BERT's lack of domain specific training data, as B+C BERT under-performs or matches BERT's performance on 70% of experiments. This again follows intuition as, while this task is called health conflict detection, the addition of knowledge from sources like biomedical corpora and clinical notes are intuitively not relevant to this task. Health advice contains common language alongside occasional references to medications or physiological effects (as shown in Table 1). Across all experiments for which scores were higher than random guess, results on the synthetic test set outperform results on real health conflicts -often by a significant margin. This confirms our understanding of synthetic data outlined in the dataset description -where we hypothesize that nonorganic conflict data may be easier to understand as they often contain simpler and less diverse sentence semantics, using phrases specific to label definitions (as discussed in the Dataset Section). Label Specific Performance Analysis Direct Conflicts: Our results show there is a clear and impactful variance in the data distributions between real and synthetic direct conflicts, as performance on their respective test sets has the highest discrepancy across all conflict types. We additionally note the significant improvement provided by DebERTa-v3 on direct conflict understanding. We believe the DeBERTa-v3 architecture is well-suited for this conflict type for the following reasons. Direct conflicts, by definition, will not contain overt textual clues in a single advice statement which facilitate better label prediction. This in contrast to temporal conflicts, for example, where a statement such as "before bed" found in Advice 2 is a strong in- Table 6: Average F1 score across 3 experimental trials using all training data to predict each conflict type on the real (F r ) and synthetic (F s ) test sets respectively. dicator that there may be a temporal conflict independent of Advice 1. Thus, in order to understand direct conflicts, longrange dependency modeling is required, where a common topic must be identified in both advice statements as well as their relative polarities. DeBERTa-v3 improves upon BERT and RoBERTa in a variety of ways that facilitate long-range dependency modeling. Specifically, DeBERTa-v3 uses (i) disentangled position embeddings, (ii) global position information, and (iii) replaced token detection pre-training, which has been shown to be a more efficient pre-training objective than those used by BERT and RoBERTa (He, Gao, and Chen 2021a). We find evidence of DeBERTa-v3's improved ability to reason about pair-wise inputs in their paper where DeBERTa-v3 outperforms BERT on the pairwise Recognizing Textual Entailment task by an astounding 20.3 accuracy points (He, Gao, and Chen 2021a). We visualize the relationship between input length and F1 score in Figure 3, where we observe how BERT and RoBERTa are much more sensitive to token length than DeBERTa-v3 is for direct conflicts. A common error made when predicting Direct Conflicts was the over-prediction of other conflict samples which contained opposite topic polarities alongside conditional or temporal constraints. For example, if Advice 1 has positive sentiment towards coffee yet Advice 2 expresses negative sentiment towards coffee only for pregnant women, false positives tend to occur as the model is unable to understand that the opposing sentiments are tied to a conditional constraint. Sub-typical Conflicts: Our experimental results find that sub-typical conflicts are the most challenging to predict. Similar to direct conflicts, sub-typical conflicts by definition have less-overt textual clues than conditional and temporal conflicts. However, unlike direct conflicts, which rely on understandings of polarity with respect to each topic, subtypical conflicts often rely on world-knowledge for proper understanding. For example, the advice pair ("Don't eat cheese", "Only some cheeses are bad for you, like Goat cheese") contains a sub-typical conflict regarding cheese type. It may be easy for a pre-trained language model to understand the topic (cheese) and sentence polarities (negative, negative), but challenging to know that "goat" is a type of cheese, and that the input isn't referring to the animal itself, but rather altering the topic-type. Temporal and Conditional Conflicts: Pre-trained language models find the most success in the prediction of conditional and temporal conflicts. This is particularly true of the synthetic test sets, where mTurk workers could add if or similar conjunctions or temporal clauses to the end of the original advice statements to generate conditional or temporal conflicts, respectively. Thus such samples are less challenging for the model to classify correctly. As previously stated, these conflict types contain more consistent textual patterns that make their detection easier. Interestingly, these two conflict types are the only experiments where DeBERTa-v3 does