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241308832 | pes2o/s2orc | v3-fos-license | Optimization of Model and Structural Analysis Hypoid Gear
The purpose of this work is to analyze structural behaviour of differential hypoid gear. For the studying of the structural analysis of the gear is made up of the different composite materials & as well as alloy materials compared with conventional metallic materials. As, composite materials provide enough strength and emerged as a good alternative for replacing metallic gears which is Ni-Cr steel. The alloy materials considered here is Aluminium alloy, Grey cast iron, and the composite material is Epoxy E-Glass unidirectional and compare with existing gear material Ni-Cr steel. This work makes an attempt to replace the metallic gears of Ni-Cr steel with alloy & composite materials based on static structural analysis results. For the analysis purpose the solid model is created by using CATIAV5R20 design software and ANSYS 17.1 is used to determine the Total deformation, Von-Mises stress and the equivalent elastic strain for the different selected materials at three different speeds.
INTRODUCTION
Now a day's gears are used in many applications for transmission of motion from one shaft to another shaft. The gears drives are positives which are transmit exact velocity ratio, the gear drives are transmit motion when two shafts distances are less than compared to the belt, chains, rope drives. The gear drive is used when the shaft having long centre distances this time some idler gears are using between driver and driven gears f or transmission of motion from one to another shaft. Thus providing the idler gears the co st also in creased and maintained cast also high than compared to the other power transmission drives like belt, rop es and chain drives, for this reason the gear drives not recommend for transmission of motion from large cen tre d istan ces from driving shaft to the driven shaft. Now a day's gears are various types are available in the market, based on the type of application, purpose and working condition the gear materials is selected, because th e selection o f the material is most important for manufacturing of the any component, because the tensile strength, young's modules, corrosion resistance, efficiency and life of the material etc. can be depended into the selection of the material.
This work makes an attempt to replace the conventional metallic gears of Ni-Cr steel gear material with the alloy & composite materials having application for high power transmission system & reducing of th e stress same as a gearbox were in automobile industries. For this purpose 3-D solid model o f th e d ifferential hypoid gear made in CATIA V5R20 design software and ANSYS 17.1 finite element method analysis software were used to carry out the static analysis in order to identify the behaviour of the conventional Ni-Cr steel gear material and identified replacements of different alloy & composite materials under the different loading criteria's. Then the simulation of the results determines the total deformation, Equivalent Vo n-Misses Stress, and Equivalent Strain of the different selected materials at three different rpms.
FEM ANALYSIS OF THE GEAR
In this project numerical method of the FEM based static structural analysis carried out by using ANSYS 1 7.1 analysis software. FEM (Finite element analysis) a computer-based analysis technique is used of the studying the behaviour of structures under the applied blundered conditions. The ANSYS 17.1 analysis software determine results like the total deformation, Equivalent Von -misses Stress, and Equivalent elastic strain under the applied boundary conditions. The 3D model for numerical analysis developed in CATIA V5R20 design and these models have been imported in ANSYS as IGES file format for further.
TORQUE CONSIDERATION
For the analysis purpose of the differential hypoid gear the applied boundary conditions are friction less supports, that means all gears are rotated in applied rpms and three different rpms were uses for conducting of the static structural analysis. Based on the selection of the different materials and applied boundary conditions the results are generated. The various torques and rpms for the analysis is mention in the below table .
STATIC STRUCTURAL ANALYSIS
This analysis, which is numerical method of the ANSYS software analysis, the static structural analysis wh ich is a system based analysis, that means, resulting the static structural analysis is generated by computer based on the selection of various different materials an applied boundary conditions the computer solve the results with the help of preloaded formulas and theorems the results will be generated. The static structural analysis is mainly conducted to find the selected material for gear manufacture can withstand or not for the applied lo ads and generation stress, temperatures etc. during motion. To finding the best material for the manufacturing of the differential hypoid gear is mandatory.
In this work, the FEA tool were used to identify the structural behaviour of various alloy materials and composite material under certain boundary conditions there by determining total deformation, equivalent Vo nmises stress and maximum strain for each alloy material and as well as composite material then the comparison was done with previous existing metallic gear material which is Ni-Cr steel. In this research work FEM structural analysis simulation results were shown the behaviour of Ni-Cr steel, Aluminium Alloy, Grey Cast Iron alloy materials and Epoxy E-Glass_UD composite material at the applied boundary conditions th e results of the static structural analysis were shown below as: reduced and the aluminium alloy has low density, based on low density the overall weight of the gearbox was reduced. 4. By decreasing of the weight of the gearbox the efficiency of the gear will be increased.
5.
After the aluminium alloy material the next suitable material is Epoxy E-glass unidirectional composite material is suitable for manufacturing of the hypoid gear.
FUTURE SCOPE OF THE WORK
In the present investigation of static analysis of hypoid gear is analyzed by single composite material in as a future work, the number of composites and some advanced alloy, nano materials with different rpms was also tested for manufacturing of the hypoid gear to overcome of the gear teeth failure by applying of the excessive loads. | 2021-08-31T16:28:14.726Z | 2020-12-23T00:00:00.000 | {
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"oa_url": "https://doi.org/10.1088/1757-899x/998/1/012038",
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232386045 | pes2o/s2orc | v3-fos-license | Strategies Targeting Hemagglutinin as a Universal Influenza Vaccine
Influenza virus has significant viral diversity, both through antigenic drift and shift, which makes development of a vaccine challenging. Current influenza vaccines are updated yearly to include strains predicted to circulate in the upcoming influenza season, however this can lead to a mismatch which reduces vaccine efficacy. Several strategies targeting the most abundant and immunogenic surface protein of influenza, the hemagglutinin (HA) protein, have been explored. These strategies include stalk-directed, consensus-based, and computationally derived HA immunogens. In this review, we explore vaccine strategies which utilize novel antigen design of the HA protein to improve cross-reactive immunity for development of a universal influenza vaccine.
Introduction
Seasonal influenza epidemics infect between 10-15% of the global population each year [1]. Symptoms typically last between 5-15 days and include fever, headache, myalgia, and respiratory distress [2]. However, in at-risk patients such as the elderly and immunocompromised, influenza infection can result in severe morbidity and even mortality [3,4]. In addition to the substantial disease burden from seasonal influenza epidemics, there is a significant threat to global health from influenza pandemics. Highlighting this, the 2009 H1N1 swine influenza pandemic infected an estimated 24% of the global population [5].
Influenza virus is a negative-sense, single-stranded RNA viruses with a genome of 8 segments. These segments encode viral proteins including hemagglutinin (HA), neuraminidase (NA), nonstructural 1 (NS1), NS2, matrix 1 (M1), M2, nucleoprotein (NP), nuclear export protein (NEP), polymerase acid (PA), polymerase basic 1 (PB1) and PB2 [6]. There is substantial viral diversity in influenza virus, both through antigenic drift from the error-prone RNA polymerase and through antigenic shift from reassortment of the segmented viral genome resulting in novel reassorted viruses [6,7]. The major proteins on the surface of the virion are HA and NA. Influenza A viruses (IAVs) are classified based on 18 HA subtypes and 11 NA subtypes while influenza B viruses are classified into two lineages, Yamagata and Victoria. The 18 HA subtypes of IAV are divided into phylogenetic groups 1 and 2 ( Figure 1). H1N1, H3N2, and both influenza B lineages currently circulate in the human population and cause seasonal epidemics. The H2N2 subtype caused a pandemic in humans in the 1957-1959 influenza seasons, but has, for the most part, been absent from the human population ever since [8,9]. Importantly, avian influenza strains H5, H7, and H9 subtypes have infected humans from zoonotic transmission but have shown limited transmission between humans [10]. However, the possibility of future mutations which enhance human-to-human transmission led the World Health Organization to recognize these subtypes as potential pandemic viruses [11][12][13]. Group 1 IAVs (blue) and group 2 IAVs (green) phylogenic groups are indicated. Subtypes which are known to circulate in humans (or have previously like H2) are circled in red, while subtypes which infect avian species but are recognized to have significant pandemic potential for zoonotic transmission are circled in yellow. Representative strains for each HA subtype were aligned using ClustalW alignment and a maximum-likelihood phylogenetic tree was constructed using PhyML3.3 on Geneious 11.1.5.
Development of influenza vaccines is challenged by the substantial viral diversity of influenza virus [14]. The RNA polymerase of influenza virus has no proof-reading activity, which results in high mutation rates and substantial antigenic drift [15]. Therefore, current seasonal influenza vaccines are updated yearly and rely upon global surveillance to predict the future circulating seasonal strains [16]. The quadrivalent formulation contains an H1N1, H3N2, and two influenza B viruses, one from the Victoria and Yamagata lineage [2]. Current seasonal influenzas vaccines are effective at reducing morbidity and mortality from seasonal influenza infections [17], however vaccine effectiveness estimates range from 10-60% [16]. Vaccine efficacy relies on the correct prediction and close antigenic match between the chosen vaccine strain and the circulating influenza strain [16]. In addition, these seasonal vaccines are unlikely to provide protection from novel emergent pandemic strains (such as the 2009 H1N1 reassorted swine influenza virus).
Many strategies have been explored to increase the cross-reactivity of influenza vaccines, with the goal of developing a universal influenza vaccine. Importantly, HA is the predominant antigenic protein of influenza viruses and antibodies directed at HA are correlated with protection against influenza virus infection [18,19]. In this review, we explore vaccine strategies which target the HA protein for development of a universal influenza vaccine, with a particular focus on novel antigen design of HA to improve cross-reactive immunity.
Hemagglutinin Structure and Function
Hemagglutinin is the most abundant protein on the surface of influenza and functions in viral entry through receptor binding and membrane fusion [6]. HA is also the predominant antigenic protein and therefore shows the highest rates of adaptive evolution out of all the influenza proteins [20]. Although there are high levels of protein sequence diversity between the subtypes, the HA protein maintains required elements, such as the cleavage site, secretory signal, fusion domain, transmembrane domain, and cytoplasmic tail as well as common protein structural motifs [21].
HA is a glycoprotein which assembles as a homotrimer on the surface of the virion ( Figure 2) [6]. Each monomer starts initially as a single polypeptide precursor (HA0) which is later cleaved into HA1 and HA2 subunits by host proteases. This cleavage is essential for maturation of the virus to an infectious virion [22]. The HA2 subunit is composed mostly of the stalk region of HA and the C-terminus which has a transmembrane domain with a cytoplasmic tail that anchors the HA protein to the envelope of the influenza virion. The HA1 subunit contains the signal peptide at the N-terminus and the globular head domain. This globular head domain contains the receptor binding site which binds sialic acid on the surface of the host cell and facilitates viral entry [7]. Upon internalization of the virion, acidification of the endosome induces a conformational change of HA, which exposes the N-terminus fusion peptide of the HA2 subunit. The fusion peptide then facilitates membrane fusion and release of the viral RNA into the cytoplasm of the host cell [22,23]. The HA trimer of an H3N2 virus was downloaded from the Protein Data Bank (PDB: 1HGF; A/X-31) and visualized with PyMOL. Two monomers are colored in grey while the third monomer shows the head region in green and the stalk region in blue. An enlarged view of the HA monomer is further colored to show the fusion peptide in red, the long alpha helix (LAH) in orange, and the receptor binding site (RBS) on the head circled in yellow. (B) A linear schematic of the HA molecular is shown below. The head domain (green) is on the HA1 subunit, while the stalk domain (blue) spans the C-and N-terminus of HA1 along with most of HA2. At the N-terminus of HA1 is the signal peptide (SP) while at the N-terminus of HA2 is the fusion peptide. The transmembrane domain (TMD) and cytoplasmic tail (CT) are at the C-terminus of HA2.
The globular head of influenza also contains the antigenic sites determined for both H1 and H3 [7]. Neutralizing antibodies for influenza are typically directed against these highly antigenic sites on the globular head and interfere with HA binding to sialic acid [7]. The HA protein of influenza virus has the ability to agglutinate red blood cells. Anti-influenza virus antibodies which bind to HA and inhibit the hemagglutination activity of HA are used as a surrogate measure of determining neutralizing antibody titers (HI titers) [18]. Human serological studies have demonstrated that HI titers of at least 1:40 correlate with protection from influenza infection [18,19]. Antibodies directed against the stalk domain of HA have different mechanisms of action, as discussed below, and cannot be measured using an HI assay.
General Principles of Stalk-Directed Stratgies
One strategy to stimulate broadly reactive antibodies against the large diversity of HA is to target the more conserved stalk region. The stalk region of HA is occluded on the surface of the influenza virion and is therefore under less selective pressure from the immune system [24]. Consequently, the stalk region is more conversed as compared to the globular head of HA, although there is still protein sequence variability within subtypes, as measured by Shannon entropy (Figure 3). Bioinformatic analysis has shown that the stalk domain of HA is evolving at a slower rate than the head domain [25]. Protective efficacy mediated by head-directed antibodies primarily work through direct binding to HA in order to inhibit viral attachment to sialic acid on host cells, thereby preventing viral entry. In contrast, stalk-directed antibodies have been proposed to work through alternative mechanisms, such as inhibiting conformational changes at low pH to prevent virus release from the endosome and preventing maturation of virus by inhibiting cleavage of HA0 [26]. Additionally, indirect Fc-mediated functions have been reported, such as antibody dependent cell-mediated cytotoxicity (ADCC) and triggering of complementdependent cytotoxicity (CDC; reviewed in [26]). This has been further supported by results indicating that stalk antibodies require Fc-mediated interactions for in vivo efficacy [27,28]. While antibodies directed to the head of HA are typically measured by HI titer, the nonclassical effector functions of stalk-directed antibodies require alternative methods to measure antibody titers, such as ELISA or ADCC reporter assay. The conservation scores of the pre-pandemic seasonal H1N1 (sH1N1) influenza viruses were calculated using the ConSurf server (https://consurf.tau.ac.il/ (accessed on 13 March 2021)) and visualized using PyMOL. Protein sequence variability was determined with the Shannon entropy server (https://www.hiv.lanl.gov/content/sequence/ENTROPY/entropy_one.html (accessed on 13 March 2021)) using ClustalW aligned HA protein sequences from pre-pandemic seasonal H1N1 ((B); sH1N1), pandemic H1N1 ((C); pH1N1), and H3N2 (D). All human isolates with complete HA protein sequences (duplicates and lab strains excluded) were downloaded from the Influenza Research Database, resulting in 962 strains for sH1N1 (strains up to 2009), 7423 strains for pH1N1 (strains during and after the 2009 pandemic), and 9043 strains for H3N2. The stalk, head, and fusion peptide are colored in blue, green, and red, respectively. Stalk-directed antibodies have shown cross-reactivity within subtypes and between multiple subtypes within a group and even between group 1 and 2 viruses. A comprehensive list of stalk-directed antibodies discovered have been previously described [29]. Researchers have explored many vaccination strategies to induce these stalk-directed antibodies (Figure 4), however induction of antibodies against the immunosubdominant stalk domain remains challenging in the presence of the immunodominant head domain. Efforts to overcome this challenge include development of "headless" HA constructs, hyperglycosylation of the head, expression of just the long alpha helix (LAH) domain of the stalk, and development of chimeric and mosaic HAs. . Vaccine strategies to increase stalk-directed immunity. Strategies used to induce stalkdirected immunity, such as hyperglycosylation of the HA [30][31][32], development of a headless HA [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47], expression of the LAH fragment alone [48][49][50], and chimeric [51][52][53][54][55][56][57][58][59][60][61][62] and mosaic [63,64] HA proteins, have been illustrated and briefly described here.
Headless HA Constructs
One strategy to increase antibodies directed to the immunosubdominant stalk region is through removal of the immunodominant head domain creating a "headless" HA. Importantly, HA is a metastable protein which undergoes extensive conformation changes at low pH during the infection cycle of influenza [35]. Removal of the HA1 head domain destabilizes the HA2 structure resulting in loss of antibodies targeting the native conformational epitopes. This was demonstrated in the first reported experiment developing a headless HA in 1983, which removed the HA1 domain of HA through chemical treatment with acid and a reducing agent [33]. However, this vaccine did not show protective efficacy, likely due to denaturation of conformational stalk epitopes. Other efforts to express only the HA2 subunit in systems such as recombinant baculovirus or E. coli have resulted in stalk antigens which lack the native confirmation and are not recognized by neutralizing anti-stalk antibodies [34][35][36].
Multiple strategies have since been explored to express the HA stalk region in a nativelike, neutral-pH conformation. One group stabilized the stalk through inserted specific mutations intended to destabilize the low pH conformation of HA2 thereby pushing the protein to a neutral pH conformation. This approach has been applied to both H1 and H3 proteins [37,38]. The stabilized HA2 protein was expressed in E. coli and folded into a neutral-pH conformation. However, mice vaccinated twice with the stabilized H1 stalk protein were protected from mortality, but not morbidity (~18% weight loss), after challenge with a lethal homologous strain [38]. Another group stabilized the H1 or H3 HA2 domain through inclusion of stabilizing linker peptides and vaccinated mice with two doses of DNA protein expression plasmids followed by a virus-like particle (VLP) formulation [65]. Vaccination with this headless HA completely protected mice from a challenge with a homologous virus strain (~5% weight loss) and induced greater in vitro heterosubtypic cross-reactive antibodies.
Other groups have aimed to express stable headless HA in a trimer conformation either as a soluble protein [39][40][41][42][43] or on the surface of virus-like particles (VLP) [44] or nanoparticles [45][46][47]. One group developed a soluble "mini-HA" H1 stalk trimer through multiple structure-based mutations [39]. Three immunizations with this "mini-HA" H1 stalk completely protected mice from weight loss and death after lethal challenge with either heterologous H1 or heterosubtypic H5 influenza virus. Sera from these mice had both neutralizing and ADCC effector functions. Another group stabilized an H1 stalk trimer through six iterative cycles of structure-based mutations and displayed the stalk on the surface of a nanoparticle [45]. Three immunizations with these nanoparticles completely protected mice from heterosubtypic challenge with H5 influenza virus but showed only partial protection in ferrets. Vaccinated mice and ferrets showed strong in vitro antibody binding against group 1 subtypes H1, H2, H5, and H9 with some weak responses to group 2 subtypes H3 and H7. However, there was limited neutralizing antibodies detected against heterosubtypic strains, indicating that protection is likely mediated by other stalk antibodydependent mechanisms, such as ADCC or CDC. This stabilized H1 trimer nanoparticle vaccine has since progressed into a phase I clinical trial with 52 participants and is expected to conclude December 2021 (NCT03814720).
Chimeric HA
To overcome the instability of headless stalk constructs while still boosting stalkdirected antibodies, a chimeric HA protein prime/boost strategy was developed. In this strategy, multiple sequential immunizations of chimeric HA proteins containing the same stalk region, but 'exotic' HA heads, results in a boosting of stalk-directed immunity. A major benefit of this approach is that the full-length HA is expressed, thereby presenting the stalk domain in the correct conformation. This chimeric strategy has been explored for use as H1 [51][52][53][54][55][56][57][58], H3 [59], and influenza B virus vaccines [60].
To boost stalk immunity against H1, mice were sequentially vaccinated with three doses of chimeric HA which all had the same H1 stalk but head regions from H9, H6, or H5 [52]. Mice were completely protected from lethal challenge with three homosubtypic H1 viruses. The authors also explored heterosubtypic protection and, to rule out the contribution of head-directed antibodies, immunized mice with a similar chimeric prime boost strategy but replaced the head domain of the corresponding challenge strain with a H1 head instead. Vaccination with this strategy completely protected mice from death after lethal heterosubtypic challenge with H5, H6, and H3 viruses, however weight loss data is not reported, although the authors state that only minimal weight loss was observed. Efficacy of this vaccine was demonstrated in a preclinical ferret model, where vaccination with the chimeric prime/boost strategy reduced viral nasal wash titers after challenge with a heterologous H1 virus [51] and heterosubtypic H6 virus [57], and demonstrated durability of protection against homologous H1 challenge up to 1 year after immunization [58]. Delivery of the H1 chimeric antigens has been explored utilizing multiple vaccine platforms, including a DNA prime with recombinant protein boosts [52], recombinant live-attenuated virus and inactivated virus [55][56][57], vesicular stomatitis virus (VSV) viral vectors [51,53], and adenovirus vectors [51].
Importantly, results from a phase I clinical trial of 65 participants have been reported [61,62]. Participants received two immunizations with different combinations of chimeric HA live-attenuated virus, inactivated virus, or inactivated virus plus adjuvant vaccines. Vaccination was found to be safe and to successfully induce anti-stalk H1 antibodies. The best results were observed in participants who received two doses of chimeric HA inactivated adjuvanted virus, in which anti-stalk antibodies persisted at 4-fold above baseline for up to 1.5 years. Mice who received a passive transfer of sera from vaccinated participants showed a trend towards reduced weight loss as compared to mice who received sera from the placebo group after challenge with recombinant H1 stalk virus. This clinical trial supports the ability of this chimeric HA vaccine strategy to induce anti-stalk antibodies in humans, however clinical trials are needed to demonstrate the efficacy of these stalk-directed antibodies in protecting humans from infection. While the most progress has been made using the H1 subtype vaccine, this strategy has also been explored for H3 [59] and influenza B [60] with promising results. Combining these candidate immunogens into a multivalent vaccine has not yet been explored.
Hyperglycosylated HA
Another strategy to increase stalk-specific responses is to hyperglycosylate the HA1 head region in order to 'mask' the immunodominant epitopes, thereby directing the response to the stalk [30]. Three immunizations of mice with a hyperglycosylated H1 protein induced stronger anti-stalk antibodies against the homologous H1 stalks than a wild type HA [32].The hyperglycosylated H1 also induced greater cross-reactive antibodies to two heterologous H1 viruses and a heterosubtypic H5 virus. Hyperglycosylated H1 vaccinated mice were protected from mortality, but not morbidity (~10% weight loss), after challenge of mice with a chimeric H1 stalk virus.
This strategy has also been applied to target the H5 stalk [30,31]. Immunization of mice with a replication-defective adenovirus vector expressing a hyperglycosylated H5 clade 1 HA and boosted with recombinant protein induced greater cross-reactive antibodies against three heterologous clade 2 viruses as compared to a wildtype virus [31]. While this vaccination induced homosubtypic antibodies against H5, it did not result in significant antibody responses to other group 1 viruses (H1, H3, H9). Vaccination with hyperglycosylated H5 protected mice from mortality, but not morbidity (~13% weight loss), after challenge with a heterologous clade 2 virus, with no significant difference in weight loss observed between the hyperglycosylated H5 or wildtype HA vaccine. Therefore, while hyperglycosylation of the HA1 head does appear to increase anti-stalk antibodies as compared to the wildtype HA, vaccination does not protect mice from severe influenza morbidity, even against a homosubtypic challenge.
"Mosaic" HA
Another strategy to silence the immunodominance of the HA1 head subunit and direct the immunity to the stalk region involves substitution of the immunodominant antigenic sites on the HA1 head, yielding a "mosaic" HA1 region. One strategy silenced the immunodominant antigenic sites of the H3 head by replacing them with amino acid sequences from an avian HA protein (H10 or H14) [63]. Mice vaccinated with this recombinant mosaic H3 protein induced more H3 stalk-directed antibodies than the commercial inactivated seasonal vaccine. Passive transfer of sera from mice vaccinated with this adjuvanted mosaic recombinant H3 protein protected mice from death after lethal challenge with two heterologous H3 viruses, however mice still experienced severe weight loss (~20% weight loss).
This strategy was also applied to an influenza B vaccine, where immunodominant antigenic sites of an influenza B Yamagata lineage virus was replaced with amino acid sequences from H5, H8, H11, or H13 [64]. Vaccination of mice with a DNA prime and two recombinant protein boosts of this mosaic head protein resulted in complete protection from weight loss and death after challenge with a lethal Yamagata and Victoria strain. Antibodies induced after vaccination with the mosaic HA showed cross-reactive ELISA antibodies against three Yamagata lineage viruses and three Victoria lineage viruses. No HI antibodies or neutralizing antibodies were detected, but there were antibodies detected through an ADCC reporter assay, indicating that efficacy of this mosaic vaccine was conferred primarily through Fc-mediated effector functions.
LAH Fragment
In an attempt to overcome the challenges with expression of a stable headless stalk and the immunodominance of the HA1 head, some strategies instead target only a small portion of the stalk region, such as the long alpha helix (LAH) or fusion peptide. One strategy conjugated the LAH of H3 to a carrier protein [48]. Two immunizations of mice with this LAH vaccine showed complete protection from death after a lethal challenge with heterologous H3 virus, however mice still exhibited severe weight loss (~15% weight loss). This LAH vaccine only partially protected from death after lethal heterosubtypic H5 challenge and did not protect against lethal heterosubtypic H1 challenge. There was similar findings by another group, which expressed the LAH and fusion peptide from H5 in E. coli and refolded the protein from inclusion bodies [50]. Mice immunized with this protein were completely protected from weight loss and death after challenge with the homologous H5 virus but only partially protected from death after challenge with a heterologous H5 virus or a heterosubtypic H1 virus. Additionally, vaccination of mice with the LAH of H1 expressed on a hepatitis B virus-like particle (VLP) demonstrated complete protection from mortality after challenge with a lethal homologous H1 virus, heterologous H1 virus, and heterosubtypic H9 virus, although mice still showed severe weight loss of 15%, 7%, and 19%, respectively [49]. Therefore, although vaccination with the LAH does increase heterosubtypic protection, the morbidity after challenge is often still severe.
Challenges Facing Stalk-Directed Strategies
Although these stalk-directed vaccination strategies do induce greater cross-reactive immunity, the weak immunogenic nature of the stalk domain necessitates as many as three immunizations to induce this immunity. In addition, animals still exhibit severe morbidity even after challenge with homosubtypic strains, often losing more than 10% of their initial starting weight, although they are protected from mortality. This is likely due to the Fc-mediated mechanism of action found to be a large contributor to protection in many stalk-directed strategies, as these mechanisms are not neutralizing but rather contribute to viral clearance after infection [26]. This indicates that although the breadth of reactivity is increased with these stalk-directed approaches, the robustness of the protection is diminished [66].
Additionally, although the stalk region of HA is more conserved than the head region, there is still variability within subtypes ( Figure 4) and the stalk region is still susceptible to selective pressures from the immune system [67]. This is highlighted by the discovery that pre-existing stalk-directed antibody titers select for a stalk-antibody escape mutant after human influenza challenge [67]. Furthermore, while these stalk-directed antibodies do confer protection from mortality in animal models, there is still conflicting literature about the efficacy of these anti-stalk antibodies in humans. One study utilizing a human household transmission experimental design found a correlation between HA stalk-directed antibodies and protection from infection [68]. In contrast, another study using human samples demonstrated that there was no significant correlation between stalk antibodies and protection from influenza virus infection after adjustment for head-specific antibodies [69]. In addition, a phase II trial examining the therapeutic efficacy of a monoclonal stalk-directed antibody showed no clinically significant effect on influenza disease [70]. These conflicting results draw into question the protective efficacy of these anti-stalk antibodies in humans.
General Principles of Consensus-Based Stratgies
Unlike stalk-directed strategies, consensus-based approaches target the full-length HA protein and aim to design an HA protein which is broadly representative of the diverse HA population. These HA genes are computationally designed and therefore are synthetic in nature. Although the central concept behind consensus-based strategies is similar, many different approaches have been developed ( Figure 5). Importantly, unlike stalk-directed strategies, consensus-based approaches typically induce HI antibodies directed against the head region of HA, which is generally accepted as a correlate of protection against influenza infection in humans [29,71]. Consensus-based approaches are typically developed for a single subtype of influenza, with the intention of expanding the approach to multiple subtypes and combining the vaccine constructs into a single multivalent vaccine. Consensus-based antigen design strategies. Consensus strategies are designed by aligning the target HA protein sequence population and determining the most common amino acid at each position. 'Consensus' immunogens are designed using all the HA protein sequences from the phylogenetic tree. 'Micro-consensus' immunogens are created by taking a consensus of each major branch of the phylogenetic tree and delivering the immunogens as a cocktail. 'Centralized' immunogens aim to reduce sampling bias by taking a representative wildtype HA (red stars) from each branch of the phylogenetic tree and designing a consensus of those HA protein sequences. 'COBRA' immunogens also aim to reduce sampling bias and use multiple rounds of consensus building.
Consensus Hemagglutinins
Consensus HA genes are constructed by taking the most common amino acid at each position of the HA after aligning a protein sequence population. A consensus strategy has been explored by multiple groups to target highly pathogenic avian influenza (HPAI) H5N1 virus [72][73][74][75], which poses a substantial pandemic threat. In addition, H5N1 has high viral diversity, with multiple clades and subclades [76]. Two immunizations of mice with a consensus H5 gene expressed in a DNA plasmid induced cross-reactive antibodies against multiple different H5 viruses from two clades [72]. Vaccination conferred complete protection from morbidity and mortality after challenge with two H5 clades but only partial protection after challenge with more distantly related H5 viruses. Additionally, a consensus H5 protein expressed in VLPs completely protected chickens from lethal challenge with two H5 viruses from separate clades [74]. Importantly, these consensus immunogens are designed using all H5 protein sequences in the database, which can lead to geographical sampling/sequence bias and result in a consensus HA that might not accurately reflect the HA diversity of the population.
Micro-Consensus Hemagglutinin
While other consensus designs develop a single consensus HA per subtype, Elliott et al. (2018) explored the efficacy of four micro-consensus immunogens to improve crossreactivity to the H3 subtype [77]. These four micro-consensus genes were expressed in a DNA plasmid and administered twice as cocktail. Vaccination of mice with this cocktail induced strong antibody responses against 8 strains circulating between 1968 and 2014 as measured by ELISA and induced significant HA-specific cellular immunity. Vaccination also protected mice from lethal challenge with two H3N2 strains, with 5-10% weight loss. Importantly, this study finds that a cocktail of consensus immunogens might improve cross-reactivity of highly diverse HA populations, such as the H3 subtype.
Centralized Hemagglutinins
Another consensus-based strategy aims to develop a synthetic HA which localizes to the central node of the phylogenetic tree, thereby minimizing genetic and antigenic differences of unmatched strains. An important limitation to consensus strategy described above is the threat of sampling bias leading to generation of a synthetic HA gene which does not accurately represent the diversity of the population and is biased towards an overrepresented geographical location. Weaver et al. (2011) overcame this by developing a centralized H1 gene using selected representative wildtype HA protein sequences from each major branch of the phylogenetic tree and designing a consensus of those sequences [78]. In this way, each branch is equally represented to prevent sampling/sequencing bias and, as a result, this synthetic HA gene localizes to the central node of the tree. This centralized H1 gene was expressed in a replication-defective adenovirus vector and vaccination of mice resulted in better cross-protection against three H1 strains as compared to mismatched wild type HAs or traditional influenza vaccines after lethal influenza challenge [78]. This strategy was then applied to develop H3 and H5 centralized genes which also demonstrated improved homosubtypic cross-protection [79]. Importantly, a combination of H1, H2, H3, and H5 centralized genes into a single multivalent vaccine did not diminish the increased cross-reactive protection [80]. A single immunization at the high dose of this multivalent formulation demonstrated complete protection from morbidity and mortality after lethal challenge with three H1 strains, three H5 strains, and one H3 strain with partial cross-protection against another two H3 strains. This validates the approach of designing multiple subtype-specific broadly reactive HA immunogens which can then be combined into one multivalent vaccine without reduction in cross-reactivity or interference between immunogens.
COBRA Hemagglutinins
Like the centralized HA design, another antigen design method called computationally optimized broadly cross-reactive antigen (COBRA) aims to minimize sampling/sequencing bias in the target population. The COBRA strategy achieves this through multiple rounds of consensus generation. A COBRA vaccine strategy has been developed and tested for H1 [81][82][83][84][85], H2 [86], H3 [87,88], H5 [89][90][91][92][93][94][95][96], and swine H1 [97]. These COBRA immunogens have primarily been expressed using a VLP platform but have also been explored using ferritin nanoparticles [84] and recombinant live influenza virus [83]. This strategy was first explored as a H5 vaccine targeting only clade 2 and was tested in mice, ferrets [89,90], chickens [95], and non-human primates (NHP) [91]. Vaccination of NHP induced crossreactive HI antibody titers against multiple clade 2 viruses and against a clade 1 and 7 influenza virus. This strategy was then expanded to target the entirety of the H5 diversity by using a cocktail of three COBRA immunogens designed for human clade 2, human and avian clade 2, and all H5 clades [92]. Vaccination of mice with two doses of this cocktail induced protective HI titers to twenty-five H5 viruses from eleven H5 clades/subclades, however cross-protection was only evaluated after immunization with individual COBRA immunogens and was not evaluated after vaccination with the cocktail.
The COBRA strategy was also evaluated for protection against seasonal and pandemic H1 strains [82]. Nine different COBRA H1 immunogens were designed and four immunogens were selected for further investigation based on their increased cross-reactivate HI titer and protection. These immunogens were evaluated in sequential prime/boost immunizations strategies or as a heterologous cocktail. Results found that the broadest HI titers were induced by a combination of COBRA immunogens designed using both seasonal and pandemic H1 strains, with a trivalent cocktail demonstrating protective HI titers to ten of the fifteen H1 strains [82]. This increased HI cross-reactivity extended to a pre-immune ferret model [81]. However, cross-protection was only evaluated against a single pandemic strain.
A similar strategy was used for the H3 subtype, in which seventeen CORBA immunogens were designed with only four of these immunogens showing promising HI titers in mice [87]. The four immunogenic H3 COBRA immunogens were further explored in a ferret model [88]. Although vaccination of naïve mice showed promising cross-reactivity, vaccination of naïve ferrets showed low HI titers and narrow cross-reactivity, with the most cross-reactive immunogen showing protective HI titers to only six of the thirteen H3 strains. In contrast, ferrets that were pre-immune to a historical H3 strain prior to vaccination showed increased cross-reactive HI titers against the entire panel of 13 viruses after vaccination with COBRA immunogens as compared to vaccination with a wildtype HA. This led the authors theorize that, although there was limited efficacy in naïve ferret, the COBRA immunogens were more effective than wildtype HA at recalling broad crossreactive memory B cells from previous influenza infection. Therefore, while this H3 COBRA vaccine might boost cross-reactive immunity in pre-immune adults, more research needs to be performed to examine potential efficacy in naïve populations, such as children. In addition, although the COBRA strategy has been explored for multiple influenza subtypes individually, COBRA has not yet been explored as a multivalent formulation targeting multiple subtypes.
Challenges Facing Consensus-Based Approaches
Consensus-based approaches target the full-length HA protein and therefore typically induce antibodies directed against the variable head region. For this reason, these vaccines are subtype-specific and do not show the cross-reactivity between subtypes of the same phylogenetic group, as is observed in stalk-directed strategies discussed above. However, antibodies induced by these consensus-based approaches have improved cross-reactivity within a subtype and often demonstrate HI activity which is a correlate of protection against influenza infection in humans [29,71]. Therefore, what these approaches lack in breadth of immunity between subtypes, they make up for in robustness of protection within subtypes.
Consensus-based influenza vaccines based on the HA protein are a more recent concept than the stalk-directed strategies and therefore have not progressed to human clinical trials yet. In addition, unlike stalk-based strategies, little work has been performed to evaluate the potential for escape mutants from pre-existing immunity induced by consensus vaccines. The subtype-specific immunity induced by consensus vaccines will necessitate a multivalent cocktail of HA immunogens to protect against the multiple subtypes currently circulating in humans. Further work to demonstrate that a multivalent vaccine containing many immunodominant antigenic sites from different subtypes does not reduce efficacy through interference. However, the centralized HA approach has shown that vaccination of mice with a quadrivalent vaccine showed no reduction in efficacy, indicating that this a promising strategy to induce a robust protective immune response against multiple subtypes relevant to human health.
General Prinicples of Computational Algorithm Approaches
Similar to consensus-based strategies, approaches using computational algorithms target the full-length HA protein and aim to develop a synthetic HA which is broadly representative of the diverse influenza virus population. However, these strategies use complex computational algorithms in order to logically design a broadly reactive HA immunogen in silico.
Ancestral Hemagglutinin
A phylogenetic algorithm called ANCESCON reconstructs the ancestral HA gene using marginal and joint reconstruction methods [98]. Ducatez et al. (2011) used this algorithm to develop a cross-reactive H5 vaccine, in which the putative ancestral gene for avian H5 was predicted and expressed in a recombinant, replication-competent influenza virus [76]. Vaccination of ferrets with the ancestral gene induced in vitro cross-reactivity to five H5 clades and protected ferrets from death after lethal challenge with three H5 viruses from two clades. However, a wild type H5 reference virus also showed similar levels of cross-reactive immunity and protection. Crucially, this study demonstrated a proof-ofconcept that synthetic HA genes could be expressed using an inactivated recombinant virus which is a currently licensed influenza vaccine platform.
This ANCESON algorithm was also used to develop an ancestral avian H9 immunogen that was expressed in a Modified Vaccinia Ankara (MVA) vector [99]. Intramuscular vaccination of chickens resulted in poor cross-reactive HI titers, with measurable titers against only one of the ten avian influenza virus strains and did not reduce viral shedding after challenge. Therefore, although reconstruction of the ancestral HA gene aids in our understanding of the evolutionary processes underlying the HA protein, the improved cross-reactive efficacy of these immunogens has yet to be demonstrated. In addition, phylogenetic uncertainty from long branches and genetic drift might weaken this approach [100].
Mosaic Algorithm
Another algorithm, called the Mosaic Vaccine Designer tool, aims to maximize the potential epitope coverage of the target viral population [101,102]. A graphic visualization of the algorithm approach has been previously described [102]. Simplified, full-length HA protein sequences undergo repeated rounds of in silico recombination and the recombinant HA protein which has the best epitope coverage of the original target population is selected as a single mosaic HA immunogen. Crucially, this algorithm results in a mosaic immunogen that typically has better epitope coverage than a consensus strategy. Mosaic HA immunogens have been designed for H5 [103][104][105][106] and H1 [107]. A mosaic H5 expressed in MVA completely protected mice from morbidity and mortality after lethal virus challenges with four H5 viruses from three clades and provided protection from mortality, but not morbidity (~10% weight loss), after challenge with a heterosubtypic H1 virus [103]. T-cell depletion and passive transfer studies found that homosubtypic protection is primarily from cross-reactive antibodies, while the heterosubtypic protection to H1 is from cross-reactive CD8+ cells [104]. Vaccination with the mosaic H5 immunogens was also shown to increase early viral clearance in the lungs of NHP after challenge with H1 virus [105] and eliminate viral shedding in chicks after challenge with H5 virus [106]. This mosaic algorithm was also used to design a H1 mosaic immunogen which was expressed in a replication-defective adenovirus vector [107]. Vaccination of mice showed increased cross-reactive ELISA antibodies and T-cell responses to a panel of four H1 viruses as compared to mismatched wild type HA immunogens. In addition, the high dose immunization of mosaic H1 provided complete protection from morbidity and mortality after challenge with three H1 viruses. These studies demonstrate the efficacy of a computational algorithm which creates a vaccine antigen optimized to include the most common epitopes from the target population.
Epigraph Algorithm
Similar to the mosaic algorithm, the epigraph algorithm aims to design vaccine antigens which contain the most common epitopes from the target population, thereby repre-senting the diversity of HA protein sequences and biasing towards antigen recognition by the immune system [108,109]. The epigraph algorithm uses a faster graph-based approach and creates a user-defined cocktail of computationally designed HA proteins. A graphic visualization of the algorithm approach has been previously described [108]. The epigraph algorithm has been used to design a swine H3 immunogen, with the goal of reducing the pandemic potential of H3 from zoonotic transmission events [110]. This epigraph HA was expressed in a replication-defective Adenovirus vector and vaccination of mice induced increased cross-reactive antibodies as compared a wildtype HA or the commercial vaccine, with protective HI titers to fourteen of the twenty diverse swine H3 influenza virus strains. Importantly, this improved cross-reactive antibody response induced by the swine H3 Epigraph immunogens was also seen in swine, the target animal of this vaccine. Of interest, this swine H3 epigraph vaccine also induced cross-reactive antibodies with human H3 strains, suggesting the possibility of reduced reverse zoonotic events. Challenge studies in mice showed greater cross-protective efficacy after challenge with three swine H3 strains, however challenge studies in swine are needed to demonstrate this protective efficacy in the target animal. The broad cross-reactive immunity induced by this vaccine demonstrated the efficacy of a cocktail of computationally designed HA immunogens.
Challenges Facing Computational Algorithm Approaches
Computational algorithm approaches face many similar challenges as the consensusbased approaches, such as the possibility of escape mutants and subtype specific immunity induced by vaccination. Computational power continues to progress, and consequently computational algorithm approaches are the most recent advances in novel antigen design for influenza vaccines. Therefore, these approaches are still in their infancy and further research is needed to demonstrate their efficacy. However, the promising results discussed above lend support towards a computational algorithm improving cross-reactive immunity against the HA protein of influenza.
Perspectives
In this review we have explored multiple novel immunogen design strategies which aim to improve cross-reactive immunity against the HA protein for the development a universal influenza vaccine. These strategies target either the more conserved stalk region of HA or aim to design a synthetic full-length HA which is representative of the influenza viral diversity, either through a consensus-based approach or computational algorithms. Some of these strategies have been explored in depth, with headless stalk constructs and chimeric HA progressing to human clinical trials. Other strategies are more recently developed but still present a promising approach towards a universal influenza vaccine. Importantly, the strategies presented here all utilize different vaccine platforms, from viral vectors, to purified recombinant protein, to VLPs. The vaccine platform and immunization strategy can have a profound effect on the cross-reactive immunity induced by vaccination, which therefore makes these strategies difficult to compare head-to-head. However, in general, stalk-directed strategies induce antibodies which cross-react with multiple subtypes within a phylogenetic group and protect from mortality, but still result in significant morbidity after challenge. In contrast, full-length synthetic HA vaccines induce only subtype-specific immunity, but protect mice from both morbidity and mortality. This is likely due to the different mechanisms of action of stalk-directed versus head-directed antibodies. Therefore, a tradeoff is made in which increased cross-reactivity leads to a reduction in the robustness of protection. Overall, many of these novel antigen design strategies have shown promise in improving the cross-reactive immunity to influenza and should be further explored as a universal influenza vaccine. Data Availability Statement: No data was generated in this study. All review material was accurately referenced.
Conflicts of Interest: Eric A. Weaver is an inventor of the H1 mosaic and swine H3 Epigraph immunogens discussed in this review and has a patent application in progress. (Application: 62/734,791, International Application: PCT/US19/52137). | 2021-03-29T05:25:42.744Z | 2021-03-01T00:00:00.000 | {
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6680295 | pes2o/s2orc | v3-fos-license | Human Tissue Kallikrein 5 Is a Member of a Proteolytic Cascade Pathway Involved in Seminal Clot Liquefaction and Potentially in Prostate Cancer Progression*
Human tissue kallikreins (hKs) are a family of fifteen serine proteases. Several lines of evidence suggest that hKs participate in proteolytic cascade pathways. Human kallikrein 5 (hK5) has trypsinlike activity, is able to self-activate, and is co-expressed in various tissues with other hKs. In this study, we examined the ability of hK5 to activate other hKs. By using synthetic heptapeptides that encompass the activation site of each kallikrein and recombinant pro-hKs, we demonstrated that hK5 is able to activate pro-hK2 and pro-hK3. We then showed that, following their activation, hK5 can internally cleave and deactivate hK2 and hK3. Given the predominant expression of hK2 and hK3 in the prostate, we examined the pathophysiological role of hK5 in this tissue. We studied the regulation of hK5 activity by cations (Zn2+, Ca2+, Mg2+, Na2+, and K+) and citrate and showed that Zn can efficiently inhibit hK5 activity at levels well below its normal concentration in the prostate. We also show that hK5 can degrade semenogelins I and II, the major components of the seminal clot. Semenogelins can reverse the inhibition of hK5 by Zn2+, providing a novel regulatory mechanism of its serine protease activity. hK5 is also able to internally cleave insulin-like growth factor-binding proteins 1, 2, 3, 4, and 5, but not 6, suggesting that it might be involved in prostate cancer progression through growth factor regulation. Our results uncover a kallikrein proteolytic cascade pathway in the prostate that participates in seminal clot liquefaction and probably in prostate cancer progression.
Proteolytic cascade pathways are implicated in many physiological functions such as blood coagulation, fibrinolysis, apoptosis, digestion, among others (1). Proteases are usually synthesized as inactive zymogens and require limited (auto)proteolysis of their propeptide to become active (2). Activation of a zymogen by the activated form of another protease can give rise to proteolytic cascades. This allows rapid amplification of the initial signal, followed by downstream control through inhibitors or (auto)digestion. Serine proteases, the second largest family of proteases, are known to participate in proteolytic cascade pathways, e.g. factors VII, X, and XI, during the coagulation and fibrin formation cascade (1).
Human tissue kallikreins are 15 homologous serine protease genes that co-localize in tandem to chromosome 19q13.4 (3)(4)(5). The associa-tion of multiple members of this family with many cancer types, such as prostate, breast, and ovarian, as well their diagnostic/prognostic value have been extensively studied (6 -8). Human kallikrein 3 (hK3/prostate-specific antigen) 2 is a valuable biomarker for prostatic adenocarcinoma (9). Recently, it has been realized that human kallikreins may function through proteolytic cascades (10,11). KLK1 genes reside at a single locus, and many are regulated by steroids and co-expressed in various tissues and fluids and concurrently up-or down-regulated during tumor progression (5,12). Similarly to hKs, several serine proteases involved in sequential steps during the coagulation cascade are encoded by tandemly co-localized genes, and some may share a common ancestor (1,13,14). Furthermore, the facts that 14 of the 15 kallikreins (except hK4) require cleavage of their propeptide after lysine (hK6, hK7, hK8, hK12, hK13, hK14, and hK15) or arginine (hK1, hK2, hK3, hK5, hK9, hK10, and hK11) by a trypsin-like enzyme, and 12 of them are predicted to have trypsin-like activity (except hK3, hK7, and hK9, which have chymotrypsin-like activity), strengthen the possibility that hKs are part of an as yet elusive proteolytic cascade.
Currently, it is known that hK2, hK4, and hK15 can activate pro-hK3 in vitro and that they may be involved in a proteolytic cascade in prostate tissue and seminal plasma (15)(16)(17)(18)(19). Other members of the family, such as hK5, hK8, hK11, hK13, and hK14, are also expressed in the prostate and are secreted in seminal plasma, so they might also participate in related cascades. Brattsand et al. (11) have recently suggested that a proteolytic cascade of kallikreins operates in the stratum corneum and that hK5, in vitro, can activate pro-hK7. In addition, some hKs, i.e. hK2 (20,21), hK5 (11), hK6 (22,23), and hK13 (24), are capable of autoactivation and may, therefore, be involved in the initiation and maintenance of a cascade, similar to factor XI of the intrinsic coagulation (25).
Human kallikrein 5 (hK5, encoded by the KLK5 gene) is a relatively new member of the human kallikrein family of serine proteases (26,27). Studies have shown that KLK5 is differentially regulated in a variety of hormone-dependent malignancies, including ovarian (28), breast (29), prostate (30), and testicular (31) cancers. By using an hK5-specific enzyme-linked immunosorbent assay, we have recently shown that hK5 is a potential biomarker for ovarian and breast cancer (32,33). We have previously shown that hK5 has trypsin-like activity with strong pref-erence for Arg over Lys for the P1 position and that its activity is inhibited by the serpins ␣ 2 -antiplasmin and ␣ 1 -antithrombin (34). Furthermore, we proposed that hK5 is implicated in tumor progression by degrading components of the extracellular matrix such as collagen types I-IV, fibronectin, and laminin and by releasing angiostatin4.5 from plasminogen (34).
In this study we examined the interaction of hK5 with the remaining members of the kallikrein family and its ability to activate them. We show that hK5 is able to activate pro-hK2 and pro-hK3 and subsequently deactivate them. Our data also indicate that hK5, along with other members of the human kallikrein family, may participate in a proteolytic cascade pathway that plays a role during seminal clot liquefaction and potentially in prostate cancer progression.
Cleavage of Heptapeptides by hK5-25 g of the heptapeptides were incubated with 1 g of hK5 (1500:1 molar ratio) in assay buffer (hK5 optimal buffer, 100 mM phosphate buffer, 0.01% Tween 20, pH 8.0), at a final volume of 150 l. The reaction was incubated at 37°C for 0.5, 1, 2, 4, and 8 h and terminated by freezing the samples with liquid nitrogen. 100 l from each time point was diluted 1.5-fold with loading buffer (distilled H 2 O, 0.1% trifluoroacetic acid) and loaded to a C18 column connected to an high-performance liquid chromatography system at a flow rate of 0.8 ml/min. Elution was performed by using Buffer A (distilled H 2 O, 0.1% trifluoroacetic acid) and Buffer B (acetonitrile 0.1% trifluoroacetic acid) with a linear gradient of 0 -100% acetonitrile at a flow rate of 0.8 ml/min.
Activation of Pro-hK3 and Pro-hK2 mut by hK5-The activation of pro-hK2 mut and pro-hK3 was monitored by complex formation of hK2 and hK3 with the serpin ACT, an inhibitor for the aforementioned kallikreins but not for hK5. After incubating pro-hK2 mut and pro-hK3 with hK5 at different molar ratios and incubation times, ACT and assay buffer for hK2 and hK3 (hK2 optimal buffer, 0.1 mM Tris-HCl, 0.1 mM NaCl, 0.01%Tween 20, pH 7.5, and hK3 optimal buffer, 0.1 mM Tris-HCl, 3 mM NaCl, 0.01% Tween 20, pH 7.5) were added in a final volume 150 l. The reaction was incubated for 4 h at 37°C and terminated by freezing in liquid nitrogen. Positive and negative controls were included as well. 25-l samples of each reaction were run on SDS-PAGE under reducing conditions and stained with Coomassie Blue to monitor the hK2/3⅐ACT complex formation.
The activation of pro-hK3 by hK5 was also monitored by the release of AMC or pNA and the increase of fluorescence or absorbance, respectively, from hK3-specific substrates, i.e. AAPF-AMC and RPY-pNA. Pro-hK3 was incubated with hK5, at different molar ratios and incubation times, in hK5 assay buffer at 37°C. The final volume was 50 l, and the reaction was terminated by the addition of aprotinin (1:100 molar ratio). The activation of pro-hK3 was monitored by adding AAPF-AMC, as above, and assay buffer in a final volume of 200 l. Reactions were set up in microtiter wells and incubated at 37°C. Fluorescence or absorbance increase was measured for 20 min on a Wallac Victor fluorometer (PerkinElmer Life Sciences) set at 355 nm for excitation and 460 nm for emission for the AMC substrate and absorbance at 405 nm for the pNA substrate. Enzyme-free reactions, for all substrate concentrations, were used as negative controls, and background fluorescence or absorbance was subtracted from each value. A reaction with hK5 alone and pro-hK3 without incubation with hK5 were used as negative controls. Duplicate reactions were run on SDS-PAGE under reducing conditions in two different gels. The first was stained with Coomassie Blue, whereas the second was electroblotted onto nitrocellulose membranes (Hybond TM -C Extra). Western blots were performed by using a polyclonal hK3 as primary antibody.
Western Blotting for Detection of the hK3 Fragments in Seminal Plasma and Prostate-Seminal plasma from healthy individuals were leftovers of samples submitted for routine biochemical testing that had been stored at Ϫ80°C. The prostate extracts were the healthy samples from matched healthy cancerous prostatic tissue pairs were obtained from prostate cancer patients who had undergone radical retropubic prostatectomy for prostatic adenocarcinoma at the Charite University Hospital (Berlin, Germany). The patients had not received hormonal therapy before surgery. The tissue samples were dissected from cancerous and noncancerous (healthy) portions of the prostate immediately after surgical removal. The samples were stored in liquid N 2 until needed. To determine the malignant or benign nature of the tissue samples, a histological analysis was performed as described previously (36). These samples were approved for research purpose use by the Ethics Committee of the Charite Hospital. The samples were resolved by SDS-PAGE (NuPAGE 4 -12% Bis-Tris gels, Invitrogen) and subsequently electroblotted onto nitrocellulose membranes (Hybond TM -C Extra). Western blots were performed as above.
Effect of Cations (Zn 2ϩ , Ca 2ϩ , Mg 2ϩ , Na ϩ , and K ϩ ) and Citrate on hK5 Activity-To determine the effect of the cations (solutions made from salts of ZnCl 2 , NaCl, KCl, MgCl 2 , and CaCl 2 ) and citrate on hK5 activity, reactions mixtures were set up as follows: purified recombinant hK5 (final concentration of 12 nM) was incubated with each cation and citrate (final concentrations of 0, 12, 60, 120, 1,200, and 12,000 nM) diluted in the assay buffer (0.1 mM Tris-HCl, 0.1 mM NaCl, 0.01% Tween 20, pH 8.0) at a final volume of 100 l in microtubes for 10 min at 37°C with gentle agitation. After incubation, the fluorogenic substrate VPR-AMC (final concentration of 1 mM) was applied to each hK5-cation/citrate mixture separately. Reactions were set up in microtiter wells and incubated at 37°C. Fluorescence was measured for 15 min as described before. Enzyme-free reactions, for all cations/citrate and substrate, were used as negative controls, and background fluorescence was subtracted from each value. All experiments were done in triplicate.
Reversal of Zn 2ϩ Inhibition of hK5 Activity by Semenogelin I and II-hK5 (final concentration, 10 nM) was incubated with Zn 2ϩ (final concentration, 5 M) to a final volume of 100 l in microtubes for 10 min at 37°C with gentle agitation. After incubation, the fluorogenic substrate VPR-AMC (final concentration of 0.8 mM) was added. Reactions were set up in microtiter wells and incubated at 37°C. Fluorescence was measured for 20 min as described before. At certain points, during measurement, semenogelin I or II (final concentration, 0.05 M) and EDTA (final concentration, 10 mM) were added in each microtiter well. Enzyme-free reactions were used as negative controls, and background fluorescence was subtracted from each value. All experiments were done in triplicate.
Cleavage of Semenogelins I and II by hK5-Purified semenogelins I and II (5 g) were incubated separately with 165 ng of hK5 in 100 mM Na 2 HPO 4 , 0.01% Tween 20, pH 8.0, 0.2 M urea or with 165 ng of hK2 in 0.1 mM Tris-HCl, 0.1 mM NaCl, 0.01% Tween 20, pH 7.5, 0.2 M urea at 37°C for 2 and 8 h. The reactions were terminated by freezing in liquid nitrogen. Subsequently, the reactions were run on SDS-PAGE under reducing conditions, and the gels were stained with silver staining.
Cleavage of IGFBPs by hK5-IGFBP1-6 (450 ng) were incubated separately with 50 ng of hK5 in 100 mM Na 2 HPO 4 , 0.01% Tween 20, pH 8.0 at 37°C for different time points. The reactions were terminated by freezing in liquid nitrogen. The reactions were run on SDS-PAGE under reducing conditions, and the gels were stained with silver staining.
N-terminal Sequencing-N-terminal sequencing was performed with the Edman degradation method. Proteins were transferred by electroblotting to a polyvinylidene difluoride membrane and visualized with Coomassie Blue stain.
RESULTS
Cleavage of Heptapeptides by hK5-Because fourteen out of the fifteen human kallikreins (except hK4) require cleavage after Arg (hK1, hK2, hK3, hK5, hK9, hK10, and hK11) or Lys (hK6, hK7, hK8, hK12, hK13, hK14, and hK15) for propeptide release and activation, and hK5 has trypsin-like activity (34), we designed heptapeptides encompassing the putative P1-P4 and PЈ1-PЈ3 positions of the activation site of each kallikrein. We incubated these peptides with hK5 for various time intervals, and the reactions were monitored with high-performance liquid chromatography chromatography, using a C 18 column. The time-dependent decrease of the height and the area under the main peak representing the intact heptapeptide and the generation of one or two new peaks representing the P1-P4 and PЈ1-PЈ3 fragments was indicative of the efficiency of hK5 to cleave each heptapeptide. hK5 was able to cleave the hep-tapeptides encompassing the cleavage sites for hK1, hK2, and hK3 with high efficiency; the heptapeptides for hK5, hK9, hK11, and hK12 with moderate efficiency and the heptapeptides for hK7, hK8, and hK15 with low efficiency. No cleavage was observed for the heptapeptides for hK4, hK6, hK10, hK13, and hK14. The data are summarized in Table 1.
Activation and Deactivation of Pro-hK3 and Pro-hK2 mut by hK5-Given the above findings (Table 1) we examined the ability of hK5 to activate recombinant pro-hK2 and pro-hK3. After incubating both pro-hK2 mut and pro-hK3 with hK5 we monitored their activation through binding to the serpin ACT. Both pro-hK2 mut and pro-hK3, after incubation with hK5, became active and formed a complex with ACT (Fig. 1, A and B).
Because hK3 has chymotrypsin-like activity, we monitored its activation by hK5, by adding a chymotrypsin-like fluorogenic or chromogenic substrate. The activation was dependent on the pro-hK3:hK5 molar ratio and the time of incubation (Fig. 2, A and B). At a pro-hK3:hK5 molar ratio of 10:1, the reaction rate decreased in a time-dependent manner (Fig. 2C). By SDS-PAGE and Coomassie Blue staining we observed generation of degradation products of hK3 (Fig. 3A). The bands 1, 2, and 5 had N-terminal sequence of IVGGWE due to activation of pro-hK3 by hK5 (cleavage of the peptide bond at the activation site between Arg Ϫ1 and Ile 1 ), bands 3 and 4 had a sequence of FLRPGDD due to internal cleavage of hK3 between Arg 85 and Phe 86 , whereas band 6 had a sequence of STCSGDS due to internal cleavage of hK3 between Lys 182 and Ser 183 . These results indicate that hK5 is able to activate and subsequently deactivate hK3 by internal cleavage. Table 3. The open arrowhead represents the low molecular weight fragment cleaved from ACT after the ACT⅐hK2 complex formation. B, hK5 was incubated with pro-hK3 (0.5 M) at 1:5 molar ratio for 5 min at 37°C (lanes 6). Subsequently, ACT at an hK3:ACT molar ratio of 1:10 was added, and the reaction was incubated for 2 h at 37°C. The activation was monitored by the ACT⅐hK3 complex formation (arrow). hK5 and pro-hK3 were incubated with ACT (without hK5) as negative control (lanes 4 and 5, respectively). The ACT inhibitor and the kallikreins hK5 and pro-hK3 alone are shown in lanes 1-3, respectively. The open arrowhead represents the low molecular weight fragment generated from ACT after the ACT⅐hK3 complex formation. M, molecular mass standards with shown masses in kilodaltons. Clipped forms of the PSA/hK3 in seminal plasma have been previously described. Typically, 20 -30% of hK3 in this fluid is clipped between residues Arg 85 and Phe 86 , Lys 145 and Lys 146 , and Lys 182 and Ser 183 (37)(38)(39). By using a polyclonal antibody against hK3, we performed Western blots of seminal plasma, prostate extracts and at different time points of the activation of pro-hK3 by hK5. We were able to detect four of the five predicted fragments generated by the incubation of pro-hK3 with hK5 (one fragment is probably not recognized by our antibodies) (Fig. 3B). In seminal plasma, we detected all four bands generated by the cleavage of pro-hK3 by hK5 at the peptide bonds Arg 85 -Phe 86 and Lys 182 -Ser 183 plus two bands generated by the cleavage at the peptide bond Lys 145 -Lys 146 (Fig. 3C). Interestingly, we were able to detect the same fragments in prostate tissue extracts (Fig. 3D), indicating that the deactivation of hK3 may initiate within the prostate, after its secretion.
During the activation of pro-hK2 mut by hK5 we also observed the generation of two fragments (Fig. 1A, lane 6). The N-terminal sequence of band 1 was IVGGWE due to activation of pro-hK2 mut by hK5 (cleavage of the peptide bond at the activation site between Arg Ϫ1 and Ile 1 ). The N-terminal sequence of band 2 was SLQCVSL due to internal cleavage of hK2 mut between Arg 145 and Ser 146 . hK2 has been shown to internally cleave itself after autoactivation (40). However, the overall catalytic efficiency of the mutated hK2 form, i.e. hK2 mut , used in this study, is Ͻ0.01% of wild-type hK2 (41). In the same study, it has also been shown that hK2 mut has a slightly altered P1 from Arg to Tyr. These data led us to conclude that hK2 mut fragmentation is due to hK5 activity and not to autolysis.
Effect of Cations and Citrate on hK5 Activity-The cations Zn 2ϩ , Ca 2ϩ , Mg 2ϩ , Na ϩ , and K ϩ and the anion citrate are present at high levels in seminal plasma and prostatic fluid and play an important role in the regulation of protease activity (42). After incubating each of the ions with hK5, we added the fluorogenic substrate VPR-AMC to monitor residual enzyme activity. Citrate seems to enhance hK5 activity at relatively high amounts (Table 2). From the cations tested, Zn 2ϩ showed strong inhibition of hK5 activity (Fig. 4A and Table 2). At a 1:10 molar ratio (hK5: Zn 2ϩ ) the inhibition was 97.5%, indicating that the high levels of Zn 2ϩ in seminal plasma and prostatic fluid could regulate hK5 activity. The ability of Zn 2ϩ to efficiently inhibit hK2 (20) and hK3 (43,44) has been previously investigated. Sodium and other tested cations had no effect on hK5 activity (Table 2), in contrast to hK3 whose activity increases with increasing Na ϩ (44). 1 and 2, respectively. The fragments that have been subjected to NH 2 -terminal sequencing are indicated by arrows. The hK3 fragments generated by hK5 can be detected by Western blots in tissue extracts and seminal plasma (closed arrowheads; panels C and D, respectively). The open arrowheads represent hK3 fragments generated by unknown protease(s). For more information see Table 2 and "Discussion."
Reversal of Zn 2ϩ Inhibition by Semenogelins I and II-Semenogelins
and regulate the activity of hK3. In this study, after incubating hK5 with Zn 2ϩ , and accessing hK5 residual activity with the fluorogenic substrate VPR-AMC, we demonstrated that addition of semenogelins I and II was able to rapidly reverse the inhibition, likely by sequestration of Zn 2ϩ by semenogelins (Fig. 4B).
Cleavage of Semenogelins I and II-Semenogelins have the tendency to aggregate, therefore we used urea (0.2 M final concentration) to keep them in solution. hK2 and hK3 are known to cleave semenogelins and play a crucial role in the processes that leads to the liquefaction of the seminal clot after the ejaculation (20,43,47). In this experiment the same amounts of hK5 and hK2 wt (internal control) were incubated in separate reactions with semenogelins I and II. After 8-h incubation, hK5 was able to fully digest semenogelin I and II (Fig. 5, A and B). The generation of fragments was observed within 5 min after initiation of the reaction (data not shown).
Cleavage of IGFBP1-6 by hK5-IGFBPs comprise a family of six soluble proteins with a primary physiological role to interact and regulate the bioavailability of insulin-like growth factors (IGF-I and IGF-II) by forming complexes and sequestering IGFs away from their receptors, i.e. IGF-IR (48,49). Numerous studies have shown that their ability to bind IGFs is due to their N and C termini, which are highly conserved among them (50). The middle (or linker) domain is the least conserved region of this protein family and is the target of many proteases, e.g. human kallikrein 3, plasmin, thrombin, MMP1-3, and cathepsin L (48,51). Cleavage within this domain will result in the release of the two termini and abolishment of the ability of IGFBPs to bind IGFs. We examined if hK5 is also able to cleave IGFBPs in vitro. Indeed, hK5 was able to cleave all IGFBPs (Fig. 6) except IGFBP6 (data not shown). All major cleavage sites were located within the linker domain ( Table 3). Two of these sites, i.e. IG3c and IG5c, were also cleavage sites for thrombin (52,53).
DISCUSSION
Human tissue kallikreins represent the largest group of serine proteases in the human genome. Members of this family are primarily known for their clinical applicability as cancer biomarkers (6 -8). Their involvement in pathological (e.g. cancer progression, neurodegeneration, and skin diseases) and physiological processes (e.g. seminal plasma liquefaction and skin desquamation) is recently becoming apparent and represents a challenging area of investigation (3,4). Ample evidence suggests the existence of cross-talk among members of the human kallikrein family (10). Here, we examine the involvement of human kallikrein 5 in a kallikrein proteolytic cascade and the role of this pathway during physiological and pathological conditions in the prostate. We show, for first time, that hK5 is able to efficiently activate both pro-hK2 and pro-hK3. Previous reports have demonstrated that hK2 (15)(16)(17), hK4 (18), and hK15 (19) are also able to activate pro-hK3. However, the activation by hK2 seems to be significantly lower than that of hK4 and hK15 (18,19). Human kallikrein 5, along with hK4, are the most efficient potential activators of pro-hK3, because both are able to activate it within 5 min at a pro-hK3:hK4/5 molar ratio of 10:1; with hK5 being able to completely convert pro-hK3 to its mature form. Takayama et al. (17), after fractionating seminal plasma with gel filtration, were unable to activate pro-hK3 with the fraction predicted to contain hK2. Instead, the activator was in the fraction of the range 60 -120 kDa, indicating that the physiological activator has a higher molecular mass. Human kallikrein 5 is a strong candidate, because recombinant hK5 has a molecular mass of ϳ50 kDa when produced in a mammalian system. Previous studies have shown that hK2 is able to become autoactivated (21,40). In addition, we here show that hK2 is also activated by hK5.
Interestingly, hK5 had the ability to also deactivate both hK3 and hK2 by internal cleavage. 20 -30% of human kallikrein 3 isolated from seminal plasma is known to be clipped between residues Arg 85 and Phe 86 , Lys 145 and Lys 146 , and Lys 182 and Ser 183 (54 -56). Until now, the enzyme that was responsible for its cleavage and inactivation was unknown.
Here we showed that hK5 is able to internally cleave and inactivate hK3 between residues Arg 85 and Phe 86 , and Lys 182 and Ser 183 (55). Furthermore, we show that these clipped forms, in addition to seminal plasma (54 -56), also exist in prostate tissue extracts. Human kallikrein 2, once autoactivated, is then autodeactivated by internal cleavage between residues Arg 145 and Ser 146 , and Arg 226 and Lys 227 (40). Our results suggest that hK5 can also internally cleave between residues Arg 145 and Ser 146 and deactivate hK2 mut , which abolishes its ability to autodeactivate (41). Deactivation by internal cleavage seems to be a common way of regulation of human kallikrein activity. hK6, -7, -13, and -14 have been shown to autodeactivate (23,24,57,58), hK11 to be internally cleaved, probably by plasmin, 3 and hK5 to exist in clipped forms in vivo (27).
One of the main characteristics of the prostate epithelial cells is their ability to accumulate cellular levels of zinc that are 3-to 10-fold higher than other mammalian cells (59). Previous studies have shown that zinc has the ability to inhibit the activity of hK2 (20) and hK3 (43,44). Our results suggest that zinc efficiently inhibits hK5 activity as well. This inhibition is reversible, and for the first time we showed that semenogelins I and II are able to revert it. Jonsson et al. (46) extensively studied Table 2 and "Discussion." the ability of semenogelins to bind zinc and regulate hK3 activity. The redistribution of zinc to semenogelins from human kallikreins may have an important physiological role during ejaculation and liquefaction of the seminal clot, as discussed below. Furthermore, zinc seems to also have a major role in the pathogenesis of prostate cancer (59). Its levels decrease 10 and 20 times in the prostate tissue and prostatic fluid, respectively (60,61). This is primarily due to the down-regulation of the zinc-accumulating apparatus (ZIP1 and ZIP2), which are responsible for the uptake and accumulation of zinc in prostate cells, in the malignant loci as compared with the adjacent normal tissue (62). The decrease of zinc levels during prostate cancer initiation will lead to higher human kallikrein activity, which could contribute in prostate cancer progression. Semenogelins I and II are dominant proteins of human seminal plasma, which, together with fibronectin, give rise to the gel-like coagulum of newly ejaculated semen (35,45,63). Various studies have shown that both hK2 (20) and hK3 (43,47) are able to degrade semenogelins I and II. Here, we show that hK5 is also able to efficiently degrade semenogelins. Given that hK5 is able to also degrade fibronectin (34), it is possible that hK5, along with other human kallikreins, contributes in the liquefaction of the seminal plasma shortly after ejaculation.
Overall, the results of this study point to the existence of a proteolytic cascade pathway in the prostate, in which human kallikreins play a predominant role (Fig. 7). Under physiological conditions, hKs are activated in the prostate but are "silenced" by an allosteric reversible inhibition by Zn 2ϩ . After ejaculation, hKs are reactivated due to Zn 2ϩ redistribution to semenogelins, and liquefy the seminal clot, leading to the release of motile spermatozoa (70). On the other hand, during prostate cancer initiation and progression, the decrease of Zn 2ϩ levels may result in an increase in hK activity and increased degradation of extracellular matrix components and insulin-like growth factor-binding proteins. These events could enhance prostate cancer progression and metastatic potential. In addition to Zn 2ϩ regulation, this pathway seems to be regulated by two additional mechanisms: (auto)degradation by internal cleavage and inhibition by serpins (4).
The predominant role of serine proteases in many enzymatic cascade pathways is well established. Here, we propose the involvement of FIGURE 7. Human kallikrein physiology and pathobiology in the prostate. A, role of the proteolytic cascade pathway after ejaculation and during prostate cancer. At ejaculation, the sperm-rich epididymal fluid mixes with prostatic fluid (including hK2, -3, -4, -5, -8, -11, and -15) along with the secretions of the seminal vesicles, (i.e. semenogelins I and II, and fibronectin), constituting the seminal plasma. The seminal vesicle secretion constitutes ϳ60% and the prostatic secretion ϳ30% of the ejaculated volume. Semenogelins I and II along with fibronectin are the predominant structural proteins of seminal plasma aggregate together to form a gelatinous mass. Semenogelins have the capacity to capture Zn 2ϩ , leading to hKs activation and subsequent degradation of the semenogelins and fibronectin, resulting in seminal clot liquefaction. During prostate cancer both the facts that zinc levels decrease, primarily due to down-regulation of zinc transporters, and hK levels increase, leading to increased serine protease activity and subsequent degradation of extracellular matrix components and IGFBPs. B, regulation of the proteolytic cascade pathway by autodegradation of hKs and by inhibition of hKs by serpins. This model was developed based on data presented here and previous literature reports. For more details refer to the "Discussion." | 2018-04-03T03:32:10.390Z | 2006-05-05T00:00:00.000 | {
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6428997 | pes2o/s2orc | v3-fos-license | A role for human Dicer in pre-RISC loading of siRNAs
RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3′ overhangs and the thermodynamic properties of 2–4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.
The RNAse III family member Dicer, along with co-factor RNA-binding proteins, processes long dsRNAs and pre-miRNAs into the functional 21-23-nt siRNA or miRNA duplexes. The functional roles of Dicer have been most well studied in Drosophila where two distinct enzymes are involved in parallel pathways for small regulatory RNA biogenesis. Dicer-2 (Dcr-2) processes long-double-stranded precursors and generates siRNAs (19,20) while Dicer-1 (Dcr-1) processes pre-miRNAs into mature miRNAs (20). Importantly, these two enzymes utilize different co-factors for their respective functions. Dcr-1 is associated with the dsRNA-binding protein Loquacious (Loqs) (21,22) whereas Dcr-2 is associated with the dsRNA-binding protein R2D2 (19,23). In contrast, mammals possess only a single species of Dicer in association with the dsRNA-binding protein TRBP, which participates in both si-and miRNA biogenesis (24,25). Given this fundamental difference between human and fly RNAi pathways, we sought to characterize the mechanism by which highly functional siRNAs are selected in humans. *To whom correspondence should be addressed. Tel: +1 626 301 8360; Fax: +1 626 301 8271; Email: jrossi@coh.org In humans, the HIV-1 TAR RNA-binding protein (TRBP) has been characterized as one of the factors that assemble Dicer-generated small RNAs into RISC by interacting directly with Dicer and Ago2 (26)(27)(28)(29). TRBP has been shown to have versatile roles in small regulatory RNA biogenesis. TRBP has high-sequence similarity to Drosophila Loqs and mammalian PACT (30), and is required for both si-and miRNA silencing (27,29). The Dicer-TRBP interaction is RNA independent (29). TRBP is required for the recruitment of Ago2 to the siRNA-Dicer complex (27) and is proposed to participate in coupling of the RNAi initiation and effector steps. Other studies have shown that MOV10 (31,32) and RNA Helicase A (33) are additional factors that associate with human RISC, forming an active complex. However, little is known about their modes of action in RNAi.
The selection of the guide strand which serves as a sequence-specific RNAi trigger largely determines the efficacy of RNAi (3). Effective siRNAs most often have a thermodynamically unstable 5 0 guide strand end (34), and often have asymmetric loading of the guide versus passenger strands (35). In Drosophila, the orientation of the guide strand of the siRNA is determined by the Dcr-2/ R2D2 heterodimer siRNA binding, with Dcr-2 binding at the 5 0 -end of the guide strand and R2D2 binding the 5 0 -end of the passenger strand (36). R2D2 is also sensitive to the 5 0 -end stability and the presence of 5 0 phosphates on the siRNA (36).
In comparison to observations with the siRNAs, which often show a wide range of potency (37)(38)(39), Dicer processing of its substrates during the initiation step of RNAi was shown to result in better programming of RISC (9,19,28). Furthermore the polarity of Dicer processing preferentially defines which strand serves as the guide (40). Conversely, it is known that Dicer processing is dispensable for RISC assembly and siRNA-mediated cleavage of target-RNA transcripts when siRNAs were exogenously introduced into cells lacking functional Dicer (41)(42)(43). Interestingly, in Drosphila, an organism which expresses both siRNAs and miRNAs as regulatory small RNAs, the Dcr-2/R2D2 heterodimer was shown to act as a gatekeeper promoting the incorporation of siRNAs for assembly with Ago2 while disfavoring miRNAs as loading substrates for Ago2 (44). The sorting process depends upon the structure of the small RNA duplex and not upon Dcr-2 cleavage.
Aside from the appropriate selection of the guide strand for incorporation into RISC, there is the concern of off-targeting in which either the sense or antisense strand binds to non-targeted sequences triggering inhibition of expression of these transcripts (45)(46)(47)(48). Therefore in order to enhance the efficacy of RNAi, it is essential to better understand the mechanism of siRNA selection and strand incorporation in the RNA-silencing pathway.
Here we show that siRNAs associate with a high-molecular weight complex in HEK293 and HCT116 whole cell extracts. The degree of this association varies from siRNA to siRNA, in some cases dramatically between two siRNAs differing by only a single base pair. We observe that Dicer is the core siRNAduplex-binding component of the whole cell extract complex. Dicer preferentially recognizes the 2-nt 3 0 overhangs and thermodynamically unstable ends. Since Dicer can bind to a siRNA duplex in either orientation, either strand of the duplex can be selected as the guide strand based on the thermodynamic end properties of the duplex. Thus, when siRNAs are delivered exogenously to cells, human Dicer (most likely in the form of a complex with TRBP and Ago2) serves as the primary sensor for the selection of highly functional siRNAs leading to handoff to Ago2. This selective process is based upon the presence of ribose 2-nt 3 0 overhangs and the thermodynamic end properties of the siRNAs. When siRNAs cannot interact with Dicer, such as in Dicer null cells, they can bypass the Dicer-binding step and directly enter RISC. Nevertheless when Dicer is present, it serves as a primary sensor for siRNA selection when the siRNAs contain an appropriate structure.
Synthetic siRNAs
All siRNAs used in this study were synthesized by IDT (Coralville, Iowa). The siRNAs were purified using high-performance liquid chromatography. The sequences are listed in Figures 1 and 3.
Cell extracts
HEK293 cells were grown to confluency in 10-cm dishes, harvested and washed with 1x PBS. The cell pellet was re-suspended in 0.5 ml of buffer D (20 mM HEPES, pH. 7.9, 0.2 mM EDTA, 0.5 mM DTT, 50 mM KCl, 10% Glycerol, 0.2 mM PMSF) and sonicated three times for 20 s on ice with a Sonifier 450 (Branson, Danbury, CT) at a setting of 60. The supernatants were collected after 15 min of microcentrifugation for direct use in subsequent experiments.
Dual luciferase reporter assays
To generate reporter plasmids psi-hnRNPH-S (sense reporter) and psi-hnRNPH-AS (antisense reporter), a 343-bp PCR fragment of hnRNPH cDNA (Acc.: NM_005520) was cloned in the 3 0 -UTR of the humanized Renilla luciferase gene in the psiCHECK TM -2 vector (Promega) in either the sense or antisense orientation (40). HCT116 cells at 60-80% confluence in 24-well plates were transfected in duplicate or triplicate with 100 ng of reporter DNA, 25-200 pM siRNA and 0.5 ml Lipofectamine2000 per well, in a total transfection volume of 500 ml. Cells transfected with an irrelevant siRNA were used as mock controls, and an average was calculated from the replicates to set Renilla/Firefly luciferase expression to 100%.
For EGFP repression, psi-EGFPS1-S (sense reporter) or psi-EGFPS1-AS was generated by cloning a 245-bp PCR fragment of EGFP in either the sense or antisense orientation in the 3 0 -UTR of the psiCHECK TM -2 vector (Promega) (40). Forty nanograms of reporter DNA, 20 or 200 pM siRNA and 0.5 ml Lipofectaimine2000 per well were used to transfect HEK293 cells in duplicate at 60-80% confluency in 48-well plates, in a total transfection volume of 200 ml. HEK293 cell lysates were collected 24-h post-transfection and subjected to Dual Luciferase assays (Promega). Transfection was done in duplicate and repeated at least three times. Renilla luciferase expression was normalized to internal control Firefly luciferase expression. Cells transfected with an irrelevant siRNA were used as mock controls, and an average was calculated from the replicates to set Renilla/Firefly luciferase expression to 100%.
For siDicer, siAgo2 or siTRBP treatment, HEK293 cells were seeded on 10-cm dishes and transfected with 40-nM siRNA-targeting Dicer, Ago2 or TRBP. The sequences of the siRNAs used in this study are as follows: Dicer, 5 0 -UUUGUUGCGAGGCUGAUU CdTdT-3 0 ; Ago2, 5 0 -GCACGGAAGUCCAUCUGA AdTdT-3 0 ; TRBP, 5 0 -UCUACGAAAUUCAGUAGG AdTdT-3 0 . Forty-eight hours later, the siRNA treated cells were seeded on either 48-well plates or 6-well plates for Dual Luciferase assays or RT-qPCR, respectively. In the second-round transfection, the same procedure described above was used for Dual Luciferase assays. Either with or without 20-nM siDicer, siAgo2 or siTRBP in the second round of transfection produced the same results.
IC 50 measurements
To generate 50% inhibitory concentration (IC 50 ) curves, HEK293 or HCT116 cells at 60-80% confluency in 48-well plates were transfected in duplicate with 40 ng of reporter DNA (either psiEGFPS1-S or -AS with HEK293 cells and psi-H1-S or -AS with HCT116 cells), siRNAs ranging from 0.2 pM to 15 nM in serial dilutions (a minimal of 10 points measurements) and 0.5 ml Lipofectaimine2000 per well in a total transfection volume of 200 ml. Cell lysates were collected 24-h post-transfection and subjected to the Dual-Luciferase Reporter Assay System (Promega). For each siRNA, transfection was carried out in duplicate and repeated at least three times. Normalization of Renilla luciferase expression to internal control Firefly luciferase expression was as described above. IC 50
Recombinant proteins
N-terminally His 6x -tagged TRBP2 was expressed from the pET11a (+) vector (Novagen) in the Escherichia coli BL21DE3 strain. Following 4-h induction with 0.5 mM IPTG, purification of His 6x -TRBP2 was performed with Nickel-Nitrilotriacetic acid (Ni 2+ -NTA) (the QIA expression system, Qiagen) under denaturing conditions. Efficiency of elution steps was determined with 12% SDS-PAGE/coomassie blue staining. The protein was further purified by dialyzing with a 3500 MWCO dialysis cassette (#66110, PIERCE, Rockford, IL), concentrated with an Amicon Ultra centrifugal filter 30KNMWL and stored in buffer D containing 10 mM TCEP. Purity of His-TRBP was also determined by Coomassie blue staining and western-blot analyses (49) with an a-His tag antibody (ab18184, Abcam Inc., Cambidge, MA). Recombinant human Dicer was purchased from Ambion, and its concentration was determined with A 205 measurements. Titration-binding assays were performed to evaluate optimal concentrations of the recombinant proteins (data not presented), ranging from 0.1 mg to 5 mg for Dicer and 4 ng to 300 ng for TRBP, 2 mg of rDicer enzyme and 80 ng of rTRBP were used in subsequent gel shift assays ( Figure 5).
Immunodepletion assays
Immunodepletion analyses were carried out as previously described (49). To immunodeplete Dicer, TRBP or Ago 2 from HEK293 whole cell extracts, 2 mg of pre-cleared extract were incubated with each primary antibodya-Dicer (15 mg of sc-30226, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), a-TRBP [25 ml of TRBP-JBX, (50)], a-Ago2 (15 mg of ab57113, Abcam Inc., Cambridge, MA). In the case of Dicer/TRBP or Ago2/ TRBP double immunodepletion, TRBP precipitation was performed following Dicer or Ago2 precipitations. Pre-immune serum (2.5 ml of sc-2007 or sc-2025, Santa Cruz Biotechnology, Inc.) was used as a control. Following 2-h incubation, protein A/G plus agarose beads (Santa Cruz Biotechnology, Inc.) were added and incubated overnight at 4 C with gentle agitation. The following day, supernatants were collected for additional incubation with the beads for 6 h. After removing the beads by a brief microcentrifugation, buffer D was added to bring the cell extracts to a final concentration of 4 mg/ ml. Of total protein extracts, 15 mg were used in the Gel Shift assays. Of the cell extracts, 150 mg were also used in western-blot analyses to evaluate the efficiency of the immunodepletions. Equal amounts of total protein were electrophoresed in SDS-PAGE gels (16 cm  16 cm), and each protein was detected sequentiqlly. After confirming the reproducibility of the procedure, the membrane was cut in three pieces to detect Dicer ($215 kDa), Ago2 ($100 kDa), TRBP ($40 kDa) and b-actin ($37 kDa), and the whole membrane was scanned at once for consistency using two different wave lengths. Antibodies a-Dicer (ab14601, Abcam Inc.), a-Ago2 (ab57113, Abcam Inc.), a-TRBP (ab42018, Abcam Inc.) or a-b-Actin (A5441, Sigma-Aldrich, St. Louis, MO) were used to detect the corresponding proteins. Additionally, the molecular weights of TRBP and Beta-actin are very close. Thus in order to avoid stripping the membrane between each probing, the secondary antibodies-anti-mouse-800nm (610-132-121, Rockland, Gilbertsville, PA) or antirabbit-680nm (A21109, Molecular Probes/Invitrogen)were detected at two different wavelengths on an Odyssey Infrared imaging system (LI-COR biosciences, NE). Scanned results in green, red or yellow were visualized as black and white. The reproducibility of the immunodepletions was verified four times.
Gel-shift assay
For incubations with radio-labeled siRNAs, singlestranded siRNAs were 5 0 -end labeled with T4 polynucleotide kinase (New England Biolabs) and g-32 P-ATP (MP Biochemicals) for 1 h and purified with a G25 column (GE Healthcare). For siRNA duplexes, independently radiolabeled sense and antisense strands were mixed and annealed at 95 C in a heat block for 2 min and allowed to cool slowly at room temperature by removing the heat block from the heat source until they reached ambient temperature. SiRNA duplexes that were radiolabeled after annealing yielded identical results. Unlabeled EGFPS1-A, -B, hnRNPH-1 and -3 siRNA duplexes were used as cold competitors. For siRNAs with only one 5 0 -end-labeled strand, the sense or antisense strand was annealed with the unlabeled complementary strand to generate S*/AS or S/AS* EGFPS1A or unlabeled sense and antisense strands were annealed and used as a cold competitor. Of total protein in buffer D, 15.0 mg were incubated with 1 nM (4 Â 10 4 c.p.m.) of labeled siRNA duplexes for 30 min at room temperature. In competition assays, cold siRNA competitors of concentrations at 2, 5, 10, 25, 50 or 100 nM were incubated in addition to 1 nM of labeled EGFPS1A siRNA duplex. The samples were mixed with 4X native gel loading dye and resolved in a 4.5% non-denaturing polyacrylamide gel (29:1) (20 Â 30 cm glass plates, 1.5 mm spacers) with 1X TBE for 3.0 h at 200V and 4 C. The gel was dried at 80 C for 1 h for autoradiography. Quantifications of the RNA-protein complexes were performed with densitometric scanning of the gel (Typhoon scanner). The percent complex binding was calculated as percent bound [bound/ free+bound) Â 100] of siRNAs relative to input siRNA incubated without the cell extract. Percentage bound of the HEK293 WCE sample was set to 100%, and relative intensities of complexes formed in immunodepleted cell extracts were calculated accordingly.
For the gel supershift assays, HEK293 cell extract (15.0 mg of total protein) was incubated with 1 mg of antibody specific for Dicer (ab14601), Ago1 (07-599, Upstate, Lake Placid, NY), Ago2 (07-590, Upstate) or 1 ml TRBP antibody Ab672 (51) for 15 min prior to the incubation with siRNA duplexes. The samples were resolved in a composite polyacrylamide (3%)-agarose (0.5%) gel and run for 7 h at 100V at 4 C. The gel was fixed with a 10% Acetic Acid: 10% methanol mix for 20 min and dried at 80 C for 1 h for autoradiography. The antibodies used in the supershifts were also used in western blot analyses (49) to detect the proteins in the cell extract. The assays described above were repeated multiple times and representative gel figures are shown in this study.
RT-qPCR
Twenty-four hours after the second round of transfection with 20-nM siRNA-targeting Dicer, Ago2 or TRBP, and 200-pM siEGFPS1A variants, RNA was extracted with RNA STAT60 (TEL-TEST), treated with Turbo DNA-Free Kit (Ambion, Austin, Texas) and reversetranscribed into complementary DNA (cDNA) using random hexamer primers and Maloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen, Carlsbad, California). One RNA sample of each preparation was processed without MMLV RT as a negative control in subsequent real time PCR reactions. Quantitative analysis of Dicer, Ago2 and TRBP expressions was performed by real time PCR SYBR Green I (Bio Rad) analysis (C1000 Thermal Cycler, Bio Rad, Hercules, California). Dicer, Ago2 and TRBP expression was detected using 50 ng of cDNA, amplified with corresponding primer sets Dicer- . GAPDH expression was detected as internal control using 50 ng of cDNA, with primers GAPDH-A (5 0 -CGCTCTCTGCTCCTCCTGTT-3 0 ) and GAPDH-B (5 0 -CCATGGTGTCTGAGCGATGT-3 0 ). PCR conditions are as follows: (i) Dicer, Ago2 and GAPDH PCR: 95 C for 5 min, followed by 40 cycles of 95 C for 30 s, 60 C for 30 s and 72 C for 1 min, and (ii) for TRBP and GAPDH: 95 C for 5 min, followed by 40 cycles of 95 C for 1 min, 60 C for 1 min and 72 C for 1 min.
RESULTS
Differential whole-cell-extract complex formation and target knockdown correlate with siRNA end structure To test the structural requirements for binding of siRNAs to components of whole cell extracts and target knockdown, various 3 0 -end modified versions of a single EGFP-targeting siRNA (EGFPS1) were incubated in whole-cell extracts for evaluation of complex formation and in cell culture for target down-regulation assays. The sequence of the antisense (guide) strand of the EGFPS1A variants was kept constant and only the sense strand was varied to create the differing types of overhangs ( Figure 1a). The results of the binding assays revealed that differences in complex formation are sensitive to the 3 0 -end structure. When the siRNA duplexes in Figure 1a were incubated with a HEK293 whole-cell extract, the strongest binding was observed with the 2-nt 3 0 overhang (EGFPS1A or 19 + 2 in Figure 1a and b) and was reduced with subsequent alterations in the siRNA end structure. Binding reached <40% with the 21+0 (blunt 3 0 -end) siRNA and complex formation and was further reduced with 5 0 overhangs (20-1 or 19-2 in Figure 1a and b), indicating that the complex is formed preferentially with siRNAs via the 3 0 overhangs, consistent with binding being initiated with siRNA 3 0 overhang-PAZ domain interactions.
To determine whether the 3 0 -end modifications introduced in the EGFPS1A variants affect RNAi efficacy, the above siRNA variants were tested in the Dual Luciferase reporter assays (Figure 1c). Silencing efficiency for many siRNAs is known to be concentration dependent. Since a high concentration of a given siRNA in an experimental setting could result in masking the effects of various structural features, we chose to use concentrations of 20 and 200 pM, or a concentration at the EGFPS1A IC50 value (Figure 3) versus one 10-fold above this. The variant siRNAs were co-transfected with the psiEGFPS1-S reporter plasmid in HEK293 cells and the effects on Renilla luciferase activity were monitored. The 19+2 siRNA was most potent, consistent with it being the best binder in the extracts. The other siRNAs followed in graded fashion with the +1, blunt, À1 and À2 siRNAs being progressively worse in target knockdown. These Without HEK293 whole-cell extract, 19+2 was included as a negative control. The band shifts were quantified by densitometric scanning of the gel (Typhoon scanner) and percent bound siRNAs were calculated by [bound/ (free+bound) Â 100] of siRNAs relative to input siRNA without cell-extract incubation. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Dual Luciferase assays. To determine the efficiency of the 3 0 -end modified siRNAs in intracellular target knockdown efficiency, silencing by the antisense strand of the end modified EGFPS1A siRNA duplexes was assayed in HEK293 cells by co-transfecting the psi-EGFP-S1 sense reporter and 20-or 200-pM siRNAs. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). cellular assays were consistent with their binding activities in the cell extracts.
We next tested the specificity of complex formation with single-or double-stranded siRNAs to determine if the high-molecular weight complex observed is an RNA duplex specific (Figure 2). When either the EGFPS1A labeled sense (S*) or antisense (AS*) strands were tested individually in the binding assays, no binding to the high-molecular weight complex was observed ( Figure 2) whereas a faster migrating complex was observed with both of the single-strand RNAs. We further tested the duplex requirement of the complex using EGFPS1A containing either one (S*/AS or S/AS*) or both (S*/AS*) 5 0 -radiolabeled strands. Both S*/AS and S/AS* were incorporated into the complex, and cold EGFPS1A (S/ AS) efficiently competed with the S*/AS* siRNAprotein complex, confirming that the complex in this study is duplex specific (Figure 2a). These observations demonstrate that the complex formation in this study is preferentially with the duplex form of siRNAs. Our observations are also consistent with reported observations that the human RLC forms exclusively with the duplex form of siRNAs (27)(28)(29).
To test the complex formation with several different canonical siRNAs harboring 19-base duplexes and 2-nt overhangs, we created a set of ten siRNAs targeting EGFP (EGFPS1-A-J, Figure 3a) in which each sequence is shifted by 1 nt towards the 5 0 -end of the target RNA. Several, but not all of the siRNAs incorporated published siRNA features that contribute to the overall efficiency of RNAi (such as an A at position 3 or 19 and 30-50% overall GC content in the passenger strand) (37,39,(52)(53)(54). Duplex end free energies-from 2 to 5 bp on both the 5 0 -and 3 0 -ends-of the siRNAs were also calculated and are listed in Table 1, panel a. When the 10 32 P-labeled anti-EGFP siRNA duplexes were incubated in HEK293 cell extracts and subjected to EMSA analyses, the duplexes formed complexes that migrated similarly. However, while the majority of siRNAs formed strong complexes, EGFPS1B, I and J formed weak complexes (Figure 3b). We next examined the relative levels of siRNA-directed mRNA knockdown using Dual Luciferase assays. IC 50 values for both sense (S) and antisense (AS) targets of the siRNAs were determined using dose-dependent target knockdown measurements (Figure 3c and Supplementary Figure S1). When the EGFPS1 siRNAs were co-transfected with Renilla luciferase target reporter plasmids-psiEGFPS1-S or psiEGFPS1-AS (40)-into HEK293 cells, differential knockdown efficiencies of the siRNAs were observed (Figure 3c). The general trend observed was that the siRNAs with the stronger complex formation percentages (Figure 3b) had better overall combined IC50 values (Figure 3c). In contrast, the three siRNAs with the poorest complex formation percentages (EFGPS1B, I and J, Figure 4b) had high-combined IC50 values (Figure 3c), and in the case of S1J there was no measureable target knockdown for the psi-EGFPS1-AS target ( Figure 3c).
As representatives of good and poor binders in the HEK293 cell extract assay, EGFPS1A and B were subsequently incubated with HCT116 cell extracts. Consistent with the observation from the HEK293 cell extract, EGFPS1A and B formed strong and weak complexes, respectively, in the HCT116 cell extract (data not shown), validating that the siRNA-binding differentials are not extract dependent.
To further explore the relationship between complex formation in the whole-cell extracts and RNAi efficacy, we created an additional set of siRNAs targeting a human gene encoding the heterogeneous nuclear ribonucleoprotein (hnRNP) H and tested these for complex formation in HCT116 whole cell extracts ( Figure 4). Seven 21-nt-long siRNAs (hnRNP H1-7) were designed with their sequences shifted 1 nt toward the 3 0 -end of the target RNA hnRNP H (Figure 5a) (40) and these were evaluated in the extracts by EMSA. As with the first set of siRNAs, duplex end-free energiesfrom 2 to 5 bp on both the 5 0 -and 3 0 -ends-of the siRNAs were also calculated and are listed in Table 1, panel b. When the seven 32 P-labeled anti-hnRNPH siRNA duplexes were incubated in HCT116 cell extracts and subjected to EMSA analyses, the duplexes formed complexes that migrated similarly (Figure 4b). However, among the seven anti-hnRNP H siRNAs, which were shifted by a single base from each other, we observed a range of binding efficiencies: hnRNP H1, H2, H4 and H7 were effectively incorporated into the complex, whereas Upper strand is sense; bottom strand is antisense. (b) Gel-shift assays. Anti-EGFPS1-A to -J siRNAs were incubated with HEK293 cell extract and resolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Determination of EGFPS1 siRNA IC50 values. HEK293 cells were co-transfected with either psiEGFPS1-Sense or -Antisense reporter plasmid and an siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 points measurements). Cell lysates collected 24-h post-transfection was subjected to the Dual-Luciferase Reporter Assay System (Promega) (dose-dependent target knockdown measurement curves are shown in Supplementary Figure S1). The n.a. indicates knockdown data were not obtainable for the passenger strand of S1J.
To determine the target knockdown efficacies of the siRNAs in intact cells the relative levels of target inhibition in HCT116 cells was evaluated using Dual Luciferase assays. The reporter system contained an hnRNPH PCR fragment in either a sense (psi-H1-S) or antisense (psi-H1-AS) orientation in the 3 0 -UTR of a Renilla luciferase reporter gene (40). The individual siRNAs were co-transfected with the sense or antisense oriented target plasmids and IC50 values were determined for both siRNA strands (Figure 4c and Supplementary Figure S2). The three siRNAs that showed poor complex formation (H3, H5 and H6) had one or both strands with high-IC50 values, whereas those that had stronger complex interactions (H1, H2, H4 and H7) had overall lower combined S/AS IC50 values (Figure 4c).
The differential strength of complex formation between EGFPS1A versus EGFPS1B and hnRNPH1 versus hnRNPH3 were examined in greater detail using cold siRNAs as competitors for complex formation ( Figure 5). Using EGFPS1A as a radio-labeled ligand, unlabeled EGFPS1A, EGFPS1B, hnRNPH1 or hnRNPH3 at 1:2 to 1:100 molar excess were incubated in the HEK293 cell extracts. When hnRNPH1 (IC 50S and AS = 9.03 and 5.04 pM, Figure 4c
Examination of the roles Dicer, TRBP and Ago2 in complex formation
The results described above were carried out using either the HEK293 or HCT116 cell lines. In order to further explore the complex composition, we carried out additional biochemical experiments in the HEK293 cell extracts. The human RLC consists of Ago2, Dicer and TRBP (26)(27)(28)(29). To determine whether any of these components are involved in the siRNA-complex binding observed in this study, we carried out antibody-mediated super-shift assays with HEK293 cell extracts using antibodies against Dicer, TRBP, Ago2 (and Ago1) to monitor complex formation with the EGFPS1A and hnRNPH1 siRNAs ( Figure 6). Addition of the anti-Dicer antibody resulted in a strong super-shift of the complex whereas the anti-TRBP antibody yielded a weak but reproducible supershift. These results indicate that the complex observed in Figures 1-3 contain at least Dicer and TRBP, consistent with the previous observation by Pellino et al. (55). Although no supershifts were observed with the anti-Ago2 or anti-Ago1 antibodies, these antibodies did recognize the cognate proteins in the HEK293 whole cell extract ( Figure 6).