not outperform other pre-trained language models. This is likely due to the lesser dependency on long-range dependency modeling required by these two conflict types. With these two conflict types, the presence of labelspecific semantic phrases in non-conflicting samples can cause false positives. Additionally, our conditional classifier was stumped by samples such as "Don't exercise on an empty stomach" where the condition is expressed in an implicit, more compact form. That is to say, the model may have predicted it correctly had it been phrased "Don't exercise if you have an empty stomach". Problems such as these can be mitigated by collecting more diverse samples in future works. Relationship to Natural Language Inference: Figure 3: Plot visualizing the relationship between token length and model performance. The x-axis represents token number bins and the y-axis shows the corresponding F1 score on that data subset. Results were generated using all training data in a one-vs-all setting on Direct Conflicts. instructions given to MNLI annotators when writing contradictions: Given a sentence 1, write a sentence 2 which is "definitely incorrect about the event or situation" in sentence 1. In the case where Advice 1 is Don't consume alcohol and Advice 2 is Don't drink alcohol if you're pregnant, we have a conditional conflict which is not at all related to the requirements for an MNLI contradiction. The NLI F T experiment highlights how NLI may be useful to some conflict types as an intermediate fine-tuning task. Specifically, Temporal and Conditional conflicts benefited significantly from this pre-training strategy. However, given the previous discussion on the difference in definitions between conflict and contradiction, we suppose the performance increase is due to a combination of the following reasons: 1) The introduction of DeBERTa-v3 to a pairwise inference task and 2) These conflict types have overt textual clues, making them easier to classify. In other words, we can classify more of the simpler sample types with additional pair-wise inference pre-training. However, this had no effect on the comparison of complex sample types such as Direct and Sub-Typical. RQ2: Are synthetic health conflicts able to aid in the prediction of real health conflicts? A core goal of this paper is to expand the size of the original HCD datasets presented in a scaleable manner as collecting real data on conflicting advice is prohibitively expensive (Preum et al. 2017a,b). We run experiments using different combinations of training data to quantify the contribution of synthetic samples on the real-world test set. Table 8 displays the results of this experiment. The synthetic data helped all transformer based models to classify conditional and temporal conflicts. This validates our hypothesis that, even when there exists severe stylistic drift 7 between real and synthetic data, models can benefit from examples of LSSP's, regardless of their context. All models additionally benefit from synthetic sub-typical conflicts. In fact, each model found synthetic data more useful than real data when predicting sub-typical conflicts. However, the synthetic data was distracting when predicting direct conflicts for BERT and RoBERTa, while DeBERTa-v3 found synthetic data useful. This can be explained by DeBERTa-v3's superior long-range dependency modeling (as discussed in RQ1). Consider that the mean token lengths for a synthetic direct conflict and real direct conflict are 55 tokens and 86 tokens, respectively. In terms of sentence length, this on average translates to real direct conflicts being 1 full sentence longer than synthetic conflicts. Thus, synthetic direct conflicts are significantly shorter and perhaps not helpful in aiding BERT and RoBERTa to resolve the long-range dependency in real world data. We find that DeBERTa-v3 is, in general, able to best utilize synthetic samples as they improve DeBERTa-v3's performance on real test data across all conflict types. RQ3: Do language models agree with humans when identifying features most influential to conflict detection? Table 7 displays the results of our experiment in which three human annotators are asked to confirm if DeBERTa-v3 identifies what tokens are most important to understand a conflict. For all experiments, the Topic score displayed in Table 7 is defined the same -the human annotator is asked to confirm if the model has attributed a high score to the conflict topic. LSSP's however are defined per conflict type. In the following sub-sections, we define the LSSP for each conflict type and analyze the results. Direct Conflicts: In this experiment, LSSP points were awarded to samples where DeBERTA-v3 identified relevant polarity tokens associated with each conflict topic. In Figure 1, we see that the topic Grapefruit is not emphasized -which is surprising given the importance of conflict understanding in humans. However, the model correctly attributes "Avoid" and "safe to take", the two opposing polarity phrases, as being more important to classification. On average, Topics were attributed highly in 18% of samples, while LSSP's were attributed highly in 75% of examples. Subtypical Conflicts: In this experiment, the model got points for LSSP identification if tokens which altered the type of the conflict topic were given high attribution scores. Our results found that 66% of samples identified Topic and 63% of samples identified LSSP's. In the sub-typical example provided in Figure 1, the topic "Milk" is identified in both Advice 1 and 2, while the topic altering tokens "full -" are highlighted in Advice 2. Given the need to identify topic to understand sub-typical conflicts, it is expected that the topic attribution rate is high for this conflict type. Conditional and Temporal Conflicts In these two experiments, LSSP attribution was credited when the model highlighted either conditional or temporal clauses respectively. For example, in Figure 1, the conditional example gives strong attribution to the statement "if you have sleep apnea". Additionally, in the temporal example, we observe strong attribution given to "between the hours of 1 and 3 pm". LSSP's for these two label types are shown to be easy to understand as conditional and temporal identify LSSP's 95% and 86% of the time respectively. However, topic attribution for these two conflict types are low, which is expected given the strong textual patterns associated with LSSP's for these conflict types. Pre-trained Language Models From their inception when introduced in (Vaswani et al. 2017), the use of the Transformer architecture has evolved greatly over the years. (Devlin et al. 2018) released BERT, a transformer-based language model pre-trained on a 3.4 billion word corpus which produced state-of-the-art results on popular NLP benchmarks. Modern adaptations of the BERT pre-training paradigm such as RoBERTa and DeBERTa-v3 (He, Gao, and Chen 2021b) improve upon BERT by optimizing the pre-training tasks used to generate high-quality contextual token representations. We explore all three transformer models in this study. Additionally, we investigate the benefits of medical training data on HCD via Bio+Clinical BERT (Alsentzer et al. 2019), which is the BERT framework described above, but instead of being pre-trained on common texts, it is trained on biomedical corpora and clinical notes. Finally, we evaluate the performance of static sentence embeddings using SBERT (Reimers and Gurevych 2019). This experiment provides a highly regularized transformer baseline where only the classification head is fine-tuned for HCD. Natural Language Inference The task of Natural Language Inference (NLI) is a multiclass pairwise classification problem for which, given a premise and a hypothesis, the model is tasked with predicting if the premise, Entails, Contradicts, or is Neutral towards the hypothesis. NLI data sets such as MNLI (Williams, Nangia, and Bowman 2018) are commonly used to evaluate a language model's ability to reason about complex language understanding tasks. NLI and HCD both depend on the compare and contrast of two disjoint texts. Additionally, it is reasonable for one to posit that there is a relationship between the notion of a pairwise contradiction and conflict. Thus in this study, we explore the relationship between notions of conflict and contradiction in effort to identify if NLI can be useful in the prediction of conflicting health information. Health Misinformation Detection Recently there has been increasing interest in misinformation detection for specific health topics from social media data (Sager et al. 2021;ElSherief et al. 2021;Weinzierl, Hopfer, and Harabagiu 2021). ElSherief et al. focus on detecting misinformation regarding medications used for opioid use disorder (OUD) treatment from multiple social media using traditional machine learning models and BERT (ElSherief et al. 2021). They formulate the problem as a binary classification task where a positive class refers to a post discussing a piece of misinformation (or myths) challenging OUD treatment and the negative class refers to any post that was not relevant. They report that logistic regression on TF-IDF based features perform better than BERT. Weinzierl et al. focus on identifying adoption or rejection of COVID-related misinformation on Twitter to better understand the effect of exposure to misinformation on COVID-19. They formulate the problem as stance classification and develop an ensemble architecture consisting of graph attention networks and BERT (Weinzierl, Hopfer, and Harabagiu 2021). Both HCD and health misinformation detection aim to measure information quality and improve public interactions with online health texts. Limitations and Future Work Limitations: Although the human-in-the-loop approach using mTurk yielded reasonable conflicting health advice pairs, the resulting dataset has a couple of limitations. First we found that mTurk workers do