To further analyze the complex components, Dicer, TRBP, Dicer/TRBP, Ago2 or Ago2/TRBP, immunodepletions were carried out and subsequently used in EMSAs (Figure 7). The efficiencies of the immunodepletions were also evaluated by western-blot analyses (Figure 7). HEK293 cell extracts immunodepleted of Dicer and/or TRBP (Id Dicer, Id TRBP, Id Dicer/TRBP), and Ago2 and TRBP (Id Ago2/TRBP) resulted in a loss of siRNA binding to the major complex. We were unable to immunodeplete Ago2 in the cell extract (Id Ago2, Figure 7), suggesting poor recognition of Ago2 by this antibody, perhaps due to interactions of Ago2 with other proteins in the extracts. Nevertheless, residual levels of Ago2 in Id Dicer, Id TRBP and Id Dicer/ TRBP cell extracts had no effect on complex formation, consistent with the previous report that Ago2 does not associate with siRNA duplexes by itself (27). It is of interest to note that the combined use of anti-Ago2 and TRBP antibodies resulted in depletion of Ago2 in addition to Dicer and TRBP and this also abolished complex formation. This result is most likely a consequence of tight interactions among TRBP, Dicer and Ago2 (27). Most importantly we observed loss of siRNA gel shifts when the cell extracts were immunodepleted of Dicer, or the Dicer/TRBP heterodimer (Figure 7, lanes 4-6 and 8 in the left panel, and lanes 3-5 and 7 in the right panel). In total, these observations support Dicer as the siRNA-binding factor in the cell extracts.
To further explore the role of Dicer in the complex formation we replenished the Dicer immunodepleted extracts using purified recombinant Dicer and/or TRBP and tested for EGFPS1A complex formation (Figure 8). While rTRBP alone had no effect on complex restoration in both Id Dicer (lane 5) or Id TRBP cell extracts (lane 13), addition of rDicer or rDicer in combination with rTRBP (lanes 6, 7, 12 and 14) resulted in restoration of the siRNA/complex formation (Figures 8a-c). . Competition assays. The specificity and requirement for siRNA duplexes for complex formation were examined using competition assays. Duplex requirements for complex formation were tested with radio-labeled EGFPS1A siRNA mixed with 2-, 5-, 10-, 25-, 50or 100-fold molar excess of cold EGFPS1A, -B, hnRNPH1 or H3. Quantifications of the RNA-protein complexes were performed using densitometric scanning of the gel (Typhoon scanner).
Given the differential complex formation of several of the siRNAs described above, we next asked if Dicer alone can discriminate binding of a strong versus weak complex forming siRNA. To do this rDicer was incubated with either the EGFPS1-A or -B siRNA duplexes. As we observed in the high-molecular weight complex formation, rDicer alone showed differential binding affinities between these two siRNAs, forming a much stronger interaction with the EGFPS1A than with the EGFPS1B duplex (Supplementary Figure S3). Although rDicer aggregated and was poorly resolved in the composite gel, the differential binding was reproducible.
To dissect the relative roles of Dicer, Ago2 and TRBP in selective incorporation of siRNAs in vivo, we used RNAi to knockdown each of these proteins and assayed for EGFPS1A-mediated target knockdown using the panel of EGFPS1A siRNAs that differ in their 3 0 -end structures (depicted and tested in Figure 1). If Dicer binding initially determines the 3 0 -end structure and guide strand selection, the differential efficiencies of the EGFPS1A variants should be absent in Dicer knockdown cells. Of course knockdown of Ago2 would also reduce RNAi overall since it is required for the RNAi effector step of cleaving the target transcripts. We compared the knockdowns of EGFP via the EGFPS1A panel in HEK293 in cells pre-treated with siRNAs targeting Dicer, Ago2 or TRBP (Figure 9). Consistent with a key role for Dicer in siRNA selection, the previously observed differences in knockdown efficiencies of the EGFPS1A variants were substantially reduced in cells pre-treated with the anti-Dicer siRNA. In these cells, we determined that levels of Dicer and Ago2 expression were repressed $85% and 65%, respectively (Supplementary Figure S4). Although TRBP mRNA levels were reduced by $60%, the effects of this reduction on the differential knockdown efficiencies by the EGFPS1A panel were minimal, which is Figure 7. Analysis of RISC components required in high-molecular-complex formation. Gel-shift assays. EGFPS1A duplex was incubated with HEK293 cell extracts immunodepleted of Dicer (Id Dicer), TRBP (Id TRBP), Dicer and TRBP (Id Dicer/TRBP), Ago2 (Id Ago2) or Ago2 and TRBP (Id Ago2/TRBP). Evaluation of immunodepletion of RISC proteins with western blot analyses was shown on the right. Of Id cell extracts, 150 mg were loaded on a 8% SDS-PAGE gel. Pre-immune serum was used as an immunodepletion control. Figure 6. Analysis of proteins comprising the complex. Super shift assays. a-Dicer, a-TRBP, a-Ago2 or a-Ago1 antibodies were added to the HEK293 cell extract and incubated for 15 min prior to addition of either EGFPS1A or hnRNPH1 siRNA. Western analyses of proteins detected with the antibodies were also shown on the right. Of total protein, 50 mg for detection of Dicer and Ago1 or 150 mg for Ago2, TRBP and b-actin was loaded on 8% SDS-PAGE gels. consistent with our EMSA results (Figures 7 and 8). To further validate these results, we also tested the effect of Dicer knockdown on siRNA-triggered gene silencing by comparing knockdown efficiencies of hnRNPH5 and H6 in siDicer-treated HEK293 cells (Supplementary Figure S5). When the siRNAs were co-transfected with the psi-H1-S or psi-H1-AS reporter plasmid at 200 pM, asymmetric knockdowns of the sense or antisense targets by the siRNA duplexes were observed; however knockdown efficiencies of hnRNPH5 and H6 siRNAs were substantially reduced in siDicer treated cells (Supplementary Figure S5). Our observations indicate that the overall efficacies of the siRNAs were diminished in the Dicer knockdown cells, consistent with previous observations (27,56), which is most likely a consequence of disruption of the RLC.
To discriminate the selective binding aspect of Dicer from direct incorporation of the siRNAs into Ago2, we modified EGFPS1A to generate an asymmetric 27/ 25-nt-long Dicer substrate form (DsiRNA)-a blunt end with two deoxyribonucleotide bases at the 3 0 -end of the passenger strand-forcing the duplex to be bound and undergo Dicer cleavage activity. EGFPS1B was also modified to a DsiRNA form for direct comparison, and the IC 50 values were determined for both DsiRNAs (Figure 10a and b). For both EGFPS1A and B DsiRNAs, the strand harboring the 2-nt 3 0 overhang was predominantly selected as the guide strand (IC 50 , Dsi-S1A-AS = 31.9 pM, IC 50 , Dsi-S1A-Sense = 101 pM; IC 50 , Dsi-S1B-AS = 27.5 pM, IC 50 , Dsi-S1B-Sense = 433 pM), consistent with previous observations (40). In contrast, the dominant selection of the antisense strand (relative to the sense EGFP target) was absent or less pronounced with the use of 19+2 siRNAs. Both the dsiRNAs and siRNAs form dicer-dependent complexes in whole-cell extracts (57) (Supplementary Figure S6). These results support a model in which the orientation of Dicer binding to its substrates sets the preference for which strand will be chosen to act as the guide strand. Taken together, our observations suggest that the initial Dicer-DsiRNA interaction is an important determinant for establishing siRNA-strand selection in RISC and ultimately
DISCUSSION
We have used siRNA binding to a high-molecular-weight complex in whole-cell extracts to better understand what cellular factors are involved in the initial binding and discriminatory selection of highly functional versus poorly functional siRNAs. In particular, we have been interested in what happens to siRNAs following transfection into cultured cells. We find that the extent of siRNA complex formation reflects siRNA-directed overall target knockdown efficiency in cultured cells. Using antibodies to core RNAi proteins to determine the key proteins of the complex, we find that it minimally consists of Dicer/TRBP, presumably as the pre-RLC. Our observations suggest that human RISC silencing efficiency with exogenously supplied siRNAs reflects the extent of siRNA-pre-RLC formation.
Previously, mammalian Dicer was shown to be dispensable for RISC assembly and siRNA-mediated cleavage of target RNA transcripts (41)(42)(43). In contrast, the absence of a functional mouse Dicer resulted in a lack of shRNA-mediated RNAi (43), showing that Dicer-cleavage activity was necessary to generate the siRNAs from this precursor. The lack of a Dicer requirement for pre-formed siRNA function led to the conclusion that only Ago2 was required for siRNA selection. Conversely, two groups demonstrated that the absence of Dicer abolished gene silencing effects (27,56). Our results suggest that when present, Dicer itself binds siRNAs that mimic the products of Dicer cleavage (19 bp + 2-nt 3 0 overhangs) and Dicer can serve as a 'gate-keeper' discriminating between potent and non-potent siRNAs by selective binding of the siRNAs in the pre-RLC.
Schwarz et al. reported that 2-4 bp on both ends of a siRNA are important for their relative loading efficiency into RISC in Drosophila (35). Our calculated duplex end free energies for siRNAs used in this study (Table 1) are consistent with these observations. Moreover, we observed that in general, siRNAs with thermodynamically unstable base pairs at both ends effectively form high-molecular-weight complexes in cell extracts and have better overall silencing efficiencies. These observations suggest that the thermodynamic properties of 2-4 bp on both ends of effective siRNAs are used as functional determinants by the pre-RLC and determine overall silencing efficiency in humans as well.
It is widely known that siRNAs targeting the same region of a target mRNA can have a wide range of Figure 1) were evaluated using Dual Luciferase assays with siDicer, siAgo2 or siTRBP treated HEK293 cells. To determine effects of Dicer, Ago2 or TRBP knockdowns on the silencing activity of the siEGFPS1A variants described in Figure 1, HEK293 cells were treated with 40 nM siDicer, siAgo2 or siTRBP for 2 days prior to the co-transfection. For the second round of transfection, Dicer kd, Ago2 kd or TRBP kd cells were transfected with the psi-EGFP-S1 sense reporter and 200 pM siEGFPS1A variants. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from four co-transfections).
potencies. To circumvent this obstacle, reported siRNA features such as an A at position 3 or 19 and 30-50% overall GC content in the passenger strand (37,39,(52)(53)(54) are widely used to predict potent siRNAs in addition to thermodynamic end properties. However, the importance of the AU base pair at these positions remained elusive. Using two sets of siRNAs targeting EGFP site I and hnRNP H site I (40), we observed that siRNAs containing an A:U base pair at positions 1 and/or 19 result in more effective binding to the high-molecular-weight complex than those with C:G pairs at these positions. This finding is consistent with the previous report of Katoh and Suzuki (58) which suggested that an AU base pair at these positions makes the siRNA a good substrate for the pre-RLC interaction and selection as an effective siRNA for target downregulation.
The manipulations of the 2-nt 3 0 overhangs, which resulted in reductions in target downregulation ( Figure 1) verify that the presence of a 1-or 2-nt 3 0 overhang is required not only for efficient binding to the high-molecular-weight complex, but is also required for efficient target downregulation. Previously, Pellino et al. (55) reported that the complex observed with HEK293 cell extracts contained Dicer. Our siRNA gel shift and binding assays along with the immunodepletion assays in HCT116 and HEK293 cell extracts demonstrate that Dicer is required for siRNA binding in these extracts, a result which is consistent with reports that the initial siRNA-Dicer interaction is via the 2-nt 3 0 overhang (59,60).
The marked differences in Dicer/TRBP binding to siRNAs and the correlations with efficacy of target Figure 10. Comparative analyses of guide strand selection for 21-mer and 25/27-mer Dicer substrate siRNAs. Target knockdowns for sense and antisense strands of siRNA EGFPS1A (a) and EGFPS1B (b) in 21-mer and Dicer substrate formats were carried out using co-transfections of the siRNAs with either the psi-EGFP-S sense reporter (red) or psi-EGFP-AS antisense reporter (black) in HEK293 cells. For both targets the 21-nt EGFPS1 siRNAs (left) or 25/27 DsiRNAs (right) were tested at various concentrations. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). Sequences and IC 50 values of each siRNA are shown. For the Dicer substrate siRNAs, the lower case letters represent deoxyribonucleotide containing bases which block Dicer entry. knockdown in cell culture, even for siRNAs differing by only a single nucleotide in sequence, point to the importance of understanding how siRNAs are selected for entry into RISC. Understanding the rules for effective siRNA design is further complicated by the fact that siRNAs which do not bind to Dicer can also be incorporated into RISC (41)(42)(43). We propose though that when Dicer is present it will preferentially bind and select siRNAs harboring structurally favorable features, and this interaction leads to the subsequent handoff step to Ago2.
Selective binding of Dicer to siRNAs could potentially determine the strand to be incorporated as a guide strand. The binding of the PAZ domain to a Dicer substrate allows the catalytic domain to interact with the opposite end of the dsRNA. Studies of the interaction of the human Dicer catalytic site with the PIWI domain of Ago2 (61) and the binding of the Archaeoglobus fulgidus PIWI protein with the 5 0 -end of the guide strand in the crystal structure (62) suggest the coupling of the initiation and effector steps of RNAi. The handoff of the guide strand from Dicer to Ago2 via the Dicer-catalytic domain could explain not only why Dicer processing of longer precursors often increases the efficacy of processed siRNAs (40,63), but also why the 5 0 -end of the siRNA within RISC leads to and determines the stable association of RISC and the target RNA (64). In this regard, Dicer substrates in the studies of Rose et al. (40) have a blunt end with two deoxybases at the 3 0 -end of the passenger strand, which precludes Dicer PAZ domain interactions from that end of the duplex. The polarity of Dicer entry from the end harboring the 2-base 3 0 overhang results in substantially reduced function of the passenger strand relative to that observed with the corresponding 19+2 siRNA ( Figure 10). Moreover, a single-nucleotide shift in the siRNA relative to the target site also resulted in a strong bias in guide strand selection for the dsiRNAs as demonstrated by EGFPS1A-25/27 (IC 50 , Dsi-S1A-Sense = 101 pM) and EGFPS1B-25/27 DsiRNAs (IC 50 , Dsi-S1B-Sese = 433 pM) ( Figure 10). Thus, Dicer interaction with the 2-nt 3 0 overhang is an important step in Dicer binding, and for 19+2 siRNAs Dicer can bind in two orientations. Nevertheless, the thermodynamic end properties of the siRNAs also influence Dicer binding as we have demonstrated. Recent single-particle EM analyses of the RLC-containing Dicer/TRBP/Ago2, show that Ago2 is associated with the C-terminal domain of Dicer, while TRBP is associated with the N-terminal or helicase domain of Dicer (65,66). This structural configuration would facilitate Dicer handoff of the 5 0 -end of the guide strand to Ago2. Our observations that Dicer binds selectively to 2-base 3 0 overhangs, most likely via the PAZ domain, would orient the enzyme such that the C-terminal portion of the enzyme is associated with the 5 0 -end of the guide strand to facilitate handoff of the guide strand to Ago2. An important observation is that the stability of Dicer binding to an siRNA is not solely mediated by the 3 0 2-base overhang, but obviously involves interactions with the duplex itself. This is made evident by the fact that many of the siRNAs used in our binding analyses contain the 2-base overhang but are not bound strongly to the Dicer-containing complex.
Thus far, the binding efficiency of Dicer/TRBP to pre-miRNAs appears to be maintained regardless of whether or not the pre-miRNA can be cleaved by Dicer (67). Our work and that of Pellino et al. (55) demonstrate that Dicer/TRBP can bind to siRNAs as well. However, these observations are in contrast to a previous study that human Dicer is incapable of siRNA binding (68). One possible explanation for this discrepancy is that the siRNA used in the previous study may fall into the group of siRNAs that are weak binders to the pre-RLC-similar to the poor binding siRNAs tested in our study.
While immunodepletion of Dicer (Id Dicer) left-residual TRBP, Id of TRBP resulted in nearly complete depletion of Dicer in the HEK 293 cell extracts, as did co-Id of Dicer/ TRBP. These results indicate that the majority of Dicer is bound to TRBP and that distinctive roles of Dicer and TRBP augment each other to function as the pre-RLC. Id of Ago2/TRBP resulted in immunodepletion of Dicer, TRBP and Ago2 (Figure 7), suggesting that TRBP functions as a bridge between Dicer and Ago2.
We have demonstrated that for many siRNAs what we deemed as the passenger strand could be more effective at target knockdown than the desired antisense guide strand. Since Dicer/TRBP binding to a siRNA duplex determines the strand to be selected as the guide strand, off-targeting will often be a consequence of both strands being incorporated into RISC. Thus the two strands of a siRNA can also compete with one another for incorporation into RISC following Dicer selection, thereby potentially reducing the potencies of both.
In conclusion our results demonstrate that human Dicer plays an important role in selection of efficacious siRNAs via direct binding to siRNAs that harbor certain structural features, which include the 2-nt 3 0 overhangs and the thermodynamically unstable base pairing at the ends of the duplex. These results are similar yet distinct from Drosophila in which R2D2 and Dcr-2 bind to the thermodynamically stable and unstable ends of siRNA duplexes, respectively. Our results demonstrate that the human Dicer enzyme can recognize and selectively bind siRNAs with thermodynamically favorable termini. Our results demonstrate that an often overlooked feature of 19+2 siRNAs is the ability of both strands to be chosen for RISC entry. This is sometimes observed for miRNAs as well, but often only one strand serves as the guide. In this regard, Dicer substrate siRNAs mimic the miRNA pathway, providing strong polarity for guide strand selection, perhaps a useful attribute for minimizing off target effects and competition between the two strands for entry into RISC. | 2016-05-04T20:20:58.661Z | 2010-10-23T00:00:00.000 | {
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252093141 | pes2o/s2orc | v3-fos-license | Next-generation sequencing based detection of BRCA1 and BRCA2 large genomic rearrangements in Chinese cancer patients
BRCA1/2 mutation is a biomarker for guiding multiple solid tumor treatment. However, the prevalence of BRCA1/2 large genomic rearrangements (LGRs) in Chinese cancer patients has not been well revealed partially due to technical difficulties in LGR detection. This study utilized next-generation sequencing (NGS) to analyze the BRCA1/2 mutation profile, including LGR, in 56126 Chinese cancer patients. We also reported that two ovarian and breast cancer patients with NGS-determined BRCA1/2 LGR benefited from PARP inhibitors (PARPi). DNA sequencing identified BRCA1/2 variants (including LGR, pathogenic and likely-pathogenic variants) in 2108 individuals. Seventy patients were discovered to harbor germline LGRs in BRCA1 and 14 had germline LGRs in BRCA2. Among the LGRs detected, exon 1-2 deletion was the predominant LGR (14/70) in BRCA1, and exon 22-24 deletion was the most frequent LGR (3/14) in BRCA2. Notably, the BRCA1 exon 7 deletion was a novel LGR and was identified in six patients, suggesting a specific LGR in Chinese cancer patients. The prevalence analysis of BRCA1 and BRCA2 LGRs across multiple cancers revealed that BRCA1 LGR more frequently occurred in ovarian cancer (1.31%, 33/2526), and BRCA2 LGR was more commonly seen in cholangiocarcinoma (0.47%, 2/425). Two ovarian and breast cancer patients with BRCA1/2 LGR benefited from PARPi therapy. This is the first study to reveal the BRCA1/2 LGR profile of a Chinese pan-cancer cohort by using an NGS-based assay. Two breast and ovarian cancer patients harboring NGS-determined BRCA1/2 LGR benefited from PARPi, indicating that NGS-based detection of BRCA1/2 LGR has the potential to guide PARPi treatment.
Introduction
BRCA1 and BRCA2 are tumor suppressor genes that protect genomic stability via homologous recombination repair (HRR). Germline mutations in BRCA1/2 are associated with a high risk of cancers, particularly breast and ovarian cancers. PARP inhibitors (PARPi), such as olaparib and niraparib, induce synthetic lethality in tumors with HRR-deficiency and are currently available in the clinic (1,2). Accurately characterizing BRCA1/2 mutational status, including BRCA1/2 somatic and germline mutations, can guide treatment for patients with these gene alterations and help assess the risk of developing hereditary breast and ovarian cancer (HBOC). Different from the well-studied single nucleotide variations (SNVs) and small insertions or deletions (InDels) of BRCA1/2 genes, large genomic rearrangements (LGRs) refer to the genomic reconstruction of large DNA fragments that affect one or more exons. Most of these chromosomal changes are pathogenic. In particular, BRCA1/2 germline LGR can be inherited, which may lead to an accumulation of HBOC in a familial manner (3). Thus, accurate characterization of LGR is of great clinical significance.
Indeed, LGR cannot be well identified using conventional methods but can be detected with copy-number sensitive assays such as multiplex ligation-dependent probe amplification (MLPA) and quantitative multiplex PCR of short fluorescent fragments (QMPSF). Their shared limitations are the requirements of high-purity DNA samples and high-order multiplex PCR to guarantee wide coverage. The nextgeneration sequencing (NGS)-based method is a newcomer with high-throughput that has the potential to detect various types of mutations in a one-set test (4). Given the increasing awareness of LGR significance, the LGR profile of different racial populations has been studied in recent years. According to data from Caucasian populations, LGRs account for approximately 4%-28% of BRCA1/2 mutations and 10% of BRCA1/2 germline mutations (4,5). Partially due to the technical difficulties in detecting LGR, the profile of BRCA1/2 LGR in Asian cancer patients has not been well elucidated (6)(7)(8). Data from the Chinese mainland are extremely limited (9,10). This study deciphered the BRCA1/2 variant profile, including LGR, in a large population of 56126 pan-cancer patients from the Chinese mainland; the largest Chinese cohort analyzed using NGS for BRCA1/2 LGR cancers to date. We also explored the utility of NGS-based detection of BRCA1/2 LGR in guiding the treatment of breast and ovarian cancer patients.
Method Data source and patients
We analyzed the sequencing data of tumor specimens from 56126 pan-cancer patients in the 3DMed database. All breast cancer and ovarian cancer patients included in this study had both FFPE tissues/plasma samples and paired white blood cell samples for screening paired somatic and germline variants in BRCA1/2. Ovarian and breast cancer patients reported in the case presentation section were identified according to their electronic medical records at First Affiliated Hospital of Nanchang University and National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital. This study was approved by each site's ethics committee.
Sample preparation and next-generation sequencing
Formalin-fixed paraffin-embedded (FFPE) tissue or blood samples were obtained from patients and subjected to somatic mutation profiling using NGS. Paired white blood cells were also collected to identify germline mutations. NGS was performed at 3D Medicines Inc., a College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA)certified laboratory. The sequencing coverage and quality statistics of each sample are summarized in Supplementary Table S1. The details of sample processing, library preparation, and sequencing data analysis are shown in the Supplementary Methods. Pathogenic and likely-pathogenic germline variants were identified according to the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines (11)(12)(13). Pathogenic and likelypathogenic somatic variants were identified as per AMP/ American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines (14).
Development of the LGR detection assay
Germline LGRs in BRCA1 and BRCA2 were detected using a capture-based NGS method developed by 3D Medicines Inc. The details of LGR detection method have been submitted for a Chinese patent (Patent NO. CN111534579A; https://pss-system. cponline.cnipa.gov.cn/). The capture probes of the invention comprise exon probes and single nucleotide polymorphism (SNP) probes. The exon probes comprise at least three probes aiming at each exon of the targeted gene and 60 bp regions at two ends of the exon. The SNP probes comprise SNP loci with a population occurrence frequency of 30%-70% in the gene intron region and the upstream and downstream within the 5000 bp ranges of the gene. Meanwhile, the invention further provides an algorithm formed by integrating a read depth algorithm module, an allelic imbalance algorithm module, and a discordant sequence algorithm module. The algorithm is used for carrying out large genomic rearrangement analyses on the NGS sequencing data. False positives caused by single-base variation were reduced, and high detection performance was achieved for exon amplification. (Supplementary Figures S1, S2). A total of 38 patients with solid tumors who had FFPE tissues/plasma samples and paired white blood cell samples were included for technical validation. NGS was performed on FFPE tissues/plasma samples and paired white blood cell samples, and MLPA, the gold standard for LGR detection, was performed on white blood cell samples. The NGS-based LGR detection assay had 100% concordance with MLPA in detecting germline LGRs (Supplementary Tables S2, S3).
Characterization of BRCA1/2 LGR
The median age of patients with BRCA1 and BRCA2 LGR was 54 and 63 years, respectively. Female was the predominant subpopulation in patients with BRCA1 LGR and BRCA2 LGR. No significant difference was found in age or sex distribution between patients with BRCA1 LGR and BRCA2 LGR (Supplementary Table 7).
The prevalence of BRCA1 and BRCA2 germline LGRs across multiple cancer types is shown in Figure 2. Table S11).
Ovarian cancer case presentation
On June 5, 2019, a 69-year-old woman with ovarian cancer underwent cytoreductive surgery consisting of bilateral salpingooophorectomy, hysterectomy, omentectomy, partial rectectomy, and removal of adhesion between the ovary and pelvic/ peritoneum. She was considered to have FIGO stage II disease according to the pathologic results and findings observed during surgery. The pathological results revealed high-grade serous carcinoma in the right adnexa. Tumor lesions were also involved in the epiploic appendix and the rectum anterior wall. She received six cycles of adjuvant intravenous chemotherapy (120 mg paclitaxel + 50 mg lobaplatin) after surgery. On July 16, 2020, the patient visited our clinic and presented with a high level of CA125 (3775 U/mL) and a high ROMA index. Enhanced computed tomography (CT) revealed multiple nodular soft-tissue density shadows, with obviously non-uniform enhancement. She was considered to have disease recurrence and was therefore treated with adjuvant intravenous chemotherapy (50 mg doxorubicin hydrochloride + 50 mg lobaplatin) for six months. On February 17, 2021, pathological examination of the vaginal stump revealed ovarian-derived highgrade serous cystadenocarcinoma vaginal metastasis. Magnetic resonance imaging (MRI) on March 5, 2021 showed a soft-tissue density mass shadow in the anastomotic stoma of a previous rectal resection, suggesting that the disease relapsed ( Figure 3A). Molecular profiling of vaginal stump biopsy identified a BRCA1 germline exon 8-11 deletion. Accordingly, the patient was administered the PARPi fluzoparib, which led to a drastic decrease in CA125 (decreased to a normal level with a reduction of 85%) and a shrinkage in the pelvic lesion ( Figure 3A), achieving partial response (PR). After six weeks of fluzoparib treatment, the patient experienced a grade 3 decrease in platelet number in peripheral blood, for which she discontinued fluzoparib administration. Upon discontinuation of fluzoparib, MRI revealed an enlarged irregular soft-tissue nodular shadow in the anastomotic stoma of previous rectal resection and a drastic increase in the level of serum CA125, which was nine folds higher than the normal level. The spectrum of large genetic rearrangements (LGRs) detected in BRCA1 and BRCA2 genes. Exon numbers are indicated on the top. The characteristics of the detected LGRs are listed on the left. The affected exons are shown as bars, among which, grey represents deletion, and orange indicates duplication. Actual locations of breakpoint are not implied.
Discussion
In clinical practice, MLPA is the most widely used method to detect BRCA1/2 LGRs (19,20). The primary weaknesses of MLPA are as follows: a high requirement for the purity of nucleic acid samples, due to which alcohol, metal ions, phenol, and TRIzol all affect the performance of MLPA; false-positive result tends to happen when SNV occurs at the probe binding site; poor performance to detect duplication. A wide range of other methods are available for LGR detection, but no one has overwhelming advantages. Southern blotting, real-time PCR, dual-color fluorescence in situ hybridization (FISH), comparative genomic hybridization, and multiplex amplifiable quantification all have disadvantages such as being timeconsuming, having a high false-positive rate, and having low sensitivity (21). In addition, BRCA1/2 has a long gene sequence and multiple pathogenic mutation types, including missense, frameshift, nonsense, and LGR, rendering it challenging to comprehensively detect BRCA1/2 mutations. Herein, we utilized a capture-based NGS method to reveal the profile of BRCA1/2 mutation, making it accurate and time-saving to characterize point mutations and small or large insertdeletions in a one-set test. The well-designed and strictly validated NGS method has full coverage for all exons and sufficient sequencing depth, demonstrating 100% concordance with MLPA.
Ovarian and breast cancers are the leading causes of gynecologic cancer-related death. BRCA1/2-mutated cancer patients represent a typical molecular subset. For ovarian cancer patients, germline and somatic BRCA1/2 mutations have been utilized as biomarkers for the use of PAPRi (1). For breast cancers, National Comprehensive Cancer Network (NCCN) guidelines recommend that recurrent or metastatic patients with germline BRCA1/2 mutations receive PARPi therapy (2). In this study, we presented two cases with BRCA1/2 LGR as determined by NGS (ovarian cancer case: BRCA1 germline exon 8-11 deletion; breast cancer case: BRCA1 germline exon 2 deletion), and both patients benefited from PARPi therapy, providing evidence for the feasibility and significance of NGS-based LGR detection in guiding treatment. Further studies are warranted to confirm the findings.
In this study, the frequency of co-occurring variants in both the BRCA1 and BRCA2 genes in the pan-cancer cohort was approximately 3%, which was somehow higher than that of most previous studies (22)(23)(24)(25). The possible reason for the relatively high frequency in our study might be the fact that except for pathogenic variants, we also included likely-pathogenic variants and LGRs, which were not included in some studies. Another possibility was that we analyzed the sequencing data in a pantumor cohort, which might have led to the discrepancy among studies.
One of the limitations of this study was that the NGS platform employed in our study could not identify the accurate breakpoints of LGR. Breakpoints frequently occur in introns, the length of which can be thousands of bp. The NGS assay applied for LGR detection was designed with capture probes covering the whole exons and extending 60 bp into intron regions at both ends of every exon, which was incapable of identifying accurate breakpoints in introns. Due to a lack of breakpoint information for these LGRs, predicting the impact of these LGRs on BRCA1/2 proteins was theoretically not feasible. Based on previous reports, deletions in BRCA1/2 exons could result in premature termination of the BRCA1 protein, an in-frame deletion, and prevention of transcription. Rearrangements involving different exons in distinct domains may have distinct effects on protein functions (26).
Most of previous studies regarding BRCA1/2 LGR have primarily focused on ovarian and breast cancer. This study examined the prevalence of BRCA1/2 germline LGRs in multiple solid tumors. Of note, the prevalence of BRCA1/2 germline LGR in other cancers, except for ovarian and breast cancers, might be underestimated because a few cases were screened only with tissue specimens. To the best of our knowledge, this was the first and largest study to depict the BRCA1/2 LGR profile of Chinese pan-cancer patients by using an NGS-based assay. We also reported two cases who had BRCA1/2 LGR benefited from PARPi, supporting the feasibility of NGS-based detection of BRCA1/2 LGRs to guide treatment.
Data availability statement
The VCF files of 84 LGR cases were uploaded to the GVM database (Accession NO. GVM000365, Project NO. PRJCA010855, https://ngdc.cncb.ac.cn/gvm/). Other datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics statement
The studies involving human participants were reviewed and approved by The ethics committees of the First Affiliated Hospital of Nanchang University and National Cancer Center/ National Clinical Research Center for Cancer/Cancer Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Author contributions
XZ, DH, and PY conceptualized the study; DH, QT, and XW, BZ, YB, and CB designed the Methods; DH, QT, XW, and LC collected, analyzed, and interpreted the patient data regarding the discovery and validation cohorts. TB and DH were major contributors in writing the manuscript. TB, XZ, and PY conducted critical revision of the manuscript for important intellectual content. DH, QT, and XW contributed equally and served as co-first authors. All authors read and approved the final manuscript.
Funding
This study was funded by the Beijing Hope Run Special Fund of Cancer Foundation of China (LC2019B16 to Xue Wang). | 2022-09-07T13:45:21.685Z | 2022-09-06T00:00:00.000 | {
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270246461 | pes2o/s2orc | v3-fos-license | A Decentralized Consistent Approach for Multi-Robot Simultaneous Localization and Object Tracking
This study focuses on the problem of multi-robot simultaneous localization and object tracking (MR-SLOT), which involves the collaborative effort of multiple mobile robots tracking a moving object on a plain. To address this challenge, this paper introduces a fully decentralized approach based on the extended Kalman filter and covariance intersection (CI), denoted as CI-SLOT. In the proposed approach, each robot can precisely track the robot-to-object correlations. Moreover information exchange occurs solely among one-hop communicating robots, and each robot only maintains the latest estimate of the object and its pose. The simulation results have shown that the performances of CI-SLOT are better than those of the state-of-the-art approaches of comparable processing and communication costs.
Introduction
Simultaneous localization and object tracking (SLOT) involves tracking an object of interest by a moving robot equipped with various sensors.This area of research holds significant scientific and engineering importance.In GPS-denied environments and when environmental characteristics are unavailable, a single robot is constrained to utilize only an odometer to localize itself, leadings to increasing position error due to sensor noise accumulation.Fortunately, within a multi-robot system, the integration of relative measurements among robots serves to reduce the cumulative error [1].The cooperative localization performance can be further enhanced when these robots collaborate to track a moving object [2,3].This gives rise to the concept of multi-robot SLOT (MR-SLOT), where multiple robots work together to track and localize objects.The applications of MR-SLOT are diverse, including cooperative transportation [4], search and rescue operations [5], etc.
Generally speaking, the architecture of existing MR-SLOT systems can be categorized into centralized architecture and decentralized architecture.Regarding the centralized architecture, all measurements from distinct robots are directly sent to the central node and subsequently fused for state estimation.This IOP Publishing doi:10.1088/1742-6596/2762/1/012067 2 architecture suffers from high computation and communication costs.Moreover, it is vulnerable to the failure of the central node.By contrast, the decentralized architecture incorporates multiple fusion centers.With each robot exclusively managing local information, this design is robust against single-point failures and demonstrates favorable scalability.
An essential problem for the decentralized MR-SLOT (DMR-SLOT) is how to get a consistent estimate of the object in the presence of an unknown inter-estimate correlation.Covariance intersection (CI) [6] is a well-established approach for addressing unknown correlations and has been widely applied in DMR-SLOT [7][8][9].However, if the fusion process exclusively employs CI to combine states of objects without addressing robot-to-object (R2O) correlation terms, it cannot ensure the positive definiteness of the joint covariance matrix encompassing both the robot and object components.Additionally, neglecting R2O correlations may result in an inconsistent estimate in the extended Kalman filter (EKF).To solve this problem, Zhu et al. [7] proposed an interleaved update-based DMR-SLOT method, in which each robot maintains 2N-1 EKFs to track robot-to-robot (R2R) and R2O correlation terms with teammate robots.Despite its decentralized nature, this method exhibits notable drawbacks, particularly concerning its high computational complexity and significant memory demands.Lyu et al. [8] proposed a recursive DMR-SLOT approach where the R2O correlations are taken into account within the upper bound through the utilization of CI-based EKF method.Afterward, a similar approach was presented by [9], employing the CI-based extended information filter (EIF) algorithm.However, due to the consideration of cross-correlation terms in the upper bound, both the CI-based EKF algorithm and CI-based EIF perform much worse than the EKF algorithm.Wang et al. [10] presented a DMR-SLOT approach based on EKF and covariance union (CU).They demonstrated that when CU is utilized to fuse the object state estimate with the object state estimated by teammate robots, the positive definiteness of the robot and object joint covariance matrix is guaranteed without calculating the R2O correlation terms.Nevertheless, in comparison to CI, using CU as a fusion algorithm may lead to a conservative estimation.
In this paper, we propose a DMR-SLOT approach based on EKF and CI approach, denoted as CI-SLOT.The purpose of this approach is to address the challenge of tracking a moving object using multi-robots with unknown poses.In the proposed approach, each robot maintains a local EKF to estimate the state of itself and the object jointly.When a robot takes relative measurements for a teammate robot, the EKF is employed to estimate the state of itself and its neighbor robot jointly.Upon receiving a local estimate from a communicating robot, CI is utilized for a robot to fuse the received estimate with its local estimate.The main contributions of this paper are summarized as follows: 1) Consistent estimates both of the robots and objects can be achieved by the proposed approach in a decentralized manner; 2) Due to the recursive property of our approach, the storage of measurements is not required.In the case of N robots, each robot leverages its local belief and 3(N -1) matrices containing the estimated correlations with teammates; 3) Our approach can efficiently handle asynchronous communication, with information exchange occurring exclusively between one-hop communicating robots.
System model
We refer to the moving object as O and N robots as . The robots and object are all moving on a two-dimensional flat terrain, and the object moves according to a constant velocity (CV) model [3,11,12].We assume that each robot is equipped with (i) communication devices that can exchange information between robots; (ii) an exteroceptive sensing system, which identifies and obtain relative measurements to the teammates and object; (iii) a proprioceptive sensing system that measures egocentric motion, including linear velocity and angular velocity.The state of robot i R at time k is denoted as . The motion model is provided as follows: where f(.) is a non-linear function.
are nominal linear and angular velocities, respectively.k is independent zero-mean white Gaussian noise with known covariance k Q .
We denote state of object O at time k as . The state transition function of O is given as follows: 1 (2) where and are the motion model and the noise Jacobian matrix which depends on the CV model, k is independent zero-mean white process noise with known covariance k .
At time k+1, the relative measurements of Ri are respectively denoted as: denotes the true relative state between A and B at time k+1.
CI-SLOT
At time k, Ri needs to maintain the estimated state ˆi R k X and covariance ˆi R k P , which includes the state and covariance of itself and the object, as follows: , where Due to the R2R measurements that exist in this system, Ri needs to maintain the cross-correlation with the teammate robot j R .
To improve the tracking accuracy and get a consistent estimation of the object with Rj, Ri needs to fuse the state and covariance of the object estimated by Rj.However, if only fuse the state and covariance of the object, without fusion the R2O correlations, the ˆi R k P will be singular.In section 3.3.2,we give a fusion rule of the R2O correlations.In this rule, Ri and Rj need to maintain cross-correlations Since communication between robots is restricted, tracking correlations of R2R and R2O in a decentralized approach is a significant challenge.Within the context, Luft et al. [13] introduced a matrix-split method.In this approach, the cross-correlation is split into two terms and maintained by two robots, which is shown as follows: However, if A and B are not in the same dimension, this spilt cannot work.In this paper, we propose a QR decomposition method to replace (7), as follows: can be decomposed by QR decomposition and distributed between robots Ri and Rj, as follows: where Ri maintains . The correlations between Ri and Rj are shown in Figure 1.
In this approach, except the ˆi R k P , Ri needs to maintain: 1) N -1 matrices encapsulating information about the correlations with the estimates of the teammate robots, which is denoted as ; 2) N -1 matrices containing correlations with the object estimated by teammate robots, which is denoted as ; 3) N -1 matrices that contain information about the correlations between the object estimate by itself and the teammate robots, which is denoted as . In summary, Ri needs to maintain 3(N -1) matrices which are the correlations with the estimates of the teammates.
Propagation
According to the motion models in (1) and ( 2), the state prediction equations for Ri and O estimated by Ri are given as follows: where The covariance terms estimate by Ri are propagation as follows:
ˆˆˆ, ,
ˆˆˆ, , ˆˆˆ, , are the Jacobian matrix of (3) To avoid communication, inspired by [13], other local correlation terms which are maintained by Ri can be updated as follows: ˆˆˆ, , ˆˆˆ, , Here, if Ri communicates with Rj, the updated state and covariance of the object can be sent to Rj to enhance the tracking accuracy, the specific algorithm refers to Section 3.3.
Relative robot measurement update .
Given that the communication range surpasses the sensing range, the establishment of communication from Ri to Rj occurs when Ri takes a relative measurement to Rj.The is firstly reproduced by the matrix multiplication, ˆ, The state and covariance are updated as follows: ˆˆˆˆˆˆˆ, , , , ˆˆˆˆˆˆˆ, , , , ˆˆˆ, , ˆˆˆ, , ˆˆˆ, , ˆˆˆ, , are the Jacobian matrix of ( 4) Formally, other local correlation terms which are maintained by Ri can be updated by ( 24), ( 31)-(32).
Object information fusion
At time k+1, if Ri communicates with Rj and , the state and covariance of the object estimate by Ri can fuse with Rj's local estimate to improve tracking accuracy.The object information fusion includes two parts: 1) the object state fusion; 2) the R2O correlations fusion.
The R2O correlations fusion.
In the previous section, the CI algorithm is used to fuse the state of the object.However, if only the state and covariance of the object are fused, neglecting the cross-covariance between the robot and the object, the 1 ˆi R t P will be singular.In this section, we propose the CI-based for the correlations between robot and object.
Before fusing the R2O correlations, we first reproduce cross-correlation , we can get the fusion rule of the R2O correlations as follows where , , ˆˆˆ, , .
To avoid communication, the correlations between the object estimate by itself and the teammates can be updated in an approximate form, which is given in (25).Then the cross-correlation
X
X is decomposed by (39) and distributed between robots Ri and Rj.
Simulations
In this section, numerical simulation experiments are conducted to evaluate the performance of CI-SLOT approach.To maintain generality, a team with three mobile robots is employed to track a moving object in formation.The control law can be seen in [14].To keep the leader-follower formation, R1 keeps the desired separation of 1m and the desired bearing of 0 with the object.R2 keeps the desired separation of 4m and the desired bearing of 3 with the object.R3 keeps the desired separation of 4m and the desired bearing of 3 with the object.To compare the fairness of different approaches, the true state of robots and object are used in the calculation of the control law.In the simulation experiment, we conducted 20 Monte Carlo runs.One of the trajectories of robots and object in simulation is shown in Figure 2. At each time step, each robot propagates its state and the measurement is updated correspondingly every tenth time step.Table 1 gives the simulation parameters.
Evaluation
To evaluate the performance of CI-SLOT comprehensively, the following three MR-SLOT approaches are selected for comparison: DMR-SLOT based on EKF and CU (CU-SLOT): when a relative measurement occurs between Ri and Rj,
8
EKF is applied to estimate the states of Ri and Rj simultaneously.When a relative measurement occurs between Ri and O, EKF is used to estimate the states of Ri and O simultaneously.Once Ri receives object state estimate Rj, it fuses the received estimate with its local object state estimate through CU, see [10].
Naive DMR-SLOT (Naive-SLOT): it is identical to the CI-SLOT approach expect for the difference, that is in Naive-SLOT, when a relative measurement occurs between Ri and O, EKF is used to estimate the states of Ri and O with , , ˆ, 0 simultaneously.Centralized EKF-based MR-SLOT (CE-SLOT): the state of each robot and the object are augmented into a single state vector.The R2R measurements, R2O measurements and egocentric measurements of robots are fused using EKF.Due to this approach being centralized, it can track the R2R and R2O correlations accurately, which can serve as the benchmark to evaluate the proposed CI-SLOT approach.
In this simulation, the sensor data processed by all approaches are the same.Additionally, the root mean square error (RMSE) and normalized estimation error squared (NEES) are employed to measure the accuracy and consistency of these four approaches.
In this simulation, is defined as: The measurements noise of relative orientation are assumed to be corrupted by white Gaussian noise 3 and 4 illustrate changes in RMSE of robots and object position estimation over time, respectively.Table 2 summarizes the averages results over the whole simulation time.As expected, the centralized method CE-SLOT proven to be the most accurate.This is attributed to the approach considering three robots and object as a joint system, precisely maintaining correlations of both R2R and R2O.Besides, the performance of the CI-SLOT closely aligns with the CE-SLOT and significantly surpassed that of CU-SLOT and Naive-SLOT.The Naive-SLOT produces estimates with large errors than other approaches due to ignoring correlations between robot and object, and produces estimates with large errors than other approaches.In Figure 4, the RMSE of the object position estimated by three robots in the CI-SLOT, CU-SLOT and Naive-SLOT approaches are almost identical.This can be attributed to the data fusion and communication between robots adopted.
Figures 5 and 6 show the NEES trend of the robots and object position estimation over time, respectively.Table 3 shows the average errors over the whole simulation time.From Figures 5 and 6, it is evident that the NEES for each robot and object hovers around the ideal value of 2 in the CI-SLOT and CE-SLOT approaches.
IOP Publishing doi:10.1088/1742-6596/2762/1/0120679 For the CU-SLOT, the NEES values of the object are considerably smaller than 2. This suggests that the object position estimated by the CU-SLOT approach is conservative as using the CU algorithm [15,16].Besides, the NEES for the Naive-SLOT approach is much larger than 2, indicating that this approach is inconsistent.
Conclusion
In this paper, a novel fully decentralized EKF and CI based approach is presented for the MR-SLOT problem.
The proposed algorithm demonstrates accurate tracking of R2O correlations while satisfying the following essential conditions: each robot retains only the latest estimate of its pose and the state of the object, obviating the necessity to store measurements, and information exchange only occurs between one-hop communicating robots.Through simulations, we have validated the performance and superiority of our proposed algorithm in comparison to state-of-the-art approaches with comparable processing and communication costs.The results highlight the effectiveness and efficiency of the proposed method, establishing it as a promising advancement in the field of MR-SLOT.The future extension of this work would be multi-robot simultaneous localization and tracking with regard to an unknown number of moving objects.
s state and covariance estimated by Ri at time k.C(A,B) denotes the cross-correlation matrix between A and B, C(A,B)= C(B, A) T .
Figure 1 .
Figure 1.The correlations between Ri and Rj.The ellipses indicate the estimation covariance, the solid dots represent the estimation state, and the lines are the cross-correlations.
1 R 2 R 3 TFigure 2 .
Figure 2. The trajectories of three robots tracking a moving object in one simulation
Figure 3 .
Figure 3. Simulation results for RMSE of robot position estimation
Figure 4 .Table 2 .
Figure 4. Simulation results for RMSE of object position estimated by R1, R2 and R3
Figure 5 .Figure 6 .Table 3 .
Figure 5. Simulation results for NEES of robot position estimation Object measurement update .When Ri conducts a relative measurement to O at time k+1, the estimated state and covariance of system are updated as follows:1
Table 1 .
Summary of simulation parameters. | 2024-06-05T15:21:16.792Z | 2024-05-01T00:00:00.000 | {
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264303119 | pes2o/s2orc | v3-fos-license | Key aspects defining the development and implementation of a regional genomic surveillance strategy for the Eastern Mediterranean Region
Abstract The COVID‐19 pandemic highlighted the critical role of pathogen sequencing in making informed public health decisions. Initially, the Eastern Mediterranean Region faced limitations in sequencing capacity. However, with robust WHO and stakeholder support, the situation significantly improved. By 2022, COVID‐19 sequencing was underway in 22 out of 23 regional countries, with varying throughput and capacity. Notably, three genomic hubs were established in Oman, UAE, and Morocco, playing a key role in providing expanded genomics training and support across the region. While primarily for COVID‐19 surveillance, this sequencing capacity offers an opportunity to integrate genomic surveillance into existing networks. This integration can enable early detection and response to high‐threat pathogens with pandemic potential. To advance this, WHO/EMRO collaborated with stakeholders to formulate the Eastern Mediterranean Regional Genomic Surveillance Strategy for Emerging Pathogens of Pandemic Concern. Consultative meetings with regional and international genomic surveillance experts identified strategy focal points, key partners, priority pathogens, and implementation steps. As the strategy awaits member states' ratification in Q4 2023, this manuscript outlines pivotal facets defined by member states and the strategic document's key deliverables and opportunities. These efforts aim to yield a substantial positive impact within the region.
monitoring SARS-CoV-2 since the first sequence was published on January 10, 2020. 13,14The analysis of SARS-CoV-2 using robust and increasingly affordable next-generation sequencing (NGS) technologies 15 has been used to complement, augment, and support strategies to reduce the burden of COVID-19. 16,17It continues to inform improved public health policies through monitoring, detecting, and characterizing SARS-CoV-2 variants.
In May 2021, the World Health Assembly (WHA) urged countries to increase their capacity to detect new threats beyond COVID-19, including through laboratory techniques such as genomic sequencing. 18Recognizing the global momentum and drive for investment and continual improvements in the cost, ease, and speed of sequencing, 19 WHO released the 10-year global genomic surveillance strategy for pathogens with pandemic and epidemic potential to support the expansion and integration of genomics into national, regional, and international pathogen surveillance programs. 20The implementation of this strategy aims to improve the speed of detection of pathogens as they emerge by encouraging collaboration between member states (MS) for avenues such as sample and data sharing, improving logistics and training programs and improving the integration of genomic data into existing pathogen surveillance networks.
Developing this global roadmap will require national and international cooperation, with a focus on developing countries and the global South.The UNESCO World Academy of Science advisory committee highlighted that 55% of the genomes in the Global Initiative on Sharing All Influenza Data (GISAID) platform come from the United Kingdom or United States, which account for 5% of the global population. 21A review of the GISAID repository shows that Africa, Asia, the Middle East, and South America combined have contributed 5% of the genomes, illustrating the disparity.
Regional networks, centers for excellence, and multi-sectoral collaboration between national public health institutes, academia, and commercial laboratories will be required to build sustainable genomic capacity, sharing costs, data, expertise, and infrastructure to support regional public health.In this paper, we document the approach and steps taken by the WHO Regional Office of the Eastern Mediterranean (WHO/EMRO) and MS of the Eastern Mediterranean Region (EMR) to develop and operationalize the Regional genomic surveillance strategy in the EMR, in-line with the Global genomic surveillance strategy.
| Landscape review of regional genomic sequencing capacity during the COVID-19 pandemic
The WHO/EMR comprises the occupied Palestinian territory and 21 MS: Afghanistan, Bahrain, Djibouti, Egypt, Iran, Iraq, Jordan, Kuwait, Lebanon, Libya, Morocco, Oman, Pakistan, Qatar, Saudi Arabia, Somalia, Sudan, Syrian Arab Republic, Tunisia, United Arab Emirates, and Yemen.They have diverse cultures, socioeconomic conditions, and demographic characteristics.[24] An assessment of MS capacity has been conducted over the past 24 months, both before and during the COVID-19 pandemic.The results showed a wide range of capacity and investment, with diverse regional capacities.This is not unexpected, as countries have a wide the Central Public Health Laboratory in Oman.These laboratories will be supported both nationally and by the WHO/EMRO to continue expanding capacity for sequencing, with the goal of having recognized reference laboratories for high-risk pathogens that can facilitate training and testing support for countries as they develop their in-country capacity.
Of the remaining seven countries, while sequencing capacity was a priority, conditions were not conducive to a rapid investment or expansion of sequencing laboratories due to complex emergencies, conflicts, and logistical challenges associated with getting reagents or samples into or out of the country.Lack of cold-chain and courier capacity for sample transport, limited information technology infrastructure, trade restrictions, and a lack of expertise in sequencing, both from the laboratory protocols, bioinformatics and interpretation of genomic data, resulted in delays in the implementation of sequencing in these countries, presenting a clear health risk to the populations (Table 1).6][27][28] This portable NGS platform is easy to use in any environment, enabling real-time DNA and RNA sequencing and analysis inside and outside the laboratory.Incountry and regional training in both laboratory techniques and bioinformatics were provided by international experts and stakeholders, including the UKHSA and US CDC.As a result of this investment in capacity, seven of these countries were actively sequencing by the end of 2022 and have started contributing or are intending to contribute to global repositories in the next 3 months.An exception is Sudan, who are currently experiencing severe civil unrest and consequently have limited access to laboratory infrastructure.An overview of all countries with national sequencing capacity pre-and post-2020 is shown in Figure 1.
A timeline for when countries began sharing data internationally through the GISAID database indicates regional contributions and preparedness (Figure 1).WHO declared COVID-19 a pandemic in March 2020, and within 3 months, national and sub-national laboratories from 10 countries were depositing genomes to GISAID.While this serves as a response metric, other countries worked with international collaborators in Europe, United States, and Asia, ensuring the region contributed to the global response (Figure 2).By the end of 2022, 15 countries were depositing sequences to GISAID, emphasizing the improvements in national capacities for COVID-19.
| Consultative fact-finding to determine member state opinions and requests of a genomic surveillance network
The key aim of the regional genomics strategy is to take advantage of the investment in genomics capacity rolled out during the COVID-19 pandemic to establish a sustainable regional genomic surveillance network for high-threat pathogens.This network would directly respond to many of the challenges found in Table 1 and allow for better coordination and information sharing for quality, timely, and appropriate public health actions locally and globally. 29,30For this to be successful and to encourage membership to the strategy, it is important that MS goals and feedback need to be taken into consideration when developing the network.As mentioned previously, the diverse range of socio-economic conditions means that national capacity varies greatly across the region and support needs to be given at all levels to ensure tangible outputs are achieved by the strategy.
While a framework of the regional genomic surveillance strategy was in development, a regional meeting took place in Amman, Jordan, between 1 and 2 February 2023, under a title of "Genomics technical workshop to explore a strategy and roadmap for the creation of a sequencing surveillance network in EMR for emerging and reemerging infectious diseases."WHO/EMRO, regional and international partners, including directors and managers of human, veterinary, and environmental health agencies from MS attended the meeting to review developments on the strategic framework, provide feedback on goals, objectives, and key activities, including operational steps that would encourage regional participation and implementation of the strategy.Other key international stakeholders include (but are not limited to) funding agencies, such as the World Bank, Pandemic Fund, donors such as the Rockefeller Foundation, international agencies such as World Organization for Animal Health (WOAH), Food and Agriculture Organization (FAO), US Center for Disease Control and Prevention (US CDC), and UK Health Security Agency (UKHSA).
Cross-sector collaboration was encouraged by the inclusion of academic institutions and commercial laboratories.
The key challenges and opportunities defined by the collaborative meetings in the Eastern Mediterranean Region.The full report on this meeting will be released through the WHO website in Q4, but the key findings and challenges are summarized in (Table 1).Network-level support to drive engagement and national policy advocacy supporting the network, data sharing, harmonization of training and operating protocols, and increasing group bargaining power for purchasing and logistics are all key aspects requiring support, and tangible recommendations supporting these will need to be incorporated into the strategy.
MS requested that the strategy advocates for establishing steering committees and technical working groups to help with the definition and operationalization of both the strategic network and national capacities.Several pillars were proposed, with each needing defined goals and key performance indicators, as outlined in Figure 3, contributing to the overarching goal of ensuring that a sustainable regional genomic surveillance network for emerging or re-emerging pathogens that can respond rapidly to outbreaks as they occur and to ensure genomics is integrated into the public health framework to provide support for decision making in the region and globally.Stakeholders will provide input and support to specific goals, all working to ensure the network is sustainable.Priority support areas, including building national capacity, supporting regional hubs, and specific support for countries with complex emergencies, are considered key.
MS also observed that the outcomes of the strategy and network needed to be clear and tangible to encourage membership.Continued expansion of existing country capacities, such as trained workforces operating with GISRS and NICs, and the expansion of capacity for non-respiratory pathogens designated as priorities within the region.
F I G U R E 1 NGS capacity for SARS-CoV-2 surveillance has evolved rapidly in EMR and is now supported in 21/22 countries in the region.WHO/EMRO, with the support of national and international stakeholders supported the expansion of NGS capacity for SARS-CoV-2 sequencing across the EMR.Within 6 months of the pandemic declaration, 10 countries were actively contributing to GISAID, and by the end of 2022, 21/22 countries had deposited data into the repository.
A clear priority of the MS was focusing on an integrated, cross-cutting approach to surveillance, incorporating food safety, agriculture, environmental genomics, and public health, with a true One Health approach to the strategy.
In response to these observations, a list of priority pathogens was assembled by the attendees through group discussion and consensus, covering high-threat pathogens of regional concern.
While the formal list needs to be ratified before publication, the breadth of responses highlights that there are a broad range of concerns in the region, ranging from bacterial (causing acute watery diarrhea, or food safety concerns), viral (including respiratory pathogens, but also arboviral such as dengue fever, or hemorrhagic fever causing viruses such as Crimean Congo Hemorrhagic Fever that threaten both human and animal health) and parasitic pathogens (including malaria and schistosomiasis) that threaten human, agricultural, and animal populations.The optimal pathways to ensure surveillance of this range of pathogens needs to be considered, as at present, there is no single laboratory in the region with the capacity to sequence all the pathogens listed, making this a key focus of the developing strategy.
| APPROACHES AND KEY DELIVERABLES FOR DEVELOPING THE REGIONAL GENOMIC SURVEILLANCE STRATEGY
3.1 | Regional genomics surveillance strategies should align with both international and national strategies and priorities to strengthen, rather than dilute, collaboration Based on the landscape review, consultative meetings, and regional experience, a series of key principles for development and subsequent implementation of a regional genomic surveillance strategy were assembled.A key goal of the network will be the capacity to rapidly respond to high-threat, priority pathogens as they emerge or reemerge in the region.Attempts have been made previously to establish these networks, with varying degrees of success, and lessons need to be learned from the successes or limitations of these networks, and the feedback gathered in the consultative meetings has highlighted the key issues being faced by all genomics projects in the region.The Global Genomic Strategy was established based on input from experts in the human, animal, and environmental health sector and strongly advocates for cross-sectoral engagement between governments, intergovernmental agencies, global, regional and national networks, technical partners, donors, academia, and the private sector.It provides high level guidance on the operation of a sequencing network, with guiding principles that need to be adapted to reflect the situation in regional and national settings.This strategy has been considered during the operationalization of several initiatives, including the International Pathogen Surveillance Network (IPSN), 31 BioHub, 32 and other pathogen-specific initiatives, and the regional strategy will seek to align as closely as possible with the recommendations within this guidance to streamline interactions with international partners both within and external to WHO.
F I G U R E 3 Proposed operational structure for the EMR Genomic Surveillance Network.The regional genomic strategy aims to support a multi-sectoral, cross-cutting genomics surveillance network, with collaborative input from MS and stakeholders.A steering committee providing high-level strategic and policy guidance, composed of multi-sectoral experts in genomics, health policy and strategy, and pathogen surveillance, will be recommended.Pillars to support the technical operation of the network will be convened, with MS and stakeholder experts in each pillar to ensure achievable key performance indicators are established and routinely achieved.Maintenance and expansion of regional and national hubs will be strategic priorities, as will continued expansion of support for complex emergency countries.Outcomes and partners will be defined and expanded on in the strategy, for ratification with MS once a draft strategy is finalized.
While the developments of both the regional and international genomics strategies are important milestones in the expansion of genomic surveillance, it is important to maintain focus on the priority of ensuring the strengthening of national surveillance systems, as the first line of response to pathogens as they emerge.This is particularly important with changes in funding policies from USAID and the implementation of the Pandemic Fund, which provides funding avenues for projects managed at the national level, rather than regional or international.With this focus in mind, WHO/EMRO is collaborating with both WHO International and MS to deliver training and support for the development of national genomic surveillance strategies containing clearly defined goals and deliverables, supported by detailed costing analyses to ensure the long-term sustainability of genomics in MS, while also aligned closely with both the regional and international strategies.
3.2 | Genomic surveillance needs to be crosscutting and inclusive, with a One Health approach to monitor the emergence of high-threat pathogens A key lesson from the COVID-19 pandemic is that pathogens can emerge from nature more at any time.The sequencing strategy needs to incorporate a "One Health" approach to improve linkage between sectors, including human, veterinary, food health, and environmental sectors, ensuring that genomic information draws on a wide network of sampling strategies and improves surveillance outcome.This will also improve the sustainability of genomics in the region by expanding the available pool of talent and expertise for sequencing.
With this objective in mind, several key action points need to be finalized before the regional strategy can be implemented.Initially, the pathogens of concern need to be established and ratified by the MS.The inclusion of specific pathogens in the strategy has a threefold purpose: First, this will allow for a secondary landscape review of pathogen-specific surveillance networks, to ensure that the sequencing and genomic data provide a useful benefit to public health responses, and second to guide the implementation of protocols, training packages, and bioinformatics analyses that are needed to operationalize sequencing at the national or regional level.And third, this will aim to minimize the duplication of effort between technical working groups and other already established networks, to meet the observations of the previous section regarding collaboration and cooperation between teams.
To ensure that the genomic surveillance network incorporates as many partners as possible, and in accordance with the results of the consultative meetings, a key deliverable of the strategy will be the convening of a steering committee for regional genomic surveillance.This committee should be comprised of high-level experts in policy for areas including public health, agriculture, environment, and legal experts with knowledge of complex areas such as cross-border data sharing, which are crucial to the function of a genomic surveillance system.Members of this committee should be drawn from the region, to ensure knowledge of local conditions, but be supported by international experts with experience and expertise in disease surveillance, including genomic surveillance.The purpose of the committee will be to provide high-level guidance to MS and the network on goals, funding, key performance indicators, and the direction the network should take to implement policy across the region.It should also provide advocacy at the highest possible levels to ensure that MS are engaged with genomic surveillance, to ensure the sustainability of genomics in the region.
The steering committee should be supported by technical working groups, composed of national and international subject matter experts in areas of critical importance to the function of genomic surveillance (outlined in Figure 3).While some of the areas are clear,
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The network should capitalize and expand on existing development and investment in sequencing throughout the region by implementing a "hub-andspoke" style genomic network As evidenced by the landscape analysis, there is a significant amount of sequencing capacity now available in the EM region.While the strategy aims to expand the scope of genomics in the region, it will look to build on the existing capacity established both before and during the COVID-19 pandemic.
A case in point for this expanded capacity was the implementation and operationalization of the three regional genomics hubs, established in United Arab Emirates, Oman, and Morocco.These facilities have a high level of technical expertise, capacity, and knowledge, which has been applied to support the region throughout the pandemic.As the burden of COVID-19 has eased in previous months, the focus is now turning to how to further expand the capacity of these laboratories to support the region for surveillance of other pathogens.
While most countries now have capacity for sequencing, it is not feasible in the short term to roll out training and support for 10 or more pathogens to all national systems simultaneously.The strategy instead aims to operate on a "hub-and-spoke" model, with central locations acting as reference and support laboratories ("Hubs") providing support, training and monitoring to decentralized, national locations ("Spokes") who over time will develop standalone capacity and expertise.
As mentioned in the previous section, there is an extensive list of pathogens of concern for the region.The strategy aims to capitalize on the regional hubs by allocating specific pathogens to each hub based on the needs of the region.While the steering committee will have the task of finalizing the allocations, it is envisioned, for example, that a hub could be responsible for viral hemorrhagic fevers sequencing.The hub would then be the primary focus for training with international partners, such as the US CDC or UKHSA NVAP program, providing intensive support for areas operationalization of laboratory and bioinformatic protocols, surveillance and sampling strategies, risk assessments, and reporting frameworks to ensure that each hub can function as a reference laboratory for the pathogen of concern.
While functioning primarily as referral laboratories, the hubs would also be responsible for supporting the region in a wider scope, bringing the ownership of the processes and protocols directly into the region, rather than relying on support from external agencies, who are often under extreme pressure due to the wide range of responses they undertake.This would also improve the cost efficiency and sustainability of sequencing, as training courses would be managed directly by the region and countries would be operating with consistent protocols and reagents allowing for improved bargaining capacity when approaching suppliers.Regional quality would also be improved, as auditing expertise would be in the hubs, allowing for more frequent, sustained assessment of quality in the region.
3.4 | Focusing on quality, monitoring, and evaluation of key performance indicators will not only ensure high-quality data but build trust in genomic surveillance as a tool for public health A surveillance system with a well-defined sampling and sequencing strategy, including integrated genome sequencing, will ensure the systematic nature of sample collection, representativeness, quality of genetic sequencing data, 33 completeness, timeliness, reliability of data generated, continuity, and sustainability of the sequencing activity as part of ongoing national surveillance.Quality control will be advocated for at multiple levels, including at the laboratory level, by following recommended good laboratory practices, enrollment in EQAP, providing regular assessment of staff training and competency, and providing support for continuing professional development of staff to ensure a sustainable workforce in the longer term.
Beyond the laboratory, bioinformatic quality assurance will also be supported and monitored.The implementation of standard quality checks on raw data and using protocols shared across the region will ensure trust in the data and genomics as a tool.Support for bioinformatics training and continued knowledge sharing and exchange across the region will be encouraged and facilitated by regional and international stakeholders to build a cohort of bioinformatics expertise for the network.
Bioinformatics expertise has been identified as a critical aspect requiring support, and continued investment in training, standardization, and infrastructure will benefit the region in the long term.Training for regional hubs, training-the-trainer initiatives, staff exchanges, and twinning initiatives with collaborative centers for pathogen surveillance will continue to build on existing expertise beyond COVID-19.Training will be multi-sectoral ensuring that key stakeholders in human and veterinary health, as well as surveillance and policy experts, know how genomic data are generated and can be used for supporting health responses.
Key performance indicators will be established, with metrics agreed upon and ratified by MS involved in the network.Suggested metrics include sample processing and turnaround time, integration with surveillance networks, risk assessment, and alert responses, EQAP results, staff competency, and retention, with an overriding metric of providing a rapid response to pathogens as they emerge.
| DEVELOPMENT, PRESENTATION, AND IMPLEMENTATION
Over the past 12 months, the key elements outlined in this manuscript have been incorporated into a document entitled "Eastern Mediterranean Regional Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential."This strategic document outlines the vision for the surveillance network and outlines the key elements that will be critical to the successful implementation of genomic surveillance in the region.This document has been submitted for internal review, with the goal of presenting the strategy to the region for further consultation and consideration at a meeting to be held in October of 2023.At this meeting, select MS will be given another opportunity to review and ratify the strategy for the region.
Once the strategy is ratified, it will be published for distribution, while the implementation plan will be developed based on the strategy.The key aspect of the composition of the steering committee and technical work groups will be discussed, with proposals for membership presented by stakeholders from the region.
| CONCLUSION
At the onset of the COVID-19 pandemic, GISRS network was leveraged to integrate SARS-CoV-2 into sentinel systems for influenza-like illness, acute respiratory infection, and severe acute respiratory infection to inform policy and the national responses to the outbreak for sustainable and reliable analysis and interpretation of genome data.
WHO/EMRO has made substantial investments in NGS and the computing infrastructure required for incorporating pathogen genomics into public health in EMR.The aim of the EMR genomic surveillance strategy is to build on the gains made by countries thus far and in parallel, equip the remaining laboratories in complex countries with much-needed capacities and capabilities for genomics sequencing.for sequencing and bioinformatics, sequencing 'nodes' at the country level (where feasible) and regional referral hubs will establish communities of practice.This will also lead to better access to supplies and consumables, including data management solutions.
EMR has gained immense experience and insight as innovations have impacted program performance over time, and the lessons learned should be considered a valuable resource.Therefore, the performance of an ever-evolving public health system emphasizes the need for continual improvement, connectivity, and close monitoring. 34 conclusion, challenges notwithstanding, the feasibility of realizing an expanded near real-time regional genomics surveillance system for multiple pathogens will better prepare EMR countries for the next big outbreak without any significant negative impact on existing surveillance systems.
range of socioeconomic conditions and health and emergency responses that place stress on existing infrastructure, affecting the ability to respond to new crises as they emerge.By the time the pandemic was declared in 2020, 11 countries had invested proactively in capacity for routine genomic surveillance through National Public Health Laboratories and National Influenza Centers (NIC).An additional four countries provisioned genomic capacity for COVID-19 in 2021, investing in sequencing equipment, training, data, and sample management.Three regional reference laboratories were established during the COVID-19 pandemic, with strong logistical and technical links to countries across the region, to provide support for genomics and sample testing, continued training and support with bioinformatics and data analysis.These are Sheikh Khalifa Medical City (SKMC) Reference Laboratory for Infectious Diseases, United Arab Emirates; Laboratoire de Virologie at the Institut National d'Hygiene in Morocco; and
F I G U R E 2
An overview of reference laboratories and WHO collaborating centers across the Eastern Mediterranean Region.Reference laboratories, national influenza centers, and WHO collaborating centers have been set up across the region to actively support genomic surveillance.The map indicates facilities with NGS, PCR, and virus isolation capacity.Sample management and logistics, reagent logistics, establishment of minimal data and metadata requirements, data collection and analysis training, harmonization of training materials, and operating procedures are recurring themes in MS member state feedback, and broadly aligns with the recommendations outlined in the Global Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 2022-2032, 20 (referred to as the Global Genomic Strategy for brevity from here on).
technical areas such as protocol development and implementation, documentation, quality assurance, training, and harmonization, other areas require significantly more development, particularly data sharing agreements and the implementation of Memoranda of Understanding to support sharing and transfer of data, expertise and equipment throughout the network.The working groups and steering committee should work together to drive genomics throughout the region and to maintain the focus on the vision of integrating a crosscutting, One Health approach to genomic surveillance with existing surveillance networks.
WHO/EMRO continues to provide leadership, oversight, and coordination to enhance sustainable surveillance systems for emerging epidemic and pandemic-prone infectious diseases.In doing so, countries will have an established framework for effective genomic surveillance strategy and capacity-strengthening plans allowing for a clear set of priority genomic surveillance use cases.Use cases could include avenues such as defining sampling strategies, protocols, analyses, and reporting strategies to guide implementation of genomics in a timely manner.Training centers and workforce training programs for sequencing and bioinformatics, quality assurance programs Weak supply chain capacityBest practices for forecasting and procurement need to be developed and adaptedWeak sample packaging and transportation capacityInvestments to improve sample transportation domestically and internationally to reduce turnaround time, respect cold chain, and increase testing Consider expansion of WHO catalog to include wide range of emerging epidemic and pandemicprone infectious diseases kits to hasten procurement mechanisms currently hampered by the lack of these items; short shelf-life of emerging epidemic and pandemic-prone infectious diseases kitsLimited fundingThe consideration by national disease control programs, emergency responses and other surveillance programs of genomic surveillance as an integral part of the broader public health and thus commit sufficient resources to it.Embargo for some countries Considering limited cold chain storage at country level | 2023-10-20T05:12:30.834Z | 2023-10-01T00:00:00.000 | {
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24032194 | pes2o/s2orc | v3-fos-license | Expression of Matrix Metalloproteinase-2, but not Caspase-3, Facilitates Distinction between Benign and Malignant Thyroid Follicular Neoplasms
Thyroid tumors with a follicular growth pattern include a broad range of lesions from benign hyperplastic nodules to malignant neoplasms (Carling & Udelsman, 2005). Follicular thyroid adenoma (FTA), a benign encapsulated tumor with evidence of follicular cell differentiation, is the most common thyroid neoplasm (Rosai, 2011). True follicular thyroid carcinoma (FTC) (not including follicular variant of papillary carcinoma) is the malignant thyroid tumor exhibiting evidence of follicular cell differentiation (Rosai, 2011). One of the most challenging tasks in pathologic evaluation of thyroid lesions, is the differentiation between FTC and FTA. Diagnosis of FTC is dependent on the presence of capsular or vascular invasion (Rosai, 2011; Bryson et al., 2008) .Fine needle aspiration biopsy is not capable to discriminate between FTA and FTC based on the mentioned criteria (Goldstein et al., 2002). Methods for distinguishing between FTA and FTC would obviate many unnecessary thyroid surgeries. However, to date no definitely reliable and feasible discriminatory marker has been introduced. Matrix metalloproteinase-2 (MMP-2) is a zinc-
Introduction
Thyroid tumors with a follicular growth pattern include a broad range of lesions from benign hyperplastic nodules to malignant neoplasms (Carling & Udelsman, 2005). Follicular thyroid adenoma (FTA), a benign encapsulated tumor with evidence of follicular cell differentiation, is the most common thyroid neoplasm (Rosai, 2011). True follicular thyroid carcinoma (FTC) (not including follicular variant of papillary carcinoma) is the malignant thyroid tumor exhibiting evidence of follicular cell differentiation (Rosai, 2011). One of the most challenging tasks in pathologic evaluation of thyroid lesions, is the differentiation between FTC and FTA. Diagnosis of FTC is dependent on the presence of capsular or vascular invasion (Rosai, 2011;Bryson et al., 2008) .Fine needle aspiration biopsy is not capable to discriminate between FTA and FTC based on the mentioned criteria (Goldstein et al., 2002).
Methods for distinguishing between FTA and FTC would obviate many unnecessary thyroid surgeries. However, to date no definitely reliable and feasible discriminatory marker has been introduced.
In this study, expression of MMP-2 and caspase-3 was examined by immunohistochemistry (IHC) in sections of FTAs and FTCs. The aim was to examine expression of MMP-2 and Caspase-3 in thyroid follicular neoplasms and to determine their usefulness for differential diagnosis between adenoma and carcinoma.
Materials and Methods
Paraffin blocks of formalin-fixed samples representing 60 cases of FTA and 41 cases of FTC diagnosed during the years 1988 -2010 in the Department of Pathology, Shariati Hospital, Tehran, Iran, were examined to determine the expression of MMP-2 and Caspase-3. Cases of FTC were diagnosed based on capsular and/ or vascular invasion. The study was conducted according to the protocol of the Ethics Committee of Endocrinology & Metabolism Research Center.
Two 3-micrometer sections from representative blocks of every tumor were deparaffinized, rehydrated, placed in 0.5% hydrogen peroxide for 10 minutes. Antigen retrieval and unmasking was performed according to manufacturer's instruction. Sections were separately incubated with primary antibodies, washed with TBS buffer, and then incubated with the appropriate biotinylated secondary antibody. Washing in TBS buffer, incubation in ABC reagent, washing in TBS buffer and incubation in suitable peroxidase substrate were then consecutively performed. Finally the slides were counterstained with hematoxyline, dehydrated and mounted. Expected staining pattern was cytoplasmic for both markers. For negative controls, the primary antibodies were substituted with normal serum at appropriate concentration. Tonsil and inflamed large bowel were used as positive controls for caspase-3 and MMP-2, respectively, as recommended by the manufacturer.
Immunoreactivity of tumor cells for each of the two markers were semi-quantitatively scored as to extent (Table 1).
After collection of data, the analysis of qualitative and quantitative data was done using Statistical Package for Social Sciences (SPSS) version 15.0 (Chicago, Illinois, USA). P value < 0.05 was considered significant.
Results
Results of IHC study of FTAs and FTCs are summarized in Figure 1.
Statistically significant relationship was observed between MMP-2 expression and malignant tumor behavior (i.e. diagnosis of FTC) (p=0.025).
Discussion
Differential diagnosis between FTA and FTC is based on presence of capsular or vascular invasion in carcinoma (Rosai, 2011;Bryson et al., 2008) which cannot be evaluated in material obtained from thyroid fine needle aspiration biopsy (Goldstein et al., 2002). Suspicious nodules must therefore be surgically removed in subtotal or total thyroidectomy. With the diagnosis of follicular carcinoma confirmed in formal pathologic evaluation, a second operation may be necessary (Stolf et al., 2006).
Regarding the fact that 5-10% of the population will develop a clinically significant thyroid nodule during their lifetime (Mazzaferi, 1993;Barden et al., 2003) and that by fine needle aspiration biopsy approximately 20% of thyroid nodules are follicular neoplasms (Carling & Udelsman, 2005), methods for distinguishing between adenoma and carcinoma would obviate many unnecessary thyroid surgeries.
Furthermore, such methods would be of great value in cases of minimally invasive (or encapsulated) follicular carcinoma, where discrimination from adenoma based on vascular or capsular invasion suffers inter-observer variation among pathologists (Hirokava et al., 2002;France et al., 2003) and also, in pathology specimens in which capsular detachment, tissue fragmentation or distortion, or subtleness of vascular or capsular invasion makes the distinction between carcinoma and adenoma difficult.
It is suggested that in molecular analysis, follicular thyroid adenoma and carcinoma have different characteristic microarray expression profiles (Barden et al., 2003;Cerutti et al., 2004, Finley et al., 2004Griffith et al., 2006). However, DNA microarray gene profiling is expensive, time consuming, and operator dependent and therefore not practical for clinical use (Bryson et al., 2008).
To overcome the difficulty in distinguishing between FTA and FTC by IHC study as a less costly and more available method, various markers have been evaluated. Despite some overlapping results, Dipeptidyl aminopeptidase IV (CD26) was reported to be useful for distinction between FTA and FTC, primarily in cytological specimens (Kotani et al., 1992;Maruta et al., 2004). Survivin (Hagh et al., 2006), HBME-1, CD15, thyroid peroxidase, CD10 (a zinc-dependent metalloproteinase, that may share certain functions with some MMPs) and cell proliferation markers have also been reported to be useful ( Cho et al., 2006). In a previous experience, we failed to show any discriminating role for nm-23 in this regard (Moradi et al., 2009).
In a study by Chen et al, EMMRPIN has been implicated in the metastatic potential of FTCs (Chen et al., 2001), Barden et al. (Barden et al., 2003) confirmed the differential expression of EMMRPIN by means of immunoblot analysis; however, no evaluation of its diagnostic potential was analyzed. A multicenter study demonstrated that IHC detection of galectin-3 (an enzyme regulating cell-cell communication and implicated in the initiation and regulation of cell growth and malignant transformation) in FNA biopsy samples from thyroid nodules served as a highly specific and sensitive means of identifying malignant lesions of the thyroid ( Bartolazzi et al., 2001). Other studies disprove the utility of galectin-3 as a single protein marker, especially in the case of follicular lesions (Bryson et al., 2008). Prasad et al. reported that a panel consisting of galectin-3, fibronectin-1 and HBME-1 may enhance the sensitivity and specificity for diagnosis of FTC versus FTA ( Prasad ML, et al 2005). Some studies have suggested trefoil factor 3 (TFF3) as a useful biomarker at the RNA level in thyroid cancer, and this gene was listed as 1 of 12 (of a total of 1795 genes) to have the most clinical promise (based on results of the array analysis) for the preoperative thyroid diagnosis (Griffith et al., 2006). IHC results of another study did not substantiate the anticipated promise of TFF3 as a clinical marker (Bryson et al., 2008).
Despite all the studies, of which only some were mentioned, no definitely reliable and feasible discriminatory marker has been introduced to date.
Matrix metalloproteinases (MMPs), including MMP-2, are a group of zinc-dependent endopeptidases with extracellular matrix-degrading properties that affect early events in carcinogenesis and tumor invasion and ⁄or metastasis (Nelson et al., 2000;Nabeshima et al., 2002;Cho et al., 2006).
Over expression of MMP-2 in tumor tissue compared to adjacent normal tissue has been documented in many types of solid tumors, including breast, colorectal and pancreatic carcinomas, as well as squamous carcinomas of the head and neck. Human thyroid carcinoma tissues have been also reported to express MMP-2 (Nakamura et al., 1999;Dubravka et al., 2000). Others proposed MMPs, could not be shown to definitely distinguish between widely invasive and minimally invasive follicular thyroid carcinomas or between the latter and adenomas (Cho et al., 2006).
However, in a study by Liang et al. 113 patient samples were analyzed for expression of several biomarkers including MMP2, which was determined as independent factor associated with the metastatic status in thyroid cancers. Finally, MMP2 was considered as a potential tumor marker to identify greater risk of metastasis (Liang et al., 2011). Moreover, MMPs are known to promote tumor metastasis and are determined as targets of 3,3'-di indolylmethane(DIM),which is believed to have antiestrogen like property and can be considered as a potential novel preventive/therapeutic dietary supplement in thyroid cancers (Rajoria et al., 2011).
Caspases, including caspase-3, are cystein-dependent aspartate-directed proteases and important mediators of apoptosis. Many studies confirm contribution of deregulated apoptotic pathways in cancer development and progression; particularly, an important feature in thyroid carcinogenesis is represented by aberrations in control of apoptosis (Veidinger et al., 2010). Inactivation of proapoptotic and/ or activation of antiapoptotic components, including decreased expression of caspase-3 and other caspases has been documented in many cancers shown to correlate with response to treatment and prognosis in some cancers, including those of breast, lung colon, uterine cervix and thyroid. (Kotani et al., 1992;Devarajan et al., 2002;Philchenkov et al., 2004;Fennel et al., 2005;Furuya, 2007;Moradi et al., 2009;) .
It could be inferred from the mentioned facts that expression of MMP-2 and caspase-3, could potentially be related to -respectively-aggressive and indolent tumor behavior. In this study, expression of MMP-2 and caspase-3 was examined by IHC in samples of FTA and FTC in order to determine their usefulness for preoperative differential diagnosis between FTA and FTC.
We have observed a statistically significant relationship between positive expression of MMP-2 and malignant behavior of thyroid follicular neoplasm. This means that positive expression of MMP-2 in a follicular thyroid neoplasm could be equal to the diagnosis of FTC (versus FTA) with 9.7% sensitivity, 100% specificity,100% Predictive value of a positive test (PPV) and 61.8% predictive value of a negative test (NPV) ( Table 2).
We failed to show any relationship between expression of caspase-3 and the probability of a thyroid follicular neoplasm, being diagnosed as FTA or FTC.
In this study, MMP-2 was differentially expressed by FTAs and FTCs. Alas, because of low sensitivity of positive expression of MMP-2 , this is not a suitable test for screening follicular thyroid neoplasms for diagnosis of FTC. However, in cases with equivocal criteria of malignancy (vascular or capsular invasion) or in a panel with potential sensitive marker, positive IHC result for MMP-2 could be of great value to confirm the diagnosis of FTC with 100% specificity and 100% PPV. | 2017-06-18T03:07:13.844Z | 2012-01-01T00:00:00.000 | {
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18766223 | pes2o/s2orc | v3-fos-license | Knowledge, Attitudes and Practices Regarding Cervical Cancer and Screening among Haitian Health Care Workers
It is estimated that Haiti has the highest incidence of cervical cancer in the Western Hemisphere. There are currently no sustainable and affordable cervical cancer screening programs in Haiti. The current status of screening services and knowledge of health care professionals was assessed through a Knowledge, Attitudes, and Practices survey on cervical cancer screening and prevention. It was distributed to Project Medishare for Haiti health care workers (n = 27) in the Central Plateau. The majority (22/27) of participants stated pre-cancerous cells could be detected through screening, however, only four had ever performed a pap smear. All of the participants felt a screening program should be started in their area. Our data establishes that knowledge is fairly lacking among healthcare workers and there is an opportunity to train them in simple, cost effective “screen-and-treat” programs that could have a great impact on the overall health of the population.
Introduction
In Haiti roughly 353 women die of cervical cancer each year, the second highest cancer-related mortality rate among Haitian women (7.1 per 100,000) [1].In 2000, the International Agency on Research for Cancer estimated that Haiti has the highest incidence of cervical cancer in the Western Hemisphere at 93.8 per 100,000 [2].It is likely that these estimates are significantly lower than the actual number of cervical cancer cases and deaths in Haiti, since there is a low level of awareness, limited access to screening services and no national cancer registry.Globally, cervical cancer is the third most common cancer in women; in 2008 there were an estimated 529,000 new cases [3].Low-resource countries experience 85% of the global burden, and in regions such as Eastern Africa and South-Central Asia, cervical cancer is the most common cancer in women accounting for 13% of all female cancers [3].If detected early cervical cancer is usually curable.However, the global mortality: incidence ratio is 52% [3].In 2008, cervical cancer was responsible for 275,000 deaths, 88% of which occurred in low-resource regions of the world [3].The majority of cervical cancer deaths occur in women who were never screened or treated, as well as those who had an early sexual debut, a history of multiple sexual partners, and a high number of live births [4].
Cervical cancer has several unique characteristics that make prevention through screening and the treatment of precancerous stages relatively straightforward [5].The cause of virtually all cervical cancer cases is known to be the persistent infection with a restricted set of human papillomaviruses (HPVs) [6,7].Cervical cancer also exhibits an identifiable precancerous condition and the time window from dysplasia to carcinoma is long (approximately 10 years on average).Several screening methods to detect precancer and cancer are available, and can be performed safely and inexpensively in an outpatient setting [5].These methods include, but are not limited to, visual inspection with acetic acid (VIA), and careHPV.In addition to being used throughout many resource-limited countries around the world, these methods are also effective at treating precancerous findings, thus further decreasing the burden of disease [5].Primary prevention through vaccination against the prevalent carcinogenic HPV types, HPV-16 and HPV-18, is also possible, but is significantly more expensive [8].In order to best address the overwhelming disease burden in Haiti, the current status of screening services and health professional knowledge must be assessed.
"A Knowledge, Attitudes, and Practices (KAP)" survey is a representative study of a specific population that aims to collect data on what is known, believed and done in relation to a particular topic.KAP survey data are essential to help plan, implement and evaluate programs, and can identify knowledge gaps, cultural beliefs, or behavioral patterns that may facilitate or impede the success of a program.
Experimental Section
A KAP survey on cervical cancer screening and prevention was distributed to Project Medishare for Haiti staff, community health workers, Haitian physicians and nurses in the Central Plateau.
It was provided to these health care workers at several Haitian medical clinics throughout the Central Plateau to assess baseline KAP about cervical cancer, screening and treatment.This project received approval to be carried out by the Emory University Institutional Review Board and the In-Country Coordinator for Project Medishare for Haiti, Marie Chery.Our project was modeled after a previous KAP study, conducted by Dr. Henry Blumberg, Dr. Jennifer Goedken, and others performed in Ethiopia [9].
Project Medishare for Haiti is a US-based Non-Governmental Organization that runs mobile and permanent clinics throughout the Central Plateau region, including Thomonde Clinic, Casse Maternity Ward and Clinic, and Marmont Clinic.The Central Plateau is a rural region of Haiti, home to several hundred thousand people.Communities exist as mostly small town centers around subsistence farms.While recent relief efforts have attempted to improve healthcare in the region, the problem of accessibility between and within communities remains a problem.Large paved roads exist between communities, but travel to rural destinations is impeded by lack of roads altogether.In many communities, the closest source of healthcare is more than a half-day's walk away.
A cross sectional, observational study design was used to assess the knowledge, attitude and practices of cervical cancer, screening methodologies such as visible inspection of the cervix with acetic acid and Pap smear (see Supplementary Information).More specifically the survey addresses healthcare worker's knowledge of risks, treatment and prognosis of cervical cancer, awareness of available screening methods, and their experience and attitudes to the different methodologies.The survey was conducted between June 22nd and 29th, 2013.All adult health care workers in the Central Plateau region were eligible to participate.Surveys were made available in French, Creole or English and were chosen to the participant's preference.Data was anonymous and no personal identifiers were collected.Basic statistical analysis of survey results was performed in SAS.The survey in English is presented in the Supplementary Information.
KAP surveys were offered to nursing staff, midwives, community health workers and physicians at mobile and permanent clinic sites.All subjects gave their informed consent for inclusion before they participated in the study.The study was conducted in accordance with the Declaration of Helsinki.The protocol was approved by the Ethics Committee of the Emory University Institutional Review Board (IRB00067076).Time was allotted during breaks so as not to interfere with daily work commitments.The survey was also allowed to be taken home and completed.Surveys were collected on site or retrieved at local clinics from a safe and secure location.Those participants included were current health care workers (nurses, midwives, community health workers and physicians) and Project Medishare for Haiti staff in the Central Plateau region that were at least 18 years old.The study excluded any persons who were younger than 18 years old.No exclusion was made with respect to gender, race, ethnicity or social status.
Knowledge
The study population included 27 participants in total.Table 1 presents the demographics of the study population.The mean age of the participants was 34 years old.95% of participants were female and 100% identified themselves as Haitian.The majority (19/27) of participants spoke Creole, and 9/27 had only worked for 1-4 years.With regards to their knowledge about cervical cancer and prevention, 69.2% (18/26) stated they did not feel they had adequate knowledge.100% of participants correctly stated that cervical cancer is one of the leading causes of death in women worldwide.Also, 52.2% (12/23) correctly stated that cervical cancer is preventable.When asked whether cervical cancer was curable, 45.5% (10/22) of the study sample correctly answered that question.When asked if it is possible to detect pre-cancerous cervical cancer cells, 81.5% (22/27) correctly stated that was true.74.1% (20/27) of participants also recognized that cervical cancer is not most common among women in their 20 s.When asked whether cervical cancer can usually be found at an early stage because of the obvious symptoms such as bleeding and pain, 18.5% (5/23) correctly stated that was false.Two-thirds (18/27) correctly recognized that if cervical cancer is left untreated it is fatal.When participants were asked whether cervical cancer is caused by a virus that is spread sexually, 77.8% (21/24) correctly stated that was true.When asked whether there is a vaccine that can prevent cervical cancer, one-third (9/27) stated that was true.Almost all of participants (25/27) correctly recognized the purpose for screening is to detect pre-cancerous changes.When asked whether screening for cervical cancer should begin when a woman is in her twenties, 51.9% (18/27) stated this was false.When the women surveyed were asked whether they felt they were themselves at risk for cervical cancer, 71.4% (10/14) stated they did not think they are at risk.The risk factors for cervical cancer most often chosen by participants were: HIV infection (38.5%), smoking (53.9%), multiple partners (57.7%), and HPV infection (73.1%).Of those participants that stated they were not at risk for cervical cancer themselves, 80% (8/10) stated multiple partners and 90% (9/10) stated HPV infection were risk factors for cervical cancer.Of those who stated they were at risk for cervical cancer, 100% (4/4) stated poor hygiene and 75% (3/4) stated IUD use were risk factors for cervical cancer.Among those participants who earlier stated they felt they were knowledgeable about cervical cancer, 83.3% (5/6) correctly identified HPV infection and multiple partners as risk factors for cervical cancer.Among those participants that stated they were not knowledgeable or required additional education about cervical cancer, 66.7% correctly identified HPV infection and 44.4% correctly identified multiple partners as risk factors for cervical cancer.When asked what they thought were symptoms of advanced cervical cancer, 20% (5/25) identified abdominal pain, abnormal bleeding, foul-smelling discharge, and loss of appetite, 20% (5/25) identified abdominal pain, abnormal bleeding, and loss of appetite, and 16% (4/25) identified abnormal bleeding as symptoms.Finally, when asked whether they felt cervical cancer screening is an essential part of women's health, 92.3% (24/26) strongly agreed.Note: Some participants did not complete all questions so numbers may not add to 27 due to missing answers.
Attitudes
Among our participants, 52.4% stated they were very willing to do visual screenings on their patients.Of those participants who stated they needed more knowledge in order to perform screening, 72.2% (13/18) were willing to do visual screening.When asked whether a cervical cancer screening program should be started in their community, 100% of participants agreed or strongly agreed.Of those who had worked less than one year, 25% (1/4) stated that screening was "too difficult".Of those who had worked 1-4 years, 57.1% (4/7) stated screening was "too difficult".Of those who had worked more than 10 years 50% stated screening was "quite a bit" difficult.Of those who felt screening was "too difficult" (defined as having answered "a lot", "somewhat", or "quite a bit") 61.5% (8/13) felt it was also too expensive to screen.Among those who did not feel screening was "too difficult" 42.9% (3/7) felt it was too expensive to screen.Additionally, 15.8% (3/19) of participants strongly felt their patients have more important issues than cervical cancer.The majority of participants felt that they need "a lot" of additional training in order to successfully perform screenings.All of those who had been working longest, more than 10 years, felt they needed additional training.Only 26.7% felt they were too busy to screen women.With regards to access to supplies needed to screen (defined as having answered, "a lot", "somewhat", or "quite a bit") 61.1% (11/18) felt they did not have the supplies needed to screen and 40.0%(6/15) felt they did not have a lab necessary to screen.The majority of those without access to supplies also did not have access to the necessary laboratory resources.Of those participants who felt that screening was too expensive, 45.5% stated they did not have the necessary lab resources, 81.8% stated they had trouble with supplies.Of those who stated they did not feel they had adequate supplies or lab resources to perform visual inspections, 71.4% were willing to perform cervical cancer screening by visual screening.
Practices
With regards to current and past practice, when asked how long they had been working, 5/27 stated they had worked less than 1 year, 9/27 stated they had been working for 1-4 years, 4/27 stated they had worked 5-10 years, 3/27 stated they had worked longer than 10 years and 6/27 did not respond.Within the participants, 25% (6/24) stated they had performed cervical cancer screening of any kind.Only 16.7% (3/18) stated they had ever performed pap smears.Of those who had performed pap smears, only one stated they had performed more than 10 in their career.Figure 1 presents the percentage of respondents who have performed pap smears distributed by the length of time they have worked.Of those participants who stated they had been working for less than a year, 33% (1/3) stated they had performed a pap smear.Of the participants who stated they had been working more than 5 years, none of them had performed pap smears at all.No one stated they had performed visual inspection (with either acetic acid and Lugol's solution).Note: In those who have been working <1 year, the additional 33.3% is accounted for because one person stated they were unsure whether they had performed a pap smear.
Discussion
Our survey results indicated that there was a strong willingness within the healthcare workers of the Central Plateau to participate in a cervical cancer screening prevention campaign but they believe that screening is difficult and expensive.It also indicates that the health care workers feel that they need additional training, access to supplies, and a functional laboratory in order to perform the examinations.In addition, hardly any of the health care workers had performed cervical cancer screening and none had performed VIA.In terms of their knowledge base, only around half believed that cervical cancer is preventable, and a little less than half believed it was curable.However, the majority of participants believed that pre-cancerous cells could be detected.This discrepancy identifies a knowledge gap, most notably with the most recent data about the role of HPV in cervical cancer.A great number of the participants felt that cervical cancer could be identified early only through symptoms.With regard to knowledge of symptoms, a number of participants felt that a patient would have abdominal pain and/or loss of appetite in addition to abnormal bleeding.This shows that the health care workers are adept in recognizing more advanced symptoms of cervical cancer but may not recognize more subtle symptoms such as bleeding after intercourse or intermenstrual bleeding as concerning symptoms of early cancer.This is especially concerning because of the significant asymptomatic phase that accompanies cervical cancer that would surely be missed if this type of assessment were utilized.In addition, only a third of participants correctly answered there was a vaccine that could prevent cervical cancer.One reason for this significant knowledge gap could be the prohibitive cost of the HPV vaccine in a low-resource country.There was also a great discrepancy in the knowledge of risk factors between those who felt they were at risk for cervical cancer and those who felt they were not at risk.Those who felt they are at risk for cervical cancer correctly identified HPV infection and multiple partners as risk factors, whereas, those who did not admit to risk for self, identified poor hygiene and IUD use as risk factors for cervical cancer.The lack of recognition of risk factors is not only detrimental to the healthcare workers themselves but also to their patients that they are counseling.With that said, a large number of participants did recognize their own lack of knowledge and felt they needed additional training to feel comfortable with cervical cancer screening and prevention.Therefore, a great deal of education needs to be undertaken to help dispel myths and adequately train the healthcare workers.The highlighted knowledge gaps present a clear opportunity for education and training of healthcare workers about cervical cancer with provision of adequate resources.With regards to their attitudes, all of our participants felt a cervical cancer-screening program should be started in their community.This is very encouraging considering the barriers they felt were limiting their ability to have a screening program in their community.Even the majority of those who stated they need additional education about cervical cancer were still willing to participate in a "screen and-treat" program.The barriers that were recognized by the health care workers were difficulty of performing screening, expense, and access to supplies and a functional laboratory.The benefit of VIA and the careHPV DNA tests is that both are very cost-effective and do not require any extensive supplies or laboratory functions.These are the best options for our resource-limited communities and the health care workers in the Central Plateau that were surveyed most definitely recognized themselves as resource limited.So in addition to adequate education and training, once shown how cost-effective and simple these techniques truly are for their resource-limited settings, we have an excellent opportunity to educate and train healthcare workers in the Central Plateau.
Lack of experience was recognized as a major barrier to providing cervical cancer prevention services.Only one out of the 27 participants had performed more than 10 pap smears and no one had performed visual screening.This suggests that the health care workers are not being trained on cervical cancer screening, which most likely has to do with the fact that pap smears are high-resource and high-expense screening practices.However, we did note that those who have been trained more recently were more likely to have performed cervical cancer screening although the number of respondents here was small.This is encouraging and may suggest a shift towards the training of healthcare workers in the technique of performing pap smears.Health care workers, as stated before, would need a great deal of additional training; fortunately there are standardized training efforts available in order to provide health care workers with the knowledge and confidence to successfully and efficiently perform visual screening and/or pap smears.
Cytology screening programs involving repeated screenings are highly effective at detecting precancerous lesions and preventing cervical cancer.The introduction of routine Papanicolaou (Pap) smears (and diagnostic biopsy coupled with treatment of precursor lesions) into high-resource European countries and North America was followed by a substantial reduction in disease burden.Despite the success of cytology-based screening programs, these programs rarely exist in low-resource regions and where they do exist are frequently ineffective, primarily due to inadequate technical, manpower and financial resources [10].The multiple follow-up visits required following cytology-based screening is also a significant barrier to success in low-resource settings due to difficulties regarding contacting patients for follow up, as well as patient related issues of transportation, clinic hours, costs, child care needs and other factors.Studies suggest that cervical cancer screening programs are most successful and cost-effective in low-resource settings when they require few visits and offer a "screen and treat" for (single-visit) approach [10].Visual inspection with acetic acid (VIA) [11] and a new rapid, low-cost simple version of HPV DNA testing (careHPV; QIAGEN, Gaithersburg, MD, USA) [12] are two cervical cancer screening alternatives to cytology that have been used in low and medium resource settings [12][13][14][15][16][17][18][19][20].The VIA method involves applying a 3% to 5% solution of acetic acid (ordinary table vinegar) to the cervix followed by a naked eye assessment (no light source or magnification required) of the cervix a minute later [11].Precancerous lesions and other abnormalities are apparent to the naked eye as areas of whiteness.VIA has been found to be low-cost, simple to perform, and can be incorporated into a single-visit approach [13,15,20].
In addition, the newly available careHPV test can be performed without electricity or running water in as little as 2.5 h [12] and therefore it may prove to be effective as part of a single-visit approach also.Cost-effectiveness analysis of cervical cancer screening approaches in less-developed countries have concluded that VIA as part of a well-organized "screen-and-treat" program can reduce cervical cancer mortality at low costs [21,22].It demonstrated clinical effectiveness in over 1 million women in clinical studies conducted in China, Nigeria, Rwanda and Thailand.In terms of cost per life year saved, an analysis based on a rural Chinese setting found VIA to be more cost-effective than careHPV DNA testing, although VIA was not quite as effective at reducing cancer incidence [23].In Nigeria, careHPV test was performed in challenging remote settings with minimal interventions and training from skilled professionals [24].The Nigeria trial showed high specificity and adequate sensitivity with local investigators without any research background.This is very encouraging considering the knowledge and resource limitations of the Haitian healthcare workers.
A single-visit approach combining VIA and cryotherapy when assessed through a demonstration project in rural Thailand was found to be safe, acceptable and feasible [16], and such an approach performed by nurses has also been found to be effective, safe and acceptable in India [20].This is an excellent option for our population in Haiti.The cure rate for cryotherapy has been reported to be between 70%-88% depending on the setting and the progression of the precancerous lesions identified [20,25].Due to VIA having a relatively high false positive rate in some settings (specificity reported to range from 64%-98% [13] there is a real possibility of over-treatment with cryotherapy; however, since cryotherapy is effective and safe, the overall risk-benefit-ratio is considered favorable.With this study and our study in mind, we feel most strongly that VIA and cryotherapy would be the most effective and cost-conscious "screen-and-treat" option for rural Haiti.Figure 2 illustrates a proposed algorithm for screen-and-treat programs that we feel would be most beneficial to rural Haiti.
A limitation to our study is our small sample size of participants.Due to the rural setting in which we performed this study there are fewer health care workers in the region to begin with, leading to a lower potential sample size overall.Many of these health care workers in the Central Plateau of Haiti take care of an entire neighborhood of individuals and work with other community members to disseminate information.Another limitation is missing information, whether due to lack of understanding of the question or inability to answer, a number of our questions had a large number of missing participants.In addition to missing information, each question had a variable number of participants answer them.This is likely due to the fact there were a significant number of incomplete surveys due to health care workers leaving questions blank.Due to such a significant number of incomplete surveys there are a number of questions for which the sample size is very low, which impacts our ability to make strong conclusions regarding the content of the question.With the limitations in mind it is very important to remember that one of the greatest strengths of this study is that we are the first to perform a KAP study about cervical cancer screening in the Central Plateau of Haiti.Due to Haiti's limited resources, cervical cancer is not being treated very often and therefore, the feeling prior to recent policy changes in the Haitian Ministry of Health, was that screening was not a priority for the patient population.Despite our small sample size, we feel that the qualitative information we elicited is extremely valuable.With improving access to treatment and the ever-growing presence of aid organizations such as Project Medishare and Partners in Health in the rural areas of Haiti, we have an opportunity to make a difference with a cost-efficient and simple method of "screen-and-treat".
Conclusions
Our data establishes the fact that knowledge is fairly lacking even among healthcare workers and there is a clear opportunity to train healthcare workers in simple, cost effective "screen-and-treat" programs that could have a very great impact on the overall health of the population.Future plans include putting together a financial plan comparing visual screening and cryotherapy with HPV DNA testing and treatment to decide which is most cost effective and best suited for our specific population and setting.Then we plan to partner with local aid groups to provide training and supplies in order to pilot a "screen-and-treat" program in the Central Plateau.
Figure 1 .
Figure 1.Percentage of respondents who have performed pap smears by amount of time worked.
Table 1 .
Demographics of Haitian Healthcare Workers. | 2016-03-22T00:56:01.885Z | 2014-11-01T00:00:00.000 | {
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119153695 | pes2o/s2orc | v3-fos-license | Kulikov surfaces form a connected component of the moduli space
We show that the Kulikov surfaces form a connected component of the moduli space of surfaces of general type with p_g=0 and K^2=6. We also give a new description for the surfaces, extending ideas of Inoue. Finally we calculate the bicanonical degree of a Kulikov surface, and prove that they verify the Bloch conjecture.
Introduction
In 1966, Burniat [Bu] constructed some new examples of surfaces of general type with p g = 0 as (Z/2) 2 -covers of the plane branched in a certain configuration of lines. Then in 1994, Inoue [I] constructed examples with the same numerical invariants as Burniat's examples by taking a (Z/2) 3 -quotient of a hypersurface inside a product of three elliptic curves. It seemed to be well known that the two constructions were equivalent, and a proof was written down in [BC]. The Inoue construction was then used in [BC] to explicitly calculate the fundamental group and homology of the Burniat-Inoue surfaces. This enabled a proof that primary Burniat-Inoue surfaces (those with K 2 = 6) form a connected component of the moduli space of surfaces of general type, and further, this component is actually closed under homotopy equivalence (see below for an explanation of this terminology). The first proof that Burniat-Inoue surfaces form a connected component of the moduli space appeared in [MP2].
Kulikov [K] exhibited a new example of a surface of general type with p g = 0 and K 2 = 6 as a (Z/3) 2 -cover of the plane branched in a different configuration of lines (see Figure 1). In this paper we show how the Kulikov surfaces fit into the same framework as Burniat-Inoue surfaces by exhibiting an Inoue-type construction. We go on to prove that Kulikov surfaces form a 1-dimensional connected component of the moduli space, which is closed under homotopy equivalence.
The layout and main results of this paper are as follows: in Section 2 we discuss preliminary results on abelian covers of the plane branched in line configurations, and use these to define the Kulikov surface. In Section 3 we prove that there is an Inoue-type construction for Kulikov surfaces, and that they form a 1-dimensional irreducible component of the moduli space of surfaces of general type. We also describe three interesting degenerate Kulikov surfaces, which can be viewed as the boundary points of the compactified moduli space.
In Section 4, following ideas employed in [BC], we use the Inoue-type construction to explicitly calculate the fundamental groups and homology of our surfaces: Theorem 1.1 If X is a Kulikov surface, then π 1 (X) = Γ and H 1 (X, Z) = (Z/3) 3 .
Here Γ is a certain infinite subgroup of the affine group A(3, C), decribed in terms of generators in Section 4. Having determined the fundamental group, we are able to prove our second main result:
Theorem 1.2 The moduli space of Kulikov surfaces is closed under homotopy equivalence.
In other words, any compact complex surface which is homotopy equivalent to a Kulikov surface is given by our construction. We say that the moduli space is closed under homotopy equivalence. This implies the pencil of Kulikov surfaces forms a connected component of the moduli space of surfaces of general type with p g = 0 and K 2 = 6. One might conjecture that any surface with the same π 1 is given by our construction. We have not been able to prove this, because the proof of Proposition 4.5(2) relies on homotopy equivalence.
Section 5 contains a calculation of the degree of the bicanonical map of the Kulikov surfaces:
Theorem 1.3 The bicanonical morphism of a Kulikov surface is birational.
This is the expected result; by [MP], [MP2] the possible values are 1 or 2, but it would be surprising if the degree did not divide 3. In the course of calculating the degree of the bicanonical map, we prove Proposition 5.1, which gives a formula for the eigenspace decomposition of the bicanonical sheaf ω 2 X for an abelian cover. This complements the standard formulas of [P] Proposition 4.1, and we believe it is of independent interest.
In our last section, we verify the Bloch conjecture on zero cycles for Kulikov surfaces. Let A 0 0 (X) denote the group of zero cycles of degree 0 on X, modulo rational equivalence.
The theorem is proved using a well known method of [IM], which involves a careful examination of various quotient surfaces associated to the maximal abelian cover X → X. Unfortunately the final step in our proof is done by computer algebra. During the final preparation of this manuscript we were informed by V. Alexeev that he and R. Pardini also computed the degenerate Kulikov surfaces of Section 3.4, using similar methods to those of [AP].
2 Surfaces as abelian covers
Abelian covers
We briefly review the preliminary results needed for our calculations. More details can be found in several articles, including [BC4], [C], [P]. Assume Y is nonsingular and let ϕ : X → Y be a finite morphism with a faithful action of the abelian group G on X so that Y is the quotient. Let ∆ be the branch divisor of ϕ, then ϕ is determined by the surjective homomorphism Φ : π 1 (Y ∆) → G, which factors through Φ : H 1 (Y ∆, Z) → G since G is abelian. In this paper Y is always P 1 , P 2 or a nonsingular del Pezzo surface, and ∆ is a configuration of points or lines, hence where ∆ i are the irreducible components of ∆ so that ∆ = n i=1 ∆ i . Now suppose P is a point of intersection of two or more branch lines l 1 , . . . , l k . Then X is nonsingular over P if and only if there are exactly two lines intersecting transversely at P , and (1) If we blow up P to obtain the exceptional curve E, then there is an induced group homomorphism on the blow up, which we still call Φ, defined by Note that the induced cover on the blow up may be unramified over E, and that the blow up may or may not resolve the singularity on X over P .
We have where G * = Hom(G, C * ) is the group of characters of G, and G acts on L −1 χ with character χ. Fix χ ∈ G * and choose a generator for the image of χ in C * , so that we view χ : G → Z/d, where d is the order of χ. Then the following formula determines the eigensheaf L χ = O Y (L χ ): If X is nonsingular, the ramification formula for K X gives where d i is the order of Φ(∆ i ) in G, and
The Kulikov surface
In this paragraph we construct the Kulikov surface. We also define the groups G 0 , G 1 , G 2 which appear throughout this article. Let ϕ : X → P 2 be the (Z/3) 5 -cover of P 2 branched in the Kulikov configuration ∆ = 6 i=1 ∆ i of Figure 1. Then ϕ is determined by the group homomorphism Φ : H 1 (P 2 ∆) → (Z/3) 6 , represented by the 6 × 6 matrix of rank 5. Since ∆ is supported on six lines, Φ is unique up to choice of generators for (Z/3) 6 . Thus X is maximal, which means any other (Z/3) kcover of P 2 branched in ∆ factorises ϕ.
Note that by (1), X is singular as a cover of P 2 , so we blow up the three singular points P 1 , P 2 , P 3 of ∆, and view X as a nonsingular (Z/3) 2 -cover of the degree 6 del Pezzo surface Y . Then computations using (2-5) show that the Kulikov surface X is a minimal surface of general type with p g = 0 and K 2 = 6.
Elliptic curves as abelian covers
This digression on elliptic curves and abelian covers is required for the Inoue construction of the subsequent section.
Let ϕ : E → P 1 be a Galois (Z/3) 2 -cover branched in ∆ = P 0 + P 1 + P ∞ . Note that E is the (unique) Fermat cubic curve, see Remark 3.1. There are several ways to study ϕ, but for consistency we prefer to use the techniques of abelian covers. Now Φ : H 1 (P 1 ∆) → (Z/3) 2 is unique up to choice of generators, and is given by Φ = 1 2 0 0 2 1 .
Using (3) to study the various eigensheaves and quotients of the cover, we establish the following diagram: Here π 1 is the isogeny induced by taking the quotient by the group η of translations by the 3-torsion point η. There are three possibilities for π 2 corresponding to the remaining nontrivial subgroups of (Z/3) 2 . We choose the subgroup ω which fixes the three points ϕ −1 (P 1 ) on E, and we label these points 0, η, 2η. Since we have now fixed the origin of E, we may consider ω = e 2πi/3 as a rotation of E, under which η is fixed. The map ρ 1 is the Z/3-cover of P 1 branched in ∆, and ρ 2 is the Z/3-cover of P 1 branched over P 0 + P ∞ . Note that in terms of the original basis for (Z/3) 2 , η = 1 2 and ω = 2 2 .
The Inoue-type construction
In this paragraph we give an alternative description of the Kulikov surface, which is called the Inoue-type construction, after [I]. This sets the Kulikov surface into context alongside the Burniat-Inoue surfaces. First note that after blowing up P 1 , P 2 , P 3 we may think of the Kulikov configuration in Figure 1 as an element of the pencil i are the coordinates on the ith factor. Now let E π 2 − → P 1 ρ 2 − → P 1 be a (Z/3) 2 -cover of P 1 as described in Section 3.1. Taking a direct product of three copies of E, we obtain the following diagram: where π = π i 2 , ρ = ρ i 2 . Fix an element Y of our pencil of del Pezzo surfaces. Then ρ −1 (Y ) splits into three components Z i , i = 0, 1, 2, each of which has stabiliser isomorphic to (Z/3) 2 . We fix Z = Z 0 , with stabiliser The quotient of X by G 1 is Y , and by construction, the branch locus of X → Y is exactly the blown up Kulikov configuration. Thus X is the maximal (Z/3) 5 -cover of Section 2.2.
Lemma 3.2 There is a subgroup G 2 ∼ = (Z/3) 3 of G 1 such that G 2 acts freely on X, and the quotient X is a Kulikov surface.
Proof Since X → Y is the maximal (Z/3) 5 -cover of Y , we see that the quotient map (ρ • π)| X = ϕ, where ϕ is determined by the matrix (6) of Section 2.2. Hence the group G 1 is generated by the columns δ i , ω i of (6) as stated in Definition 2.1. By Definition 2.2, the subgroup G 2 is generated by For completeness, we reconcile the above construction of G 0 with Definition 2.2. Observe that we subdivided the rows of matrix (6) into pairs, each of which corresponds to the action of G 1 restricted to the respective factor of E × E × E. For example, by Definition 2.2, ξ 1 = t 2, 1, 1, 2, 0, 0 , and comparing this with the definition of η = 1 2 from (11), we see that where η i now denotes translation by the 3-torsion point η on the ith factor of the product. We also have This is called the Inoue-type construction of Kulikov surfaces, cf. [BC], [I] for the original Inoue surfaces.
The moduli space of Kulikov surfaces
Using the Inoue-type construction, it is clear that we obtain a pencil of Kulikov surfaces as the pullback of the pencil of del Pezzo surfaces. In fact we have: Theorem 3.3 The pencil of Kulikov surfaces forms a 1-dimensional irreducible component of the moduli space of surfaces of general type with p g = 0 and K 2 = 6.
Proof Let X be a Kulikov surface. We calculate h 1 (T X ) = h 1 (ψ * T X ) using the splitting of ψ * T X into eigensheaves according to Proposition 4.1 of [P]: curve. The remaining cases are Given the decomposition (13), it is clear that We use several methods, which we describe subsequently, to calculate the value of h 2 ((ψ * T X ) (χ) ) for each χ. For convenience, we present the results in Table 1. Now since h 0 (T X ) = 0 for a surface of general type, we have So using this equality, Table 1 and equation (14) we get h 1 (T X ) = 1, which proves the theorem. Table 1: Decomposition of ψ * T X .
The remainder of this section deals with Table 1. The table contains the data required to write down the eigenspace decomposition of ψ * T X according to (13). We also remark that the divisors ∆ g are always composed of a number of disjoint rational curves. Thus there is no need to distinguish between logarithmic poles along D 1 and D 2 , and logarithmic poles along D 1 + D 2 for any curves D 1 , D 2 . The data are grouped into cases (0), (a), . . . , (d) by symmetry considerations, avoiding repetition in our calculations.
First observe that by Serre duality, where A = K Y + L χ . Thus the foundation for our calculations of the various h 1 ((ψ * T X ) (χ) ) is the standard logarithmic residue sequence twisted by A, and its associated long exact sequence of cohomology, with connecting homomorphism δ : Thus it is possible to calculate the rank of δ directly, because of the Lefschetz (1, 1) Theorem.
In turn this gives us the value of h 0 (Ω Y (log ∆ g : g ∈ S χ )(A)) using the long exact sequence. The crucial part of our proof is to adapt this argument to situations where A is nontrivial. Note that it is possible to calculate the value of h 1 ((ψ * T X ) (χ) in each case, using the Riemann-Roch theorem together with the logarithmic residue sequence (16). However, this is not needed for the proof so we omit this calculation.
The basic method
In this paragraph we compute the last column of Table 1 in cases (0), (b) and (d). We first give the set up: suppose that |−A| has no fixed part, so there is a morphism O Y (A) → O Y which is nonzero on each irreducible component of the branch divisor ∆. Then we obtain the following commutative diagram: The connecting homomorphisms for the corresponding long exact sequences fit into the commutative square , c 1 is the Chern class map, δ is the connecting homomorphism whose rank we wish to calculate, and β has kernel As an illustration, consider case (b), so that χ = (0, 1) and A = −2H + E 1 + E 2 + E 3 . Clearly A has no fixed part, and moreover Now since ∆ 1 , ∆ 2 and ∆ 3 are linearly independent in Pic Y , rank α = 3. Thus rank δ = 3, and so h 0 Ω Y (log ∆ g : g ∈ S (0,1) )(A) = 0.
The contraction lemma and fibration method
This paragraph deals with case (a), where the basic method does not work. We contract some branch divisors on Y and then calculate directly. A more general version of the following lemma appears in [BC2], [BC3]. We quote a simplified version, which is sufficient for our purposes: In case (a), with χ = (1, 0), we have A = −H + E 1 . Note that the basic method does not work here: the rank of α is 3, which is not maximal, so we do not have sufficient information to calculate rank δ. Instead we contract ∆ 3 and E 3 via π : Y → Q = P 1 × P 1 , and let f : Q → P 1 be the projection to the second factor. Then by Lemma 3.4, where F 1 = π * ∆ 2 , F 2 = π * A, F 3 = π * E 2 are fibres of f , and B 1 = π * ∆ 1 , B 2 = π * ∆ 5 are sections. Now, the standard short exact sequence Thus twisting by −F 2 and observing that f * ω P
Final case (c)
In case (c), A = −H + E i , so |−A| is empty and it is not possible to use the basic method. We can not use the contraction lemma either, because when we contract ∆ i , we still have poles along E i , and further contractions are complicated. Instead we use the natural inclusion Hence h 0 (Ω Y (log ∆ g : g ∈ S (0,2) )(A)) = 0.
Degenerate Kulikov surfaces
In this paragraph we constuct three degenerate Kulikov surfaces arising as (Z/3) 2 -covers of special elements of the pencil of del Pezzo surfaces These degenerate surfaces are members of the boundary of the compactified moduli space, in the sense of [KSB].
A hyperelliptic surface
When λ = µ = 1, the three lines ∆ 4 , ∆ 5 and ∆ 6 of the Kulikov configuration meet in a point P 0 . This configuration is called the complete quadrangle and it is illustrated on the left side of Figure 2. The (Z/3) 2 -cover X → Y has an elliptic singularity of degree 9 over P 0 . If we blow up Y at P 0 , then the induced (Z/3) 2 -cover X is the resolution. Figure 2: The complete quadrangle and the cube Note that the pencil of conics in P 2 passing through P 0 , . . . , P 3 gives rise to a pencil of Fermat cubics on X, so X is an elliptic surface. Moreover, the minimal model of X is the (Z/3) 2 -cover of P 1 × P 1 obtained by contracting the strict transforms of ∆ 4 , ∆ 5 and ∆ 6 . The branch locus is transformed to six lines (three in each ruling), and the minimal model of X is the hyperelliptic surface (E × E)/(Z/3) 2 .
One can also see this degeneration in terms of the Inoue-type construction of Section 3.2. The action of G 2 is free on E × E × E outside the orbit of the point (0, 0, 0), which has stabiliser Z/3 generated by g 1 g 2 g 3 . The degenerate Kulikov surface described above is obtained when the hypersurface X 3,3,3 ⊂ E × E × E contains the point (0, 0, 0).
Two reducible surfaces
When µ = 0 or λ = 0, the del Pezzo surface Y breaks into three copies of P 1 ×P 1 . We only consider the case µ = 0 as shown on the right side of Figure 2, because λ = 0 has a similar treatment by symmetry. The components of Y are called Y i : (x i = 0), and Y has normal crossing singularities along the lines D k : (x i = x j = 0) for i, j, k = {1, 2, 3}, indicated by dotted lines in the figure. The divisors ∆ 4 , ∆ 5 and ∆ 6 break into pairs of lines, for example ∆ 4 consists of the two lines joining E 1 to ∆ 1 , intersecting in a single point Q 1 . For clarity, we do not label ∆ 4 , ∆ 5 , ∆ 6 in the figure.
The other components of X have similar singularities over the points Q i . Perhaps the most interesting aspect of this example is that when we glue the components of X back together, we see that over each Q i , we must attach a 1 3 (1, 2) point to a 1 3 (1, 1) point. This gives rise to orbifold normal crossing singularities, which can be expressed locally as where the double curve is given by (x = y = 0).
Fundamental groups and homotopy equivalence
In this section we give an explicit description of the fundamental group of the Kulikov surface. Using this description we show that the 1-dimensional irreducible component of the moduli space constructed in the previous section is actually closed under homotopy equivalence. Hence the Kulikov surfaces form a connected component of the moduli space.
Fundamental group and homology
Using the Inoue-type construction of Section 3.2, it is possible to lift the action of G 2 = g 1 , g 2 , g 3 to the Fermat cubic curves. Indeed, let z i be a uniformising parameter for the ith curve in the direct product (12). Recall that in the proof of Lemma 3.2, we showed that ξ i = η 2 i η i+1 for i = 1, 2, 3. Now by Definition 2.2, we have g i = ξ i+1 ω i+1 . Hence the action of G 2 lifts to the product E × E × E as Now write e i , e ′ i for the lattice generators of the ith factor in E×E×E. In particular, we fix e i = 1, e ′ i = ω so that the 3-torsion points η i = 1 3 (2e i + e ′ i ) and 2η i = 1 3 (e i + 2e ′ i ) are fixed under rotation by ω. We lift g i to affine transformations γ i in A(3, C), and define Γ ⊂ A(3, C) to be the subgroup generated by γ i , t i , t ′ i for i = 1, 2, 3, where t i , t ′ i are affine translations by e i , e ′ i respectively. Note that since γ 3 , where i is taken modulo 3, Γ is in fact generated by the γ i and t i .
Proof By the Inoue-type construction of Section 3.2, X has anétale (Z/3) 3 -cover X, which is a smooth hypersurface of tridegree (3, 3, 3) in E × E × E. Therefore, by Lefschetz's theorem, π 1 ( X) = π 1 (E × E × E), which is generated by translations t i and t ′ i for i = 1, 2, 3. Now, Γ acts on the universal cover C 3 of E × E × E, and it acts freely on the universal cover X ⊂ C 3 of X. By construction X = X/Γ, so X is the universal cover of X and π 1 (X) = Γ. Clearly, we have also shown that there is an exact sequence The group H 1 (X, Z) is the abelianisation of Γ, so we calculate its centre [Γ, Γ]. Observe that where the generators are the residue classes of γ i modulo [Γ, Γ].
The moduli space is closed under homotopy equivalence
In this paragraph we prove that the moduli space of Kulikov surfaces is actually a connected component of the moduli space of surfaces of general type with K 2 = 6. In fact we prove a stronger result:
Theorem 4.3 Any compact surface S which is homotopically equivalent to a Kulikov surface X is itself a Kulikov surface.
We prove the theorem in several steps, mostly by an explicit analysis of the fundamental group. A similar theorem for Burniat-Inoue surfaces is proved in [BC]. For the remainder of this section, X is a Kulikov surface and S is a compact surface homotopically equivalent to X. Now consider the three subgroups of Γ defined by These are of index 3 in Γ and using the commutation relations (17) as well as we see that they are normal subgroups. Thus each Σ i corresponds to ań etale Z/3-cover S i → S.
Lemma 4.4 The Albanese variety Alb(S i ) of each S i is the Fermat cubic curve. In other words, Alb(S i ) is the unique elliptic curve which admits complex multiplication by a primitive cube root of unity.
Proof We prove the lemma for S 1 , since the other cases are the same by symmetry. Using commutation relations (17) and (18), we have and since Σ 1 / t 2 2 t ′ 2 , t ′ 2 t −1 2 , t 3 , t ′ 3 is abelian, the reverse inclusion holds. Hence where t 1 , γ 2 denote the classes of t 1 , γ 2 respectively, and the torsion summand (Z/3) 2 is generated by the classes of γ 1 and t 2 . This shows that Alb(S 1 ) is an elliptic curve. Now the Γ/Σ 1 -action associated to the cover S 1 → S descends to a nontrivial Z/3-action on H 0 (S 1 , Ω S 1 ). Indeed, by Hodge theory. Hence Alb(S 1 ) admits an automorphism of order 3. This proves that Alb(S 1 ) is the Fermat cubic curve. Now write E ′ i = Alb(S i ), and let Λ ′ i = t i , γ i+1 be the lift of the torsion free part of H 1 (S i , Z) to Γ. Then t i , γ 3 i+1 = t i , t ′ i is an index 3 normal subgroup of Λ ′ i , which we call Λ i . Viewed as a lattice, Λ i corresponds to an elliptic curve E i and a Z/3-cover E i → E ′ i . Since E i and E ′ i are isogenous, E i is also a Fermat cubic curve.
Proposition 4.5 Let S → S be the maximal abelian cover of S. Then (1) the Albanese variety of S is the product of elliptic curves E 1 × E 2 × E 3 ; (2) the Albanese map alb : S → E 1 × E 2 × E 3 is a birational morphism onto its image A S , and A S is a hypersurface of tridegree (3, 3, 3).
Proof Define Λ = Λ 1 ⊕ Λ 2 ⊕ Λ 3 , and let ψ : S → S be theétale (Z/3) 3cover of S with fundamental group Λ ⊳ Γ. Note that H 1 (S, Z) = (Z/3) 3 , so S → S is maximal and thus ψ factors through each S i . Now composing these factorisations with alb : S i → E ′ i and taking the direct product, we obtain a morphism f : , this factorisation of f is the Albanese map of S. This proves part (1) of the proposition.
To prove part (2) we compare H * ( S, Z) with H * ( X, Z), where X is the maximal (Z/3) 3 -cover of a Kulikov surface X. First let us factor the Albanese map of S through its image A S as follows: Then recall from Section 3.2 that X is a hypersurface of tridegree (3, 3, 3) in E × E × E. Now following the procedure described in part (1), we may also factor the Albanese map of X through its image A X : Here we have defined T = Alb( X) ∼ = Alb( S). Now a * X is an isomorphism, and we also note that i * X can not be trivial, because A X is a hypersurface of tridegree (3, 3, 3) in T . This implies A S is codimension 1 in T , because H 4 (A S , Z) = 0 by commutativity. Let [A S ] and [A X ] be the fundamental classes of A S and A X in H 2 (T, Z) respectively. Then for any λ ∈ H 4 (T, Z), we have By Poincaré duality, there exists λ ′ ∈ H 4 (T, Z) such that [A X ] · λ ′ = 1, which implies that deg a S = 1. Thus S and A S are birational, and a * S is an isomorphism. Therefore, , which implies A S is a hypersurface of tridegree (3, 3, 3) in T . This proves part (2) of the proposition.
To complete the proof of the theorem, we prove that the quotient of A S by Γ/Λ is in the same moduli space as S. This follows from the following: Lemma 4.6 The quotient of A S by Γ/Λ is a surface with p g = 0, K 2 = 6 and at worst rational double points.
Proof We compare the invariants of A S and S, and show that A S has at worst rational double points. Since = 3 3 · 6 because S → S is anétale (Z/3) 3 -cover. Note that p g ( S) = 29 because χ( S) = 3 3 χ(S) = 3 3 and q( S) = 3. The short exact sequence Since O T (A S ) is very ample, we see that |ω A S | is base point free and , so ω S = alb * ω A S and A S has at worst rational double points as singularities. Since the induced action of Γ/Λ on A S is free, the quotient has only rational double points, so p g = 0 and K 2 = 6.
Thus we have recovered the Inoue-type construction of our surface S using only the fact that S is homotopy equivalent to a Kulikov surface. This proves the theorem.
Degree of the bicanonical map
Let k 2 : X → P K 2 X be the bicanonical map of a Kulikov surface X. Then k 2 is a morphism by Reider's theorem [Re], and the image is a surface by [X]. In this section we prove that the degree of k 2 is 1.
Let r be the degree of k 2 , and s the degree of its image in P K 2 X . Then clearly 4K 2 X = rs. Moreover, r ≤ 4 because s ≥ K 2 X − 1, and r = 3, 4 by [MP], [MP2] respectively, so the only possible values for r are 1 or 2. Now to calculate the value of r, we use the following proposition, which is a natural extension of [P], Proposition 4.1: Proposition 5.1 Let ψ : X → Y be a finite abelian cover with group G and branch divisor ∆, where X and Y are nonsingular varieties of dimension n. Then the direct image of the bicanonical sheaf ψ * ω 2 X splits into eigensheaves where S χ = {g ∈ G| m d χ(g) = m − 1}, m is the order of g, d is the order of χ, and ∆ g is the sum of the components ∆ i of ∆ such that Φ(∆ i ) = g.
Proof By Lemma 4.1 in [P], locally free sheaves on Y which coincide on the complement of a codimension ≥ 2 subset are in fact equal on Y . Thus it suffices to prove the statement in a neighbourhood of every point which lies on a single irreducible component of ∆. Therefore we assume ∆ is irreducible and ∆ = ∆ g for some g ∈ G.
Let W = X/ g . Then ψ factors through W as ψ = ρ • π, where π : X → W is a cyclic cover branched over ρ −1 (∆) and ρ : W → Y is unramified. Let M = O W (M ) be the line bundle on W corresponding to the cyclic cover π, so M m = O W (ρ * ∆) and the formula Choose local coordinates b, w 2 , . . . , w n for W , so that b is a local equation for ρ * ∆, and let z be a local generator of M −1 as an O W -module. Then X is defined by the equation z m = b, so that mz m−1 dz = db, and we have the following local basis for π * ω 2 X as an O W -module: As in Section 2, we identify g * with Z/m so that the dual character to g is 1. Then g acts on z by g · z = εz, where ε = exp( 2πi m ), and by considering the action of g on our basis, π * ω 2 X splits into eigensheaves: Now since ρ is unramified, we have and clearly ψ * ω 2 X = ρ * π * ω 2 X . Combining this with (22), we obtain the required decomposition of ψ * ω 2 X . In Table 2 we list the eigensheaves of ψ * ω 2 X as calculated using Proposition 5.1. For example, when χ = (2, 2) so that χ −1 = (1, 1), we have S (1,1) = 1 0 , 0 1 , 2 1 and ∆ ( 1 0 ) + ∆ ( 0 1 ) + ∆ ( 2 1 ) = 5H − 2E 1 − E 2 − 3E 3 . Now by equation (3), Proof It suffices to show that k 2 separates points in X. It is clear from Table 2 that the only summands of ψ * ω 2 X with global sections are the eigensheaves with characters (0, 2), (2, 2), (2, 0) and (2, 1) together with the invariant eigensheaf. Note that these characters generate (Z/3) 2 * . Now choose a basis of H 0 (X, 2K X ) comprising eigenfunctions for the decomposition, so that k 2 is defined via this basis. Then for a generic point x in X and any g in (Z/3) 2 , if x and gx have the same image under k 2 , we have (0, 0)(g) = (0, 2)(g) = (2, 2)(g) = (2, 0)(g) = (2, 1)(g), which implies g = 0 0 . Therefore, k 2 separates points in each fibre of ψ. Finally we show that k 2 separates fibres of ψ. Note that (ψ * ω 2 X ) (0,2) = O Y (H), and therefore k 2 separates points in Y (E 1 ∪ E 2 ∪ E 3 ). It follows that k 2 separates fibres of ψ, and so k 2 is birational.
Bloch conjecture
In this section, we use methods of [IM] to show that the Bloch conjecture is verified for Kulikov surfaces. Let A 0 0 (S) denote the group of rational equivalence classes of zero cycles of degree 0 on a surface S. The conjecture is that for a surface S with p g = 0, A 0 0 (S) is canonically isomorphic to Alb(S). We note that the conjecture is proved for surfaces with Kodaira dimension κ < 2 in [BKL]. Since the Kulikov surface X is a surface of general type, we must show that A 0 0 (X) = 0. We briefly outline the approach: suppose G is a finite group of automorphisms of a surface S. Then every element g in G induces an endomorphism g * : A 0 0 (S) → A 0 0 (S), and this extends linearly to a homomorphism Γ : CG → End(A 0 0 (S)). Now for a subgroup H of G, we define Then by [IM] we have A 0 0 (S/H) = 0 if and only if Γ(z(H)) = 0. This very elegant result was further refined in [Ba]: Lemma 6.1 Let H, H i , i = 1, . . . , n be subgroups of G, and let I be the ideal of CG generated by z(H i ).
Now, this lemma is used together with the Inoue construction in [IM] to prove that Burniat-Inoue surfaces verify the Bloch conjecture. We adapt their method to Kulikov surfaces: Theorem 6.2 Kulikov surfaces verify the Bloch conjecture.
The proof for Kulikov surfaces is slightly more involved than in [IM] because the groups used are larger, so we present the full calculation. First we establish some notation to streamline the algebraic manipulations which are used in the proof. Recall from Section 3.2 that a Kulikov surface X is a G 2 -quotient of X 3,3,3 ⊂ E × E × E, where G 2 = g 1 , g 2 , g 3 ∼ = (Z/3) 3 . Now G 2 is contained in the larger group G 1 ∼ = (Z/3) 5 of automorphisms of X. Following Lemma 3.2 we choose generators ξ 1 , ξ 2 , ξ 3 , ω 1 , ω 2 , ω 3 for G 1 subject to the relation ξ 1 ξ 2 ξ 3 = 1. By Lemma 3.2 and Definition 2.2, ξ i = η 2 i η i+1 are composite translations generating G 0 , ω i are rotations on the ith factor E and g i = ξ i+1 ω i+1 . Lemma 6.3 Let H ⊂ G 1 be one of the following collection of subgroups: ω i , ξ l n ω j , ξ m n ω k for 0 ≤ l, m ≤ 2, where {i, j, k} = {1, 2, 3} and n = i or i − 1. Additionally we may choose H = ξ 3 ω 1 , ξ 2 ω 2 , ξ 2 ω 3 . Then the surface X/H is rational.
Remark 6.4 This is not an exhaustive list of subgroups H which give rise to a rational quotient, but it is sufficient to prove the Theorem. The additional subgroup ξ 3 ω 1 , ξ 2 ω 2 , ξ 2 ω 3 is necessary for the proof, and this is precisely what makes the calculation harder than the elementary one in [IM].
Now suppose H is a subgroup of G 1 . The quotient X/H is a G 1 /H-cover of P 2 branched in the Kulikov line configuration of Figure 1. Thus we may use the techniques of abelian covers to prove that X/H is rational. This involves a series of repetitive calculations, and various cases are related to one another by symmetry. As an illustration, we calculate a typical quotient: Let H = ω 1 , ξ 2 3 ω 2 , ξ 3 ω 3 , then G 1 /H ∼ = (Z/3) 2 is generated by the classes δ 1 and ω 3 . We list the class of each generator of G 1 under the quotient: δ 2 ≡ ω 2 3 , δ 3 ≡ δ 2 1 ω 3 , ω 1 ≡ 1, ω 2 ≡ ω 2 3 . Viewing X/H as a (Z/3) 2 -cover of P 2 branched in the Kulikov line configuration, we must blow up P 2 and P 3 to remove singular points on the cover. Moreover, the point of intersection of ∆ 5 and ∆ 6 fails the nonsingularity condition of equation (1). Thus we blow up this point, introducing another −1-curve which we call E. Having done this, we note that by equation (2), the induced cover is unramified over E 2 and E. Finally, we contract the strict transforms of ∆ 1 and ∆ 5 . We are left with a nonsingular (Z/3) 2 -cover of P 1 × P 1 branched in four distinct lines, two in each ruling. This is a rational surface.
Proof of theorem Let I be the ideal generated by z(H) for all H listed in Lemma 6.3. Then the quotients X/H are rational surfaces, so by [BKL], A 0 0 ( X/H) = 0. Thus by Lemma 6.1, it is sufficient to prove that z(G 2 ) is an element of I. Now, the verification of the Bloch conjecture for Burniat-Inoue surfaces [IM] is a series of elementary polynomial manipulations. Unfortunately, the corresponding manipulations do not suffice for the Kulikov surface.
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14646661 | pes2o/s2orc | v3-fos-license | Induction of Epigenetic Alteration by CPUK02, An Ent- kaurenoid Derivative of Stevioside
Background: Dietary polyphenols, such as those found in green tea and red wine, are linked to antitumor activity. They are known to influence many signaling pathways epigenetically within the human body. In this regard, CPUK02 (15-Oxosteviol benzyl ester) is a new ent-kaurenoid derivative of stevioside and exhibits strong anti-cancer activity in vitro and in vivo. Nowadays, the role of epigenetics in cancer has been the subject of intensive study and DNA methylation targeting represents a relevant strategy for cancer treatment. There are no reports regarding the effects of CPUK02 on epigenetic alterations in colorectal cancer cell line. This study was an attempt to compare CPUK02 with 5-AZA as DNMT inhibitor agent and evaluate whether it can induce its anti-cancer effects via altering the level of DNMT3b mRNA, MGMT and SFRP2 methylation pattern in HCT 116 cell line. Methods: To evaluate DNMT3b expression, DNMT3B mRNA levels in HCT116 CRC cell line were quantified by real-time reverse-transcriptase Polymerase Chain Reaction (PCR) assay after 24 hr of incubation time with CPUK02 and 5-AZA. In addition, the methylation patterns of 2 CpG islands in this cell line were examined by methylation-specific PCR methods. Results: CPUK02 surprisingly, decreased the DNMT3b mRNA level. The average expression levels of DNMT3b in HCT116 treated with CPUK02 and 5-AZA relative to the GAPDH expression level in control were 0.16 and 0.5%, respectively. Furthermore, CPUK02 could decrease the methylated allele of MGMT and SFRP2 genes in HCT 116 after 24 hr. Conclusion: In this study, positive correlation was found between mRNA expression of DNMT3b and gene promoter hypermethylation after treatment with CPUK02 and 5-AZA. Our data confirmed that CPUK02 like 5-AZA exhibits demethylating properties.
Introduction
Colorectal Cancer (CRC) is a multistep process with accumulation of genetic and epigenetic errors which causes a normal cell transformation to an invasive tumor cell 1 . Three distinct pathways including chromosomal instability, microsatellite instability, and CpG island gene methylation pathways have been recognized in colon cancer initiation and progression. Alterations in DNA methylation patterns are the best understood epigenetic cause of the disease which occurs usually via either hypomethylation of global DNA or hypermethylation of tumor suppressor genes 2 . DNA methylation is performed by at least two DNA methyltransferases (DN-MT; 3a and 3b) and maintained by DNMT1 3 . Overexpression of DNMTs has been also detected in a variety of malignancies, including lung, prostate, and colorectal tumors 4 .
DNA hypermethylation is also associated with gene silencing and is often observed in CpG islands of cancer-related genes. Transcriptional silencing by the hypermethylation of CpG islands is an early event in tumor progression. In this regard, researchers suggest that hypermethylation of O6-methylguanine-DNA methyltransferase (O6-MGMT) is a pacemaker for certain gene mutations such as K-ras 5 . On the other hand, aberrant methylation in genes related to WNT signaling pathway has crucial roles for cancer progression especially in CRC 6 . Among specific genes in WNT signaling, SFRP2 methylation is considered as a marker with high sensitivity and specificity in serum and stool for colorectal adenomas and CRC screening [7][8][9] . 5-Aza-CR and 5-Aza-CdR potentially inhibit the DNA methyltransferases (DNMTs) [10][11][12][13] . Nowadays, therapeutic targeting via the DNA methylation machinery has been the subject of intensive study 14 .
Recently, natural compounds, such as curcumin, Epigallocatechin Gallate (EGCG) and resveratrol, have been considered to increase sensitivity of cancer cells to conventional agents and induce tumor growth inhibition via epigenetic mechanisms 15 . One of the plants that has recently attracted the attention of researchers is stevia rebaudiana bertoni and its glycoside compounds. Among stevia derivatives, isosteviol and derivative compounds inhibit human cancer cell growth 16 . Furthermore, only isosteviol potently suppresses both mammalian DNA polymerases and human DNA topoisomerase 17,18 . Paul et al also reported that stevioside is a potent inducer of apoptosis via intracellular ROS generation in MCF7 breast cancer cell line 19 . In this regard, the Chinese researcher made a new ent-kaurenoid derivative of stevioside known as CPUK02 20 . A large number of ent-kaurane diterpenoids isolated from different natural plants have been reported to display anticancer activities 21 . The anticancer activity of CPUK02 was evaluated in in vitro and in vivo models. CPUK02 strongly inhibits cell proliferation and induces apoptosis in several human cancer cell lines. Furthermore, CPUK02 demonstrates its anti-tumor effects much better than fluorouracil (5-FU) in mouse xenograft cancer model 22 .
Lack of information about the effects of CPUK02 on epigenetic alteration in colorectal cancer mandates more research in this field. Therefore, the aim of this study was to further elucidate the anti-cancer effects of CPUK02 via epigenetic alteration. In this regard, the level of DNMT3B mRNA expression, MGMT and SFRP2 methylation pattern as representative of epigenetic changes in HCT 116 cell line were assessed.
Materials and Methods
The human colon cancer cell line, HCT 116 was obtained from the National cell bank of Iran (Pasteur Institute, IRAN). Cell culture medium, penicillin, streptomycin medium supplement, glutamine and fetal bovine serum were obtained from Gibco Life Technologies (UK). Tripure Isolation Reagent was purchased from Roche Applied Sciences (USA).
DNA Synthesis Kit was purchased from Fermentas, EU. SYBR green DNA PCR Master Mix was purchased from the Applied Biosystem (ABI) Company, (Foster City, CA USA). CPUK02 was kindly provided as a gift from Drug Research Institute, China Pharmaceutical University.
Cell line
The human colon cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and incubated at 37C in a humidified 5% (v/v) CO 2 incubator.
Treatment with CPUK02
Cells were seeded for 12 hr and then washed with PBS and medium was replaced with serum-free RPMI medium. Finally, non-growing, confluent cells were incubated with CPUK02 and 5-aza dissolved in DMSO to a final concentration of 4.5 µM and 1 µM, respectively for 24 hr. Optimum concentrations were determined according to MTT assay. At the end of the incubation, cells were harvested for RNA and DNA isolation.
Methylation specific-PCR analysis
DNA was extracted from cells according to the standard phenol/chloroform method 23 . The status of promoter methylation of the MGMT-B, and SFRP2 genes was determined by Methylation -Specific PCR (MSP-PCR). The sequences of primers and annealing temperatures used for amplification of the promoter regions of genes are listed in table 1.
The genes promoter methylation status was determined by chemical treatment of DNA samples with sodium bisulfite and subsequent MS-PCR as previously described 24 .
In every reaction, DNA from peripheral blood lymphocytes was considered as a negative control. PCR products were analyzed by electrophoresis on 2% agarose gel.
RNA extraction and semi-quantitative PCR
The total RNA was extracted from the treated cells using the TRIZOL Reagent Kit, according to the manufacturer's instructions. The RNA concentration was quantified by measuring the absorbance at 260 nm in a spectrophotometer. Ratios of absorption (260/280 nm) of all samples were between 1.8 and 2.0. RNA samples were subjected to electrophoresis through a 1.4% agarose-formaldehyde gel to verify their integrity. The total RNA samples were stored at -80°C until analysis. The expression of DNMT3b mRNA was determined by semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). Two μg of total RNA were reverse transcribed by incubation at 37°C for 1 hr in a 25 μl reaction mixture consisting of 100 U M reverse transcriptase, 8 U RNase inhibitor, 0.5 μg of oligo (dT), 50 mmol/L Tris-HCl (pH=8.3), 3 mmol/L MgCl 2 , 75 mmol/L KCl, 10 mmol/L DDT, and 0.8 mmol/L each dNTP. The reaction was terminated by heating at 95°C for 5 min and quickly cooling on ice. Two μl of the RT reaction mixture were used for PCR in a final volume of 25 μl, containing 2.5 μl of 10×PCR buffer, 2 μl of each 2.5 mmol/L dNTPs mixture, 1 unit of Taq DNA polymerase, and 10 pmol/L of each forward and reverse primer. The conditions of PCR amplification were as follows: one cycle at 95°C for 5 min, 33 cycles at 95°C for 30 s, 60°C for 40 s and 72°C for 1 min, and a final extension cycle at 72°C for 10 min. The PCRamplified fragments were run alongside molecular weight markers on 2% agarose gels stained with gel red. The experiments were repeated twice. The primers and PCR conditions for DNMT3b, GAPDH are listed in table 2. 232 and 113 bp amplicons were expected upon performing PCR for GAPDH and DNMT3b, respectively.
Quantitative real-time PCR
Real-time PCR was carried out using the ABI real time PCR 7500 system. The PCR reaction mixture contained 2 μl of cDNA (tenfold diluted), 0.5 μl of 5 mmol/L solutions of each of the forward and reverse primers and 10 μl of SYBR green DNA PCR Master Mix in a total volume of 20 μl. All incubations included an initial denaturation step at 95C for 10 min and 30 cycles (15 sec at 95C and 30 sec at 60C) subsequently. A melting curve analysis was achieved by performing 70 cycles of 10 s with a temperature increment of 0.5C/cycle starting from 60C. Efficiency of amplification was measured by the slope of a standard curve. Data were analyzed by using the 7500 Software v2.0.1. The relative expression level (fold changes) of DNMT3b gene was calculated by the 2 -ΔΔCT formula. GAPDH was also considered as the internal control. Expression levels of target gene were normalized using the GAPDH as the housekeeping control gene.
Statistical analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post-hoc Dunnett multiple comparison tests using SPSS [Version 16; SPSS, Chicago, IL, USA]. The values of p< 0.05 were considered statistically significant.
Methylation of SFRP2 and MGMT genes
The methylated and unmethylated sense/antisense primers for MGMT produced a 127 bp fragments. However, the methylated and unmethylated sense/antisense primers for SFRP2 produced 138 and 145 bp fragments, respectively. In this study, it was observed that CPUK02 has a positive effect on SFRP2 and MGMT methylation pattern in HCT 116 cells. CPUK-02 and 5-aza had a similar effect on the methylation of MGMT gene promoter. As shown in figure 1B, both of them moderately decreased methylated allele. However, according to our results ( Figure 1A), 5-aza completely inhibited SFRP2 methylation but CPUK02 moderately did.
DNMT3b expression analysis
The presence of appropriate bands for DNMT3b Researchers showed that CPUK02 can induce the apoptosis in different cancer cell lines via p53 22 . However, there is no data to address the effects of CPUK02 on epigenetic alteration in colorectal cancer. Pierre-Olivier and colleague have found that DNMTs are integral part of the p53 protein network. On the other hand, the levels of DNMTs, especially DNMT3a and DNMT3b, are often increased in various cancer tissues and cell lines such as HCT 116 cells 25 . Therefore, this phenomenon may increase hypermethylation of tumor suppressor genes in a variety of malignancies. Alt-hough cytosine promoter methylation is accomplished by five mammalian DNMTs (DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L), but in contrast to DNMT1, DNMT3a and DNMT3b are able to do de novo DNA methylation during normal development. DNMT1 is also required for maintenance of methylation patterns 26 .
Regarding the importance of DNMT3b, several studies demonstrated that DNMT3b is overexpressed at higher frequency than DNMT3a and DNMT1 in CRC and breast cancer [27][28][29] . In addition, DNMT3B overexpression is associated with CIMP-high phenotype in colorectal cancer 30 . Furthermore, direct interaction between Dnmt3a and/or Dnmt3b and transcription factors provides a molecular mechanism which leads to DNA hyper methylation 31 .
In this regard, Zhang et al showed that DNMT1, DNMT3b protein level decreased in HCT116 cells treated with DNA-damaging drug doxorubicin as the apoptosis-inducible agent 32 . Furthermore, Qiang Li et al showed that 5-aza-2-deoxycytidine inhibited both DNMT1 and DNMT3B protein expression in breast, colon, and other types of cancer cells 33 . Our data showed that CPUK02 was also able to decrease mRNA level of DNMT3b gene in HCT 116 cells. So, CPUK02 mimics the role of 5-AZA to decrease mRNA level of DNMT3b. However, considering CPUK02 as an epigenetic drug, further investigation is needed.
In the next part of our study, the effect of CPUK02 on the methylation status of two key genes in colorectal cancer initiation and progression was evaluated.
SFRP2 as a negative regulator of Wnt signaling has important implications in tumorigenesis, and its down regulation has been correlated with CRC [34][35][36] . It has been also postulated that lack of MGMT expression increases the spontaneous G:C.A:T mutation rate in tumors in vivo. In addition, MGMT methylation is also a central member in cancer development from normal adenoma to carcinoma 37 .
According to the importance of SFRP2 and MGMT role in cancer progression, promoter methylation of these two genes was investigated in HCT 116 treated with CPUK02. It was revealed that CPUK02 and 5-AZA had a similar effect on methylation pattern of MGMT gene. Both of them induced moderate demethylation of MGMT gene. In case of SFRP2, it was shown that 5-aza completely restored unmethylated allele but CPUK02 moderately did.
In this case, the ER-negative breast cells were also treated with demethylating agent 5-aza and CPUK02. As shown in supplement figure, our results showed that CPUK02 like 5-AZA may effectively restore ER aberrant hypermethylation. Thus, inactivation of ER gene has a close relationship with the abnormal methylation of ER gene promoter which can be restored by 5-AZA 38 and probably CPUK02.
However, 5-aza and CPUK02 were not able to completely restore the unmethylated pattern of MGMT gene in CRC cell line. In this regard, Wodarz et al revealed that methylation rate of MGMT gene in HCT-116 cell line is slower than methylation rate of SFRP2 gene 39 . Also, they showed that MGMT promoter methylation in HCT 116 cell line started with a delay. Given this finding, it can be concluded that remethylation rate of MGMT gene will be slower than SFRP2. Therefore, increasing the incubation time during treatment from 24 hr to 48 might help the unmethylated allele of MGMT to appear. This experiment was performed on HCT 116 cells for longer time and the same results were obtained which are seen in ER-negative breast cells.
Recent researchers showed that epigenetic pharmaceuticals could be a replacement or adjuvant therapy for currently accepted treatment methods such as radiation and chemotherapy, or could sensitize cells to the current treatments 40 . In this regard, although 5-Aza is the most potent demethylating agent in clinical usage, general side effects led to efforts for finding new DNA methylation inhibitors with greater potency and low cytotoxicity.
Recently, natural compounds, such as curcumin, EGCG, and resveratrol, have been shown to increase sensitivity of cancer cells to conventional agents via alteration in epigenetic mechanisms, which may lead to tumor growth inhibition 41 . It has been shown that the epigenetic control of the proto-onco regions and the tumor suppressor sequences by conformational changes in histones directly affects the formation and progression of cancer [42][43][44] . CPUK02 as a new semi natural compound which exhibits strong anti-cancer effect in in vitro and in vivo models might also manipulate epigenetic pathways and our data confirmed that CPUK02 like 5-AZA exhibits demethylating properties.
Conclusion
According to our data, CPUK02 is a promising molecule with epigenetic/anti-cancer properties and it might be a front-runner candidate for new pharmaceutical targets. However, little information exists about performance of CPUK02 and further investigations are needed to introduce it as a new epigenetic drug. | 2018-04-03T05:31:49.253Z | 2017-01-01T00:00:00.000 | {
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246771502 | pes2o/s2orc | v3-fos-license | Effects of Nalbuphine Combined with Anterior Serratus Plane Block in Elderly Patients Undergoing Thoracoscopic Surgery
Postoperative pain in elderly patients with lung cancer after thoracoscopic surgery is still an important factor affecting the prognosis of patients. In this study, 200 elderly patients with lung cancer who were positive and planned to undergo video-assisted thoracoscopic surgery were randomly divided into four groups: control group, SAPB (serratus anterior plane block) group, Nalbuphine group and Nalbuphine + SAPB group. The effects of drugs and nerve block on the perioperative indexes of elderly patients were observed. The results showed that ① The VAS and SAS scores of postoperative analgesia in the Nalbuphine + SAPB group were lower than those in the single group and the control group. ② The postoperative spontaneous respiratory recovery time, extubation time, resuscitation room stay time, extubation cough, restlessness and respiratory depression in the Nalbuphine + SAPB group were lower than those in the single group and the control group. ③ The heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and blood oxygen saturation (SpO2) of patients in Nalbuphine + SAPB group before induction, T2 after intubation, T3 before skin incision, T4 after skin incision, T5 after chest closure and T6 after extubation were lower than those in single group and control group. Therefore, this study concluded that Nabufine combined with SAPB can make the vital signs of intraoperative patients more stable, which is worthy of clinical promotion.
Introduction
VATS (Video-assisted thoracoscopic operation) has grown gradually and allows mediastinal tumor resection and wedge resection. Widespread utilization of modern VATS provide postoperative retrieval of the lungs. e VATS also potentially decreases cost, operative trauma, and allows earlier ambulation. e pain experienced is normally average and is mostly pain at the incision in the chest tube site, elastic tissues and intercostal muscles. Over-all management of Nalbuphine analgesia treatment, that is widely the core of pain monitor procedures, may involve intravenous opioids regional blocks and oral non-steroidal anti-seditious medicines [1]. Perfect Nalbuphine treatment offers effectual pain assistance, lessens side effects, and augments patient comfort. With progress in ultrasonic technology, regional block technologies happen to be more popular and involve erector spinal block, as well as thoracic epidural block (TEB). TEB is a technique in which analgesia is produced by injecting local anesthetic agent by itself or combined with additives or alone into the epidural space. TEB is presently the best paradigm for Nalbuphine treatment in thoracic operation, though it needs usual coagulation work, and the letdown rate in medical work is up to 29 percent because of problematic catheter detachment and catheterization.
Furthermore, extreme epidural anesthesia (Nalbuphine) simply results to hypotension necessitating fluid change and possibly administration of vasoactive medicines that may result to the damage of microcirculation, in order to reinstate hemodynamic strength. Wang et al [2] adds that other probable extreme outcomes include pruritus, nausea, and vomiting, maybe due to the epidural opioids administration. In relieving thoracic pains, paravertebral can work well but needs many injections, spinal cord or nerve injury pneumothorax and this potentially increases the dangers of an overdose of local anesthetics. Ultimately, erector spinae block usually employed for the cure of chest pains at the posterior, has painful implications on post-operation stage of the anterolateral and lateral chest walls. SAPB is a new chest slab methodology started in 2013. SAPB is a local block initially established for analgesia after breast operation, and more recently as Nalbuphine treatment particularly for the elderly undergoing thoracoscopic surgery. Serratus plane block is a simple, effective and safe thoracic fascial plane block. SAPB process depends on the presence of two possible gaps on the face on the anterior of the serratus tissues or muscles, a deep muscle plane between the anterior regions of the serratus and the intercostal muscles, and the latissimus dorsi [3]. Nalbuphine is injected at the block of the two planes, hence alleviating pain at the chest walls [4]. e aim of the SABP is on the T9 and T2 intercostal branches at the nerve system to augment Nalbuphine at the posterior chest walls. VATs are steadily adopting this technique, though there are a few yet randomized managed testing to back its utilization in this situation [5]. At present, the thoracoscopic surgery makes a comparison of the time, mostly within a period of 48 hours postoperatively to the overview of the analog pain notch (VAS) ≥4.01 between local infiltration Nalbuphine treatment and SABP. is is mostly among the elderly persons undergoing VATS for lung malignance to see how it affects them over a specific period.
is aids in correlating the usage of postoperative Nalbuphine treatment among the elderly patients. is is particularly crucial in ascertaining the SAPB in combination with Nalbuphine treatment in offering more safeguards among the elderly patients undergoing thoracoscopic surgery.
Subject-Elderly Patients.
is research was done conforming to the Helsinki Declaration for research encompassing human subjects. All the elderly patients gave written informed consent to back their participation in this research.
is research was undertaken with full consent from the Chinese Clinical Trial Registry way before enrolling the elderly patients to participate in this research.
Setting and Study Design.
e elderly patients were put in the control group, SAPB group, Nalbuphine group and Nalbuphine + SAPB group through an arbitrary minute table, as well the group work kept in a closed wrapper, referred just to the doctors doing the nerve block surgeries [6]. e grouping of the patients was based on the simple randomization. e surgeons and elderly patients undergoing the intraoperative anesthesia (local Nalbuphine treatment) procedure, as well as the doctors doing postoperative pain, and valuations were unaware of the task assignments, as they were not opened and the lumps were done away from the operating theater. ① Control group (routine) regimen: midazolam 0.08mg/kg, sufentanil 0.4ug/ kg, etomidate 0.3mg/kg, vecuronium 0.1mg/kg, propofol 6mg/kg/h, remifentanil 8ug/kg/h, 30min before suture, sufentanil 0.1ug/kg for analgesia during operation. ② SAPB group: midazolam 0.08mg/kg, sufentanil 0.4ug/kg, etomidate 0.3mg/kg, vecuronium 0.1mg/kg, SAPB after induction, propofol 6mg/kg/h, remifentanil 8ug/kg/h, 30min before suture, sufentanil 0.1ug/kg for analgesia during operation. ③ Nalbuphine group: midazolam 0.08mg/kg, sufentanil 0.4ug/kg, etomidate 0.3mg/kg, vecuronium 0.1mg/kg, propofol 6mg/kg/h and remifentanil 8ug/kg/h were used during operation. Before anesthesia induction, Nalbuphine 0.2mg/ kg was given to relieve pain. ④ Nalbuphine + SAPB group: midazolam 0.08mg/kg, sufentanil 0.4ug/kg, etomidate 0.3mg/kg, vecuronium 0.1mg/kg, SAPB was performed after induction, propofol 6mg/kg/h and remifentanil 8ug/kg/h were used during operation, and nalbuphine 0.2mg/kg was given before anesthesia induction. Keep the perioperative heart rate and blood pressure in the normal range. If the blood pressure and heart rate rise or decrease by 30% in the basic value, give the corresponding vasoactive drugs for treatment; if the hemoglobin is lower than 100g/L, blood transfusion products should be selected according to the actual situation.
Main Research Indicators.
e main indicators were heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and blood oxygen saturation (SPO 2 ) in patients T1 before induction, T2 after intubation, T3 before skin incision, T4 after skin incision, T5 after chest closure, and T6 after extubation. e time of recovery when it comes to impulsive breathing, residence time in the resuscitation room, extubation time, the frequency of extubation choking, respiratory depression and agitation. e SpO2 a lesser amount of 90% for 15s or the rate of respiration less than 12 times for a period of 10 minutes. e VAS score was recorded before the onset of the operation, instantaneously after 4 hours, 8 hours, 12 hours, and 24 hours after extubation. Additionally, we compared the adverse events in the present study, and the adverse events were defied as the vomiting and nausea.
Statistical Analysis.
e research examined quantitative data of abnormal or normal distribution with Mann-Whitney or t-test.
is qualitative data was made in comparison to the set data utilizing Fisher's exact trial or χ 2 test. Additionally, the assessing of the occasion-time data was with record-position test and curve of Kaplan-Meier.
Sample
Size. In this study, 200 elderly patients with lung cancer who were positive and planned to undergo videoassisted thoracoscopic surgery were randomly divided into four groups: control group, SAPB group, Nalbuphine group and Nalbuphine + SAPB group. e effects of drugs and nerve block on the perioperative indexes of elderly patients were observed. e sample size for this research was 200 elderly patients. e main pointer was the period to VAS count four through the first two days post-operation. e valued median period following surgery to the first VAS count of four was 4 hours (SD � 9.3401) with indigenous Nalbuphine permeation in thoracoscopic operation. In theory the fruitful SAPB lasts for 12 hours (91% power and a 6% importance degree), which would need 50 elderly patients per unit. To report for lost patient figures, there was a composition of 20 patients in each unit.
Results
ere were around 200 elderly patients qualified for the research, of which 80 were left out (40 did not endorse informed consent, 28 elderly patients had a history of chest surgeries, and 9 of the elderly patients were using Nalbuphine drugs. Whilst 3 of the elderly patients had difficulties with language expression). Figure 1 is the flow chart of the study.
As articulated earlier, the main indicators were heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and blood oxygen saturation (Sp02) in patients T1 before induction, T2 after intubation, T3 before skin incision, T4 after skin incision, T5 after chest closure, and T6 after extubation. e elderly patients were presented with hypoxaemia, for managing severe respiratory distress. Ninety percent Sp02 to manage shock, through titrating to Sp02 ≥ 90% as well as initiating oxygen therapy at 5 L/min and titrate to SpO2. Cardiac output and oxygen consumption (VO2) measurements were affected through utilizing a CPX/D Medical Graphics metabolic analyzer. e determination of cardiac output was after 6 hours through employing the ancillary CO2 rebreathing technique. e data and statistical data encompassed the analysis of variance with repeated measures, that indicates that at the 6 hours, there was increased heart rate (HR) due to increase in metabolism, that resulted to a rise in VO2. e rise was occasioned by the HR (heart rate) response and the fall in SVR (systemic vascular resistance). e SBP (systolic blood pressure) and MAP (Mean arterial pressure) decreased while on the other hand there was increase in blood oxygen saturation.
ere were no major variations in the T2 after intubation, and skin incision. However, T5 after chest closure and extubation was vital in increasing metabolism during the thoracoscopic surgery.
ere were no significant differences in terms of sex, BMI, or the time of operation (p > 0.05 in Table 1). e projected median period following the thoracoscopic surgery to the initial point VAS count was 5 hours (1.45 to 7.75) in the set of control and 12 h (7.45 to 16.35) in the SAPB set (log order test) (survival curve study): P � 0.009, suggesting protracted lasting postoperative Nalbuphine with ultrasound-led SAPB in comparison to indigenous infiltration Nalbuphine. e number of elderly patients necessitating additional Nalbuphine at 7 hrs and 12 hrs after thoracoscopic operation was marginally greater( Journal of Healthcare Engineering control versus the set of SAPB, but there was no major variance among the sets in Nalbuphine prerequisite after the 12 hrs. ere was no major variation among the sets in the dosage after interoperation of propofol and remifentanil (Table 3) (P > 0.05) or in the prevalence of post-operation vomiting as well as nausea (Table 4) (P > 0.05). e postoperative spontaneous respiratory recovery time, extubation time, resuscitation room stay time and the incidence of extubation cough, restlessness and respiratory depression in the Nalbuphine + SAPB group were lower than those in the single group and the control group (Table 5)(P < 0.05). e heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and blood oxygen saturation (SpO2) of patients in Nalbuphine + SAPB group before induction, T2 after intubation, T3 before skin incision, T4 after skin incision, T5 after chest closure and T6 after extubation were lower than those in single group and control group.
Discussion
e outcome of this research implies that SAPB with Nalbuphine is essential in minimizing initial pain of post operation after the lung resection of VATS with minimal side effects. is study has been critical in analyzing the effective outcomes of SAPB in randomized regulated attempts or trials after thoracoscopic surgery for postoperative Nalbuphine [7].
is research proved significance in demonstrating that there was no major difference in the scores of VAS score between the placebo group and the SAPB group during the initial 24 hours after the process of operation. Concurring with Piccioni et al [8], there was efficacy of the SAPB when it came to the postoperative reprieve of the chest walls of the elderly patients as the medicinal doses of Nalbuphine in the SAPB set or group were marginally lower, and the time required to utilize the first doses of Nalbuphine was palpably longer [9]. e research judging from the results was significant in attesting the efficacy of SAPB in reducing postoperative pain around the chest wall whilst minimizing the postoperative prescription of opioids [10]. e overriding difference is that VATS does not function effectively as SAPB when it comes to minimizing postoperative pain [11].
From this research study, there was contrast in that local infiltration Nalbuphine and SAPB for postoperative analgesia after the VATS established that elderly people in the SAPB group possessed superior VAS scores during the initial 8 hours postoperatively; this is despite the dosage of Nalbuphine being significantly lower. Chen et al [12], avers that a preliminary meta-analysis on the Nalbuphine result of SAPB in elderly people going through thoracoscopic surgery demonstrates that Nalbuphine amalgamated with SAPB minimizes the perioperative chest pains among the elderly patients. is is particularly when compared to the VATS treatment. Centered from the results of this research, the Nalbuphine score on the SAPB group at 6-, 12-and 24 hours after the thoracoscopic surgery was lower compared to that of the control set. is proves that that Nalbuphine treatment has better efficacy levels at 6 hours compared to when it was at 24 hours. Deng et al [13] asserts the period to the VAS score four after the process of operation was considerably reduced, (median 12 hours) in the control group versus the SAPB group.
is ostensibly shows that the number of elderly patients necessitating advance Nalbuphine during the initial twelve hours after the process of operation was considerably lower. Moreover, there are similarities both at home and abroad in that SAPB is effective compared to local infiltration at the site of incision for postoperative management of chest pains among the elderly patients after VATS resection of the lung [14]. e difference is that period of Nalbuphine abroad in our SAPB group surpassed the initial levels suggesting that higher concentrations of Nalbuphine were used [15].
Initially, even though local Nalbuphine or nerve block, among the elderly patients was not done in the presence of the surgical anesthetists and operating surgeons, the anterior serratus plane block method was arbitrated in accordance with the needle puncture mark [16]. Nonetheless, the surgeons adjusted the Nalbuphine centered on PTi monitoring and sedation threshold index that has minimal effect on the management of opioids and Nalbuphine depth [17]. e other significant difference is severity and presence of pain by elderly patients suffering cognitive disorders. Hence, over the years, there has been more and more objective assessment techniques. Nalbuphine not only considerably constrains the actions of the cerebral cortex but also maintains integral the actions of the subcortical autonomic nervous system among the elderly patients. As attested through this research when undergoing thoracoscopic surgery, the major signs that damages the stimuli might encompass variations such as diaphoresis, pulse wave amplitude, pupil diameter, blood pressure, and increased heart rate among the elderly people [18]. e entirety of this condition is because of changes or suppression when it comes to the balance of parasympathetic and sympathetic nerve actions. e present study did not have the comparison on the analog pain notch due to lack of the data. e future studies should perform the analysis on this topic.
Conclusion
In conclusion, SAPB combined with Nalbuphine is a convenient and safe regional nerve block method for VATS, which in effect alleviates initial wound pain among the elderly, offers protracted-lasting analgesia compared to local infiltration at the incision, and constrains the requirement for initial postoperative advance Nalbuphine.
Data Availability e simulation experimentation data utilized to back the conclusions of this research are accessible from the agreeing writer upon demand.
Conflicts of Interest
ere was no conflict of interest of this research. Journal of Healthcare Engineering 5 | 2022-02-12T16:18:17.806Z | 2022-02-10T00:00:00.000 | {
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248909553 | pes2o/s2orc | v3-fos-license | The perioperative anaesthetic management and outcomes of COVID-19 associated mucormycosis patients
This retrospective study was approved by the hospital ethics committee (RMHEC/AL/06/2022). Medical records of all patients admitted with CAM at a single tertiary care centre between April 2021 and August 2021 were analysed for patient demographic data, presenting complaints, COVID-19 status, all investigation reports, vitals monitoring recorded throughout the hospital stay, the number and type of surgeries performed on each patient, duration of anaesthesia, duration of hospital stay, risk scores, post-operative sequelae, and outcomes.
INTRODUCTION
During the second wave of the coronavirus disease (COVID)-19 pandemic, many of the delta variant cases were associated with rhino-orbital cerebral mucormycosis. Due to the shortage of healthcare infrastructure and unavailability of amphotericin B, mucormycosis became more fulminant, warranting emergency radical surgical debridement, orbital 'exenteration', and maxillectomies.
Post-COVID-19 systemic sequelae like residual respiratory insufficiency, adrenocortical suppression, cardiac dysfunction, and difficult airway complicated by mucormycosis, adverse effects of amphotericin B treatment, dysregulated innate immune response, microvascular coagulation can affect the general anaesthetic management and the outcomes. [1] We conducted a study to analyse the perioperative anaesthetic management of COVID-19 associated mucormycosis (CAM) patients and to describe their pre-anesthetic evaluation and risk stratification for emergency surgical debridement.
METHODS
This retrospective study was approved by the hospital ethics committee (RMHEC/AL/06/2022). Medical records of all patients admitted with CAM at a single tertiary care centre between April 2021 and August 2021 were analysed for patient demographic data, presenting complaints, COVID-19 status, all investigation reports, vitals monitoring recorded throughout the hospital stay, the number and type of surgeries performed on each patient, duration of anaesthesia, duration of hospital stay, risk scores, post-operative sequelae, and outcomes.
Analytical Statistical Package for the Social Sciences (SPSS) for windows version 25.0 was used for data analysis. The mean ± standard deviation (SD) and range values were calculated for quantitative variables and the percentages were calculated for categorical variables.
RESULTS
Eighty-three individuals were hospitalised with COVID-19 associated mucormycosis. Out of these, records of 67 patients could be obtained. The American Society of Anesthesiologists (ASA) physical status was grade I in one patient, II in 55 patients, III in 6 patients, and IV in 5 patients, with a median age of 48.3 ± 14.1 (range, 23-84) years.
Out of six patients with ASA grade III, five had Quick Sequential Organ Failure Assessment (qSOFA) score of 1 and Quick Systemic Inflammatory Response Syndrome (qSIRS) score of 2, while one had qSOFA score of 2 and qSIRS score of 4. For five patients of ASA grade IV, the qSOFA score was >2 and qSIRS score was >4 [ Figure 1].
Six (8.9%) patients had COVID-19 complications, out of whom, one patient had severe COVID-19 illness with septic shock. Another patient had bilateral cortical venous thrombosis, while one more patient was admitted with ischaemic stroke and altered sensorium. Furthermore, in one patient, acute superior cerebellar artery infarct, and in one patient post COVID-19 bilateral axonal neuropathy and upper trunk plexopathy was diagnosed. One patient with myocardial infarction was also admitted to the intensive care unit (ICU) in a gasping state and had a cardiac arrest for which cardiopulmonary resuscitation was administered, after which spontaneous circulation was restored. This patient was planned for surgical debridement but died pre-operatively.
Ten patients (14.9%) had COVID-19 associated pneumonia and required oxygen therapy at the time of admission [ Figure 2]. At admission, the mean peripheral oxygen saturation (SpO 2 ) was 96% (66%-100%) among the patients in the study. The mean random blood Among 67 patients, 15 patients were newly detected with diabetes during COVID-19 illness, 41 patients were known cases of diabetes (pre-COVID-19), and 11 patients were non-diabetic. 25 patients had a history of hospitalisation with COVID-19 and had received steroids as a part of treatment, 12 patients had been home quarantined (no steroids received), and the rest of the 30 patients' files did not contain data regarding the same.
Sixty-five patients were operated upon. Two patients consented to non-surgical management due to advanced COVID-19 rhino-orbital cerebral mucormycosis. The computerised tomography of the paranasal sinus (CT-PNS) was done in 51 (76.11%) patients, magnetic resonance imaging (MRI) was done in 16 (23.88%) patients pre-operatively, and a repeat MRI was required in 8 (11.94%) patients post-operatively.
Thirty-two patients received various forms of amphotericin B, 10 patients received liposomal amphotericin B, 4 patients received lipid emulsification, and the remaining 18 received amphotericin B deoxycholate. Sixty-five (97.01%) patients received a parental/oral form of posaconazole. All patients who received amphotericin B had alternate day renal profile monitoring. Twenty-five patients received potassium correction for hypokalaemia perioperatively.
Three patients had thromboembolic events in our study, one patient had multiple cerebral infarcts, one patient developed middle cerebral artery infarct, hemiplegia, and aphasia post-operatively, and one patient was admitted with acute cerebral infarct with a Glasgow coma scale (GCS) score of 7/15.29 patients received both injection enoxaparin 40 mg subcutaneously once daily (OD) and tablet aspirin 150 mg OD. Twenty-five patients received tablet aspirin 150 mg OD. Thirteen patients did not receive tablet aspirin.
With the combined treatment of emergency radical surgical resection and rapid initiation of high-dose antifungal therapy treatment, the mortality rate was reduced to 8.9% and also it decreased the duration of hospital stay. The mean duration of hospital stay was 10.7 ± 6.3 (1-30 days). The mean duration of ICU stay was 8.5+/-4.56 (4-17 days).
DISCUSSION
Literature review shows that rhino-orbital cerebral mucormycosis, mainly associated with COVID-19, has a poor prognosis. [2,3] Historically, mucormycosis is associated with high mortality rates of 25%. [3] However, our study showed a better outcome with a mortality rate of 8.9%. The post-operative outcome and improved survival rates in the mucormycosis patients in our study were influenced by aggressive pre-operative risk stratification, correction of predisposing factors, radical surgical resection, and rapid initiation of high-dose antifungal therapy.
Coronavirus has been linked to stress-induced cardiomyopathy and myocardial damage; according to conventional protocols, a comprehensive cardiac examination, including echocardiography, is recommended before surgery. [4] Post-COVID-19 individuals have been shown to have abnormal pulmonary function tests and symptoms of restrictive lung disease. [5] The risk of thromboembolic events linked with COVID-19 may impact all the systems including the central nervous system, warranting the patient's complete coagulation profile and a brain CT scan. Hence, the protocols and methods mentioned [6] needed revision and customisation as these may not always be feasible for COVID-19 positive patients and may delay the emergency surgical debridement. We hastened pre-operative optimisation and risk stratification for emergency surgeries by use of the room air saturation, arterial blood gas (ABG) analysis, previous chest radiogram or high-resolution computed tomography scans done during the patient's past COVID-19 illness, and simple bedside breath-holding tests to assess the patient's pulmonary condition. The echocardiogram was not available for emergency surgeries 24/7, so we used the 12-lead electrocardiogram, and other clinical signs like raised jugular venous pressure, and pedal oedema to stratify the cardiac condition.
qSOFA score containing alertness (GCS score <15), respiratory rate >20 breaths/min, systolic blood pressure <90 mm of Hg; and SIRS score -temperature >38.3 0 C or <36 0 C, HR >90 beats/min, and white blood cell count >12,000 or <4,000/mm 3 were taken into consideration for pre-operative risk assessment and admission into the ICU by the attending anaesthesiologists. qSIRS (the combined qSOFA and SIRS) can improve the accuracy of the individual scores in the prediction of mortality. [7] ASA physical status grades III and IV patients with qSOFA >2, and qSIRS scores >2 with fever, and persistent hypotension in our study were pre-operatively optimised in the ICU to treat the sepsis and related multiple organ dysfunction syndrome. Arterial blood gas analysis and biochemical studies were performed at the appropriate juncture, depending upon which treatment of acidosis, electrolyte imbalances, and coagulation abnormalities was done in these patients.
Under standard anaesthetic monitoring, general anaesthesia was administered for all types of surgeries as per the post-COVID-19 mucormycosis guidelines. [8] Personal protective equipment and an intubation box were used for 23 patients within 14 days of being diagnosed as COVID-19 positive. The intubation box was devised by anaesthesiologists of our institute. Whenever intubation of a difficult airway was anticipated, C-Arm machine cover (sterile plastic cover) was used to cover the head, and neck up to the chest of the patient. Oral intubations were performed as per detailed protocols to minimise the dissemination of respiratory droplets and aerosols. [9] Patients who achieved spontaneous ventilation (tidal volume >5 ml/kg, inspiratory pressure ≥25 cm H 2 O, vital capacity >10 ml/kg) and maintained oxygen saturation were extubated. Patients who demonstrated post-anaesthetic Aldrete recovery score <9 were shifted to the ICU with an endotracheal tube for elective post-operative ventilation. [10] They were ventilated according to the National Institutes of Health, National Heart, Lung, and Blood Institute (NIH NHLBI) therapy for acute respiratory distress syndrome (ARDS) mechanical ventilation protocol. For patients with post-COVID-19 ARDS changes, lung-protective ventilation was given with a tidal volume of 4-6 ml/kg, and P plat <35 cm H 2 0. Patients received ABG analysis-guided spontaneous breathing trials followed by weaning and extubation. For patients who had the partial pressure of oxygen (PaO 2 ), divided by the fraction of inspired oxygen (FiO 2 ) (P/F Ratio) <150, prone ventilation was done according to the ICU protocols.
A stress dosage of corticosteroids was given to all our patients intraoperatively, irrespective of the history of corticosteroid therapy. Blood and blood product transfusions and intraoperative inotropes were used as required depending upon the extent of surgery and pre-operative abnormalities in ASA physical status class III and IV patients to maintain intraoperative haemodynamic stability.
Perioperative hyperglycaemia management was done according to the latest updated protocols. [11] Antifungal treatment was initiated early with a high dose regimen as per the guidelines. [12] All patients had mandatory renal function tests conducted on admission and post-operatively on alternate days. Patients with amphotericin B-related side effects like nephrotoxicity, hypokalaemia, uncontrolled random blood glucose levels, and diabetic ketoacidosis were medically managed stringently throughout the perioperative period.
Patients who underwent maxillectomy and orbital exenteration were provided with prostheses and a palatal obturator. Continuous follow-up for better aesthetic, functional rehabilitation, and further psychological counselling was provided.
CONCLUSION
Superior outcomes in terms of reduced mortality in CAM patients can be achieved by aggressive pre-operative optimisation, organised anaesthetic management, and careful post-operative planning with the help of a dedicated CAM care group involving anaesthesiologists, surgeons, and intensivists. A detailed clinical examination with quick basic investigations, bedside tests, ABG analysis, and scores like qSIRS and qSOFA in those COVID-19 patients in whom conventional protocolised tests are not possible can help for emergency CAM surgeries. This can reduce the time interval between the patient's hospital admission and proceeding for surgery which can significantly improve patient recovery and survival rates. However, these results deserve validation in the context of a prospective study. | 2022-05-20T15:21:22.005Z | 2022-05-01T00:00:00.000 | {
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270919785 | pes2o/s2orc | v3-fos-license | Azelnidipine prevents cardiac dysfunction in streptozotocin-diabetic rats by reducing intracellular calcium accumulation, oxidative stress and apoptosis
Background Numerous evidences suggest that diabetic heart is characterized by compromised ventricular contraction and prolonged relaxation attributable to multiple causative factors including calcium accumulation, oxidative stress and apoptosis. Therapeutic interventions to prevent calcium accumulation and oxidative stress could be therefore helpful in improving the cardiac function under diabetic condition. Methods This study was designed to examine the effect of long-acting calcium channel blocker (CCB), Azelnidipine (AZL) on contractile dysfunction, intracellular calcium (Ca2+) cycling proteins, stress-activated signaling molecules and apoptosis on cardiomyocytes in diabetes. Adult male Wistar rats were made diabetic by a single intraperitoneal (IP) injection of streptozotocin (STZ). Contractile functions were traced from live diabetic rats to isolated individual cardiomyocytes including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR90), maximal velocity of shortening/relengthening (± dL/dt) and intracellular Ca2+ fluorescence. Results Diabetic heart showed significantly depressed PS, ± dL/dt, prolonged TPS, TR90 and intracellular Ca2+ clearing and showed an elevated resting intracellular Ca2+. AZL itself exhibited little effect on myocyte mechanics but it significantly alleviated STZ-induced myocyte contractile dysfunction. Diabetes increased the levels of superoxide, enhanced expression of the cardiac damage markers like troponin I, p67phox NADPH oxidase subunit, restored the levels of the mitochondrial superoxide dismutase (Mn-SOD), calcium regulatory proteins RyR2 and SERCA2a, and suppressed the levels of the anti-apoptotic Bcl-2 protein. All of these STZ-induced alterations were reconciled by AZL treatment. Conclusion Collectively, the data suggest beneficial effect of AZL in diabetic cardiomyopathy via altering intracellular Ca2+ handling proteins and preventing apoptosis by its antioxidant property.
Background
Individuals with diabetes develop cardiomyopathy independent of coronary artery disease, hypertension or atherosclerosis [1][2][3]. This 'diabetic cardiomyopathy' is characterized in the early stages by reduced relaxation rates (diastolic dysfunction) while in later stages the systolic dysfunction becomes more prominent [4][5][6]. Also, hyperglycemia-induced defective intracellular Ca 2+ ([Ca 2+ ] i ) homeostasis and increased reactive oxygen species (ROS) production have been implicated in this impaired electromechanical performance [7,8]. A combination of these events ultimately leads to diabetic cardiomyopathy [9,10]. Accumulating evidences implicate that ROS plays pivotal role in the pathogenesis of cardiac dysfunction during diabetes, and is likely to be a causative agent for the disturbance in intracellular Ca 2+ signaling. Several ion-transport pathways are highly sensitive to redox regulation and oxidative stress directly impedes intracellular Ca 2+ homeostasis [11].
In the diabetic heart, abnormal Ca 2+ handling during the contractile cycle results in a decreased upstroke phase of the Ca 2+ transient due to reduction in the release of Ca 2+ from the sarcoplasmic reticulum (SR) by ryanodine receptor (RyR2) [12]. In addition, the diastolic decline of the Ca 2+ transient is diminished due to reduced activity of the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA)2a pump [13]. Recent evidences indicate that ventricular dysfunction secondary to myocardial infarction in diabetic rat model was attenuated by restoring the balance of calcium regulatory proteins [14]. As far as the endogenous sources of ROS are considered, NADPH oxidase and mitochondria are the important centers of ROS production and essentially determine the redox state of the myocardium [15][16][17][18][19][20]. Also, higher myocardial NADPH oxidase activity and increased mitochondrial ROS generation have been detected in diabetes way before diastolic dysfunction is detected indicating a subtle role of hyperglycemia in generation of ROS [21][22][23][24]. More importantly, NADPH oxidase activity is markedly increased by high glucose levels [25]. Therefore, improving the abnormal Ca 2+ flux in the heart with calcium channel blockers (CCBs) that possesses additional antioxidant property is an attractive strategy to effectively normalize the disturbed Ca 2+ transients and improve contractile function.
Long-acting CCBs have been reported to be effective in treating ischemic heart disease; however, their effects on diabetic cardiomyopathy are still unclear. Our previous study showed beneficial effects of AZL in the animal model of STZ-induced diabetes on the circulating markers of cardiac damage, oxidative stress, homocysteine, pro-and anti-inflammatory cytokines [26]. The present study was designed to examine the effect of AZL on cardiomyocyte contractility and intracellular Ca 2+ homeostatic defects in the streptozotocin (STZ)-diabetic rat model with special relevance to oxidative stress and apoptosis.
Development and characterization of diabetic rats
Six to eight-week-old male Wistar rats (NCCS, Pune, India), weighing 250 to 280 g, were made diabetic by single intra-peritoneal (IP) injection of streptozotocin (STZ) (55 mg/kg, Sigma, St. Louis, MO). Control animals were treated with vehicle (0.1 mol/L sodium citrate buffer, pH 4.5). Hyperglycemia (blood glucose > 200 mg/dL) was confirmed 3 days post STZ injection using a glucometer (AccuCheck; Roche, Germany). Diabetic animals were treated with single dose of 5 mg/kg AZL suspended in 1% carboxy methyl cellulose, administered orally by gavage, starting the 4 th day of STZ treatment (n = 12) daily for a period of 12 weeks. Blood glucose and body weight were measured weekly and at the end of the study. All procedures were approved by Institutional Animal Care and Use Committee and were performed in accordance to the standards for the care and use of animal subjects, as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Resources, National Academy of Sciences, Bethesda, MD).
Measurement of cardiac contractility in vivo
Urethane (1 g/kg bw IP) was selected as an anesthetic agent as its single dose induces long-term anesthesia and analgesia with minimal cardiovascular and respiratory system depression [27]. The right carotid artery was cannulated with a microtip pressure transducer (SPR-671, Millar Instruments) connected to 8-channel PowerLab instrument via bridge amplifier (AD Instrument). The pressure-tip transducer catheter was then advanced into the left ventricle for the evaluation of ventricular pressures. LV systolic and end-diastolic pressures, the maximum rate of LV systolic pressure rise (+ΔP/Δt max ) and minimum rate of LV systolic pressure decay (-ΔP/Δt min ) were monitored and recorded using Chart 5.5. Rectal temperature was maintained at 36-38°C throughout the procedure [28].
Measurement of cell shortening/relengthening and intracellular Ca 2+ fluorescence in isolated cardiac myocytes
Adult rat ventricular myocytes (ARVMs) were isolated as described previously [29]. The mechanical properties of ventricular myocytes from control and treated rats were assessed using a SoftEdge MyoCam system (IonOptix Corp., Milton, MA, USA) [29]. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS)-indicative of peak ventricular contractility, time to PS (TPS)-indicative of contraction duration, time to 90% relengthening (TR 90 )representing cardiomyocyte relaxation duration, and maximal velocities of shortening (+dL/dt) and relengthening (-dL/dt)-indicators of maximal velocities of ventricular pressure rise/fall. Briefly, myocytes were loaded with Fura-2AM (0.5 μM) for 10 min and fluorescence measurements were recorded. Resting calcium, qualitative changes in the intracellular calcium, and fluorescence decay time (Tau) were also measured. Both single-and bi-exponential curve-fit programs were applied to calculate the intracellular Ca 2+ decay constant [29]. At least 25 individual myocytes were used for data collection. Changes in [Ca] i were calculated by determining the rise in [Ca] i relative to basal levels measured immediately before that particular experimental maneuver.
Determination of intracellular superoxide (O 2 · -) levels in diabetic ARVMs
ROS generation was measured using fluorescent probe DHE, an O 2 · --sensitive probe [30,31]. DHE at a final concentration of 2 μM was added to the ARVMs from control and diabetic rat, and the staining was carried out at 37°C. The cells were washed using phosphate-buffered saline (PBS) and fixed with 4% buffered paraformaldehyde. The coverslip was mounted with antifade on a glass slide and observed using a confocal laser-scanning microscope (Zeiss 510; Zeiss GmbH, Oberkochen, Germany). Quantitative determination of DHE fluorescence was done using fluorimetry. Briefly, post treatment, the cells were washed with PBS, and re-suspended in HEPES buffer (5 mM HEPES, pH 7.4; 5 mM KCl, 140 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 and 10 mM glucose), stained with DHE for 20 min and their fluorescence intensities were acquired by fluorimetery (SpectraMaxPro, USA).
Terminal Transferase dUTP Nick End Labeling (TUNEL) Assay
Apoptotic cell death in cardiomyocytes in heart was detected by in situ terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling. TUNEL staining was performed on the cardiac tissue sections using the fluorescent In situ Cell Death Detection Kit (Roche Diagnostic GmbH, Mannheim, Germany) according to the manufacturer's instructions. TUNEL-positive nuclei were counted in a minimum of 150 cells per group by fluorescence microscopy and an apoptotic index (AI) was determined as the percentage of TUNEL-positive nuclei which was scored blindly by two evaluators. The statistical analysis revealed a good correlation (Pearson's correlation coefficient 0.91, p < 0.0001).
Statistical analyses
At least six to seven rats were used per group for each treatment (control and diabetic with or without AZL) for mechanical and intracellular Ca 2+ recordings. For each experimental series, data are presented as means ± SE. Statistical significance (P <0.05) for each variable was estimated by ANOVA by using Tukey-Kramer post tests using Prism 4.0 GraphPad software (GraphPad, San Diego, CA, USA). A p value less than 0.05 was considered statistically significant.
Results
AZL improves cardiac contractile dysfunction in diabetic rat heart I) In vivo STZ-induced diabetic animals showed stable signs of diabetes, including hyperglycemia, reduced levels of insulin. Also there was a noted increase heart/body weight ratio (H/BW). Diabetic rats treated with AZL showed improvement in these physiological parameters. STZ group showed reduced rate of contraction (+ Δp/Δt) and rate of relaxation (-Δp/Δt) as compared with control group. A significant reduction in the heart rate and impairment in left ventricular pressure (LVP) as well as left ventricular end diastolic pressure (LVEDP) was observed in STZ-diabetic animals. Table 1 shows AZLtreated diabetic rats significantly improved left ventricular parameters.
II) In isolated single myocytes from diabetic rat
The contractile dysfunction observed in vivo could be partly due to extrinsic factors, such as changes in circulating metabolites or hormones. In isolated myocytes, the influence of extrinsic factors is eliminated, which allows for the evaluation of intrinsic contractile dysfunction. Therefore, contractile function was examined in isolated myocytes from control, diabetic and AZL-treated groups.
Peak shortening (PS) amplitude normalized to cell length was significantly decreased in ventricular myocytes under STZ-induced diabetes (27.82% ± 8.17, p < 0.05). Myocytes from the diabetic group also demonstrated significantly prolonged time-to-peak shortening (TPS, (29.30% ± 11.2, p < 0.05) and time-to-90% relengthening (TR 90 , (25.72% ± 20.67, p < 0.05) compared with control. AZL treatment completely abolished the diabetesinduced abnormalities of PS, TPS and TR 90 ( Figure 1A-D). The maximal velocities of shortening (+dl/dt) and relengthening (-dl/dt) were significantly reduced by diabetes and AZL treatment restored the diabetes-induced dysfunction ( Figure 1A and 1B). Myocytes isolated from 12-week AZL-treated diabetic rats had significantly smaller deviation from corresponding values when compared to the myocytes isolated from the control group suggesting role of AZL in maintaining ventricular function of the heart along with preserving the contractile properties of individual myocytes.
AZL maintains global Ca 2+ homeostasis in diabetic rat heart
Our data indicated enhanced level of resting intracellular Ca 2+ in STZ induced diabetic rat myocytes. The rise of intracellular Ca 2+ in response to electrical stimuli was significantly reduced. "Diabetic" myocytes showed reduced intracellular Ca 2+ clearing rate (single and biexponential decay). Furthermore, 12-weeks of AZL treatment significantly ablated intracellular Ca 2+ abnormalities in STZ treated diabetic rats. Consistent with its response in cardiomyocyte shortening, AZL treatment improved diabetes induced changes in Ca 2+ homeostasis including elevated resting intracellular Ca 2+ levels, depressed intracellular Ca 2+ rise in response to electrical stimuli and prolonged intracellular Ca 2+ decay ( Figure 2 A-C).
AZL reduces superoxide (O 2 · -) from diabetic ARVMs
Superoxide overproduction in the cellular systems is an important feature of diabetic cardiomyopathy. A significant increase in the DHE fluorescence was observed in isolated myocytes from the STZ diabetic rats indicating generation of superoxide radicals in comparison to the myocytes isolated from control rats. Myocytes from 12week AZL-treated diabetic rats showed significant decrease in the fluorescent levels indicating that AZL can reduce the superoxide production ( Figure 3A).
Qualitative analysis of DHE fluorescent intensity showed that there was a 5.6 ± 0.5 fold (p < 0.001) increase in the DHE fluorescence in the "diabetic" myocytes when compared to the "control" myocytes. The fluorescence intensity of DHE showed a 2.7 ± 0.8 fold (p < 0.001) decrease in the AZL treated group when compared to the STZ diabetic rats ( Figure 3B). These results suggest that AZL treatment prevents diabetes-induced accumulation of superoxide in the myocytes.
AZL neutralizes the increased expression levels of contractile proteins (Troponin I) in diabetic heart
Since mechanical dysfunction that characterizes diabetic cardiomyopathy plays an essential role in the Ca 2+ regulation of muscle contraction, we studied the effect of AZL on the expression level of cardiac Troponin I in our experimental model. Our western blot results showed a 2.15-fold increase (p < 0.05) in the expression of Troponin I in the heart of STZ treated animal when compared with the control. The increased cardiac Troponin I expression counter-balanced in the cardiac tissue from AZL treated diabetic rats ( Figure 4A). These findings suggest that AZL treatment under diabetic condition prevents cardiac damage by reducing the expression of cardiac Troponin I.
AZL regulates the expression of calcium regulatory receptors and channels in diabetic heart
Downregulation of key Ca 2+ -handling proteins like sarco (endo)plasmic reticulum Ca 2+ -ATPase (SERCA)2a and ryanodine receptor (RyR2) is one of the major cause of abnormal Ca 2+ homeostasis in diabetic cardiomyopathy [13,[32][33][34]. The alteration in SERCA2a and RyR2 expression results in altered cytosolic Ca 2+ transients, leading to abnormal contraction. Our western blot results indicated a complete loss of expression of RYR2 and a significant reduction in SERCA-2a in STZ-treated diabetic rats compared to the controls ( Figure 4B). Reduction in the RyR2 expression induced by uncontrolled diabetes was attenuated with AZL treatment. Similarly, the expression of SERCA-2a was also restored after AZL treatment ( Figure 4B). These findings indicate that AZL treatment under diabetic condition prevents dysregulation of calcium regulatory proteins in the heart.
AZL induces downregulation of NADPH oxidases and prevents oxidative stress
We determined effect of AZL treatment on the endogenous pro-oxidants and antioxidants like p67 phox and Mn-SOD. Our western blot results showed increased expression of p67 phox in diabetic heart. This indicated that endogenous pro-oxidant system was triggered under diabetic condition which may further aggravate oxidative stress. Treatment with AZL significantly attenuated the p67 phox expression ( Figure 4C). On the other hand, a significant decrease (0.46 fold, p < 0.05) in the expression of Mn-SOD in STZ-diabetic heart was observed as compared to control hearts. Treatment with AZL resulted in a 2.5-fold (p < 0.05) increase in the expression of Mn-SOD in the diabetic condition. These results indicate that AZL exerts its protective effects by targeting the NADPH oxidase and mitochondrial redox enzymes ( Figure 4C).
AZL prevents STZ-induced cardiac apoptosis
Apoptosis was also evaluated in the cardiac tissue by TUNEL assay. Diabetic rats showed significant myocardial apoptotic cell death manifested by a 6 fold increase in the percent TUNEL-positive cell labeling compared with control rats ( Figure 5A and 5B). The counts of TUNEL-positive nuclei significantly decreased in AZL-treated group. Under oxidative stress, mitochondria play an important role in apoptosis and a decrease in the level of bcl-2 is observed. This decreasing bcl-2 expression is one of the hallmarks of apoptosis through mitochondrial pathway. STZ diabetic animal showed elevated cardiac apoptosis, as indicated by decreased bcl-2 protein expression, compared to the control animals. AZL-treated diabetic rats expressed enhanced level of bcl-2 in the heart lysates indicating that AZL plays protective role in cardiac apoptosis.
Discussion
The key findings of our present study demonstrated that AZL treatment for 12 weeks in diabetic animal inhibits Figure 1 Contractile properties of cardiomyocytes isolated from control, diabetic rats and diabetic rats treated with AZL (5 mg kg -1 day -1 ).
A: Maximal velocity of shortening (+dL/dt, A) and relengthening (-dL/dt, B) of ventricular myocytes isolated from control, diabetic rats and diabetic rats treated with AZL (5 mg kg -1 day -1 ). B. Graph illustrates Peak shortening (PS, normalized to cell length) of the myocytes isolated from control, diabetic rats and diabetic rats treated with AZL. C. Graph illustrates time-to-peak shortening (TPS) of the myocytes isolated from control, diabetic rats and diabetic rats treated with AZL. D. Graph illustrates time-to-90% relengthening (TR 90 ) of the myocytes isolated from control, diabetic rats and diabetic rats treated with AZL. Values are means ± SE; n = 151-163 cells from 5-7 rats per group, *P < 0.05 vs. control group; **P < 0.05 vs. diabetic group.
the development of early characteristics of diabetic cardiomyopathy, such as, prolonged relaxation and abnormal E-C coupling in vivo in the intact myocardium as well as in the isolated ventricular myocytes. Delayed diastolic relaxation in diabetic cardiomyopathy is related to diminished removal of [Ca 2+ ] i from cardiomyocytes after the systolic contraction event. Treatment with AZL showed improvement in the systolic and diastolic duration. Also the markers for diastolic dysfunction, viz., maximal rise and decay in the blood pressure showed improvement. These mechanical abnormalities may be underscored by altered intracellular Ca 2+ homeostasis that was associated with enhanced oxidative stress. We found that AZL prevents maladaptive ventricular remodeling. Untreated diabetes further accelerated oxidative stress at molecular level by upregulating the endogenous NADPH oxidases like p67 phox and downregulation of Mn-SOD. Treatment with AZL normalized the p67 phox and Mn-SOD expression. Also, AZL treatment normalized the levels of cardiac troponin I, RyR2, and SERCA2a. The ability of AZL to restore all the parameters to the control level provides a plausible explanation for its ability to prevent diabetes-induced defects in calcium signaling. The intrinsic antioxidant activity of AZL might thus contribute to its beneficial effects on LV dysfunction and cardiac failure. Abnormalities in the myocardial calcium handling can contribute to deranged cardiac mechanics in the diabetic heart. Diabetes impairs sarcoplasmic reticular calcium pump activities, which reduces the rate of Ca 2+ removal from the cytoplasm in diastole [7]. Such alterations may contribute to the increased diastolic stiffness characteristic of diabetic cardiomyopathy. Intracellular retention of calcium in diabetes is associated with the depletion of highenergy phosphate stores, derangement of cellular ultrastructure and can lead to cardiac dysfunction. Calcium AZL is a novel dihydropyridine calcium channel blocker which has a long lasting anti-hypertensive action [35]. It is generally well tolerated and its use is not Figure 3 Generation of O 2 ·in ventricular myocytes isolated from control, diabetic rats and diabetic rats treated with AZL (5 mg kg -1 day -1 ). A. Representative confocal laser scanning microscopy images of cells fluorescently stained with DHE. B. Graph shows quantification of DHE fluorescence emission (Arbitrary Units) of the myocytes isolated from control, diabetic rats and diabetic rats treated with AZL after staining with DHE. Data represents Means ± SEM; n = 104 to 126/group. *P < 0.05 vs. control group; **P < 0.05 vs. diabetic group.