not generate health conflict data which stylistically reflects real-world data. Specifically, mTurk workers struggle to write diverse sub-typical conflicts and often generated repetitive instances of conditional or temporal conflicts with no or limited stylistic and semantic variation. In general, synthetic samples have much more textual overlap than real samples, as mTurk annotators can often change/add a small number of tokens to generate Fever Medication Direct If the left breast has been getting larger and developing those veins recently, and if you feel firmness of the left breast that is different from the right, then I would get it checked out by your PCP or GYN. They're normal. Breasts (like eyebrows) are "sisters, not twins" -no one has perfectly symmetrical breasts. Breast Cancer Conditional Table 9: Conflicting health information found on reddit.com/r/AskDocs -A social media forum where anyone can get medical advice from a professional whose medical certifications are verified by the forum moderators. We show that suggestions from medical professionals can be conflicting. a conflict. These themes are common and produce a stylistic drift between real and synthetic samples in our dataset. However, this is an expected limitation of mTurk-generated samples as they were only tasked with mimicking the linguistic phenomena of an advice conflict, not to generate realistic samples. 8 Another limitation of this work is that the distribution of conflict-types found in this dataset will be biased towards the official data sources used to collect real advice statements. As the HCD task moves to new data domains such as social media data, different conflict types/distributions may emerge. The solution provided in this paper is limited to understanding the problem linguistically. In the real world, health conflicts are often subjective to user physiology, diagnosis and prognosis. Effects of health conflicts might differ in terms of degree of risk and potential severity. A usercentric conflict detection system should be personalized to avoid false alarms and cause user anxiety. This demands continuous user sensing, as information of the users' medical context is required for the personalized system to detect what is conflicting for a given user. For example, the HCD system can only flag potentially dangerous drug-to-drug interactions if it knows what medications the end-user is currently taking. Future work: Future work on HCD should investigate better use of medical knowledge. While experimental results on Bio+Clinical BERT showed HCD does not benefit from pre-training on scientific texts and clinical notes, it may be the case that HCD requires a medical knowledge integration solution that is well-suited for non-clinical text related to public health. Medical knowledge graphs like UMLS (Bodenreider 2004) may aid BERT-like models in the understanding of rare diseases and medications not common in language model vocabularies. Additionally, our attempt at organic HCD dataset expansion only targeted professional sources. Future works may 8 For more details on how Turkers were instructed to write each conflict type, please refer to the supplementary materials. consider non-professional sources such as social media data. However, advice extraction from social media data is itself a separate research question. Additionally, non-professional sources may provoke differing data distributions with new conflict types. Exploration of such data sources should be the subject of future works. Broader Impacts and Ethical Considerations Broader Impacts New dataset with high quality and reliability: We provide a new public dataset on health conflict detection. The data is reliable as our use of multi-stage human-review helps mitigate annotation artifacts and human errors commonly found in large-scale crowd-sourced datasets. Additionally, our work on expanding the original HCD dataset uses data sourced from a wide variety of topics on various diseases and conditions. This makes HCD more health topic inclusive. Providing consistent online health information: An accurate health conflict detection system will protect consumers of online health information by providing a cautionary signal upon detection of potential health information conflicts. In addition, such technology can reduce the number of errors in medical decisions made by patients using online medical information sources. This is particularly important for individuals who rely on online sources for decisionmaking due to limited access to healthcare, an increasingly common case since the onset of a global pandemic. Reducing cognitive overload and aiding patient-provider communication: In general, health information inconsistency checking is a challenging task for most humans. It can be overwhelming for patients to memorize all of the health advice they receive and cross-reference it with all the new pieces of advice they encounter. Automatic conflict detection can help patients