Kain et al. Cardiovascular Diabetology 2011, 10:97 http://www.cardiab.com/content/10/1/97 associated with reflex tachycardia [35]. AZL has recently been approved in Japan for the treatment of patients with hypertension. Very recently, the results from OSCAR trial revealed that regimen that included AZL showed less composite fatal and nonfatal cardiovascular events compared to the group treated with other CCBs like amlodipine. Another study demonstrated the novel beneficial aspect of azelnidipine, whereby azelnidipine could play a protective role against atherosclerosis by suppressing monocyte chemoattractant protein-1 overexpression in endothelial cells [36]. Very recently, azelnidipine treatment have been shown to be useful in conditions like glucose tolerance, insulin sensitivity, inflammation, and number of circulating progenitor cells in non-diabetic patients with essential hypertension [37]. Another comparative study between azelnidipine and olmesartan revealed that AZL was equally effective in reducing the blood pressure but also reduced the urinary albumin/ creatinine ratio and 8-hydroxydeoxyguanosine and renal fatty acid binding protein levels significantly compared with the amlodipine group. Also, AZL group showed lower plasma aldosterone levels indicating that AZL is far more effective in preventing albuminuria and oxidant stress in hypertensive diabetic patients with CKD than amlodipine [38]. In the settings of oxidative stressinduced hepatotoxicity mice model, Azelnidipine significantly decreased inflammatory cell infiltration, profibrotic gene expressions, Hematopoietic stem cell activation, lipid peroxidation, oxidative DNA damage and fibrosis and also prevented the decrease in the expression of antioxidant enzymes [39]. It has been now well known that AZL can produce some beneficial effects independent of its anti-hypertensive effect [26], so the direct pharmacological effect of AZL on the initial management and prevention of diabetic cardiomyopathy are paid more attention.
Our study was an attempt to identify the effect of AZL on alteration of cardiomyocyte contraction and related calcium regulatory proteins, which might explain the effect of AZL on cardiac performance under diabetic conditions. Diabetes is characterized by consistently elevated blood glucose levels, decreased insulin levels and increased heart/body weight ratio indicative of hypertrophied heart was observed in the STZ diabetic rat. Cardiac hypertrophy involves remodeling of entire heart especially in the left ventricular region which eventually leads to impaired diastolic function, further causing deterioration of cardiac morphology and function. The important finding of the present study is that STZ-induced hyperglycemia leads to dilapidated cardiac function further leading to diabetic cardiomyopathy. Our STZ diabetic rats showed left ventricular dysfunction. This study along with our previous report [26] provides evidence that hyperglycemia-induced left ventricular dysfunction due to oxidative stress induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) and defective antioxidant system contributing to the development of cardiomyopathy. Apoptosis induced by hyperglycemia is an early event in the pathophysiology of diabetic cardiomyopathy [40]. Hyperglycemia and insulin resistance independently contribute to functional alteration in the heart [41][42][43][44]. AZL treatment in streptozotocin diabetic rats has been shown to improve these functional cardiac abnormalities perhaps through tyrosine kinase-dependent increases in intracellular [Ca 2+ ] i removal after systole. In the present study, treatment with AZL showed improvement in the systolic and diastolic duration. Also the markers for diastolic dysfunction, viz., maximal rise and decay in the blood pressure showed improvement. In the present study, we found that AZL prevents ventricular remodeling accompanied by cardiac dysfunction. We also demonstrated that AZL did not alter blood pressure and Figure 5 Apoptosis in diabetic rat hearts and treated with AZL. A: representative photographs of in situ detection of apoptosis in heart tissue from controls, diabetic rats treated with vehicle, and diabetic rats treated with AZL (5 mg kg -1 day -1 ). Total nuclei were labeled with DAPI (blue), and apoptotic nuclei were detected by TUNEL staining (green). B: average number of percent TUNEL-positive nuclei in tissue sections from each group (n = 4 to 5 sections each per group). *P < 0.05 vs. control group; **P < 0.05 vs. STZ treated group. C: Western blot showing bcl-2 expression in hearts from the same groups. Image is representative of the best of three separate experiments. β-actin served as loading control. Normalized densitometric quantification has been depicted by numerical value below the bands. this suggests that AZL has preventive effects on cardiac dysfunction beyond its antihypertensive effects. Oxidative stress might play an important role in the progression of LV dysfunction and failure, the data somewhat is consistent with previous finding using STZ diabetic models [45][46][47]. These mechanical abnormalities may be underscored by altered intracellular Ca 2+ homeostasis that was associated with enhanced oxidative stress. The intrinsic antioxidant activity of AZL might thus be a contributor to its beneficial effects on LV dysfunction in diabetic cardiomyopathy. Although these findings are of interest, no clinical trials to date have investigated the effect of AZL on the development and progression of congestive heart failure in diabetic patients.
In our study, certain diabetes-induced mechanical defects were not improved or protected by AZL treatment. For example, AZL improved the diabetes-induced reduction in PS but not Ca 2+ -induced Ca 2+ release. Although the underlying mechanism is largely unknown, the ability of AZL to enhance myofilament Ca 2+ sensitivity may play a role. This is somewhat supported by the results shown in Figure 1, where myocytes from the AZL-treated group exhibit an improved PS compared with the myocytes from the diabetic group. The results from this study revealed that AZL treatment lowered the resting intracellular Ca 2+ levels in the diabetic group. This AZL-induced reduction in resting intracellular Ca 2+ level may be associated with an enhanced SERCA Ca 2+ clearing ability in the AZL-treated group ( Figure 2) and is consistent with the vasodilatory and cardioprotective effect against Ca 2+ overload under pathological conditions such as heart failure. The frequency-PS relationship was improved by AZL-treated diabetic group (Figure 1), indicating a preserved sarcoplasmic reticulum (SR)-replenishing function in diabetic hearts. One possible explanation is that AZL may significantly augment the basal SR Ca 2+ load in the diabetic group. The impaired intracellular Ca 2+ homeostasis may be associated with a reduction in the main Ca 2+ -regulating protein SERCA2 and ryanodine receptor (RyR) proteins indirectly with reduced levels of Troponin I under the diabetic state [48]. Interestingly, the STZ diabetes-induced oxidative stress, apoptosis and alterations in oxidative stress-related signaling molecule p67 phox NADPH oxidase were effectively alleviated by AZL treatment. It also improved the levels of Troponin I, RyR2, and SERCA2a. Because SERCA and RYR2 contributes to~92% of the cytosolic Ca 2+ removal workload in rat hearts [49], our finding of an overt reduction in SERCA2a protein level in STZ-induced diabetic hearts should have provided one of the most compelling explanations for the slowed intracellular Ca 2+ clearing and prolonged duration of relaxation (TR90). The ability of AZL to restore all the parameters to the control level provides a plausible explanation for its ability to prevent diabetes-induced defects in calcium signaling.
Further, restoration of TPS after AZL treatment indicates that AZL may have a significant effect on the key rate-limiting components determining the length of contraction duration such as SR Ca 2+ release, troponin, tropomyosin, and actin-myosin cross-bridge linking. These observations are consistent with the functional data of improved intracellular Ca 2+ clearing and duration of relengthening (TR 90 ) after AZL treatment. These results suggest that AZL treatment may ameliorate contractile disturbances in cardiomyocytes from diabetic animals and could provide therapeutic potential in the treatment of diabetic cardiomyopathy.
Since AZL did not affect the hyperglycemic condition in diabetes, our data suggest that STZ-induced diabetes may elicit cardiac contractile dysfunction and intracellular Ca 2+ mishandling likely through enhanced oxidative stress and cell injury.
Increased oxidative stress is believed to be an initial and important step in the development of cardiac dysfunction and cardiomyopathy. NADPH oxidase and mitochondria are considered to be important sources of ROS [15][16][17][18][19][20] and are critical determinants of the redox state of the diabetic myocardium. Previous studies reported that membrane translocation of p67 phox and the increased expression of p22 phox was prevented by N-acetyl L cysteine [50]. Therefore, we further tested whether AZL exerts its antioxidative properties by modulating the expression and function of NADPH oxidase subunit p67 phox and mitochondrial ROS-eliminating enzyme Mn-SOD. The results from the isolated cardiomyocytes study showed that hyperglycemia leads to increased oxidative stress by enhancing the O 2 ·generation, by decreasing the expression of antioxidant enzyme Mn-SOD and by increasing expression of p67 phox . The main new findings of this study are that AZL treatment prevents the increased expression of p67 phox , and enhances Mn-SOD expression, thus reducing myocardial superoxide formation in the diabetic rat hearts. This reduction of O 2 ·generation and normalization of p67 phox and Mn-SOD after AZL treatment indicate that AZL reduces diabetic cardiac damage by targeting the redox signaling pathways. Moreover, an increase in the expression of p67 phox and decrease in the expression of Mn-SOD as well as bcl-2 and normalization of these expressions by AZL indicates a mutual functional relationship between NADPH oxidase and mitochondria. AZL in our study not only improves cardiac contractile function but also offers protection against oxidative stress, apoptosis and ultimately leading diabetic cardiomyopathy.
Limitations of the study
We did not test the effect of known antioxidants in comparison to AZL in the present study. We observed the changes after the treatment duration of 12 weeks. Some of the changes could be result of the overall functional improvement due to AZL treatment and may not be directly attributable to AZL treatment. The hemodynamic parameters were evaluated at the end of the study, and comparisons were made with comparing with diabetic and non-diabetic control in the experimental design. As a result of which the signals were recorded only at the end of the experimental period, leading to the lack of baseline values in the same animals at the start of the study.
Conclusion
In conclusion, the present study reveals the beneficial effects of AZL treatment on diabetes induced early left ventricular dysfunction. AZL exhibited additional antioxidant properties in addition to its calcium channel blocking activity. This intrinsic antioxidant property of AZL may provide a promising advantage over other calcium channel blockers in the management of compromised heart function especially under diabetes. | 2018-04-03T00:21:54.265Z | 2011-11-04T00:00:00.000 | {
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269581023 | pes2o/s2orc | v3-fos-license | Hyperspectral and Fluorescence Imaging Approaches for Nondestructive Detection of Rice Chlorophyll
Estimating and monitoring chlorophyll content is a critical step in crop spectral image analysis. The quick, non-destructive assessment of chlorophyll content in rice leaves can optimize nitrogen fertilization, benefit the environment and economy, and improve rice production management and quality. In this research, spectral analysis of rice leaves is performed using hyperspectral and fluorescence spectroscopy for the detection of chlorophyll content in rice leaves. This study generated ninety experimental spectral datasets by collecting rice leaf samples from a farm in Sichuan Province, China. By implementing a feature extraction algorithm, this study compresses redundant spectral bands and subsequently constructs machine learning models to reveal latent correlations among the extracted features. The prediction capabilities of six feature extraction methods and four machine learning algorithms in two types of spectral data are examined, and an accurate method of predicting chlorophyll concentration in rice leaves was devised. The IVSO-IVISSA (Iteratively Variable Subset Optimization–Interval Variable Iterative Space Shrinkage Approach) quadratic feature combination approach, based on fluorescence spectrum data, has the best prediction performance among the CNN+LSTM (Convolutional Neural Network Long Short-Term Memory) algorithms, with corresponding RMSE-Train (Root Mean Squared Error), RMSE-Test, and RPD (Ratio of standard deviation of the validation set to standard error of prediction) indexes of 0.26, 0.29, and 2.64, respectively. We demonstrated in this study that hyperspectral and fluorescence spectroscopy, when analyzed with feature extraction and machine learning methods, provide a new avenue for rapid and non-destructive crop health monitoring, which is critical to the advancement of smart and precision agriculture.
Introduction
Rice is a vital human food crop with a long history of production and consumption.Rice is consumed as a staple food by more than half of the world's population because it is extremely adaptable to varied environments, making it an essential staple crop worldwide [1].Rice production is the world's third highest in terms of food production, only after corn and wheat [2].The primary yield-limiting factors in rice farming are water and fertilizers, and a lack of fertilizers will cause changes in external morphology and internal structure, such as the thickness and color of leaves, which will result in variations in the reflectance properties of rice leaves and the spectrum of tree crowns [3].Traditional indicators of crop growth include chlorophyll content and nitrogen content.The main methods of measurement are manual measurement, indoor chemical analysis [4], and the rapid determination of leaf chlorophyll by SPAD (Soil and Plant Analyzer Develotrnent) device.Molina L et al. used atomic absorption spectrometry to determine the content of heavy metals in rice [5].Atomic absorption spectrometry can be used to assess magnesium content and, consequently, chlorophyll content indirectly.The concentration of chlorophyll in the extract can also be calculated using a spectrophotometer to determine the absorbance value of the chlorophyll extract at the maximum absorption wavelength [6].Traditional detection methods are laborious and frequently require a significant amount of time and work, which hinders the development of plant growth monitoring and precise management to some extent.An ultra-portable SPAD assessment system for leaf SPAD distribution analysis was proposed by Tan LH et al. [7], which was used to further calculate leaf chlorophyll content.The collection of plant growth information by traditional analysis methods has gradually evolved remote sensing technology and its application in the field of agriculture, particularly with the advent of hyperspectral remote sensing technology.
Chlorophyll is the principal pigment involved in photosynthesis in plants, with its concentration directly affecting a plant's capacity to absorb light energy; therefore, monitoring chlorophyll levels in plants is of paramount importance [8].A deficiency in chlorophyll within rice leaves can arise from various factors, such as a lack of nitrogen, a deficiency in essential trace nutrients, or unsuitable soil acidity.Timely interventions can prevent yield losses caused by insufficient chlorophyll.Upon detecting a deficiency in chlorophyll, it is imperative to promptly analyze the cause and take corrective measures such as adjusting irrigation management, conducting soil tests, and swiftly replenishing nutrients.Given that chlorophyll content affects the spectral reflectance of rice, particularly within the visible light spectrum, reflectance spectra can be utilized to estimate chlorophyll content.Kandpal, K.C. and colleagues compared various methods of chlorophyll detection in leaves under laboratory and field conditions, concluding that Arnon's spectrophotometric method is most suitable for laboratory settings, while machine learning methods are widely employed in chlorophyll detection tasks based on hyperspectral data [9].Zhao, J.W. employed hyperspectral technology to detect chlorophyll content in tea leaves, with the constructed MSAVI2 model achieving an optimal result of RMSE = 8.60 on the test set, proving the viability of measuring chlorophyll content in tea leaves through hyperspectral imaging technology [10].Jang, S.H. established a combined model of stepwise multiple linear regression and partial least squares to measure chlorophyll content in cucumber seedling leaves, identifying nine critical bands including those at 501 and 505 nm, enabling accurate and non-destructive detection of chlorophyll content in the leaves [11].Yang, Y.C. and colleagues targeted 335 wheat varieties to establish a non-destructive model for detecting the SPAD value of wheat leaves, with experimental results indicating that the first-order reflectance at 549 nm and 735 nm had the strongest correlation with SPAD values.Cao, Y.L. et al. utilized drones equipped with spectral sensors to collect rice spectral images, successfully estimating chlorophyll content using a mathematical inversion model, thereby providing research ideas and data support for related research fields [12].Feng, H. et al. developed an automatic spectral data analysis system using spectral imaging technology, discovering that chlorophyll, among four rice pigments, exhibited the strongest correlation in the 700-760 nm range [13].
Theoretical support exists for spectroscopic detection of chlorophyll concentration in rice leaves [14].However, since different samples and experimental settings result in distinct chlorophyll sensitive bands, there is a need for a quick, non-destructive detection approach with broader applicability.As artificial intelligence technology advances, more scientists are employing machine learning to hyperspectral detection.Liu, H.H. et al. employed a hyperspectral imager on a drone to predict the chlorophyll content of a rice canopy by analyzing the obtained hyperspectral images [15].Yang, Y.C. et al. proposed a more precise approach for non-destructive detection of chlorophyll using hyperspectral images and then studied the mechanism of photosynthesis in wheat drought tolerance [16].Ruszczak, B. et al. examined 15 machine learning methods and chose the most effective model for detecting chlorophyll content in the hyperspectral data of rice leaves [17].The utilization of machine learning algorithms aims to construct a non-invasive detection model.In the research presented herein, a system capable of rapidly and non-destructively determining the chlorophyll content in rice leaves through spectral data has been developed.With this model, spectral collection from rice leaves suffices to obtain the corresponding chlorophyll values, obviating the need for destructive measurement methods.The accurate detection of chlorophyll content can effectively detect the nutritional state of rice, which aids in the scientific management of agricultural production, fertilization, and grain yield.
Fluorescence is a powerful tool to study photosynthetic performance, especially when coupled with other noninvasive measurements such as absorption spectroscopy, gas analyses, and infrared thermometry.The use of chlorophyll fluorescence measurements to examine photosynthetic performance and stress in algae and plants is now widespread in physiological and ecophysiological studies [18].Malenovsky, Z. posited that supplementing the measurement of vegetation physicochemical characteristics with fluorescence measurement methods, based on existing spatial platforms, can help eliminate errors and uncertainties in the interpretation of recent remote sensing data.Remote sensing of plant fluorescence signals can contribute to a better understanding of the photosynthesis process in vegetation [19].Mishra, A. employed chlorophyll fluorescence imaging and compared eight classifiers and four feature selection methods, resulting in a species discrimination model with enhanced resolution efficiency [20].Mattila, H. utilized a pulseamplitude-modulated fluorescence camera imaging technique, which enabled the capture of fluorescence induction curve characteristics for each pixel in an image, achieving a 92.2% identification accuracy rate in the recognition of oat leaves [21].Codrea, M.C. used a chlorophyll fluorescence kinetics imaging technique that allows for the simultaneous use of multiple fluorescence traits to determine photosynthetic mutants, facilitating rapid and non-destructive screening of these mutants [22].Tyystjärvi, E., through the construction of a chlorophyll fluorescence fingerprint spectrum, successfully differentiated corn and barley from six weed species, extracting 17 features from fluorescence induction curves based on a neural network classifier with an accuracy rate ranging from 50.2% to 80.8% [23].Fluorescence spectroscopy has demonstrated considerable promise in a host of applications, ranging from growth monitoring to the identification of diseases and stressors.Empirical evidence attests to the capability of fluorescence spectroscopy in pinpointing various stress factors affecting rice, including hydric stress, nutritional shortages, and pathogenic invasions.For example, research indicates that fluorescence metrics can exhibit marked variations under differing conditions of water scarcity, yielding invaluable data for the optimization of irrigation strategies [24].In a similar vein, deficiencies in key nutrients such as nitrogen and phosphorus induce discernible changes in the fluorescence profiles of rice foliage, facilitating the prompt detection and rectification of these insufficiencies [25].
The utility of fluorescence spectroscopy has also been harnessed for the differentiation of rice cultivars [26].Yang et al. advocated for the application of Laser-Induced Fluorescence (LIF) in conjunction with a multivariate analytical approach, incorporating Principal Component Analysis (PCA) and Support Vector Machine (SVM), to distinguish various paddy rice varieties [27].Zhang et al. endeavored to delineate the correlation between the chlorophyll fluorescence spectra of rice and its growth dynamics, employing the PCA to facilitate the prognostication of rice development [28].
To achieve the accurate, rapid, and non-destructive detection of chlorophyll content in rice leaves, this study focuses on rice cultivars, and employs spectral technology as the foundational research tool.The study utilizes machine learning methodologies and information extraction techniques to identify characteristic bands and parameters for chlorophyll content in rice leaves.We have compared various analytical algorithms and investigated the predictive efficacy of hyperspectral and fluorescence spectral imaging methods to determine chlorophyll content in rice leaves.A fused CNN+LSTM model was employed, which accurately predicts the chlorophyll content in rice leaves.This research contributes to the non-destructive automatic monitoring and rational management of plant growth, thereby promoting the advancement of precision agriculture.
Measurement of Hypespectral and Fluorescence Data
Figure S1 depicts the experimental region, which is located on a farm in the Yucheng District, Ya'an City, Sichuan Province (29.9890N, 102.9820E).The type of rice we selected was Ya5You5217, which is a rice variety selected and bred by the College of Agriculture of Sichuan Agricultural University.The climatic type is that of the western boundary of the Sichuan Basin-humid subtropical monsoon.The annual average temperature ranges from 14.1 • C to 17.9 • C , and rainfall is abundant, with most places receiving more than 1000 mm.There are 10 sampling fields in the test area, with a single sampling field size of 12 × 8 m.
The GaiaSorter Hyperspectral Sorter is used to capture hyperspectral image data from rice in situ.Its key components are a unified tungsten-bromine light source, a spectral camera, and an electronically controlled mobile platform with a spectral band range of 387 nm to 1034 nm.Gaia series and aFluo series fluorescence spectral detection systems have been used to acquire fluorescence spectral image data of rice.The core components include a Gaiafluo VN-HR spectral camera, xenon light source, and fluorescence filter.Spectral data were measured directly with two sets of four LSTS-200 tungsten bromine lamp uniform light sources.A white reference plate with 99% reflectance was used for calibration before and after measurements.The spectral reflectance of three parts of rice leaves, namely, leaf tip, leaf center, and leaf occiput, were measured and their average values were used as the spectral reflectance of the samples.The sampling interval was 0.1 s and the spectrometer was calibrated every 15 min with a white reference plate.
The working principle is to irradiate the object to be measured (sample) placed on the electronically controlled moving platform (or conveyor belt) through the light source, the reflected light of the sample is captured by the spectral camera through the lens, and a one-dimensional image as well as spectral information is obtained, and with the electronically controlled moving platform (or conveyor belt) to drive the sample to run continuously, so as to obtain continuous one-dimensional image as well as the real-time spectral information.For both hyperspectral and fluorescence spectra measurements, the diffuse light source was placed directly above the measured sample at the same distance from the sample.The spatial uniformity of illumination was greater than or equal to 90%.
We cut normal leaves from rice plants during normal growth.Because of the differences in chlorophyll content in the tip, occiput, and middle of the leaf of rice leaves, we sampled these three positions separately to ensure the reasonableness of our data.Three areas of interest (ROI) were chosen for each rice leaf based on the tip of the leaf, the middle of the leaf, and the back of the leaf.Each ROI was 15 × 15 pixels in size, and the raw spectral values of the samples were calculated by averaging the ROI values of the three portions of each sample.Figure 1a depicts the hyperspectral curves of the 90 rice samples.The hyperspectral has 256 spectral bands ranging in wavelength from 387.15 nm to 1034.99 nm.According to Figure 1a chlorophyll and other pigments in rice leaves absorb weakly near the red part of the spectrum, creating absorption valleys at 500 and 670 nm.The reflection peaking at 550 nm are mostly caused by the rice leaves' high absorption of green light.
The fluorescence spectrums contained 125 spectral bands ranging in wavelength from 376.80 nm to 1011.05 nm.This wavelength range is green light (wavelength 500-560 nm), which is mainly absorbed by chlorophyll.The studies were carried out with a 495 nm fluorescence filter, and after passing through the excitation filter at 390 nm, the resulting fluorescence spectrum pictures revealed obvious wave peaking at about 510 nm, as well as inconspicuous wave peaking at around 690 nm and 740 nm.Varied samples contain varied chlorophyll contents in the hyperspectral and fluorescence spectra, resulting in variances in the spectral curves.Before building a model with machine learning algorithms, the data was frequently separated into samples, which helps to increase the predicted accuracy of the model while also maintaining the model's stability [29].The Kennard Stone model was employed in this investigation [30,31].The Kennard Stone algorithm first chooses the two samples with the greatest Euclidean distances from the data to be included in the training set, then computed the Euclidean distances between the remaining data and the chose data, and finally included the two samples with the greatest distances from the known samples in the training set.The above calculation is repeated to select representative samples until the required number of training samples is met, which helps to improve the model's computational efficiency and generalizability.Table 1 shows the specific information for the 90 samples, which are separated into the training set and the test set in a 2:1 ratio.
Invasive Measurement of Chlorophylls
Three sections of each rice leaf sample were taken from top to bottom in the experimental plot for the measurement of chlorophyll content in the rice samples; each section was cut into pieces of less than 1 cm, and samples weighing 2 g were selected and placed in triangular vials.As diffusate, 95% ethanol was poured into a 100 mL volumetric flask and filled to capacity.A total of 5 mL of the solution was pipetted into a 50 mL volumetric flask and fixed at 50 mL with 95% ethanol after vigorous shaking.Once the chlorophyll had been completely extracted, its colorimetric content was evaluated using a spectrophotometer.The chlorophyll content of the three regions of each sample leaf was averaged to determine the average chlorophyll content of the rice leaf [32].
Experimental Techniques and Protocols
Savitzky-Golay convolutional smoothing (SG) [33], a common preprocessing approach for hyperspectral data, was used to reduce noise and interferences of image acquisition environment.Figure S2 illustrates the data before and after preprocessing.
Mean-variance normalization (Z-score normalization) is a commonly used data processing method in machine learning [34].Data normalization is a common machine learning method.The values of different bands of different samples have obvious differences, and using data normalization cannot change the data distribution while limiting the data to a small range, as depicted in Figure S3.Normalizing the spectral data of the samples facilitates the convergence of the model.The scaled data size helps to reduce the running memory during model training and improve the training efficiency.The normalized data helps the model find a better solution.
Feature Extraction Methods
Preprocessing only changes the data, but not the dimension of the data, and the processed data still has high dimensionality and redundant variables.Different bands show different correlations for chlorophyll content, so it is necessary to use feature extraction algorithms to filter out the feature variables with higher correlations and eliminate irrelevant variables.In this study, the bootstrapping soft threshold method bootstrapping soft shrinkage (Boss) was chosen overall [35], which is used to filter out noise, highlight key features, and improve the resolution of the data.A total of six feature extraction algorithms, competitive adapative reweighted sampling (CARS), Iteratively Variable Subset Optimization (IVSO), Model Adaptive Space Shrinkage (MASS), and Interval Variable Iterative Space Shrinkage Approach (IVISSA) were selected to process the spectral data and increase the prediction accuracy [36].
Modeling 2.5.1. Convolutional Neural Networks and Long Short-Term Memory
Convolutional neural networks (CNNs) are multilayer perceptrons for simulating neurons that can be deepened by continuously deepening the layers of the perceptron, which is why they are also referred to as deep learning [37].Convolutional neural networks are powerful and can be used for image classification [38], text analysis [39], microplastics in soil [40], and other areas.A complete convolutional neural network consists of an input layer, an output layer, a convolutional layer, and a fully connected layer.Generally, the input layer receives the preprocessed data and passes them to the subsequent convolutional layer for feature extraction.The convolutional layer is the central functional layer of the convolutional neural network, which contains multiple convolutional kernels for feature extraction.The pooling layer is a special type of convolutional layer that usually pools the data after each convolutional operation.The pooling layer can play a role in compressing the parameters and data, which helps to improve the accuracy of the model and increase the efficiency of the model operation.Long short-term memory (LSTM) is a network specialized in sequential data processing and used in stock price prediction [41], text comprehension and other prediction tasks.The use of LSTM networks for accurate spectral predictions also has good potential as a result of the fact that spectral data are also continuous between 400 and 1000 nm.Convolutional neural networks have higher feature extraction capabilities; however, they are generally independent in computation and can only extract features for a section of the data, frequently neglecting the correlation before and after the data.In this study, a fusion network comprising CNN and LSTM is utilized to assure the effectiveness of feature extraction while boosting the model's ability to capture the information before and after the data, which helps increase the accuracy of model's prediction.Figure 2 depicts the network.Furthermore, this study conducts a comparative analysis of three prevalent machine learning algorithms-linear regression, decision tree regression, and XGBoost (eXtreme Gradient Boosting)-to elucidate the superiority of the proposed method in assessing the chlorophyll content within rice leaves ( [42][43][44]).
Model Analysis
When there is a significant linear relationship in the data, the predictive accuracy of the model constructed using the linear regression method is very accurate.The measured hyperspectral/fluorescecne spectral features contain a large number of nonlinear relationships, which is the reason why numerous experiments in linear regression perform generally.In contrast, the regression tree can learn non-linear relationships and is also highly robust to outliers.Therefore, the regression tree model basically outperforms the linear regression model in all experimental metrics.And XGBoost adopts the idea of integrated learning, which effectively avoids the overfitting problem that occurs in the regression tree model.Therefore, its performance is optimal among these three comparative methods.For the CNN+LSTM fusion network method proposed in this paper, it achieves very impressive performance with the same input features as the above comparison experiments.Meanwhile, drawing on previous ideas, we have conducted secondary feature extraction experiments on the combination of CNN+LSTM+IVSO.Since CNN itself has certain feature extraction ability, more information can be extracted for prediction when there are more feature bands.Therefore, we obtain the optimal experimental combination: CNN+LSTM+IVSO−IVISSA.
Evaluation Indicators
To analyze the classification outcomes in this work, the training set root mean square error (RMSE-Train), test set root mean square error RMSE-Test), and relative analysis error RPD are utilized as assessment metrics ( [45][46][47][48]).The formula for the metrics used is as follows: The square root of the ratio between the square of the predicted value's divergence from the true value and the total number of samples N is RMSE.In Equation (1), f t is the predicted value of the sample and y t is the actual value of the sample determined by the standard method.In Equation (2), Std p is the standard deviation.The formula is as in (3), where y is the average of the actual measured value of the sample.The lower the RMSE score is, the greater the model's prediction accuracy will be.RPD is the ratio of the data standard deviation to the root mean square error.4.2 Effectiveness of feature extraction.
Feature Extraction Results
In this study, 90 rice leaf samples were employed, each of which had spectral information from 256 different bands, and a substantial quantity of redundant information is always present in sample data at high latitude.Six feature extraction techniques (BOSS, CARS, IVSO, MASS, IVISSA, UVE) are utilized for feature extraction of hyperspectral data to increase operating efficiency and prediction accuracy.The approach with the best results is chosen for the next trial based on the experimental findings, and the results are shown in Figure 3.As shown in Figure 3a, 21 feature variables are generated following feature extraction of the spectrum data with the BOSS algorithm, accounting for 8.2% of the total number of hyperspectral variables.The retrieved feature bands are primarily in the 500-650 nm and 800-1000 nm ranges.The feature variables are concentrated near the wave crests and troughs, but their distribution is insufficiently uniform.As shown in Figure 3b, after applying the CARS algorithm, a total of 37 feature bands are created, accounting for 14.4% of the total hyperspectral variables.The feature bands are predominantly concentrated between 400-600 nm and 750-900 nm, and the frequency of the feature bands outside the wave crest is higher.As shown in Figure 3c, the IVSO algorithm recovers 46 feature bands for the original data, accounting for 17.9% of the total number of hyperspectral variables.The feature distribution is quite uniform, with the majority of features gathered between 800 and 1000 nm.The MASS algorithm selects 58 feature bands from the original data, accounting for 22.6% of the total hyperspectral variables, as shown in Figure 3d.As illustrated in Figure 3e, the IVISSA method yields 76 feature bands, accounting for 29.6% of the total number of hyperspectral variables.Among all feature extraction techniques, it obtains the most feature bands, and the feature bands are uniformly distributed.The EIS method generates 64 feature bands, accounting for 25% of the total number of hyperspectral variables; the feature band distribution is illustrated in Figure 3f.
The results of six feature extraction techniques used to extract features from fluorescence spectrum data are displayed in Figure S4.As illustrated in Figure S4a, when BOSS feature extraction is performed on the original fluorescence spectral data, the minimum number of feature bands obtained is only 11, accounting for 8.8% of the total number of feature bands in the fluorescence spectra.The main feature bands are clustered near the 510 and 700 nm peaks.Figure S4b depicts the results of CARS feature extraction from the original fluorescence spectrum data.A total of 14 feature bands were discovered, accounting for 11.2% of all feature bands in the fluorescence spectrum.The majority of the feature bands are approximately 70 nm in wavelength.As shown in Figure S4c, the IVSO algorithm discovers 19 feature bands from the original fluorescence spectral data, accounting for 15.2% of the total number of fluorescence spectral features.The feature bands are mostly spread uniformly near the wave crests and troughs.The MASS algorithm extracted a total of 24 feature bands for the original data, accounting for 19.2% of the total number of hyperspectral variables, as shown in Figure S4d, with the main feature bands focused at the wave crests at 510 nm and 700 nm.The IVISSA feature extraction of the original fluorescence spectral data produced the most feature bands, with a total of 38 feature bands accounting for 30.4% of the total number of fluorescence spectral features, and the feature band range is between 475-600 nm and 700-800 nm, as shown in Figure S4e.Following the application of the EIS method, a total of 27 feature bands are retrieved, accounting for 21.6% of the total number of feature bands in the fluorescence spectrum, as shown in Figure S4f.
Linear Regression
The six types of hyperspectral feature variables collected above were separately entered into the linear regression model, and the prediction results obtained are displayed in Table 2.As shown in Table 2, the linear regression models built with the six types of feature spectra are generally less effective.The IVISSA algorithm has the highest number of features after extraction, but the RMSE train indicator is the highest at 0.82.The reason for this is that there are still some redundant data in the feature variables extracted by the IVISSA algorithm.The most effective feature extraction method is the IVSO algorithm with an RMSE train of 0.60 and an RPD of 2.04, which is also the highest value.The linear regression model, which can only capture the relationship between variables based on a linear relationship between data, has weak performance on data with a hierarchical structure.The results show that the linear regression method has a better predictive ability for high-dimensional and complex data.The results of prediction performance obtained by inputting each of the six types of fluorescence spectral features extracted above into the linear regression model are shown in Table 3.The IVSO algorithm, which performs better on hyperspectral data, performs poorly on fluorescence spectral data, and the RMSE train reaches the highest value of 0.74 among the comparison algorithms.From the RMSE train index, IVISSA is the algorithm with the best feature extraction effect, with the smallest RMSE train of 0.54.However, the corresponding RMSE train of the IVISSA algorithm is the highest at 0.83, indicating that the prediction model is overfitted and has poor generalization performance.In terms of RPD index, the best effect of feature extraction is the Boss algorithm, which achieves a maximum RPD of 2.11.The prediction effect of the fluorescence spectral data is better than that of the hyperspectral data.
Regression Tree
The constructed regression tree model is better at capturing the nonlinear relationship in the data, and it is obviously lower than the above model in terms of training and prediction speed.However, the prediction accuracy is relatively low, and due to the complex structure of the tree model, more data is often required to determine the best parameters.The indices of the prediction results are shown in Table 4.The RMSE train index of the regression tree model is significantly lower than the RMSE test index, which proves that the regression tree model has a generalization ability and the possibility of overfitting.The CARS feature extraction algorithm performs optimally in the regression tree model, and both RMSE-Train and RMSE-Test reach the minimum, and the RPD value is the highest among the comparison models, which proves the effectiveness of the selected features.The IVISSA algorithm, on the other hand, performs poorly in this section of the experiment.The results of the regression tree prediction model based on fluorescence spectral data are shown in Table 5.Overall, the indicators of each algorithm are better than the regression tree prediction model for hyperspectral data.The best feature extraction algorithm is IVSO with the lowest RMSE train metric of 0.60, and the RMSE test metric also performs well, with the largest RPD value of 2.12 among the algorithms, while the IVISSA algorithm performs poorly, with the highest values of both the RMSE train and RMSE test metrics and the lowest corresponding RPD value of 1.93.also has the smallest value of 1.93.
XGBoost
The predictive performance of the six types of hyperspectral feature variables input to the XGBoost model is shown in Table 6.XGBoost uses the practice of random forests to sample different columns for training, which is able to avoid overfitting while reducing the training time.The XGBoost model has the lowest performance on spectral features extracted by the IVSO algorithm, and both the RMSE train and RMSE test are the lowest among the comparison methods.The CARS algorithm is the second most effective and the UVE feature extraction algorithm is the most ineffective.The six types of fluorescence spectral variables were input into the XGBoost model, whose prediction results are shown in Table 7.For fluorescence spectral data, the IVSO algorithm again performs well, with RMSE-Train and RMSE-Test achieving the best results in all comparison experiments.While the IVISSA feature extraction algorithm did not perform well, the regression prediction model for fluorescence spectral data performed better than the regression prediction model for hyperspectral data.
CNN+LSTM
In this work, when the fusion network based on convolutional neural network and LSTM is developed, a learning rate of 0.01 is selected, the relu function is used as the activation function, and the cross entropy loss function is used as the objective function of the model.The six types of spectral feature variables extracted as described previously are respectively input to the convolutional neural network, and the obtained prediction result indices are shown in Table 8.As seen from Table 8, the lowest RMSE train of the convolutional neural network built with six types of spectral feature variables is 0.32, which corresponds to the IVISSA feature extraction method, while the RMSE train of the IVSO algorithm, which performs better in the previous model, is 0.36.The lowest value of the RMSE train is also found in the features extracted by the IVISSA algorithm, while the maximum RPD value is 2.2.The suggestion that this round of experiments seems to contradict the previous experiments is that the convolutional neural network also plays a role in feature extraction during the convolutional process.Again, feature extraction is more beneficial for samples with a larger number of features to obtain more valid information.For samples with a smaller number of features, re-feature extraction may eliminate the effective information, which in turn affects the results.To further investigate this hypothesis, a secondary feature extraction experiment is conducted.
Based on fluorescence spectral data, the results of the different prediction indices of the convolutional neural network constructed with six types of spectral features are shown in Table 9.When spectral data are used to construct the model, the results obtained are generally better than those obtained with the hyperspectral data.Consistent with the results of the hyperspectral data, the IVISSA feature extraction algorithm is the most effective, with the lowest RMSE train value of 0.30, the lowest RMSE test value of 0.34, and the highest corresponding RPD value of 2.56, while the Boss algorithm performs the worst.In the conventional realm of hyperspectral prediction, it is posited that the amalgamation of features derived from the extraction of multifaceted attributes can enhance the veracity of the resultant predictions.This assertion is predicated on the premise that integrating diverse spectral characteristics can provide a more comprehensive representation of the data, thereby facilitating improved analytical outcomes [49].In Table 10, the IVSO feature extraction algorithm was chosen, which performs well in most models, as the benchmark and perform feature extraction again on the data after performing feature extraction once and input it to the convolutional neural network for training.The results are shown in Table 6.The number of feature bands obtained after the second feature extraction is reduced.From Table 6, it can be seen that the feature extraction algorithm obtained by combining IVSO-IVISSA performs the best, with the number of feature bands at 37 and the maximum RPD value at 2.57.Compared with the other experiments, the effect of the feature bands obtained by the secondary feature extraction algorithm is much better than that of the feature extraction algorithm using only the primary feature extraction algorithm.Compared with the experiments in Table 9, although the RMSE values all decrease, the degree of change in the RMSE values of the IVSO-IVISSA method is less than that of the other centralized feature extraction algorithms, while the IVSO-IVISSA method obtains more bands.This shows that the CNN+LSTM network is still superior, but when the number of features decreases, it has a certain negative impact on the performance of the network.To validate the performance of the fusion network chosen for this study, a regression analysis were conducted independently for each model based on the selection of the ideal feature extraction algorithm, and the chlorophyll prediction results are displayed in Regression analysis was carried out separately for each model using fluorescence spectral data, and the results are displayed in Figure 5.In a direct comparison, the CNN+LSTM fusion algorithm surpasses the control group in both the training and test sets.The model that utilizes solely CNN and LSTM performs better than the regression tree and XG-Boost algorithms in the training set and maintains better overall performance in the test set.The regression tree approach, coupled with the XGBoost training process, performed poorly, failing to effectively learn the probable relationships within the data.When the models are developed with fluorescence spectrum data, the six regression prediction models exhibit a slight improvement over those developed with hyperspectral data.Given the limited experimental data available, we used five-fold cross-validation within the established context of fluorescence spectral data and the dual feature extraction methods of IVSO-IVISSA to compare the performance of the models discussed in this paper.The results, as depicted in Table 11, confirm the superior performance of the methodologies proposed in this study.
Discussion
This study collects hyperspectral and fluorescence spectral data from rice leaves to examine the spectral features of chlorophyll.Six feature extraction algorithms, including regression trees, linear regression, and the XGBoost model, are employed to assess the nitrogen content in rice leaves.The gathered data are normalized, and convolution smoothing is applied to reduce noisy samples.The findings suggest that fluorescence spectroscopy is superior in detecting chlorophyll concentration in rice leaves.Utilizing the strengths of both CNN and LSTM for learning non-linear and multidimensional data, the CNN+LSTM fusion model presented in this paper outperforms other machine learning models in accurately predicting chlorophyll content.This study also verifies the effectiveness of secondary feature extraction and identifies the optimal combination for chlorophyll prediction in rice leaves: the CNN+LSTM+IVSO−IVISSA method based on fluorescence spectral data.A comparison with other studies on chlorophyll prediction using machine learning methods-most of which involve standard approaches such as XGBoost, SVM, and Random Forest-demonstrates the capability of machine learning to effectively predict chlorophyll content.Beyond rice, the task of chlorophyll prediction is relevant to fields such as water quality, corn, tea, and more, as shown in Table 12.The paper underscores that spectral imaging technology combined with machine learning can facilitate rapid, accurate, and nondestructive detection of chlorophyll in rice leaves, offering a simpler and time-saving measurement method.Limited by equipment constraints, it is challenging to acquire leaf spectral band information in field conditions during practical applications.Consequently, designing portable spectral sensors is an issue that urgently needs to be addressed.Future research will expand the types of samples, including the measurement of rice canopies.Moreover, while this study focuses on a single developmental stage of rice, future work will develop multiple growth monitoring models for different developmental stages to elucidate the rice growth process.The current study, based on field experiments, examines the physiological and biochemical parameters of rice at the leaf level using hyperspectral diagnostics.Its practical application awaits further validation.Due to the commonality of spectral mechanisms, the findings will be verified using unmanned aerial vehicles (UAVs) and satellite remote sensing data to ensure the broader applicability of the study's conclusions.
Conclusions
This study collected hyperspectral and fluorescence spectral data of rice leaves and analyzed the spectral characteristics of chlorophyll in rice leaves.Six feature extraction algorithms, utilizing regression trees, linear regression, XGBoost models, and other machine learning models, were employed to detect the chlorophyll content in rice leaves.The collected data were normalized, and convolutional smoothing was applied to reduce noise samples.The results indicate that fluorescence spectra have advantages in detecting chlorophyll content in rice leaves.In comparison to existing machine learning models, the CNN+LSTM fusion model used in this study more accurately predicts the chlorophyll content in rice leaves.The CNN+LSTM fusion model, applicable to fluorescence spectral data, exhibited optimal RMSE-Train, RMSE-Test, and RPD indicators, with values of 0.26, 0.29, and 2.64, respectively.This demonstrates that the combination of spectral imaging technology and machine learning regression prediction methods enables the rapid and accurate non-destructive detection of chlorophyll in rice leaves, providing a simpler and time-saving method for measuring chlorophyll in rice leaves.The detection method devised in this study relies on specific spectral equipment, and obtaining real-time spectral band information from rice leaves in the field presents certain challenges.In future work, we will further diversify sample types, considering chlorophyll content measurements in both rice leaves and the rice canopy.Additionally, this study focused solely on a specific
Figure 2 .
Figure 2. Schematic representation of the CNN+LSTM network structure.
Figure 4 .
The CNN+LSTM fusion technique, among various techniques, outperforms the other networks in terms of prediction accuracy.The linear regression algorithm and the LSTM-only algorithm outperform the other three networks in the training set, but the linear regression approach performs poorly in the test set, while the LSTM prediction set outperforms them.The CNN algorithm performs relatively well in the training and prediction results of test set.The preceding experiments demonstrate the fusion network's advantage.
Table 1 .
Summary of hyperspectral and fluorescence spectroscopy data.
Table 1
shows that the training set's chlorophyll content ranges from 32.4 µg/cm 2 to 47.8 µg/cm 2 while the test set's chlorophyll content ranges from 33.6 µg/cm 2 to 48.2 µg/cm 2 .The test set's standard deviation, 1.62 µg/cm 2 , is lower than the training set's, 1.95 µg/cm 2 , indicating that the data distribution in the test set is more concentrated.
Table 2 .
Linear regression prediction results based on hyperspectral data.
Table 3 .
Linear regression prediction results based on fluorescence spectral data.
Table 4 .
Regression tree regression prediction results based on hyperspectral data.
Table 5 .
Regression tree prediction results based on fluorescence spectral data.
Table 6 .
XGBoost regression prediction results based on hyperspectral data.
Table 7 .
XGBoost regression prediction results based on fluorescence spectral data.
Table 8 .
CNN+LSTM prediction results based on hyperspectral data.
Table 9 .
CNN+LSTM prediction results based on fluorescence spectral data.
Table 10 .
CNN+LSTM and quadratic feature extraction prediction results based on fluorescence spectral data.
Table 11 .
Model performance comparison.
Table 12 .
The comparison of the work of this paper with the work of other researchers. | 2024-05-05T15:04:01.114Z | 2024-05-01T00:00:00.000 | {
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54707266 | pes2o/s2orc | v3-fos-license | Film Reception by Means of New Media or How the Film Escaped from the Cinema
The biggest change in film reception occurred in the moment when the film audience began to watch films over the Internet, or with devices that can connect to it, i.e. computers, laptops and smartphones. The paper redefined notions of contemporary audience and different horizons of expectations, and a new horizon of expectations has been established, and that is the horizon of device expectations. Also, we have redefined the conative function i.e. the empty spaces function, individual and social reception of the film, while the theory of production relations in the case of the production of art has been applied to the film industry. The paper applies to the film received through social networks requirements of all characteristics of new media, which is also a novelty in scientific thinking.
Introduction
Motivated by a lack of attention that is given to the systematization of theory postulates of media reception in Croatia and its environment, and consequently with almost the total lack of their implementation, we decided to deal with the issue of the film reception in more detail, at the moment in time when the way in which the film can be accessed in terms of technology significantly changed.This significant change is reflected primarily in the mobile, Internet transmission of films and hence the film 'consumption' gets relocated from the controlled conditions of cinemas and homes and becomes available at any moment.This change is followed by the change of the audience, its characteristics, expectations and requirements that should also be considered from the aspect of the reception theory.As the reception theory has met with different interpretations over the years, we decided to check out contemporary events through the postulates of original theorists in order to find out if they are applicable in the new media challenges as well.We have opted for this approach due to the especially noticeable lack of scientific production in the environment related to the film reception, which is not surprising given that, unfortunately, scientists have transferred dealing with film onto professional levels that, when the film reception is in question, almost exclusively deal with how the audience received a particular film.
On the other hand, the world's scientific journals (mostly American and British) intensively deal with the problems of film, literature and other media's reception, although their systems do not draw a line between scientific and professional approaches, but still this professional approach is at much higher level than the commercially-semi-scientific approach which we are witnesses of in countries of the region.And although it served to us as preliminary research in the form of content analysis, in this paper we will use only small parts of the analysed corpus that can be considered somewhat deficient from the methodological point of view because it is too broad and not fully defined: the study included the search of online content i.e. scientific journals, which are allowed in free access and those that are available to the Croatian academic community in the temporary access (such as a collection of research and academic journals 'Oxford Journals' that was available until December 4, 2016).The main corpus of the paper will consist of the application of the basic concepts of the reception theory of its primary theorists (Jauss, Isser, Mandelkov, Marx and Nauman) to the medium of film, or to the reception of the film through the devices connected to the internet, while the paper will also indicate the possible ways of their redefining.A review of the global reception-theoretical production will constitute a smaller part of the paper.
The third motivational aspect is the expected functionality of the initiated researches and scientific work on the definition of legality of media reception shown and customized for the specific medium of film, or in the narrow sense for the film reception by means of information and communication technologies.This paper does not intend in any way to represent a new reception-film manifesto, but rather it tends to 'update' and adapt the existing standpoints to the contemporary film and social changes.The biggest change occurred in the moment when the film audience began to watch films over the Internet, or with devices that can connect to it, i.e. computers, laptops and smartphones.Therefore, this change is necessarily reflected on the film reception as well.
The hypothesis of this paper was that, through the darting changes brought by the advancement of information and communication technologies, which caused a reversal in the understanding and functioning of all forms of human communication, the biggest change was experienced by the audience which represents one of the basic concepts and approaches of the reception theory.The change of such an important factor places the whole reception theory in imbalance and comes to standstill, that is, it also includes the changes of other factors in a greater or lesser extent, and searches for redefining of the theoretical assumptions so that the balance can be established again.
Examples of the Application of Literary Reception Theory to the Film Medium
As we have said in the introduction, the application of the theory of literary reception to the medium of film is not by any means a new approach.We will show here only a few randomly selected examples from the extensive corpus dealing with similar issues, but they do not deal with the change in the reception influenced by ICT, which we consider our own contribution to science.
In their work, 'Narrative', Thomas Albrecht and Celine Surprenant give a book review by Karl Kroeber 1 stressing his study as extremely important for the portraying of the novels of the nineteenth century in the films of the twentieth century.'Kroeber's approach to this topic is a combination of a casual formalist analysis and what might be called reception theories/…//…/ (in this portion of his demonstration, he makes reference to classical reception theory, for instance to the work of Wolfgang Iser)./…/ Some of these points have been addressed more systematically, more rigorously, and in greater detail by current and past scholarship in narrative theory'.
In her work 'Film Theory', Lisa Trahair 2 also refers to contemporary research of reception theory on examples of actual, empirical viewers of the film with the assumed future, 'secondary' audiences and deferred meanings of the film text.'In these terms, cinema, like other arts and technologies, is not bound to the predictability of the future (le future) but the unpredictability of l'avenir, the time to come'.
In the work of the same name ('Film Theory') Lisabeth During and Deborah Levitt are doing a review of expert books on film 3 which recreate the historical approach to the film in very different ways, and in doing so, they move away from the so 1 "Make Believe in Film and Fiction: Visual vs. Verbal Storytelling is a study of considerable value to anyone interested in nineteenthcentury novels, in twentieth-century narrative cinema, and in the question of how novels and films tell stories differently.Kroeber's approach to this topic is a combination of a casual formalist analysis and what might be called reception theories.On the one hand, he discusses and compares passages from well-known European realist novels and sequences from classical Hollywood and postwar European narrative films in order to demonstrate how each constructs a narrative using very distinct techniques (his first chapter, for example, opens by suggestively juxtaposing the ax murder scene from Dostoevsky's Crime and Punishment with the killing of Marion Crane in Alfred Hitchcock's Psycho [1960])./…/ (in this portion of his demonstration, he makes reference to classical reception theory, for instance to the work of Wolfgang Iser)./…/ Some of these points have been addressed more systematically, more rigorously, and in greater detail by current and past scholarship in narrative theory, " (Downloaded from http://ywcct.oxfordjournals.org/at University of North on November 22, 2016) 2 :"Instead of countering the ideological conception of the cinematic spectator with the notion of real empirical viewers, as so many recent studies in reception theory do, the tropes of the ghost, hauntology, after-effects and deferred meaning make it possible to think of film texts as engaging with the alterity of future audiences as much as current ones.In these terms, cinema, like other arts and technologies, is not bound to the predictability of the future (le future) but the unpredictability of l'avenir, the time to come,"( Downloaded from http://ywcct.oxfordjournals.org/at University of North on November 22, 2016) 3 "Reviewed in this section are a set of books which, in rather different ways, reanimate the historical approach to cinema: they are not film histories perse, but they each take us further into the rich ground where history, culture, and the philosophic imagination meet, and far established models stemming from psychoanalysis, cultural studies, reception theory or cognitivism.It is exactly this emphasis on the move-away from, among others, the reception theory that proves to us to what extent the reception theory application in film studies used to be generally accepted.
Ernest Mathijs dealt with specific reception in his work 'Bad reputations: the reception of 'trash' cinema 1 .Mathijs believes that marketing and reception contexts, both historical and topical, play a crucial role in determining the public meaning of a film text and believes that the discourse of its reception actually starts long before it is received (which can be considered the epoch's horizon of expectations (author's comment).Such considerations are fully justified on the reception theory.
Further, similar considerations are discussed in the work 'Film history terminable and interminable: recovering the past in reception studies' by Barbara Klinger in which she states 2 that 'Reception studies scholars almost exclusively come to terms with a film's meaning by considering the impact that its original conditions had on its social significance.'Klinger points out the presence of contextual analyses which hope to reveal the impact of discursive and social situations on the film, and they examine a network of relationships between a film and filmic element with intertextual fields such as censorship, exhibition practices, star publicity and reviews, and the dominant or alternative ideologies of society at a particular time, that is, reception theories again.
As we can see, most of these works and studies (which represent only a small part of the researched sample which also has similar characteristics) quite competently apply the legality of the theory of literary reception to the medium of film, but in doing so, they mostly concentrate on the historical reception of the film.This approach, of course, cannot be applicable for this research on contemporary synchronic level, since the mobile reception of films is a new concept in the ways of showing and reception of the film and it is not possible in the diachronic analysis.
Redefining of Film Reception Legality
The film is as a mass medium, but with the existing and very present artistic component (in the paper we will not insist on the division of the film into artistic and commercial film, because the debate about it would take us in a completely different direction and would greatly complicate the planned development guidelines relating to redefining of exclusively reception terms) much more suitable for questioning of different horizons of expectations, which, we believe, have changed more than, for example literature, which still represents a largely individual experience.The film is, therefore, chosen as the primary investigated medium although the studied theories focused primarily on literature or on the written text.But the film, with its literary template in the basis, represents a kind of upgrade to the literature which is more approachable to the inform the appreciation of the cinema./…/"One of the tendencies in this rethinking is a move away from the models that have historically dominated theories of spectatorship, whether these emerge from psychoanalysis, cultural studies, reception theory, or cognitivism."(Downloaded from http://ywcct.oxfordjournals.org/at University of North on November 22, 2016) 1 "marketing and reception contexts, both historical and topical, play a crucial role in determining the public meaning of a film text and explaining the creation of reputation, hypes and controversy./…/the pursuit of information from the time of the film'sinception (which is an obvious influence on the film's further development) and its significance for the ways in which the film is eventually presented and received raise the theoretical problem that the discourse of its reception actually starts long before it is received./…/This provides a view of the complex pattern of influences and opinions that makes up a film's reception.(Downloaded from http://screen.oxfordjournals.org/at University of North on November 22, 2016) 2 "the object of literary analysis from the text to the intertext -the network of discourses, social institutions, and historical conditions surrounding a work -they helped inspire the development of historical reception studies in film.Those engaged in reception studies typically examine a network of relationships between a film or filmic element (such as a star), adjacent intertextual fields such as censorship, exhibition practices, star publicity and reviews, and the dominant or alternative ideologies of society at a particular time.Such contextual analysis hopes to reveal the intimate impact of discursive and social situations on cinematic meaning, while elaborating the particularities of cinema's existence under different historical regimes from the silent era to the present./…/ On occasion, reception studies focused on the industry fail to raise the question of how the industrial context connects to surrounding social and historical processes./…/ Reception studies scholars almost exclusively come to terms with a film's meaning by considering the impact that its original conditions had on its social significance.(Downloaded from http://screen.oxfordjournals.org/at University of North on November 22, 2016) nowadays audience than the book itself.In a culture of the visual and virtual 1 2 , which today more than ever is emphasized by the visual quality of all-pervasive Internet, the audience has even more pronounced demands and assumed changes must be more clearly shown in the visual media 3 , such as film, which is therefore, with the necessary respect for the obvious differences, fully adequate to show the reception transformations.
In the scientific literature this approach is not new, but his opponents appear frequently.Thus, for example, the professor Milivoj Solar in his 'Lectures on bad taste' points out that 'At the same time, the image accompanied by speech radically narrows the scope of imagination.The very fact that the image prevails over the speech in advance cancels what theorists call places of uncertainty.What you get on the screen does not give you anything to suggest further imagining and thinking, but you get a concrete form product that is in the consequences exclusively confined to affirmative suggestion' (Solar, 2004: 22.).If we accept such a position, and partly we have to agree with him, one more fundamental concept of the reception theory requires redefinition, and that is (according to Isser 4 ) the function of empty spaces.However, the coming of age of a new generation of film audiences, which is used to receive non-linear content, selecting by interest and scrolling content that they do not read, but scan, they fill empty places that were not selected in a completely new way.Because they are so used to it and have learned to access the media in that way, their particular approach will be applied to the chronological, non-interactive media by inserting empty spaces into sequences that did not predict such course of actions by extending their meaning and creating no more mass media hypnotic effect, but individual experience.In that way, the film will be a medium that will most satisfy their still existing artistic demands that social networks and television cannot meet.In such an approach conative structure of the film 5 is more prominent, so it is understandable that the film has a big impact on social issues and particularly on the actions of an individual.
By transferring the film through this relatively inadequate equipment, the quality of the recorded film will be much lower than on the cinema screen for which it was originally recorded.On the mostly small screens (let us even take into account cell phone screens) you simply cannot discern what would be seen in the cinema or even on television.Therefore, there are new empty spaces that the viewer has to complement by his/her imagination.This leads to an almost absurd situation that in fact the poor quality of the film reproduction leads to its greater artistic value!The filmmakers will probably never agree with this thesis (we are not sure if we ourselves can agree with it), but it certainly gives us food for thought.
When we talk about new ways/techniques of receiving/watching the film, we must be aware that the horizons of expectations have fundamentally changed.The very term horizon of expectations can thereby remain unchanged from the original Jauss's definition 6 , or the one by Charles Robert Mandelkov 7 .
Moving the film on the medium of the Internet (although thus the Pandora's box of copyright relating to almost all Internet content has been opened), the film has received another form of its appearance, or another transmission medium to the audience.In doing so, the individual reception is further influenced by social reception, but to a lesser extent because the online film viewer has the opportunity to, without the mediation of cinemas and distributors, choose a film on his own, but the selection will still probably be influenced by the film's advertising campaigns.However, low-budget films become available as well which owe their promotion almost exclusively to positive evaluation of the audience that evaluates and/or shares them with other users.Therefore, an individual viewer takes more than an active role in promoting a particular film that, in the above case, does not have to invest abundant resources in advertising campaign.Also, film criticism is reduced here to amateur comments, which are short and mostly unskilled, but the audience trusts them much more than the 'professional' ones whose authors, as the impression has been created, are often members of particular film interest lobbies.The possibility of evaluating, sharing, commenting and discussing the film through the Internet and social networks gives the film added value and meets the need of the audience for the immediacy and interactivity that is expected of all content on the Internet, and also of the film, which, in itself, does not have this feature.A new layer of expectations horizon appears here, and that is the horizon of device expectations.As the audience responded to the need that any content posted online must have an interactive content as well, the film industry has done the same.It already started with films on DVD, where they quickly responded to the online media's third request, and that is connectivity, and they offered a choice of content related to the film; shots from the filming, unlisted parts of the final film, uncensored parts, interviews with the cast and director, music videos with film music, link to an online store offering T-shirts, scarves, cups, figurines ... with characters and situations from the film which opens the possibility of creating additional indirect financial gain.
The fourth feature of online media, and that is archivism, will also come into focus while searching the films so that, after watching a particular film, the device 'itself' (using the 'cookies') offers similar films under the assumed affinity; director, actor, genre ... from the vast archive of films on the Internet.
The expectations of the audience, which is understandable, experience the most complete change.Since the film is received through computers, laptops and mobile phones (smartphones), the audience expects of the film similar stimuli and similar forms of presence as in other contents they receive through their gadgets.These expectations will also be linked to the resolution, design, photography and film syntax, and expectations will be extended to the form of film's narrative as well.Since a large number of viewers were raised on computer games, they will look for similar stimuli in the film, remaining less empathic for the fate of the characters, but also for the violence in the film, which, indeed, appears less brutal on a small screen than on the big screen.And although it seems fundamentally different, the requirements of the audience have been changing for centuries in similar direction: 'We do not state that the audience is more corrupt than before: it just has more experienced nerves, so it needs rougher and cruder stimulations.'(Ujević, 2004: 200.)Hereby, we come to another postulate of the reception theory, and this is a film audience.V. Žmegač wrote about the phenomenon of the audience, but of the literary audience, declaring: 'From the methodological point of view it is necessary to draw attention to the lack which is often accompanied by theoretical considerations about the reception (for example Jauss's considerations), and this lack is reflected in the generalized, abstract understanding of 'literary audience'/.../ The audience, as we conclude, is definable as a sociological and also literary-aesthetic category: in the literary process the reader is economic factor, and partly a selective one, but he is one of the unknown heroes of literature, anonymous or present, elusive figure whom the author either gives in and pleases or throws down a challenge to him.' (Žmegač, 1976: 71-73.)With the film audience this is even more prominent since one entire, very profitable industry, that is the film industry, is based precisely on the whims of the audience and it is very important, from the standpoint of profit, to assess what film audiences 'love' and what they do not like.Of course, just as the case is with literature, but also with the news media, this pandering to audiences usually goes towards loosening and adjustment and reduction of challenges, rather than its referral that is still present in the non-commercial art film.
Talking of the industry in the context of the reception, and it is more than necessary with the film, it is impossible to bypass the categories of production and consumption that are woven into the critique of political economy of Karl Marx the categories of production and consumption 1 , Marx also mentions the way of consumption which proved to be a special and new in the case of the film reception through ICT.Another theorist of receptions refers to Marx, and that is Manfred Naumann: 'If we do not forget its 'substantial difference', then 'the production of art' is nothing more than just a 'special form' of production, and hence the same provisions apply to it as the ones 'that apply to production in general.' (Nauman, 1978: 138).When we talk about the film industry, even the reception via the Internet, we cannot disagree with these two theorists from (before) the last century, even though, at first glance, it seems impossible and bizarre, primarily because of the exceptional time delay, but also all the connotations associated with Marx, especially in this region.
Conclusion
The darting changes brought by the progress of information and communication technologies, which caused a reversal in the understanding and functioning of all forms of human communication, the biggest change (with naturally the way or technology of receiving) has been experienced exactly by the audience.The assumed hypothesis at the beginning of this paper that the change of such an important factor, such as the audience, placed the whole reception theory in imbalance has been demonstrated in terms of the necessity of redefining certain settings.
The paper redefined notions of contemporary audience and different horizons of expectations, and a new horizon of expectations has been established, and that is the horizon of device expectations.Also, we have redefined the conative function i.e. the empty spaces function, individual and social reception of the film, while the theory of production relations in the case of the production of art has been applied to the film industry.The paper applies to the film received through social networks requirements of all characteristics of new media, which is also a novelty in scientific thinking.All contemporary changes were compared with root settings of reception theory, rather than with contemporary interpretations.
Bibliography
1 .Besides conceptually distinguished that determine the process of reception and influence it.Mandelkov called these foils the epoch's horizons of expectations, acts expectations and expectations of the author (according to Mandelkov, 1978: 120.)1Introduction to the Critique of Political Economy was created in 1857 and first published in the journal Die Neue Zeit, 1903, in its unfinished form. | 2018-12-14T23:43:34.619Z | 2017-01-01T00:00:00.000 | {
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8693469 | pes2o/s2orc | v3-fos-license | Lamivudine plus adefovir is a good option for chronic hepatitis B patients with viral relapse after cessation of lamivudine treatment
Aim Currently, there is no consensus on the retreatment recommendation of chronic hepatitis B (CHB) patients with viral rebound after cessation of treatment. In the search of reasonable treatment, we compared the efficacy and safety of adefovir (ADV) plus lamivudine (LAM) and LAM alone for the retreatment of patients with viral relapse but without genotypic resistance after cessation of LAM. Methods This is a prospective controlled study, and a total of 53 hepatitis B e antigen (HBeAg)-positive patients with viral rebound but without resistance were received either LAM plus ADV or LAM alone treatment. Results After 1-year treatment, more patients who received LAM plus ADV than those who received LAM alone had ALT normalization (84% versus 53.6%, P = 0.018) or HBV DNA levels below 1000 copies/mL (80% versus 42.9%, P < 0.006). Seven patients receiving LAM plus ADV had HBeAg seroconversion, as compared with 0 in patients receiving ALM alone (28% versus 0%, P = 0.003). During 1-year retreatment, five patients receiving LAM alone had virological breakthrough and all of them had LAM resistance strains (rtM204V/I), while no LAM- or ADV- associated resistance strains were detected in patients receiving LAM plus ADV. All patients receiving LAM plus ADV were well tolerated, and no serious side effects were noted. Conclusions Patients treated with LAM plus ADV exhibited significantly greater virological, biochemical and serological responses compared with LAM alone. These data suggested that combination of LAM plus ADV would be a good option for the retreatment of CHB patients with viral relapse after cessation of LAM.
Background
Estimated 350~400 million individuals worldwide are chronically infected with hepatitis B virus (HBV) [1], and chronic hepatitis B(CHB) can progress to cirrhosis, hepatocellular carcinoma (HCC), and death [2]. Antiviral therapy is used in CHB to minimize the liver damage and progression of disease [3], but total eradication of the HBV is seldom achieved by current treatment, especially for patients who infected HBV in early childhood.
Nucleos(t)ide analogs approved for treatment of CHB include lamivudine (LAM), adefovir (ADV), entecavir, telbivudine and tenofovir, and they have been considered to have a good effect in suppressing virus replication with few side effects [4]. In general, HBeAg seroconversion is an important end point for HBeAg-positive patients, which is associated with sustained remission [5,6]; and international guidelines suggested that early withdrawal of antiviral therapy, without reaching sustainable serological response, would lead to a very high risk of relapse [4]. In fact, a wealth of clinical experience also suggested the problem of viral relapse after drug withdrawal could not be ignored [7][8][9]. Some investigators had reported that viral load rebound is common in CHB patients receiving nucleos(t) ide analogs and the relapse rates would be high to 70% [8,10,11]. However, nearly 40% of the relapses or virological breakthroughs were not related to antiviral drug resistance. For example, due to the poverty, many CHB patients in Asia cannot afford their long-term medication, and they always terminated their treatment just when reaching virological response irrespective of the occurrence of HBeAg seroconversion. How to therapy those patients with viral relapse has become an urgent problem that we have to face [12]. Unfortunately, there is no consensus on the retreatment of viral relapse with or without resistance after cessation of nucleos(t)ide analogs treatment. And it is still unknown whether there is significant difference between original drug monotherapy or combined with other drugs without cross-resistance.
Current study was designed to compare the efficacy and safety of LAM plus ADV with that of LAM alone again in CHB patients with viral relapse after cessation of previous LAM treatment.
Characteristics of the study patients
A total of 53 HBeAg-positive CHB patients were analyzed in the study, which comprised of 25 patients in LAM plus ADV and 28 patients in LAM alone retreatment ( Figure 1). The demographic and disease parameters were well matched at baseline of retreatment between two groups, which were described in detail in table 1.
Biochemical response
Serum ALT levels declined in both treatment groups, but ALT level was normalized in a higher proportion of LAM plus ADV than LAM alone retreatment in total. At 6 and 12 months after retreatment, normal ALT was achieved in 76% (19/25) and 84% (21/25) of patients receiving LAM plus ADV, and in 35.7%(10/28) and 53.6% (15/28) of patients receiving LAM alone, respectively ( Figure 2). The difference in ALT normalization between two groups was significant at year 1(84% vs 53.6%, p = 0.018).
Virological response
The reduction in serum HBV DNA levels from baseline of retreatment during the first year of observation period was greater in LAM plus ADV than in LAM alone retreatment ( Figure 3). At 6 months, the proportion of patients with virological response (HBV DNA < 3 log copies/ml) was achieved in 60% (15/25) of patients receiving LAM plus ADV retreatment as compared to 32.1% (9/28) of patients receiving LAM alone. At 12 months, the proportion of patients with virological response was achieved in 80% (20/25) of patients receiving LAM plus ADV as compared to 42.9% (12/28) of patients receiving LAM alone. The difference in virological response between two groups was statistic significant at 6 and12 months, respectively (P = 0.042 for month 6 and P = 0.006 for month 12).
HBeAg response
More patients receiving LAM plus ADV retreatment had HBeAg seroconversion as compared to patients receiving LAM alone retreatment at month 12 ( Figure 4), and the difference in HBeAg seroconversion between two groups was significant (7/25 versus 0/28, P = 0.003). Additionally, significant more patients receiving LAM plus ADV obtained HBeAg loss as compared to patients receiving LAM alone at month 12(9/25 versus 1/28, P = 0.004) ( Figure 4).
Breakthrough and resistance
Viral breakthrough was found in 5 patients receiving LAM alone retreatment during the 1-year observation period, and the mutations of rtM204V were found in 3 patients and rtM204I were found in 2 patients at year 1 of retreatment. In LAM plus ADV retreatment patients, no viral breakthrough were found and no LAM or ADV associated mutations were detected.
Safety
Majority of patients were well tolerated in two retreatment groups. Among patients receiving LAM plus ADV, only1 patient had serum creatinine slightly increasing but there was no discontinuation due to this adverse event. Elevations in ALT were observed less frequently in LAM plus ADV group than LAM alone in group. ALT flares during the first year of treatment were in 5 patients with LAM alone and 1 patient with LAM plus ADV. In LAM plus ADV group, all the ALT flares were associated with toil; while in LAM alone retreatment group, all the ALT flares were associated with HBV DNA flares. Between two groups, none hepatocellular carcinoma was reported.
Discussion
Although LAM is not recommended as first line therapy for CHB in the American Association for Study of Liver Disease (AASLD) CHB guidelines [4], and the European Association for Study of Liver (EASL) CHB guidelines [13], which recommend oral nucleosides or nucleotides with high genetic barrier (entecavir and tenofovir) as first line monotherapy, LAM is still widely used in Asia due to cost constraints [14]. Thus, it is necessary for us to concern about the re-treatment issues of patients with viral relapse after discontinuation of LAM monotherapy. Unfortunately, the retreatment options for viral relapse are still in doubt. In clinical practice, a variety of retreatment strategies have been applied, including back to their original drug, switch to another drugs, or two or more drugs in combination. Theoretically, if there were no evidence of antiviral resistance of original agent, it seemed reasonable to back to their original drug for retreatment. However, this hypothesis was recently questioned by clinicians. Evidence already had showed that HBV exists in form of quasi-species in patients with chronic hepatitis B [15], and because of the sensitivity and specificity limitation of detection, drug-resistance strains sometimes could not be detected in time, especially when its proportion was less than 20% in the pool of viral quasi-species [16,17]. Thus, it is not difficult to conclude that before the drug-resistance strains reach a high number, they may not be detected in patients with viral relapse after cessation of antiviral treatment [16]. But it is worth mentioning that those inferior strains would be easily developed to predominant strains with antiviral resistance by positive selection of antiviral agents [18,19]. Taking into account of the above reasons, when viral relapse occurred after cessation of treatment, switch to agents with high genetic barrier or combination of two or more agents without cross-resistance may be good options for preventing the occurrence of drug resistance [9,20].
In this study, we explored and compared retreatment options for chronic hepatitis B patients with viral relapse after cessation of LAM. After 1 year retreatment, the proportion of undetectable HBV DNA was achieved in 80% of patients receiving LAM plus ADV as compared to 42.9% of patients receiving LAM alone. Moreover, higher proportion of ALT normalization and HBeAg seroconversion were also reached in LAM plus ADV retreatment group. Several other studies also had reported that the combination of LAM and ADV could lead to effective viral suppression in most cases after development of viral breakthrough due to LAM monotherapy [21,22]; and patients receiving LAM plus ADV combination therapy have a lower risk of developing genotypic resistance to ADV [22]. In our study, patients receiving combination therapy of LAM and ADV were well tolerated, and no viral breakthrough was reported and no LAM-or ADV-associated resistant strains were detected. In contrast, among 28 patients receiving LAM alone, five patients experienced a viral breakthrough and LAM-associated resistant strains were detected in all of them. Those findings gave us another hint that the combination of LAM plus ADV was associated with lower antiviral resistance compared with LAM alone. In fact, for some special populations with chronic hepatitis B, the combination of agents without cross-resistance had been clearly put forward by authoritative guidelines [4,13], and data from many clinical observational trials also suggested combination therapy would bring more benefits to nucleosides or nucleotides-refractory CHB patients.
At present, majority of clinical studies on LAM plus ADV combination therapy were for HBeAg-negative patients, and few studies on HBeAg-positive patients reported the combination of these agents had no synergistic on HBeAg seroconversion. But in our study, among 25 patients receiving combination therapy of LAM plus ADV, we delighted to found that 9 patients obtained HBeAg loss and 7 patients obtained HBeAg seroconversion, which were significant higher to that of patients receiving LAM alone. However, because of the limited sample size and short observation time, we currently cannot conclude that the combination of ADV plus LAM could increase the rate of HBeAg seroconversion as compared to single nucleoside or nucleotide analogue.
In conclusion, the current study showed that CHB patients with viral relapse after cessation of LAM retreated by LAM plus ADV exhibited significantly greater virological, biochemical and serological responses compared with LAM alone. These findings indicated that combination of LAM plus ADV would be a good option for the retreatment of CHB patients with viral relapse after cessation of LAM.
Patients
Outpatients from Chengdu Military General Hospital, with viral relapse after cessation of LAM treatment were screened, of who included in this study should also meet the following criteria: serum hepatitis B e antigen (HBeAg) positive; serum HBV DNA load above 1000 copies/ml; alanine aminotransferase (ALT) levels between 2 and 10 times the upper normal level. And patients who fulfilled one of the following criteria should be excluded: evidence of LAM associated resistance (rtM204V or rtM204I); presence of serum antibodies against hepatitis C virus (HCV), or human immunodeficiency virus (HIV); breast-feeding, pregnancy or inadequate contraceptive measures; other acquired or inherited causes of liver disease; coexisting serious medical disease; advanced liver disease(including decompensated cirrhosis with ascites, severe hepatitis, and hepatic carcinoma).
Study design
This is a prospective controlled study, aimed to evaluate and compare the efficacy of LAM plus ADV with that of LAM alone retreatment in patients with viral relapse after cessation of LAM antiviral therapy. All included patients were administered 100 mg of LAM plus 10 mg of ADV (GlaxoSmithKline) or 100 mg of LAM alone daily according to patients' choice, and they were followed up in outpatient clinic of hospital. Clinical data were collected at baseline, and every 3 months after retreatment. The primary efficacy outcomes were ALT normalization, reduction in HBV DNA, and seroconversion of HBeAg. Second efficacy outcome was antiviral resistance.
This study was approved by Chengdu Military General Hospital's institutional review board and was conducted in accordance with the 1975 Declaration of Helsinki.
Serum assay methodology
HBeAg and serum HBV DNA were measured with the use of Enzyme-Linked Immunosorbent Assay (InTec Technology Co., Ltd., Xiamen, China) and real-time PCR-based quantification assays(DA AN gene Co., Ltd., Guangzhou, China) according to the manufacturer's instructions, respectively. Serum ALT and Creatinine (Cr) were measured using automatic biochemistry analyzer according to standard laboratory procedures. Antiviral resistance was analyzed using PCR sequencing assay if HBV DNA no less than 1000 copies/mL after 1-year antiviral treatment.
Definition
Biochemical response was defined as a decrease in serum ALT to within the normal range. Virological response was defined as a decrease in serum HBV DNA to undetectable levels by PCR assays (<1000 copies/mL). HBeAg response was defined as a loss or seroconversion of HBeAg in patients who were initially HBeAg positive. Virological breakthrough was defined as an increase in serum HBV DNA by 1 log10 (10-fold) above nadir, or to detectable level (≥ 1000 copies/mL) after achieving virological response during retreatment. Viral relapse was defined as an increase in serum HBV DNA to detectable level after achieving virological response.
Statistical analysis
Quantitative variables were expressed as mean, and categorical variables were presented as counts and percentages, and HBV DNA levels were presented as log transformation. Comparisons between groups of quantitative and qualitative variables were performed using the student t test and Chi-square test (or Fisher's exact test), respectively. A P-value of less than 0.05 (two-tailed) was considered to indicate a significant difference. All statistical analyses were performed using the SPSS software package version 13.0 (SPSS Inc., Chicago, IL). | 2017-08-03T02:11:54.593Z | 2011-08-04T00:00:00.000 | {
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58960419 | pes2o/s2orc | v3-fos-license | Understanding Roles and Responsibilities of Data Curators : An International Perspective
Data curation has emerged as a new area of responsibility for researchers, librarians, and information professionals in the digital library environment. This paper presents the preliminary findings of a large research project sponsored by the International Federation of Library Associations (IFLA), under the auspices of its Library Theory and Research (LTR) Section. The primary objective of the project is to identify the characteristic tasks and responsibilities of data curators in both international and interdisciplinary contexts. The ultimate objective, however, is to develop a “data curation ontology” that will better define the profession and support the development of educational curricula to train future data curators.
Introduction
The variety and sheer volume of research data that must be processed, preserved, and made available to the scholarly community and public at large is creating new challenges, both technical and theoretical, for researchers and the librarians and information professionals who support them. In the past few years, national funding agencies have begun to require researchers to prepare data management plans and to make data sets available in open access repositories. In addition, the demands for open data publishing are coming from scientific publishers. The concept of data curation has roots in the management of scientific information, but its usage has branched out to other disciplines, including digital humanities.
The IFLA Section Library Theory and Research (LTR) international team has conducted preliminary research using multiple data collection techniques, including literature review, questionnaires, in-depth interviews with professional data curators, as well as content analysis of data curation job announcements.
The research questions addressed in this study are: • How is data curation currently defined by its practitioners working in the field?
• What specific professional vocabularies are used to describe the data curator's core competencies?
• What are the data curator's primary roles and responsibilities?
• What are the key educational qualifications and areas of expertise required of successful data curators?
• How do these vocabularies, roles, skills, and responsibilities vary by country and region around the world?
First, the team has conducted a broad, comprehensive, and systematic survey of the literature related to the area of digital curation. Next, a subgroup conducted content analysis of job descriptions, disseminated a questionnaire, and conducted interviews with data curators working in the field. The first workshop during BOBCATSSS 2016 conference presented the initial findings and was conducted with the active participation of the audience. Since then the scope of the research project has expanded and more data has been collected using a combination of quantitative and qualitative data collection techniques.
The preliminary findings of this study provide a starting point for what we hope will be an in-depth description of the roles and responsibilities of data curators.
Terminology Data curation has emerged as a new area of responsibility for researchers, librarians, and information professionals in the digital library environment (Heidorn 2011, Witt. 2008. In a 2011 article published in the International Journal of Digital Curation, Higgins (2011) proposes that digital curation has made great strides in establishing itself as a new discipline. However, identifying digital curation as a newly emerged discipline may be premature. Although there is a visible growth in educational programs, it is still unclear how the education for this knowledge and skills set fits within the educational landscape. Digital curation is instead mostly embedded in practice.
A number of alternative vocabularies have been deployed to describe the same or similar practices, reflecting the diverse environments in which data is now archived, as well as an evolving understanding of what the practice means in the field.
An exhaustive list of search terms was used to conduct the literature review: • data curator Terms such as digital curation, digital archiving, or digital preservation are often used interchangeably (Beagrie 2008). The term digital curation is increasingly being used for the actions needed to add value to and maintain these digital assets over time for current and future generations of users. Digital curation refers to the actions people take to maintain and add value to digital information over its lifecycle, including the processes used when creating digital content. Digital preservation focuses on the series of managed activities necessary to ensure continued access to digital materials for as long as necessary (Walters and Skinner 2011).
Diverse vocabularies are also invoked to describe the various roles for professionals working with organizing, managing, preserving, and disseminating data. Moreover, there are important differences in understanding and usage of basic data curation terminology among countries and regions around the world.
Multidisciplinary engagement
The digital curation is an area of inter-disciplinary research and practice, and different disciplinary trends are influencing its development (Beagrie 2008). The traditional roles in library and information science (LIS) relating to work with digital assets are in transition. Information professionals don't only require skills and knowledge from the LIS field, but also skills in collaboration and professional approach to interdisciplinary work.
Poole (2013) considers and evaluates digital curation work undertaken in the sciences and in the humanities. In theory and in practice digital curation has benefited substantially from practices developed and tested first in the natural sciences and subsequently adapted for and extended in the humanities. The roles of libraries and data centres are not easy to define. Traditionally positioned at opposite ends of the research lifecycle, the convergence of data and publications and interdependencies between both has modified this traditional scope of duties. Both libraries and data centres are in a transition process.
Education and training
There are two Frameworks providing a common language and helping to define the skills, knowledge and abilities that are necessary for the development of digital curation training and for promoting the continuing production, improvement and refinement of digital curation training programmes.
The DigCCurr (Digital Curation Curriculum) project (Lee, Tibbo, and Schaefer 2007) has developed a graduate level curricular framework, course modules, and experiential components to prepare students for digital curation in various environments.
In 2013, the DigCurV collaborative network (Molloy et al. 2014) completed the development of a Curriculum Framework for digital curation skills in the European cultural heritage sector. DigCurV synthesised a variety of established skills and competence models in the digital curation and LIS sectors with expertise from digital curation professionals, in order to develop a new Curriculum Framework.
Librarians need opportunities to learn more about these services either on campus or through attendance at workshops and professional conferences (Tenopir et al. 2014).
Preliminary findings
The role of data curator has become an important information management responsibility worldwide, stimulated by the growing need to organize and preserve large volumes of data generated by scholars, governments, and research institutions. A number of research studies have examined the roles of librarians and information professionals in research data management, but have focused primarily on U.S. libraries and research institutions (Akers et al. 2014, Kim, Warga and Moen 2012, 2013, Palmer et al. 2014). Our study builds upon this prior research and expands it by providing an inclusive international perspective.
The empirical part of this study included two phases of data collection and was designed using a mixed-method strategy. The first phase focused on quantitative content analysis of job announcements derived from a variety of library and information science job posting sites, including American Library Association (ALA) Job List, International Association for Social Science Information Services and Technology (IASSIST), and Code4Lib. IASSIST and Code4Lib provided international coverage and were the main sources of data. In the second phase of the study, interviews were conducted with data curators working at academic libraries and research centres. The following section presents the initial findings from the two phases of the study completed as of May 2016. As more data collection activities are scheduled for summer 2016, all results reported here are preliminary.
Phase I: Content analysis of job announcements
Phase one of the IFLA Data Curator research project consisted of a quantitative content analysis of job listings for data curators and related positions. The source data for this analysis were extracted from multiple sources, with a majority (more than 98%) scraped from the Code4Lib and IASSIST websites. The initial data set included 6051 job announcements. From there the database was winnowed to 441 data curator positions using both automated and hand coding, with 54% classified as composed of "primary" data curation responsibilities and 46% as "secondary". This final database is preliminary in nature as in likely to change as our research deepens.
Because of their source these results showed a selection bias towards positions located in the United States, though 34 countries in all were included in the database, and 12 countries were represented among the positions coded as data curators.
Perhaps the most interesting result of the analysis was a comparison of data curator and non-data curator positions by their location. Only 11.9% of non-data curator job listings were located in countries outside of the United States. Yet 26.4% of data curator jobs (including both "primary" and "secondary" positions) were located in other countries. These data suggest that data curation, as well as the data curator job market, is a more international practice than other kinds of work in the library and information science sector.
Other findings from the analysis show that the title "Data Curator" is a poor indicator of data curation responsibilities. There were 280 unique titles across the 441 positions coded as "data curator. " No single title covered more than 3.5% of the total jobs. The most common titles were "Data Librarian", "Data Services Librarian", "Data Curator", and "Digital Scholarship Librarian". The prevalence of "librarian" in these titles indicates that typical responsibilities of librarianship, including reference, instruction, and outreach, are common for data curators as well.
Among the types of organizations employing data curators, universities (and university departments) were by far the most common (74.6%), which possibly reflects the North American bias in the source dataset. After universities, university libraries were second most frequent (9.8%), followed by research centres (6.1%) and government agencies (4.8%).
Phase II: Interviews with data curators
The second phase of the study provided an opportunity to gain insight into the practice of data curation and look at the roles and responsibilities from the perspective of information professionals working in the field. Interviews were a primary source of data and were supplemented by questionnaires and documentary evidence. Interviews followed a semi-structured protocol and focused on the participant's' job functions, work activities, distribution of responsibilities, and skills and competencies. Participants were recruited from academic libraries, research centres, and data curation centres in Australia, Canada, and the United States. Convenience and snowball sampling were used in the recruitment. Nine participants were interviewed as of May 2016. The interviews were conducted over Skype or phone and lasted between 30 and 45 minutes. All interviews were recorded and transcribed. The research team plans to conduct additional interviews in summer 2016.
The participants recruited for the study held different position titles, including coordinator of data curation and scholarly communications, data curation librarian, data librarian, data curation scientist, digital curation coordinator, e-research project officer, project scientist, research data management librarian, and research services coordinator. A phrase "data curation" and a word "librarian" appear in several titles. However, all titles are unique, which confirms the findings from the first phase about a wide variety of titles for positions with data curation responsibilities. Six participants in the sample worked at the university libraries, one at the research centre, one at the university department, and one at the newly funded, campus-wide research services unit. The participants came from a wide variety of backgrounds with Bachelor education in computer science, engineering, health sciences, and history. All participants held Masters in library and information science (MLIS) and three had PhDs.
The importance of outreach and training responsibilities emerged as a pattern in all interviews, regardless of the disciplinary or international contexts. A number of participants discussed a mismatch between perceptions of data curation and their actual work activities. As it turned out, most professionals interviewed for this project have not been involved in managing data directly. Instead, their efforts have been focused on outreach, consultation, and training of researchers. As indicated by Participant E, "data curation is more about providing information about good data curation practices to the people who need to curate their data or could be curating data. " While more time is devoted to reaching out to researchers in the early stages of data curation programs, outreach remains a key responsibility for data curators working for more than 2 years. Outreach efforts are focused on: • Informing researchers about new data curation services available their institutions • Educating about data life cycle and good data management practices • Promoting open access and data sharing through data repositories • Consulting on metadata standards, data formats, and citation standards.
Data curators are involved in teaching workshops and providing one-onone consultations to faculty and graduate students. Workshops cover a wide spectrum of topics from data management plans to data citation and sharing. In addition to expertise in data management practices and standards, data curators need to have good communication and instruction skills to deliver effective presentations. Although data curators may not work directly with research data, their work requires some technical skills and knowledge of new technological solutions since they often make recommendations to researchers and lead data curation initiatives at their institutions.
Data curation, or perhaps more appropriately research data services, emerges as a new specialty that combines technical and public services skills. As one of the study participants emphasizes, "It's almost like multiple jobs in one really. I'm a technical services librarian, as well as an outreach librarian" (Participant C). The emerging specialty requires expertise in multiple areas of data creation, organization, publishing, and preservation. It also involves collaboration -often a cross-campus collaboration -and building bridges between a library, IT unit, campus departments, or specialized centres. In a traditional library environment, technical and public services librarians tend to work in separate departments and have been educated using different tracks in LIS programs. Data curation as a new area of responsibility for librarians and information professionals requires a combination of technical, reference, and instruction skills and poses new challenges to the development of LIS curricula.
Conclusion
The IFLA Data Curator research project is a work in progress. Moving forward, we hope to find additional sources for non-North American data curator positions, engage a natural language analysis of the entire position database, conduct additional interviews, and perform cross-analysis of job descriptions, questionnaires, and interview data. The ultimate goal of the project is to identify key responsibilities of data curators and to develop a glossary that should help to better define the profession and develop appropriate educational curricula. The preliminary findings of this research study indicate that different terms are used to describe various roles for professionals working with organizing, managing, and preserving data. Data curation is an emergent field and precise vocabulary has yet to mature. | 2017-05-19T16:48:10.399Z | 2017-03-02T00:00:00.000 | {
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237250692 | pes2o/s2orc | v3-fos-license | Interrogating Strategies of Justice and Racial Politics: A Post- colonial Reading of Abir Mukherjee’s A Rising Man
The present article begins with a brief historical account of the exclusionary politics of Western crime fiction, with most of the works representing the East as ‘exotic other’ while assuming the subject position themselves. A post-colonial analysis of Abir Mukherjee’s A Rising Man (2016) is conducted to study how the novel deals with questions of justice and racial politics, and further encompasses a brief inquiry into it can be positioned as an anti-colonial text which advocates a move towards decolonization. The text can be seen as representing the body of work by writers who give voice to the oppressed within colonial contexts and vehemently refuse the idea of being inferior.
cultural powers that upheld the colonial dominance and that which prevails even after political freedom is attained. It operates as a counter-discourse that gives voice to the indigenous people and helps them discover their misplaced subjectivity (Sharma et al., 2015, p. 11). According to Prasenjit Duara, the term embodies not only the transference of legal jurisdiction but also an undertaking towards "moral justice and political solidarity against imperialism" (Duara, in Gunde 1994).
Post-colonial crime fiction refers to crime novels which examine the crimes of colonialism and neo-colonialism; it includes "the perspectives and values of previously colonized subjects, whilst utilizing a literary aesthetic and epistemology which are modified by a specific postcolonial context" (Naidu, 2018, p. 83). Crime fiction produced in the West has an extensive history of representing Eastern nations as the 'exotic other' while taking up the subject position themselves. This is particularly evident in the representation of violence in some of these works; more often than not, the element of violence is increasingly associated with the Orient. Even when Oriental malefactors are absent within works such as Arthur Conan Doyle's Sherlock Holmes series, "spaces like South America, Africa and India are synecdochically represented through strangely horrific indigenous poisons (or even a poisonous snake), all of which are used in murders within England" (Roy, 2020, p. 121). As stated by Maureen T. Reddy, early works of hard-boiled crime 1 fiction of the 1920s and 1930s forged a cultural image of the sleuth which presented whiteness, heterosexuality and conventional masculinity as the normative standards of the genre (2003, p. 7). However, over the past couple of decades, several white (most often male) writers have centered their fictional crime narratives on Asian protagonists but more often than not they have fallen into the same Orientalist trap of accentuating numerous stereotypes. Some of these writers and their Asian protagonists include Eliot Pattison and his Beijing Justice Department investigator Shan Tao Yun; John Burdett and his Royal Thai police force detective Sonchai Jitpleecheep; and Colin Cotterill and his French-trained physician Dr. Siri Paiboun (Makhijani, 2017). These contemporary writers of crime fiction have been condemned for representing imported and exoticized perspectives, which lack authenticity, through their writings. In short, their representations of the Asian settings were considered artificial and lacking credibility by some critics. For instance, even though Biggers claimed to have created his protagonist in reaction to anti-Chinese stereotypes that he witnessed in California, his fictional representation speaks otherwise. Chan's character embodies a mixture of stereotypes -he is "a portly and effeminate man who speaks faux-Confucian aphorisms" (Makhijani, 2017). In Charlie Chan: The Untold Story of the Honorable Detective and His Rendezvous with American History (2010), Yunte Huang posits, "Make no mistake: Charlie Chan is an American stereotype of the Chinaman. Anyone who believes that Chan is Chinese would probably also believe that the fortune cookie is a Chinese invention" (p. 19). While deliberating on John Burdett's novels, Laotian American writer Bryan Thao Worra rebukes that Jitpleecheep's mysteries are always set in or adjacent to Thailand's red-light districts and portray "lots of exotic deaths and kinky eroticism" (in Makhijani, 2017).
The notion of the foreign and the post-colonial have long existed in the area of crime and detective fiction, with works set in the colonies having a long and complex history within the British tradition (Morgan, 2020, p.3). Prabhat K. Singh calls attention to the rise of genre fiction in English in India and urges the readers to recognize the significance of genre fiction (2013, p.8). The development of crime fiction in India can be categorized into three stages, and they correspond to the phases in the cultural trajectory of the colonized intellectual as outlined by Frantz Fanon in his seminal work The Wretched of the Earth (1965, p. 222). The first stage is one of mimicry where the colonized mimic the colonizer and conforms to the latter's desires. In other words, the colonized attempts to be like the Europeans, assimilating the various constituents that comprise Western culture. This is the stage where writers of crime fiction in India wrote fiction that was more or less exact replicas of their Anglo-American varieties. The local fictional detectives like Sharadindu Bandyopadhyay's Byomkesh Bakshi, Asrar Ahmad's (popularly known as Ibn-e-Safi) Ali Imran and Kalpana Swaminathan's more contemporary Lalli were modelled on Sherlock Holmes, Nick Carter and Miss Marple respectively. The second stage is characterized by the colonized revolting against the colonizer. Parallel to this lies the Indian writers' attempt to adapt the genre and make minor changes to it, even though a torrent of Western elements were still retained as part of colonial hangover. The final phase is the one where the colonized finally embrace its culture and contest for emancipation from the colonial regime. They write "a fighting literature, a revolutionary literature and a national literature" (p. 223) in the hopes of discovering their own true identity and subjectivity in the process. This is the phase where writers produced post-colonial metafiction, "a narrative mode that accommodates the self-questioning ambiance of the postmodern and the politicised stance of the post-colonial" (Gamal, 2011, p. 1). According to Gamal, metafiction operates as a postcolonial tool of rewriting and hence recuperating the history of the colonized (p. 1). In the arena of Indian crime fiction, while most of the earlier works tried to emulate Western works without much experimentation or representation, the modern writers have taken up the task of redefining the genre by incorporating the innate 'Indian-ness' into their works. Indian culture is more heterogeneous in comparison to its Western counterpart and this heterogeneity adds to its diversity. This cultural diversity leads to a thematic diversity as well as diversity in terms of characterization, and consequently, the cultural imageries in the Indian detective fiction are more vibrant and dynamic.
One of the significant developments that can be seen when it comes to the arena of Indian crime fiction is the multifarious collection of historical works with colonial or pre-colonial settings. Abir Mukherjee's Sam Wyndham series, Sujata Massey's The Widows of Malabar Hill (2018, also published as A Murder on Malabar Hill), Arjun Raj Gaind's A Very Pukka Murder (2016), and Madhulika Liddle's Muzaffar Jang series are some works that belong to this category. Such hybridization of genres reflects a "postmodern tendency towards rejecting boundaries between (socalled) high and low art forms and blurring, when not altogether dissolving, generic distinctions" (Humann, 2020, p. 57). In crime fiction, genre hybridization opens up novel avenues that turn it into a more appropriate medium for challenging existing social conventions, raising awareness about global issues and critiquing existing socio-political constructs (p. 59).
Abir Mukherjee's debut novel, A Rising Man was first published in 2016 and was shortlisted for both the Crime Writers' Association (CWA) Gold and CWA Historical Daggers (which he eventually won), the Historical Writers' Association Debut Crown in 2017, and the Mystery Writers of America (MWA) Edgar Award for Best Novel in 2017 (Majumdar, 2018). Perhaps what makes his writing stand out from other crime narratives that deal with similar themes is the fact that even though his writing gets political; it turns away from being polemical. The representation of postcolonial elements in the novel is subtle, balanced and does not interfere with the central plot that involves the murder investigation of a British official. According to Shampa Roy, the investigation of crimes in detective novels entailing post-colonial elements is invariably tied to examining the crimes of colonial regimes and what has followed in their wake from the perspectives of previously colonized subjects (2020, p.120). The protagonists of such novels serve as a medium for the transmission of the author's response to colonialism and its aftermath. What makes Mukherjee's writing particularly interesting is the unbiased way in which he can account for the events concerning the colonial rule in India. Born to Indian parents who immigrated from Calcutta and having spent most of his life in West Scotland (currently lives in London), Mukherjee takes up a dual identity using which he represents the standpoints of both the nations in a neutral, unprejudiced manner. Mukherjee's fascination with the tartan noir, a popular form of noir in Scotland, is evident in his representation of the binaries of good and evil and social commentary in A Rising Man. In an interview with Samhita Chakraborty, he reveals his fondness for tartan noir and how he has always been captivated by the idea of representing a good man upholding a bad system in which he does not believe (2019). This is exactly what we get to see in the novel under study -both the main characters, Captain Sam Wyndham, former Scotland Yard detective and a World War I veteran working for the Calcutta Police under the British rule, and Sergeant Surendranath Banerjee of the Calcutta Police, represent the same dilemma. The present article entails a post-colonial analysis of Abir Mukherjee's A Rising Man (2016) to study how the novel deals with questions of justice and racial politics, and it further encompasses a brief inquiry into how the novel positions itself as an anti-colonial text and advocates a move towards decolonization. The text can be seen as representing the body of work by writers that give voice to the oppressed within colonial contexts and categorically refuse the idea likening being colonized to being inferior, thereby situating themselves within a body of works with a similar agenda of freeing minds from colonial, Eurocentric ideology.
The novel begins with the corpse of an Englishman being discovered in an alley located within a shady part of Calcutta. Inspector Sam Wyndham, a World War I veteran and a former Scotland Yard detective, is assigned to investigate the case. The year is 1919 and Sam being new to the city means that he has to seek the help of his colleagues -Sergeant Digby and Sergeant Surendranath Banerjee -to navigate the city and better understand its workings. Digby is an arrogant Englishman who embodies all the abominable attitudes of imperial authority and holds a personal grudge against Sam for landing the job he believes should be his own. Surendranath -or "Surrender-Not" Banerjee as he is often referred to by the Brits as they find the former too hard to pronounce -is an Oxford-educated Indian from a prosperous family. There is a note written in Bengali stuffed into the corpse's mouth which translates to: "No more warnings. English blood will run in the streets. Quit India!" (Mukherjee, p. 6). The victim is soon identified as Alexander MacAulay, an aide and fixer to the Lieutenant-Governor, one of the many officers working under the colonial government. Since MacAulay holds a prominent position in the empire, his murder is suspected of having a political angle to it and the initial speculation point towards an attack carried out by 'terrorists' seeking independence. This incident is what drives the plot of the novel and the remaining portion of the text deals with the investigation of MacAulay's murder.
Throughout the novel, Mukherjee examines the compelling question of the moral legitimacy of the colonial regime. While making enquiries about MacAulay's murder, Wyndham and Banerjee come across a sign etched on the gates of The Bengal Club that reads, "No dogs or Indians beyond the point" (p. 82). Seeing Wyndham's discomfort, Banerjee says, "Don't worry, sir. We Indians know our place. Besides, the British have achieved certain things in a hundred and fifty years that our civilisation didn't in over four thousand […] We never managed to teach the dogs to read" (p. 82). Here, Abir Mukherjee makes use of Banerjee's character to condemn the colonial administration which restricted Indians from entering various spaces within their own country.
Charles J. Rzepa and Lee Horsley, in their introduction to A Companion to Crime Fiction (2010) states that "detective fiction has remained a resilient and versatile genre because […] it represents the investigation of individual crimes but can also work to expose the failures, traumas and brutalities of political and social life" (p. 1). Ed Christian too reiterates this idea when he posits that "as a genre, detective fiction often moves from the interrogation of suspects to the interrogation of society" (2010, p. 284). In post-colonial crime fiction, the detective fights crime on two levels simultaneously -besides solving the immediate, obvious crime, they also deal with the 'larger' crime of colonialism and its aftereffects on the colonies. These writings question the very definition of 'crime' and emend it to incorporate the "violations and abuses of colonialism, slavery, [and] genocide […]" (Naidu, 2020, p. 114). In Mukherjee's novel, the immediate crime is MacAulay's murder and the train robbery which is solved at the end of the novel; however, the greater crime of paramount importance brought about by the colonial hegemonic system remains unsolved. These are crimes that cannot be solved within a short period and require constant negotiation over several years. The representation of justice in the novel is obscure and by depicting this perverted form of justice, the author aims to highlight the oppression faced by the colonized throughout history. From the very moment the corpse is discovered, suspicion arises against the native 'terrorists', their struggle for freedom being the crime. As the scene of the crime is near Mrs. Bose's brothel, Wyndham and his colleagues decide to question her and she responds, " […] people are killed in this part of the city every day [...] Normally, the unfortunate wretch is simply carted off to the morgue and that's the end of it. Why all the fuss this time?" (Mukherjee,p. 11). The difference this time lied in the fact that it was an Englishman who had been murdered and not a native. This represents the level of disparity when it comes to justice; in their own land, Indians were subjected to a form of justice which was completely different from the one applicable for the English folk. As Surendranath asks later in the novel, "Does British justice mean justice only for the British?" (p.
288).
Mukherjee's narrative also makes frequent references to various other injustices such as the Rowlett Act and laws against people organizing peaceful gatherings. The Rowlett Act (or The Anarchical and Revolutionary Act of 1919) was passed by the Imperial Legislative Council in 1919. According to this act, the government could arrest and imprison citizens without a formal trial; it aimed to suppress and deprive revolutionary groups of their right to individual expression and liberty. Similarly, most gatherings involving any mention of freedom struggle were labelled seditious and the organizers, as well as the participants, were jailed. When Benoy Sen, an Indian revolutionary was arrested on suspicions of murdering MacAulay, Inspector Wyndham asks him, "Do you know why you've been arrested" to which Sen responds, "Do you need a reason?" (p. 204). When questioned about his role in organizing a gathering in favor of the need for independence, he responds, "Under this law, Indians are banned from meeting in their own homes to discuss their desire for freedom in their own country. It was passed by Englishmen without the consent of the Indians to whom it applies. Wouldn't you agree that such a law is unjust? Or do you believe that an Indian, unlike a European, should not have the right to determine his own destiny?" (p. 206).
Through the above-mentioned instances, Mukherjee uses Sen's character to represent the extent to which Indians were oppressed by the British; a myriad of laws was imposed to limit any form of dissent against the British rule. While elaborating on his concept of Justice, Plato refers to Thrasymachus, an ancient Greek sophist who first articulated that "Justice is in the interest of the stronger" or "might is right" (Plato & Bloom, 1968, p. 15). This is exactly what we get to see in the novel under study; the guilt of an Indian is often at the predisposition of the ruling English, whether the charges are true or not is irrelevant. When Sen is accused of the charges, Inspector Digby says, "The man is guilty. Whether he admits it or not is irrelevant […] He would be pronounced guilty and hanged" (Mukherjee,p. 217).
In several parts of the novel, the natives are represented as violent and untrustworthy while the English are depicted as requiring protection from these "nefarious natives" (p. 139). This association of violence with natives runs parallel to the linking of violence with the East in Western crime fiction. The irony lies in the fact that when the English Brigadier-General Dyer shot down a peaceful, unarmed crowd without warning or a chance to disperse in Jallianwala Bagh in Amritsar, he is hailed and glorified as a hero and the Lieutenant Governor of Punjab credits him with "averting an armed insurrection" (p. 283). Throughout the narrative, locals are suspected of murdering MacAulay and it's only towards the end that it is revealed that the crime is committed by the Lieutenant Governor himself through Inspector Digby, yet another Englishman. The ending can be seen as a backlash against the British overlooking their own countrymen as potential perpetrators of violence. Even after the perpetrators are identified, no serious action is taken against them. Inspector Digby dies in the end but is given a posthumous promotion for the part he played in the investigation. Similarly, Wyndham and the Commissioner are helpless when it comes to holding the Lieutenant Governor culprit as his power is beyond their reach. They decide to make use of the situation as leverage and prevent the L-G from interfering with the police cases in the future. In a setup where Indians are imprisoned and hanged for the smallest of crimes, this resolution brings forth the discrepancy in terms of justice in colonial India.
Through the course of the narrative, Mukherjee sheds light on the racial politics within colonial India. Statements such as "In India, it seemed, even the forces of the law and order were subordinate to the hard fact of race" (p. 77) and "[w]hile the colour of a man's skin should have no bearing on the importance of the case, the reality was that it generally did […]" (p. 72) demonstrate the extent to which race was a deciding factor in the kind of justice received by the individual. The plight of Anglo-Indians is way worse; they are ousted by both the Indians and the English. As a result, they fail to attain a sense of belonging to either of the categories. (2006) by Christine Matzke and Susanne Muehleisen presents how post-colonial crime fiction has expanded and reworked the genre of crime fiction to discuss issues related to community, race, gender and various other sociopolitical issues (Matzke and Muehleisen, in Naidu 2020, p. 113). The result is a newly emerging postcolonial crime fiction which is characterized by hybridity, especially among the detective figures, making them more effective in their respective settings (p. 113). These detectives combine "western police methods and indigenous cultural knowledge" (Christian, 2001, p. 13) throughout their investigation. This trend is evident in Mukherjee's novel too; as Captain Wyndham is new to the city of Calcutta, he soon realizes the importance of indigenous cultural knowledge during the process of investigation and that he cannot sustain in this new terrain with his mere Western policing methods alone.
Postcolonial Postmortems: Crime Fiction from a Transcultural Perspective
One of the several ways in which Indian writers of crime fiction writing in the English language strive towards decolonization is through their very use of language itself. According to Salman Rushdie, "[t]he language like so much else in the colonies, needs to be decolonized, to be remade in other images, if those of us who use it from positions outside Anglo-Saxon culture are to be more than artistic Uncle Toms" (in Schröttner 2009, p. 295). The writers of crime fiction in India often use a hybrid form of English intermingled with words from their native, regional languages (Hindi and Bengali being the most popular choices). This 'chutnification' 2 of the English language with words from regional languages like Hindi and reflect India's hybrid culture. For instance, Abir Mukherjee's A Rising Man makes use of several Indian words like wallah 3 , sahib 4 , gullee 5 , lathi 6 , and aschee 7 . The entire narrative is coalesced with such words, thus serving to illustrate how linguistic aspects function in the process of literary decolonization. Ngũgĩ wa Thiong'o is of the view that 7 Interrogating Strategies of Justice and Racial Politics: A Post-colonial Reading of Abir Mukherjee's A Rising Man "language carries culture and culture carries, particularly through orature and literature, the entire body of values by which we come to perceive ourselves and our place in the world" (1986, p. 13). As a result, a difference in the use of language can be seen as a positive step towards freeing our indigenous culture from indirect colonial domination. A post-colonial reading of Abir Mukherjee's A Rising Man (2016) exposes the ways in which the crime genre is used to address issues of social concern such as injustice and racial prejudice. By constantly engaging in a dialogue on colonialism and its implications on the Indian society, such texts propagate a move towards liberating our psyche from imperialist ideology and thereby recover our true identities. | 2021-08-19T20:00:41.178Z | 2021-01-01T00:00:00.000 | {
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15441884 | pes2o/s2orc | v3-fos-license | Cholecystomucoclasis: revaluation of safety and validity in aged populations
Background We evaluated the safety and validity of cholecystomucoclasis (CM) and compared its intraoperative characteristics with those of standard cholecystectomy (SC). Methods We enrolled 174 patients who underwent cholecystectomy and retrospectively evaluated the outcomes of patients in the SC and CM groups. Results Significant differences in age (71.1 vs. 61.9 years), American Society of Anesthesiologists physical status (ASA-PS), and serum C-reactive protein levels (CRP) (18.1 vs. 4.7 mg/dL) were observed between the CM and SC groups. Conversely, no significant differences were observed in the operation time (129 vs. 108 min), amount of blood loss (147 vs. 80 mL), intraoperative complications (0% vs. 5.7%), or duration of hospital stay (13.2 vs. 8.9 days) between the 2 groups. A high conversion rate (35.3%), postoperative complications (33%), and frequent drain insertions (94%) were observed in the CM group. Conclusions CM is a safe and valid surgical procedure and surgeons should not hesitate to transition to CM for patients who are of advanced age, in poor general condition (high ASA classification), or have high levels of serum CRP.
Background
Cholecystectomy, particularly laparoscopic cholecystectomy (LC), has become the standard treatment for patients with benign gallbladder disease [1][2][3]. Although the indications for surgery in patients with acute cholecystitis have been expanding [4][5][6], there are cases where standard cholecystectomy (SC) is difficult owing to the presence of acute or chronic inflammation, strong omental adhesion, or gangrenous cholecystitis. As patients with benign gallbladder disease tend to be of advanced age, safer and more feasible surgical techniques are required for difficult cases [7][8][9].
Cholecystomucoclasis (CM) is a traditional method that is combined with subtotal cholecystectomy and involves the cauterization of the posterior gallbladder wall preserved in the liver bed that remains after anterior wall resection, in patients with advanced inflammatory cholecystitis [10]. CM, also called deroofing of the gallbladder [11,12], is a useful procedure that includes partial [13] or subtotal cholecystectomy [14,15]. Recently, increased experience in laparoscopic surgery and other advanced techniques have shown that CM is a safe and feasible option [16][17][18][19][20][21]. However, preoperative conditions and intraoperative technical characteristics have been less well described. Hence, we revaluated the safety and validity of CM and compared its intraoperative characteristics with those of SC.
Patients
As shown in Table 1, 174 patients (93 men and 81 women) underwent cholecystectomy at the Department of Surgery, Asanogawa General Hospital, Kanazawa, Japan, between January 2007 and December 2011, excluding those who were diagnosed with malignancy and underwent choledocholithotomy or simultaneous resection of other organs.
The average age of the patients was 62.9 ± 14.3 years (range, 23-95 years). Preoperative clinical diagnoses of cholecystitis were made on the basis of each patient's history of right upper abdominal pain and tenderness, fever, leukocytosis, increased C-reactive protein (CRP) levels, and positive signs on computed tomography or ultrasonography (thickened gallbladder wall and pericholecystic fluid collection). All resected specimens were also evaluated histopathologically. This study was approved by the Institutional Review Board of the Asanogawa General Hospital (AGH-IRB No. 22).
Operative technique
LC was performed by the standard 3 or 4-trocar technique. Briefly, the anesthetized patient was placed in the standard supine, crucifix, reverse-Trendelenburg position, with the surgeon on the patient's left side. Pneumoperitoneum was achieved by visually guided, cannular CO 2 insufflation. The dissection was started at the Calot's triangle, and the cystic duct, the common bile duct, and the cystic artery were exposed and divided between clips. Intraoperative cholangiography was routinely performed to detect residual calculus and bile duct injury. The gallbladder itself was carefully mobilized from the liver bed using electrocautery. An endobag was always used to remove the gallbladder, thus preventing wound infection. The abdominal cavity was irrigated before the trocars were removed, and the fascial defects were closed.
In April 2010, single-incision laparoscopic cholecystectomy (SILC) was instituted at our institution and subsequent LCs were performed using this technique. For the SILC technique, the patient was positioned supine on the operating table with the legs widely separated. The surgeon stood between the patient's legs, and the cameraperson stood to the right of the surgeon (near the patient's left leg) [22]. A 2-cm vertical transumbilical incision was made, and either a SILS port (Covidien Inc., Norwalk, CT, USA) or the hand-made glove method was used [23]. A 5-mm flexible endoscope (Olympus, Tokyo, Japan) was used for intraabdominal visualization. Cholecystectomy was then performed as mentioned above and as described previously [24,25].
Open cholecystectomy (OC) was performed through a right subcostal or a midline incision. In almost all cases, decompression of the gallbladder was performed using needle aspiration. Dissection was performed to identify the cystic duct and the common bile duct. In cases of advanced local inflammation, the dissection was performed from the fundus towards Calot's triangle. Intraoperative cholangiography was routinely performed. Finally, the gallbladder was removed from the liver bed.
In principle, the gallbladder should be totally resected. However, CM was considered, at the discretion of the surgeon, to prevent massive bleeding or bile duct injury. In particular, CM was applied in cases of a thinned necrotic gallbladder wall due to advanced inflammation, a thickened sclerotic gallbladder wall because of advanced inflammation, the inability to determine the exact orientation of the Calot's triangle, and the burial of the gallbladder deep within the liver bed. In severe, gangrenous cholecystitis, the dissection began from the fundus to the neck of the gallbladder, after decompression of the gallbladder. The residual gallbladder mucosa was cauterized by electrocautery or argon plasma coagulation. If the gallbladder wall was accidentally damaged, the gallbladder was continuously dissected from the orifice (Figure 1, 2).
Data collection
The data were retrospectively collected from medical records, operative reports, and histopathological reports. The outcomes for the SC and CM groups were evaluated. The outcome measures included the conversion rate (laparoscopic to open), operation time, loss of blood (low bleeding was defined as 0 mL), maximum preoperative CRP level, American Society of Anesthesiologists physical status (ASA-PS) score, success rate of intraoperative cholangiography, treatment of the proximal bile duct stump, intraoperative complications, postoperative complications, presence or absence of a drain, and length of hospital stay.
Statistical analysis
The values were expressed as means ± standard deviations (SD). The statistical analysis was conducted using the 2-sided Student's t test and the Mann-Whitney U test for continuous data or Fisher's exact test and the chi-squared test for categorical data. All statistical analyses were performed using the SPSS 10.0 software package (SPSS, Chicago, IL, USA). Significance was defined as P < 0.05.
Patient characteristics and preoperative findings
The 174 patients who underwent cholecystectomy were divided into the CM and SC groups. Data from the 2 The gallbladder mucosa and gallstones were exposed (arrowhead). c: The gallbladder was incised consecutively from the site of the exposed gallbladder mucosa. d: Overview after gallbladder removal showing the residual gallbladder posterior wall (arrowhead).
groups are summarized in Table 3).
Complications and adequacy
The intraoperative and postoperative complications are described in Table 4. Although the 2 groups did not differ significantly in this regard (P = 0.578), the CM group had no intraoperative complications, whereas the SC group had a relatively high incidence of complications ( Figure 3). Three cases of massive bleeding (>1000 mL) and 6 cases of bile duct injury were observed in the SC group.
Postoperative complications were observed in 4 patients of the CM group. Bile leakage and wound infection occurred in 2 patients and a residual calculus, which was subsequently removed endoscopically, was observed in 1 patient. Additional surgical treatment was not required for any of the CM patients. In the SC group, postoperative complications were observed in 10 patients. Two patients experienced intra-abdominal hemorrhage: 1 patient required reoperation and the other was treated conservatively with blood transfusions. Bile leakage was observed in 4 patients: 1 was treated conservatively, 1 needed endoscopic drainage, 1 required immediate reoperation, and 1 required reoperation even though endoscopic drainage and percutaneous transhepatic cholangial drainage were both attempted.
A drain was inserted into the liver bed in almost all CM patients (17/18), but only in some of the SC patients (61/156, P < 0.001). The average length of the hospital stay was 13.2 ± 6.4 days (range, 5-27 days) in the CM group and 8.9 ± 26.1 days (range, 3-330 days) in the SC group (P = 0.495).
Histopathological examination was performed for all patients, with gallbladder cancer being diagnosed in 2 patients, who received additional treatment. No mortality was associated with the procedures in either group.
Discussion
Difficulties in performing cholecystectomy include a lack of clarity of the anatomical orientation of the Calot's triangle, resulting from severe, acute inflammation or chronic atrophic sclerotic change. In this situation, subtotal cholecystectomy is recommended for a safe surgery. Subtotal cholecystectomy can be performed using 2 methods: dissecting the neck of the gallbladder rather than approaching the Calot's triangle to prevent bile duct injury or not removing the posterior wall of the gallbladder to prevent bleeding from the liver bed. In the latter method, ablation of the remaining mucosa is common and is referred to as CM.
In principle, the gallbladder should be completely resected, as gallbladder carcinoma has been reported in 0.3-1.5% of patients who have undergone cholecystectomy [26,27]; therefore, it is necessary to check for any signs of malignancy during preoperative diagnostic imaging. Because CM or subtotal cholecystectomy may result in an intraoperative bile leak into the abdominal cavity, these procedures are associated with potential dissemination. Although there are no reports of residual gallbladder cancer following CM or subtotal cholecystectomy, Shimizu et al. reported a case of biliary tract cancer in the liver bed after subtotal cholecystectomy. They considered the possibility of a peripheral type of intrahepatic cholangiocarcinoma as well as carcinomas from the residual gallbladder mucosa or the Luschka duct [28]. Therefore, regular follow-ups with diagnostic imaging are needed, even when the patient does not show pathological evidence of malignancy.
The frequency of postoperative complications in subtotal cholecystectomies has been reported to be 6.7-20.7% [17][18][19][20][21], with the patients having an average age of 53-62.9 years. In the current study, a relatively high incidence of postoperative complications (33% [6/18]) was observed, and the average age of patients in the CM group was 71.1 ± 8.4 years. In particular, 2 patients were found to have postoperative bile leakage, both of whom were treated with drainage only, without processing of the bile duct stump. One patient required endoscopic biliary drainage (EBD) and the other recovered spontaneously without any additional treatment. The reason for the spontaneous closure was presumed to be the result of postoperative firm adhesion and scar formation, which is expected in patients with advanced inflammation. The SC group had 4 patients with postoperative bile leakage. Two patients required a reoperation for bile leakage closure, and the other 2 were treated by diversion of bile from the leakage site by EBD or percutaneous transhepatic cholangial drainage. There were no significant differences between the two groups in additional treatment (P = 0.06), but the treatment for bile leakage in SC group was more difficult.
Few detailed studies have reported on intraoperative findings in these types of cholecystectomies. In the CM group, there was no evidence of intraoperative damage to the biliary tract, a finding that was similar in the SC group. Additionally, there was no significant difference in the amount of bleeding between the 2 groups, and the CM group did not include patients exhibiting massive bleeding (>1000 mL). During cholecystectomy, bleeding primarily originates from the cystic arteries and the liver bed; CM and subtotal cholecystectomy help prevent bleeding from these important locations.
The conversion rate to OC was 35.3% in the CM group, which was higher than that in previous reports (1.7-7.7%) [18.19.21]. Thus, we emphasize on safety in our surgical procedures and convert to OC without hesitation in difficult cases. The lack of intraoperative complications in the CM group is probably the result of these efforts. Furthermore, patients who needed reoperation were observed only in the SC group, highlighting the need for a flexible approach according to patient characteristics rather than adherence to a particular policy of laparoscopic surgery in order to avoid unnecessary intraoperative complications and conversion to laparotomy.
OCs are typically more closely associated with advanced age, poor general condition, or a high inflammatory response than are laparoscopic surgeries [29]. These same characteristics are also associated with an increased severity of acute cholecystitis, which may result in a more difficult surgery. The aforementioned patient characteristics are collective, but not independent, risk factors for postoperative complications of acute gangrenous cholecystitis [30,31]. In addition, Schäfer et al. have reported that advanced age and a high serum CRP level may be predictive factors for the surgical procedures that are used in patients undergoing laparotomy and patients who were converted to laparotomy [32]. The current study was a retrospective study, with a selection bias based on intraoperative findings; the CM group showed an advanced age and a high serum CRP level. With the transition to CM and open surgery in mind, the conversion to a safe surgical procedure should be considered in elderly patients with high inflammation of the gallbladder neck.
Conclusions
The intraoperative technical characteristics of SC and CM were analyzed, and a low rate of handling of the cystic duct stump and a low success rate for intraoperative cholangiography were observed in the CM group. However, no significant differences were observed in the operative time, amount of bleeding, and number of intraoperative complications. Compared with SC, high conversion rates and drain insertion rates were noted in patients undergoing CM, but the length of hospital stay did not differ significantly. CM is thus considered to be a safe and valid surgical procedure. In addition, surgeons should not hesitate to transition to CM for patients with advanced age, poor general condition (high ASA classification), and a high serum CRP level. | 2016-05-04T20:20:58.661Z | 2012-08-21T00:00:00.000 | {
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52016632 | pes2o/s2orc | v3-fos-license | Sorafenib in patients with progressed and refractory bone tumors
Patients with metastatic, progressive, or recurrent bone tumors have a dismal outcome. Sorafenib has been proposed as an effective salvage regimen for some malignancies. Thus, we sought to evaluate this approach for young patients with relapsed or refractory bone tumors. Twelve patients with refractory bone tumors (two with Ewing sarcoma, two with chondrosarcoma, and eight with osteosarcoma) received salvage treatment with sorafenib. All patients had standard tumor imaging and laboratory evaluation. All toxicities were documented. At the time of the beginning of sorafenib treatment median age among 12 patients was 18 years (range 4.1–27.9 years), eight were male, and eight had osteosarcoma. All received sorafenib because of relapse. Seven patients were treated parallel to other standard chemotherapy. Overall response rate was 75%. Median time to sorafenib time to progression for patients with osteosarcoma was 4 months (range 1.8–7.9 months). Four patients (33%) are alive, in that two with no evidence of disease with a median follow-up of 41 months (range 26.5–60.9 months). The estimated 5 year overall survival (OS) for the whole group was 64.49%. There were no serious toxicities. Sorafenib is well-tolerated in young patients with bone tumors, and particularly could be an option for patients with metastatic disease and refractory osteosarcoma. Sorafenib only allows to extend OS and different procedures are needed to achieve permanent remission. This regimen deserves further investigation in the upfront management of patients with high-risk bone tumors.
Introduction
Approximately 1100-1200 new cases of malignant neoplasm in children are diagnosed in Poland every year. Out of that group all bone tumors consist about 7%. The frequency of occurrence increases with age. Adolescents and young adults are the most common group of patients, although appearance of such diseases is also not rare among the children's early age group. The most commonly diagnosed bone cancers are osteosarcoma (OS) and Ewing sarcoma (ES)-56% and 34%, respectively. With advances in multimodal therapy, survival rates for patients with primary localized bone disease approaches 65-75% [1,2]. However, patients with metastatic, progressive, or recurrent diseases have a dismal outcome [1][2][3][4][5].
For some malignancies sorafenib has been proposed as an effective drug, particularly primary kidney cancer, liver cancer, and thyroid carcinoma. Furthermore, it has been proposed as an oral agent in the therapy of high grade progressing osteosarcoma in adult patients and in refractory solid tumors in children and young adults [6][7][8][9][10][11]. Sorafenib is a kinase inhibitor drug and is available as an oral formulation; compared to other drugs from the same group sorafenib inhibits also Raf, Mek, and Erk kinase pathways. Both the moderate toxicity profile and the promising results observed in a few studies allow to consider sorafenib as a reasonable treatment option also for heavily pretreated young patients with bone tumors [7,8,12]. Thus, we sought to evaluate this schedule, particularly the response rate and progression-free survival for patients with refractory or relapsed bone tumors.
Patients
Twelve patients with histologically confirmed primary bone tumors were treated with sorafenib during the period 2015-2017 at the Mother and Child Institute (Warsaw, Poland). Prior to treatment informed consent was obtained from all patients. In cases when minors were involved the consent was obtained from their legal guardians. Approval for this retrospective study was obtained in compliance with the international regulations for protection of human research subjects (Bio-ethical Committee at the Mother and Child Institute in Warsaw, opinion issued under number 35/2018).
Treatment
Sorafenib was administered at a dose of 400 mg twice a day in patients older than 15 years and/or heavier than 50 kg. In younger patients the dose was calculated in proportion to their body weight and was 100 mg twice a day for patients weighing 15-20 kg, and 200 mg twice a day if the weight was 20-30 kg. Treatment was to be continued until disease progression or unacceptable toxicity. Dose reduction was undertaken in case of CTCAE v. 4.0 grades 3 and 4. Patients developing allergic symptoms received steroids and antihistaminic drugs. Patients developing leucopoenia or thrombocytopenia were treated symptomatically with G-CSF and transfusion of blood products.
Assessment of Response and Toxicity
All patients had standard tumor imaging using CT, MRI, bone scan, or PET, as indicated, prior to starting sorafenib and every 3 months afterwards. Physical examination and laboratory evaluation were performed prior to each cycle of standard chemotherapy, every month, or weekly when necessary. All toxicities were documented from day 1 of the first day of sorafenib until end of therapy. WHO criteria were used to evaluate the response.
Statistical methods
Overall survival (OS) was defined as the time interval from the date of diagnosis to the date of death or to last follow-up date. Time to the first relapse was defined as the time interval from date of initial biopsy to the date of the first day of the first relapse's treatment. Sorafenib OS was defined as the time interval from the first day of sorafenib treatment to the date of death or to the last follow-up date. Sorafenib time to progression (TTP) was defined as the time interval from the start date of sorafenib to the date of disease progression. Response rate was defined as the percentage of patients who achieved stable disease (SD), partial response (PR), and complete remission (CR) during sorafenib treatment. Results' distribution was estimated using the Kaplan-Meier method. Log-rank test was used to compare groups. P ≤ 0.05 was regarded as significant. Statistical analysis was performed using STATA 10.0 for Windows.
Patients
Between 2015 and 2017, 12 patients (8 males, 4 females) with histologically confirmed primary bone tumors (eight with osteosarcoma, two with Ewing sarcoma, and three with chondrosarcoma) were treated with oral sorafenib. Patients' clinical characteristics are shown in Table 1. Median age at the time of diagnosis was 13.4 years (range 2.6-19.9 years). Ten patients had metastatic disease at diagnosis (seven of them only to the lungs, one of them to the bones, and two of them to the lungs and bones). Median time to the 1st relapse was 16.5 months (range 1.2-41.8 months). Sites of relapse were as follows: isolated lung metastases 6 patients (50%), isolated bone metastases 1 patient (8.3%), combined lung and bones 3 patients (25%), and local with lung metastases 2 patients (16.7%).
Treatment
Sorafenib was used in 5 patients as 1st, in 3 as 2nd, in 1 as 3rd, in 1 as 4th, in 1 as 5th, and in 1 as 6th line of relapse's treatment. Median age at the start of sorafenib treatment was 18 years (range 4.1-27.9 years). Median time from the initial diagnosis to the start of sorafenib was 33.8 months (range 1.9-113.4 months). Median time of sorafenib treatment was 65.5 days (range 15-299 days). Seven patients were treated parallel to other standard chemotherapy (Table 1).
Outcome and toxicity
Median follow-up from start of sorafenib was 9.6 months (range 2-24.8 months). Partial response was achieved in eight patients (six with osteosarcoma), SD in two patients (both with osteosarcoma), and PD in two pts, with an overall response rate of 75%. Median time to sorafenib TTP for the whole group was 2.2 months (range 1.3-7.9 months). Median time to sorafenib TTP for the patients with osteosarcoma was 4 months (range 1.8-7.9 months). Four patients (33%) are alive including two with no evidence of disease with a median follow-up of 41 months (range 26.5-60.9 months) and median follow-up from start of sorafenib was 22.1 months (range 8.4-24 months). 5-years OS estimate for the whole group was 64.49%. The efficiency of sorafenib appears to be higher when it is used in the first recurrence, however, p is not significant (p = 0.125) (Fig. 1). Three patients had documented grade 3 skin toxicity and for that reason treatment was discontinued. There were no other significant toxicities.
Discussion
The dismal prognosis of recurrent or progressive bone tumors highlights the importance of the development of new treatments. Over the last years, several drugs and combinations have been explored in this setting, but enough satisfactory results have not been achieved [13,14].
Here we have presented our results with the use of sorafenib in the management of progressive or recurrent bone tumors. Importantly, there is a very small number of reported studies using sorafenib in young patients with that kind of tumors. In our group, we had four patients under the age of 15, and the youngest patient on the day of starting sorafenib treatment was only 4 years old. To our knowledge, this is the youngest patient with bone tumor who has used this type of therapy.
Some studies have described the efficacy of sorafenib in refractory bone tumor. A moderately successful response was reported in Coventon's study regarding the application of sorafenib in advanced, relapsed, and refractory to standard chemotherapy osteosarcoma in 35 patients over 14-years-old; in 46% patients 4 months progression-free survival was observed, and 29% pts had SD over 6 months. The other study described by Coventon included 4 patients; 3 of them had SD over 3 months. Our study contained eight patients with osteosarcoma. Four of them achieved PR, two had SD. Median time to sorafenib TTP for patients with osteosarcoma was 4 months (range 1.8-7.9 months) similarly to Conventon's study.
Preclinical data have shown the possibility of better results in combination of sorafenib with other drugs, included mTOR inhibitors [15]. Grignani has shown a study, where overall-response rate was 10% in sorafenib and everolimus combination in unresectable, advanced osteosarcoma patients older than 17 years. Unfortunately, clinical data available in the literature are still not satisfactory. Fig. 1 Comparison of OS between patients treated with sorafenib when it was used in the first recurrence vs. when it was used in the next recurrence According to Saletta's study in an early pediatric clinical trial sorafenib has shown 30% response in solid tumors and 75% in AML. A high response rate (75%) was noted in our group. Most likely the difference derives from the fact that our group was less heterogenic and included only patients with bone tumors, in majority osteosarcoma.We have not observed serious toxicity in our group (this is compatible with other different studies) although, those skin adverse events were not acceptable to our patients and their families, and they were the reason for treatment's discontinuation.
Unfortunately, in our study the median treatment's response for the whole group was 2.2 months. Therefore, it seems that other additional procedures (like surgery), new drugs, or strategies are necessary to achieve permanent remission in this type of patients, and at this moment sorafenib allows only to extend OS. Our study is limited and can be bias because of the small heterogeneous group and retrospective data. Nevertheless, our study confirms that sorafenib is well-tolerated in young patients with bone tumors and particularly could be an option for patients with metastatic disease and refractory osteosarcoma. Further prospective studies are needed to better define the use of sorafenib in the upfront management of these groups of patients. | 2018-08-18T21:15:57.275Z | 2018-08-16T00:00:00.000 | {
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59456822 | pes2o/s2orc | v3-fos-license | Tunable mechanical filter for longitudinal vibrations
This paper presents both theoretically and experimentally a new kind of vibration isolator called tunable mechanical filter which consists of four parallel hybrid periodic rods connected between two plates. The rods consist of an assembly of periodic cells, each cell being composed of a short rod and piezoelectric inserts. By actively controlling the piezoelectric elements, it is shown that the periodic rods can efficiently attenuate the propagation of vibration from the upper plate to the lower one within critical frequency bands and consequently minimize the effects of transmission of undesirable vibration and sound radiation. In such a filter, longitudinal waves canpropagate from the vibration source in the upper plate to the lower one along the rods only within specific frequency bands called the “ Pass Bands” and wavepropagation is efficiently attenuated within other frequency bands called the “ Stop Bands”. The spectral width of these bands can be tuned according to the nature of the external excitation. The theory governing the operation of this class of vibration isolator is presented and their tunable filtering characteristics are demonstrated experimentally as functions of their design parameters. The concept of this mechanical filter as presented can be employed in many applications to control the wavepropagation and the force transmission of longitudinal vibrations both in the spectral and spatial domains in an attempt to stop/attenuate the propagation of undesirable disturbances.
Appropriately partitioned matrices of the stiffness Kp Total stiffness of the piezo-insert
Introduction
The periodic rods in the proposed mechanical filter act as the transmission paths of the vibration from the upper plate where the source of vibrations is located to the lower plate.Therefore, proper design of these rods is essential to the attenuation of the vibratory energy of and the noise radiated into any structure connected to the lower plate.A periodic rod consists of an assembly of identical cells connected in a repeating array which together form a 1-D periodic structure.The study of periodic structures has a long history.Wave propagation in periodic systems related to crystals and optics has been investigated for approximately 300 years.Brillouin [1] developed the theory of periodic structures for solid state applications and then, in the early seventies, the theory was extended to the design of mechanical structures [2,3].Since then, the theory has been extensively applied to a wide variety of structures such as spring-mass systems [4], periodic beams [2,[4][5][6][7][8][9][10][11][12], stiffened plates [9,10,13], ribbed shells [14] and space structures.Examples of such structures are found in many engineering applications.These include bulkheads, helicopter drive shafts [11], airplane fuselages, vehicle engine mounting systems [15], and helicopter gearbox supporting systems [16,17].Each such structure has a repeating set of stiffeners which are placed at regular intervals.Sackman et al. [18] presented a layered notch filter device that is limited only to high-frequency vibrations.Such a filter which was developed theoretically based on the Floquet theory is a periodically layered stack of two alternating materials with widely different densities and stiffnesses.Wave propagation in a periodic truss-work beam was investigated computationally by Signorelli and Flotow [19] based upon the transfer matrix approach.
The work presented here is an experimental implementation of the work of Baz [20] in which an active periodic spring mass system is employed theoretically, in a quasi-static manner, to control the wave propagation of longitudinal vibration.Periodic rods in passive mode of operation exhibit unique dynamic characteristics that make them act as mechanical filters for wave propagation.As a result, waves can propagate along the periodic rods only within specific frequency bands called the "Pass Bands" and wave propagation is attenuated within other frequency bands called the "Stop Bands".The spectral width and location of these bands are fixed for a 1-D passive periodic structure [16], but are tunable in response to the structural vibration for active periodic structures [20,21].The shape memory alloy has been used as a source of irregularities to attenuate the wave propagation in periodic rods [21].
The spectral finite element analysis and transfer matrix method [20][21][22][23] will be used to analyze the hybrid periodic rod and determine the propagation parameter, µ which indicates the regions of stop bands and pass bands.
This paper is organized in four sections.In Section 1, a brief introduction is given.Section 2 presents the theoretical background of the hybrid periodic rod and Section 3 demonstrates the performance characteristics of the tunable mechanical filter.Comparisons between the theoretical and experimental characteristics are also presented in Section 3. Section 4 summarizes the findings and the conclusions of the present study.It outlines also the direction for future research.
Overview
In this section, the emphasis is placed on studying the dynamics of the tunable mechanical filter in order to demonstrate its unique filtering capabilities.The dynamics of one-dimensional hybrid periodic rods in their active and passive modes of operation are determined using the transfer matrix method.The basic characteristics of the transfer matrices of periodic rods are presented and related to the physics of wave propagation along these rods.The methodologies for determining the pass and stop bands as well as the propagation parameters are presented.
In this paper, the focus is placed on hybrid periodic rods consisting of a straight rod with periodically placed piezoelectric inserts as shown in Fig. 1.
Spectral finite element method of the rod
The first development of the spectral Finite Element (SFE) method occurred in the early eighties [23].The main distinct between the SFE and Finite Element (FE) stems from the type of the shape functions used to approximate the model equations.In FE method, the shape function is only a function of a spatial variable, whereas in SFE the shape function has two independent variables: the spatial variable (x) and the spectral variable (ω).As a result, the SFE method is much more accurate than FE method.For example, one can show that the exact natural frequencies for a rod can be found by one element using SFE method, whereas a large number of elements needs to be used to get the same accuracy using FE method.
Consider the rod shown in Fig. 2. The equation of motion of the rod is given by: where u is the longitudinal deflection, ρ is the density and E is Young's modulus.Then, assuming a solution u(x, t) = U (x)e iωt where ω is the frequency, reduces the equation of motion to: where k = wave number = ρ/E ω.
Using the following spectral shape function: U (x) = Ae −ikx + Be ikx , which is also a solution of Eq. ( 1), yields the spectral finite element description of the dynamics of the rod.This results in the following dynamic stiffness matrix of the rod: Where L, E, and A are the length, Modulus of Elasticity, and the area of the rod.The corresponding Transfer Matrix [T ] takes the following form: where and the subscript L and R represent the left and right end of the cell of the rod.
Dynamics of the hybrid rod
Consider now the dynamics of the hybrid periodic rods which consists of a passive sub-cell and an active piezoelectric sub-cell as shown in Fig. 3.
Passive sub-cell
As shown in Eq. ( 4), the dynamic characteristics of the passive sub-cell (a) can be described as follows: where u and F define the deflection and force vectors with subscripts Land I denoting the left and interface sides of the passive sub-cell and K dij a defines the elements of dynamic stiffness matrix of the sub-cell a which can be calculated from Eq. ( 5).
In a more compact form, Eq. ( 6) can be rewritten as: where T a is the transfer matrix of the passive sub-cell a.One can show that the transfer matrix is a simplectic matrix [16].
Active sub-cell
The constitutive equations of the active piezoelectric insert are given by [20]: where E p , D p , T p and S p are the electrical field intensity, electrical displacement, stress and strain of the piezo-insert.Moreover, ε S , h p and C D define the electrical permittivity, piezo-coupling constant and elastic modulus.Equation ( 8) can be rewritten in terms of applied voltage V p , Interface piezo-force F I , electrical charge Q p and net deflection (u R − u I ) as follows: where D b and L b are the diameter and the length of the piezo insert.
The equation of the charge Q p can be formulated from the first raw of Eq. ( 9) as follows: Substituting Q p into the second raw of Eq. ( 9) gives: Let the piezo-voltage V p be generated according to the following control law: where K g is the control gain which can be complex if phase shift is allowed.Appropriate phase shift means that the piezo-insert can act as a damper.Then, Eq. ( 11) reduces to: where with k pc and k ps denote the active piezo-stiffness due to the control gain K g and the structural piezo-stiffness respectively.Equation ( 13) can be used to generate the force vector {F I F R } T acting on the piezo-insert rewritten as: with k p = (k pc + k ps ) is the total stiffness of the piezo-insert which is complex if the gain is complex by virtue of the phase shift.Hence, the dynamic equation of the active sub-cell is given by: where K dij b defines the elements of dynamic stiffness matrix of the sub-cell b which can be calculated from Eq. ( 13).Now, the state vectors at ends R andI of the passive sub-cell b are related through the transfer matrix derived from Eq. ( 15) as follows: In a more compact form, Eq. ( 16) can be rewritten as: where T b is the transfer matrix of the active sub-cell b.
Interface I (b) Interaction between two consecutive cells Fig. 4. One-dimensional periodic rod.
Dynamics of entire cell
The dynamics of the entire cell can be determined by the assembly of the dynamic equations of the passive and active sub-cells which are given by Eqs ( 7) and ( 16) respectively.This yields the following dynamic equations: where also T a and T b represent the transfer matrix of subcell a and the stiffness matrix of the subcell b respectively.In a more compact form, Eq. ( 16) can be rewritten as: where Y and [T k ] denote the state vector = {u L F L } T and the transfer matrix of the k cells.
For exactly periodic rods, the transfer matrices are identical so [T k ] = [T ] and the eigenvalue problem of [T ] can be written as: where λ is the eigenvalue of [T ] Combining Eqs (18) and (19) gives: indicating that λ of the matrix [T ] is the ratio between the elements of the state vectors at two consecutive cells.Hence, the magnitude of λ determine the nature of wave dynamics in the periodic rod as follows: If |λ| = 1 , the wave propagates along the rod without any change of amplitude, indicating a Pass Band and if not then the wave will be attenuated, indicating a Stop Band.
A further explanation of the physical meaning of the eigenvalue λ can be extracted by rewriting it as: where µ is defined as the "Propagation Factor" which is a complex number whose real part (α) represents the logarithmic decay of the state vector and its imaginary part (β) defines the phase difference between the adjacent cells [28].
One can rewrite Eq. ( 20) as: Now, let us assume a harmonic motion and consider only the j th components u Lj of the deflection vector u L , at cells k and k + 1, which yields to: where U Lj n and φ jn denote the amplitude and phase shift of thej th component u Lj at the n th cell.
From Eqs ( 22) and ( 23), we get: Equation (24) indicates that: α = ln(U Lj k+1 /U Lj k ) = Logarithmic decay of amplitude, and β = (φ j k+1 − φ j k ) = phase difference between the adjacent cells Therefore, the equivalent conditions for the Pass and the Stop bands can be written in terms of the propagation constant parameters (α and β) as follows: 1.If α = 0 (i.e.µ is imaginary), then we have "Pass Band" as there is no amplitude attenuation.2. If α = 0 (i.e.µ is real or complex), then we have "Stop Band" " as there is amplitude attenuation defined by the value of α.
The propagation factor, µ, can be calculated directly from the diagonal elements of overall transfer matrix as following: In case of rod element, the transfer function has two eigenvalues λ and 1 / λ and according to the properties of the simplectic matrix (or transfer matrix), the trace is equal to the summation of the eigenvalues, so one can show that: A better insight into the physical meaning of the eigenvalues λ and λ −1 can be gained by considering the following transformation of the cell dynamics into the wave mode component domain: where Φ is the eigenvector matrix of the transfer matrix [T ].Also, W k is the wave mode component vector which has the right-going wave component w r and left-going wave component w L .Substituting Eq. ( 27) into Eq.( 17), it reduces to: Note that the matrix Φ −1 [T ] Φ reduces to the matrix of eigenvalues of the matrix [T ], i.e.: But, because of the particular nature of the eigenvalues of [T ] as they appear in pairs (λ, λ −1 ), then the above equation reduces to: Direction Amplification High Amplitude Low Amplitude where 30) can be expanded to give: For the j th component of w, we have: It is clear from Eq. ( 32) that the eigenvalue λ j is the ratio between the amplitude of the right-going waves whereas λ −1 j defines the ratio between the amplitude of the left-going waves.Hence, if (λ j < 1) , then λ −1 j > 1 and the pair of (λ j , λ −1 j ) denote attenuation of the amplitude of wave propagation from cell k to cell k + 1.This can be clearly understood by considering Fig. 5.
Note that the right-going wave (w r Lj k ) is attenuated by λ j as it propagates from cell k to k + 1 and the resulting wave (w r ) is amplified by λ −1 j as it propagates from cell k + 1 to cell k.Hence, the left-going wave (w L Lj k+1 ), at cell k+1, has a lower amplitude than that at k. Accordingly, the amplitudes of both the right and left-going waves (w r ) at left end of cell k + 1.
Overview
In order to demonstrate the feasibility of the theoretical concepts presented, experimental investigations are conducted.These investigations were carried out on two main steps.In the first step, the vibration attenuation characteristics of the hybrid periodic rod with two actuators on the shaker was studied and evaluated.In the second step, the tunable mechanical filter is used to evaluate its performance for attenuating the vibration transmission from the vibration source, the motor, to the lower plate.
In the present study, piezo actuators model 712A02 (see the index Table 1) from PCB piezotronics, Inc. (Depew, NY) are used.The actuator shown in Fig. (6-a) has two built-in side bolts so that the short aluminum rods can be bolted from both sides.Figure (6-b) shows the frequency response characteristics of the actuator authority between 150-5000 Hz with a sensitivity of 0.015 pound/volt and a peak excitation voltage of 100 Volts.
Experimental facilities
Figure 7a shows the dimensions of the hybrid periodic rod used to build the tunable mechanical filter.It consists of three short aluminum rods with shown in (see for properties Table 2 in the appendix) bolted together by the actuator as shown in Fig. 7b.The fundamental longitudinal natural frequency of the periodic rod is 640 HZ.The frequency response of such a rod is obtained both in its passive (open-loop) and active (closed-loop) modes of operation.Two experimental test rigs have been employed in this study to evaluate the performance of the tunable mechanical filter.The first test rig aims at monitoring the vibration transmission characteristics of the hybrid rod alone as influenced by geometrical and material discontinuities.Figure 8 shows the details of the employed test facility.
The objective is to measure the transfer function between the input excitation, the shaker, and the output response of the tip of the rod.
First, for the passive plain rod (i.e.no periodicity and open circuit), second, for the passive periodic rod (i.e. with periodicity and open circuit), and third, for the hybrid periodic rod (i.e. with periodicity and short circuit).The phase shifter has been used to introduce a feedback signal to the actuator to produce the destructive interference of waves.
The experimental setup shown in Fig. 9 was used to demonstrate the feasibility of using the tunable mechanical filter to isolate the vibrations induced by a motor.The experiment has been done for the three cases described in first test rig.Two phase shifters have been used to produce the negative feedback signals to the two actuators.
Single hybrid rod
Figure 10a shows the magnitude of the transfer functions of plain, passive periodic, and hybrid periodic rods when they are subjected to random excitation in the axial direction.These transfer functions quantify the response of the free end of the rods to input excitations at the other end of the rods.Figure 10a indicates clearly that the passive periodic rod exhibits experimentally a broad stop band between 650-4000 Hz whereby the vibration transmission through the rod is almost eliminated.Furthermore, Fig. 10a indicates that the hybrid rod provides an effective means for attenuating the vibration transmission over a very broad frequency range.It is particularly effective for stopping the low frequency vibration in the range below 600 Hz where the passive periodic rod has been ineffective.This result is particularly important in using a hybrid rod that combines both the passive and active strategies to stop high as well as low frequency wave propagation.Figure 10b shows that the widths of the stop bands can be clearly predicted theoretically by plotting the real part α of the propagation parameter µ.For values of α =0, the stop bands can be clearly identified and match closely the experimental results for both the passive and hybrid rods.
In all the reported results, the hybrid periodic rod has provided a viable means for extending the width of the stop band between 0-4000 Hz.Also, attenuations of more than 20 dB are obtained with control voltages less than 5 volts as shown in Fig. 11 for the two control actuators.Note that frequency range higher than 4000 Hz was not considered because of the limitation imposed by the actuator bandwidth.Figure 12 displays the lateral vibration distribution over plain, passive periodic, and hybrid periodic rods at four different frequencies using the PSV-200 scanning laser vibrometer.Figure 12 shows that the lateral vibration transmission is attenuated also in addition to the longitudinal waves whenever the frequencies lie inside the stop band with the hybrid periodic rod out-performing the plain and the passive periodic rods.
The tunable mechanical filter
Figure 13 shows the transfer function of the tunable mechanical filter with plain, passive periodic, and hybrid periodic rods when it is subjected to broad band excitations from vibration source, the motor.The excitation from the motor can be considered as a random excitation and the geometrical information of the configuration of rods is the same as shown in Fig. 7.The periodic rods in active mode are very efficient in the low frequency range while in high frequency range they have the same efficiency of the passive mode.That is way the blue and green lines in Fig. 13 have the same trend above 1500 Hz.The corresponding control voltage of the upper control actuators is shown in Fig. 14.
Conclusions
This paper has presented the tunable mechanical filter as a new tool for isolating the vibration.It consists of four hybrid periodic rods installed between two plates.The theory governing the operation of this class of rods has been presented.The factors governing the design of effective periodic rods have been identified.The performance characteristics of passive and hybrid periodic rod alone have been measured experimentally and compared with the theoretical predictions of the real part of the propagation factor.Close agreement between theoretical predictions and experimental results has been achieved.The performance of the tunable mechanical filter with a motor assembly has also been monitored experimentally.The predictions of the stop bands have also been found to be in close agreement with the experimental results.The periodic rods in active mode are very efficient in the low frequency range while in high frequency range they have the same efficiency of the passive mode.As a result, the mechanical filter with hybrid periodic rods is found to be more effective in attenuating the transmission of vibration from the source of vibrations to the lower plate over a broader frequency range extending from 150-4000 Hz with control voltages not exceeding 5 volts.
This concept of the tunable mechanical filter can be implemented efficiently in many applications.For instant, the gearbox support system of helicopter and automotive vehicle engine mounting systems.With such unique filtering characteristics, it would be possible to control the wave propagation both in the spectral/spatial domains in an attempt to stop/confine the propagation of undesirable disturbances.
Length of cell b M ij Appropriately partitioned matrices of the mass N Number of cells Qp Electrical charge Sp Strain of the piezo-insert tp Thickness of piezo-insert T Transfer matrix of the unit cell T k Transfer matrix of the k th cell Tp Stress of the piezo-insert u Longitudinal deflection V Applied voltage Y State vector = {u L F L } T Y k Eigenvetor of transfer matrix of the unit cell Y k Eigenvetor of the transpose of transfer matrix of the unit cell α Logarithmic decay of amplitude of state vector β Phase difference between the adjacent cells ε S Electrical permittivity λ Eigenvalue of transfer matrix of the unit cell ρ Density µ Propagation Constant ω Excitation frequency
Fig. 5 .
Fig. 5. Attenuation of right and left-going waves in the propagation direction.
Lj k+1 )
has a lower amplitude as w r Lj k+1 = λ j w r Lj k from the first part of Eq. (32).The left-going wave (w L Lj k+1
)
at the left end of cell k are higher than the right and left-going waves (w r Lj k+1 and w L Lj k+1
Fig. 9 .
Fig. 9. Experimental setup used to demonstrate the feasibility of using the tunable mechanical filter to isolate the vibrations induced by the motor.
Fig. 10 .
Fig. 10.The transfer functions of the plain, passive periodic, and hybrid periodic rods with the propagation factor in both passive and active modes.
Fig. 11 .
Fig. 11.The control voltage of: (a) the lower actuator, and (b) the upper actuator.
Fig. 12 .
Fig. 12.The vibration of plain, passive periodic and hybrid periodic rods at.
Fig. 13 .
Fig. 13.The transfer function in case of collocated sensor/actuator arrangement with broadband excitation from the motor.
Fig. 14 .
Fig. 14.The control voltage applied to the upper control actuator.
Table 1
The main geometrical and perofrmance parameters of the actuators
Table 2
Material properties of the aluminum rods | 2018-12-28T18:26:34.140Z | 2007-01-01T00:00:00.000 | {
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252863846 | pes2o/s2orc | v3-fos-license | Inhibition of mitochondrial complex I leading to NAD+/NADH imbalance in type 2 diabetic patients who developed late stent thrombosis: Evidence from an integrative analysis of platelet bioenergetics and metabolomics
Type 2 diabetes mellitus (T2DM) is a strong indicator of late stent thrombosis (LST). Platelet bioenergetic dysfunction, although critical to the pathogenesis of diabetic macrovascular complications, remains uncharacterized in T2DM patients who developed LST. Here, we explored the mechanistic link between the alterations in platelet bioenergetics and LST in the setting of T2DM. Platelet bioenergetics, metabolomics, and their interactomes were analyzed in a nested case-control study including 15 T2DM patients who developed LST and 15 matched T2DM patients who did not develop LST (non-LST). Overall, we identified a bioenergetic alteration in T2DM patients with LST characterized by an imbalanced NAD+/NADH redox state resulting from deficient mitochondrial complex I (NADH: ubiquinone oxidoreductase) activity, which led to reduced ATP-linked and maximal mitochondrial respiration, increased glycolytic flux, and platelet hyperactivation compared with non-LST patients. Congruently, platelets from LST patients exhibited downregulation of tricarboxylic acid cycle and NAD+ biosynthetic pathways as well as upregulation of the proximal glycolytic pathway, a metabolomic change that was primarily attributed to compromised mitochondrial respiration rather than increased glycolytic flux as evidenced by the integrative analysis of bioenergetics and metabolomics. Importantly, both bioenergetic and metabolomic aberrancies in LST platelets could be recapitulated ex vivo by exposing the non-LST platelets to a low dose of rotenone, a complex I inhibitor. In contrast, normalization of the NAD+/NADH redox state, either by increasing NAD+ biosynthesis or by inhibiting NAD+ consumption, was able to improve mitochondrial respiration, inhibit mitochondrial oxidant generation, and consequently attenuate platelet aggregation in both LST platelets and non-LST platelets pretreated with low-dose rotenone. These data, for the first time, delineate the specific patterns of bioenergetic and metabolomic alterations for T2DM patients who suffer from LST, and establish the deficiency of complex I-derived NAD+ as a potential pathogenic mechanism in platelet abnormalities.
Introduction
Despite successive refinements in stent technologies, stent thrombosis (ST) remains the most severe complication of percutaneous coronary interventions (PCI) with high morbidity (up to 45%) and a high recurrence rate (15-20%) [1]. Recently, multiple registry studies and meta-analyses have reported type 2 diabetes mellitus (T2DM) as a strong predictor for ST, especially for late ST (LST) occurring more than 30 days after implantation [2][3][4]. Platelet dysfunction, as a hallmark of T2DM [5], may be the potential pathological basis linking T2DM with LST, due to its direct effects on thrombus formation in coronary stents [6].
Platelets, as cytoplasmic fragments of megakaryocytes that contain abundant mitochondria, are considered the most metabolically active organelle, even in the resting state [7]. Upon platelet activation, the cells Abbreviations: T2DM, type 2 diabetes mellitus; LST, late stent thrombosis; PCI, percutaneous coronary intervention; CI, complex I; OCR, oxygen consumption rate; ECAR, extracellular acidification rate. need a greater amount of chemical energy, most of which is from the oxidative phosphorylation (OXPHOS) occurring in mitochondria [8]. As mitochondrial impairment progresses during diabetes [9], platelets may initiate a complex sequence of bioenergetic changes involving altered mitochondrial complex activities [10], metabolic switch between OXPHOS and glycolysis [11], and oxidant overproduction [12], all of which may help maintain energy homeostasis but potentially increase platelet sensitivity to thrombotic stimuli [13]. In addition, these bioenergetic changes may also remodel the platelet metabolome, dysregulating the key metabolic pathways controlling platelet activation and aggregation [14]. However, despite the pivotal roles of mitochondrial bioenergetics in platelet function, very little is known about how platelet bioenergetics, including profiles of mitochondrial respiration and glycolysis, contributes to LST, especially in the context of T2DM. Notably, although studies of healthy platelets have suggested significant impacts of bioenergetic changes on the global metabolome [15], the exact role of the bioenergetics-metabolite interactome in platelet thrombotic function and LST risk is still largely unknown.
Herein, using a high-throughput extracellular flux analyzer, we first identified the specific pattern of bioenergetic changes in both the resting and activated states in platelets from T2DM patients who developed LST. Then, by performing untargeted metabolomics in the same population, we uncovered the dysregulated metabolic pathways underlying the bioenergetic aberrancies in LST platelets, and constructed the differential networks of platelet metabolomics and bioenergetics. Finally, we provided ex vivo evidence that the bioenergetic aberrancies observed in LST platelets were at least partially due to inhibition of mitochondrial complex I (CI) and further explored the possibility that targeting mitochondrial respiration by rebalancing the NAD + /NADH redox state might protect LST platelets against CI inhibition.
Study population
This was a nested case-control study within a prospective cohort of 1165 type 2 diabetic patients who received primary PCI from September 2017 to September 2019 at the First Affiliated Hospital of Zhengzhou University. The study design of the prospective cohort has been reported previously [16][17][18]. Briefly, the prospective cohort included subjects who had T2DM but did not suffer from other systemic diseases including cancer, hepatic insufficiency, serious infection, and type 1 diabetes. All participants underwent primary PCI for obstructive coronary artery disease at baseline, and then completed clinical follow-up at 30 days, 1 year, 2 years, and the final follow-up visit (in September 2021) after PCI. During a mean follow-up of 2.5 ± 0.3 years, a total of 15 patients with definite late (>30 days after PCI, n = 9) or very late (>1 year after PCI, n = 6) ST were identified and enrolled in the LST group. Based on the Academic Research Consortium criteria, definite ST was defined as angiographic confirmation of thrombus within the stent with acute onset of ischemic symptoms or ischemic ECG changes or typical rise in cardiac biomarkers [19]. The non-LST group included 15 diabetic patients who did not develop ST and other cardiovascular events (including death, repeated revascularization, myocardial infarction, and stroke) at least 2 years from PCI. LST and non-LST patients were matched on clinical and procedural data at baseline using the propensity score matching method (Table 1). To minimize the influence of antiplatelet therapy on platelet testing, the proportions of non-LST patients on dual antiplatelet therapy (80%, n = 12), aspirin alone (13%, n = 2), and clopidogrel alone (7%, n = 1) were set to exactly match those of the LST patients at the time of platelet isolation. In addition, 15 age-(62.1 ± 7.4 years) and sex-matched (male:female, 9:6) healthy subjects, who had no history of vascular diseases, diabetes, or other systemic diseases and had not used medications known to perturb platelet function in the prior 2 weeks before enrollment [20], were also recruited as healthy controls. Blood collection for platelet isolation was performed at the final follow-up visit for all participants. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. All participants gave written informed consent.
Platelet isolation
Platelet isolation was performed according to the Abcam protocol with minor modifications [20]. Briefly, whole blood samples were collected in citrate by standard venous puncture without a tourniquet to avoid platelet activation [21]. Platelet rich plasma (PRP) was obtained from whole blood by centrifugation (200 g for 20 min), diluted 1:1 with HEPES buffer (Gibco, cat# 15630080) containing 1 μM PGE 1 (also for preventing platelet activation, Sigma, cat# 900100P), and centrifuged (100 g for 20 min) to discard contaminated cell pellets. The obtained platelet rich supernatant was further centrifuged (800 g for 20 min) to pellet platelets, which were washed twice with platelet wash buffer
Platelet metabolomic profiling
Details of the metabolomic profiling have been described in our previous reports [25] (also introduced in the Supplementary Methods). Briefly, platelets (3 × 10 8 per well) were incubated with cold extraction solution (acetonitrile: methanol, 1:1, v/v) containing a mixture of internal standards, and centrifuged (13000 g, 15 min) to remove protein precipitates. The remaining metabolite extracts were further separated using a Vanquish UHPLC system equipped with an UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm). Mass spectral detection of the eluted samples was completed on a Q Exactive mass spectrometer (Thermo, Waltham, USA) operated in positive or negative ion mode. The acquired MS raw data were converted to mzXML files, which were uploaded onto the XCMS platform for feature detection and peak alignment [26]. Metabolite annotation was performed by matching the MS2 data of the detected samples to a reference library using the MetDNA webserver [27]. The raw counts for each metabolite were normalized using the best-matched internal standard normalization method [28]. Platelet samples were analyzed in randomized order, with a pooled quality control sample injected at the beginning and end of every 10 samples to qualify instrument performance ( Supplementary Fig. 1).
Platelet aggregation
Light transmittance aggregometry (Chrono-Log Model 700, Havertown, USA) was used to measure platelet aggregation under constant stirring (1000 rpm). Briefly, suspended platelets were mixed with 1 mM calcium (Sigma, cat# GF353280607) and 5% autologous platelet-poor plasma (as a source of fibrinogen) [29], followed by the addition of thrombin, ADP, or collagen. For each measurement, unaggregated platelets (0% light transmission) and blank buffer (100% light transmission) served as references [29]. Platelet aggregation was expressed as the maximal percent change in light transmittance over 6 min after stimulation with the agonists.
Flow cytometry for detecting platelet activation
The platelet surface expression of P-selectin was detected by flow cytometry to reflect the extent of platelet activation [20]. Briefly, suspended platelets (1 × 10 6 ) were incubated with APC-labeled anti-CD41a (Invitrogen, cat# 17-0419-42) and PE-labeled anti-CD62P (Invitrogen, cat# 12-0626-80) for 30 min, fixed with 1% paraformaldehyde HEPES saline, and centrifuged at 800 g for 5 min. The platelet pellets were resuspended in stain buffer, and analyzed on a FACSCalibur flow cytometer (BD Bioscience, Franklin Lakes, USA). Platelets were identified by their characteristic light scattering and specific binding of anti-CD41a. Isotype-matched antibodies were used to control non-specific fluorescence. Data are expressed as the percentage of 10, 000 CD41a + platelets expressing CD62P.
Detection of the rate of mitochondrial reactive oxygen species (mtROS) production
For quantification of mtROS, platelets were stained with 5 μM of the non-fluorescent probe dihydrorhodamine 123 (Sigma, cat# D1054), which can freely penetrate through the cell membrane, localize in mitochondria, and irreversibly react with mtROS to form fluorescent rhodamine 123 [30]. The fluorescence intensity of rhodamine 123 (λex/em: 490/530 nm) was continuously monitored on a PheraStar FS plate reader (BMG LABTECH, Ortenberg, Germany) every 4 min for 20 min [30]. The acquired raw data were fitted by linear regression to calculate the regression slope, which represented the rate of mtROS generation over time [31].
Biochemical assays for determining mitochondrial complex activity, the NAD + /NADH ratio, and ATP contents
The activities of mitochondrial complexes I-V were measured in suspended platelets using spectrophotometric methods as summarized by Rodenburg [32].
The concentrations of NAD + and NADH in platelets were measured with the EnzyFluo NAD/NADH assay (BioAssay Systems, cat# EFND-100), which utilizes a non-fluorescent probe to specifically react with NADH to form a fluorescent product. The fluorescence intensity of this product at λex/em = 530/585 nm was read by a PheraStar FS plate reader to quantify NAD + , NADH, and their ratio following the manufacturer's instructions.
The ATP concentrations of platelets were determined using the EnzyLight ATP assay (BioAssay Systems, cat# EATP-100). Briefly, platelets were lysed by a single working reagent to release ATP, which reacted with the ATP-dependent luciferin-luciferase system. The light intensity, as a direct measure of the ATP contents, was detected on a PheraStar FS plate reader within 1 min after adding the assay reagent.
Statistical analysis
For the bioenergetic data, individual group samples were compared using the unpaired t-test; ex vivo comparisons between the treatment group and the vehicle group were made by the paired t-test; multiple comparisons were performed using one-way ANOVA with the LSD post hoc test.
For the metabolomic data, a supervised model of orthogonal partial least-squares discriminate analysis (OPLS-DA) was conducted to compare the global differences between groups, and to assess the importance of each metabolite in OPLS-DA by calculating the Variable Influence on Projection scores. The goodness of fit and predictability of OPLS-DA were evaluated by a 200-permtation test. Differential comparisons of single metabolites were performed using the Wilcoxon ranksum test, with the Benjamini-Hochberg procedure to control the false discovery rate (FDR) [33]. Metabolites with FDR <0.05 and fold changes >4/3 or <3/4 were considered differentially abundant [34]. All the above analyses were conducted using R (v 3.5.3).
The integration of bioenergetics and metabolomics was performed using the xMWAS tool [35], in which sparse partial least-squares regression was conducted to calculate the pairwise correlations between bioenergetic and metabolomic features. Then, the multilevel community detection algorithm was executed to identify a series of clusters containing metabolic and bioenergetic nodes that were highly connected with each other (|r| > 0.4, P < 0.05). We used MetaboAnalyst 5.0 [36] to conduct metabolic pathway analysis for each bioenergetic feature independently. Significant pathways had to match at least 4 metabolites and have a P value of <0.05 [37].
Bioenergetics in resting platelets
For standardized testing, platelets of all study participants were freshly isolated, and seeded in XF DMEM at a density of 1 × 10 7 per well [15,22]. Both OCRs and ECARs of platelets were stable for at least 3 h (Supplementary Fig. 2A). Detecting the surface expression of CD62P did not reveal platelet activation induced by seeding or bioenergetic measurement ( Supplementary Fig. 2B).
Then, we sought to measure the mitochondrial respiration profiles in resting platelets ( Fig. 1 A and Supplementary Table 1). The basal respiration was first measured, then oligomycin (complex V inhibitor) was added to quantify OCRs linked to proton leak and ATP synthesis [7]. Diabetic patients with LST exhibited no significant alterations in basal respiration, proton leak, or ATP-linked respiration compared with diabetic patients without LST (non-LST, Fig. 1B) or healthy subjects (Supplementary Table 1). Further addition of FCCP for uncoupling electron flux resulted in a slightly lower rate (by 12%, Fig. 1B) of maximal respiration in LST platelets than in non-LST platelets. Final injection of rotenone (CI inhibitor) and antimycin A (complex III inhibitor) completely blocked the mitochondrial electron transport chain to allow monitoring of the non-mitochondrial OCR [7], which was not significantly altered in LST platelets. Additionally, we did not find significant differences in glycolytic profiles between the LST, non-LST, and healthy control groups ( Fig. 1C and D and Supplementary Table 1).
Bioenergetics in activated platelets
To further describe the bioenergetic characterization of platelets in the activated state, the Mitochondrial Stress test was also performed in thrombin-activated (0.1 U/mL) platelets. As presented in Fig. 2A and B, adding thrombin induced a subtle increase (by 28%) in basal OCR in non-LST platelets, while the thrombin-induced increase (by 5%, P = 0.007) in basal OCR was almost eliminated in LST platelets. Furthermore, we found a much stronger effect of oligomycin on inhibiting OCR and a much weaker effect of FCCP on releasing maximal OCR ( Fig. 2A), compatible with the substantial decreases in ATP-linked (P = 0.008, Fig. 2C) and maximal respiration (P = 1.8E-11, Fig. 2D), in platelets from LST patients. Consistent with a recent report by Avila et al. [38], we observed a slight but significant reduction in both basal and maximal respiration in diabetic patients without LST compared with those of healthy subjects (Supplementary Table 2), which suggested a certain degree of bioenergetic impairment in diabetic platelets. In the comparison of diabetic patients with LST with healthy subjects, the changes in platelet bioenergetics became more substantial, but the change patterns were generally similar to those in the comparison of LST with non-LST patients (Supplementary Table 2).
Considering that aerobic glycolysis may compensate for impaired mitochondrial respiration to sustain the energy need during platelet activation [39], we continued to monitor the glycolytic profiles of activated platelets. We first observed a strong effect of thrombin on increasing the basal ECAR (by 94%, Fig. 2E), which suggested an increased energy demand during platelet activation. Then, following the addition of glucose and oligomycin, activated platelets from LST patients displayed significant increases in both basal glycolysis and maximal glycolytic capacity compared with non-LST ( Fig. 2F and G) or healthy platelets (Supplementary Table 2). This was potentially the reason for the unchanged ATP contents in activated platelets from LST patients compared with those in non-LST platelets (Fig. 2H).
To investigate whether the aberrant mitochondrial respiration in LST platelets was linked with a specific enzymatic deficiency, we measured the enzymatic activity of each mitochondrial complex (I-V). Although the activities of complexes II-V were similar between LST and non-LST platelets, we did observe a significant reduction (by 33%, P = 1.3E-7, Fig. 2I) in CI activity in LST patients. Congruently, the CI-dependent OCR was also decreased by 30% (P = 1.1E-7, Fig. 2J and K) in LST platelets when respiring on CI-selective substrates. As CI is the major site controlling NADH oxidation [40], we reasoned that the reduced CI activity might alter the NAD + /NADH redox state. Indeed, platelets from LST patients exhibited a substantial reduction in NAD + levels, a subtle increase in NADH levels, and a more than 60% decrease in the NAD + /NADH ratio (P = 1.8E-6, Fig. 2L), accompanied by a higher production rate of mtROS (Fig. 2M).
In parallel with these bioenergetic aberrancies, thrombin seemed to induce greater platelet aggregation in LST platelets than in non-LST platelets, especially at low to intermediate doses ( Fig. 2N and Supplementary Fig. 3). A similar tendency for greater aggregation was also observed in LST platelets in response to ADP and collagen (Supplementary Fig. 3). As granule secretion plays essential roles in the amplification of platelet signaling and consequent aggregation [41], we continued to measure α-granule secretion by detecting surface P-selectin expression. Thrombin-induced P-selectin exposure was found to be significantly increased in LST platelets, further validating the hyperreactive status of LST platelets (Fig. 2O).
Partial inhibition of CI activity mimics the bioenergetic changes in LST platelets
To explore the mechanistic link of CI inhibition with platelet bioenergetic dysfunction, we analyzed the dose-response effects of CI inhibition by using different doses of rotenone to culture platelets from diabetic patients without LST (non-LST). Exposure to high-dose rotenone (500 nM) led to an almost complete loss of CI activity, accompanied by an entirely inverted ratio of NAD + /NADH, a more than 80% decrease in OCRs, and an up to 3-fold increase in mtROS (Supplementary Fig. 4). Such severe damage to mitochondrial bioenergetics has been reported to be more likely to induce platelet apoptosis rather than platelet activation [42], which was highly different from that observed in LST platelets. This might explain the observation of no significant effects of high-dose rotenone treatment on thrombin-, ADP-, or collagen-initiated platelet aggregation (Supplementary Fig. 4E).
In contrast, treatment with rotenone at a low dose of 50 nM caused a 34% reduction in CI activity (Fig. 3A), which was a fall similar to that in LST platelets. This degree of CI inhibition led to 20-60% decreases in ATP-linked OCR (by 40%), maximal OCR (by 24%), and the NAD + / NADH ratio (by 60%, Fig. 3B-E) as well as significant increases in glycolysis, mtROS, and platelet aggregation (Fig. 3F-J), thus mimicking the bioenergetic changes in LST platelets. Interestingly, the effect of lowdose rotenone exposure might be associated with diabetic status, because in healthy platelets, treatment with low-dose rotenone had a much smaller effect on the bioenergetic profiles and platelet aggregation ( Supplementary Fig. 5). Collectively, these data suggest that in the context of T2DM, partial but incomplete inhibition of CI activity can recapitulate the pattern of bioenergetic aberrancies observed in LST platelets.
Metabolomics in activated platelets
To further elucidate the metabolic changes underlying the bioenergetic aberrancy in LST patients, we further performed untargeted metabolomic profiling in platelets preactivated by thrombin. Similar to previous reports [15], we annotated up to 943 metabolites in activated platelets. The OPLS-DA plot depicted a significant difference in global metabolome profiles between diabetic patients with and without LST (Fig. 4A). The goodness of fit and predictability of OPLS-DA were validated by 200-permutation testing, which showed small variances in cross-validated Q 2 and R 2 (Fig. 4B). At the thresholds of FDR <0.05 and fold changes <3/4 or >4/3, a total of 155 metabolites were differentially abundant in LST versus non-LST platelets, with 84 downregulated and 71 upregulated metabolites ( Fig. 4C and Supplementary Table 3).
The downregulated metabolites strongly affected pathways of the tricarboxylic acid (TCA) cycle, NAD + biosynthesis, and glutamine metabolism (Fig. 4D). Specifically, almost all TCA cycle metabolites were significantly decreased in LST platelets (Fig. 4E). Of note, isocitrate, α-ketoglutarate, and malate, as the key metabolites of the TCA cycle involved in NADH generation, were among the top 5 downregulated metabolites (Supplementary Table 3). The downregulation of the TCA cycle in LST platelets was also accompanied by reduced abundances of several NAD + precursors (nicotinic acid, nicotinamide riboside, nicotinamide mononucleotide, and nicotinamide, Fig. 4F), which was in agreement with the observation of an imbalanced NAD + /NADH ratio in LST platelets. In addition, L-glutamine, as a precursor of α-ketoglutarate that fills the TCA cycle [22], was also downregulated (73%) in LST patients (Fig. 4D).
Then, we analyzed the effects of CI inhibition on the global metabolome in non-LST platelets. As shown in Fig. 5A and B, CI inhibition by low-dose rotenone led to a significant change in global metabolomic profiles, including differential changes in the abundances of 134 metabolites (78 downregulated and 56 upregulated metabolites, Supplementary Table 4), in rotenone-treated non-LST platelets. Of note, the rotenone-treated non-LST platelets shared up to 46% (n = 61) of differential metabolites with LST platelets (Fig. 5C). Specifically, rotenone treatment induced widespread downregulation of TCA cycle metabolites and NAD + precursors as well as upregulation of several proximal glycolytic metabolites (Fig. 5D), generating a metabolomic profile similar to that observed in LST platelets. Collectively, these results concur with the bioenergetic data, suggesting that both the bioenergetic and metabolomic aberrancies observed in LST patients are at least partially caused by deficient CI activity.
Integration of bioenergetics and metabolomics in activated platelets
We next sought to construct the integrative networks of bioenergetics and metabolomics using xMWAS. In diabetic patients without LST (non-LST), we identified a total of 313 highly connected nodes (|r| > 0.4, P < 0.05) between 175 metabolites and 10 bioenergetic features within 5 clusters (Fig. 6A and Supplementary Table 5). Of them, Clusters 1 and 2 encompassed 80 (46%) metabolites having high connections with 6 features of mitochondrial respiration, whereas Clusters 3-5 included 95 (54%) metabolites highly correlated with 4 glycolytic parameters. In contrast, there were much fewer metabolites (n = 58, 32% vs. 54%) connected with glycolytic function in LST platelets. Instead, strong correlations with mitochondrial respiration were identified in up to 125 metabolites within 4 clusters (68% vs. 46%, P = 1.6E-5, Fig. 6B and Supplementary Table 6). Congruently, when calculating the centrality measure to assess the importance of connected nodes, all 6 features of mitochondrial respiration had higher centrality values in LST platelets than in non-LST platelets (Supplementary Table 7), demonstrating a more important role of mitochondrial respiration in integrative networks of LST patients. Interestingly, when the non-LST platelets were pretreated with low-dose rotenone, the proportion of metabolites connected with mitochondrial respiration significantly increased to 72% (vs. 46%, P = 5.5E-8, Supplementary Fig. 6 and Supplementary Table 8), which was statistically more similar to that in LST platelets (68%, P = 0.38).
Then, we performed pathway enrichment analyses for metabolites within each cluster. In non-LST platelets (Fig. 6C), both mitochondrial respiration and glycolytic features were connected with a series of metabolic pathways, generally involving metabolism of carbohydrates (TCA cycle, PPP, glycolysis, etc.), vitamins (nicotinamide, folate), amino acids (alanine, aspartate, glutamate, etc.), and lipids (arachidonic acids, glycerophospholipids). In LST platelets (Fig. 6D), the impacts of mitochondrial respiration on metabolic pathways were still strong and broad, while pathways associated with glycolytic features were mainly limited to glycolysis-related pathways (i.e. glycolysis, gluconeogenesis, and PPP). Taken together, the integrative network analysis suggests that the metabolomic alterations in LST patients may be attributed primarily to mitochondrial respiratory impairment rather than increased glycolysis.
Protection against CI inhibition: role of mtROS scavenging and NAD + /NADH rebalance
Understanding how CI inhibition induces bioenergetic aberrancies may provide insight into the causal link between CI inhibition and platelet abnormalities in LST patients. One potential explanation is that CI inhibition causes increased production of mtROS, which may impair mitochondrial respiration and boost platelet aggregation [7,44]. However, treating LST platelets with MitoQ, a mitochondria-targeted ROS scavenger, failed to improve ATP-linked or maximal respiration but markedly blunted platelet aggregation ( Supplementary Fig. 7). These results validate the role of mtROS as a second messenger inducing platelet activation [7], but cannot establish the direct link between mtROS and impaired mitochondrial respiration.
Having observed that inhibiting CI could substantially decrease NAD + levels and disrupt the NAD + /NADH balance, we next questioned whether normalization of the NAD + /NADH ratio might reverse impaired mitochondrial respiration in LST platelets. To test this hypothesis, LST platelets were supplemented with NR, a NAD + precursor that maintains NAD + synthesis in platelets [45]. Indeed, exposure to 1 mM NR for 30 min was able to increase the NAD + /NADH ratio to a level similar to that in non-LST platelets (Fig. 7A). Moreover, NR supplementation effectively improved mitochondrial respiration while suppressing mtROS generation and platelet aggregation (Fig. 7C-G).
Another orthogonal method to increase NAD + bioavailability may be the inhibition of NAD + consumption [46]. Thus, we incubated LST platelets with olaparib, a clinically approved inhibitor of the major NAD + consuming enzyme PARP1 [47]. The inhibition of PARP1 by 20 μM olaparib successfully increased the NAD + /NADH ratio (Fig. 7B), accompanied by significant upregulation of mitochondrial respiration and partial inhibition of mtROS production and platelet aggregation in LST platelets (Fig. 7C-G). Importantly, a similar effect of NR or olaparib treatment on normalizing mitochondrial respiration, mtROS production, and platelet aggregation was also found in non-LST platelets pretreated with low-dose rotenone (Fig. 7H-J). Collectively, our data suggest that rebalancing the NAD + /NADH redox state may protect diabetic platelets against the proaggregatory effects of CI inhibition.
To further examine the relationship between NAD + metabolism, mtROS production, and platelet aggregation, we incubated the rotenone-treated non-LST platelets with MitoParaquat, a mitochondrial ROS generator that selectively induces redox cycling at CI [48]. As shown in Fig. 7H-J, addition of MitoParaquat (0.1 μM) abolished the inhibition of platelet aggregation by NR or olaparib, (Fig. 7H), but did not interrupt the NR-or olaparib-induced improvements in mitochondrial respiration (Fig. 7J). Together, our data support the idea that partial inhibition of CI may cause impaired homeostasis of NAD + metabolism first, which further induces the leakage of mtROS at CI and consequently boosts platelet aggregation.
Interestingly, although LST platelets had impaired mitochondrial respiration in the activated state, their total ATP contents were not decreased. The maintenance of ATP production in LST platelets might be attributed to the significant upregulation of glycolytic profiles. This metabolic shift to glycolysis, in the presence of impaired mitochondrial respiration, may represent a conserved immunometabolic pattern that essentially reflects the nature of the high metabolic flexibility of platelets [39,52]. Indeed, despite being a less efficient energy-producing pathway, glycolysis may better support a rapid need for ATP production in response to enhanced platelet aggregation in LST platelets [39].
Importantly, the imbalance of mitochondrial respiration and glycolytic function in LST platelets could be reflected at the metabolic level. To be specific, the untargeted metabolomics revealed a dramatic change in global metabolic profiles in LST platelets, with proximal glycolytic metabolites being raised and mitochondrial TCA cycle being substantially impaired. Considering that studying each metabolic pathway may not fully reflect the complexity of platelet metabolism [53], we further integrated metabolomics with bioenergetic data using a multi-omic approach, which may elucidate the differential networks of metabolic pathways in response to the given bioenergetic changes [14]. In the instance of LST platelets, a large number of metabolic pathways, involving a series of carbohydrates, vitamins, amino acids, and lipids, were found to be connected with the profiles of mitochondrial respiration, but few of them were correlated with glycolytic parameters. The non-LST platelets, by contrast, exhibited a distinct network composition that linked mitochondrial respiration and glycolytic profiles together through a number of shared metabolic pathways. These results are meaningful, because they promote the understanding of how the platelet metabolome interacts with bioenergetics, highlighting that in the context of LST, the metabolomic alterations of platelets are probably driven by impairment of mitochondrial respiration, but not by compensatory increase in glycolysis.
The observation of the substantial declines in both CI activity and CIdependent respiration in LST platelets may provide an enzymatic basis for the observed bioenergetic aberrancies. Furthermore, partial inhibition of CI in non-LST platelets by low-dose rotenone successfully recapitulated the bioenergetic aberrancies observed in LST platelets and significantly enhanced platelet aggregation, suggesting a possible mechanistic link between CI inhibition, bioenergetic aberrancies, and platelet hyperaggregability. CI, as the gatekeeper of the electron transport chain, catalyzes the oxidation of NADH, which provides up to 40% of the proton-motive force utilized for mitochondrial ATP production [54]. If the process of NADH oxidation is inefficient, protons may escape from CI and react with ambient oxygen to produce mtROS [40], which is closely related to platelet hyperactivation possibly through modulation of arachidonic acid metabolism [7]. Our study further supported the linkage between mtROS and platelet aggregation by showing that both mtROS and proaggregatory metabolites of arachidonic acids were upregulated in LST platelets and that scavenging mtROS could blunt rotenone-induced platelet aggregation.
Another direct consequence of CI inhibition may be the dysregulation of NADH oxidation, which damages NAD + turnover and causes an imbalanced NAD + /NADH redox state [55]. Generally, NAD + turnover is maintained by common controls of NAD + -regenerating (CI, LDH, and malate-aspartate shuttle), NAD + biosynthetic (salvage or de novo synthesis), and NAD + -consuming pathways (PARP1 and CD38) [56]. We decided not to modulate NAD + regeneration to balance the NAD + /NADH ratio, because targeting LDH or the malate-aspartate shuttle may exert additional impacts on glycolysis and OXPHOS [57]. Considering that platelets contain an enzymatic system to metabolize NR into NAD + [45], we directed our attention toward NR, a NAD + precursor that has cardioprotective effects in animal models of heart failure and cardiomyopathy [58,59]. We found that supplementation with NR could preserve mitochondrial respiration, in conjunction with the inhibition of mtROS production and platelet aggregation, in both LST and rotenone-treated non-LST platelets. Congruently, we observed a similar effect of reducing NAD + consumption by the PARP1 inhibitor (olaparib) against CI inhibition. These findings provide ex vivo evidence that pharmacologically targeting NAD + metabolism may be effective against platelet hyperaggregability induced by CI defects.
It is well known that diabetes may accelerate the production of ROS and damage mitochondrial function, possibly through transduction of the aldose reductase pathway [42,50]. Excessive ROS can then stimulate platelet aggregation through activation of the PLCγ2/PKC/p38α MAPK pathway, a hyperactive Ca 2+ response to stimulation, and further induction of thromboxane [7,50], while the severity of mitochondrial dysfunction may determine whether diabetic platelets are prone to be apoptotic (severe) or hyperactive (modest) [42]. Here, our study deepens the understanding of the relationship between impaired NAD + metabolism, mtROS overproduction, and platelet aggregation in an ex vivo model of mitochondrial dysfunctiondiabetic platelets with partial inhibition of CI. We found that pharmacological induction of CI-sourced mtROS abolished the NR-or olaparib-induced inhibition of platelet aggregation, while downregulating mtROS by MitoQ did not improve mitochondrial respiration. Taking all the ex vivo data together, we suggest that the NAD + /NADH imbalance resulting from CI inhibition may be the direct cause of mitochondrial respiratory dysfunction, whereas mtROS is more likely downstream of impaired mitochondrial bioenergetics, mediating the effects of dysregulated NAD + metabolism on platelet aggregation.
Our study is the first to report the mechanistic link between platelet mitochondrial respiration and LST, with comprehensive delineation of both bioenergetic and metabolomic characterizations in platelets from LST patients. Nevertheless, our study has limitations. First, although the LST and non-LST groups were carefully balanced with respect to a series of clinical, procedural, and pharmacological variables relevant to LST incidence, the case-control design still has the limitations inherent to retrospective inclusion of participants into the 2 groups. We adopted the case-control design mainly because of the rarity of ST occurrence and the large number of platelet samples that would be needed. Second, we elected to collect platelet samples at the end of follow-up, in order to monitor platelet bioenergetics in the stable quiescent phase, but not in the acute phase of the ST episode when many temporary changes of platelet bioenergetics occur [60]. This study design also allowed for a longer follow-up to preclude ST development in the non-LST group. However, it is, therefore, not possible to investigate the role of mitochondrial bioenergetics just prior to the ST episode in the thrombotic process of platelets. Third, antiplatelet treatment, as the standard of care after PCI, was applied in every included patient. Although the use of antiplatelet medications was matched between the LST and non-LST groups, the treatment strategy was still heterogeneous, ranging from dual antiplatelet therapy to aspirin alone and clopidogrel alone. Fourth, due to the reduced availability of platelet samples, some ex vivo experiments were performed only in 7-8 samples in each group. Finally, out study was nested within a prospective cohort of type 2 diabetic patients. This may limit the generalizability of our findings to nondiabetic subjects.
Conclusions
Collectively, our study reveals that platelets from type 2 diabetic patients who developed LST possess a specific pattern of bioenergetic aberrancies characterized by an imbalanced NAD + /NADH redox state, reduced mitochondrial respiration, increased glycolysis, and mtROS overproduction. Subsequent ex vivo experiments suggest that this pattern of bioenergetic aberrancies is probably driven by CI inhibition and leads to platelet hyperaggregability. Further integration of bioenergetic data with metabolomics uncovers the metabolic pathways underlying the observed bioenergetic aberrancies in LST patients, which may have significant implications for using the bioenergetics-metabolite interactome to elucidate the pathogenesis of complex conditions such as LST.
Funding
This study was supported by the National Basic Research Program of China (82170343 and 81800317 to X.W.) | 2022-10-13T15:11:32.622Z | 2022-10-01T00:00:00.000 | {
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245529097 | pes2o/s2orc | v3-fos-license | Foot Wounds and the Reconstructive Ladder
Background: Foot soft tissue coverage represents a challenge to reconstructive surgeons due to a lack of donor sites for this specialized skin. This glabrous tethered thick skin is designed to withstand weight bearing stress and is hard to replace. The limited arch of rotation of foot local flaps contributes to further difficulties. In this study, we share our experience in foot soft tissue loss coverage using techniques tailored to each wound presentation. Methods: This case series presents eight patients with wounds of the plantar and dorsal surfaces of the foot, heel, and ankle. Closure techniques were selected and planned based on wound presentation and comorbidity status. Results: Patients’ mean age at surgery was 61 years. Etiologies of wounds include trauma, frostbite, diabetic ulceration, malignancy, pressure ulcer with osteomyelitis, and necrotizing infection. Coverage techniques included split and full-thickness skin graft, medial plantar arch pinch graft, cultured epithelial autograft, Hyalomatrix wound device, EpiFix tissue matrix, pedicle flap, and free rectus flap. Complete soft tissue coverage was achieved in each case within reasonable postoperative periods, and ambulation was preserved and/or restored. Conclusions: Foot soft tissue reconstruction is challenging and should be planned carefully due to the required specialized skin replacement. Primary closure should be considered first and attempted if possible. Technique escalation in accordance with the reconstructive ladder should be undertaken based on wound etiology, presentation, amount and nature of tissue loss, available resources, and surgeon experience.
INTRODUCTION
Soft tissue reconstruction on the foot is a unique surgical challenge for several reasons. The function of the plantar foot as the surface of weight bearing and ambulation means that the skin requires a distinctive composition capable of withstanding the pressure and force associated with bipedal movement. The skin of the plantar surface of the foot is a thick, glabrous skin and is largely free of melanocytes, and contains a substantially thickened stratum corneum. Additionally, it contains cytokeratin 9 (CK9), a cytokeratin species found exclusively in the suprabasal layers of the plantar foot and palm of the hand. 1 The dermis and epidermis of the plantar foot are also firmly tethered to the underlying plantar fascia via fibrous connective tissue, another adaptation to the trauma associated with ambulation. 2 The unique composition and microanatomy of the plantar surface of the foot, combined with limited donor-site availability, makes it difficult to adhere to the fundamental reconstructive tenet of "replacing like by like." 1,3 Skin of this composition is exclusive to the sole of the foot and the palmar surface of the hand, and the challenge is further compounded by the limited arch of rotation of local plantar flaps. Thus, restoration of the plantar foot's full functionality following trauma, including withstanding weight and compression during ambulation, is an elusive goal. Defects of the heel and ankle present a substantial challenge to surgeons as well. In addition to being of a unique, intricate form, these structures are the crux of ambulation and movement and, thus, play an integral functional role. 4 Foot injury can present as a result of direct trauma, surgical excision of malignancies and growths, infection, ischemia, diabetic ulcers, burns, and more. 5 There are numerous treatment modalities and strategies for soft tissue reconstruction depending on the nature and extent of foot trauma. In this case series, we present techniques for foot soft tissue coverage, including tissue replacement products, cultured epithelial autografts, split and fullthickness grafts, and pedicle and free flaps.
CASE 1
Here, we present a case of a 65-year-old man with diabetes with a nonhealing, 6-week-old right foot ulcer. This ulcer spanned roughly one-third of the dorsum of the foot and contained exposed tendons. EpiCord was applied following debridement in the OR. Figure 1A shows EpiCord covering the wound 2 weeks after surgery. One week later, the tendons were partially covered. Two applications of EpiFix were performed in the next month in the outpatient clinic (Fig. 1B). Complete healing was achieved in 8 weeks (Fig. 1C). A satisfactory aesthetic outcome was achieved, and the patient was fully ambulatory following wound closure.
CASE 2
In this case, we present a 67-year-old man with diabetes mellitus type II with peripheral neuropathy S/P surgical debridement of a necrotizing soft tissue infection. He was referred to plastic surgery for skin grafting and amputation of the left fourth toe. He had partially exposed necrotic extensor tendons shown in Supplemental Digital Content 1. (See figure, Supplemental Digital Content 1, which shows the wound bed preparation for skin grafting with hyaluronic acid-based matrix. A, Partially exposed necrotic extensor tendons of left foot. B, Two weeks later, all wounds were covered with granulation tissue and were ready to receive skin grafts. C, Wounds 10 days after receiving split-thickness skin grafts. D, Healed wounds, including the fourth toe at 4 weeks post grafting and 9 weeks from presentation. http://links.lww.com/ PRSGO/B926.) The medial left ankle and leg wounds were partially covered with slough. The patient was taken to the OR and underwent debridement of necrotic tissue, which included part of the extensor tendons followed by application of a hyaluronic-acid-based 3D matrix covered with a silicone layer. Five days postoperatively, the patient developed a Pseudomonas infection treated with oral ciprofloxacin and twice-a-day dressing changes using wet to moist one-fourth strength Dakin's solution and gauze after removing the silicone sheets. Two weeks later, another application of hyaluronic acid matrix was done at the wound center over the partially exposed tendon of the fourth toe. Two weeks later, all wounds were ready to receive skin grafts (Supplemental Digital Content 1B). Supplemental Digital Content 1C shows the wound 10 days after split-thickness skin grafting. All wounds were healed, including the fourth toe, at 4 weeks post grafting and 9 weeks from presentation (Supplemental Digital Content 1D).
CASE 3
Simman et al presented a case of a 30-year-old man presenting with severe bilateral frostbite of the feet requiring necrotic tissue debridement to the level of the plantar fascia ( Fig. 2A). The authors cultured palmar keratinocytes taken from a biopsy of the hypothenar area, and applied a palmar keratinocyte autograft to cover the patient's right sole (Fig. 2B), while they used a split-thickness graft from the thigh for the patient's left sole and the remaining open area of the right forefoot. 1 Each sole was fully covered, and the patient was fully and freely ambulatory Fig. 1. treatment of a diabetic foot ulcer with epicord and epiFix. a, epicord 2 weeks after application over exposed tendons of the right foot. B, epiFix is being applied. c, Healed wound 8 weeks after presentation.
Takeaways
Question: What approaches can plastic surgeons take when presented with complex wounds of the foot?
Findings: By tailoring reconstructive techniques to each of eight unique foot wound presentations, we were able to provide complete coverage and preserve ambulatory capability using various modalities, including tissue replacement products, cultured epithelial autografts, split and full-thickness grafts, and pedicle and free flaps Meaning: Despite the unique challenges associated with reconstruction of the plantar foot, heel, and ankle, plastic surgeons have a multitude of treatment modalities at their disposal to achieve a durable, lasting, and aesthetically pleasing result.
with orthotic shoes 5 months later. Biopsies from each sole were taken for histological and immunohistochemical analysis after each graft was completely healed. The cultured graft displayed histochemical features consistent with the thick, glabrous skin of the palmar and plantar surfaces, including a thickened stratum corneum, absence of melanocytes and the presence of cytokeratin 9. The split-thickness thigh graft did not show these characteristics. Both grafts healed in the same amount of time, and both were durable and capable of bearing weight and withstanding the forces of ambulation.
CASE 4
In a previous case report by Simman, a 45-year-old male patient presented with a traumatic wound of the right lateral heel. 5 For this wound, primary closure was not possible, and healing by secondary intention would have involved excessive time, pain, and potential scarring. Split-thickness pinch grafts were taken from the MPA of the foot ipsilateral to the wound (to localize any potential morbidity to one foot and retain full integrity on the contralateral side). 5 The medial arch bears little to no weight relative to other areas of the sole, making it an optimal donor site for a graft. 6 The wound was covered with 12 pinch grafts. Figure 3A shows the pinch grafts 1 week postapplication. The donor site healed in 10 days, and full wound coverage was achieved in 14 days when the grafts reached confluence. Figure 3B shows the healed wound 1 month later. Both recipient and donor sites received Xeroform gauze and Kerlex roll for coverage. The patient showed pain-free ambulation, with little reservations regarding weight placed on the operative sole. The grafts also displayed features consistent with the surrounding native skin at 14-weeks follow up, matching it with respect to pigmentation and texture, and were free of scarring, contracture, and complications of any nature. Patient survey indicated strong aesthetic results and high patient satisfaction. Additionally, pain completely disappeared within 3 weeks after surgery and sensation returned to the graft area within 6 months. 5
CASE 5
Here, we present a relatively unusual scenario of classical Kaposi sarcoma (KS) developing in an older woman in the absence of HIV or other immunocompromised status. This case involves a 91-year-old woman with recurrent bleeding lesions on the right plantar forefoot and great toe. (See figure, Supplemental Digital Content 2, which shows the full-thickness skin graft to cover plantar forefoot excision site. A, Bleeding KS at the right plantar forefoot and great toe. B, S/P excision and full-thickness skin graft 6 weeks later. http://links.lww.com/PRSGO/ B927.) Biopsy of the lesions showed KS. She also tested positive for HHV 8. She underwent excision of the lesions, and defects were subsequently closed with a full-thickness skin graft. The graft healed 6 weeks later (Supplemental Digital Content 2B). In a case of classic KS such as this, in which the cutaneous lesions are localized and benign, local treatment was most effective. 7 Surgical excision was performed in this case, which provided an aesthetically satisfactory result and allowed the patient to retain ambulatory capability.
CASE 6
This case involves a 56-year-old man S/P Whipple procedure for pancreatic cyst removal. He was placed on heparin postoperatively and developed HIT (heparin induced thrombocytopenia) syndrome. He was placed on IV Argatoban and stabilized. He was then sent to the wound clinic with dry gangrenes shown here and was subsequently cleared by vascular surgery. Then he underwent left transmetatarsal amputation and right foot fifth and fourth ray amputations. After a few months, his right foot wounds were not healing (Fig. 4A). Due to residual chronic osteomyelitis in his metatarsal bones, chronic pain, and forefoot deformity shown here, he was offered below knee amputation or Lisfranc amputation. He chose the latter, and his wound was closed with an MPA myocutaneous rotational flap. He healed 6 weeks later and was ambulating with orthotic shoes (Fig. 4B).
CASE 7
Here we present a 67-year-old man with diabetes with a history of CABG who presented with right forefoot gangrene. Vascular status was assessed and found to be satisfactory. He was admitted and placed on IV antibiotics and underwent excision of the necrotic area, which included soft tissue and the distal first metatarsal head with osteomyelitis (Fig. 5A). PICC line was placed for the administration of IV vancomycin to treat his staphylococcus infection. Daily dressing changes were done using wet and moist N/S and gauze. Two weeks later, he was taken back to the OR and his wound was debrided, the proximal part of the defect was closed with undermined skin flaps over a drain and the fillet of right great toe pedicle flap was used to close the distal part of the defect (Fig. 5B). The wound healed a month later and the patient was able to ambulate (Fig. 5C).
CASE 8
Here, we present a 65-year-old man with a history of spinal chordoma, producing bilateral neuropathy of the lower extremities that led to pain, weakness, and numbness. He was admitted to the hospital with a left heel stage IV pressure ulcer with necrosis and osteomyelitis of the calcaneus (Fig. 6A, B). He received IV antibiotics via PICC line and underwent excisional debridement, which included a partial calcanectomy. Dressing changes using calcium alginate silver were started with complete offloading of the heel. Several weeks later, he was taken back to the OR and underwent right rectus muscle free flap and split-thickness skin graft over the muscle. The flap healed 3 weeks later. He subsequently received physical therapy and was then able to ambulate with a walker and braces a few weeks later.
Placental Matrix Replacement in Diabetic Foot Ulcers (EpiCord, EpiFix)
The EpiFix allograft is a placenta-derived tissue matrix obtained from dehydrated human amnion/chorion membrane that is rich in growth factors, cytokines, and metalloproteinase inhibitors, which are conducive to cell proliferation and wound coverage. 6 EpiFix has also demonstrated competency in the recruitment of human mesenchymal, adipose-derived, and hematopoietic stem cells in vitro, and has been shown to upregulate both their migration and activity. 8 By stimulating endogenous wound healing pathways, EpiFix has a role as an adjunct to the standard of wound care and a tool for expedited coverage of chronic, nonhealing wounds. Randomized, multicenter clinical trials have concluded that allografts such as EpiFix are superiorly effective in providing timely, complete closure of chronic diabetic and venous ulcers of the lower extremity compared with conventional methods. [9][10][11] The lifetime risk of foot ulceration for diabetic patients is estimated to be about 15%. Roughly 60%-80% of diabetic foot ulcers will heal via standard approaches, with the remainder progressing to chronicity, putting many patients at risk for amputation. 12 Dehydrated human amnion/chorion membrane tissue matrices have potential value as a reinforcement when the standard of care fails, thus presenting an opportunity to improve outcomes for diabetic patients.