identify potential conflicts and consider when to raise questions to their medical providers. This is especially important for caregivers of individuals with cognitive challenges such as dementia or those who suffer from extreme stress, where mental cross referencing be-tween sources becomes increasingly difficult. Even for the general population, HCD reduces the burden to understand and memorize all of the fine-grained details of their medical conditions/medications by automatically detecting conflicts which pertain to a patient's specific condition. Also, the goal of this work is to flag potential conflicts found in textual health information so an end-user can bring them to the attention of their medical provider. Thus there is no conflict resolution currently in-place, as it is beyond the scope of this work. Extending HCD task for detecting and characterizing misinformation: We additionally note that, while the nonaugmented HCD samples in this study were taken from verified medical sources, the ideas proposed in this work extend to non-official sources such as text collected from Twitter, Reddit, Facebook or other social media (Sager et al. 2021;ElSherief et al. 2021;Weinzierl, Hopfer, and Harabagiu 2021). For example, one can formulate the comparison of health advice between official social media sources (e.g. @CDCgov on twitter) and non-official sources aiming to spread health misinformation. This differs from other health misinformation formulations where the veracity of a claim is verified using the text itself, or an external scientific resource. In the HCD formulation, we not only better permit the direct comparison of two pieces of advice, but can predict fine-grained labels that provide better insight into how two advice texts differ. Thus it can help to characterize misinformation from a linguistic standpoint. Additionally, fact checking often uses knowledge base triplets for claim verification. Under this paradigm, it may be hard to extend misinformation detection to new/niche health domains for which knowledge triplets are unavailable. Exploring misinformation detection in the context of HCD and language models may help keep predictive models more robust to lowresource domains or outdated evidence. Ethical Considerations This research involved human subjects only for data annotation, review, and prompt-based synthetic data generation. The project did not provide any intervention to any human subjects nor did it collect any user level data. So there is no risk to human subjects. The Amazon mechanical turk workers were recruited only for prompt-based synthetic data generation using formal procedure and no personal data was collected from them. The authors took careful measures to avoid annotation errors and maintain the quality and reliability of the data. Future progressions of HCD studies, in effort towards providing contextualized user-centric health information, may require use of end-user medical data. This might raise various ethical and privacy concerns and will require careful consideration. However, this is not a concern of the current study. The intended audience of the collected advice statements are often for the general population. Thus, the HCD dataset may not contain many advice statements directed at underrepresented communities/populations. Future rounds of data collection should focus on both diversification of medical condition coverage and target audience. User-generated medical content from social media can be a good source to collect health advice targeted to a specific sub-population, e.g. heart disease related advice targeted to African Americans. Conclusion Detecting conflicting health information makes human interaction with online health platforms safer. In this work, we introduce the HCD task in the context of pre-trained language models. We provide a detailed analysis of five different language models, examining the challenges brought about when predicting different health conflict types. Our experimental results find that the DeBERTa-v3 architecture performs better on challenging conflict types with less obvious semantic patterns. However, for simpler conflicts like conditional and temporal, BERT and RoBERTa provides comparable performance. We additionally confirm that the token attribution scores provided by a deep interpretability model, Captum, align with human judgement regarding input importance for predicting different conflict types. Finally, we show that the addition of synthetic conflict data does help with the prediction of real-world data, irregardless of differences in style and content accuracy.	2022-09-23T06:43:00.255Z	2022-09-22T00:00:00.000	{ "year": 2022, "sha1": "b40bdedc0e5ac9b3f048d3a5afea04d0f5c0aaeb", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "b40bdedc0e5ac9b3f048d3a5afea04d0f5c0aaeb", "s2fieldsofstudy": [ "Computer Science", "Medicine" ], "extfieldsofstudy": [ "Computer Science" ] }

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