Hyaluronic Acid-based Matrix Replacement (Hyaff) over Exposed Tendons
Skin grafts placed over complex wounds containing exposed musculoskeletal structures in the absence of a perfused, granulating wound bed can result in contracture, deformity, and graft failure. [13][14][15] Additionally, factors such as age and comorbidity can preclude a patient from being a suitable candidate for free flap microsurgery, which is often used in these wounds. 13,15 Esterified hyaluronic acid matrix technology provides an alternative avenue for closure of complex lower extremity wounds in patients for whom flap coverage is contraindicated. Hyaluronic acid is a glycosaminoglycan that is found throughout the body as a principle component of extracellular matrix and joint capsules, a cell signaling molecule with receptors on almost all human cells, and a mediator in inflammation, immune response, and tissue repair. 16 It has been posited that its roles in wound repair include cell migration and infiltration, inflammatory attenuation, and stimulation of angiogenesis, in addition to its role as a key constituent of extracellular matrix. 17,18 Hyalomatrix is an esterified hyaluronic acid matrix product consisting of esterified hyaluronic acid, called Hyaff, and an outer silicone sheet. 19 This matrix, when placed on a tissue bed with exposed tendons that is devoid of granulation tissue, helps generate a richly vascularized scaffolding for grafting and ultimately wound closure. 15
Grafting Techniques
Coverage of large, full-thickness injuries to the sole of the foot can be achieved via cultured epithelial autograft techniques. This technique can provide large epithelial sheets from relatively small tissue samples, and is a useful alternative in cases where there is limited donor-site availability, defects are large, there are several traumas present, or when the patient is not a suitable candidate for long, invasive surgery. 1,20 MPA pinch grafts are effective in resurfacing wounds of the heel, toes, thumb, and palm of the hand. However, this technique is limited in its application. The donor site itself is limited, as the medial arch cannot yield enough soft tissue to provide full coverage of large defects. 5 Additionally, for defects on the plantar surface of the foot containing exposed vessels, tendons, or bone, flap coverage is necessary. 21 Still, there remains a place for splitthickness grafts such as the MPA pinch graft in the foot reconstructive ladder.
Right MPA Flap after Lisfranc Amputation
The MPA flap is derived from the MPA of the foot, receiving vascular supply from the medial plantar artery and innervation from cutaneous branches of the medial plantar nerve. 22 Current literature describes the MPA flap and the reverse sural artery flap as the two most prominent modalities for hindfoot reconstruction. 23 The MPA flap provides the glabrous skin of the plantar foot that is crucial in weight bearing and ambulation. Additionally, the MPA flap remains sensate and provides protective sensation to the covered area in the majority of reconstructions. [23][24][25] Finally, the MPA flap is associated with lower rates of complication and avoids latent venous congestion and edema that often occurs in patients years after receiving a sural artery-based reconstruction. 24,25 In the first systematic review of MPA-based reconstruction, Opoku-Agyeman et al found a 98.2% flap survival rate, 9.4% flap complication rate, 5.2% donor-site complication rate, and retention of protective sensation in 99% of MPA flap reconstructions. 23 Thus, several features of the MPA flap confer distinct advantages to this approach.
Fillet of Toe Flap
Fillet flaps are a product of the "spare parts" concept, in which limbs or digits that cannot be salvaged are repurposed as a source of material for the repair of large defects. 26 In this case, amputation of the patient's hallux was necessary due to extensive forefoot necrosis and surgical debridement of the distal first metatarsal head and surrounding soft tissue. However, his vascular supply was good, and his digit was repurposed into a well perfused, ideally suited source of soft tissue for reconstruction of his forefoot defect. Fillet flaps such as the one used herein are a strong choice for coverage of forefoot defects because they provide comparable tissue, have limited donor-site morbidity, and avoid many of the complexities associated with a forefoot reconstruction of this nature. 27
Free Flap Coverage of Foot Defects
Treatment of extensive calcaneal osteomyelitis with soft tissue involvement and skin ulceration continues to challenge reconstructive surgeons. Systematic review of the various therapeutic strategies that exist for this have struggled to identify specific bone treatment, soft tissue coverage, and wound healing modalities that lead to superior results relative to others. 28 Although below the knee amputation is often considered the definitive treatment, partial and total calcanectomy have been identified as viable, potentially limb-sparing alternatives. Furthermore, partial calcanectomy has been associated with lower rates of postoperative complications and secondary amputation compared with total calcenectomy. 29,30 Following extensive debridement and partial calcanectomy, a free flap is often necessary to completely permeate the vacated space with well-vascularized tissue. 28 Given that calcaneal osteomyelitis combined with heel ulceration is often a multifactorial disease process that is hindered by comorbidity, significant prognostic variation exists in these cases. More investigation is necessary and future efforts should aim to develop a more standardized, empirically backed algorithm for management.
CONCLUSIONS
Foot soft tissue reconstruction can be a puzzling task for reconstructive surgeons. Defects in this area can be complicated by comorbidity, vascular insufficiency, neuropathy, and superimposed infection. Furthermore, this area is anatomically and histologically unique due to the presence of specialized glabrous skin, a thickened stratum corneum, thickened fascial layers and unique molecular composition, including lack of pigmentation and presence of CK9. We have highlighted eight cases of foot soft-tissue defects resulting from trauma, surgical excision of malignancy, diabetic ulceration, vascular deficiency, frostbite and more, and have detailed various reconstructive modalities for their correction. Although cases are heterogenous in presentation, and outcome and case series are limited in their application, we understand the limitations and do not intend to generalize our results. Rather, we intend to document unique situations and demonstrate the therapeutic modalities that experienced reconstructive surgeons are armed with that can be effective in treating the broad range of defects observed in the foot. | 2021-12-29T16:07:52.492Z | 2021-12-01T00:00:00.000 | {
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18521362 | pes2o/s2orc | v3-fos-license | Actin-myosin–based contraction is responsible for apoptotic nuclear disintegration
Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.
Introduction
Apoptosis leads to the death and subsequent removal of damaged or redundant cells. Cysteine-proteases called caspases are responsible for the apoptotic "execution" phase, which is characterized by morphological changes including cell contraction, dynamic membrane blebbing, and nuclear disintegration. Contractile force generated by actin-myosin cytoskeletal structures is the driving power behind cell contraction and the formation of membrane blebs (Coleman and Olson, 2002). In the apoptotic cell, the disintegrated nucleus is found within blebs and packaged into membrane-clad apoptotic bodies that facilitate uptake by neighboring cells or by specialized phagocytic cells. The release of nuclear fragments from apoptotic cells is believed to be the source of antigens in autoimmune diseases such as systemic lupus erythematosus (Rosen and Casciola-Rosen, 1999;Stollar and Stephenson, 2002).
The dynamic contraction and membrane blebbing seen in apoptotic cells are dependent on intracellular force generated by the actin-myosin cytoskeleton. These morphological events are controlled by the Rho effector ROCK I, a serine/ threonine kinase that plays a key and central role in the regulation of actin cytoskeletal structures. We and others showed that caspase-mediated cleavage of ROCK I results in constitutive activation and consequent myosin light chain (MLC) phosphorylation leading to contraction and membrane blebbing (Coleman et al., 2001;Sebbagh et al., 2001). Inhibition of ROCK activity with the small molecule inhibitor Y-27632 attenuated blebbing in a variety of cell types, independent of the type of apoptotic stimulus. Inhibition of ROCK activity also prevented the relocalization of fragmented DNA into membrane blebs and apoptotic bodies (Coleman et al., 2001), suggesting additional roles for ROCK in the morphological changes that occur during apoptosis.
In addition to the gross external morphological responses, there are significant effects on the morphology and integrity of organelles, the most obvious being nuclear disintegration. Separating the nucleus from the cytoplasm is the nuclear envelope, which is comprised of outer and inner nuclear membranes. Giving the nucleus form, structure, and rigidity is a filamentous meshwork called the lamina, which is made up from intermediate filament A-type (A and C, alternately spliced products from the Lmna gene) and B-type (B1, B2, and B3) lamins. Caspasemediated cleavage of lamins A/C and B1 is thought to contribute to nuclear fragmentation during apoptosis (Neamati et al., 1995;Rao et al., 1996;Broers et al., 2002).
Ultrastructural analysis has shown that the nucleus is surrounded by a meshwork of actin (Clubb and Locke, 1998b), with "knots" of actin physically associated with the nuclear envelope (Clubb and Locke, 1998a). Disruption of the actin cytoskeleton alters nuclear morphology (Zhen et al., 2002), while mutations to Anc-1/Syne family actin-binding proteins result in aberrant nuclear anchoring (Starr and Han, 2003), indicating that the actin cytoskeleton influences nuclear positioning, shape, and structure. Therefore, one possibility is that during apoptosis, active caspase-cleaved ROCK I leads to shortening of actin-myosin filaments that are tethered to the nucleus at one end, resulting in nuclear envelope tearing and disintegration, thereby allowing for the relocalization of fragmented DNA to membrane blebs and apoptotic bodies (Coleman et al., 2001). Mitotic nuclear envelope breakdown also requires weakening of the nuclear lamina and a pulling force, but is mediated by phosphorylation-induced depolymerization of the nuclear lamina (Heald and McKeon, 1990) and microtubule-anchored pulling force generated by the minus-end-directed motor, cytoplasmic dynein, and components of its associated regulatory complex, dynactin (Beaudouin et al., 2002;Salina et al., 2002).
In this work, we examined the contribution of ROCK activity and MLC phosphorylation to nuclear disintegration during apoptosis. We found that ROCK activity, intact actin filaments, MLC phosphorylation, and MLC ATPase activity are each required for the breakdown of nuclear structure, whereas intact microtubules are dispensable. Caspase-mediated cleavage of lamins A/C and B1 is not sufficient for nuclear disintegration in the absence of ROCK and MLC ATPase activity. In addition, conditional activation of ROCK I induces nuclear breakdown in nonapoptotic cells only in the absence of lamin A/C expression. These results indicate that apoptotic nuclear breakdown requires weakening of the nuclear lamina by proteolytic cleavage and the contractile force generated by ROCK on actin-myosin filaments. Thus, apoptotic nuclear breakdown parallels mitotic nuclear breakdown in the requirements for lamina disassembly and generation of pulling force, but differs in the means by which these events are achieved.
ROCK activity and nuclear disintegration
We previously observed by TUNEL that in apoptotic cells treated with the ROCK inhibitor Y-27632, fragmented DNA was located within discrete foci and not in membrane blebs or apoptotic bodies as in untreated apoptotic cells (Coleman et al., 2001). Therefore, in addition to plasma membrane blebbing, we hypothesized that ROCK activation by caspase-mediated cleavage promotes apoptotic nuclear disintegration, thereby allowing fragmented DNA to be released from the nuclear compartment.
We used transmission EM (TEM) to obtain fine-detailed photomicrographs of nuclear morphology during apoptosis. NIH 3T3 mouse fibroblasts were treated with 25 ng/ml TNF ␣ plus 10 g/ml cycloheximide (CHX) for 2 h as the apoptotic stimulus, and then processed for TEM. A typical apoptotic cell is shown in Fig. 1 A, with membrane blebs, chromatin condensation, and a significantly fragmented nucleus with large invag-inations of the nuclear envelope and evidence of electron-dense heterochromatin within the cytoplasm and in blebs ( Fig. 1 A , arrows). In contrast, cells given the ROCK inhibitor Y-27632 (10 M) in conjunction with TNF ␣ /CHX did not bleb and the nucleus remained intact, although chromatin condensation and nuclear envelope dilation were evident ( Fig. 1 B). Consistent with our previous observations (Coleman et al., 2001), heterochromatin was retained within the nucleus, indicating that the nuclear envelope remained a barrier to macromolecules.
Phosphorylation of ROCK substrates
We and others previously demonstrated that ROCK I becomes cleaved and activated in apoptotic cells (Coleman et al., 2001;Sebbagh et al., 2001). The cleavage of ROCK I ( Fig. 1 C, cleaved form indicated by gray arrow and ⌬ ) is accompanied by increased phosphorylation of type II MLC on residues Thr18 and Ser19 ( Fig. 1 D), which is critical for association of MLC with actin filaments and generation of contractile force. MLC phosphorylation could be blocked with the ROCK inhibitor Y-27632 ( Fig. 1 D) or by blocking ROCK I cleavage ( Fig. 1 C) by pretreating cells with the caspase inhibitor z-VAD-fmk before the induction of apoptosis with TNF ␣ . The myosin binding subunit (MYPT1) of the MLC phosphatase has been reported to be phosphorylated by ROCK on Thr696, which results in inhibition of MLC dephosphorylation (Feng et al., 1999;Kawano et al., 1999). Using a phospho-specific antibody against Thr696, no significant change in phosphorylation was observed ( Fig. 1 E, top); however, one major and one minor ( Fig. 1 E, gray arrows) proteolytic fragment were observed. MYPT1 cleavage was not apparently changed by Y-27632 but could be prevented with z-VAD-fmk. An antibody raised against the amino terminus of MYPT1 revealed a loss of immunoreactivity in apoptotic cells, indicating that the cleavage occurred at the amino-terminal end ( Fig. 1 E, bottom). Analysis of protein mobility on SDS-PAGE revealed that ف 80 amino acids were removed from the cleaved fragment. Deletion analysis of MYPT1 has shown that the amino-terminal region is responsible for binding both MLC and the catalytic subunit of the phosphatase complex. Therefore, cleavage of the ف 80 amino acids from the MYPT1 amino terminus would likely inactivate the MLC dephosphorylating activity and commit the apoptotic cell to irrevocable MLC phosphorylation.
Additional downstream substrates of ROCK that may contribute to actin-myosin contractility are the LIM kinases (LIMK) 1 and 2 (Ohashi et al., 2000;Sumi et al., 2001). Overexpression of LIMK2 has been reported to induce the formation of stress fibers and membrane blebs (Amano et al., 2001), suggesting that LIMK1/2 activation contributes to the apoptotic morphology. Using an antibody that detects phosphorylation of a Thr residue in the conserved activation loops of LIMK1 (Thr508) and LIMK2 (Thr505), we observed increased phosphorylation following the induction of apoptosis, which could be blocked with ROCK inhibitor Y-27632 or by inhibition of ROCK I cleavage with z-VAD-fmk ( Fig. 1 F). These results indicate that LIMK activation may contribute to ROCKmediated morphological effects in apoptotic cells.
We also examined how the induction of apoptosis affected the phosphorylation state of Ezrin, which acts as a linker between actin filaments and transmembrane proteins such as CD44, thereby providing an anchoring point in the plasma membrane for actin-myosin fibers. ROCK-induced contraction of NIH 3T3 cells was reported to be dependent on phosphorylation of Ezrin Thr567 (Tran Quang et al., 2000), which allows for dissociation of inter-and/or intramolecular interactions and association with actin filaments . Using an antibody specific for Ezrin phosphorylated on Thr567, no changes in the phosphorylation status were observed in apoptotic cells or after Y-27632 treatment ( Fig. 1 G), indicating that ROCK-mediated phosphorylation of Ezrin on Thr567 does not contribute to apoptotic cell contraction. Treatment of cells with the general kinase inhibitor staurosporine (0.2 M) for 30 min eliminated Ezrin T567 phosphorylation, suggesting that kinases other than ROCK are responsible for basal phosphorylation.
Nuclear disintegration and the actin and microtubule cytoskeletons
Next, we determined whether or not, in addition to ROCK activity, an intact actin cytoskeleton was required for nuclear disruption. Pretreatment with the actin-destabilizing compound cytochalasin D (2 M; Sampath and Pollard, 1991) for 2 h before treatment with TNF ␣ /CHX disrupted the actin cytoskeleton ( Fig. 2 A) and blocked fragmentation of the nucleus (Fig. 2 B). Similar results (unpublished data) were obtained when the actin cytoskeleton was disrupted with swinholide A (0.1 M; Bubb et al., 1995) or latrunculin B (0.5 M; Spector et al., 1983). These results indicate that an intact actin cytoskeleton is necessary to mediate nuclear disintegration. Disruption of the cytoskeleton with cytochalasin D did not affect caspase 3 activation ( , the common epitope of phospho-LIMK1 (Thr508; F) and phospho-LIMK2 (Thr505; F), and Ezrin (G, bottom) and phosphorylated Ezrin (Thr567; G, top). Phosphorylation of MLC and LIMK1/2 in apoptotic cells was blocked by ROCK inhibitor Y-27632 and by preventing caspase-mediated ROCK I cleavage with z-VAD-fmk. No differences in MYPT1 or Ezrin phosphorylation were evident, although MYPT1 was cleaved in a z-VAD-fmk-sensitive manner in apoptotic cells, removing 08ف amino acids from the amino-terminal end.
To determine whether or not an intact microtubule network is required for apoptotic nuclear breakdown, microtubules were disrupted by pretreatment with nocodazole (1 M) for 2 h before treatment with TNF ␣ plus CHX for an additional 2 h. Despite efficient disruption of the microtubule network ( Fig. 3 B), nocodazole treatment did not prevent apoptotic membrane blebbing or nuclear breakdown (Fig. 3 C). These data indicate that nuclear disruption in apoptotic cells differs from mitotic nuclear breakdown in that the actin, and not microtubular, cytoskeleton provides the framework for mechanical force that leads to nuclear tearing and rupture.
MLC phosphorylation and ATPase activity
Intact actin filaments and MLC phosphorylation have been shown to be required for plasma membrane blebbing (Coleman and Olson, 2002). Given that ROCK I is cleaved and MLC phosphorylation is elevated in apoptotic cells, the requirement for MLC phosphorylation in apoptotic nuclear disintegration was examined. Expression of MLC with alanine substitutions at Thr18 and Ser19 has previously been shown to block actin bundling and contractility (Iwasaki et al., 2001). NIH 3T3 cells were transfected with plasmids encoding GFP, wild-type rat MLC with carboxyl-terminally fused GFP, or the MLC(TASA) double mutant fused with GFP ( Fig. 4 A). Approximately 10-12% of GFP-positive cells were selected (Fig. 4 A, R3), all of which had greater fluorescence intensity than the autofluorescence of untransfected cells (Fig. 4 A, R4, blue peak). Post-sorting analysis revealed the GFP-positive population to be 99% pure (unpublished data). Equal aliquots of GFP-, MLC-GFP-, and MLC(TASA)-GFP-positive cells were Western blotted with GFP and  -tubulin antibodies (Fig. 4 B) revealing approximately equal protein expression. Sorted cells were plated in medium containing 10% fetal calf serum for 5 h, placed in serumfree medium for 18 h, and apoptosis induced with TNF ␣ and CHX treatment, and then cells were collected, fixed, and processed for TEM. Cells expressing GFP (not depicted) or MLC-GFP (Fig. 4 C) were typically apoptotic, with plasma membrane blebs and disintegration of nuclei, often with portions of hetero-chromatin visible within blebs (Fig. 4 C,arrows). In marked contrast, cells expressing MLC(TASA)-GFP were typified by an absence of plasma membrane blebs and possessed intact nuclei (Fig. 4 D). In addition, MLC(TASA)-GFP-expressing cells contained long, apparently membrane enclosed, structures. The origin of these membranous structures is currently under investigation. These results demonstrate that MLC phosphorylation is required for nuclear disintegration in apoptotic cells.
Cell contraction is dependent on the myosin ATPase, which through an ATP-dependent process catalyzes the shortening of actin-myosin filaments. Blebbistatin is a recently developed inhibitor of nonmuscle myosin II function that will most likely supplant the nonselective inhibitor butanedione monoxime (BDM) as the inhibitor of choice. We tested whether the ATPase activity of myosin was required for apoptotic nuclear disintegration by coadministering blebbistatin (50 M) along with the TNF ␣ plus CHX apoptotic stimulus for 2 h. As can be seen in Fig. 5 A, inhibition of myosin ATPase activity inhibited apoptotic nuclear breakdown and resulted in the formation of elongated cytoplasmic structures, similar to those seen in MLC(TASA)-GFP-expressing cells. Blebbistatin did not affect ROCK I cleavage (Fig. 5 B) or LIMK phosphorylation ( Fig. 5 C), which is consistent with the protective effect on nuclear integrity being the result of myosin ATPase inhibition.
Caspase cleavage of nuclear proteins
The nucleus is separated from the cytoplasm by a nuclear envelope composed of outer and inner nuclear membranes that are joined at nuclear pore complexes (for review see Burke and Ellenberg, 2002). Underlying the inner nuclear membrane is the nuclear lamina, composed of A-type and B-type lamins that form a fibrous meshwork. Chromosomes associate with the lamina via lamin-associated proteins (LAP), which are either integral membrane proteins or soluble chromatin-associated proteins such as LAP2 ␣ . Apoptotic nuclear disintegration is associated with caspase-mediated proteolysis of nuclear proteins, particularly the lamins B1 and A/C, which leads to the destabilization of the structural elements that preserve the integrity of the nucleus (Neamati et al., 1995). Given that lamin phosphorylation by protein kinases C ␣ (Shimizu et al., 1998),  II (Chiarini et al., 2002), or ␦ (Cross et al., 2000) has been reported to be required for subsequent caspase-mediated cleavage, we wished to determine if inhibition of ROCK activity with Y-27632 prevented apoptotic nuclear disintegration by interfering with nuclear protein cleavage. Lysates from NIH 3T3 cells that were untreated, treated with TNF ␣ /CHX either without or with Y-27632, or treated with TNF ␣ /CHX after a 2-h pretreatment with the general caspase inhibitor z-VAD-fmk (50 M) were prepared and analyzed by Western blotting. Lamins A/C (Fig. 6 A) and B1 (Fig. 6 B) levels were significantly reduced after TNF ␣ treatment with the generation of a faster mobility fragment of lamin B1 (indicated by ⌬ ), even in the presence of Y-27632. Inhibition of caspase activity with z-VAD-fmk fully protected lamins A/C and B1 from cleavage. Treatment with cytochalasin D or blebbistatin also had no effect on lamin B1 cleavage (unpublished data). We also observed that the generation of cleaved forms ( ⌬ ) of LAP2 ␣ (Fig. 6 C), nuclear pore protein Nup153 (Fig. 6 D), and PARP (Fig. 6 E) were unaffected by Y-27632 administration but fully protected by z-VAD-fmk. These results indicate that degradation of the constituent proteins of the nuclear lamina and nuclear pore complexes, as well as soluble nuclear proteins, does not require ROCK activity. By inference, these results indicate that degradation of nuclear proteins is not sufficient for apoptotic nuclear disintegration in the absence of contractile force induced by ROCK. along with the TNF␣ plus CHX apoptotic stimulus for 2 h before collection and processing for TEM. Bar, 2 m. NIH 3T3 fibroblasts were untreated or treated with 50 M blebbistatin to inhibit myosin ATPase activity or 50 M z-VAD-fmk to inhibit caspase activity as indicated. Apoptosis was induced with TNF␣/CHX where indicated. Lysates were prepared and Western blotted with antibodies against ROCK I and PARP (B) and phospho-LIMK1 and 2 (C). Blebbistatin affects apoptotic nuclear morphology without inhibiting caspase-mediated proteolysis or ROCK activity.
Active ROCK I affects nuclear morphology
We wished to determine whether or not the contractile forces generated by active ROCK I were sufficient to induce nuclear disintegration in nonapoptotic cells. NIH 3T3 cells were transduced either with a retroviral vector encoding amino acids 2-537 of human ROCK I fused to the hormone-binding domain of the estrogen receptor (ROCK I:ER) or with a kinase-dead version (KD:ER) in which Lys105 was mutated to glycine, similar to ROCK II:ER fusion proteins we have used previously (McMullan et al., 2003;Croft et al., 2004). Estrogen receptor fusion proteins have been previously used to produce conditionally active kinases (McMahon, 2001) that can be stimulated by estrogen analogues such as tamoxifen or 4-hydroxytamoxifen (4-HT). The addition of 1 M 4-HT for 18 h specifically activated ROCK I:ER resulting in MLC (Fig. 7 A) and LIMK1/2 phosphorylation (Fig. 7 B), which could be blocked by the ROCK inhibitor Y-27632 (10 M). Further confirmation that the effect of 4-HT on MLC and LIMK1/2 phosphorylation resulted from activation of ROCK I:ER was the lack of effect in parental NIH 3T3 or KD:ER-expressing cells (Fig. 7, A and B). Expression of the ROCK I:ER fusion protein was confirmed by Western blotting with anti-ER ␣ antibody, revealing no expression in parental NIH 3T3 cells and comparable levels in KD:ER-and ROCK I:ER-expressing cells that were elevated approximately twofold by 4-HT treatment (Fig. 7 C), whereas blotting with anti-ERK2 antibody confirmed equal protein loading (Fig. 7 D). These results indicate that conditional ROCK I:ER activation resulted in phosphorylation of proteins also found to be phosphorylated in apoptotic cells in a Y-27632-sensitive manner, which is consistent with the conclusion that these modifications during apoptosis are a consequence of ROCK I cleavage and activation.
We next used ROCK I:ER to examine the effects of ROCK activation on nuclear morphology and integrity. Consistent with previous findings (Coleman et al., 2001;Sebbagh et al., 2001), conditional activation of ROCK I:ER with 4-HT, in the absence of apoptosis (cells were pretreated with z-VADfmk), resulted in contraction and induction of membrane blebbing in ف 50% of ROCK I:ER-expressing NIH 3T3 cells (Fig. 8 A, F-Actin left panels), which could be blocked by coadministration of ROCK inhibitor Y-27632 (Fig. 8 A, F-actin bottom panels). Treatment of parental NIH 3T3-or KD:ER-expressing cells with 4-HT did not affect actin structures or cell morphology (not depicted), which is consistent with the lack of effect on MLC or LIMK1/2 phosphorylation (Fig. 7). Staining with anti-lamin B1 antibody revealed that nuclei in contracted, ROCK I:ER-activated cells were smaller and occasionally distorted, similar to previous findings that expression of active ROCK II can alter gross nuclear morphology (Leung et al., 1996). These morphological changes were blocked by Y-27632. Although sufficient to induce contractility and membrane blebbing, these data illustrate that ROCK activation is not sufficient for nuclear disruption.
Next, we tested whether or not ROCK I:ER-induced contractility was sufficient for nuclear disruption under conditions in which the tensile strength provided by an intact lamina is reduced. Lamin A/C null mouse embryo fibroblasts (MEFs), which have a significant frequency of distorted nuclei with herniations (Sullivan et al., 1999), and wild-type MEFs were transduced with ROCK I:ER or KD:ER retrovirus. Treatment of ROCK I:ERexpressing cells with 4-HT plus z-VAD-fmk resulted in increased actin stress fibers, and frequent cell contraction accompanied by membrane blebbing, which could be blocked by the ROCK inhibitor Y-27632 (Fig. 8, B and C, F-actin). Similar to NIH 3T3 cells, KD:ER-expressing MEFs did not display changes in actin structures or morphology after 4-HT treatment (unpublished data). Nuclei in contracted blebbing wild-type MEFs were smaller than spread or untreated cells (Fig. 8 B), similar to nuclei in 4-HT-treated ROCK I:ER-expressing NIH 3T3 cells (Fig. 8 A). In marked contrast, lamin A/C null MEFs treated with 4-HT plus z-VAD-fmk that contracted and blebbed had significantly disrupted nuclei (Fig. 8 C), an effect that was blocked by Y-27632. Staining by TUNEL showed no detectable DNA fragmentation (unpublished data), which is consistent with the nuclear disruption being a direct effect of ROCK and not resulting from the induction of apoptosis. These results indicate that nuclei weakened by deletion of an important component of the lamina are disrupted by ROCK-induced cell contraction. Therefore, two elements are required for nuclear disruption, contractile force generated by active ROCK and weakening of the nuclear lamina, both of which occur in apoptotic cells through caspase-mediated cleavage of ROCK I and nuclear lamins.
Cell contraction, blebbing, and nuclear disruption in lamin A/C null MEFs occurred after 4-HT-induced activation of ROCK I:ER in the presence of caspase inhibitor z-VADfmk, which is consistent with these effects being directly induced and not secondarily through the induction of apoptosis. Western blotting of lysates prepared from ROCK I:ER-and KD:ER-expressing wild-type and lamin A/C null fibroblasts revealed no evidence of lamin B1 (a caspase 6 substrate; Fig. 9 A) or PARP (a caspase 3 substrate; Fig. 9 B) cleavage, which were readily detectable in cells made apoptotic by TNF ␣ /CHX treatment. Western blotting for lamin A/C confirmed the absence of expression in the lamin A/C null cells (not depicted), whereas blotting for  -tubulin and ERK2 showed protein loading (Fig. 9, A and C). These results reinforce the conclusion that ROCK I:ER-mediated contraction is sufficient to induce nuclear disruption in nuclei with compromised lamina structures independent of caspase activation.
Discussion
Nuclear fragmentation is one of the classical hallmarks of apoptosis originally described by Kerr et al. (1972) three decades ago. Early studies concluded that caspase-mediated cleavage of nuclear structural proteins was sufficient for apoptotic nuclear disintegration (Neamati et al., 1995). However, we now show that in the absence of ROCK activity, MLC phosphorylation, or an intact actin cytoskeleton, the proteolysis of lamins, lamin-associated, and pore proteins is not sufficient for nuclear disintegration during apoptosis. Caspase-mediated cleavage of lamins B1 and A/C, Nup153, LAP2 ␣ , and PARP occurred to the same extent in apoptotic cells with disintegrated nuclei or in ROCK inhibitor-treated cells with intact nuclei, indicating that proteolysis of nuclear proteins is not sufficient to rupture the nucleus during apoptosis. In addition, expression of conditionally active ROCK I:ER induced membrane blebbing and distortion of nuclear morphology without causing nuclear disintegration in NIH 3T3 and primary MEFs. However, genetic deletion of lamin A/C, which weakens the nuclear lamina occasionally resulting in spontaneous herniations (Sullivan et al., 1999), allowed ROCK-induced contraction to disrupt nuclear integrity. We propose that during apoptosis, cleavage of nuclear lamins A/C and B1 weakens the lamina and makes the nuclear envelope susceptible to tearing Coadministration of ROCK inhibitor Y-27632 blocked the effects on actin structures and morphological responses (bottom panels). Nuclear morphology and integrity was assessed by staining for lamin B1 (middle column), ROCKinduced contraction and blebbing also resulted in contracted and distorted nuclei that remained intact (middle row), which could be blocked by ROCK inhibitor Y-27632 (bottom row). (B) Primary wild-type mouse embryo fibroblasts (MEFs) transduced with ROCK I:ER responded to 4-HT treatment with increased filamentous actin structures and many contracted, blebbing cells (middle row), responses that could be blocked by ROCK inhibitor Y-27632 (bottom row). ROCK-induced contraction and blebbing also resulted in contraction of nuclei (middle column) as determined by lamin B1 staining. (C) Primary lamin A/C null MEFs transduced with ROCK I:ER also responded to 4-HT with increased filamentous actin structures, cell contraction, and membrane blebbing (middle row), which could be blocked with ROCK inhibitor Y-27632. Nuclei (middle column) in contracted blebbing cells were severely distorted and often disrupted, even in the presence of z-VAD-fmk (middle row), which could be blocked by ROCK inhibitor Y-27632.
by actin-myosin filaments, which contract due to caspase-activation of ROCK I. This proposed mechanism is analogous to, but distinct from, mitotic nuclear breakdown, which is mediated by phosphorylation-induced depolymerization of the nuclear lamina (Heald and McKeon, 1990) and microtubuleanchored pulling force generated by the minus-end-directed motor, cytoplasmic dynein, and components of its associated regulatory complex, dynactin (Beaudouin et al., 2002;Salina et al., 2002).
One unresolved question is the identity of the protein or proteins that couple actin-myosin filaments to the nuclear envelope, thereby facilitating the disruption of nuclear integrity during apoptosis. The leading candidates for this function are the spectrin family proteins Syne-1 and Syne-2 (of which there are numerous alternative splice variants and names including Myne-1/Nesprin-1/Enaptin/CPG2 and Myne-2/Nesprin-2/NU-ANCE) that have been shown to associate with actin filaments at their amino-terminal ends (Zhang et al., 2001(Zhang et al., , 2002Zhen et al., 2002;Padmakumar et al., 2004) and insert into the nuclear membrane at the carboxyl-terminal ends (Apel et al., 2000;Zhang et al., 2001;Mislow et al., 2002b;Zhen et al., 2002;Padmakumar et al., 2004), where they may directly bind A-type lamins (Mislow et al., 2002a,b). Previous research has demonstrated that disruption of the Syne homologue ANC-1 in Caenorhabditis elegans leads to defects in nuclear tethering, which result in aberrant spacing of nuclei in multinucleated syncytial cells (Starr and Han, 2002). Therefore, we propose that during apoptosis the Syne proteins contribute to nuclear disintegration by serving as the physical link that couples the contraction of actin-myosin filaments to the physical tearing of the weakened nuclear lamina and membrane. Efforts in our laboratory are currently underway to test this proposal.
Data from the lmna knockout mouse (Sullivan et al., 1999) and from RNAi studies (Harborth et al., 2001) have shown that Lamin A/C is not essential for viability. However, data from the knockout mouse and from human laminopathies (Burke and Stewart, 2002) indicate that Lamin A/C function is critical in specific tissues, most notably in skeletal and cardiac muscle where actin-myosin contractile forces are considerable. Therefore, we speculate that the viability of cells with mutated or deleted lmna is compromised in these tissues because actinmyosin contractile forces are sufficient to disrupt nuclear integrity, leading to cell death. Other tissues are apparently unaffected, likely due to actin-myosin contractile force being insufficient to significantly disrupt nuclear integrity.
Certain autoimmune diseases such as systemic lupus erythematosus are believed to arise due to immunological responses to nuclear autoantigens released from apoptotic cells. Treatment with ROCK inhibitor Y-27632 reduced the translocation of the 681 (Shiratsuchi et al., 2003) and D56R/S76R (Cocca et al., 2002) nuclear autoantigens to plasma membrane blebs and apoptotic bodies without affecting phosphatidylserine externalization (Coleman et al., 2001). The results of this work indicate that ROCK activity is required for actinmyosin contractile force generation and consequent disruption of nuclear integrity, which is required for the movement of DNA and proteins from the nucleus into blebs and apoptotic bodies. Therefore, ROCK inhibition may have some prophylactic and/or ameliorative benefits for the treatment of specific autoimmune diseases.
Materials and methods
Cell culture and plasmids NIH 3T3 and primary MEFs were cultured in DME containing 10% FCS (Sigma-Aldrich) at 37ЊC in a 10% CO 2 atmosphere. To induce apoptosis, cells were placed in serum-free medium for 18 h, and then treated with 25 ng/ml TNF␣ (R&D Systems) plus 10 g/ml CHX (Sigma-Aldrich) for 2 h. For EM, cells were scraped in media and processed as described in the section TEM. For Western blotting, cells were scraped in media, lysed in 1ϫ Laemmli buffer, briefly sonicated to shear DNA, and heated to 100ЊC for 5 min before loading equal portions onto SDS-PAGE.
Rat MLC cDNA (provided by L. Machesky, The University of Birmingham, Edgbaston, Birmingham, UK) was subcloned into the EcoRI restriction site of pEGFP-N3 (CLONTECH Laboratories, Inc.). Mutation of Thr18 Ser19 sites to Ala was done using a QuikChange site-directed mutagenesis kit (Stratagene) according to instructions. Human ROCK I:ER was made by subcloning a blunt-ended PCR product encoding amino acids 2-537 into filled-in ScaI-EcoRI sites of pBABE puro EGFP:Raf-1:ER (Woods et al., 1997; provided by M. McMahon, University of California San Francisco Comprehensive Cancer Center, San Francisco, CA). Mutation of Lys105 to Gly was done with QuikChange.
BOSC 293 ecotropic retroviral packaging cells were transfected with pBABE puro ROCK I:ER or KD:ER plasmid using Effectene (QIA-GEN) according to instructions. After 36 h, supernatant was collected, centrifuged at 14,000 rpm for 15 min, and aliquots were stored at Ϫ80ЊC. Exponentially growing NIH 3T3 or MEFs were infected with undiluted retroviral supernatant mixed with 4 g/ml polybrene (Sigma-Aldrich) and selected with 2.5 g/ml puromycin (Sigma-Aldrich) to establish transduced pools.
Cytochalasin D, 4-HT, and swinholide A were from purchased from Sigma-Aldrich, z-VAD-fmk and staurosporine were purchased from BIO-MOL Research Laboratories, Inc., and Y-27632 and Latrunculin B were purchased from Calbiochem. Figure 9. ROCK I does not activate caspases. Wild-type and lamin A/C null MEFs expressing ROCK I:ER or kinase dead KD:ER were left untreated or treated with 1 M 4-HT, either in the absence or presence of z-VAD-fmk as indicated. As a positive control for caspase activation, cells were treated with 25 ng/ml TNF␣ plus 10 g/ml CHX. Western blotting revealed that 4-HT treatment of KD:ER-or ROCK I:ER-expressing wild-type and lamin A/C null fibroblasts did not induce cleavage of lamin B1 (A) or PARP (B) although each was cleaved in response to TNF␣ treatment. (C) Western blotting for ERK2 reflects protein levels across samples.
TEM
Cell suspensions were pelleted by centrifugation, resuspended in 1 ml of fixative in an Eppendorf tube and immediately spun down again to form a pellet, and stored overnight at 4ЊC. Samples were postfixed in 2% osmium tetroxide, dehydrated through a graded series of ethanols, infiltrated, and embedded in Epon. For light microscopy, 1.0-m sections were cut, dried onto microscope slides, and stained with toluidine blue. For EM, 75-nm sections were collected onto copper grids, double stained with uranyl acetate and bismuth subnitrite, and examined in a transmission electron microscope (model 1010 Jem; JEOL) at 80 Kv accelerating voltage. Digital images were collected using a CCD camera (model 2k; Hamamatsu) aided with AMT 12-HR software.
Western blot analysis
After treatment as described in the figure legends, cells were lysed in 1ϫ Laemmli sample buffer, sonicated, and electrophoresed on 10% SDS-PAGE before electro-transfer to nitrocellulose membranes. Blots were probed with antibodies against lamins B1 and A/C (Santa Cruz Biotechnology, Inc.
FACS sorting and analysis
NIH 3T3 cells were transfected with pEGFP-N3 (CLONTECH Laboratories, Inc.), pEGFP-N3 MLC-GFP, or pEGFP-N3 MLC(TASA)-GFP using Lipofectamine as described previously (Coleman et al., 2001). GFP-expressing cells were detached with trypsin, sorted, and collected using a FACSVan-tageSE cell sorter (BD Biosciences). The machine has a Coherent 90 C-4 argon ion laser that is tuned to 488 nm. This light was used to excite GFP, which was measured at 515-545 nm. | 2014-10-01T00:00:00.000Z | 2005-01-17T00:00:00.000 | {
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17091479 | pes2o/s2orc | v3-fos-license | Suicide and Suicide Risk in Lesbian, Gay, Bisexual, and Transgender Populations: Review and Recommendations
Despite strong indications of elevated risk of suicidal behavior in lesbian, gay, bisexual, and transgender people, limited attention has been given to research, interventions or suicide prevention programs targeting these populations. This article is a culmination of a three-year effort by an expert panel to address the need for better understanding of suicidal behavior and suicide risk in sexual minority populations, and stimulate the development of needed prevention strategies, interventions and policy changes. This article summarizes existing research findings, and makes recommendations for addressing knowledge gaps and applying current knowledge to relevant areas of suicide prevention practice.
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A. P. Haas et al. Despite strong indications of elevated risk of suicidal behavior in lesbian, gay, bisexual, and transgender people, limited attention has been given to research, interventions or suicide prevention programs targeting these populations. This article is a culmination of a three-year effort by an expert panel to address the need for better understanding of suicidal behavior and suicide risk in sexual minority populations, and stimulate the development of needed prevention strategies, interventions and policy changes. This article summarizes existing research findings, and makes recommendations for addressing knowledge gaps and applying current knowledge to relevant areas of suicide prevention practice. KEYWORDS LGBT, risk factors, suicide, suicide attempts, suicide prevention Relatively little attention has been given to the problem of suicidal behavior in lesbian, gay, bisexual, and transgender (LGBT) populations, despite reports of elevated risk for over four decades. The U.S. National Strategy for Suicide Prevention (U.S. Surgeon General, 2001) and the Institute of Medicine's Reducing Suicide: A National Imperative (Goldsmith, Pellmar, Kleinman, & Bunney, 2002) defined gay and bisexual youth as a risk population but provided little information about contributing factors and did not address whether targeted interventions, prevention strategies or public health policies are needed to reduce suicide risk in this population.
In November 2007, the American Foundation for Suicide Prevention in partnership with the Suicide Prevention Resource Center and the Gay and Lesbian Medical Association convened a conference to address the need for better understanding of suicidal behavior and suicide risk in LGBT populations. The two dozen invited participants, including suicide and mental health researchers, clinicians, educators, and policy advocates, discussed findings from relevant research and their implications for reducing suicidal behavior in the target populations, and made recommendations to address knowledge gaps. Additional studies have been reported since the conference, further expanding our knowledge base. Increased national as well as international attention has also been given to the need to use our extant knowledge to reduce risks and prevent suicidal behavior in LGBT populations.
This article was developed by the 2007 conference participants to review the findings from relevant research, identify knowledge gaps, and stimulate the development of strategies, interventions, and policy changes to reduce suicidal behavior and suicide risk among LGBT people. It seeks specifically to: 1. Summarize what is currently known about completed suicide, suicide attempts and suicide risk across the lifespan, 2. Identify knowledge gaps most in need of future research, and make recommendations for how these can be addressed, and 3. Offer recommendations for applying what is already known to reduce suicidal behavior and suicide risk in sexual minority populations.
DEFINITIONS
Sexual minorities are defined with reference to two distinct and complex characteristics: sexual orientation and gender identity. Sexual orientation is generally defined as having at least three dimensions: sexual selfidentification, sexual behavior, and sexual attraction or fantasy (Saewyc et al., 2004;Sell, 1997). Researchers have tended to define sexual orientation by one or another of these dimensions, most often using as the defining criterion either self-identification as gay/lesbian, bisexual or heterosexual, or the gender of one's sexual partners (same sex, both same and opposite sex, or opposite sex).
Estimates of the prevalence of gay, lesbian, and bisexual people in the United States vary according to how they are defined (Pathelal et al., 2006). Studies using representative school-based samples have found that about 3% of students in grades 9-12 identify themselves as gay, lesbian, or bisexual (Garofalo, Wolf, Kessel, Palfrey, & DuRant, 1998;Garofalo, Wolf, Wissow, Woods, & Goodman, 1999). Data from the third wave of the National Longitudinal Survey of Adolescent Health (Add Health), collected in 2001-2002, similarly found that 3.2% of young adults aged 18-26 described themselves as mostly or exclusively homosexual or bisexual, with more females (3.6%) than males (2.6%) using these labels (Silenzio, Pena, Duberstein, Cerel, & Knox, 2007). One early representative survey of U.S. adults, the 1992 National Health and Social Life Survey (NHSLS), reported that 2.8% of men and 1.4% of women identified themselves as homosexual, slightly lower than the 3% of men and 1.6% of women who reported current sexual behavior exclusively with same-sex partners, and considerably lower than the 7.7% of men and 7.5% of women who indicated same-sex sexual attraction (Laumann, Gagnon, Michael, & Michaels, 1994). More recent studies of adults have generally confirmed these figures and have consistently shown that more respondents indicate same-sex sexual behavior, and especially same-sex attraction, than identify themselves as gay or lesbian (Black, Gates, Sanders, & Taylor, 2000;Pedersen & Kristiansen, 2008;Sell, 1997;Wells, McGee, & Beautrais, 2010). Increasingly sophisticated methods are being used to determine the prevalence of lesbian, gay, and bisexual people in the U.S. population through merging and cross-validating responses from multiple national surveys. Based on pooled sexual behavior data from the NHSLS and six waves (1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996) of the nationally representative General Social Survey, 4.7% of adult men and 3.6% of adult women are estimated to have had at least one same-sex sex partner since age 18, with 2.5% of men and 1.4% of women having exclusively same-sex sex partners during the past year (Black et al., 2000).
There is limited understanding of which dimensions of sexual orientation are most meaningfully related to suicidal behavior. One recent adolescent study that incorporated multiple measures of sexual orientation found suicidal behavior to be significantly higher in youth who identified as gay, lesbian or bisexual, compared to those who identified as heterosexual (Zhao, Montoro, Igartua, & Thombs, 2010). Those who indicated same-sex attraction or behavior but identified as heterosexual, however, did not report a higher rate of suicide attempt than heterosexual youth without same-sex behavior or attraction. Data from a large national survey of U.S. adults that included multiple questions related to sexual orientation showed that rates of mood and anxiety disorders, key risk factors for suicidal behavior, were more strongly linked to gay, lesbian or bisexual identity than to sexual behavior or attraction, particularly in women.
In contrast to sexual orientation, gender identity refers to a person's internal sense of being masculine, feminine, or androgynous. Rather than a binary concept, gender identity includes gradations of masculinity to femininity and maleness to femaleness, as well as identification as neither essentially male nor female (Fausto-Sterling, 2000). Transgender is an umbrella term that is broadly used to describe people with gender identities, expressions or behaviors which differ from their biological sex at birth (Feinberg, 1992;Kirk & Kulkarni, 2006). Although the term transgender is sometimes used synonymously with transsexual, the latter more commonly describes a subset of transgender individuals who undergo gender reassignment surgery and/or hormone treatment to align physical sex and gender identity. For varying reasons that include cost and lack of access to appropriate health care services, an unknown proportion of transgender people do not elect to obtain surgery or hormonal therapies. Terms such as "genderqueer" are increasingly used by younger transgender people, as well as some who do not identify as transgender, to describe a wide range of gender identifications, behaviors and expressions other than exclusively male or female (Nestle, Howell & Wilchins, 2002). As with sexual orientation, gender identity is not an entirely fixed characteristic, and many transgender people move fluidly between identities over time, often without any specific labels (Whittle, Turner, & Al-Alami, 2007).
Inconsistent definitions contribute to the lack of clarity in our knowledge about the prevalence of transgender people, and particularly transgender youth, in the population. To date, no general population-based survey of the adolescent or adult U.S. population has attempted to measure transgender identity. In the United States, 1 in 30,000 assigned males and 1 in 100,000 assigned females are estimated to seek gender reassignment surgery at some point in their lifetime (American Psychiatric Association, 2000). The Amsterdam Gender Dysphoria Clinic, which has collected data on the Dutch transsexual population for more than four decades, has estimated the prevalence to be substantially higher at 1 in 10,000 assigned males and 1:30,000 assigned females ( van Kesteren, Asscheman, Megens, & Gooren, 1997). Either set of figures is certainly an underestimate of the broadly defined transgender population. One large internet survey found that 0.2% of respondents described themselves as transgender (Mathy, Schillace, Coleman, & Berquist, 2002).
Sexual orientation varies among transgender individuals, just as it does among people who perceive their gender identity to be aligned with their biological sex. Although precise numbers are lacking, one survey of 515 self-identified transgender persons found that 31% of male-to-female respondents and 65% of female-to-male respondents identified as gay, lesbian, or bisexual (Clements-Nolle, Marx, Guzman, & Katz, 2001).
In this article, we focus first on summarizing the accumulated research literature on suicidal behavior and suicide risk in lesbian, gay, and bisexual people. Next, we summarize the far more limited findings from comparable research among transgender people. Consistent with widespread usage in the research literature, we frequently use the acronyms LGB (lesbian, gay, bisexual) and LGBT (lesbian, gay, bisexual, and transgender) while recognizing that they do not adequately reflect the heterogeneity of self-identifications or behaviors within these populations.
Suicide Deaths
Because death records do not routinely include the deceased person's sexual orientation, there is no official or generally reliable way to determine rates of completed suicide in LGB people. Some researchers have attempted to determine whether these groups are overrepresented among those who die by suicide, using "psychological autopsy" reports of family and friends to determine the decedents' sexual orientation. Several studies using this method have been published, focusing on young adult male suicides in San Diego (Rich, Fowler, Young, & Blenkush, 1986) and adolescent suicides in the New York metropolitan area (Shaffer, Fisher, Hicks, Parides, & Gould, 1995) and the province of Quebec (Renaud, Berlim, Begolli, McGirr, & Turecki, 2010). Each of these studies has concluded that same-sex sexual orientation is not disproportionately represented among suicide victims. 16 A. P. Haas et al. To date, psychological autopsy studies that have examined sexual orientation have used relatively small samples and have identified very few suicide decedents as having minority sexual orientation. In the New York study, 3 of 120 adolescent suicide decedents and none of a similar number of living community control subjects with whom the suicide victims were compared, were found to have a same-sex orientation (Shaffer et al., 1995). The Quebec study similarly identified same-sex orientation in 4 of 55 suicide adolescent suicide victims and none of the community control subjects (Renaud et al., 2010). Minority sexual orientation may have been underreported by key informants in these studies because they were not aware of, or chose to withhold this information (Renaud et al., 2010). In any case, conclusions based on the small numbers reported must be regarded as tentative.
The San Diego study lacked a living control group and has been criticized based on the researchers' assumption that the 11% of young male suicide decedents who were identified as gay approximated the expected prevalence rate for young gay men in the population under study (McDaniel, Purcell, & D'Augelli, 2001). Using a more likely prevalence rate of 3-4% would have suggested that young gay men were overrepresented among suicide decedents by a factor of at least three.
Recent studies have used Denmark's extensive registries of vital statistics and other sociodemographic data to examine whether people in same-sex registered domestic partnerships (a proxy indicator of sexual orientation) were overrepresented among suicide decedents. The Danish data can be matched fairly easily because individual information recorded in the various registries uses unique identification numbers assigned to citizens at birth. One study that linked Danish mortality and sociodemographic data (Qin, Agerbo, & Mortensen, 2003) noted that same-sex registered domestic partners were 3-4 times more likely than heterosexual married persons to die by suicide, although this was not a key focus of the study and corroborating data were not presented. A subsequent study that was designed explicitly to examine suicide risk in Denmark by sex and relationship status (Mathy, Cochran, Olsen, & Mays, 2009) found that elevated risk of suicide in same-sex partnered people was concentrated almost exclusively among men. Men who were currently or formerly in same-sex domestic partnerships were eight times more likely to die by suicide compared to men with histories of heterosexual marriage, and almost twice as likely as men who had never married. Although small numbers of cases limited the precision of the analyses, same-sex partnered men appeared to have an elevated risk of suicide across the lifespan. Women in current or former same-sex domestic partnerships did not show significantly higher risk of suicide mortality compared to heterosexually married or never-married women. A limitation of the approach used in the Danish studies is that it captures suicide deaths only among partnered and officially registered LGB people. Further, opportunities for replication in other countries have been limited, but these may expand as more as more countries and U.S. states officially recognize and record same-sex marriages and partnerships (Strohm, Seltzer, Cochran & Mays, 2009).
An 18-year follow-up study of the mortality status of over 5,000 U.S. men aged 17-59 who were interviewed in the Third National Health and Nutrition Examination Survey (1988)(1989)(1990)(1991)(1992)(1993)(1994) found no suicide deaths among the 85 men who reported having any lifetime same-sex sexual partner (Cochran & Mays, 2011). Findings from this study, in stark contradiction to the Danish registry studies, suggest that suicide mortality may not be elevated among U.S. men who have sex with men. The authors cautioned, however, that the number of men who reported same-sex sexual behavior in this survey was quite small, and that elevated risk of suicide mortality among U.S. men may yet be observed in studies with larger samples and a longer follow-up period.
Suicide Attempts
In contrast to the data on death by suicide, a relationship between sexual orientation and nonfatal suicidal behavior has been observed worldwide (Mathy, 2002a). Studies in the United States and abroad provide strong evidence of elevated rates of reported suicide attempts among LGB individuals. We draw primarily on "population-based studies," in which sexual orientation has been assessed in randomly selected samples, allowing comparisons to be made among sexual orientation groups within a defined population. From a scientific perspective, these methods yield the best estimate of the prevalence of suicidal behavior (and associated risk factors) in groups, without the biases that can occur in convenience or other nonrepresentative samples.
Since the early 1990s, population-based surveys of U.S. adolescents that have included questions about sexual orientation have consistently found rates of reported suicide attempts to be two to seven times higher in high school students who identify as LGB, compared to those who describe themselves as heterosexual (DuRant, Krowchuk, & Sinal, 1998;Falkner & Cranston, 1998;Garofalo, Wolf, Kessel, et al., 1998;Garofalo, Wolf, Winssow, et al., 1999;Remafedi, 2002;Russell & Joyner, 2001). Gender-specific analyses have found sexual orientation to be a stronger independent predictor of suicide attempts in young males than in young females (Garofalo et al., 1999). These findings are consistent with reports of elevated rates of suicide attempts among LGB adolescents and young adults from other random and nonrandom studies (Kulkin, Chauvin, & Percle, 2000;Russell, 2003;Suicide Prevention Resource Center, 2008). Although it has been speculated that LGB youth may exaggerate the extent and seriousness of their suicidal behavior (Savin-Williams, 2001), methods used in over half of all suicide attempts reported in one nonrandom sample of LGB adolescents and young adults were classified as moderate to lethal, with 21% resulting in a medical or psychiatric hospital admission (Remafedi, Farrow, & Deisher, 1991).
Using another population-based approach, a longitudinal study of a large New Zealand birth cohort found that at age 21, those who identified as LGB were six times more likely than those who identified as heterosexual to report one or more lifetime suicide attempts (Fergusson, Horwood, & Beautrais, 1999). When interviewed again at age 25, LGB individuals in this cohort reported a significantly higher rate of suicide attempts since age 21 than did heterosexual respondents (Fergusson, Horwood, Ridder, & Beautrais, 2005).
Health-related surveys of U.S. males aged 17-39 (Cochran & Mays, 2000a) and Dutch males and females aged 18-64 (de Graaf, Sandfort, & ten Have, 2006) found higher rates of lifetime suicide attempts among respondents who reported same or both-sex sexual behavior compared to those who reported only opposite-sex sexual behavior. A similar finding emerged from the population-based Vietnam Era Twin Registry, consisting of 4,774 male-male identical or fraternal twin pairs born between 1939 and 1957 (Herrell et al., 1999). This study found that middle-aged men who reported any male sex partners after age 18 were six times more likely to have made a lifetime suicide attempt than were their male twins who reported only opposite-sex sexual behavior. Among U.S. urban gay and bisexual men, about 12% reported making a lifetime suicide attempt, about three times the rate among American adult males overall (Paul et al., 2002).
A recent meta-analysis of 25 international population-based studies that measured suicidal behavior in LGB adolescents and/or adults (variously defined) concluded that the lifetime prevalence of suicide attempt in gay/bisexual males was about four times that of comparable heterosexual males (King et al., 2008). Based on the relatively small number of studies in this meta-analysis that included substantial numbers of women, lesbian/bisexual women were found to have lifetime suicide attempt rates almost twice those of heterosexual women. Overall, LGB adolescents and adults were also more than twice as likely as comparable heterosexual persons to report a suicide attempt in the past 12 months.
Many of the studies that have investigated suicide attempts in LGB groups have also measured suicidal ideation, with combined results showing LGB respondents to be twice as likely as comparable heterosexual respondents to report suicidal ideation (King et al., 2008). Several studies have reported that the gender pattern for suicidal ideation is opposite that for suicide attempts, with risk of suicidal ideation higher among lesbian/bisexual women and risk of suicide attempts higher among gay/bisexual men. One large-scale U.S. survey (Gilman et al., 2001) found a three times higher rate of reported suicidal ideation in lesbian/bisexual women compared to heterosexual women, but no higher rate in gay/bisexual compared to heterosexual men. Thus, reported suicidal ideation does not appear to be a stable predictor of LGB suicidal behavior.
Demographic Factors
In most Western countries, suicide attempts occur more frequently among adolescents and young adults (Goldsmith et al., 2002), and some studies suggest this is also true for LGB people (de Graaf et al., 2006;Paul et al., 2002;Remafedi et al., 1991). One recent analysis of data from four waves of the National Longitudinal Study of Adolescent Health (Add Health), which tracked a large national cohort of youth from an average age of 15 to their late 20s, found that the risk for suicide attempts in young men who reported same-sex romantic attractions was largely confined to the adolescent years (Russell & Toomey, 2010).
Understanding of age-related patterns of suicide attempts in LGB adults has been limited by a lack of information from population-based surveys about respondents' age at the time of reported suicide attempts. Further, surveys have identified few LGB participants over the age of 60. One study of 416 LGB adults aged 60-91 who were attending social and recreational programs (D'Augelli, Grossman, Hershberger, & O'Connell, 2001) suggests that suicidal behavior in LGB populations may be more widely distributed across the lifespan than has been reported. In that study, 52 respondents (13% of the sample) reported a total of 97 lifetime suicide attempts; of these 27% occurred at or before age 21, 69% between the ages of 22 and 59, and 4% at or after age 60. There is some evidence that suicide attempts may be more closely linked to the ages at which lesbian women (Hughes, 2003) and gay men (Paul et al., 2002) recognize and disclose their sexual orientation to others than to chronological age.
Studies have generally found lifetime suicide attempt rates to be higher in gay/bisexual men than in lesbian/bisexual women (King et al., 2008). This represents a clear departure from the population overall, in which women are three times more likely than men to make a lifetime suicide attempt (Kessler, Borges, & Walters, 1999). The Danish registry data (Mathy, Cochran, et al., 2009), which showed an increased risk of completed suicide among same-sex-partnered men but not same-sex-partnered women, suggests that LGB suicide deaths may occur disproportionately among men, similar to the gender pattern found in the general population.
Little is known about the relationship of race/ethnicity and other demographic characteristics to LGB suicidal behavior, largely because the size of the LGB sample obtained in many population-based surveys has been too small to discern significant differences among these LGB subgroups. One study reported suicide attempt rates in LGB adolescents to be especially high among African-American males (Remafedi, 2002). Among adults, suicide attempt rates have been reported to be highest among gay/bisexual men of lower socioeconomic status (Paul et al., 2002) and among LGB Latinos (Meyer, Dietrich, & Schwartz, 2007). In a national probability study of Latino and Asian-American adults (Cochran, Mays, Alegria, Ortega, & Takeuchi, 2007), gay and bisexual men were more likely than heterosexual men to report a recent suicide attempt.
Mental Disorders
In the population as a whole, mental disorders constitute the single largest risk factor for suicidal behavior, and studies have also reported a generally strong association between mental disorders and suicide attempts in LGB adolescents and adults. In the New Zealand birth cohort study (Fergusson et al., 1999), researchers used structured interview schedules to assess for several psychiatric diagnoses, including major depression, generalized anxiety disorder, conduct disorder, and alcohol/substance use disorders. They found that elevated rates of reported suicide attempts in youth who identified as LGB were associated with significantly higher rates of depression, generalized anxiety disorder and conduct disorder than were observed among heterosexual youth.
LGB youth were also six times more likely to have multiple disorders. Follow-up of the cohort during their mid-20s (Fergusson et al., 2005) found that elevated suicidal behavior among LGB members was associated with depression, anxiety disorders and substance use disorders, and that the associations were more marked in males than females.
Elevated rates of mental disorders, including substance use disorders, have also been reported in one-quarter to one-third of LGB adult respondents in large-scale health surveys that have defined sexual orientation based on self-identity Cochran, Mays, & Sullivan, 2003;Cochran, Mays, Alegria, et al., 2007;Conron, Mimiaga, & Landers, 2010;Hughes, Szalacha, & McNair, 2010;Jorm, Korten, Rodgers, Jacomb, & Christensen, 2002;McCabe, Hughes, Bostwick, West, & Boyd, 2009) or gender of sexual partners (Cochran, Ackerman, Mays & Ross, 2004;Gilman et al., 2001). Combining results from 25 international adolescent and adult studies, researchers found depression, anxiety disorders, and substance use disorders to be 1.5 times more common in LGB people than in comparable heterosexual individuals (King et al., 2008). Although findings for most disorders were similar for males and females, lesbian/bisexual women had especially high rates of substance dependence, more than three times the rate for heterosexual women. The findings of higher rates of depression and panic disorder in gay/bisexual men, and higher rates of substance use disorders in lesbian/ bisexual women point to different gender patterns among LGB people, compared to the population as a whole.
The National Epidemiologic Survey on Alcohol and Related Conditions (NESARC), a population-based survey of U.S. adults aged 18 and older, provided a unique opportunity to look at the relationship of sexual orientation and mood and anxiety disorders. In the latest wave of this survey, occurring in 2004-2005, almost 35,000 nationally representative Americans completed an extensive in-person interview that included separate questions on sexual identity, behavior and attraction. The percentage of NESARC respondents who identified as LGB (1.4%) was lower than other population estimates, possibly because the data were collected using face-to-face interviews. However, the 577 LGB respondents identified constitute the largest nationally representative sample of LGB adults in any mental health survey to date (Hatzenbuehler, McLaughlin, Keyes, & Hasin, 2010).
A recent analysis of these data confirmed a higher prevalence of lifetime mood and anxiety disorders among participants who identified as LGB, compared to those who identified as heterosexual. Men who reported same-sex sexual behavior or attraction reported a higher prevalence of most mood and anxiety disorders. Among women, however, those who reported only female sexual partners had a lower prevalence of every disorder examined compared to women who reported only male or both male and female sexual partners, or who were not sexually active. Similarly, women who reported sexual attraction to only females had the lowest rates of most mood and anxiety disorders compared with other attraction-defined groups (only male, mostly male, both male and female, and mostly female). Confirming findings of an earlier large-scale Australian survey (Jorm et al., 2002), this analysis found that bisexual behavior and identity were strongly associated with elevated risk of mood and anxiety disorders in both men and women. Similar to men who identified as gay or bisexual, men who reported being unsure about their sexual identity were significantly more likely to have mood or anxiety disorders than heterosexual men. In women with unsure sexual identity, however, rates of these disorders were generally not significantly higher than among heterosexual women. The findings of this study point to the complexity of defining sexual orientation, especially in women, and illuminate differences among female subgroups that past surveys have subsumed within a single female category.
Most studies have shown an association between mental disorders and suicide attempts in LGB respondents who report suicidal behavior. Mental disorders, however, do not appear to entirely explain elevated rates of suicide attempts in these individuals. An unpublished analysis of the NESARC data found that after adjusting for mental disorders, suicide attempt rates in LGB respondents overall remained two-to-three times higher than among heterosexual respondents (Belik & Sareen, 2010). Relative to comparable heterosexual respondents, suicide attempt rates ranged between just over twice as likely among lesbian women to more than three times more likely among bisexual men. This finding is consistent with reports from studies of 22 A. P. Haas et al. U.S. adolescents and young adults who identified as LGB (Silenzio et al., 2007), as well as U.S. (Herrell et al., 1999) and Dutch adult men with same-sex sex partners (de Graaf at al., 2006).
Stigma, Prejudice and Discrimination
Over the past decade, consensus has grown among researchers that at least part of the explanation for the elevated rates of suicide attempts and mental disorders found in LGB people is the social stigma, prejudice and discrimination associated with minority sexual orientation (Cochran, Mays & Sullivan, 2003;de Graaf at al., 2006;King et al., 2008;McCabe, Bostwick, Hughes, West, & Boyd, 2010). The terms gay-related stress (Rosario, Schrimshaw, Hunter, & Gwadz, 2002;Rotheram-Borus, Hunter & Rosario, 1994) and minority stress (Meyer, 1995(Meyer, , 2003 have been used to describe a range of stressors resulting from individual and institutional discrimination against LGB people.
INDIVIDUAL DISCRIMINATION
There is ample evidence that across the lifespan, LGB people commonly experience discrimination in the form of personal rejection, hostility, harassment, bullying, and physical violence. One especially powerful stressor for LGB youth is rejection by parents and other family members. Several nonrandom studies have found an association between parental rejection because of sexual orientation and higher risk of suicide attempts among LGB youth (D'Augelli, Grossman, Salter, et al., 2005;D'Augelli, Hershberger, & Pilkington, 2001;Remafedi et al., 1991;Ryan, Huebner, Diaz, & Sanchez, 2009). One study of White and Latino LGB young adults aged 21-25 (Ryan et al., 2009) found that those who experienced frequent rejecting behaviors by their parents or caregivers during adolescence were over eight times more likely to report making a suicide attempt than those with accepting parents. Young Latino gay and bisexual men reported the highest number of rejecting behaviors and were more likely than Latina females or White respondents to report suicide attempts. The impact of parental and family rejection is suggested by the alarmingly high number of LGBT adolescents and young adults who are homeless, estimated to constitute 20-40% of the almost 2 million homeless youth in the United States (Ray, 2006).
Analyses of data from large public health surveys of U.S. adults have also demonstrated a link between discrimination and hostile treatment based on sexual orientation, and increased risk of substance use and other mental disorders. Data from the National Survey of Midlife Development showed elevated anxiety, depression and other stress-related mental health problems in LGB adults aged 25-74 who reported personal experiences with discrimination . Data from the National Epidemiologic Survey of Alcohol andRelated Conditions (2004-2005) further documented the association between personal experiences of discrimination and interpersonal violence on elevated rates of substance use disorders and posttraumatic stress disorder (Roberts, Austin, Corliss, Vandermorris, & Koenen, 2010) in LGB adults over the age of 20.
There is some evidence that the interrelationship among gay-related stressors, mental disorders and suicidal behavior may vary between different racial and ethnic groups. A nonrandom study of almost 400 ethnically diverse, self-identified LGB adults aged 18-59 living in New York City (Meyer et al., 2007) found that White participants had significantly higher rates of mood disorders than Black or Latino individuals. Black and especially Latino individuals, however, reported significantly higher rates of lifetime suicide attempts than did whites, with most attempts occurring before the age of 20. A key hypothesis emerging from the study, which is currently being tested, is that suicide risk among Black and Latino LGB people is more strongly related to major stressful events associated with coming out, such as assault, abuse and homelessness, than to depression and other mental disorders.
INSTITUTIONAL DISCRIMINATION
Institutional discrimination results from laws and public policies that create inequities or fail to provide protections against sexual orientation-based discrimination. Using the NESARC data, Hatzenbuehler, Keyes, and Hasin (2009) found that LGB adults who lived in one of 19 states that lacked specific protections against sexual orientation-based hate crimes or employment discrimination had significantly higher prevalence of mood, anxiety, and substance use disorders, compared to heterosexual adults living in those 24 A. P. Haas et al. states and LGB adults living in states that extended protection in at least one of these areas.
LGB respondents in states without protective policies were almost five times more likely than those in other states to have two or more mental disorders.
A subsequent study (Hatzenbuehler et al., 2010) examined the effects of state constitutional bans on same-sex marriage on the mental health of LGB adults. Such amendments gained impetus following the passage of the 1996 Federal Defense of Marriage Act or DOMA, which affirmed that states are not required to treat a relationship between persons of the same sex as a marriage, even if the relationship is considered a marriage in another state. DOMA also defined marriage as a legal union exclusively between one man and one woman (The 'Lectric Law Library, 1996). Using the NESARC data from 16 states that enacted constitutional amendments against same-sex marriage during 2004 and 2005, the researchers found significant increases in mental disorders among self-identified LGB respondents in these states between wave 1 (2001-2002) and wave 2 (2004-2005) of the survey. Specifically, mood disorders increased by more than one-third, from 23 to 31% of LGB respondents. Increases were also found in generalized anxiety disorder, from 3 to 9%, and alcohol use disorder, from 22 to 31%. By contrast, no comparable increases in mental disorders between the two waves of the survey were observed in heterosexual respondents living in these 16 states. Noting that the constitutional amendments largely underscored preexisting state laws, the researchers hypothesized that the negative mental health impact on LGB citizens stemmed primarily from the hostile political campaigns and public discourse that preceded their passage, which further promulgated stigma and reinforced the marginalized social and legal status of LGB people.
Among LGB respondents living in the 34 states where constitutional amendments against same-sex marriage were not enacted during the period examined, increases in generalized anxiety disorder and substance use disorders were also found between the two waves of the survey, possibly related to extensive national media coverage of the amendment campaigns and the associated anti-gay rhetoric. Again, comparable increases in mental disorders were not found in heterosexual respondents living in the same states.
Prohibiting same-sex marriage has also been found to result in significant disparities in health insurance coverage between heterosexual and same-sex couples (Buchmueller & Carpenter, 2010;Carpenter & Gates, 2008;Heck, Sell, & Gorin, 2006;Ponce, Cochran, Pizer, & Mays, 2010). One recent study in California found that partnered lesbians and gay males were more than twice as likely to be uninsured as married heterosexuals, primarily because of lower rates of employer-provided coverage of dependent partners (Ponce et al., 2010). Using data from the California Health Interview Survey in 2001, 2003, and 2005, the study found that partnered gay men were less than half (42%) as likely to have dependent health insurance coverage as married heterosexual men, and partnered lesbians were only 28% as likely to have coverage as married heterosexual women. Even when insurance coverage is offered to domestic partners, this study noted that the benefit is not financially equivalent to that provided to heterosexual married spouses because federal law requires unmarried partners to pay income tax on the value of employer-sponsored health insurance. Because of the Defense of Marriage Act (DOMA), same-sex couples who have been legally married in a U.S. state or other jurisdiction are treated as unmarried for this and all other federal tax provisions.
Lack of health insurance coverage among persons with mental disorders has been related to delays in treatment-seeking (McLaughlin, 2004) and to self-treatment with alcohol and other substances and the development of psychiatric and physical comorbidities (Wang, Berglund, Olfson, & Kessler, 2004). It is not clear whether the Patient Protection and Affordable Care Act of 2010 (Government Printing Office, 2010) will close the insurance gap currently faced by many same-sex couples. While requiring large employers to provide health insurance to employees and their dependents, the law does not specify that domestic partners be included as covered dependents, and does not address the tax burden imposed on domestic partners who are covered by employer-sponsored health insurance (Ponce et al., 2010).
HIV/AIDS
Among some urban men who have sex with men, elevated risk of HIV/AIDS has been found to be associated with depression, substance abuse, and elevated risk of suicidal behavior (Paul et al., 2002;Stall, et al, 2003). Although increased rates of completed suicide and suicide attempts in persons with HIV/AIDS have been reported by numerous U.S. and international studies, understanding of any possible direct effect of the virus on suicidal behavior has been limited by a lack of longitudinal studies and inconsistencies in illness definitions and characteristics of the samples studied. One comprehensive review (Komiti et al., 2001) suggested that substance abuse, other psychiatric disorders, and previous suicide attempts may be more predictive of suicidal behavior in HIV-seropositive individuals than the diagnosis per se. Risk of suicidal behavior in HIV-positive individuals appears to have decreased as more effective antiretroviral treatments have offered a better prognosis. The psychological impact of HIV/AIDS on lesbian/bisexual women has not been sufficiently studied (Mays, Cochran, Pies, Chu, & Ehrhardt, 1996). In one report of HIV-positive U.S. Air Force personnel, which did not identify sexual orientation, suicidal behavior was found to be reported by a much smaller percentage of females than males (G. Brown & Rundell, 1989).
PROTECTIVE FACTORS
In spite of an increased risk of suicide attempts among LGB compared to heterosexual respondents, those reporting suicidal behavior are a clear minority of the LGB individuals who have been studied, estimated at 12-19% of gay/bisexual males, and a smaller percentage of lesbian/bisexual women (King et al., 2008). Relatively little research has been done on factors that protect the large majority of LGB people from suicidal behavior. Analysis of data from a statewide survey of 6th, 9th, and 12th grade students in Minnesota (Eisenberg & Resnick, 2006) found three factors to be significantly protective of reported suicide attempts in youth with samesex sexual experience: family connectedness, perceived caring from other adults, and school safety. A nonrandom study of self-identified young and middle-aged LGB adults in New York City found connectedness to a gay/lesbian community and positive sexual identity were associated with greater social and psychological well-being (Kertzner, Meyer, Frost, & Stiratt, 2009).
The finding that exclusive same-sex sexual behavior and attraction are associated with positive mental health outcomes in women, but not men , suggests that women may be protected by greater latitude and tolerance in regard to female sexuality. This may also be reflected in the association between unsure sexual identity and elevated rates of mood and anxiety disorders in men, but not women. Although it has not yet been empirically studied in population-based research, intimate relationship stability may also protect against suicide risk, in some of the ways that marriage functions as a protective factor among heterosexual people (Kposowa, 2000;Masocco et al., 2009).
SUICIDE AND SUICIDE RISK IN TRANSGENDER POPULATIONS
Little information is available about completed suicide among transgender individuals Mathy, 2002b). Because of researchers' greater access to transsexuals who seek medical treatments such as sex reassignment surgery or hormone therapy, studies have tended to focus on this subgroup of the overall transgender population. One clinical study reported a disproportionate number of suicide deaths among Dutch transsexual women and men receiving hormone therapy, compared to the general population (van Kesteren et al., 1997). Another international review of studies that followed over 2,000 persons in 13 countries who had undergone gender reassignment surgery identified 16 possible suicide deaths (Pfäfflin & Junge, 1998). If confirmed as actual suicides, these figures would translate to an alarmingly high rate of 800 suicides for every 100,000 post-surgery transsexuals. By contrast, the current suicide rate for the overall U.S. population is 11.5 suicides per 100,000 people. It is not clear whether this very high suicide rate still exists among transexuals.
Associated factors identified in these surveys include high rates of depression, anxiety and substance abuse (Clements-Nolle, Noelle, Guzman, et al., 2001;Mathy, 2002b;Xavier et al., 2007). Transgender youth have reported parental rejection to be a particular stressor (Grossman & D'Augelli, 2008), and frequent experiences of discrimination have been reported by transgender adults (Clements-Nolle, Marx, & Katz, 2006). Preliminary findings from a 2009 U.S. National Transgender Discrimination Survey (National Center for Transgender Equality & the National Gay and Lesbian Task Force, 2009), which included almost 6,500 transgender and gender-variant people identified through a network of 800 trans-related service and advocacy organizations, support groups, list-servs and online social networks, showed that 47% reported an adverse job action because of transgender status. This included not getting a job (44%), being denied a promotion (23%), or being fired (26%); Black and multiracial respondents were especially likely to report these events. Overall, respondents reported being unemployed at twice the rate of the population as a whole, and only 40% reported having employer-based insurance coverage, which directly impacts access to health and mental health care. Almost all (97%) reported having experienced mistreatment or harassment on the job, including invasion of privacy, verbal abuse and being purposefully referred to as the wrong gender.
Little research has compared prevalence of suicidal behavior in transgender people to other population groups. One study using a nonclinical sample of over 40,000 largely U.S. volunteers who completed an internet survey (Mathy, 2002b) found 73 individuals who identified themselves as transgender. This group's responses related to suicidal behavior were compared to those reported by six other groups: heterosexual males and females, homosexual males and females, and males and females who matched the transgender individuals on nationality, age, sexual orientation, relationship status, and population size of the area in which they resided. Transgender respondents had a higher rate of reported suicide attempts than any group except homosexual females. Although sexual orientation did not differentiate transgender attempters from non-attempters, attempters were more likely 28 A. P. Haas et al. to report past and current psychiatric treatment and problems related to substance use.
KNOWLEDGE GAPS
Population-based studies over the last decade provide firm evidence of elevated rates of reported suicide attempts in LGB compared to heterosexual adolescents and adults. Although comparably representative research on completed suicide among LGB populations is lacking, recent analyses of data from Danish registries suggest significantly higher suicide rates among men with histories of domestic partnerships with men. Many population-based studies have linked elevated risk of suicide attempts in LGB populations to higher rates of mental disorders, although there is increasing evidence that other factors-notably, sexual orientation-related stigma, prejudice and discrimination-may also play a role.
While these findings suggest a significant, largely unaddressed public health problem among LGB people (King et al., 2008), little is known about specific risk and protective factors in particular subgroups of the LGBT population. In addition, although findings from nonrandom surveys of transgender individuals have consistently found high rates of reported suicide attempts, virtually no generalizable conclusions have been generated about suicidal behavior or suicide risk in this population. Gaps in current knowledge about LGBT suicidal behavior and suicide risk result from a confluence of many factors, including the low priority and historically sparse funding given to the study of sexual minority populations, difficulties inherent in studying relatively small, largely hidden population groups, and the omission of sexual orientation and gender identity from the sociodemographic characteristics that are routinely assessed in most suicide and mental health studies.
Among the most pressing questions for future research is whether LGBT people are overrepresented among suicide deaths, and if so, why. Given the stigma and secrecy associated with minority sexual orientation and gender identity, psychological autopsy methods appear to have limited utility for this purpose (King et al. 2008), and few alternative research approaches have been developed. Better methodological approaches and routine collection of sexual orientation and gender identity data as part of the death record are needed to identify rates of completed suicide and related risk factors in different LGBT age, gender, and racial or ethnic groups.
In recent years, research on nonfatal suicidal behavior and related risk factors has relied heavily on large-scale population-based surveys, especially the increasing number of national surveys that have assessed markers of sexual orientation. On the positive side, national health surveys that have included questions related to sexual orientation have provided valuable opportunities to compare LGB and heterosexual adults on a wide range of mental health variables, including rates of reported suicide attempts and mental disorders (Cochran, 2001). Examples in the United States include the National Survey of Midlife Development, the National Health and Nutrition Examination Survey, the National Survey of Family Growth, the National Household Survey on Drug Abuse, the National Comorbidity Study, the National Latino and Asian American Survey, and the National Epidemiological Survey of Alcohol and Related Conditions. In states that have added the optional sexual orientation questions to the CDC's schoolbased Youth Risk Behavior Survey, valuable information has been obtained about rates of suicidal ideation and behavior and associated factors in LGB compared to heterosexual adolescents. Surveys that follow a representative national sample over multiple waves of data collection, notably the National Longitudinal Study of Adolescent Health and the National Epidemiologic Survey of Alcohol and Related Conditions (NESARC), have been especially helpful in tracking changing patterns of mental disorders against events and evolving attitudes in the larger society.
Whenever appropriate, federal benchmark surveys related to health and mental health should include measures of sexual orientation and gender identity. This will require special attention to issues related to confidentiality and assurances of privacy during data collection. Eventually, appropriate and reliable methods for assessing gender identity must also be developed and incorporated into population-based surveys in order to track the anticipated effects of gender identity on suicide-related morbidity.
At the same time, the limitations of general health and mental health surveys as a research method for studying LGBT suicidal behavior and suicide risk must be recognized. Most such surveys are cross-sectional, collecting data at a single point in time. By their very nature, such data are retrospective and do not effectively allow cause-and-effect relationships to be discerned, especially when different windows of time are used for different questions. Most surveys have assessed lifetime or past year suicide attempts (or both), but have used a much more recent time period, such as the last two weeks or last 30 days, in assessing symptoms of mental disorders or mental distress. While surveys can demonstrate that current mental disorders tend to co-occur with reports of past suicide attempts, they cannot irrefutably determine that mental disorders-or other potential risk factors-are causally related to temporally earlier behaviors. Sorting out the complex relationships between potential risk factors and suicidal behavior, and among various risk factors themselves, is a challenge in all suicide research. Researchers who study suicidal behavior in the general population have used many different approaches including psychological autopsies, genetic and neurobiological studies, longitudinal cohort studies and randomized controlled clinical studies to advance understanding of causal factors. In contrast, nearly all of what is known about suicidal behavior in LGBT people has come from cross-sectional surveys or other nonexperimental research.
Another limitation of national health and mental health surveys is that they typically explore a range of topics and thus are restricted in the number of questions that can be asked about suicidal behavior. Often, surveys ask a single question about suicide attempt history, for example, "Have you ever made a suicide attempt?" Because of the tendency of some respondents to report plans or preparations for suicide or self-harm behaviors without the intent to die as a "suicide attempt," researchers who focus specifically on suicide have recognized the need for multiple questions that distinguish these behaviors from actual suicide attempts (Bongiovi-Garcia et al., 2009). In addition, surveys designed for the general population are usually limited in the number of questions that assess current sexual orientation, and are rarely able to identify changes in orientation (and related factors such as partnership status) that may occur over the lifespan (L. Diamond, 2008;Fergusson et al., 2005).
Much has been learned from population-based surveys, but researchers studying LGBT suicidal behavior and suicide risk need to augment this research method with other approaches. Identifying LGBT people among samples already being studied by suicide researchers would be a potentially valuable, low-cost first step toward improving assessment of suicidal morbidity in this higher-risk population. Even when relatively small samples are used, as is the case in many neurobiological and genetic studies, identifying research subjects with minority sexual orientation or gender identity may over time provide valuable clues about factors that are related to elevated rates of suicidal behavior in these populations.
In-depth studies of methodologically sophisticated samples of LGBT (and comparable heterosexual) populations are needed to identify pathways to suicidal behavior in different age, gender, and racial and ethnic groups within the overall sexual minority population. Longitudinal studies that follow representative samples of the population over different time points such as the New Zealand cohort study (Fergusson, Horwood, & Beautrais, 1999;Fergusson, Horwood, Ridder, et al., 2005) and the Add Health cohort (Russell & Toomey, 2010) have considerable promise. More studies are needed to identify the processes that create and maintain the vulnerability of sexual minority populations for suicide-related morbidity. These should focus on LGBT people in different cultural settings, beginning early in life and tracking participants through adolescence, adulthood and into the elder years so that time sequences of factors related to suicidal behavior can be established. Such studies are costly, however, and will require a significant expansion of the funding for LGBT research.
More studies are also needed to illuminate the relationship of suicidal behavior to LGBT developmental milestones such as recognition of samesex attractions and/or transgender identity, or to stressors that are unique to LGBT individuals. Subgroups for which solid research data related to suicidal behavior and suicide risk are especially lacking include older LGB adults, transgender people of all ages, and LGBT people who are further marginalized by homelessness, juvenile justice detainment or incarceration, or serious mental illness. In addition, very little research has been directed toward identifying factors that protect the large majority of LGBT people from suicidal behavior. Better understanding of protective factors and how LGBT individuals develop and sustain resilience in the face of the challenges inherent to sexual minority status, may contribute to reducing risk of suicidal behavior in these populations.
Recommendations for Future Research
To advance knowledge and understanding of LGBT suicide and suicide risk: • Expand public and private funding for research on LGBT suicide and suicide risk over the lifespan, including funding from LGBT foundations and donors. • Identify and test new ways of determining sexual orientation and gender identity among people who die by suicide. Explore the possibilities of linking existing records on same-sex marriages, civil unions or registered domestic partnerships with suicide mortality data in appropriate countries and U.S. states. • Continue to test which aspects of sexual orientation and gender identity are most strongly related to negative mental health outcomes, including suicide attempts, and on that basis, establish empirically supported, standardized measures of sexual orientation and gender identity for use in suicide and mental health research. In this task, Best Practices for Asking Questions about Sexual Orientation on Surveys (Badgett, 2009) provides an excellent start. • Develop and test measures to determine partnership status and intimate relationships among LGBT people in different age, racial and ethnic, geographic, and cultural groups. • Encourage inclusion of measures of sexual orientation and gender identity and partnership status in all suicide and mental health research, including clinical, neurobiological and genetic studies, with appropriate safeguards for privacy and confidentiality. Public and private agencies that fund suicide and mental health research can play a critical role in this regard. • Educate key stakeholders about the need for questions on sexual orientation and gender identity in suicide and mental health research. These include funding agencies, researchers, institutional review boards that ensure the protection of human research subjects in research, as well as coroners, medical examiners, police investigators and others who develop or create records of fact used by suicide researchers. • Develop and conduct longitudinal studies, using large cohort designs to ensure sufficient numbers of LGBT participants, in order to: • identify prevalence, causes, and pathways to suicidal behavior for LGBT individuals in all gender, age and racial and ethnic groups; • assess both risk and protective factors related to sexual/gender developmental milestones (e.g., feeling different, self-recognition, disclosure to others); and • examine the relationship between LGBT suicidal behavior and stigma and discrimination, and document the impact of evolving policies and laws related to LGBT people.
• Conduct studies of the prevalence of suicidal behavior and contributing contextual factors in LGB racial, ethnic, cultural and religious groups. • Conduct studies of LGB midlife and older adults to establish the prevalence and patterns of past and current suicidal behavior. Examine the role of specific risk factors including depression, drug and alcohol abuse, discrimination, long-term effects of HIV/AIDS and other chronic illnesses and social isolation in different developmental life stages. These studies should also include potentially protective factors such as positive sexual/gender identity, family and community connectedness, and access to affirming health and mental health services. • Conduct studies of suicidal behavior and suicide risk in diverse groups of transgender people across the lifespan. • Conduct studies of suicidal behavior and suicide risk among high-risk LGBT subgroups, including LGBT youth who are homeless, in juvenile detention facilities or incarcerated. • Conduct studies of factors that protect against or mitigate the impact of suicide risk factors in the large majority of LGBT people, and factors that contribute to the development of resiliency in each of these populations. • Mandate inclusion of empirically valid measures of sexual orientation and gender identity in all federally supported benchmark surveys related to health and mental health in which privacy and confidentiality can be appropriately ensured. Include assessment of sexual orientation and gender identity in existing large or longitudinal surveys such as the National Institute of Child Health and Human Development (NICHD) Study and the National Health Interview Survey (NHIS). • In health surveys and other research studies, use consistent time periods to assess sexual orientation and gender identity, suicidal behavior and suicide risk factors, in order to more precisely identify interrelationships.
GAPS BETWEEN KNOWLEDGE AND PRACTICE
Although much remains to be learned, what is already known has not yet been applied to practices aimed at reducing suicidal behavior and suicide risk in LGBT people. In this section, we discuss three areas in which improved practices may contribute towards this goal: mental health interventions, suicide prevention strategies, and public policy. At the conclusion of each section, we offer recommendations to bring about needed change. Few mental health interventions or suicide prevention strategies have been evaluated with LGBT people, and it cannot be assumed that what has worked with predominantly heterosexual populations will have the same results with specific sexual minority groups. At the same time, as researchers have noted (King et al., 2008), the consistency of the data pointing to elevated risk of suicidal behavior and mental health problems among sexual minority people argues for taking action now rather than awaiting more research evidence. Where we recommend extending evidence-based practices for general populations to LGBT populations, we urge that careful process and outcome evaluations be conducted so that timely and appropriate adjustments can be made and differences in effectiveness determined. In this regard, it will be critical to look at outcomes among LGB individuals in different gender, age, racial and ethnic and cultural groups.
Generalizable information is lacking about suicidal behavior and suicide risk among transgender populations, and thus there is currently little empirical basis for specific recommendations for practices involving transgender individuals. Where there appears to be a logical basis for assuming commonalities across sexual minority persons, we include transgender along with lesbian, gay, and bisexual groups in our recommendations, again urging that careful evaluations be conducted to identify possible differences in outcomes among transgender compared to LGB people, and among transgender people having different sexual orientations as well as different gender identities, behaviors and expressions.
Mental Health Interventions
LGBT organizations have been at the forefront of bringing national attention to health problems that disproportionately affect LGBT people, notably HIV/AIDS, seeking remedies to associated disparities in research funding, and working to improve access to high quality, culturally appropriate health care services. Commensurate efforts have not been directed toward elevated rates of mental disorders in LGBT people and the risk these disorders pose for suicidal behavior, even though Healthy People 2010: Companion Document for Lesbian, Gay, Bisexual and Transgender Health (Gay and Lesbian Medical Association and LGBT Health Experts, 2001) provided welldocumented recommendations to support a focus on LGBT mental health. Up until 1973, when LGBT psychiatrists, other mental health professionals, and their allies spearheaded efforts at the annual meeting of the American Psychiatric Association to remove homosexuality per se as a psychiatric diagnosis, variant sexual orientation was routinely equated with mental disorder and sexual deviance, leading to a therapeutic focus on "curing" people of same-sex sexual attraction and behavior. Today, more than three decades after homosexuality was removed from the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM), a small but vocal minority among mental health professionals continues to promulgate "curative" therapies for gay men and lesbians, despite evidence that such methods are ineffective and harmful (American Psychiatric Association, 2000). In addition, since 1980 the DSM has included diagnoses that pathologize variant gender identities and behaviors. Given this legacy, it is understandable that mental disorders and suicide risk have not been propritized among the many pressing issues on the agenda of LGBT organizations. However, the strength of the empirical evidence that significant numbers of LGBT people suffer from mood, anxiety and substance use disorders compels concerted action aimed at encouraging help seeking, improving the quality and availability of culturally appropriate mental health services, and generating greater federal responsiveness to the mental health problems of LGBT people.
Although LGB people utilize mental health services more than heterosexual individuals (Cochran & Mays, 2000b;Cochran, Mays, & Sullivan, 2003;Grella, Greenwell, Mays, & Cochran, 2009), it is not clear how frequently they access evidence-based, time-limited treatments that have been established to be most effective in reducing depression and suicidal behavior (Guthrie et al., 2001;Brown, Ten Have, & Henriques, 2005). Although progress in developing culturally competent treatments has certainly been made since the 1990s when widespread dissatisfaction with mental health services was documented among LGBT people (Israel & Tarver, 1997;Liddle, 1996), access to competent and mental health care remains limited in many geographic areas. Given the high rates of substance use disorders among LGBT men and women, which in the general population increase risk for suicidal behavior, increasing access to and participation in culturally competent drug and alcohol treatment is especially critical. Particular efforts should be made to expand the participation of LGBT people in substance abuse treatment programs that incorporate approaches and protocols based on current research evidence (Center for Substance Abuse Treatment, 2006Treatment, , 2009Grella et al., 2009;Pettinati et al., 2010).
Evidence shows that targeted or modified mental health interventions for LGB individuals may increase treatment acceptability, retention, and effectiveness. One study showed that methamphetamine-dependent gay and bisexual men given "gay-tailored" cognitive behavioral therapy (CBT) showed more rapid declines in depressive symptoms and methamphetamine use, compared to those given traditional CBT or other general interventions (Jaffe, Shoptaw, Stein, Reback, & Rotheram-Fuller, 2007). A promising, empirically based approach for treating depressed and suicidal adolescents, Attachment-Based Family Therapy (G. S. Diamond, Siqueland, & Diamond, 2003), is currently being adapted and tested for use with LGBT adolescents.
While recognizing the need for individually focused treatment and support services for those who already have or are developing mental disorders, community-based programs are being developed by LGBT organizations in some European countries that emphasize LGBT-specific mental health promotion and behavioral health interventions (International Gay, Lesbian, Bisexual, Transgender and Queer Youth and Student Organization, 2007;Mule et al., 2009).
Overall, however, there is a dearth of mental health interventions specifically designed for LGBT people, which means the large majority of those who need treatment must rely on general community providers. In recent years, there has been growing recognition of the need to include more extensive information on LGBT people in educational and training programs for mental health professionals. One promising start is the development of a LGBT Mental Health Syllabus for psychiatry residents (Group for the Advancement of Psychiatry, 2007). Although this curriculum provides helpful information on such topics as LGBT psychological development and common LGBT medical and mental health problems, the lack of direct discussion of suicide or suicide risk is an unfortunate omission that should be addressed. No systematic information is available on the extent to which this or similar curricula are being implemented in psychiatry or other clinical training programs.
For current practitioners, the Association for Lesbian, Gay, Bisexual, and Transgender Issues in Counseling, a division of the American Counseling Association, provides Competencies for Counseling Gay, Lesbian, Bisexual and Transgender Clients (Association for Lesbian, Gay, Bisexual, and Transgender Issues in Counseling, n.d.). These offer excellent guidelines for ethical, culturally competent care of sexual minority clients, but do not touch specifically on heightened risk for suicidal behavior. Likewise, the American Psychological Association's Guidelines for Psychotherapy with Lesbian, Gay and Bisexual Clients (American Psychological Association, n.d.) provide helpful general principles but do not specifically address psychotherapeutic treatments for reducing suicidal behavior. The American Psychiatric Association has developed guidelines for the Assessment and Treatment of Patients with Suicidal Behaviors (American Psychiatric Association, 2003), which identify sexual orientation as a potential suicide risk factor, but provide limited information about factors associated with suicidal behavior among LGBT persons. Regarding adolescents, the American Academy of Pediatrics alerts physicians to elevated suicide risk among LGBT youth (Shain & the Committee on Adolescence, 2007), but its guidelines for the identification, assessment, and treatment of adolescent depression in primary care Zuckerbrot et al., 2007) do not address LGBT youth or young adults.
In lieu of any evidence of intrinsic pathology associated with transgender identities, mental health professionals as well as transgender and allied groups have voiced increasing concern that the DSM diagnoses of Gender Identity Disorder (GID) and transvestic fetishism stigmatize variant gender identities and behavior and discourage many transgender people from seeking mental health care (Drescher, 2010). In advance of the publication of a fifth edition of the DSM in 2013, many are calling for the removal of these diagnoses. Some fear, however, that removing GID as a recognized condition that can lead to significant distress may result in the denial of insurance coverage for expensive health care services related to transitioning, leaving many transgender individuals without needed care A number of compromise positions have been put forth, including changing the term Gender Identity Disorder to Gender Incongruence or Gender Dysphoria, and repositioning the condition in the medical rather than psychiatric realm to reduce stigma and assure access to care (Allison, 2010;Winters & Ehrbar, 2010).
Recommendations Related to Mental Health Interventions
To make the mental health needs of all LGBT people a priority within the national LGBT agenda, it is recommended that LGBT organizations and suicide prevention organizations work in concert to: • Develop LGBT-focused campaigns to: • destigmatize mental disorders, particularly mood and anxiety disorders, among LGBT people; • educate LGBT people about the relationship of mood, anxiety, and substance use disorders to suicide; • encourage help seeking among LGBT people who are suffering from mental disorders, or are suicidal; and • encourage the development of equitable, accessible, and culturally appropriate mental health and substance abuse services for LGBT people of all ages.
• Provide leadership for the development of needed mental health interventions and programs for LGBT people, including: • adaptations to LGBT people of mental health interventions and therapies that have been established to be effective among the general population; • programs for early identification of risk behaviors and mental health disorders and substance abuse, especially among LGBT youth; • LGBT-specific behavioral health interventions, related in particular to substance abuse; and • LGBT-specific mental health promotion programs that provide education and information resources about healthy sexual and gender variations and promote positive identity and family and community connectedness.
• Ensure the involvement of LGBT people of all age, racial and ethnic, and gender identity constituencies in the planning, design, development, and implementation of all new mental health interventions and programs for substance abuse treatment.
To insure the availability of professionals with the knowledge, skills, and attitudes to provide quality mental health care to LGBT individuals, including those who are at risk for suicide: • All professional educational and training programs that prepare students to provide mental health care or administer mental health programsincluding physician residency programs in psychiatry and primary care; graduate programs in psychology, social work, nursing, and public health; and other mental health and human services training programs-should develop and provide comprehensive, empirically based education about LGBT mental health needs and suicide risk. • Professional organizations that accredit professional training programs and certify the competence of mental health care providers and primary care physicians, should: • determine the core body of knowledge and standards of care for the treatment of LGBT mental health problems and suicide risk within their specialty areas; • provide continuing education offerings and educational materials related to LGBT mental health needs and suicide risk to all current practitioners; • update existing guidelines for the treatment of LGBT people within their specialty areas, based on up-to-date research findings related to LGBT mental health and suicide risk and evidence-based treatments for those who are suicidal; and • develop guidelines for the treatment of LGBT people in all disciplines where they are currently lacking.
To reduce stigma of transgender people within mental health professions: • Revise DSM-V diagnoses related to transgender people to: • affirm that gender identity, expression and behavior that differ from assigned birth sex is not indicative of a mental disorder; and • establish the medical necessity of transition treatments for those who perceive biological characteristics to be incongruent with their gender identity.
Suicide Prevention Strategies
Suicide prevention strategies focus on four key domains, in addition to mental health treatment (Mann et al., 2005): • awareness campaigns and educational programs for the general public, primary care physicians, and community and organizational gatekeepers; • screening programs, hotlines, and other activities that identify at-risk individuals and direct them to treatment; • restriction of lethal means used for suicide; and • programs that promote media as an avenue for public suicide prevention education, and discourage coverage that glamorizes or normalizes suicide.
By addressing LGBT suicide and suicide risk in such a limited way, national and most state-level suicide prevention strategies have provided little guidance for the development of suicide prevention programs that specifically target LGBT groups. One comprehensive review of suicide prevention programs for LGBT youth (Suicide Prevention Resource Center, 2008) identified only one such program, The Trevor Project, which operates the only national crisis and suicide prevention lifeline for LGBT and questioning youth. The Trevor Project also provides in-school workshops, educational materials, and online educational resources for youth, and advocates for public policies to reduce LGBT stigma. No systematic data are available on the impact of these programs on reducing suicidal behavior or suicide risk among LGBT youth.
Other national organizations serving LGBT populations focus on issues that, while not explicitly addressed to suicide prevention, may contribute to this goal. Best known among such organizations are the Gay, Lesbian and Straight Education Network (GLSEN), Services and Advocacy for Gay, Lesbian, Bisexual and Transgender Elders (SAGE), and Parents, Families and Friends of Lesbian, Gay, Bisexual and Transgender People (PFLAG). Many other state and local LGBT organizations provide suicide prevention resources in conjunction with activities focused on child welfare, HIV/AIDS prevention and support, violence prevention, or health promotion (Suicide Prevention Resource Center, 2008).
A recent program, the Family Acceptance Project based at San Francisco State University, is developing family interventions based on research findings on the relationship between parental and caregiver behaviors and mental health outcomes, including suicide attempts, among LGBT youth (Ryan et al., 2009). The Family Acceptance Project is also developing training materials on working with LGBT youth and families for school personnel, mental health professionals, and child welfare, juvenile justice, and family service providers.
Suicide prevention interventions designed for the general public rarely address suicidal behavior or suicide risk within LGBT groups. As a result, unless LGBT people are the specific focus of an intervention, they are generally not included in suicide prevention programming. Although guidelines for increasing LGBT competency among human services organizations have been developed (Suicide Prevention Resource Center, 2008), there is as yet limited evidence that mainstream suicide prevention interventions are becoming more LGBT inclusive.
Recommendations Related to Suicide Prevention Interventions
To improve LGBT suicide prevention efforts: • Address LGBT suicide risk and possible interventions for reducing risk in national and state suicide prevention strategies and plans. • Provide educational and resource materials on LGBT suicide and suicide risk to LGBT organizations, and encourage consideration of how suicide prevention can be advanced within the context of each organization's mission and activities. • Incorporate well-designed outcome evaluations into all interventions aimed at reducing suicidal behavior and suicide risk among LGBT people. • Develop a wider range of interventions for reducing suicidal behavior and suicide risk in specific LGBT groups. • Encourage a focus on LGBT groups within suicide prevention interventions and programs designed for the general population. • Develop and implement educational programs for increasing competency in LGBT suicide risk within: • organizations and groups providing suicide prevention interventions for the general population; and • community gatekeepers including teachers and staff in youth programs, senior centers, aging services agencies and others who come in contact with at-risk individuals.
• Encourage training in LGBT suicide risk for staff and volunteers of suicide crisis lines, law enforcement, emergency care professionals, and others who work with suicidal individuals.
PUBLIC POLICY
Among the most salient findings to emerge from recent research are those linking public policies that discriminate against sexual minorities to elevated rates of mental disorders in LGB people (Hatzenbuehler, Keyes, et al., 2009;Hatzenbuehler, McLaughlin, et al., 2010). The wellestablished association between mental disorders and suicide attempts in at least some LGBT subgroups points to the need to include advocacy for policy change as a component of a comprehensive plan for LGBT suicide prevention.
In the United States, as in other countries, LGBT organizations have long provided the key leadership in identifying and advocating for policy and legislative changes related to protecting LGBT people from violence, hate crimes, school bullying and harassment, and for ending discrimination in housing, the workplace, the military and marriage rights. With the exception of bullying and school safety issues, LGBT advocacy efforts have not commonly linked discriminatory laws and policies to negative health or mental health outcomes in LGBT people. In recent years, however, health and mental health associations have begun to articulate this linkage in speaking out against laws and policies that discriminate against LGBT people. In approving a December 2009 motion to advocate for repeal of the current Don't Ask, Don't Tell law that restricts gay men and lesbians from serving openly in the military, the American Medical Association (AMA) was especially critical of the law's requirement that military physicians and psychiatrists report gay and lesbian service members who disclose their sexuality in the context of treatment, noting that this discourages help seeking among gay and lesbian patients in need of care, while placing an untenable burden on treatment providers (Moran, 2009). At the same time, the AMA also approved a report titled "Health Disparities in Same-Sex Partner Households," which called for the AMA to work to eliminate inequities in access to health and mental health care arising from the exclusion of same-sex couples from civil marriage and the associated difficulties in obtaining health insurance (Moran, 2009). In its Position Statement for Support of Legal Recognition of Same-Sex Civil Marriage, the American Psychiatric Association (2005) noted its history of supporting equity, parity, and nondiscrimination in matters that have an impact on mental health. In addition to the negative impact of discriminatory marriage laws on the stability of same-sex couples' relationships and their mental health, the statement noted the particular effects on older adults in same-sex relationships, including deprivation of survivorship and inheritance rights, financial benefits, and legal recognition as a couple in health care settings, which increase the psychological burden associated with aging.
Suicide prevention organizations have shown strong support for policies that seek to improve mental health outcomes, such as the federal Mental Health Parity and Addiction Equity Act of 2008 (U.S. Department of Labor, 2010), which mandated that insurance coverage for mental health treatments be comparable to that for other medical interventions. Advocating for nondiscrimination and protections for LGBT people is a logical extension of the effort to lower suicide risk through alleviating mental disorders.
Recommendations Related to Public Policy
To reduce the negative mental health outcomes of institutional discrimination against LGBT people and its associated stigma and prejudice: • Advocate for anti-bullying and safe schools legislation, and for the specific inclusion of sexual orientation and gender identity in protective legislation related to school safety. • Advocate for changes in all federal and state laws and regulations that create inequities based on sexual orientation or gender identity and have been shown to have negative mental health outcomes or otherwise heighten suicide risk for LGBT people. • Advocate for improved access to mental health services through nondiscrimination policies and expansion of health insurance coverage to same-sex partners. • Advocate for legislation requiring measures of sexual orientation and gender identity to be incorporated into federally supported benchmark surveys and other population-based databases related to health and mental health, so that the consequences of inequities affecting LGBT people can be more fully identified.
CONCLUSION
Over the last two decades, an increasing body of empirical research in the United States and other countries has pointed to significantly elevated suicide risk among LGBT compared to heterosexual people. Although many questions are as yet unanswered, there appears to be little doubt that a broad national effort will be needed to encourage and fund the needed research, raise awareness of the problem among LGBT and suicide prevention leaders, and develop the interventions, prevention strategies, and policy changes through which suicidal behavior and suicide risk in LGBT populations can be reduced. We hope this report and the recommendations it offers will contribute to stimulating this effort. | 2014-10-01T00:00:00.000Z | 2010-12-30T00:00:00.000 | {
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248302703 | pes2o/s2orc | v3-fos-license | Clinical and Molecular Validation of BAP1, MTAP, P53, and Merlin Immunohistochemistry in Diagnosis of Pleural Mesothelioma
BAP1 and MTAP immunostains play an important role in diagnosis of mesothelioma, but additional markers are needed to increase sensitivity. We analyzed 84 pleural mesotheliomas (51 epithelioid, 27 biphasic, 6 sarcomatoid) by a hybrid-capture next-generation sequencing (NGS) panel including complete coverage of coding and splicing regions for BAP1, MTAP, NF2, and TP53 and correlated molecular findings with diagnostic immunostains for BAP1, MTAP, Merlin, and p53, respectively. Fifty-seven reactive mesothelial proliferations served as benign comparators. Loss of BAP1, MTAP, and Merlin protein expression were, respectively, 54%, 46%, and 52% sensitive and 100% specific for mesothelioma. Two-marker immunopanels of BAP1 + MTAP, BAP1 + Merlin, and MTAP + Merlin were 79%, 85%, and 71% sensitive for mesothelioma, while a three-marker immunopanel of BAP1 + MTAP + Merlin was 90% sensitive. Diffuse (mutant-pattern) p53 immunostaining was seen in only 6 (7%) tumors but represented the only immunohistochemical abnormality in 2 cases. Null-pattern p53 was not specific for malignancy. An immunopanel of BAP1 + MTAP + Merlin + p53 was 93% sensitive for mesothelioma, and panel NGS detected a pathogenic alteration in BAP1, MTAP, NF2, and/or TP53 in 95%. Together, 83 (99%) of 84 tumors showed a diagnostic alteration by either immunohistochemistry or panel NGS. Adding Merlin to the standard BAP1 + MTAP immunopanel increases sensitivity for mesothelioma without sacrificing specificity. p53 immunohistochemistry and panel NGS with complete coverage of BAP1, CDKN2A/MTAP, TP53, and NF2 may be useful in diagnostically challenging cases.
Introduction
Malignant mesothelioma is a rare tumor, with approximately 3000 new cases annually in the United States 1,2 . 85-90% arise in the pleura, with most of the remainder affecting the peritoneum [3][4][5] . Pleural mesothelioma carries a poor prognosis, and accurate distinction from benign mesothelial proliferations is paramount. However, this distinction may be challenging, particularly in small biopsy specimens.
To date, there has been limited study of immunohistochemistry for Merlin (the protein encoded by NF2) and p53 in diagnosis of mesothelioma. Diagnostic fluorescence in situ hybridization (FISH) for hemizygous NF2 deletion is reportedly ~50% sensitive for mesothelioma and 100% specific in the differential with reactive mesothelial hyperplasia 12,36 . Two studies of Merlin immunohistochemistry yielded inauspicious results 19,37 ; however, newer commercially available anti-Merlin antibodies warrant further exploration.
Using arbitrary non-biological thresholds (usually 10%) to distinguish "low" versus "high" p53 expression, numerous older studies found no use for p53 immunohistochemistry to distinguish benign and malignant mesothelial proliferations 38 . However, using empirical thresholds derived from high-grade serous carcinoma 39,40 , one recent study found that p53 immunohistochemistry corresponds to TP53 mutational status in mesothelial proliferations and has a role in mesothelioma diagnosis 41 . Those findings warrant confirmation together with robust molecular correlation.
As the number and reliability of diagnostic immunostains have grown, so too has routine application of multigene NGS panels to mesothelioma diagnosis and management, offering the opportunity for orthogonal validation of immunostains. We studied 84 pleural mesotheliomas to 1) correlate immunohistochemical and molecular results for BAP1, MTAP, TP53, and NF2; 2) specifically explore the diagnostic characteristics of Merlin and p53 immunohistochemistry; and 3) re-evaluate current ancillary testing algorithms.
a. Cohort
This study was approved by the institutional review board at Brigham and Women's Hospital. The electronic pathology database was searched for mesotheliomas meeting the following inclusion criteria: 1) pleural primary site, 2) resection (decortication or extrapleural pneumonectomy) specimen, 3) analyzed by the OncoPanel next-generation sequencing assay as part of a consented protocol sponsored jointly by Brigham and Women's Hospital and Dana Farber Cancer Institute (PROFILE), 4) hematoxylin and eosin (H&E)-stained slides available, and 5) tissue blocks available in institutional archive. Localized mesothelioma and tumors reviewed only for pathological diagnostic consultation were excluded. A separate database search was carried out for reactive mesothelial proliferations with tissue blocks in institutional archives and at least one year of clinical follow-up.
b. Clinicopathologic parameters
Patient sex and age at diagnosis were extracted from the electronic medical record. Clinical and radiology reports were reviewed to confirm pleural primary site. Three representative H&E-stained slides (including, where possible, a slide from the sequenced block) were reviewed from each case to determine histotype (epithelioid, biphasic, sarcomatoid). For epithelioid tumors, predominant architecture 42 was determined. Biphasic tumors were subclassified as epithelioid-or sarcomatoid-predominant.
Immunostains were performed on freshly cut 5-micron sections of formalin-fixed paraffinembedded tissue. When possible, immunostains were performed on the sequenced tumor block (n=45); when necessary, another tumor block from the sequenced specimen was used (n=31, principally cases with sequencing on fresh frozen tissue), or tumor from a different surgical specimen (n=8, principally cancer center transfer cases). Where possible (n=25), both the epithelioid and sarcomatoid components of biphasic tumors were immunostained, with staining profile documented for each component.
BAP1 was scored as retained (positive tumor nuclear staining) or lost (negative tumor nuclear staining with a positive internal control). Staining patterns in tumors with BAP1 loss were subclassified as "negative" (no nuclear or cytoplasmic staining) or "cytoplasmic-only." MTAP was scored as retained (positive tumor cytoplasmic staining) or lost (negative tumor cytoplasmic staining with a positive internal control). Percent tumor cells with cytoplasmic staining was documented to identify tumors with "heterogeneous" MTAP expression. Any spatially discrete MTAP-negative tumor cell population was regarded as evidence for clonal MTAP deletion and classified as MTAP loss.
Merlin was scored as retained (positive tumor cell staining, irrespective of distribution or intensity) or lost (negative tumor cell staining alongside a positive internal control). To further assess molecular correlates of specific staining patterns, we also noted 1) staining intensity (classified as "weak" if evident only under 20x or 40x objective), 2) staining distribution (membranous versus cytoplasmic), and, when applicable, 3) pattern of membranous staining (linear versus granular/discontinuous).
d. Sequencing
All tumors were analyzed by tumor-only hybrid-capture NGS on the 298-gene (n=22) or 447-gene (n=62) OncoPanel platform 43 . Briefly, samples were required to contain at least 20% tumor by pathologist's visual estimate. DNA was extracted and subjected to library preparation as previously described 43 . Sequencing was performed on an Illumina HiSeq 2500 (Illumina Inc., San Diego, CA). Mutect and GATK (Broad Institute, Cambridge, MA) were used for detection of single nucleotide and insertion-deletion variants. Variants were filtered to remove technical artifacts, synonymous variants, and variants at >0.1% frequency in the Genome Aggregation Database (https://gnomad.broadinstitute.org/). Copy number alterations were determined using an internally developed tool (RobustCNV). Structural variants were detected using BreakMer 44 . Sequencing data from this study is publicly available through the AACR Genie database.
Pass-filter variants were subclassified as missense, truncating (nonsense, frameshift), or splice site and assessed for likely pathogenicity by a molecular pathologist (LMS). Genomic deletions were classified as homozygous (log 2 ratio of target copy coverage:diploid normal approximating −2); shallow deletions were not further specified in light of challenges in inferring degree of copy loss in samples with tumor content <30% or genomic heterogeneity. For loss of heterozygosity analysis, population variants detected on the panel were plotted according to their genomic coordinates and variant allele fraction. Regions of the genome showing deviation of heterozygous variant allele fractions away from 0.5 in the absence of concomitant numeric copy change were considered to have copy neutral loss of heterozygosity. Tumors were deemed to show "near-genome-wide loss of heterozygosity" when loss of heterozygosity was detected in at least 17 of 22 chromosomes (X chromosome excluded). Due to limitations in our bioinformatic analysis, we did not distinguish neargenome-wide loss of heterozygosity with versus without subsequent endoreduplication, but this distinction is of uncertain biologic significance, and prior work has grouped both cases as a singular molecular class. 17 Further, the term "near-genome-wide loss of heterozygosity" is used in this manuscript in lieu of "near-haploidization" (as previously termed by others 17 ) to acknowledge that, due to methodological differences, the tumors in our cohort with near-genome-wide loss of heterozygosity may, individually or in aggregate, differ subtly from previously described mesotheliomas with near-haploidization, as perhaps reflected by their relatively high prevalence in our study.
e. Statistical analyses
Summary statistics were tabulated in Excel (Microsoft, Redmond, WA). Contingency table (sensitivity, specificity) analyses were performed manually. Between-group differences in categorical and continuously distributed variables were assessed by Pearson's chi-squared and Mann-Whitney U tests, respectively (SAS 9.4, SAS Institute, Cary, NC, USA). Tests were two-tailed with α=0.05 for statistical significance. P values for multiple comparisons were corrected by Holm's method.
As addressed in the Discussion, neither immunohistochemical nor panel NGS can be definitively claimed as a diagnostic gold standard when comparing results for BAP1, MTAP, TP53, and NF2; accordingly, Cohen's kappa is presented for these comparisons to quantify interassay agreement. Using the standards of Landis and Koch 45 , 0.0-0.2 is classified as slight, 0.21-0.4 as fair, 0.41-0.6 as moderate, 0.61-0.8 as substantial, and 0.81-1.0 as near-perfect agreement. Because MTAP immunohistochemistry is regarded as a surrogate for CDKN2A deletion, sensitivity and specificity are reported for this comparison.
3) Merlin-All 57 reactive mesothelial proliferations showed retained immunohistochemical expression of Merlin, including 35 with strong linear membranous staining, 19 with weak granular membranous staining, and 3 with cytoplasmic-only staining. In contrast, Merlin was lost in 44 (52%) mesotheliomas ( Figure 4). Among 40 malignant tumors with retained Merlin, 15 showed strong linear membranous staining, 7 showed weak linear membranous staining, 12 showed granular membranous staining, and 6 showed cytoplasmic-only staining (Supplemental Figure 3). The intensity, distribution, and pattern of Merlin immunostaining did not differ between mesothelioma and reactive mesothelial proliferations. All sarcomatoid tumor populations (i.e., sarcomatoid tumors and sarcomatoid components of biphasic tumors) with retained Merlin (n=9) showed cytoplasmic-only staining. Among 28 epithelioid tumors with membranous Merlin staining, distribution was apico-lateral in 16 and circumferential in 12. Apico-lateral staining was associated with tubulopapillary architecture, whereas circumferential staining was associated with solid and trabecular architecture (Supplemental Table 4). Two tumors showed heterogeneous Merlin immunostaining (see below).
c. Immunostaining Patterns in Biphasic Tumors
Both the epithelioid and sarcomatoid components were present for immunohistochemical analysis in 25 of 27 biphasic tumors (Supplemental Table 5). BAP1 immunostaining was concordant in all cases reviewed. MTAP immunostaining was discordant (retained in epithelioid, lost in sarcomatoid component) in 4 (see Figure 2G, 2H), On Merlin immunostaining, 17 tumors showed concordant loss in both components, 5 showed concordant retention, and 2 showed loss in the sarcomatoid component only.
d. Molecular-Immunohistochemical Correlates
None of the studied markers showed a significant difference in molecularimmunohistochemical concordance by histotype (see Supplemental Table 2).
i. BAP1-BAP1 concordance statistics and specific molecular-immunohistochemical correlates are listed in Supplemental Table 6. Overall, BAP1 molecular and immunohistochemical results were concordant in 72 (86%) of 84 tumors (κ=0.71). Of 9 tumors with shallow BAP1 deletion only, only 3 lost BAP1 protein expression. In contrast, loss of BAP1 expression was observed in 37 (97%) of 38 tumors with two-copy deletion, structural variant, and/or point mutation. (The single case (#6) with intact protein expression despite presence of a BAP1 mutation showed a C-terminus frameshift mutation predicted to read through into the 3' untranslated region.) Conversely, in 5 (11%) of 45 tumors with protein loss, no definite deleterious alteration was detected by molecular profiling. No significant correlation was observed between type or location of BAP1 alteration and presence of cytoplasmic staining (see Supplemental Table 6).
ii. CDKN2A and MTAP-Concordance, sensitivity, and specificity statistics for MTAP immunohistochemistry and CDKN2A & MTAP deletion are in Supplemental Table 7.
iii. P53-TP53 molecular and p53 immunohistochemical concordance is reported in Table 1 (see also Supplemental Figure 4). Overall, p53 immunohistochemistry was concordant with TP53 mutational status in 67 (80%) of 84 tumors. Of 11 tumors with shallow arm-level or sub-arm-level chromosome 17p deletion, 4 showed null-pattern and 7 showed wildtype p53 immunostaining. Of 12 tumors with point mutations or subgenic TP53 deletion, 6 showed diffuse, 5 wildtype, and 1 null-pattern p53 immunostaining. All 6 tumors with diffuse p53 expression harbored a TP53 point mutation, whereas only 5 (42%) of 12 tumors with null-pattern p53 showed any TP53 molecular alteration. Tumors with null-pattern versus wildtype p53 immunostaining did not differ in the rate of TP53 truncating mutations, deletions, or molecular alterations overall. On this basis of this immunohistochemicalmolecular correlation and comparison with p53 immunostaining in reactive control samples (detailed above), null-pattern immunostaining was classified as a non-aberrant pattern alongside wildtype staining.
iv. NF2 / Merlin-NF2/Merlin concordance statistics and specific molecularimmunohistochemical correlates are listed in Table 2 Rates of immunohistochemical-molecular discordance did not differ between tumors with testing performed on the same block, different block from the same surgical specimen, or different surgical specimen (Supplemental Table 9).
f. Comparison of diagnostic algorithms
Sensitivities and comparative P values for immunomarkers and immunopanels are in Table 3. In summary, BAP1 is significantly more sensitive than MTAP for diagnosis of epithelioid (P=0.02) but not biphasic (P=0.58) mesothelioma. Merlin did not differ significantly from BAP1 or MTAP for diagnosis of epithelioid or biphasic mesothelioma. p53 is the least sensitive immunomarker for diagnosis of epithelioid and biphasic mesothelioma, though p53 was the only aberrant immunostain in 2 (4%) of 51 epithelioid mesotheliomas (see Figure 2E-2H). Among two-marker panels, there was no significant difference in sensitivity between BAP1 + MTAP, BAP1 + Merlin, and MTAP + Merlin for diagnosis of epithelioid or biphasic mesothelioma. Two-marker panels including p53 were of consistently inferior sensitivity. Adding Merlin to a panel of BAP1 + MTAP increased sensitivity from 75% to 88% for diagnosis of epithelioid mesothelioma and from 85% to 96% for biphasic mesothelioma, equating to an increase in sensitivity from 79% to 90% for all mesotheliomas (P=0.03). Seventy-eight (93%) of 84 tumors showed abnormal staining for at least 1 of the 4 immunomarkers under study.
Discussion
This study represents the first large correlation of diagnostic immunohistochemistry with clinical panel NGS in pleural mesothelioma, with four principal conclusions:
1.
Immunohistochemical loss of BAP1, MTAP, or Merlin is highly specific for malignancy in mesothelial lesions, including in cases where panel NGS does not reveal a corresponding molecular alteration.
2.
Adding Merlin significantly improves the performance of the standard BAP1 + MTAP immunopanel, and routine application of this three-marker panel may be recommended following independent validation.
3.
Diffuse p53 immunostaining is specific for TP53 mutation and for malignancy, but we did not identify a molecularly specific "null-mutant" p53 immunophenotype among mesothelial lesions. Diffuse p53 represents the only immunohistochemical abnormality in a small subset of mesotheliomas.
4.
Immunohistochemistry and panel NGS are both highly sensitive for mesothelioma and show extensive but incomplete overlap in diagnostic detection. Although immunohistochemistry remains the first-line ancillary assay for mesothelioma diagnosis, molecular testing (including panel NGS) may be required in select cases.
Despite high concordance overall, immunohistochemistry and panel NGS each detect diagnostic alterations that the other does not. In this study, immunohistochemical loss of BAP1, MTAP, or Merlin was noted in 3 of 4 tumors with no molecular alteration. Such instances are principally due to low sequenced tumor cellularity, wherein significant alterations fall below the detection threshold of panel NGS. Alternately, this discordance may be secondary to miRNA regulation 47 or to molecular alterations (including loss-offunction rearrangements, copy-neutral gene fusions, and deep-intronic splicing alterations) that abrogate protein expression but evade detection by clinical panel NGS 15 .
Conversely, panel NGS identified pathogenic alterations in 5 of 6 mesotheliomas with no abnormal immunostains. Most such discordances stem from poor correlation between immunohistochemistry and shallow gene deletion, noted across all genes under study. The cause of this poor correlation is unclear but could include haplosufficiency on the one hand or, on the other, undetected genetic or epigenetic alterations on the non-deleted allele or underappreciated two-copy deletions in tumors with low sequenced cellularity.
Alternately, some such discordances may result from pathogenic mutations sparing the immunohistochemical epitope.
a. BAP1
In keeping with published data 6,14-19 , we identified a broad spectrum of mutational mechanisms for BAP1 inactivation. Interestingly, 1 distal truncating event (p.N690Gfs*36) was undetected by immunohistochemistry. This specific frameshift extends the reading frame into the 3' untranslated region, which may result in escape from nonsense-mediated decay 48 . We observed a non-significant trend toward cytoplasmic-only BAP1 staining in tumors with truncating BAP1 mutations, which eliminate both nuclear localization signals (located at amino acids 656-661 and 717-722, or 729 total) 49,50 . An additional tumor with cytoplasmic-only BAP1 staining harbored a catalytic domain missense mutation (p.R146T) and shallow deletion, consistent with prior reports 51 , though two other cases with catalytic domain missense mutations had negative staining.
Our data indicate strong correlation between BAP1 immunostaining in the epithelioid and sarcomatoid components of biphasic tumors. Previous data on this topic are conflicting 24,52-54 , though rigorous molecular 27,52 and survival 52 data indicate that supposed biphasic mesotheliomas with BAP1 loss confined to the epithelioid component most likely represent epithelioid mesothelioma with a reactive spindle component.
b. CDKN2A and MTAP
Our data confirm growing evidence that MTAP immunohistochemistry is a reliable surrogate for CDKN2A deletion in diagnosis of mesothelioma, 8,[31][32][33] with 100% specificity in this NGS-based cohort in line with the 96-100% specificity reported in FISH-based studies 8,31,33 . Our data further support evidence that MTAP immunohistochemical loss is highly specific for malignancy in mesothelial lesions, including in lesions with no corresponding MTAP deletion detected 34 . Previous studies have generally set a threshold of at least 50% tumor cell MTAP loss for a diagnosis of malignancy 8,12,26 . However, our experience in this work and prior studies 20,31 indicates that this 50% cutoff is overly conservative, and we regard any discrete population of MTAP-deficient tumor cells as a clonal molecular alteration supporting diagnosis of malignancy.
To our knowledge, this is the first study to compare MTAP immunostaining in the epithelioid and sarcomatoid components of biphasic mesothelioma. Although the two components were concordant in 21/25, 4 tumors showed MTAP loss confined to the sarcomatoid component, including 3 with focal (heterogeneous) MTAP loss in the epithelioid component. These observations suggest that CDKN2A deletion may be associated with transition to sarcomatoid morphology in a subset of biphasic tumors. This pattern has not been observed in FISH-based studies 52,54 , possibly due to enumeration of few cells relative to immunohistochemistry. Diagnosis of biphasic mesothelioma should be regarded with skepticism if MTAP is lost in the epithelioid but retained in the sarcomatoid population 52,54 .
c. TP53
Using criteria refined in high-grade serous carcinoma 39,40 , we found diffuse "mutantpattern" p53 in 7% of mesotheliomas. Available evidence does not suggest a histotypespecific predilection for diffuse p53 15,16,41 . Prior work suggests an increased prevalence of TP53 mutations among mesothelioma with genomic near-haploidization. 17 We similarly found an enrichment of TP53 mutations in tumors with near-genome-wide loss of heterozygosity. Although our sequencing strategy cannot definitively distinguish true genomic near-haploidization (i.e., tumors with 24-30 chromosomes) from genomic nearhaploidization followed by endoreduplication of the remaining chromosomes, Hmeljak, et al., 17 grouped both of these molecular profiles under the umbrella of "genomic nearhaploidization," which they regarded as a distinct molecular subtype of mesothelioma. The 14% prevalence of near-genome-wide loss of heterozygosity seen in our cohort substantially exceeds the 3% rate of near-haploidization reported by Hmeljak, et al. The clinical significance of such extensive loss of heterozygosity in mesothelioma remains unclear.
Diffuse p53 immunostaining was 100% specific for underlying TP53 mutation and for malignancy, supporting the recent finding that diffuse p53 immunostaining may aid diagnosis in rare challenging mesothelial proliferations 41 . No wildtype tumor in our cohort showed more than 50% tumor cell staining, whereas no diffuse-pattern tumor showed <90% tumor cell staining -a quantum difference that, together with broad familiarity among practicing surgical pathologists, suggests that p53 immunostaining can be easily implemented in practice.
We did not identify a reproducible "null-mutant" p53 immunoprofile in mesothelial lesions. We observed null-pattern p53 immunostaining among reactive mesothelial proliferations and mesotheliomas alike, and we identified no difference in the rate of all mutations, truncating mutations, or deletions between mesotheliomas with wildtype versus null-pattern staining, suggesting that null p53 immunostaining is a non-specific finding in this context. This finding contrasts with a recent study 41 , which employed similar immunohistochemical methods but examined a smaller cohort with more limited molecular analysis. Further immunohistochemical-molecular correlation is necessary to resolve this discrepancy on null-pattern p53 staining in mesothelial proliferations. Until additional data are available, we advocate that null-pattern p53 staining not be regarded as evidence of underlying TP53 mutation in this context.
d. NF2
Our data indicate that Merlin immunohistochemistry could be a useful adjunct to BAP1 and MTAP immunohistochemistry in routine mesothelioma diagnosis. Merlin expression was lost in 52% of all tumors and 70% of non-epithelioid tumors; adding Merlin to an immunopanel of BAP1 + MTAP increased sensitivity across histotypes from 79% to 90%. Earlier reports found Merlin loss in just 4-8% of tumors 19,37 , but potentially non-specific cytoplasmic staining in one study 19 and absence of detailed immunophenotypic descriptions or figures in the second 37 limit confidence in those results. Those earlier studies also employed different antibodies than the present study.
Merlin loss was seen only in malignant but not in reactive mesothelial lesions. Given poor immunohistochemical correlation with shallow NF2 deletion, we sought to define a subgroup with retained but aberrant (i.e., weak, granular, and/or cytoplasmic-only) Merlin immunostaining. However, the prevalence of aberrant Merlin immunostaining did not differ significantly between mesothelioma and reactive mesothelial proliferations (which consistently lack pathogenic NF2 alterations 12,13,55 ), and published literature indicates that granular membranous and cytoplasmic Merlin staining may be the norm in meningioma 56,57 . These findings indicate that diagnostic emphasis should be placed on complete loss of Merlin immunoexpression. Validation of Merlin immunohistochemistry in independent studies is necessary for adoption in routine mesothelioma diagnosis.
Because NF2 alterations appear specific for mesothelioma in the differential with other common malignancies, Merlin immunohistochemistry (like BAP1 58,59 ) could be used to distinguish mesothelioma from both benign mesothelial proliferations and other malignancies, particularly lung 21,60-62 and ovarian cancers 63,64 . This exciting prospect warrants further validation.
e. Comparative sensitivities of immunomarkers and immunopanels
Under current guidelines, the diagnostic workup of a mesothelial proliferation begins with BAP1 and MTAP immunohistochemistry, with reported 67-89% combined sensitivity 8,12,26,34,35,[65][66][67] . In our study, BAP1 + Merlin was more sensitive than BAP1 + MTAP for both epithelioid and biphasic mesotheliomas, but these differences were not statistically significant. However, adding Merlin to a panel of BAP1 + MTAP significantly increased sensitivity among all histotypes (79% vs 90%), in keeping with prior studies with NF2 FISH 12,36 . This may support routine application of this three-marker immunopanel, pending independent validation of Merlin immunohistochemistry for this use.
Routine upfront use of p53 immunohistochemistry may be low-yield, given the low (~5-10%) rate of diffuse mutant-pattern staining in mesothelioma. However, our cohort included two tumors in which diffuse p53 was the only immunohistochemical abnormality; p53 immunohistochemistry may therefore be a useful diagnostic tool for select mesothelial lesions suspicious for malignancy but with retained BAP1 and MTAP (and, where in use, Merlin) 41 .
In this cohort, panel NGS identified a pathogenic variant in BAP1, CDKN2A/MTAP, NF2, and/or TP53 in 80 (95%) of 84 of cases. This is impressive sensitivity for a single assay; however, 1) this is not significantly greater than the 90% sensitivity achieved by a panel of BAP1, MTAP, and Merlin immunostains; 2) this figure includes shallow deletions, which should be interpreted with caution, particularly in specimens with low sequenced tumor cellularity; and 3) sequencing in this cohort was performed on resections, and it is unclear whether panel NGS would perform as well in biopsy samples, where the ability to profile potentially scant cell populations remains a strength of immunohistochemistry. Indeed, all 4 tumors with no pathogenic molecular alteration had <20% estimated sequenced tumor cellularity --3 of these showed a diagnostic immunohistochemical abnormality (indicating a probable false-negative molecular result), and none of these harbored a detectable molecular alteration in other mesothelioma-associated genes (e.g., PTCH1, SETD2). (LATS2, another gene of potential diagnostic interest, is not included in our panel, but published literature suggests LATS2 is mutated in ~10% of pleural mesotheliomas, though seldom independent of the genes examined in this study 16,17,68,69 .) This study has limitations. First, we studied only pleural mesotheliomas, and extrapolation to the peritoneum or other sites should be approached with great caution. Second, our cohort included only 6 sarcomatoid mesotheliomas, limiting our ability to draw conclusions about this rare histotype. Third, our study did not include FISH or other cytogenetic data, which plays a practical role in mesothelioma diagnosis. Our recommendations should not be interpreted as removing FISH assays from the mesothelioma diagnostic workup, when appropriate. Fourth, immunostains and molecular studies were performed on different surgical specimens in 8 cases and different tumor blocks from the same surgical specimen in 31 cases. Spatial and temporal mutational heterogeneity may therefore account for a subset of discrepant results, though this effect was not statistically significant, and available data suggest that significant heterogeneity is rare in the genes under study 16,55 , with an occasional exception for NF2 point mutations 55 . Finally, given certain discrepancies between published literature and our findings on p53 and Merlin immunohistochemistry for Chapel et al. Page 13 mesothelioma diagnosis, additional independent studies of these immunomarkers would be prudent prior to adoption into routine diagnostic practice.
In summary, our data emphasize the complementarity of immunohistochemical and molecular assays in classification of mesothelial lesions. We support the published literature on the molecular-immunohistochemical correlation for BAP1 and CDKN2A/ MTAP and have validated p53 and Merlin immunostains as novel markers of mesothelial malignancy. Our data suggest that adding Merlin to the standard BAP1 + MTAP immunopanel could significantly increase diagnostic sensitivity. P53 immunohistochemistry, cytogenetic studies, and molecular sequencing are appropriate for worrisome mesothelial lesions with retained BAP1 and MTAP (and, when/where available, Merlin) immunostaining. These considerations are reflected in a proposed updated diagnostic algorithm ( Figure 5). Additional studies are warranted to 1) validate our findings on Merlin immunohistochemistry, 2) explore the diagnostic utility of p53 and Merlin immunohistochemistry in cytology specimens (where NF2 FISH has proved fruitful 36 ), 3) assess the specificity of Merlin loss for mesothelioma in the differential with other malignancies, and 4) explore a potential role of BAP1, MTAP, and Merlin immunostains as predictive biomarkers for targeted therapies 70 . | 2022-04-22T14:04:10.355Z | 2022-04-13T00:00:00.000 | {
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225171260 | pes2o/s2orc | v3-fos-license | Increasing the Accuracy of the Difference Scheme Using the Richardson Extrapolation Based on the Movable Node Method
A one-dimensional convective-diffusion problem is considered. To improve the quality of difference schemes, the method of moving nodes is used in combination with Richardson interpolation. Approximate analytical solutions and improved schemes are obtained. Numerical experiments carried out.
Introduction
In mathematical modeling of various physical phenomena, initial and boundary-value problems arise for differential equations with small parameters at higher derivatives [1].
Due to the importance of such problems, the construction of various schemes of the convective-diffusion problem is the subject of the work of many authors [2][3][4][5][6][7][8][9][10][11][12][13][14]. The choice of the optimal sampling scheme for convective flows is one of the main problems in modeling flows.
The construction of discrete analogues of the convective-diffusion equation plays an essential role for transport processes. This is especially true when discrete analogues of the Navier-Stokes equation are constructed for large Reynolds numbers. In this regard, the movable nodes method (MNM) allows in many cases to design higher-quality discrete analogs of differential equations.
MNM arose in connection with the solution of differential equations by numerical methods [15,16]. When approximating derivatives (ordinary or partial) in a differential equation by difference relations, or by the finite volume method, we obtain a discrete equation. MMN for simple cases allows you to get an analytical representation of the solution between the nodal points of the boundary value problem. Based on this representation, it is possible to construct a higher-quality discrete scheme. In the case of a coarse mesh (one nodal point inside the region), an approximate analytical solution of the boundary value problem can be obtained. In the simplest cases, this solution is accurate. To refine the solution, you can increase the number of moved nodes.
Using MNM, it is possible to improve the quality of the difference scheme. An increase in the accuracy of various schemes of the convective-diffusion problem using extrapolation of Richardson is given. Based on the developed algorithm, numerical calculations were performed.
Problem Statement
Let's consider a boundary value problem Here is an improvement of the difference scheme for (1) using Richardson extrapolation in combination with the method of moving nodes.
In [15,16] two aspects of the application of the method of movable nodes are given. On the one hand, this method can be used to obtain an approximate analytical solution, and on the other, to obtain improved schemes. Here, the movable knot method is applied to improve the quality of the scheme using the Richardson method.
The Method of Movable Nodes for a One-Dimensional Convective-Diffusion Problem
Let inside the segment ( , ) x W E take an arbitrary one node. Consider a difference analogue of Eq. (1), in which the convective term is approximated by upwind difference scheme.
Then the upwind difference scheme has the form This scheme can be rewritten as follows: When changes ( , ) x W E the position (we will make its moved in an interval ( , ) WE , on the basis of (4) we will receive values of unknown function in each position. In other words, 1 U received by means of (4), will give us problem approximate solution. We will notice that in this case 11 ( ), ( ).
WE U Ф W U Ф E
The Superscript corresponds to an amount of moved grids.
When ( , )
x W E changes its position (let's make it moveable within the interval ( , ) WE ), based on (4) we get the values of the unknown function at each position. In other words, 1 U obtained with the help of (4), will give us an approximate solution to the problem. Note that in this case the Superscript corresponds to the number of movable nodes.
Add additional moved nodes: 12 ,. 22 x W x E xx
Now we have three moved nods 12 , , .
x x x We will note that if x changes the positions 1 x and 2 x also change the positions.
The scheme of type (3) for a segment [ , ]
Wx has the form: Here xE has the form: The upwind scheme for a segment 12 [ , ] xx : Here 33 22 () U U x In (7) we exclude 33 12 , UU using (5) and (6). Then we get the following scheme: The notation is introduced here 11 can be rewritten as follows: 3 3 3 3 3 3 3 ( ), where 3 3 Increase the number of moveable nodes: , 24 In the difference scheme (9), the unknown function appears in three nodes: W, x, E.
Function S is calculated in points 12 , , .
The graphs show that approximate solutions give good results.
Improving Accuracy with Richardson Extrapolation
Using the method described in Marchuk and Shaidurov [16], we can improve the accuracy of approximate solutions to the problem. Linear combination we get a more refined solution to the problem. Figure 3 shows the graphs of approximate solutions of problem (1), (2) obtained by (12) by Richardson extrapolation at 0, W
1.
E The solid line in Fig. 3 the exact solution. Fig-3.
Numerical Experiments
Simulation of the approximate solution to the problem given above can be used to construct difference schemes.
has an approximation order 4 () Oh .
Pe . Table 1 shows the absolute difference between the exact and approximate solutions according to the schemes. The solid curve is the exact solution, the circle obtained according to scheme (3), the circle according to U 3 , the solid rectangle according to U 7 , the diamond Q 3 , the star according to Q 7 . The solid curve is the exact solution, the circle obtained according to scheme (3), the circle according to U 3 , the solid rectangle according to U 7 , the diamond Q 3 , the star according to Q 7 .
From the graphs in Fig. 7, 8 and from Tables 1, 2 it is clear that the linear combination according to Richardson gives a more improved scheme.
Conclusions
With the help of the method of movable nodes and the method of the Richardson extrapolation, it is possible to construct a better scheme. The approach presented here can be successfully applied to other boundary value problems. | 2020-10-28T18:55:34.818Z | 2020-10-07T00:00:00.000 | {
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253992491 | pes2o/s2orc | v3-fos-license | Addressing the COVID-19 Shock: The Potential Job Creation in China by the RCEP
In 2020, coronavirus disease (COVID-19) left around 81% of the global workforce, nearly 2.7 billion workers, affected. Employment in China was the first to be hit by COVID-19. The Regional Comprehensive Economic Partnership (RCEP) is expected to bring dynamism to China’s employment market in an era of long COVID-19. This study aims to examine the number of sectoral jobs that the RCEP will create in China, with the number of skilled or unskilled labour employed in each sector. The exogenous shocks to the RCEP can be reflected in the number of jobs created through multipliers based on a social accounting matrix compiled from China’s input-output tables in 2017, combined with the employment satellite accounts compiled. The results show that the RCEP is expected to create over 17 million potential jobs in China, with unskilled labour accounting for 10.44 million and skilled labour for 6.77 million. It is even expected that there will be job losses in the metalworking machinery sector. The contribution of this paper can serve as a reference for policies to protect vulnerable sectors, further open up trade markets and strengthen cooperation among RCEP members as important measures to address the employment impact of long COVID-19.
Introduction
At the end of 2019, a few cases of unexplained pneumonia were identified in some hospitals in Wuhan, Hubei Province, which were confirmed to be acute respiratory infections caused by the 2019 novel coronavirus infection [1]. On 11 February 2020, World Health Organization Director-General Dr Tedros Adhanom Ghebreyesus announced that the new coronavirus-infected pneumonia would be named COVID-19. COVID-19 has serious implications for the productivity, performance and well-being of the workforce worldwide [2]. COVID-19 led to a sharp decline in labour demand in many sectors of the economy and resulted in significant initial labour shortages in other sectors [3]. COVID-19 left education, training and work-based learning disrupted, with increased difficulties for job seekers and new entrants to the labour market. At the same time job and income losses, as well as the quality of employment, have deteriorated [3,4]. In addition, COVID-19 has a negative psychological impact on the workforce, as people with COVID-19 may be discriminated against in employment and healthy people have a constant fear of contracting COVID-19 at work [5,6]. China's response to COVID-19 was swift, including blocking the movement of people between cities and quarantining potential patients [7]. COVID-19 has severely impacted the Chinese labour market, making it impossible or difficult for everyone to get to work [8]. By June 2020, there was still 20% of the workforce "lost", of which 11% were "unemployed", 4% were "waiting to return to work" and 5% were in employment but with "insufficient workload recovery" [9]. Even in some sectors where remote working is possible, there is still a low level of productivity and well-being [2]. On 24 September 2022, Singapore's Union-Tribune website reported that Dr Tedros Adhanom Ghebreyesus 1.
How much sectoral job creation is expected from the RCEP in China? 2.
Is there more job creation in the skilled or unskilled labour? 3.
How is the government seizing the RCEP opportunity to deal with the shock of COVID-19?
Trade in Goods
The RCEP's liberalisation of trade in goods is a two-by-two offer of tariff reduction commitments by the 15 member countries. More than 90 percent of intra-regional trade in goods will eventually achieve zero tariffs, including immediate zero tariffs and zero tariffs over the next ten years. This allowsthe RCEP to deliver its promise to liberalise the vast majority of trade in goods in the short term, significantly reducing intra-regional trade costs and commodity prices [28]. For example, Brunei has increased its zero-tariff product commitments to China on tobacco, bedding, home appliances and furniture, Thailand on paper products, copper and electric motors, and Cambodia on chicken, vegetables and fruits, food and rubber. China has also increased its zero-tariff commitments to ASEAN countries on fruits, chemicals, diesel engines, auto parts, etc. According to the Chinese Ministry of Commerce, ASEAN's actual investment in China's manufacturing sector reached USD 2.18 billion in 2020, topping all sectors. Next in line are the real estate industry, leasing and business services, transportation, storage and postal services, and wholesale and retail trade, with the number of new enterprises in these four sectors reaching 990 and the actual investment amounting to USD 4.42 billion, accounting for 52.9 percent and 55.5 percent of the total number of new enterprises and actual investment by ASEAN in China, respectively. In terms of commodity structure, electromechanics, plastics and their products, iron and steel and their products, textiles, mineral fuels and transport equipment are the main products traded by China with RCEP members [29]. The RCEP region is the primary import and export market for Chinese mineral fuels, mineral oils and their products, and China's imports and exports to RCEP members all account for more than 40 percent of the total import and export of this category of goods. The rules of origin accumulation are considered the most significant achievement of the RCEP in the area of trade in goods. Under the cumulative rules of origin, materials of origin from other RCEP member countries can be accumulated to meet the 40 percent value-added origin criterion for final exports when specifying the product's eligibility for tariff preferences. For example, if a member country supplies the raw materials for a Chinese-made household appliance at more than 40 percent, it will be exempted from 20 percent tariffs when exported to another member country. The RCEP simplifies customs clearance procedures by adopting efficient management tools such as pre-determination, pre-arrival processing, and information technology. Goods are released within 48 h of arrival whenever possible. Express cargo, perishable goods, etc., are released within 6 h whenever possible [30]. This is expected to reduce the overall customs clearance time for goods in the region.
Trade in Services
China's services trade liberalisation level reaches the highest level of existing free trade agreements [31]. Firstly, the number of committed service sectors has increased by 22 from the approximately 100 sectors committed by China's accession to the World Trade Organisation, mainly in 11 areas such as management consultancy-related services, manufacturing-related services, professional design services, elderly care services, sports and entertainment services, passenger transport services, market research services, personnel placement services, beauty and hairdressing services, building cleaning services, printing services, and 11 other areas. Secondly, the level of commitment was raised in 37 sectors, mainly in 12 areas such as legal services, construction and engineering services, environmental services, insurance services, banking services, securities services, shipping and related services, real estate services, aircraft maintenance and computer reservation system services, advertising services, software implementation services, and interpretation and translation services. In terms of service sectors that China focuses on, other RCEP member countries have committed to a greater degree of liberalisation in sectors such as construction, engineering, tourism, finance and transport [32]. China has also overall liberalised service sectors related to the installation of computer hardware, wholesale or retail trade services without a fixed location, maritime agency services, and road trucking or motor freight transport services, granting full national treatment to foreign investors. These new areas of liberalisation provide a wide market space for Chinese companies to go global and expand their regional industrial chain [33].
Investment
The investment chapter of the RCEP integrates and upgrades the investment rules of the 10+1 Agreement and provides a comprehensive and balanced investment arrangement in terms of investment market access and investment protection, forming the most extensive investment agreement arrangement in the Asian region and helping to create a more stable, open, and convenient investment environment. All RCEP member countries have adopted the negative list model to make commitments in terms of investment market access [34]. China has made high-level liberalisation commitments in five areas, including agriculture, manufacturing, fisheries, mining and forestry, to enhance transparency. In terms of the temporary movement of natural persons, the parties to the RCEP have committed to facilitate visa access for investors, intra-company transferees, contract service providers, accompanying spouses and family members, and other types of businesspersons from countries in the region who are eligible to enter and obtain residence rights in each country. The RCEP temporary movement of the natural person chapter will facilitate intra-regional commercial labour exchanges and further facilitate the "going out" of national enterprises and the "coming in" of foreign labour [35].
RCEP and COVID-19
The COVID-19 global pandemic caused severe shocks to international trade and global industry chains [36]. In January 2022, China's Vice Minister of Commerce said that the entry into force of the RCEP would greatly boost confidence in the economic recovery in the context of the epidemic, would effectively hedge the negative impact of COVID-19 on the economy, boost trade and investment confidence, and give a new impetus to the economic growth of member countries. One of the reasons for the Chinese government's proactive approach to signing the RCEP is the serious impact of COVID-19 on China's import and export trade, manufacturing chains and service industries [37]. The RCEP is expected to create more jobs in China to alleviate the current huge employment pressure in China [38]. Having considered COVID-19 and the trade war between China and the US, the RCEP could bring huge benefits to China, increasing its trade volume and national income [39]. In China's Inner Mongolia Autonomous Region, while COVID-19 has had a negative impact on employment well, the signing of the RCEP will offset the negative impact and reap greater benefits [40].
Exogenous Shocks and Employment
The RCEP is a free trade agreement that emerged out of the era of long COVID-19 to counter the shock of COVID-19. Since the outbreak of COVID-19 in late 2019, the dramatic shock has led to a decline in employment levels. This is because the government had to take measures in response to COVID-19, such as total civic silence [41], the closure of workplaces [42], the cessation of public transport [43]. Added to this was the psychological panic caused by COVID-19 [44], all of which led to a chilling winter for employment. A social accounting matrix (SAM) is considered to be an effective way of describing the impact of exogenous shocks on employment [45][46][47]. The effects of exogenous shocks are transmitted through production or consumption linkages. Production linkages depend partly on the extent and intensity of inputs from other sectors that are used to produce the output of the sector initially affected by the shock (i.e., backward linkages). The extent to which the output of the affected sector is used as an input to the production of the upstream industry is also a determinant of production linkages (forward linkages) [48]. In examining how sectoral growth affects inequality, the SAM's wage accounts were disaggregated into three education levels and ten employment sectors, showing that only growth in agriculture reduced inequality, while growth in the heavy manufacturing and service sectors increased inequality [49]. The SAM is used to examine the impact of public job creation in social care provisioning on employment creation, and the provision of social care also contributes to gender equality [50]. The regional social accounting matrix allows for an economic analysis of the impact of exogenous shocks to entrepreneurial activity on the economy of a region, the sustainability of the economy, economic growth and the contribution to reducing unemployment in the short term [51]. Based on post-Keynesian theory, the social accounting matrix is used to examine changes in demand and household income, and the conclusions suggest that the state becoming the employer of last resort will help reduce unemployment in the mainstream approach, with female income multipliers playing a role in both supply and demand [52]. The social accounting matrix (SAM) has empirical applications in several areas including tourism [53][54][55], energy [53,56,57], environment [58,59] and employment [60,61]. In the social accounting matrix (SAM) for India, it is noted that the labour demand multipliers are higher in agriculture and services. Therefore, these two sectors play a key role in employment generation in the Indian economy [62]. In the SAM study in Ecuador, modelling a 10 percent injection of international tourism demand led to a significant multiplier effect of tourism on the economy and the potential for significant benefits to labour-intensive sectors [63].
Social Accounting Matrix
The RCEP and its related government policies have a huge impact on the existing economic system. Most policies directly or indirectly affect the economic system and the labour market, as anyone can benefit or lose from them. It is, therefore, natural for China, which occupies an important position in the global industrial chain and supply chain, that one affected sector will have an impact on other sectors. For example, when RCEP policies affect the grain industry, they may have an impact on the upstream fertiliser industry through the supply chain. How to model the impact of exogenous economic shocks on the economy? Economists consider the social accounting matrix (SAM) model to be an effective tool for dynamically predicting the impact of exogenous shocks on the overall economic system [64][65][66]. Given China's current strict COVID-19 embargo, capital flows are clearly restricted. It is difficult for the RCEP to work through the endogenous market mechanism of prices. In addition, SAM can find links between one sector and another where RCEP can have a complex impact on the economic system. Results and policy changes are transparent.
To accurately estimate the amount of job creation or destruction that the RCEP may have on multiple sectors in China, and to ensure that policies and outcomes are directly linked, SAM was used in this analysis. The SAM is a square database of transaction matrices, i.e., there are an equal number of rows and columns containing information representing the economy [47]. The square transaction matrix combines an input-output table describing production with national income and production accounts. SAM provides a comprehensive picture of the economic cycle in an economic system in which production creates income, income generates demand, and demand leads to production [67]. Each cell shows the payments from its column to its row. Accounts can be divided into endogenous accounts (e.g., activity, commodities, and factors) and exogenous accounts (e.g., government and fixed capital formation). SAM does not require complex programming knowledge, and the arithmetic allows a transparent indication of how the results are linked to policy changes. As a database, SAM can be used for different types of research, using different analysis methods. One such method is backward linkages analysis, developed by Nobel Prize-winning economist Wassily Leontief [68,69]. Backward linkages reflect the additional intermediate demand generated upstream by expanding production in one sector due to the interdependence between economic sectors and actors. The impact can be due to an exogenous monetary transfer into a sector (i.e., injection) or outflow from a sector (i.e., leakage) on the upstream sector of the economy [70,71]. In this paper, this refers to the entry into force of the RCEP. The indirect effect of an exogenous shock is through production or consumption. The backward linkages through production linkages are the main ones considered in this paper.
The specific operation is as follows: The square transaction matrix is defined as T. The elements in the matrix are defined as T ij , where i and j are the individual sectors. Let T en represent the endogenous account and T ex the exogenous account. I represent the identity matrix, with all the diagonal elements of the matrix equal to 1. Dividing each element T ij by the corresponding column sum of the endogenous account (Y en ), we obtain the technical coefficient of the endogenous account matrix (A). Then, by definition: The value of Y en is calculated as follows: It may be noted that the matrix (I − A) −1 is the famous Leontief inverse matrix. Another important matrix we need is the employment matrix Z. Z is obtained by dividing the number of people employed in each sector by the value of their output and placing these calculated values on the diagonal of the matrix. The final employment effect is matrix E:
Model Scenario Design
According to our summary of the RCEP, the entry into force of the RCEP will have a comprehensive impact on the Chinese economy in terms of trade in goods, trade in services, and investment. In comparison, the Comprehensive and Progressive Trans-Pacific Partnership (CPTPP) is a more comprehensive free trade agreement, as it contains both trade-related issues and provisions for deeper integration of member countries, such as state-owned enterprises and designated monopolies, labour, regulatory consistency, transparency, and anti-corruption [72][73][74]. In contrast, the text of the RCEP agreement is more focused on trade-related issues such as trade in goods, rules of origin, and trade in services, while the annexes include a tariff schedule of commitments and a schedule of specific commitments for services, making it a more trade-oriented agreement. Therefore, without loss of generality, the simulated RCEP positively impacts scenarios on China's labour market in the 149 sectors of the SAM, mainly focused on manufacturing, including mining, processed agricultural and sideline products such as vegetables and fruits, textiles, paper, plastic products, ceramic products, base metals, construction, real estate, wholesale, retail, warehousing and postal services, leasing, and business services (29 sectors in total). China's Ministry of Commerce estimates that the RCEP will increase China's real GDP by 0.35 percent more than the baseline, exports by 7.59 percent and imports by 10.55 percent. In addition, simulations by academics at the China Development Research Institute studying the economic impact of the RCEP on sectors in China are around 10% [21]. Therefore, the positive injection rate for exogenous shocks was determined to be 10 percent.
In contrast, because crops are a relatively weak sector in China and a labour-intensive industry, we consider the agricultural products sector as a negative injection [75,76]. For other sectors with negative injection, according to our summary of the RCEP and Zhou Ling Ling's latest theory of the RCEP's extensive fan decomposition for different sectors in China, we think that the export market effect of steel, non-ferrous metals and alloys, iron and ferroalloys, metalworking machinery, automotive parts and accessories, electrical equipment and other manufactured products is low. At the same time, the domestic market effects on these sectors are shown to be detrimental [21]. Even if the RCEP is considered a possible response to the shock of COVID-19, the negative impact of COVID-19 on the sector cannot be ignored [77]. Considering the market squeeze caused by the RCEP in some sectors and the shock of COVID-19 in some sectors [78][79][80], a total of 12 sectors are considered as the injection of possible negative effects (X) of the RCEP, with a negative injection of −10 percent.
The final employment effect (Emp) is calculated as follows: The 2017 SAM was compiled from input-output tables published by the National Bureau of Statistics of China. There are 149 sectors in the SAM, including five agricultural sectors such as crops, forestry, livestock, fisheries, and agricultural services, 86 manufacturing sectors, 13 other industrial sectors, and 45 service sectors. Households are separated into two types: rural and urban. Using labour force employment data by industry and labour force data by education from the 2017 China Labour Statistics Yearbook and the 2017 China Population Statistics Yearbook, the labour force was divided into unskilled and skilled workers, supplemented with the latest data from 2020 [81,82]. Based on the actual experience in China, the labour force with junior secondary education or equivalent is considered unskilled workers, while those with senior secondary education or above are classified as skilled workers [83][84][85][86][87]. We calculated the number of employed persons using total wages from the input-output tables and wage rates by industry from the China Statistical Yearbook to measure labour force employment. Adjustment was then made to the number of employed persons in the labour force in each industry using statistical labour force employment in three sectors: agriculture, industry, and services.
Overall Potential of RCEP for Creation of Jobs
The RCEP is expected to create a significant amount of sectoral employment in China, and even if job creation is much greater than job destruction, the negative impact is still not negligible. In some sectors, such as construction installation, civil engineering construction and building construction, the RCEP is expected to generate a rate of growth in job creation of over 9%. Overall, the RCEP has had a dramatic impact on the Chinese labour market, and it may lead to the estimated potential creation of over 17 million jobs, for China, particularly in some manufacturing industries. It is the consensus of many scholars that the RCEP is likely to bring about a significant increase in employment in China. This is because a country's economic growth is mainly driven by the "troika" of investment, consumption and exports. It so happens that the RCEP, currently the world's largest free trade agreement, covers a large area of China in these areas, especially trade. This has injected new economic dynamism into China, a major manufacturing country, especially at the lower and middle end of the manufacturing spectrum. Table 1 details the rate and number of estimated potential job creation or destruction by the RCEP as an exogenous shock variable in 149 sectors in China, in descending order of job growth rate.
Potential of Job Creation According to Workforce Skills
The RCEP is not a perfect free agreement at the moment, and the amount of job creation expected to result is more concentrated in unskilled labour jobs [12]. This also has a lot to do with the fact that China occupies the middle and lower end of the world manufacturing chain [88]. Benefiting from job creation at the lower end of the sector and completing the upgrade from unskilled to skilled labour in a timely manner is key to quality employment in China. The potential job creation of the RCEP in China is quantitatively significant across the skilled labour force. The number of unskilled labour jobs potentially created by the RCEP is about 10.44 million, much larger than the 6.77 million skilled labour jobs. Why is there a 3.67 million gap? The main reason for this is that China's current industrial structure mainly focuses on products at the middle and lower end of the market. In other words, many jobs are currently in the unskilled labour category. This is also related to the RCEP agreement on China which is mainly about trade in the low-end product sector, which directly leads to the creation of more jobs for unskilled labour than for skilled labour.
The Potential of Job Creation According to the Sectors
It is clear to see that the RCEP has had the most significant impact on job creation in China's construction installation, civil engineering construction, building construction, gypsum and cement products, cement, lime and gypsum, building materials, building decoration services, non-metallic mining, refractory products and ceramics sectors, with potential job growth rates of over 6 percent. This is related to the volume and growth rate of China's industry and engineering in the RCEP region, both domestically and abroad. The Chinese government has been committed to infrastructure and engineering labour, especially in the last two decades. From a volume perspective, the potential number of jobs created in sectors such as building construction, civil engineering construction, crops, financial services, retail, professional technical services, wholesale, business services, and forestry exceeded 500,000. There may be a partial sector-to-growth mismatch because of the large labour force (over 100 million) in some specific sectors in China (e.g., agriculture).
In general, this is in line with the fact that China's infrastructure-related sectors are characterised by a large number of employees, large volumes and fast growth rates. For example, in 2020, China's contracted infrastructure projects in the RCEP region had a completed turnover of about USD 39.19 billion, with an average annual growth rate of 9.1 percent, accounting for 25 percent of China's total foreign contracting. Among them, ASEAN countries and Australia dominate, at USD 34 billion and USD 3.9 billion, respectively. Contracting work to South Korea was worth USD 730 million, to Japan USD 400 million, and New Zealand USD 100 million. This should also prove that the entry into force of the RCEP will have a more profound impact on China's infrastructure-related industries.
Negative Effects of RCEP
The damaging effects of COVID-19 on employment remain, and because the RCEP has not had a positive impact on the number of jobs in all sectors, this has allowed the RCEP to increase the restrictiveness of employment in some sectors as well. In some sectors where the domestic market has been squeezed as a result of the RCEP, the RCEP is not a solution to the COVID-19 shock. The metalworking machinery sector was the most severely negatively hit in our simulations. Estimated potential job growth was −5.72 percent, with the potential number of unskilled jobs decreasing by 33,639 and the potential number of skilled jobs decreasing by 22,542. Several sectors that are largely immune to the impact of the RCEP are public administration and organisation, other general equipment, TV and radar equipment, complete vehicles, social work, and hygiene. This is because most of these commitments are related to China's political and security interests, such as core technologies, scientific research, information technology, social work, biological resources protection, ethnic minorities, special groups, and NGOs. At the same time, state-owned enterprises, emerging companies and sectors are also retained, aiming at a higher degree of autonomy and relatively flexible policy space for China's future economic development.
Conclusions
When the world's largest free trade area agreement meets China, a country rich in labour resources, there is no doubt that it will create violent sparks. The impact of the RCEP on the output of different sectors varies greatly, mainly because of the different degrees of labour intensity in China, the different values of output of different sectors, and the different degrees of participation of different sectors in global value chains. On the whole, China needs to cooperate with other members of the RCEP to exploit its strengths, but also needs to compete to induce the transformation and upgrading of some Chinese sectors in the new free trade agreement (FTA) landscape.
Further exploiting the advantages of labour resource endowment. In this study, the positive impact of RCEP on China's labour market is mainly concentrated in the construction, real estate and related sectors. The seven sectors where the RCEP is expected to generate the highest growth rates of employment in China are, building decoration services, building materials, cement, lime and gypsum, gypsum and cement products, building construction, civil engineering construction, and construction installation; all of these sectors are expected to grow at over 8%, with a combined increase of around six million. At the same time, this impact will also extend to upstream sectors through backward linkages, such as gypsum, cement products and similar products, building materials such as bricks and stones, building decoration, decoration and other building services. For instance, the Chinese government should take the first year of the RCEP as an opportunity to expand further the number of jobs in related sectors that will be positively affected by the RCEP. Firstly, Chinese sectors such as high-speed rail, steel, building materials and construction should play a leading role in driving the supporting sectors to participate in the RCEP regional cooperation on production capacity and equipment manufacturing, to complete infrastructure projects with high quality and form a comprehensive competitive advantage in the international market. This will stimulate construction and other advantageous sectors to export, while helping RCEP member countries lagging in infrastructure to modernise their infrastructure construction. Secondly, most of the sectors positively affected by the RCEP are labour-intensive industries. The RCEP is expected to create over 10 million unskilled labour jobs in China, particularly in sectors such as crop, forestry, livestock, fishery and mining, which require a lot of labour rather than skills and equipment. It is necessary to speed up the construction of a labour interest protection and risk warning and prevention system. It is also necessary to step up efforts to protect the labour force in important trade routes and major investment projects and effectively prevent and resolve various risks in "going out" investment and international operations to safeguard the personal and asset safety of the labour force. Thirdly, sectors positively affected by the RCEP should, in turn, actively participate in the RCEP rules to facilitate future adaptation to the Comprehensive and Progressive Trans-Pacific Partnership (CPTPP) and other similar rules. By doing so, they will serve as a good model for other sectors to take the lead and will then be able to add to the "new pattern of multilateral trade and investment" opening in China.
Promote industrial upgrading and create more skilled labour jobs. China's industrial structure is currently in a transition period. Since China's reform and opening-up in 1978, its economy has lacked competitiveness in the international arena, so it relied on cheap labour to formulate an export-oriented economic policy. Today, China has become the largest industrial country in the world. From the perspective of quantity, China handed over a satisfactory answer sheet. However, in terms of quality, China lags far behind developed countries. How to solve the current problems? The RCEP may be the answer. First, the RCEP can promote China's industrial upgrading to improve the quality of labour force creation. The accumulation rules of the RCEP's region of origin are beneficial for multinational companies to rely on the resource endowments and market advantages of each member in the RCEP region to adjust the supply chain layout of the industrial chain and realize efficient allocation of factor resources in the region more flexibly. This will also promote the trade and investment of intermediate products in the region and promote the formation of a closer, stable, and competitive regional industrial chain division and cooperation system. The RCEP also helps to promote the optimal allocation of production factors in the whole region and serves the upstream and downstream industry enterprises in the value chain of final local consumption, which is expected to receive further investment attention. In particular, it will further promote the investment layout of infrastructure, electronic information, petrochemical, textile and garment, automobile and other industries in the RCEP and form a relatively complete industrial chain division pattern covering upstream and downstream, which will also create more jobs for skilled labour. Moreover, China can promote labour-intensive industries or production links to further transfer to other member countries with lower land and labour costs according to RCEP rules of regional accumulation of origin, thereby accelerating the reconstruction of the RCEP regional industrial chain supply chain. However, this may bring some competitive pressure to underdeveloped areas such as central and western China.
Nirvana for sectors negatively affected by RCEP in the long COVID-19 era. Because of the impact of COVID-19 and China's stringent response policies, labour is not naturally mobile by default, leaving some sectors deeply embedded in the RCEP regional chain negatively affected. For example, the metalworking machinery sector in China has been hit by the synergy of the RCEP agreement and COVID-19, and has seen a decline in jobs. Then again, some agricultural and livestock products and their processing sectors will also be hit by COVID-19 and the RCEP, and jobs in these sectors will likely decline. As workers involved in agricultural production probably know, the most critical factor in the agricultural sector is price. There are still many small workshops in China in the production of agricultural, pastoral, and livestock products, while more large-scale production is taking place abroad. For example, Australia and New Zealand in the RCEP have the advantage of resources for agricultural products, and their agricultural products are relatively cheaper and of better quality. When tariffs fall, consumers will be more inclined to buy from these regions. As such, some of China's producers of agricultural and livestock products will be affected or may even have to cut production, and jobs will fall. Faced with such a dilemma, the Chinese government must first and foremost avoid pricing out the land and labour factors from the industrialisation model. This also fundamentally protects employment in vulnerable sectors in China. Secondly, policies should be formulated to liberalise the market in these sectors to a certain extent. It is essential to ensure that products with advantages in the RCEP region can enter China smoothly, such as crops from Australia and New Zealand and fruits from ASEAN. It is also vital to ensure that Chinese labour is transferred to a hightech industrialised model. Finally, cooperation between countries should be strengthened in the post-epidemic era; the signing of the RCEP has had a catalytic effect in enhancing the post-epidemic economic recovery and long-term prosperity of countries. The reduction in tariffs will allow for smoother international trade between member countries, leading to a gradual increase in total international trade and strengthening economic ties between countries, providing a sustained impetus to employment in sectors involved in foreign trade in China.
Limitations and Future Research
This paper has yet to be verified in terms of the results data, as the RCEP came into force in January 2022 and the number of jobs expected to be created may not be available until 2024. In addition, the social accounting matrix for China in 2021 may likewise need to be reviewed in 2024 due to the data lag in the input-output tables. Future research will focus more on the updating of data and the application of the latest research methods. In addition, the SAM approach can be used to study the impact of exogenous shocks on the economy as a whole, for example how much it affects consumption and the impact of open and closed loops. | 2022-11-27T17:06:29.557Z | 2022-11-25T00:00:00.000 | {
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4856138 | pes2o/s2orc | v3-fos-license | Preeclampsia and Retinopathy of Prematurity in Very-Low-Birth-Weight Infants: A Population-Based Study
Preeclampsia and retinopathy of prematurity (ROP) are associated with impaired angiogenesis. Previous studies on the relationship between preeclampsia and ROP have produced conflicting results. The goal of this study was to evaluate the association between maternal preeclampsia and ROP using a large population-based cohort of very-low-birth-weight (VLBW) infants from 21 neonatal departments registered in the database of the Premature Baby Foundation of Taiwan. Multivariable logistic regression analysis was used to estimate the adjusted odds ratios (OR) and 95% confidence intervals (CI) for preeclampsia with reference to ROP and severe ROP. A total of 5,718 VLBW infants (844 cases with maternal preeclampsia) were included for analysis. The overall incidences of mild and severe ROP were 36.0% and 12.2%, respectively. Univariable analysis showed lower GA and lower birth weight, vaginal delivery, non-SGA, RDS, PDA, sepsis, transfusion, and absence of maternal preeclampsia to be associated with mild and severe ROP development. However, OR (95% CI) adjusted for the variables that were significant according to univariable analysis showed the risks of developing any-stage ROP and severe ROP for maternal preeclampsia to be 1.00 (0.84–1.20) and 0.89 (0.63–1.25), respectively. The results remained unchanged in stratified analyses according to SGA status. Our data showed that maternal preeclampsia was not associated with the subsequent development of any stage or severe ROP in VLBW infants.
Introduction
Retinopathy of prematurity (ROP), a disease associated with abnormal retinal vascular development in preterm infants [1], occurs frequently among very preterm infants [2,3]. With the improved survival of this population due to advances in neonatal care, ROP has become a leading cause of childhood blindness worldwide [4]. Several risk factors, including small gestational age, low birth weight, and postnatal oxygen therapy, are known to be associated with the development of ROP [5,6]. Preeclampsia causes maternal and fetal morbidity and is also a leading cause of preterm delivery of VLBW infants [7,8]. Dysregulation of circulating antiangiogenic factors plays an important role in the pathogenesis of both preeclampsia and ROP [9,10]. Previous studies have analyzed the relationship between maternal preeclampsia and ROP in preterm infants; however, the results have been inconsistent and conflicting due in part to the relatively small sample sizes of the studies [11][12][13][14][15][16][17].
Recently, 3 large-scale studies analyzing the association between preeclampsia and ROP have produced inconsistent results. Yu et al. found an association between preeclampsia and a significantly reduced risk of ROP in preterm infants. [18] However, they did not adjust for SGA, which was more common in the maternal preeclampsia group and was significantly associated with ROP [18][19][20]. Araz-Ersan et al. demonstrated that maternal preeclampsia was associated with decreased incidence of progression to severe ROP, but not the onset of ROP. [21]. In addition, they did not adjust their findings for other confounding factors. Lee et al. reported that maternal preeclampsia was not associated with any stage of ROP in Extremely Low Gestational Age Newborns. However, extremely preterm infants born to mothers with preeclampsia, which is associated with neonatal hyperoxemia and bacterial infection, have an increased risk of severe ROP [22]. Because of the numerous discrepancies in previous studies, we examined the association between maternal preeclampsia and ROP in a large population-based cohort of very-low-birth-weight (VLBW) infants. The goal was to demonstrate the independent association between the two variables. We also performed subgroup analysis based on SGA status, which was shown in previous studies to be strongly associated with both maternal preeclampsia and ROP.
Study subjects
A total of 8,652 VLBW infants were registered in the database of the Premature Baby Foundation of Taiwan between 1997 and 2006. All 21 NICUs in Taiwan participated in this project, making the data a truly population-based cohort. The data collected included prenatal, perinatal, and postnatal demographic and clinical variables. Patient information obtained by the database coordinator was cross-checked with our national birth registry. The exclusion criteria included GA above 36 weeks, congenital or chromosome anomalies, infants who died before the screening of ROP, and those whose ROP status was not available. The percentages of infants excluded because the ROP status was not available were 0.4% in the non-preeclampsia group and 10.9% in the preeclampsia group. There were no differences in GA, BW, incidence of transfusion requirement, or sepsis in the two groups that were excluded for analysis. In addition, infants with maternal chronic hypertension were also excluded. Preeclampsia was defined as a diastolic blood pressure of greater than 90 mm Hg accompanied by proteinuria of at least 1+ (30 mg per deciliter) on dipstick test or nondependent edema during pregnancy. The gestational age (GA) was dated by the last menstrual period or the date of embryo transfer for in vitro fertilization. The ROP status was determined by pediatric ophthalmologists in each hospital.
Ethics Statement
Written informed consent was obtained from the parents or designated relatives of the infants in the study. The study was approved by the Institutional Review Board of National Taiwan
Outcome variables
Mild and Severe ROP were defined using the criteria of an international committee [23]. Severe ROP was defined as stage 3 ROP plus any disease, and stage 4 or 5 ROP [23]. Respiratory distress syndrome (RDS) was defined by clinical diagnosis and need for surfactant therapy. Necrotizing enterocolitis (NEC) was defined according to the criteria of Bell [24]. Small for gestational age (SGA) was defined as birth weight of less than the 10 th percentile for the gestational age [25]. Transfusion was defined as requiring PRBC transfusion. Sepsis was defined as clinical sepsis with proof of causative agent in the blood culture. PDA required treatment means PDA required indomethacin/ibuprofen treatment or surgical ligation.
Statistical analysis
The chi-square test and Student's t-test were used for comparing distributions of categorical variables and the continuous variables between groups, respectively. A multivariable logistic regression model was used to analyze the association between maternal preeclampsia and ROP risk, adjusted for variables that were found to be statistically significant by univariate analysis. Adjusted odds ratio (OR) and 95% confidence interval (CI) were derived to assess the magnitude of the association between various factors and ROP risk. We performed a similar analysis to evaluate the association between maternal preeclampsia and ROP in subgroups of different severities of ROP (mild ROP, severe ROP, ROP without therapy, and ROP requiring therapy). Statistically significant levels were determined by the 2-tailed test (p<0.05). The association between preeclampsia and ROP was further examined in subgroup analysis with stratification according to SGA status.
Results
A total of 5,718 VLBW infants, including 844 (14.8%) cases with maternal preeclampsia, were included for analysis. The numbers (overall incidence) of mild and severe ROP were 2,087 (36.5%) and 698 (12.2%), respectively. Infants born to a mother with preeclampsia were more likely to have higher gestational age, higher maternal age, delivery via Cesarean section, female gender, singleton, higher Apgar score at 5 minutes, and small for gestational age (SGA). They were less likely to have respiratory distress syndrome (RDS), patent ductus arteriosus (PDA), sepsis, and transfusion. The incidence of ROP was significantly lower in infants with maternal preeclampsia than in those without maternal preeclampsia (41.4% vs. 50%) ( Table 1).
Since SGA was strongly associated with preeclampsia and ROP, we further performed subgroup multivariate-adjusted analysis with stratification according to SGA status. Maternal preeclampsia was not related with ROP in either the SGA group (0.98 (0.78-1.23), P = 0.8329) or the non-SGA group (1.06 (0.78-1.43), P = 0.7177). The risk factors of ROP included small GA, small BW, vaginal delivery, Apgar score at 5 minutes below 7, transfusion, PDA, and non-sepsis VLBW. In the SGA group, the risk factors of ROP, except for BW, Apgar score at 5 minutes below 7, and sepsis, were similar those in the non-SGA group ( Table 4).
Because of the known phenomenon that ROP occurs almost exclusively in infants < 34 weeks, we analyzed the data of 5,296 VLBW infants of < 34 weeks, including 717 (13.5%) with maternal preeclampsia. The numbers (incidence) of mild ROP and severe ROP were 2,006 (37.9%) and 692 (13.1%), respectively. The incidence of ROP was significantly decreased in
Discussion
Our population-based large cohort study showed that maternal preeclampsia was not associated with ROP risk in VLBW infants. This finding is consistent with the study by Lee et al., which showed that preeclampsia itself was not associated with ROP in ELGANs [22]. In addition, our data also support the findings of other studies showing that lower GA, NSD, SGA, PDA, sepsis, transfusion, and lower Apgar score at 5 minutes are associated with ROP [12-19, 21, 22, 26-29]. Preeclampsia occurs in 2%-7% of pregnancies worldwide and results in fetal and maternal morbidity [8,30]. Increasing circulating soluble Flt-1, a soluble form of vascular endothelial growth factor (VEGF) receptor-1, which can bind both VEGF and placental growth factor (PGF), plays an important role in the pathogenesis of preeclampsia [31][32][33][34]. VEGF signaling also plays a critical role in the pathogenesis of ROP, and anti-VEGF therapy has been shown to have significant benefits in ROP treatment [35,36]. Several reports have produced inconsistent results on the association between maternal preeclampsia and ROP [11][12][13][14][15][16][17]. Recently, 2 largescale studies reported that maternal preeclampsia significantly reduced the incidence of ROP in preterm infants [18,21]. However, they did not adjust for SGA, which is strongly associated with both preeclampsia and ROP [18][19][20], as a confounder. In agreement with the studies by Yu et al. and Araz-Ersan et al., we found in this large multicenter study that VLBW infants delivered by mothers with preeclampsia had significantly lower incidence of ROP. However, after adjusting for confounding factors, including SGA and GA, we demonstrated that maternal preeclampsia was not associated with the risk of ROP in VLBW infants. Nonetheless, with the large sample size, we were able to do the subgroup analysis according to the SGA status, GA groups, or severity of ROP. Again, multivariate logistic regression analysis showed that maternal preeclampsia was not associated with ROP in either the SGA group or the non-SGA group, or in various GA strata, nor was maternal preeclampsia associated with either mild ROP or severe ROP. These findings suggest that although preterm infants born to mothers with preeclampsia have significantly lower incidence of ROP, maternal preeclampsia itself is not a protective factor, which is supported by a report from Lee et al. [22]. However, because potential bias such as small GA, low birth weight, and SGA may contribute to both preeclampsia and ROP, it is difficult to study an independent association between maternal preeclampsia and ROP.
The strength of our study is that it was a large multicenter population-based cohort study with several subgroup analyses, allowing us to assess the association between maternal preeclampsia and ROP. However, our study also had some limitations. First, some data of interest were unavailable (see tables). However, the large size of our database and the absence of differential misclassification would minimize these influences. Second, our cohort included only infants of birth weight below 1,500 gm, so data on larger infants born above 30 weeks' gestation are not available, and thus infants born SGA are overrepresented in this group. However, we performed several subgroup analyses to minimize this bias.
Conclusions
Although there was potential bias of a link between preeclampsia and ROP, our large population-based retrospective analysis found no association between these two diseases. | 2016-05-04T20:20:58.661Z | 2015-11-20T00:00:00.000 | {
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52012773 | pes2o/s2orc | v3-fos-license | Ant Colony System for Multi-Document Summarization
This paper proposes an extractive multi-document summarization approach based on an ant colony system to optimize the information coverage of summary sentences. The implemented system was evaluated on both English and Arabic versions of the corpus of the Text Analysis Conference 2011 MultiLing Pilot by using ROUGE metrics. The evaluation results are promising in comparison to those of the participating systems. Indeed, our system achieved the best scores based on several ROUGE metrics.
Introduction
Multi-document summarization (MDS) is a type of summarization in which the contents of a set of documents are represented as a single summary. Today, this type of summarization has become a necessity due to the existence of enormous amounts of information. It reduces the quantity of text by providing a summary that contains the most relevant and important parts. The automatic summarization problem has been studied since the middle of the 20 th century. Therefore, the application of several summarization approaches, such as statistical Alguliev et al., 2013;Rautray and Balabantaray, 2017) and graph-based (Erkan and Radev, 2004;Shen and Li, 2010;Mosa et al., 2017b) approaches, have been described in the literature.
An extractive summary represents a combination of the most important sentences from the source without modifying them. Summary sentences are selected according to the two following approaches. The first is the greedy selection approach, in which the best textual units (e.g., sentences) are selected one item at a time. This approach is widely used in text summarization and is simple and fast; however, it rarely produces the best summaries (Huang et al., 2010). The second approach is the global optimal selection approach, which searches for the best summary rather than for the best sentences. It reduces the summarization task, or at least the step of selecting sentences, to an optimization problem in which the overall score of the output summary is optimized by searching for the best mixture of sentences. In the literature, several summarization objectives have been studied and optimized, such as information coverage, text diversity, and readability. In addition, different meta-heuristics have been applied in the studies to approximate the solution of the summarization problem. One group of such meta-heuristics is swarm intelligence (SI). SI is a nature-inspired population-based type of meta-heuristics that has been applied successfully to the summarization problem (Alguliev et al., 2013;Peyrard and Eckle-Kohler, 2016). Additionally, ant colony optimization (ACO) algorithms have been successfully applied to short text (Mosa et al., 2017a;Mosa et al., 2017b) and single document summarization (Tefrie and Sohn, 2018).
Motivated by the importance of the summarization task as well as by the promising results of the studies mentioned above, this paper investigates the application of the ACO algorithms to MDS for both the English and Arabic languages. It proposes an extractive MDS algorithm that maximizes the information coverage and saliency and minimizes the redundancy in the resulting summary using ACO. Specifically, it uses an ant colony system (ACS) to search for a good summary that optimizes these objectives. The implemented system (called MDS-ACS) has been evaluated on both English and Arabic versions of the corpus of the Text Analysis Conference (TAC) 2011 MultiLing Pilot (hereafter referred to as the 2011 MultiLing Pilot) and using ROUGE metrics (Lin, 2004). The results of the proposed algorithm are promising compared to those of the top-ranked participated systems. The outline of the paper is as follows: Section 2 briefly presents some related studies; Section 3 briefly describes the ACS algorithm; Section 4 presents the formulation of the MDS problem; Section 5 describes the proposed algorithm and its main steps; Section 6 presents the experimental results; and finally, Section 7 presents the main conclusion and considerations for future work.
Related Work
Due to space limitations, this section only covers text summarization studies that use SI meta-heuristics. In addition, since the results of the proposed algorithm are compared to the results of the systems that participated in the 2011 MultiLing Pilot, these systems will be briefly presented. During the last decade, SI has accompanied the other meta-heuristics used to solve the text summarization problem. Examples of these meta-heuristics are particle swarm optimization (PSO), which simulates the behavior of a fish school or a bird flock; ACO, which was inspired by the ant society; and the artificial bee colony (ABC) algorithm, which simulates the behavior of honey bees. Recently, new SI meta-heuristics such as cuckoo search (CS) has also been used in the summarization field (see Table 1.) PSO has been used by several summarization studies. For example, they have been used to assign weights for features extracted from the text to be summarized in Binwahlan et al. (2010) and as a feature selection method for Arabic single document summarization in Al-Zahrani et al. (2015). Aliguliyev (2010) proposed a multi-document method based on sentence clustering, which has been solved using a modified PSO algorithm. PSO has also been used in several summarization studies to perform the actual summary extraction process. Alguliev et al. (2013) proposed an optimization model that uses a discrete PSO algorithm to generate multi-document summaries by maximizing their content coverage and diversity. Asgari et al. (2014) proposed an extractive summarization method based on a multi-agent PSO (Ahmad et al., 2007). Foong and Oxley (2011) proposed an extractive summarization model that combines two kinds of algorithms: PSO and harmony search. PSO has also been included in summarization studies with languages other than English, such as Arabic (Al-Abdallah and Al-Taani, 2017) and Hindi (Dalal and Malik, 2018).
ACO is another SI meta-heuristics that has been used in summarization. Tefrie and Sohn (2018) proposed a summarization model that incorporates several features to calculate the heuristic value of each sentence. There are several differences between Tefrie and Sohn (2018) algorithm and our algorithm. Besides using different extracted features, several aspects related to the ACO are also different, such as the initial pheromone values, the calculated heuristics, the pheromone updating method, and the termination condition. Unfortunately, we could not compare the performance of our algorithm and of their algorithm because the exact values of the results are not provided in their paper. ACO has also been used with short text summarization problems. Mosa et al. (2017a) proposed a technique based on the use of ACO and Jensen-Shannon divergence to summarize a large number of Arabic user-contributed comments. Mosa et al. (2017b) proposed another technique to summarize comments using ACO along with graph coloring and local search to extract the summaries. Peyrard and Eckle-Kohler (2016) used ABC meta-heuristic to create an extractive multi-documents summarizer. They proposed a general optimization framework in which any objective function of input documents and an output summary can be used. Another ABC based summarizer was proposed by Sanchez-Gomez et al. (2017). The main difference between this study and all the previous ones is that the summarization problem is formulated as a multi-objective one. Finally, Rautray and Balabantaray (2017) used the CS for multi-document summarization.
The remainder of this section is devoted to describing the eight systems that participated in the 2011 MultiLing Pilot, in which they were given IDs from 1 to 8. All these systems were applied to both English and Arabic languages except system 5, which was not applied to Arabic. Liu et al. (2011) proposed a solution (ID 1) based on using the hierarchical latent Dirichlet allocation (LDA) topic model along with other traditional features to score the sentences. The CLASSY model (ID 2) (Conroy et al., (Lund and Burgess, 1996) model to estimate the probability for each word w that another word w occurs with w within a window of size K. Based on these probabilities, the system gives a score for each sentence, and the summary is created by selecting the sentences with the highest scores. Saggion (2011) proposed system (ID 6) in which summary sentences are selected based on their similarity to the centroid of the set of documents to be summarized. Das and Srihari (2011) proposed a solution (ID 7) based on the use of LDA. The solution combines several models to solve the summarization problems, including global tag-topic models (i.e., on a corpus-level) and local models (i.e., on a document set level). Finally, El-Haj et al. (2011) proposed a centroid-based summarizer (ID 8) whereby the sentences are ordered and selected based on their similarity to its centroid.
Ant Colony System
Ant colony optimization (ACO) is a family of SI meta-heuristics that mimics the collective behavior of real ants. Communication among ants in their colonies is realized by means of pheromone trails laid down while the ants search for food. Because these trails evaporate over time, the shortest path from the colony to the food source attracts more ants because it has a greater amount of pheromone. An example of an ACO method is the ant colony system (ACS) algorithm. Dorigo and Gambardella (1997) proposed this algorithm and applied it to the traveling salesman problems (TSP.) They made three modifications to the ant system (AS) algorithm, which is another example of ACO. The first modification concerns to the state transition rule that balances between the exploration of new paths and the exploitation of old ones. Formally, an ant k moves from city r to city s by following this rule: where τ and η represent the pheromone value and the heuristic value, respectively. J k (r) is a set of cities that can be reached by the ant k. q is a random number that is uniformly distributed over [0,1]. The relative importance of exploration versus exploitation in the algorithm is controlled by the parameter q 0 . β is another parameter used to control the relative weight of the pheromone verses the heuristic. S is a city that is randomly selected according to the following probability distribution: otherwise. (2) The second and third modifications concern the pheromone-trail updating process. The second modification adds a local updating rule that is applied to the pheromones of the visited edges while constructing the solutions. The third modification is applied to the global updating rule so only the ant with the best tour is allowed to deposit pheromone.
Formulating the MDS Problem
In this step, MDS problem is formulated into an optimization problem and summary sentences are selected in a way that maximizes its overall content coverage score using ACS algorithm. This optimization problem can be formulated as follows. Let D be a set of input documents to be summarized and each of these document is split into sentences. Thus, D can be written as D = {s 1 , . . . , s |D| } where |D| is the total number of sentences in D and s i represents sentence i (1 ≤ k ≤ |D|.) The extractive MDS problem imposes the generation of a sequence of sentences; summary S, with a maximum length L by selecting a number of sentences from D such that the overall information coverage of S is maximized. Formally, it asks to optimize the main objective below: where c k and l k stand for the content coverage score and the length of of sentence k, respectively. The binary variable z k is equals to 1 if s k is part of the summary and zero otherwise. The content score of each sentence is based on the weight of the words it contains. However, to maximize the information coverage and saliency as well as to minimize the redundancies, only the weight of the words that have not been covered by other sentences that already selected as a part of S will be considered and the other words are ignored. Thus, even if a word j occurs more than once in S, its weight w j is added only once to the total content coverage score. Therefore, the overall content coverage score of S can be calculated by summing the weights of words it covers: The binary variable b j is defined as: where d kj is a constant that equals 1 if sentence k contains word j and 0 otherwise.
The Proposed Algorithm
This study proposes a summarization algorithm that generates extractive MDS summaries where its overall content score is maximized. It starts by preparing the input text using four preprocessing steps. After that, it gives a score for each word. Finally, these scores are used to generate the summary by selecting the sentences that maximize its information coverage score by using an ACS algorithm. The proposed algorithm (denoted as MDS-ACS) is outlined by Algorithm 1.
input: a set of related documents the maximum summary length output: a summary begin 1 Preprocessing 1.1 Segment the text into a set of sentences 1.2 Tokenize the set of sentences 1.3 Remove the stop words 1.4 Replace each word by its stem 2 Scoring of words 2.1 Build the sentence-to-sentence, word-to-word, and sentence-to-word graphs 2.2 Apply the reinforcement algorithm (Wan et al., 2007) 3 Extracting of summary sentences 3.1 Build the graph of the input documents 3.2 Optimize the information coverage of the summary sentences by ACS end Algorithm 1: MDS-ACS
Preprocessing
Four preprocessing steps are applied to the text before conducting the summarization process. First, Stanford CoreNLP 1 (Manning et al., 2014) is used for text segmentation and sentence tokenization. The text segmentation step breaks up the text into sentences while the sentence tokenization step specifies the words in each of these sentences. After that, a stop word elimination step is applied to the text to remove the frequently occurring words that have low semantic weight (Jurafsky and Martin, 2009). A stop word list from the SMART information retrieval system 2 is used for the English text and the general stop-word list provided in El-Khair (2006) is used for the Arabic text. Finally, a stemming step is applied to obtain the stem of each word, using the Porter stemmer 3 and Khoja's stemmer 4 for English and Arabic text, respectively.
Scoring of words
In this step, the score of each word is computed by following the approach proposed by Wan et al. (2007). This iterative reinforcement approach merges ideas similar to those of two graph-ranking algorithms: PageRank (Brin and Page, 1998) and the HITS (Kleinberg, 1999). It starts by building three graphs. The first one is a bipartite graph that links each word with the sentences in which it appears and its edges are given a score based on the TF-ISF scores and cosine similarity measure. The second graph represents the relationship between each pair of sentences using also the TF-ISF scores and cosine similarity measure while the third one represents the relationship between each pair of words using the longest common substring. The reinforcement algorithm is then applied to the graphs. Specifically, the score of each word is computed by applying a method similar to that of the HITS algorithm is applied to the first graph and a method similar to that of the PageRank algorithm is applied to the second and third graphs. Formally, each graph is represented by a matrix: W for the first graph, U for the second graph, and V for the third graph. In addition, this approach has two outputs; the score of each word and the score of each sentence. These scores are computed by applying repeatedly the following equations: where v and u are two matrices that hold the scores of the words and the score of the sentences, respectively. W , U , and V are the normalized versions of W , U , and V , respectively, W is the normalized transposed version of W . u (n) and v (n) are the values of matrix u and matrix v at the iteration n. Finally, u (n−1) and v (n−1) are the values of matrix u and matrix v at the iteration n − 1. At this point, the score of each word is computed and ready to be used to generate the summary. However, due to the importance of the first sentences in the summarization, the proposed algorithm doubles the weight of the words that exist in the first sentences. Several differences exist between the proposed algorithm and the reinforcement approach. First, the proposed algorithm generates multi-document summaries while the reinforcement approach creates single document summaries. Second, while the reinforcement approach depends on the scores of the sentences to create the summary, the proposed algorithm uses the scores of the words to generate a summary that maximizes information coverage and saliency and minimizes redundancy. Third, the proposed algorithm uses the longest common substring to compute the similarities among the words. Forth, the words of the first sentences are given more weight than the other words. Finally, an ACS algorithm is used to extract summary sentences.
Extracting of summary sentences
A modified version of the ACS algorithm is used to extract summary sentences (see Algorithm 2.) input : the graph representation of input documents the maximum summary length output: summary sentences begin Initialize the pheromone trails and parameters while the maximum number of iterations is not reached do Create and position an ant on each node (i.e., sentence) Activate the ants repeat for each active ant do Choose the next sentence according to Equation (1) and Equation (2) if ant cannot include more sentences then Deactivate the ant end else Update the ant's current scores of the unvisited sentences Update the current length and score of the ant's partial summary end end for each active ant do Apply pheromone local updating rule end until all ants become inactive; Increase the current number of iterations end Apply pheromone global updating rule using the best solution found in the current iteration if a summary with a new higher score is found then Update the best-so-far summary end return the best-so-far summary end Algorithm 2: ACS This step begins by building a connected graph from the text to be summarized by adding a node to represent each sentence. Then, a modified version of the ACS algorithm proposed by Dorigo and Gambardella (1997) ), adapted to work for MDS instead of TSP, is applied to the graph. In this study, the ACS algorithm starts by creating and placing an ant on each node (i.e., sentence). After each iteration, each ant generates a solution (i.e. a summary) which is a path of nodes that represent the extracted sentences. Regarding the summary length constraint, each ant keeps its own length and stops when it reaches the maximum summary length. Therefore, MDS-ACS assigns a state for each ant; active ant if it can include more sentences to its summary and inactive ant otherwise.
Regarding the maximization of the coverage objective (i.e., minimization of the travel distance), the heuristic value to include a new sentence (i.e., to go to a new node) is the inverse of the content score of this sentence. In other words, the heuristic value to travel from sentence r to sentence u is computed as follows: where c u represents the content score of sentence u. In addition, each ant updates the current scores of its unvisited nodes (i.e., sentences) while constructing its solution based on the words that it has covered so far. Finally, the ACS parameters were set to the same values recommended by Dorigo and Gambardella (1997), except the number of ants, which was set to the total number of sentences in the documents to be summarized.
Experiments
The proposed algorithm was implemented in Java programming language and is available online 5 . The evaluations were performed on a machine running Windows 10 with with 12 GB RAM and an Intel(R) Core(TM) i7-6500U CPU 2.5 GHz processor. The corpus of the 2011 MultiLing Pilot was chosen to evaluate MDS-ACS on both English and Arabic languages. The 2011 MultiLing Pilot is a multilingual MDS corpus written in seven languages, including English and Arabic. This pilot asked the participants to test their systems on at least two languages to create multi-document summaries of between 240 and 250 words. In this study, MDS-ACS was applied to the English and Arabic versions of this corpus, each consisting of 10 clusters including 10 documents. The results of the present study were compared to the results of participating systems (eight systems for the English version and seven for the Arabic version, see Table 2) as well as to those of the topline and the baseline summaries. Topline summaries were created using a genetic algorithm with the models (human) summaries, whereas the baseline summaries were created based on the similarity between the text and the cluster centroid.
System ID
Research Group (Participant) Language ID1 (Liu et al., 2011) CIST English and Arabic ID2 (Conroy et al., 2011) CLASSY English and Arabic ID3 (Steinberger et al., 2011) JRC English and Arabic ID4 (Hmida and Favre, 2011) LIF English and Arabic ID5 (Varma et al., 2011) SIEL IIITH English ID6 (Saggion, 2011) TALN UPF English and Arabic ID7 (Das and Srihari, 2011) UBSummarizer English and Arabic ID8 (El-Haj et al., 2011) UoEssex English and Arabic In this study, ROUGE, specifically the ROUGE-1.5.5 toolkit 6 , was used to produce the results. In addition to ROUGE-L, the ROUGE metrics used in the competition (ROUGE-1, ROUGE-2, and ROUGE-SU4) are used in this study for comparison purposes. ROUGE scores are reported in terms of F-measure scores. MDS-ACS scores are reported in terms of mean F-measure of five independent runs. These scores, those of participating systems, baseline, and topline summaries of the English and Arabic versions of the corpus are presented in Tables 3 and 4, respectively. Relative improvements of MDS-ACS over the other systems are also reported in these tables. The relative improvement of MDS-ACS over another system, X, was computed as follows: The results of MDS-ACS were promising. When tested on the English version of the corpus, MDS-ACS outperformed all the eight systems based on ROUGE-1, ROUGE-SU4, and ROUGE-L scores. MDS-ACS showed improvements of 2.9%, 0.49%, and 1.31% over the top ranked systems in terms of these metrics, respectively. In addition, MDS-ACS was ranked second among other systems based on ROUGE-2 scores. It outperformed the baseline in terms of ROUGE-1, ROUGE-2, ROUGE-SU4, and ROUGE-L metrics with relative improvements of 25.02%, 62.73%, 43.09%, and 24.24%, respectively. When tested on the Arabic version of the corpus, MDS-ACS outperformed all the participating systems based on ROUGE-1 and ROUGE-L scores. In comparison to the top ranked systems ID3 and ID2, MDS-ACS showed relative improvements of 3.98% and 4.09%, respectively. The former comparison used ROUGE-1 and the latter ROUGE-L. MDS-ACS was ranked second among other systems based on ROUGE-2 scores and third based on ROUGE-SU4 scores. MDS-ACS outperformed baseline summaries based on all four ROUGE metrics used in this study. The relative improvement of MDS-ACS over the baseline was 35% (ROUGE-1), 26.56% (ROUGE-2), 33.12% (ROUGE-SU4), and 34.04% (ROUGE-L).
Paired t-tests (p-value = 0.05) were conducted to check whether performance differences between MDS-ACS and the other systems were statistically significant. The results showed that on the English version of the corpus, MDS-ACS significantly outperformed the systems ID1, ID6, ID7, and ID8 as well as the baseline in terms of all metrics used in this study. MDS-ACS outperformed the ID5 system according to ROUGE-1, ROUGE-2, and ROUGE-L metrics. However, there was no significant difference between MDS-ACS and ID5 according to ROUGE-SU4. In addition, MDS-ACS was significantly outperformed by the topline system (ID10), and there were no statistically significant differences between MDS-ACS and the ID2, ID3, and ID4 systems. Regarding the Arabic version, t-tests showed that the only significant difference was between MDS-ACS and ID9 (the baseline) in terms of ROUGE-L. This may be because ROUGE 1.5.5 has not been adapted to the Arabic language. Overall, these experiments showed that adding more weight to words occurring in the first sentences of input documents signifi- cantly improved the performance of MDS-ACS. We think that these results could be enhanced by adding other semantic and language-dependent features.
Conclusion and Future Work
This study proposed a generic extractive MDS approach based on ACO. The original ACS algorithm was adapted to search for the sentences maximizing the information coverage of the summary generated. The implemented system, MDS-ACS, was evaluated using the corpus of the 2011 MultiLing Pilot (English and Arabic versions) based on four ROUGE metrics. The results show that the performance of MDS-ACS was comparable to the performance of the best participating systems. It outperformed all participating systems based on ROUGE-1, ROUGE-SU4, and ROUGE-L for the English version and ROUGE-1 and ROUGE-L for the Arabic version. As a future work, we plan to study the influence of other semantic features on the performance of MDS-ACS. We also intend to explore other SI metaheuristics for maximizing the information coverage in the generated summaries. | 2018-08-16T13:28:09.922Z | 2018-08-01T00:00:00.000 | {
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3383038 | pes2o/s2orc | v3-fos-license | Pure ovarian choriocarcinoma: report of two cases.
Pure primary ovarian choriocarcinoma is an extremely rare condition of gestational or nongestational origin. The possibility of gestational origin can be suspected by the presence of a corpus luteum of pregnancy but definite diagnosis would be based on genetic analysis. Here, we present two cases of pure ovarian choriocarcinoma in the forth decade of life with the possibility of gestational origin.
erm cell malignancies may represent up to 15% of ovarian cancers in Asian and African-American nations, where epithelial ovarian cancers occur less commonly. 1 Pure ovarian choriocarcinoma is extremely rare, with about 40 cases described in the English language articles. 2 Choriocarcinoma may be gestational or nongestational. The gestational type arises from an ovarian pregnancy or is of metastatic origin from uterine choriocarcinoma, whereas the nongestational type is an extremely rare germ cell neoplasm. To distinguish pure nongestational choriocarcinoma of the ovary from metastatic or primary gestational choriocarcinoma, DNA polymorphism analysis is a useful method. 3,4 The gestational variants are extremely sensitive to chemotherapy with overall cure rate approaching 98% in specialized centers. 5 The prognosis correlates with the bulk of tumor as well as the sites and number of metastases. 6 We present two cases of pure ovarian choriocarcinoma with possibility of gestational origin.
Case Report
Case 1: A 31-year-old woman, with a gestational history of G9, P1, Ab8 and L1 was admitted to Mirza Koochak Khan Hospital, Tehran, Iran in June 2001 with the symptoms and signs of acute abdomen. She had 5 years secondary infertility of unknown reason following her last abortion and gave the history of a missed menstrual period as well as 50 days of spotting. The serum β-hCG level was more than 1000 mIU/ml. Pelvic sonography revealed enlarged right ovary and a 2.7 cm left ovarian cyst. No abnormal finding was detected in uterus. In view of an adnexal mass along with positive pregnancy test, a possibility of ectopic pregnancy was considered. A right salpingo-oophorectomy was carried out. Gross examination of right ovary revealed a 7×7×4.5 cm 3 mass with a hemorrhagic cut surface. Following thorough sampling of the ovarian mass (1 section per cm of the greatest dimension of the tumor), microscopic examination revealed characteristic histological fea-G tures of choriocarcinoma, composed of bilaminar structures of cytotrophoblasts and syncytiotrophoblasts with severe nuclear atypia and mitotic figures admixed with necrotic tissue. A corpus luteum of pregnancy ( Figure 1) with multiple colloid bodies and calcifications was also observed (Figures 2 & 3).The patient received Etoposide/ Metotrexate / Actinomycin D/ Cisplatin/ Etoposide (EMA-CE) regimen for treatment and serum β-hCG level decreased to 70 mIU/mL. She underwent second laparotomy for careful examination of tumoral involvement. Specimens from left ovary and omentum revealed no histologic features of malignancy. A corpus luteum cyst was detected in the left ovary. Computed tomographic scan revealed no brain or lung metastases. Serum alpha-fetoprotein (AFP) was in normal range. After the surgery, she took 4 courses of chemotherapy with EMA-CE regimen. The level of β-hCG decreased to below the cut-off value. There has been no evidence of tumor recurrence during seven years followup.
Case 2:
A 32-year-old woman with a gestational history of G3, P2, Ab1 and L2 was admitted to Mirza Koochak Khan Hospital, Tehran, Iran in September 2003 with nausea¸ vomiting and vaginal spotting. Sonography revealed a large necrotic mass in the left adnexa. Serum β-hCG level was 5500 mIU/mL and AFP level was within the normal range. A large necrotic mass in left ovary with extension to the posterior aspect of uterus was identified in laparotomy. Total abdominal hysterectomy and bilateral salpingo-oophorectomy, tumor debulkation and infracolic omentectomy were performed. On gross examination, there was a left ovarian mass measuring 13×11×10 cm 3 with lobulated and hemorrhagic-necrotic cut surface. Microscopic examination of the tumor demonstrated clusters of atypical cytotrophoblasts and syncytiotrophoblasts with mitotic figures (Figure 4) as well as a corpus luteum of pregnancy. There was no evidence of other germ cell tumors in multiple provided sections. The tumor was attached to the poste-rior surface of uterus. However, only serosal lining showed tumor extension. No tumoral involvement was identified in endomyometrium. Computed tomography of chest, abdomen and brain showed no abnormal findings. Since the initial impression of the clinician was more in favor of nongestational choriocarcinoma, the patient at first received 3 courses of Bleomycin/ Etoposide/ Platinum (BEP) regimen for 3 months. Due to incomplete response, the chemotherapy regimen was switched to EMA-CE. After 4 courses of new treatment, the serum β-hCG level reached to less than 5 mIU/mL. No recurrence has been identified during five years follow-up.
Discussion
Choriocarcinoma of the ovary is a rare and aggressive tumor arising from germ cells, constituting less than 5% of all ovarian malignancies in Western countries. 1 Ovarian choriocarcinoma can be divided into three groups: as a metastatic gestational choriocarcinoma due to a regressed or occult primary gestational choriocarcinoma in other parts of the genital tract (mostly uterine corpus); as a primary gestational choriocarcinoma arising from an ectopic ovarian pregnancy, and as a nongestational germ cell tumor differentiating to trophoblastic components. 2 All types secret β-hCG. However, β-hCG levels are usually lower in nongestational variants in comparison to gestational types. 7 Monitoring of serum β-hCG can be a useful method in evaluating response to therapy. Serum β-hCG elevation leads to isosexsual pseudopuberty in premenarchal patients, while patients in reproductive age usually present with menstrual abnormalities (mainly amenorrhea). 1 Pure nongestational ovarian choriocarcinomas are extremely rare and highly malignant tumors frequently metastasizing through the lymphatics with intraabdominal spread. 8,9 Non-gestational type choriocarcinoma involves the patients with average age of 13 and is largely confined to females under 20. The presence of a well developed corpus luteum of pregnancy adjacent to the tumor may be indicative of a gestational origin. 8 However, a search for paternal DNA in tumor allows a definite distinction between gestational and nongestational types. Tumors with gestational origin have paternal genomic structure while nongestational tumors have genomes of only maternal origin without any alleles from paternal origin. 2 Since DNA polymorphism analysis is not a generally available laboratory technique, we couldn't perform it on our cases. Gestational ovarian choriocarcinomas have a better prognosis than their nongestational counterparts. 10,11 However some studies suggest that surgical stage of pure ovarian choriocarcinoma may be more important determinant of clinical outcome compared to being of gestational or nongestational type. 12 The two presented patients were in the forth decade of life and showed no elements of other germ cell tumors but well developed corpus luteum of pregnancy with hyaline globules and calcifications adjacent to tumor. This finding is in favor of the gestational origin of the tumors. However the possibility of pronounced corpus luteum due to high levels of β-hCG can not be excluded.
Conflict of Interests
Authors have no conflict of interests. | 2018-04-03T06:04:43.074Z | 2009-05-11T00:00:00.000 | {
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252882997 | pes2o/s2orc | v3-fos-license | Two Cases of Delayed Onset Myelopathy at the Cervicothoracic Junction Caused by Spontaneous Multiple Interlaminar Bony Fusion after Cervical Laminoplasty in Patients with Ossification of the Posterior Longitudinal Ligament
Cervical laminoplasty is the standard method for cervical myelopathy; it has stable long-term results. Although spontaneous interlaminar fusion following laminoplasty frequently occurs, it is rarely clinically problematic. Here, we report two rare cases of cervical ossification of the posterior longitudinal ligament (OPLL) with a spontaneous multiple interlaminar fusion after laminoplasty and delayed onset myelopathy at C7/T1.
Cervical laminoplasty is the standard method for cervical myelopathy 1,2) ; it has stable long-term results 3,4) . Although spontaneous interlaminar fusion following laminoplasty frequently occurs, it is rarely clinically problematic 5) . Here, we report two rare cases of cervical ossification of the posterior longitudinal ligament (OPLL) with a spontaneous multiple interlaminar fusion after laminoplasty and delayed onset myelopathy at C7/T1.
Case 1
An 80-year-old man underwent a double-door laminoplasty from C3 to C7 for cervical OPLL 12 years earlier (Fig. 1A). He had no problems for 10 years after the operation. At the initial visit, he used a cane while walking due to spastic gait, and his Japanese Orthopaedic Association (JOA) score of cervical myelopathy was 10 out of 17 points.
A lateral plain X-ray showed multiple interlaminar fusion between C2 and C6, and bony fusion of the anterior longitudinal ligament (ALL) at C6/C7. The cervicothoracic junction was difficult to visualize (Fig. 1B). T2-weighted magnetic resonance imaging (MRI) showed severe spinal cord compression due to increased OPLL from C7 to T1 (Fig. 1C). Computed tomography (CT) myelography showed incomplete bony fusion of the ALL or the OPLL at C7/T1, but a complete bony fusion of the OPLL and diffuse idiopathic skeletal hyperostosis (DISH) at T1/T2 and on the caudal side of T2, respectively (Fig. 1D).
Posterior fusion from C4 to T4, including laminectomy from C6 to T2 was conducted ( Fig. 2A, B). CT images one year after the surgery confirmed complete bony fusion of the ALL and OPLL at C7/T1 (Fig. 2C). Four years after the surgery, the patient showed a stable gait with a cane, and a plain X-ray confirmed no implant failure (Fig. 2D).
Case 2
A 43-year-old man underwent a double-door laminoplasty from C3 to C7 for cervical OPLL 14 years earlier (Fig. 3A). Although OPLL compressed the spinal cord at T1/T2 (Fig. 3B), the patient exhibited no problems with daily life 13 years after the initial operation. The patient could not walk at the initial visit, and his JOA score was 7.
A lateral plain X-ray exhibited bony fusion at C3/C4 and between C5 and C7 (Fig. 3C). The cervicothoracic junction was difficult to visualize on a functional X-ray (not shown). T2-weighted MRI showed severe spinal cord compression due to the intervertebral disc and ligamentum flavum at C7/ T1 (Fig. 3D). There was no change in the degree of spinal cord compression at T1/T2. A plain CT did not show bony A. The patient underwent double-door laminoplasty from C3 to C7 for cervical OPLL 12 years earlier. B. Lateral plain X-rays showed multiple interlaminar fusion between C2 and C6 (arrowheads), and bony fusion of the ALL at C6/ C7 (arrow). C. T2-weighted MRI showed severe spinal cord compression due to increased OPLL from C7 to T1. D. CT myelography showed incomplete bony fusion of the ALL (arrow) or the OPLL (arrowhead) at C7/T1, but a complete bony fusion of the OPLL was visible at T1/T2, and DISH was present on the caudal side of T2. fusion at C7/T1 and T1/T2, but there was bony fusion by DISH on the caudal side of T2 (Fig. 3E). Posterior fusion from C5 to T5, including laminectomy from C7 to T2, was conducted (Fig. 4A, B). MRI taken one week after surgery showed that the spinal cord was well decompressed (Fig. 4C). One year after surgery, the patient could walk using a Lofstrand crutch, and CT images con-firmed that bony fusion of the ALL at C7/T1 was completed (Fig. 4D).
Although myelopathy at C7/T1 is one of the differential diseases of gait disturbance, it is a rare condition 6) . In clinical situations, a C7/T1 lesion can be initially neglected because the cervicothoracic junction is difficult to visualize on a plain X-ray 6) . Cervical OPLL and thoracic DISH are fre- A and B. Posterior fusion from C5 to T5, including laminectomy from C7 to T2, was conducted. C. MRI taken one week after surgery showed that the spinal cord was well decompressed (arrowhead). D. CT images confirmed complete bony fusion of the ALL at C7/T1 one year after treatment. quently combined 7) , and OPLL lesions may increase the following laminoplasty in cervical OPLL 8) . Moreover, longlevel cervical instrumentation tended to result in the progress of C7/T1 degeneration 9) , and thoracic DISH tended to result in the progressive degeneration between adjacent vertebrae 10) .
In our cases, thoracic DISH was present. Although instru-mentation was not conducted, the condition might be similar to that of instrumentation due to spontaneous multiple interlaminar fusion following laminoplasty. Therefore, excessive mechanical stress might have been applied at C7/T1, resulting in minor and chronic instability though it is difficult to visualize on X-ray. Long-term observation is essential after laminoplasty in patients with cervical OPLL. Coexisting thoracic DISH could induce delayed onset myelopathy at the cervicothoracic junction when multiple interlaminar fusions occur after laminoplasty.
Conflicts of Interest:
The authors declare that there are no relevant conflicts of interest.
Sources of Funding: None
Author Contributions: Toru Funayama wrote and prepared the manuscript, and all authors participated in the study design. All authors have read, reviewed, and approved the article.
Ethical Approval: Unnecessary for Clinical Correspondence.
Informed Consent:
The patients in this study provided informed consent. | 2022-10-14T15:36:42.398Z | 2022-10-13T00:00:00.000 | {
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260275634 | pes2o/s2orc | v3-fos-license | Modeling of Conductors Catenary in Power Lines: Effects on the Surge Propagation Due to Direct and Indirect Lightning
In this article, the effects of the variable height in overhead power lines are investigated with reference to the surges produced by direct and indirect lightning events. Simulations are carried out both by an implicit finite-difference time-domain scheme and by the commercial tool EMTP-RV. Differences in the waveforms computed by the two approaches are discussed and clarified. Additionally, the validity of the commonly adopted assumption of weakly varying vertical component of the incident electric field is investigated.
I. INTRODUCTION
L IGHTNING strokes represent a severe threat to industrial facilities, infrastructures, or overhead lines [1], [2], [3], [4], being natural hazards that may trigger possible subsequent technological disasters [5].Both direct and indirect lightning strikes pose severe electrical stress to installed power equipment, which should have the proper insulation strength withstanding the lightning impulse voltage to avoid insulation failure and breakdown.
Rachidi [6] proved the equivalence of the different approaches proposed in the literature for the analysis of the interaction between a lightning electromagnetic pulse (LEMP) and a transmission line (TL), namely, those by the authors of [7], [8], and [9].A comprehensive dissertation on the assumptions and derivation of the different field-to-line coupling models is reported in [10], [11], and [12].Other models, e.g., Rusck model and Chowduri model, were revised in [13] and [14].
In the present work, the results of overvoltages produced (direct lightning) and induced (indirect lightning) on multiconductor transmission lines (MTLs) are discussed considering the conductors' sags.In order to show particularly the consequences of nonuniformity of the TL [15], the line is initially considered lossless above a perfectly conducting plane (PEC).Different approximate approaches were proposed to account for the finite conductivity of the soil in the computation of the radial component of the incident electric field (e.g., the wellknown Cooray-Rubinstein correction term [16], [17]); as to the propagation of the vertical component of the incident electric field, the contribution of the soil finite electric conductivity and permittivity is commonly neglected [17].The inclusion of losses in a time-domain algorithm through accurate transient parameters accounting for losses in the conductors and in the ground valid in a wide frequency range (i.e., for wide time intervals) has been presented in [18].The finite-difference time-domain (FDTD) analysis of a lossy nonuniform line has been presented in [19].
The current research is directed towards more efficient implementation of FDTD codes for field-to-line coupling studies [20], improved interface with EMT simulators [21], and assessment of the effects of LEMP [22].The modified telegrapher's equations are usually solved through explicit FDTD algorithms because of their reduced runtime and simplicity; although other approaches, e.g., the one presented in [23], may offer a major accuracy, on the other hand, the computational process is time consuming.In this article, the implicit Crank-Nicolson FDTD scheme with second-order accuracy is implemented: its longer computational time compared with explicit schemes is compensated by the intrinsic stability [24].First, an investigation is carried out with reference to the propagation of overvoltages caused by both direct and indirect lightning events, approximating the line conductors (which have variable height) as a uniform MTL with an average equivalent height.Then, the approximation is removed by considering the height variable along the line.A comparison between the results obtained by the EMTP-RV tool and those obtained by means of the FDTD code is presented.Both implementations are based on the discretization of each span into minor sections.
Differences in the induced overvoltages computed by the FDTD code and the software EMTP-RV-LIOV were observed when modeling the conductors' catenary; the underlying reasons and the accuracy of the results by the software were investigated.An explanation is proposed by showing that a modification of the FDTD code, which alters its standard implementation, allows to reproduce the software results by enforcing a discontinuity in the scattered voltage (which is an intrinsically continuous quantity instead [25]).
The rest of this article is organized as follows.After presenting the modified telegrapher's equations in Section II, the assumptions adopted in the computation of the lightning electromagnetic field are summarized in Section III; Section IV deals with the solution of the telegrapher's equations by the implicit FDTD scheme in combination with different modeling approaches of the conductor's catenary.Numerical results are presented in Sections V and VI.Finally, Section VII is dedicated to conclusions.Appendices A and B include additional numerical results for the interested reader.
II. AGRAWAL ET AL. FIELD-TO-LINE COUPLING MODEL FOR A NONUNIFORM LINE
Agrawal et al. field-to-line coupling model is implemented in the FDTD code to simulate the effects of indirect lightning.It is also adopted by, for example, the LIOV toolbox provided by EMTP-RV [26].
The direction of propagation of voltage and current surges is assumed along the x-axis; the modified telegrapher's equations accounting for the LEMP illumination of a lossless TL read [8] (for the sake of conciseness, reported for a single conductor) The lossless line case has been considered also for comparison purposes since the LIOV code does not include the internal and ground per unit length (p.u.l.) transient impedances [27].In (1), L (x) and C (x) denote the p.u.l.external inductance and capacitance of the single-conductor TL, respectively; E x i is the component in the x-direction of the incident electric field.By means of Agrawal et al. model, propagation is assessed in terms of the scattered voltage v s .A transformation is required to obtain the total voltage at points located at height h(x) or to simulate the interaction between the TL conductors and other devices installed along the line (e.g., grounding of the shield wires (SWs) at the towers, surge arresters, etc.).In case of PEC ground, it reads (2) Herein, subscripts t, s, and i refer to the total, scattered, and incident quantities (fields and voltages), respectively.In (2), v t is the total voltage-to-ground of the conductor and E z i is the vertical component of the incident electric field.
In propagation studies along MTLs, when the points of suspension at the span terminations present the same heights, the geometrical average height h ave = h max − 2 3 S of the conductor is adopted according to the common practice; S is the average sag (which depends on the conductor p.u.l.weight, installation configuration, external temperature, and environmental conditions), and h max (h min ) is the maximum (minimum) height of the conductor within the span, i.e., in correspondence of a tower (at midspan).Nevertheless, the catenary profile of each conductor within the spans (symmetrical with respect to their midpoint) will be considered in the following discussion, approximating its height by a second-order polynomial (i.e., a parabola) [28]: h(x) is the variable height of the conductor, denotes the span length, and x refers to the longitudinal position along the span, being x = 0 and x = the left and right endpoints of the span, respectively.In case, the line includes also inclined spans above the horizontal soil, expression (3) should be replaced by the specific equation in order to account for the different conductor's arrangement and height at the span endpoints [29].
III. FIELD COMPUTATION
The relevant approximation adopted here consists in neglecting the actual tortuosity of the lightning channel in the computation of the fields produced by the return-stroke current [30], [31]; the lightning channel is modeled by a vertical cylinder above a PEC plane [32].The fields are computed by the integral forms given by Rachidi et al. [33].
Many analytical expressions are available in the literature to describe the propagation of the return-stroke current along the lightning channel (comprehensively reviewed in [32] and [34]).The TL model will be considered for comparison purposes since this model is adopted in EMTP-RV-LIOV.In this case, the returnstroke current i rs (z, t), as a function of time t and distance z from the ground, is given by where u(t) is the unit step Heaviside function, ν f is the upwardpropagating front speed (also called return-stroke speed), and ν 0 is the current-wave propagation speed.The value ν 0 = 1.5 • 10 8 m/s is chosen for simulations, being in the range of common values adopted in the literature, along with the hypothesis ν f = ν 0 [32], [35].Since engineering TL models for the propagation of return-stroke currents do not account for reflections at the upper endpoint of the lightning channel, any evaluation of (4) at time t > H/ν 0 may lead to unreliable results (values of the channel height H are expected to be within some kilometers, depending on the specific lightning event and geographical location).
A. Line Model in the Implicit FDTD Code
The Crank-Nicolson implicit scheme, equivalent to the single average alternating direction implicit (ADI) scheme [36], is Fig. 1.Piecewise linear approximation of the line catenary implemented in the implicit FDTD code.The case of a lossless single-conductor TL is displayed.adopted to solve the telegrapher's equations for the lossless line case [24].When the expressions of the FDTD method are modified to account for field-to-line coupling according to the Agrawal et al. model, the following equations are obtained for an MTL with N c conductors: and In ( 5) and ( 6), whose single-conductor equivalent circuit representation is shown in Fig. 1 for three consecutive Δx, [1] denotes the identity matrix of order N c ; L k+ 1 2 and C k are the matrices of p.u.l.external inductances and capacitances of the line; V n s k is the vector of dimension N c × 1 of conductors' scattered voltages at node k and time nΔt; and I n k+ 1 2 is the vector of dimension N c × 1 of conductors' currents flowing from node k toward node (k + 1) at time nΔt.The ith element of the 2 )Δx, y is equal to the average conductors coordinate in the y-direction, z = h i k,k+1 is equal to Expression (7) corresponds to the average height h i k,k+1 of the ith conductor, of variable height (h i ), given by an expression of the type (3) within each span, between nodes located at x = kΔx and x = (k + 1)Δx.
The quantities h i k,k+1 , with i = 1, . . ., N c , are used to derive the conductor matrix of p.u.l.external inductances L k+ 1 2 .Instead, the matrix of p.u.l.capacitances is derived by inversion of the matrix of potential coefficients, which are computed referring to the actual conductors' heights at the defined nodes.The space mesh for the evaluation of the unknown voltages and currents at time t = (n + 1)Δt in ( 5) and ( 6) corresponds to that commonly adopted by the explicit leap-frog scheme since the line nodes for the evaluation of voltages and currents are separated by half a step Δx/2; yet, they are calculated at the same time instants nΔt, disclosing the implicit nature of the method [24].
Zheng et al. proved the unconditional stability of the ADI algorithm [37]; hence, the choice of Δx and Δt for the implicit scheme may be conveniently related to considerations on the numerical burden, on the scheme dispersion error, on the geometry of the configuration under study, and on the known or predicted time constants involved in the specific transient study.
Finally, total voltages are obtained at the nodes of interest by means of the finite-difference counterpart of (2) It should be noted that an approximation is introduced in (8) since the vector of incident voltages at node k is computed by the product of the N c × 1 vector h k (whose elements are the conductors' heights at distance kΔx from the MTL left endpoint) with the quantity E n+1 z k , i.e., the vertical component of the incident electric field at x = kΔx, y (defined previously), and at z = 0.
Piecewise linear (or constant) line modeling approaches neglect any proximity effect that actually exists between the catenary segments.Rigourously, if the segment inclination was considered, the scattered electric field would not be confined in the y-z plane.Nevertheless, similar simplified approaches with two-dimensional approximations are often to be preferred in terms of implementation simplicity and shorter simulation time.
B. Line Model in EMTP-LIOV
The line is simulated in EMTP-RV-LIOV by means of multiple connected LEMP-illuminated lines.The macrostep Δx m = 10 m is chosen to discretize the line catenary.Hence, a number /Δx m of EMTP-LIOV devices connected in series is used to model each span of the line.The simulation time step Δt = 3.33 ns has been set.Since the software adopts a trapezoidal integration method with Courant factor f C = 1, the corresponding space step is equal to Δx = 1 m.The constant Authorized licensed use limited to the terms of the applicable license agreement with IEEE.Restrictions apply.height of each conductor within a macrostep is set as the average value of its actual height (accounting for the catenary profile) at x = kΔx m and x = (k + 1)Δx m .
This implementation guarantees the continuity of the total voltages-to-ground of the conductors at the point of connection between LEMP-illuminated line sections with conductors located at different heights (i.e., macrosteps).
C. Modified Line Model in the Implicit FDTD Code
Each span is discretized into segments of length Δx.However, macrosteps Δx m = k m Δx are introduced to model a coarser discretization mesh of the catenary profile, gathering k m small space steps with the same height.The height of the segment within each Δx m is computed as the average height of the catenary nodes limiting each macrostep.The discretization criterion is represented in Fig. 3.
This second approach is implemented as an additional modification to the FDTD solving scheme to compare with results by the LIOV code (as detailed in Section IV-B), which automatically sets the space discretization step to Δx = c 0 Δt (corresponding to f C = 1), c 0 being the velocity of light.The user may either represent the line through a very high number of LIOV lines with different heights (minding possible truncation errors in case the EMTP-computed Δx = c 0 Δt differs from the chosen length of the LEMP-illuminated line segment) or adopt a reduced number of line sections with length equal to Δx m ; the macrostep Δx m is successively discretized by the software into a finer mesh of segments Δx with constant conductors height within each Δx m .
Following the suggestions in [38] and the software documentation, a good approximation of the LIOV results is achieved by means of this modified FDTD approach (as discussed in Section V).The discontinuity introduced by the piecewise constant approximation of the variable conductor height in EMTP-RV is to be enforced into the modified FDTD code through two ideal voltage sources accounting for the different incident voltages at the junction point between adjacent macrosteps at different heights.
The modification to the equivalent circuit at these specific voltage nodes is schematically represented in Fig. 4, where subscripts L and R are employed to simplify the notation and denote the (constant-valued) inductances and capacitances within the
V. PRELIMINARY SIMULATIONS
In this section, the simple configuration sketched in Fig. 5 is considered.The used version of the LIOV toolbox allows for the simulation of lossless MTLs with three or four conductors; hence, three, nonfed, equal conductors (with same h max and S) were simulated for reference, displaying the overvoltages of the central conductor (which lies on the axis y = 0).The line total length is 2 km, consisting of five equal spans with length = 400 m, with symmetric catenary profile with respect to midspan and matched terminations.The average sag is S = 10 m and h max = 27 m.The FDTD code may also be used to simulate more extended TLs; increased computational time is expected due to the larger number of discretization steps (increasing the order of the solving system) and points along the line requiring the computation of the components of the incident electric field at each time iteration.For direct lightning studies (when neglecting the effects of the external field), larger Δx may be adopted to alleviate the total computational burden; nevertheless, for simulations of indirect lightning overvoltages, this choice should be preceded by the evaluation of the loss of the overall accuracy for larger Δx.In fact, besides a poorer representation of the catenary, the validity of the approximation of a constant E x i along the chosen Δx should be considered (for the presented configuration, the choice of a step equal to 10Δx could also be suitable [40]).EMPT-RV guarantees shorter simulation times, requiring rather cumbersome modeling effort when accounting for the catenary, since a considerable number of line devices with different geometrical characteristics should be individually inserted by the user.
First, overvoltages caused by a lightning current striking the central conductor, as computed by the implicit FDTD code and by EMTP-RV software with Δt = 3.33 ns, are compared when the catenary profile is simulated.In particular, Δx m = 10 m is adopted for simulations in EMTP-RV; as to the implicit FDTD code, the adopted discretization step is Δx = 1 m with Courant factor f C = 1.
Successively, results are compared with reference to an indirect lightning current striking the ground (PEC surface) at x s -y s (the coordinate reference system is shown in Fig. 5).
The same waveform is implemented as the channel base return-stroke current for both direct and indirect lightning simulations, expressed as the sum of two Heidler's functions with parameters in Table I (for a median subsequent stroke current in [39]).
A. Direct Lightning
In the simulated scenario, the lightning current strikes the central conductor at x = 1.2 km.The overvoltages computed by the FDTD algorithm and by EMTP-RV, which are displayed in Fig. 6 at different observation points along the TL and account for the conductor catenary, are practically superimposed, enabling (and validating) results from the developed code.The curves computed by the modified FDTD method are not included since the additional voltage sources in Fig. 4 are commonly neglected for the simulations of direct lightning, and results by the modified and the non-modified methods practically coincide.
The overvoltages computed with the constant height h ave = 20.3m are also shown.In this case, the predicted voltages peak values accounting for the influence of the variable height of the conductor and those computed for the corresponding uniform TL at h ave differ by less than 1%.The effect of the catenary is mainly evident in the low-frequency oscillatory behavior superimposed to the main voltage waveform, which is to be observed more easily at the tail.Its period ≈ 2.67 μs is related to 2 /c 0 , i.e., to the periodicity of the line geometry.Due to the line nonuniformity and discretization with constant Δx, the low-frequency superimposed oscillations may be more or less pronounced depending on the observation point along the line.
As expected, the results support the validity of the average height approximation, which may be adopted to study the propagation of conducted disturbances, e.g., the overvoltages produced by the direct lightning of overhead lines with typical geometric characteristics and values of the conductors sag.
B. Indirect Lightning
Two different channel base locations are simulated as to indirect lightning strokes, namely, x s = 1.0 km, y s = 0.05 km and x s = 1.0 km, y s = 0.10 km.Initially, results from the proposed FDTD code are validated by comparison of the induced overvoltages with results from the LIOV code when accounting for h ave .With reference to the observation points identified in Fig. 7, results are found to agree more than satisfactorily and highlight the natural reduction of the induced surges peak values as the return-stroke channel base is moved farther from the line.
Fig. 8 displays the influence of the conductor catenary on overvoltages computed by the implicit FDTD code (described in Section IV-A), EMTP-RV (in Section IV-B) and the modified FDTD code (in Section IV-C) with x s =1.0, 0.8 km and y s = 0.05 km.Fig. 8(a) refers to the simulated configuration when the lightning channel base is located opposite a span midpoint (i.e., where the conductor has the minimum height).Fig. 8(b) refers to simulations conducted with the lightning channel base located opposite a TL tower (i.e., where the conductor has its maximum height).Overvoltages at x = 0.6 km and x = 1.0 km Authorized licensed use limited to the terms of the applicable license agreement with IEEE.Restrictions apply.are superimposed due to the symmetric position with respect to x s ; differences are noted at t 4.7 μs since reflections from the left endpoint reach the closest observation point at x = 0.6 km first. 1 Waveforms obtained with h ave are included for reference.
The results highlight the impact of four relevant aspects.
1) Relative position between the channel base and the line:
By comparing overvoltages in Fig. 8(a) and (b), it may be deduced that harsher overvoltages are expected when the lightning channel is opposite a TL tower.Referring to results computed by the FDTD code, Fig. 9 displays incident and scattered voltages at the observation point closest to the LEMP source and at half-span distance, when the channel base is located at x s = 0.8, 1.0 km and y s = 0.05 km.The scattered voltages obtained with x s = 1.0 km and x s = 0.8 km are less sensitive to the variable conductor's height: in fact, their amplitude is comparable at the observation point opposite the lightning channel (x = 1.0 km and x = 0.8 km, respectively), and the deviation is even reduced at half-span distance (x = 0.8 km and x = 1.0 km, respectively).Hence, the conductor height h(x) contributes significantly to differentiating the total voltages at the two observation points through the incident voltage term, displayed in Fig. 9(a), which holds a predominant role in the determination of the overvoltages pattern (further details in Appendix A). 2) Average height approximation: Depending on the modeling approach, results obtained for the uniform TL with h ave present a deviation, which may be larger than 20%.At the observation point closest to the LEMP source, the average height approach over-(under-)estimates the induced overvoltages when x s -y s are opposite a point where the MTL conductors display the minimum (maximum) height.Similar deviations are not found for simulations of direct lightning that only account for the catenary by means of the height-dependent matrices of the p.u.l.capacitances and inductances of the line (not performing any additional integration of the incident field along the conductor's height).3) Chosen line model: The proposed implementation allows to represent the contribution of the incident voltages more accurately.In fact, it does not resort to any artificial discontinuity in the incident (and, as a consequence, in the scattered) voltages, overcoming this intrinsic limitation of staircase modeling; it considers the line integral of the vertical component of the incident electric field from a point at null potential (z = 0 for the PEC plane case) to the variable conductor height z = h(x).This feature of the FDTD scheme leads to nonnegligible differences against results by the modified FDTD scheme (and EMTP-RV-LIOV), which are enhanced at observation points closer to the LEMP source [e.g., at x = 0.8 km in Fig. 8(b)] and in the proximity of the spans' endpoints [e.g., at x = 0.8 km Fig. 8(a)].With reference to (8), this is justified, respectively, by the larger values of E z i and by the more pronounced height discontinuity between macrosteps (due to the reduced accuracy of the staircase model in representing the actual catenary, where |dh(x)/dx| is larger, i.e., at the span endpoints).These elements accentuate any difference in the amplitude of the additional voltage sources, as introduced in Section IV-C.4) Courant factor: Fig. 10 displays the total voltages already presented in Fig. 8(b), with lightning channel base located at x s = 0.8 km, y s = 0.05 km but with f C = 2, 5, 10, consistent with the time constants typical of lightning studies.Good accuracy of results is retained compared to curves obtained with f C = 1, speeding up the simulation time by a factor 2, 5, and 10, respectively.The differences observed at early times in the inset of Fig. 10 do not substantially affect the voltage front and peak value, relevant to the estimate of the electrical stress caused by lightning overvoltages close to the source (where the most severe stress typically occurs).At observation points located several hundreds of meters from the source, where numerical dispersion is expected to affect more significantly the computed curves, the compromise between accuracy and simulation time suggests to adopt f C ≤ 5 for the analyzed lightning events and configurations [24].
A. Test Line
The same channel base lightning current is used for the computation of induced voltages along the conductors of the lossless MTL, as depicted in Fig. 11, with geometrical characteristics in Table II.The line section has length equal to 2 km.Phase conductors are terminated in their characteristic resistance and the SW is grounded by a small-valued resistance (equal to 1 μΩ, approximating a quasi-ideal grounding condition).Two locations of the lightning channel base are simulated, i.e., x s = 0.8 km and x s = 1.0 km, with y s = 0.05 km.The II.
TABLE II CHARACTERISTICS OF THE SIMULATED LINE
distance of the channel base was chosen by application of the electrogeometric model [41].
Results from EMTP-RV-LIOV and the implicit FDTD code are compared.In the former case, the line is modeled by the cascade of LEMP-illuminated lines with different heights and length equal to 10 m; the selected time step is Δt = 3.33 ns.As to the original FDTD code, the line is discretized into segments Δx = 5 m, with f C = 0.2.The coarser space mesh adopted in the FDTD code, not affecting the results [40], allows to speed up the computations by reducing the dimension of the solving system.
B. Effect of the Conductors' Catenary
In Fig. 12, the overvoltages are displayed for two different channel base locations, accounting for the conductors catenary (computed by the proposed FDTD method and by the LIOV code) and for the corresponding uniform MTL. 2 Comparing accounting for the catenary or for the average heights, overvoltages are found to be affected predominantly (and nonnegligibly) by the simulated variable conductors' height at observation points closer to the LEMP source.However, regardless the relative position of the lightning channel with respect to the line, peak values of the overvoltages computed along the phase conductor are affected by wider deviations (27%-35%) with respect to those found for the SW (12%-21%).This might be justified by the larger sag characterizing the installation of overhead phase conductors, hence, by the rougher 2 Conversely to results in Figs.7 and 8, overvoltages for the uniform MTL by the FDTD code differ slightly from the ones by EMTP-RV, probably due to the specific derivation of E x along the conductors.In the FDTD scheme, it is evaluated at ȳ at the actual height of the ith conductor; in EMTP-RV, it is calculated at the highest point of the MTL and retrieved along the lower conductors by a linear approximation [27].
Authorized licensed use limited to the terms of the applicable license agreement with IEEE.Restrictions apply.approximation introduced by the average height approach as to indirect lightning studies.Some different assumptions in the implementation of Agrawal et al. model by the LIOV code (in the first place, the numerical aspects discussed in Section IV-B and IV-C) lead to differences in the range 5%-17% on the predicted peak value of the overvoltages at the observation point nearest to the channel base.Enhanced differences in the tail of the waveforms computed at x = 0.8 km are also noticed for results in Fig. 12(b).This is likely due to a combination of the relative position of the lightning channel base and the wider height gap between subsequent LEMP-illuminated line devices in correspondence of the span endpoints (the adopted piecewise constant approximation for the conductors catenary is less accurate in proximity of the poles, causing harsher discontinuities).
C. Effect of a Different Integration Method for the Incident Voltages
A three-point trapezoidal quadrature rule [42] was included in the code to compute the incident voltages and riser terms at the line terminations (as defined in [43]) in order to assess the validity of (8).The accuracy of the single-point integration method, based on the assumption of weakly varying E z i , may not be satisfactory for larger heights [i.e., longer integration paths in (2)].Hence, the following is implemented: where denotes the Hadamard product; for instance, the total voltage-to-ground V n+1 A,t k of conductor A, with height h A k at node k, may be computed as follows: (10) In ( 9), the quantity E n+1 z k h k denotes a vector with dimensions N c × 1; the ith element corresponds to the vertical component of the incident electric field evaluated at distance kΔx from the line left termination, at z equal to the height of the ith conductor.
Fig. 13(a) displays overvoltages along the SW and conductor B when the MTL is modeled as uniform, with conductors located at their average (constant) height.The simulation is carried out for x s = 1.0 km, y s = 0.05 km for the indirect lightning case; herein, direct lightning is not addressed since only the predominant effect of the injected current is typically considered in this type of simulation.
The waveforms obtained by the single-point (8) or by the three-point (9) quadrature rule converge at observation points farther from the lightning channel; differences of 17% and 8% are observed at x = 1.0 km in the voltage peak values along the SW and conductor B, respectively.Specifically, the improved integration results in the reduced amplitude of the computed overvoltages at points opposite the lightning channel.Already at early times, the single-point integration method overestimates the incident voltage, adopting the value of the vertical component of the electric field attained at z = 0 along the whole integration path and neglecting the time delay necessary for the external field to propagate at 0 < z ≤ h i .
The comparison between results obtained by the two integration methods when the conductors' catenary is simulated and the channel base is located at x s = 1.0 km-y s = 0.05 km or x s = 0.8 km-y s = 0.05 km is displayed in Fig. 13(b) and (c), respectively.
It is confirmed that less accurate integration methods for the calculation of the incident voltages lead to an overestimation of the total voltages-to-ground at points closer to the source.The higher is the local height of the conductors, the more relevant is the (precautionary) overestimation of the conductor voltages by the single-point integration method with respect to the threepoint quadrature; indeed, the deviation of 23.0% of SW peak voltage at x = x s = 0.8 km [in Fig. 13(c)] reduces to 14.3% for x = x s = 1.0 km [in Fig. 13(b)] and is generally wider than that for conductor B, located below the SW.
As expected, the reflected waves produced by the SW grounding at the line endpoints increase the voltage difference between the SW and conductor B, i.e., the stress across the line insulators.
VII. CONCLUSION
This article contributes to assessing the influence of the conductors' catenary as regards overvoltages caused by direct lightning currents striking a line conductor or due to coupling with incident electromagnetic fields.
The relevance of the implemented line model, i.e., uniform line, piecewise constant, or piecewise-linear discretization of the conductors' heights, has been assessed.It has been shown that neglecting the MTL nonuniformity may lead to considerable underestimation of the peak value of the overvoltages induced by external electromagnetic fields.This conclusion holds for lightning channel base locations which are opposite the line towers, where the conductor's height is maximum, due to the contribution of the incident voltages.
Despite the increased computational time, the original FDTD code guarantees several elements of flexibility.Only a few additional code lines allow to arbitrary chose finer discretization meshes in order to model the height variations accurately.Depending on the conductors' height, different numerical integration schemes may be implemented as to the evaluation of the contribution of the incident voltages.
Even though flexibility and accuracy come at the cost of an increased computational burden, measures to speed up the computation can be applied (e.g., reducing the dimension of the solving system by adopting larger discretization step Δx while ensuring results' deviations within a tolerance; adopting larger Courant factors, depending on the time constants involved in the specific study; simulating critical line sections and stroke locations, etc.) Further research may involve analogous studies on power lines with different features (height, sag, etc.); the simulation of the conductors' catenary in dealing with LEMP-induced effects may affect insulation coordination considerations, especially along the line section closest to the lightning channel base.
APPENDIX A
With reference to the configuration analyzed in Section V, overvoltages computed at additional observation points along the line, for the nonuniform and the constant height case, are displayed in Fig. 14(a) with x s = 0.8 km and y s = 0.05 km.Fig. 14(b) and (c) refer to incident and scattered voltages, respectively.
The pattern of the predicted waveforms along the line changes whether accounting or not for the conductors' catenary.For the uniform line, the voltage peak values decrease moving farther from the lightning channel; this trend is not to be found in the computed overvoltages when the catenary is simulated.In fact, in the latter case, the waveform at x 7 = 0.40 km displays larger amplitude with respect to those computed at x 5 = 0.60 km and x 6 = 0.50 km (closer to the LEMP source).This is mainly due to the contribution of incident voltages, being the scattered ones less affected by the catenary; in Fig. 14(c), when the catenary is simulated, larger amplitudes are expected for the incident voltages where the height is larger than h ave .
The percentage deviation between results for the uniform and nonuniform line also depends on x.However, the large deviation of 30% at x = x s is noticeably reduced to less than 10% at half-span distance (x ≤ 0.60 km).At larger distances from the source, a reverse in the polarity of scattered voltages is observed; the amplitude of total voltages is mainly determined by scattered voltages, and the contribution of incident voltages (predominantly affected by the local conductors' height) is limited.
Finally, in Fig. 14(c), the low-frequency oscillations of the scattered voltages, only visible when the catenary is considered, are associated to reflections from subsequent spans, hence, to propagation phenomena along the MTL conductors.
Authorized licensed use limited to the terms of the applicable license agreement with IEEE.Restrictions apply. in [18] and [24], i.e., by means of a recursive method exploiting suitable approximating functions of the internal and ground p.u.l.impedance obtained by Prony's algorithm.The towers were modeled as lossless TLs with characteristic impedance 175 Ω and propagation velocity 0.85c 0 , with grounding resistance 15.8 Ω.The same lightning current, described by the sum of Heidler's functions with parameters in Table I, was injected at x = 1.2 km, accounting for the lightning channel equivalent resistance 400 Ω [44].The effect of catenary is confirmed to be negligible for direct lightning simulations; the attenuation due to the transient internal and the ground impedance may be already observed at x = 1.0 km, with the multiple reflections due to the periodic grounding of the SW at the towers.
The inception of corona discharge along the line further supports the negligibility of the conductors' catenary in direct lightning studies.It should be noted that accurate simulations require the inception and development characteristics of impulse corona to be dependent on voltage steepness, polarity, weather conditions, etc. [45], [46], [47], [48].In this regard, due to the unavailability of experimental data for the simulated line, the corona model by Mihailescu-Suliciu and Suliciu [49] with symmetrical behavior under different voltage polarities was selected to provide a first estimate of the effect of corona discharge.The parameters given in [ [50], Sec.5.3], comparing the calculated overvoltages against measured data, were used.The Peek formula was implemented to compute the corona inception field (accounting for a position-dependent inception field due to the variable conductors' height, with surface irregularity factor m = 0.75).The corona current in the transverse plane is simulated by means of distributed voltage-controlled current sources.Fig. 15(b) reflects the sizeable impact of corona discharge on traveling voltage waves, practically overshadowing any catenary effect, when plotted against the curves accounting for all the previous simplifying assumptions (lossless MTL, conductors at average height, absence of corona).
Fig. 2 .
Fig.2.Representation of the voltage nodes within a span of a single-conductor TL, with reference to the piecewise linear approximation of the line catenary implemented in the implicit FDTD code.
Fig. 3 .
Fig.3.Piecewise constant approximation of the line catenary implemented in EMTP-RV, and in the modified FDTD code as to reproduce results from the LIOV code; the TL is subdivided into macrosteps with length Δx m (a simple line discretization with k m =Δx m /Δx =3 is shown as an example).
Fig. 4 .
Fig. 4. Modification of the equivalent circuit for discretization steps Δx located at a junction node between macrosteps of the line with different heights, as by the piecewise constant approximation of the line catenary.
Fig. 5 .
Fig. 5. Sketch of the top and lateral view of the central conductor of the simulated three-conductor line; the simulated lightning channel base locations (x s , y s ) and the striking point of the direct stroke are displayed.
Fig. 6 .
Fig. 6.Overvoltages at different observation points along the central conductor of the line in Fig. 5 due to a direct lightning stroke at x = 1.2 km.
Fig. 7 .
Fig. 7. Overvoltages at different observation points along the line central conductor due to indirect lightning strokes with channel base located at x s = 1.0 km and y s = 0.05, 0.10 km; the simulated MTL is uniform, with conductors' height equal to h ave .
Fig. 8 .
Fig. 8. Overvoltages at different observation points along the line central conductor, induced by the indirect lightning current with channel base located at x s -y s .Results account for constant and variable conductor height, computed by the LIOV code, the proposed and the modified FDTD code.Percentage deviations of the peak values are computed with respect to results by the proposed FDTD code when accounting for the catenary.(a) Channel base location point: x s = 1.0 km, y s = 0.05 km.(b) Channel base location point: x s = 0.8 km, y s = 0.05 km.
Fig. 9 .
Fig. 9. Incident and scattered voltages at different observation points along the line central conductor, with channel base location (x s , y s ), accounting for the conductor catenary.(a) Incident voltages.(b) Scattered voltages.
Fig. 10 .
Fig. 10.Overvoltages computed by the FDTD code at different observation points along the line central conductor, induced by the indirect lightning current with channel base located at x s = 0.8 km, y s = 0.05 km.Results account for constant and variable conductor height, and different Courant factors f C .
Fig. 11 .
Fig. 11.Arrangement of the conductors at the towers with geometrical characteristics in TableII.
Fig. 12 .
Fig. 12. Overvoltages at different observation points along the line (for SW and conductor B), induced by the indirect lightning current with channel base located at x s , y s .Results account for constant and variable conductor height, computed by the LIOV code and the proposed FDTD code.Percentage deviations of the peak voltage values are computed with respect to results by the proposed FDTD code when accounting for the catenary.(a) Channel base location point: x s = 1.0 km, y s = 0.05 km.(b) Channel base location point: x s = 0.8 km, y s = 0.05 km.
Fig. 13 .
Fig. 13.Overvoltages at different observation points along the line (for SW and conductor B), induced by the indirect lightning current with channel base located at x s , y s .Results account for constant and variable conductor height, computed by the original FDTD code with a single-or a three-point integration of the vertical component of the incident electric field.Percentage deviations of the peak values are computed with respect to results obtained by the threepoint quadrature.(a) Channel base location point: x s = 1.0 km, y s = 0.05 km.Conductors at constant (average) height.(b) Channel base location point: x s = 1.0 km, y s = 0.05 km.Conductors' catenary is accounted.(c) Channel base location point: x s = 0.8 km, y s = 0.05 km.Conductors' catenary is accounted.
Fig. 14 .
Fig. 14.Total, and scattered voltages at different observation points along the central conductor of the line sketched in Fig. 5, with channel base location x s = 0.8 km, y s = 0.05 km.Results are computed by the FDTD code and account for constant and variable conductor height.(a) Total voltages-toground.(b) Incident voltages.(c) Scattered voltages.
Fig. 15 .
Fig. 15.Overvoltages at different observation points along the SW and conductor B of the line sketched in Fig. 11 due to a direct lightning stroke at x = 1.2 km.Curves in (a) present the effects of losses and catenary, while curves in (b) also account for the corona discharge along the line. | 2023-07-29T15:10:03.087Z | 2023-10-01T00:00:00.000 | {
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6181714 | pes2o/s2orc | v3-fos-license | Molecular modeling and docking characterization of CzR1, a CC-NBS-LRR R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum
Plant NBS-LRR R-genes recognizes several pathogen associated molecular patterns (PAMPs) and limit pathogen infection through a multifaceted defense response. CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene isolated from Curcuma zedoaria L exhibit constitutive resistance to different strains of P. aphanidermatum. Majority of the necrotrophic oomycetes are characterized by the presence of carbohydrate PAMPs β-glucans in their cell walls which intercat with R-genes. In the present study, we predicted the 3D (three dimensional) structure of CzR1 based on homology modeling using the homology module of Prime through the Maestro interface of Schrodinger package ver 2.5. The docking investigation of CzR1 with β-glucan using the Glide software suggests that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 act as catalytic residues and are involved in hydrogen bonding with ligand β-(1,3)-D-Glucan. The calculated distance between the carboxylic oxygen atoms of Glu187–Asp355 pair is well within the distance of 5Å suggesting a positive glucanase activity of CzR1. Elucidation of these molecular characteristics will help in in silico screening and understanding the structural basis of ligand binding to CzR1 protein and pave new ways towards a broad spectrum rhizome rot resistance development in the cultivated turmeric.
cultivars available today are equally suceptible to major fungal and bacterial diseases. Crop losses to the tune of 60% has been realized in the recent times mainly due to the infection by a necrotrophic oomycetic fungus Pythium aphanidermatum causing the rhizome rot disease in turmeric [2]. Due to several constraint associated with the utilization of chemical pesticides and host resistance breeding, a genetic transformation approach using alien genes could be the most likely solution towards development of rhizome rot resistance. Our lab has recently identified and characterized a coiled-coil-nucleotide-bindingsite-leucine-rich repeat (CC-NBS-LRR) R-gene, CzR1 from wild turmeric genotype Curcuma zedoaria that exhibit constitutive upregulated expressions in response to different strains of P. aphanidermatum [3]. The conceptual protein has 906 amino acids, a predicted molecular weight of 102473.57 Da and a calculated isoelectric point (pI) of 8.55.
Before utilization in long term turmeric improvement programmes, it is essential to determine the three dimensional (3D) model of CzR1 to assess its structural integrity and biological relevance. A homology modeling based protein structure perdiction of CzR1 could be an efficient strategy taking into account the time consuming and cumbersome nature of X-ray diffraction or nuclear magnetic resonance spectroscopy (NMR) [4]. Further, several evidences suggest that various R-genes and pathogenesis related proteins play major role in neutralizing the oomycetic cell wall to kill the pathogen in line of defense signaling [5]. Beta-1,3-glucan, the major cellwall constituents of necrotrophic oomycetes often acts as elicitors of defence reactions and serve as pathogen associated molecular patterns (PAMPs) [6]. Thus, the recognition of these PAMPs by R-genes are crucial to activate defense reactions in plants. However, there is no report on the interaction between R-gene and β-1,3-glucan to study the presence of a possible glucanase activity in R-gene dependent pathogen defense. Thus the present study was designed to predict the 3D structure of CzR1 based on homology modeling and molecular docking of the R-protein with β-1, 3(D)-glucan.
Methodology:
A BLASTX search of the Protein Data Bank (PDB) using CzR1 predcited protein as query showed high sequence identity (61%) with human APAF-1 protein (PDB ID: 1Z6T) and significant homology with coiled coil domain of MLA10 gene of Hordeum vulgare (PDB ID: 3QFL) and leucine rich repeat domain from Xanthomonas oryzae (PDB ID: 4FCG). Models of CzR1 protein were constructed based on the crystal structure coordinates of 1Z6T along with other two sequences at a resolution of 2.21 Å. The pair wise alignment of CzR1 was done with 1Z6T as reference using the homology module of Prime through the Maestro interface (Schrodinger, LLC, and New York, USA). The pair wise alignment was improved manually by minor editing based on the secondary structure predictions as well as the pair wise superposition of the NB-ARC domain to develop the three dimensional structure of CzR1. The model was screened for unfavorable steric contacts and remodeled using a rotamer library database of Prime. Explicit hydrogen was added to the protein and the model was subjected to energy minimization using the force-field OPLS-2005 using 300 iterations in a simple minimization method. The steepest descent energy minimization was performed until the energy showed stability in the sequential repetition. CzR1 model structure was evaluated for its backbone conformation by inspecting the Psi/Phi angles in a Ramachandran plot generated using the software PROCHECK version 3.5 [7]. Z score of CzR1 and the template (1Z6T) was estimated using the software Prosa2003 [8]. Nevertheless, PROCHECK assured the reliability of the structure and the protein was subjected to VERIFY3D [9] available from NIH MBI Laboratory Servers. The root mean square deviation (RMSD) was calculated by superposing the predicted Cα traces and backbone atoms of Cz1R structure onto the template (1Z6T) in order to assess the the deviation of the modeled structure from the template. The protein structure image of the model was illustrated using the software PyMOL.
Molecular docking was performed in order to determine the binding affinity of polymer of β-(1,3)-D-Glucan onto Cz1R protein. The chemical structure β-(1,3)-D-Glucan was extracted from Chemical Book (http://www.chemicalbook.com) and its three dimensional structure was developed using the molecular builder tool of Maestro (Schrodinger package, ver 8.5). Initially different binding sites were predicted on CzR1 protein using SiteMap (Schrodinger, ver 2.4), out of which only the binding site from the NB-ARC domain was considered for docking study. The receptor-grid file was generated at the centre of the predicted binding site using Glide (Schrodinger, ver 2.5). A bounding box of size 14Å x 14Å x 14Å was defined and centered on the mass center of each binding site in order to confine the mass center of the docked ligand. β-(1,3)-D-Glucan was then docked into each predicted binding site using Glide XP (extra precision) docking [10][11]. Out of the 5,000 poses that were sampled initially through exhaustive search of the torsional minima 800 poses per ligand were selected for energy minimization (conjugate gradients 1,000 steps). The 10 lowestenergy poses obtained were subjected to post docking minimization (Monte Carlo sampling based on torsional minima and refining the orientation of side groups of ligand). A single best conformation of the ligand was considered for further analysis and the binding affinity between the protein and ligand was estimated using the GlideScore function [12]. The mode of protein-ligand interaction was plotted using the program LIGPLOT [13].
Results & Discussion:
The CzR1 protein from C. zedoaria with 906 amino acids contains NB-ARC domain (168 to 465) and LRR-1 domain (630 to 651) predicted from Pfam database [3]. Although Mla1 protein from Hordeum vulgare shared 62% similarity with CzR1, it could not be used as a template due to non-availability of its structure information in the PDB database. Hence, the crystal structures of MLA10 coiled coil domain (3QFL), human APAF-1 protein (1Z6T) and Xanthomonas oryzae leucine rich repeat domain (4FCG) were combinedly used as templates for developing a homology based model of CzR1 ( Figure 1A). The overall stereo chemical quality of the model was assessed by PROCHECK. The Ramachandran plot showed 96.6% amino acid residues from the main chain conformation of CzR1 within the favored or allowed regions suggesting that majority of the amino acids are in a phi-psi distribution that is consistent with a righthanded α-helix. The G-factors, indicating the quality of the covalent bond length and overall bond angles, were −0.10° for dihedrals, 0.42° for covalent bonds, and −0.11° as overall value. Although, a calculated RMSD value of 0.448 Å ( Figure 1B) was obtained by super positioning of the Cα atoms of the CzR1 and the template protein (1Z6T), the RMSD between the NB-ARC domain structures between both the proteins was only 0.284 Å. Prosa2003 analysis yielded a Z score of −4.32 for CzR1 and −4.63 for the central template (1Z6T). The final model, which we took for further analysis, consisted of 631 amino acid residues. The overall main-chain and side-chain parameters, as evaluated by PROCHECK, are all very favorable. The assessment with VERIFY3D, which derives a "3D-1D" profile based on the local environment of each residue, described by the statistical preferences for: the area of the residue that is buried, the fraction of side-chain area that is covered by polar atoms (oxygen and nitrogen) and the local secondary structure, also substantiated the reliability of the three dimensional structure. (Table S2). β-(1,3)-D-Glucan shares higher values of electrostatic interaction (Ecoul, -15.42 kcal/mol), hydrogen bonding energy (Hbond, -2.092 kcal/mol) and Van der Waals interaction (Evdw, -33.03 kcal/mol) energies Table 2 (see supplementary material) predicted by Glide-XP because of the polar and non polar interactions with the binding site amino acids. The model produced by GlideXP predicts that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 are involved in hydrogen bonding with docked polymer β-(1,3)-D-Glucan (Figure 2A & 2B). The calculated distance between the carboxylic oxygen atoms of the Glu187-Asp355 pair is 4.38Å, which is well within the distance of 5Å required between the two catalytic residues for glucanase activity [15]. Furthermore, polymer of β-(1,3)-D-Glucan showed desirable hydrophobic interactions with the hydrophobic residues within the binding cavity (Figure 3). The favourable H-bonding, columbic, and hydrophobic interaction energies make polymer of β-(1,3)-D-Glucan highly active and selective for CzR1. Previous to our studies, there is no data on the possible glucanase or hydrolytic activity of the R-genes. However, many PR-proteins with antifungal activity exhibits such interdomain surface cleft for glycoside hydrolase enzymes and binding by (1,3)-b-D-glucan [16][17]. Further research on binding interactions between variable NBS-LRR class R-genes and different pathogen /microbe associated molecular patterns (PAMPs/MAMPs) is needed to get a decisive conclusion on the global functioning and regulation of such R-genes.
Conclusion:
In conclusions, molecular modeling procedures revealed the presence of a narrow cleft near the ATP-GTP binding site of the highly charged NBS domain of CzR1, a CC-NBS-LRR class Rgene from C. zedoaria. Molecular docking predicted high affinity between CzR1 and (1, 3)-β-D-glucan, a major component of the oomycete cell wall with favorable pairing of two negatively charged amino acid residues Glu187-Asp (355) within the docked complex. This suggest that CzR1 could be possibly having a β-1, 3-glucanase activity in vivo besides the regular ATP-GTP binding function in initiating pathogen defense. Revelation of these molecular characteristic of CzR1 controlling the resistance of C. zedoaria against P.aphanidermatum together with studies on the significance of hydrolytic activity of CzR1 will be highly informative towards the development of a broad spectrum rhizome rot resistance in the cultivated turmeric. | 2016-05-12T22:15:10.714Z | 2013-06-29T00:00:00.000 | {
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209380032 | pes2o/s2orc | v3-fos-license | Semi-parametric modelling of sun position dependent solar gain using Bsplines in grey-box models
Modelling the effects of solar irradiation plays an important role in various applications. This paper describes a semi-parametric (combined grey-box and spline-based), data-driven technique that can be used to model systems in which the solar gain depends on the sun position. The solar gain factor is introduced, i.e. the absorbed fraction of measured solar irradiation, and estimated as a continuous non-parametric function of the sun position. The implementation of the spline-based solar gain factor in a grey-box model framework is described. The method is tested in two case studies—in a model of the internal temperature of a dwelling in Aalborg, Denmark, and a model of the return temperature of a solar collector field in Solrød, Denmark. It is shown that the solar gain factor as a function of sun position is able to account for structural variations in solar gain that may occur due to factors such as shading obstacles and window or absorber orientation. In both test cases, the spline-based solar gain function improved the model accuracy significantly, and largely reduced structural errors in prediction residuals. In addition, the shape of the estimated function provided insight into the dynamics of the system and the local solar input characteristics. Accurate representation of such site characteristics was not possible with any data-driven method found in the literature. Besides the grey-box models used in this study, the solar gain factor can be used in a variety of data-driven models, for example in linear regression models.
Introduction
Solar irradiation is a crucial factor in the field of building engineering, renewable energy generation, and many other applications. In many cases, measurements or predictions of the solar irradiation are available, and the effect of solar irradiation needs to be captured in a model. However, the relation between solar gain and measured solar irradiation is typically non-linear and dependent on the position of the sun and site characteristics. This paper, presents and implements a datadriven model for dynamical thermal systems, that include a sun position dependent solar gain. The model is tested in two thermal systems: a building and a solar collector field.
In both systems, the heat dynamics related to solar irradiation are modelled for various reasons. For buildings, models are used to document the energy performance and identify a potential energy performance gap (Roels et al., 2017;Johnston et al., 2016;Haldi and Robinson, 2011;Brohus et al., 2010;Socolow, 1977). For instance, this has been done using data-driven thermal dynamic building models such as grey-box models (Roels et al., 2017;Madsen and Holst, 1995). As solar heat gain can significantly affect estimated thermal building properties, the model used for solar heat gain needs to be accurate. This is especially true for well-insulated buildings with low thermal mass and large window areas.
Similarly, for solar collector fields, forecasting models need to describe solar irradiation effects accurately to improve heat output forecasts and system control. The control must respond to rapid fluctuations in solar irradiation to prevent the collector fluid from boiling, and to ensure a high and stable outlet temperature. As solar irradiation is the dominating effect on the outlet temperature, an accurate model of the absorbed solar energy can improve predictions and hence operation of solar heat systems.
The following two sections present a literature review on typical implementations of data-driven solar heat gain in thermal building and solar heat plant models. aperture, effective window area, or gA-value) can be seen as the equivalent area of a perfectly transparent surface that transmits the same amount of solar energy as the actual windows of the space.
For inverse problem solving, the location of windows and shading obstacles are often unknown. Even if the location of the shading obstacles is known, it can still be cumbersome to estimate their thermal effects, just as it is difficult to estimate the effects of other factors such as reflections or dirt on the windows. Some data-driven thermal dynamic building models disregard the solar gain (Zeifman and Roth, 2016;Coley and Penman, 1992) and consequently treat solar gain as model uncertainty and observation noise. In other models, solar gain is considered a constant fraction of the global solar irradiation (Rabl, 1988;Bauwens and Roels, 2013;Madsen and Holst, 1995;Jimenez and Madsen, 2008), for example where sol is the solar gain, I g is the global radiation, and is the solar gain factor. The solar gain factor can then be estimated from data. When assessing the energy performance of buildings, Mejri et al. (2011) describe the solar gain in buildings as a hidden forcing function. They propose a method in which the solar irradiation striking each building facade, I i , is estimated separately as where i is the facade number and k i is a constant for the i th facade. Letting the constant, k i , account for the properties of the windowpane-e.g. the solar energy transmittance (g-value) and the area-this method could be used to calculate the solar gain coming through the windows in a manner analogous to Eq. (1). However, there is no evidence that this method would improve model accuracy compared to the simpler approach in Eq. (1).
Both of the above approaches assume that the solar gain is independent of the sun position. While these approaches may be sufficient for buildings with limited window area (and therefore limited solar gain), they may fall short for buildings with higher sensitivity to solar irradiation. As suggested by Bacher and Andersen (2014) and Madsen et al. (2016), parametric grey-box models could be used in conjunction with non-parametric models of solar gain, such as splines. Hence, rather than assuming a constant solar gain factor for the entire enclosed space or the facade of interest, the factor can be described as a function of the sun position. The present paper details the specific procedure for achieving this.
Solar gain in solar heat plant models
Solar irradiation is the main factor that determines the heat production of a solar heat plant (SHP), so it is crucial to include the effects of solar radiation in detail when modelling the return temperature (also called outlet temperature) of a solar heat field. The solar incidence angle on the panel largely influences the share of reflected irradiation, and thereby the solar gain. Furthermore, the position of the sun affects the solar gain due to possible shading objects around the collectors. It is common for the collector rows to cast shade on one another at some point in the afternoon as well. Finally, dirt or snow on the panels may affect the solar gain.
The most recent standard for performance testing of solar heat collectors by the International Organization for Standardization (ISO) is described in ISO 9806:2017 (European Committee for Standardization (CEN) Technical Committee, 2017). The method is considered to be the state-of-the-art for solar collector modelling (Kicsiny, 2014). The solar gain is included in the ISO dynamic model as where I t is the total radiation in the collector plane. The parameter (usually named F ( ) en in this application) represents the fraction of Rasmussen, et al. Solar Energy 195 (2020) 249-258 solar irradiation that is absorbed by the panels at an incidence angle equal to zero, whereas the incidence angle modifier (IAM) K ( ) accounts for incidence angle dependence of the solar gain. The incidence angle is defined as the angle between the sunbeam and the normal of the collector plane. When beam and diffuse irradiation are measured separately and an incidence angle modifier is used, the solar gain is modelled as where the diffuse IAM (K d ) is a constant, as it is independent of the incidence angle. The standard incidence angle modifier (IAM) used in the ISO performance test is Depending on the applied model type, the parameter b 0 that determines the slope of the curve is manually tuned or fitted to data using statistical techniques. For most flat plate collectors, this equation is considered sufficiently accurate for describing incidence angle effects (Perers, 1997). Another IAM given in ISO 9806:2017 is the Ambrosetti function In this case, the dimensionless parameter determines the slope of the function. Almost all models intended for control of SHPs present in the scientific literature combine (partial) differential equations with parameters estimated from data, see for example Pasamontes et al. (2013) and Andrade et al. (2015). The incidence angle dependence is either neglected, or modelled using IAM functions such as in Eq. (5) and (6). The vast majority of existing models for SHP control do not take shadowing effects into account (Bava, 2017;Bava and Furbo, 2018).
Although these IAM functions may suffice for performance testing and simulation, they may not be accurate enough for control purposes. The functions used currently are tuned by a single parameter and therefore only a limited amount of function shapes are possible. Furthermore, the functions do not allow for asymmetric diurnal variations of efficiency, which may exist due to shading of the collectors.
Motivation
This paper aims to formulate and test a novel non-parametric method to estimate the solar heat gain in thermal dynamical systems such as buildings and solar collector fields. To the best of our knowledge, and as seen in the literature review, only simple data-driven methods for modelling solar gain in buildings exist, i.e. models in which the solar gain is independent of the sun position. For solar collectors, parametric incident angle modifiers (IAM) have been widely applied in the literature, in both steady-state and dynamic models. However, these IAMs are not able to account for potential shading patterns on the collectors.
As the relationship between solar gain and measured solar irradiation is sun position dependent and site-specific, it is often not feasible to choose a parametric representation of this relationship. In addition, the solar gain factor, i.e. the share of measured solar irradiation that is absorbed in the system, depends on the sun position non-linearly.
Spline functions address both of these issues. A key benefit of this non-parametric modelling technique is that no model structure needs to be specified. Furthermore, the splines can model non-linear phenomena, but the nonlinear operations in the construction of basis splines are performed before the model fitting process. Hence, only linear operations on the constructed basis splines are carried out in the model fitting procedure, as will be demonstrated later.
This study implements and tests the spline-based solar gain factor in a grey-box model setting. Grey-box models are a class of parametric physics-based statistical models, which in form lie between the deterministic white-box models known from simulation tools such as TRNSYS, and entirely data-driven black-box models such as simple linear regression and neural networks. Grey-box models are often relatively simple and computationally lightweight, they have excellent forecasting properties, and parameters that are directly related to physical properties.
In Section 2 the proposed method is discussed, including the mathematics behind the applied B-spline functions (Section 2.1), the applied grey-box model (Section 2.2), and the actual implementation of splines in state-space models (Section 2.3). In Section 3 the approach is applied to two use cases: modelling the effect of solar irradiation on the internal temperature of a dwelling in Aalborg, Denmark (Section 3.1), and forecasting the heat production from a solar collector field in the municipality of Solrød, Denmark (Section 3.2). Lastly, in Section 4, the approach is discussed and closing conclusions are drawn in Section 5.
Method
This section presents the used modelling procedure, which is also outlined in Fig. 1. First, Section 2.1 introduces spline functions and the construction and usage of a B-spline basis for data fitting, corresponding to the first step in Fig. 1. Next, Section 2.2 presents the resistance-capacity grey-box models and model selection procedure used in this article, which span all remaining steps in Fig. 1. Finally, Section 2.3 discusses in detail how the spline functions are implemented in the grey-box model setting in particular.
Splines
Splines are piece-wise polynomials that are continuously differentiable up to a certain order. The points at which these piece-wise polynomials connect are called knots. Splines are in practice used for interpolation, smoothing, and function approximation (Boor, 2007). This paper concerns the latter challenge, and aims to estimate the solar gain factor from data as a smooth function.
Splines are attractive for function approximation due to several properties. The functions are smooth up to a certain order, have C. Rasmussen, et al. Solar Energy 195 (2020) 249-258 compact support, and their formulas are explicit and rather simple (Prautzsch et al., 2002;Christensen, 2010). The proposed method is based on a specific type of spline function: the B-spline. Given a knot sequence, one can construct a set of B-splines that form a basis for all splines with this same knot sequence (Boor, 2007;Prautzsch et al., 2002). The construction of the cardinal B-spline basis will be discussed in Section 2.1.1, and in Section 2.1.2 a more general construction of B-splines is shown.
Cardinal B-splines
The is a piece-wise polynomial of order m, meaning that the spline consists of m polynomials of degree m 1. A transition point from one polynomial to the next is called a knot. If the knot sequence is uniformly spaced, it is called a cardinal B-spline or a cardinal basis spline.
The cardinal B-spline can be defined and constructed in various ways. A compact definition is the recursive convolution relation, first shown by Schoenberg (1946), where * is the convolution operator m and B 1 is defined by the characteristic function The B-spline has a number of desirable properties, one of which is continuity of the 0 th to m ( 2) th derivatives in the knots, i.e. B m m 2 (Sauer, 2006). For example, the B-spline of order = m 4 is a piece-wise polynomial, each of these polynomials is of third degree, and the function value, slope and curvature are continuous in each knot.
Another property is that infinitely many cardinal B-splines pointwise add up to unity, when evenly distributed along the x-axis with a distance corresponding to the distance between two neighbouring knots (Christensen, 2010).
As demonstrated later, the B-splines are suitable for estimating the solar gain factor as a smooth function of sun position. The estimated function is obtained by summing the scaled B-splines. In many use cases, one is only interested in a series of B-splines that covers a finite range of the x-axis. However, the sum of a finite set of cardinal B-splines will go to zero at the boundary regions as seen in Fig. 2A. Therefore, the cardinal B-splines near the boundaries need to be modified to add up to unity for the entire finite domain. The following section illustrates this modification of the B-splines.
B-splines in a finite domain
For a strictly increasing series of knots , in Eq. (7) by the alternative Cox-de Boor recursion formula: The Cox-de Boor recursion formula can be used to define B-splines with non-uniform knot distances and to shift the B-spline along the xaxis. The above criterion is relaxed from strictly increasing to non-decreasing knots, so that knots may be positioned at equal locations. In order to still use Eq. (9) in this generalised case, division by zero is considered as zero.
If the first m knots of the knot sequence … x x x , , , , and the same holds for the last m knots of the knot sequence, then the B-splines as defined in Eq. (9) will add up to unity on the finite domain. The leftmost B-spline will have m coinciding knots, the second leftmost B-spline will have m 1 coinciding knots, etc. The resulting set of B-splines has the same properties as the cardinal Bsplines, except that continuity is reduced in the coinciding knots to m n , where n is the number of coinciding knots. Figs. 2A-D illustrate how the basis splines change shape as more knots are coinciding and eventually how the resulting spline S x ( ) adds up to unity on the finite domain.
Data fitting with splines
The property that the B-spline series sums up to unity for the entire range of interest is especially useful for data fitting. By scaling and summing the individual B-splines as in Eq. (10), the spline function S x ( ) is obtained: where i is the scaling factor (and model parameter) of the i th B-spline. Typically linear ( = m 2), quadratic ( = m 3), or cubic ( = m 4) splines are used. Fig. 2E illustrates how it is possible to fit a spline to a certain data set by scaling the individual basis splines. By utilising the modified boundary basis spline as described in the previous section, it is possible to estimate a nonzero value at the left and right side of the spline domain. Section 2.3 will further discuss the implementation of the spline function S x ( ) in a grey-box setting.
Grey-box models
Grey-box models are mathematical representations of a physical system that is described by a number of inputs, outputs and state variables. The state variables are described by first order differential or difference equations for continuous and discrete time, respectively. This paper focuses on grey-box models with states in continuous time and observations in discrete time.
In a grey-box model, a noise term is added to the-otherwise deterministic-white-box model. The stochastic nature of these models furthermore allows for the application of statistical methods for structural model identification, such as the likelihood ratio test.
A grey-box model consists of a series of stochastic differential equations (the system equations) and one or more observation equations. The grey-box model in the form used in this article, namely the C. Rasmussen, et al. Solar Energy 195 (2020) 249-258 state-space form, is written where x t is the state vector, y tk is the observed output vector, A is the transition matrix, B is the input matrix, C is the output matrix, u t is the input vector, t is the time, k is the number of the discrete time step, is the measurement noise, and finally t is a vector of Wiener processes. The measurement error vector Ñ (0, ) t obs k is assumed to be i.i.d., and t and tk are assumed independent. Further theory on stochastic differentiation and integration can be found in for example (Kloeden et al., 1994 andZastawniak andBrzezniak, 1998). For the purposes of this paper, a basic knowledge of random variables suffices.
Model selection procedure
In order to choose the optimal number of B-splines to approximate a functional relationship present in the data, several possibilities are implemented and compared. For cubic splines with intercept, the minimum number of splines is 4. The maximum is kept at 12, which typically is sufficient for the specific modelling purposes.
Forward model selection is used, in which a simple model is iteratively extended. The procedure is visualised in Fig. 1. In each iteration, the most significant model extension is selected using the likelihood ratio test (LRT), a statistical test providing a p-value that expresses the significance of the extension (Madsen and Thyregod, 2010). The model selection ends when no extension is significant. A significance level of 0.05 is used throughout the model selection. The likelihood ratio test is also used to determine the optimal number of B-splines in each model extension. Fig. 2E visualised how a set of basis splines is scaled such that the resulting spline fits a certain data set. For the case of modelling the solar gain in a given energy system, the exact same approach can be used.
Implementing splines in state-space models
The position of the sun is uniquely defined by the solar azimuth angle, , and the sun elevation, , which can be determined from latitude, longitude and time. This paper deals with the solar gain factor as a function of the azimuth angle only. This is a valid assumption for sufficiently short time periods during which the sun elevation corresponding to each azimuth angle is approximately constant over the days included in the data.
In the following, the spline-based approach is described for modelling solar gain in a resistance-capacity (RC) grey-box model. Only cubic B-splines with equidistant knots are used here. When the data set covers all times between sunset and sunrise, the boundary knots are placed at these two moments. Otherwise the boundary knots are placed at the extremes of the azimuth angle present in the data.
The solar heat gain sol in an RC model can be expressed by multiplying the solar gain factor and the outdoor solar irradiation measured in a certain plane as shown in Eq. (1), (3), and (4). To include azimuth angle dependence of the solar gain, the solar gain factor is substituted with the spline, One needs to specify a sequence of knots in any sub-interval of°°[ 0 , 360 ), where is the azimuth angle of the sun. This sub-interval will typically include the azimuth angles between sunrise and sunset. Thereby the functional representation of the solar gain sol is obtained as in Eq. (13), where is the sun elevation, and I is the solar irradiation. The knots are defined as described in Section 2.1.2 to ensure that the spline curve can take any value for the entire chosen sub-interval. Depending on the complexity of the shadow pattern over the day, the number of base splines can be adjusted.
The basis splines B ( ) i m , are uniquely defined by the knot sequence and the azimuth angle time series. Therefore, the basis splines can be constructed before model fitting, as seen in the first step of Fig. 1.
Assuming that the solar radiation only affects the first state, the stochastic differential equations on state-space form in Eq. (11) can be written as: : Second input matrix )and its associated input vector )excluding solar gains.
In Eq. (14), p is the number of states, q is the number of basis splines used to model the solar gain, r is the number of inputs excluding solar irradiation, i is the scaling factor of the i th basis spline, is the heat capacity of the temperature node to which the solar gain is assigned, and I t is a time series of the measured outdoor solar irradiation. Finally, × A p p , is the transition matrix and x p is the state vector.
Note that the matrix B 1 can be modified to make the solar gain affect more than one state, however, for simplicity of the formulations this is not done here.
Case study
This Section describes the implementation of the spline-based approach for solar gain modelling of two dynamical thermal systems. In the first case (Section 3.1), the splines are included in a model of the heat dynamics of a single apartment. In the second case (Section 3.2), splines are applied to model the solar gain in a solar collector field.
In the following sections the used data and applied models are described for each case, along with results of the model selection procedure.
All models are formulated and fitted with use of the package ctsmr (Continuous Time Stochastic Modelling for R, version 0.6.17) (Juhl, 2013), which uses a Kalman filter and maximum likelihood methods for parameter estimation. The B-spline basis functions are generated with use of the R-core package splines (version 3.5.1) (R Core Team, 2017).
Modelling the thermal dynamics of a building
In this case study, the physical parameters of a building, such as heat loss coefficients, heat capacities, and time constants, were estimated. The following sections show how the models improve when the solar gain is modelled with splines. This semi-parametric model allows the estimation of the azimuth-dependent solar gain factor and the resulting solar gain.
Data
During the period from February 13 to April 1, 2018, measurements were obtained from a two-room apartment in Aalborg, Denmark. The apartment consists of an open kitchen and living room, a bedroom, a bathroom, a small corridor, and a heated weather porch. The apartment has a gross floor area of 67 m 2 and a total window area of 10.7 m 2 . Similar heated apartments are located on the north side, south side, and above the apartment. An unconditioned basement is located below the apartment. The floor plan of the apartment is shown in Fig. 3, including C. Rasmussen, et al. Solar Energy 195 (2020) 249-258 window sizes and the physical parameters used for modelling. The total space heating of the apartment was measured every 15 min, and the air temperature in eight locations in the apartment was measured every 5 min. The internal temperature used in the model was the arithmetic mean temperature of all eight sensors in each time step.
In addition to the heating power heat (kW) and the internal temperature T i (°C), the outdoor ambient temperature T a (°C) and the global solar irradiation I g (kW/m 2 ), were measured every minute at Aalborg University, 2.6 km away. All measurements were re-sampled to hourly values by averaging. A subset of the data is plotted in Fig. 4.
Models
The model that all the other models originate from is the single-state model T i as given by Note that the time indices shown in e.g. Eqs. (11) and (12) are omitted for simplicity. In the model above, the only state is the internal temperature T i , as indicated by the model name. The C R , , , i i w and the variance of are model parameters which are to be estimated. T , a heat and I g are model inputs, and T i is the observed internal temperature. For further specification, see the Nomenclature.
All models include the state variables in their names. The temperature of an internal thermal mass, the heating system, and the exterior wall/building envelope are represented by T , T m h , and T w , respectively. Finally, the postfix S q indicates that the solar gain is modelled by a cubic spline with q basis spline.
For illustrative purposes Eq. (16) presents a model with two states: the internal temperature T i , and the hidden state temperature of the building envelope T w . The inputs of model T T i w are the ambient temperature T a , space heating heat , and global irradiation I g .
To convert a model with constant solar gain factor, , to a model with azimuth-dependent solar gain factor, ( ),the spline function in Eq. (10) is substituted by The solar gain = I ( ) sol g is assigned to a single state, namely the internal temperature node T i , for all fitted models. Fig. 5 shows the model selection framework and the result of the model selection. The diagram shows that the simplest model T i is first extended with a hidden state without use of solar splines (row 2). In this case, the model T T i w was found to be the best model. No more than one hidden state has been tested, to avoid potential structural identifiability issues. After the model was fitted, it was extended further by introducing splines to estimate the azimuth-dependent solar gain factor. This is shown in the lower part of the figure.
Results
All the spline models with five or more basis splines (except the models with eight and nine, which did not converge) outperform the null model T T i w . However, no statistically significant improvement was found when more than ten basis splines were used for this particular data set.
The likelihood ratio test between the null model T T i w and the alternative model T T _ S i w 10 reveals a p-value of less than e 2.2 ·10 16 , which clearly shows a significant improvement obtained by the alternative model. The best model was therefore found to be the two-state model T T _ S i w 10 , with a cubic spline describing the solar gain based on ten basis splines. Fig. 6 shows the estimated solar gain factor of model T T i w and T T _ S i w 10 . In the azimuth interval from 110°to 260°, two distinct peaks in the solar gain factor from model T T _ S i w 10 and a flat insignificant section in-between can be seen. This is in agreement with the apartment layout as it has windows towards the east and west, as seen in Fig. 3.
It is noted that solar gain factor for the morning hours (azimuth angle from 100°to 110°) is rather high and oscillates. As the solar irradiation intensity is low in this period of the day it is expected that the scaling factor of the basis spline is difficult to estimate. Furthermore, the confidence band is underestimated, as the scaling factor in this region is reaching the upper parameter search boundary. However, due to the low irradiation intensity, the effect on the model states is limited as well. This is seen in the general low solar gain levels in the interval from 100°to 110°, in the lower plot in Fig. 6.
Forecasting solar heat generation
In the second use case, the aim was to predict the return (outlet) temperature of a solar heat field. As the solar irradiation has a dominant influence on this variable, it can be expected that accurate modelling of shading patterns improves the model performance significantly. C. Rasmussen, et al. Solar Energy 195 (2020)
Data
Measurements were collected at the Aalborg CSP Solar Heat Plant in Solrød Municipality between May 23, 2017 and May 31, 2017. The total panel area is 2569 m 2 , which yields around 1300 MWh annually with a peak power of 1.9 MW, serving a community of 350 households. A schematic overview of the site and measurements are shown in Fig. 7. The following measured inputs were used: total solar irradiation in collector plane I t , fluid flow per panel surface area Q f , fluid supply and return temperature T s and T r , and ambient temperature T a . The solar azimuth angle was computed from the longitude, latitude, and time. In Fig. 7, note the trees located west of the collectors, which shade (part of) the field in the afternoon.
A subset of the measurements is depicted in Fig. 8. The values were 1 min averages of measurements taken every second. A control of the return temperature had already been implemented at the time of data collection, which limited the variation in measured return temperature, A subset of the data was selected for parameter estimation. Periods of low or zero flow were excluded, as well as data points with large incidence angle , such that < cos( ) 0.2 (i.e. >°78.5 ). Finally, some periods in which the plant switches back and forth from preheating to operating were removed. This results in a data set with gaps and a varying number of measurements per day, including days with clear sky and days with intermittent cloud cover. This is, however, not a problem in the grey-box model setup.
Models
An initial stochastic state-space model was taken from , whose model in turn derives from Perers (1997). An extended form of the heat balance proposed by the latter is used in the current ISO standard for quasi-dynamic testing (European Committee for Standardization (CEN) Technical Committee, 2017). The resulting state-space formulation for the base model T r is The modelled quantity is the return temperature T r . The measured inputs are T Q T , , a f s , and I t .
The variable T f is the average of supply and return temperature. The factor c f is a given constant, whereas mC U ( ) , , , e fa f and obs are parameters to be estimated. The exact specification of the variables can be seen in the nomenclature.
Four extensions of the base model T r were considered, which all add detail to modelling the effect of solar irradiation. One extension was the splined solar gain factor, which is indicated by S q in the model name, where q is the number of splines. In addition, two different IAM functions were implemented. The model name includes IAM for the standard function as given in Eq. (5), and IAM a for the Ambrosetti IAM from Eq. (6). Finally, the total irradiation was split into direct and diffuse components, using the method proposed by Ruiz-Arias et al. (2010). Models including this irradiation split contain I split in their names. All applied models lump the field into a single compartment with one corresponding state variable, the return temperature T r . All extensions were applied to the base model in an iterative manner that is visualised in Fig. 9. In each iteration, the model was extended in several ways, one extended model for each of the remaining extensions. The extension with the lowest p-value were selected, and the procedure was repeated until no extensions were significant or all extensions were implemented.
Note that in cases of the split radiation being combined with the splined solar gain factor ( ), the solar gain was modelled differently to Eq. (4) to ensure identifiability. Instead, it was given by in case where the model did not include an IAM.
Results
In the first model extension, the spline was the most significant improvement to the model, in particular T _S r 8 . The next selection round C. Rasmussen, et al. Solar Energy 195 (2020) 249-258 included the radiation split to the model, where again the version with eight basis splines was most significant. Finally, the model was extended with one of the IAM functions, again with eight basis splines. The model selection ended here, as all extensions considered were accepted. Note that the final model includes both a state-of-the-art Amrbrosetti incidence angle modifier and spline functions, which together model the sun-position dependence of the solar gain factor. This result shows that several solar gain modelling components can be combined and in this way complement each other. The combined effect of the IAM, split radiation, and spline functions on the estimated solar gain factor for the (estimated) direct radiation is shown as a function of azimuth angle in Fig. 10. The models have different estimated heat capacities mC ( ) e , which scale the functions to different maximum values. In order to compare the possible function shapes of the solar gain factor function that the different models provide, for this plot the functions were scaled to a common maximum value of 1. The spline-based solar gain factor decreases faster in the afternoon (azimuth angle from 225°to 280°), which indicates that the effect of the shading forest is captured in these models, but not in the state-of-the-art model.
Model performance was compared using prediction with perfect forecasts of the inputs, that is, using the actual measurements as inputs. Fig. 11 shows how the model predictions improved due to the added solar gain function. Note that all models, including the final model, overestimate the return temperature at the end of the day due to a measurement error in the ambient temperature. The measurement error occurs due to exposure of the sensor to direct sunlight. This could be accounted for in future improvements of the model, but is outside the scope of this article. Fig. 11A compares the residuals of two of the base model extensions, T _ IAM r and the selected T _ S r 8 . It is clear that the spline-based model is able to account for shading, and thereby reduces structural prediction errors that were present when modelling incidence angle effects with an IAM only. Fig. 11B shows improvements in the residuals after the final model extension from T _ S _ I r 8 split to T _ S _ I _IAM r 8 split . The improvement was less pronounced than in 11A, but it can be seen that the residuals in the late afternoon were slightly improved by including the IAM. The remaining error in the late afternoon is explained by the ambient temperature measurement error.
With each model extension, the time of day from which the model starts overestimating the return temperature is postponed. As one might expect, the improvements are less pronounced on cloudy days than on sunny days as plotted here, but still present.
Discussion
First of all, it is noted that although the splined solar gain factor was applied in grey-box models here, the used of the method is not limited to this model type. The B-splines can also be used in other models in which the parameters are fitted from data, such as linear regressions.
In the present article, the splined solar gain factor only affected a single state in the state-space models. It is however a simple adaptation to assign the solar gain to the system in more complex ways, e.g. by assigning it to more than one state.
While only equidistant spline knot sequences are used in this article, the location of the knots can perhaps be optimised by concentrating more knots around azimuth angles for which fast changes in the shading pattern occur. One should in this case be aware of potential overfitting when spline knots are placed too closely together, as the , only the best model is shown in the diagram, however, models with four to eight basis splines were tested. C. Rasmussen, et al. Solar Energy 195 (2020) 249-258 individual basis splines would be fitted to fewer data points. When using longer periods of measurements covering several months, there will be a larger range of sun elevations associated with a single azimuth angle. As the elevation also affects shading patterns and the incidence angle, one may need to account for this as well. This can be solved in several ways. First, an additional spline curve can be introduced to account for the sun elevation variation of the solar gain factor. The downside of this approach is that it requires significant amounts of data to estimate the splines and adds parameters to the model. This will increase the computation time and may even result in an unidentifiable model.
A more convenient approach than introducing a second spline curve is to apply an adaptive model in which parameters are allowed to change over time. One example of adaptive time-varying parameters estimation is presented by Joensen et al. (2000). In that way the scaling factors of the B-splines will be specific to shorter time periods of data, so that the azimuth angle again largely determines the shading pattern within this time period.
An important note regarding the estimated spline curve is that its physical interpretation is not always straightforward. The splines may account for other effects than solar irradiation such as systematic use patterns (heat gain from people, computers etc.) or systematic measurement errors. These concerns are mostly relevant for climates or measurement periods with low daily variation in solar irradiation, i.e. climates and periods with mainly clear sky conditions. Finally, it should be noted that it can be difficult to estimate the scaling factors for periods with low solar irradiation, e.g. close to sunrise and sunset. This may be prevented by placing the boundary knots closer towards the south, and fitting a constant or linear solar gain factor to the periods outside these bounds.
Conclusion
Modelling solar gain is of crucial importance for energy system models. This paper shows that the approaches for solar gain modelling in the literature can be improved significantly by taking the position of the sun into account when modelling the thermal dynamics of an apartment and a solar collector field. This paper has proposed B-splines as a non-parametric method to estimate the relation between measured solar irradiation and the solar gain. In two case studies, this method was implemented and tested in grey-box models that included the solar gain as a function of the azimuth angle. This function is estimated using coefficients on a B-spline basis. The splines have been used on the global, total, and beam irradiation in different model versions.
A two-state model of an apartment was improved significantly by estimating the solar gain factor with use of a diurnal spline factor instead of the commonly used constant solar gain factor. The estimated solar gain curve showed good agreement with the authors expectations for the specific apartment with east-and west-facing windows, as the peak in solar gain occurred during morning and evening.
For a return temperature model of a solar collector field, it has been shown that the splines account for local shading patterns caused by the panels themselves and trees next to the field, while also accounting for incidence angle effects. The key difference compared to the methods from ISO 9806:2017 and most models in the literature is that the splinebased solar gain factor has a higher degree of freedom than frequently used single-parameter incidence angle modifier (IAM) functions. This makes it capable of accounting for site-specific shading patterns, which largely reduced structural prediction errors. In addition, the splines can be combined with standard IAM functions to achieve even better results.
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. C. Rasmussen, et al. Solar Energy 195 (2020) 249-258 | 2019-11-28T12:39:53.303Z | 2020-01-01T00:00:00.000 | {
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236000383 | pes2o/s2orc | v3-fos-license | Medical practitioner’s knowledge on dengue management and clinical practices in Bhutan
Background Dengue has emerged as a major public health problem in Bhutan, with increasing incidence and widening geographic spread over recent years. This study aimed to investigate the knowledge and clinical management of dengue among medical practitioners in Bhutan. Methods We administered a survey questionnaire to all practitioners currently registered under the Bhutan Medical and Health Council. The questionnaire contained items on four domains including transmission, clinical course and presentation, diagnosis and management, and surveillance and prevention of dengue. Participants were able to respond using an online Qualtrics survey, with the invitation and link distributed via email. Results A total of 97 respondents were included in the study (response rate: 12.7%), of which 61.86% were Health Assistants/Clinical Officers (HAs/COs) and 38.14% were medical doctors. The afternoon feeding behaviour of Aedes mosquito was correctly identified by only 24.7% of the respondents, and ~66.0% of them failed to identify lethargy as a warning sign for severe dengue. Knowledge on diagnosis using NS1 antigen and the clinical significance of elevated haematocrit for initial fluid replacement was strikingly low at 47.4% and 27.8% respectively. Despite dengue being a nationally notifiable disease, ~60% of respondents were not knowledgeable on the timing and type of cases to be reported. Respondent’s median score was higher for the surveillance and reporting domain, followed by their knowledge on transmission of dengue. Statistically significant factors associated with higher knowledge included respondents being a medical doctor, working in a hospital and experience of having diagnosed dengue. Conclusion The study revealed major gaps on knowledge and clinical management practices related to dengue in Bhutan. Physicians and health workers working in Basic Health Units need training and regular supervision to improve their knowledge on the care of dengue patients.
Study setting
Bhutan is a small landlocked country measuring 38,394 square kilometres, nestled in the Eastern Himalayas between India in the south, east and west, and China in the north. The right to free access to essential health services is mandated by the constitution, and currently, the expenditure for healthcare services including referrals to third countries is borne by the government of Bhutan. Healthcare services are delivered through 32 hospitals (one-national, two regional and the remainder district general hospitals) and 208 Primary Health Care (PHC) or Basic Health Units (BHUs-grade I and II) [16]. Primary care is delivered in each of these health centres, but medical technologies and healthcare services become more advanced and specialized from BHUs to district general hospitals, to regional hospitals and the national referral hospital. Over 903 MPs are currently registered by the Bhutan Medical and Health Council (BMHC). This includes 92 medical specialists (MD), 186 general duty medical officers (GDMO with a Bachelor of Medicine/Bachelor of Surgery), 21 clinical officers (COs) and 604 Health Assistants (HAs) [16]. HAs undergo two years in-country training for primary care based on a whole-of-society approach that includes health promotion, disease prevention, diagnosis, treatment, rehabilitation and palliative care. These HAs undertake an additional one-year diploma in clinical management to become COs. Although doctors and HA/COs work together in all health facilities, HA/COs predominantly work at BHUs.
Study design
We conducted a self-administered online survey among MPs in Bhutan from November to December 2019. This cross-sectional study sought to understand the MP's knowledge and management practices in relation to dengue.
We used Qualtrics (Qualtrics, Provo, UT) to administer the survey questionnaire. Each respondent was sent an invitation message in their email to complete the survey questionnaire, with a URL link embedded within the message. Once the respondents voluntarily agreed to participate in the study, they then navigated to the survey questionnaire. To increase our response rate, as explained by Cook et al. [17], we sent two reminders on days, 14 and 28 after sending out the initial email.
Respondent recruitment
Eligible respondents comprised of any practitioner practising in areas involved in diagnosis and management of dengue patients, including general medicine, paediatrics, emergency medicine, intensive care and laboratory medicine, at any level of the health system. Of the 903 MPs registered by the BMHC, email addresses were obtained for 777 of them. Five were excluded as they didn't work clinically in areas relevant to dengue (including health service managers, surgeons and anaesthesiologists), thus creating a potential respondent group of 772 MPs (Fig 2). Survey questionnaires were emailed to all respondents from December 2019 to January 2020. Email addresses were collected from the district health services. The district health managers contacted target respondents via telephone and email to encourage participation in the survey. Reminder telephone calls were also made by managers to enhance participation of the respondents. No incentives or compensations were offered to the respondents.
Survey questionnaire
Development of the study tool was informed by a review of the literature related to clinical practices for dengue care and past surveys on dengue management [11,12,14,18,19], and the tool was evaluated by three local experts on dengue. The tool was further modified after feedback from a pilot study which was conducted in Gomtu Hospital, Samtse district. Respondents from the pilot study were excluded in the final survey and analysis. Demographic questions included district, region, age, sex, highest qualification, number of years in service, whether they had diagnosed dengue and their specialty. The main questionnaire consisted of 25 multiple choice questions, which included the following domains: 1) Transmission; 2) Clinical course and presentation; 3) Diagnosis and management; 4) Surveillance and prevention of dengue (S1 Appendix). Since English is the official medium of pedagogy in both schools and medical institutes in the country, the questionnaire was designed in English and not translated into Dzongkha (the national language).
Data analysis
The response rate was calculated by dividing the total number of surveys returned by the total number of invitations (772). Characteristics of the respondents were described using categorical variables and presented as frequencies and proportions. For simplicity, national, regional and district general hospitals were grouped as "hospital", while BHU grade I and II were grouped as "BHU". Correct responses to each question were summarized under separate domains, using frequencies and proportions. A choropleth map was developed in ArcGIS version 10.5 (ESRI, Redlands, CA) for visualizing the proportion of respondents (%) who participated in the survey [20].
To compare knowledge across respondents with different characteristics, a cumulative knowledge score was calculated as an aggregate of all the knowledge-based questions separately for each domain. A score of one was given for the right answer. For questions with multiple-choice questions, respondents received a score of one for every correct answer. Normalization was performed to standardize domain scores into values that ranged from 0 to 1 by dividing the respondent's total score for each domain by the total domain score. Boxplots were created for each domain using the normalized scores. Since the distributions were non-normal as determined by a histogram, knowledge scores of all domains were compared across categories of respondent characteristics using non-parametric tests; i.e. the Man-Whitney U test and the Kruskal-Wallis test. All data analyses were carried out using Stata statistical software (version 15.1; Stata Corp, College Station, Texas, USA) [21].
Ethical clearance
The study was reviewed and approved by the Research Ethics Board of Health (REBH), Ministry of Health, Bhutan (2019/081) and the Human Research Ethics Committee (HREC), Australian National University (ANU), Australia (2019/808).
Response rate
Of the 772 respondents to whom the invitation was sent, we received notification that seven emails were not delivered. In addition, 52 respondents responded but did not complete the survey questionnaire, and were thus excluded from the response rate and subsequent analysis. Among those respondents who did not complete the survey, 14 were from the BHUs, two were from the national referral hospital and seven were from district hospitals. However, 27 of the respondents did not complete the health centre details. In terms of roles, 17 were HAs/ COs, five were medical doctors and the rest did not provide any information. We got a response from a total of 97 respondents who had completed the questionnaire, yielding a total response rate of 12.7% (97/765) (Fig 2).
Characteristics of the respondents
Chukha district had the highest frequency of respondents (n = 13) (Fig 3). The age group with the largest number of respondents was <30 years (44.3% of the total) and most respondents were male (72.2%). There was a greater number of respondents who were HAs/COs (61.9%) than medical doctors (38.1%). Respondents from all health centres participated in the survey, including one from a private diagnostic centre. General medicine/practice was the most commonly reported specialty (90.7%) followed by emergency services (3.1%). More than half of the respondents had personally diagnosed dengue in a patient (55.7%) ( Table 1).
Knowledge on the transmission of dengue (domain 1)
The majority of respondents (89.7%) correctly identified Aedes mosquitoes as the vector of dengue. Whilst more than half of the respondents reported mornings and evenings as times favourable for feeding behaviour of vector mosquitoes, only 24.7% correctly identified afternoons as a favourable feeding time. Only 16.7% reported that when a mosquito bites a dengue patient, it might become infected and then infect the next person it encounters during feeding time. About one-third of the respondents were aware of different dengue serotypes circulating in the country ( Table 2).
Clinical course/presentation of dengue (domain 2)
Most of the study respondents (88.7%) were knowledgeable about the duration of the incubation period of infection with dengue virus. While more than half of the respondents correctly indicated options for identifying a patient with dengue at the time of presentation, only 45.4% would ask patients about vomiting, abdominal pain, lethargy and bleeding. Persistent vomiting, severe abdominal pain and bleeding were correctly identified as warning signs for severe dengue by 54.6%, 55.7% and 83.5% respectively. Lethargy was identified as a sign of severe dengue by only 34.0% of respondents. A notable lack of knowledge was observed on the classification of dengue based on the given clinical scenario; only 27.8% were able to correctly identify the right option of "dengue with warning signs" (Table 3).
Diagnosis and management of dengue (domain 3)
Respondents less frequently identified NS1 antigen for early diagnosis (47.4%) than anti-dengue IgM antibodies (62.9%). Chest X-ray with a lateral decubitus view for pleural effusion and haematocrit for diagnosing plasma leakage were reported as parts of the diagnostic approach for dengue by 27.8% and 56.7% respectively. Only 21.7% of the respondents correctly reported that patients with pre-existing co-morbidities need to be closely monitored before discharging them. Of the indications identified for administering IV crystalloids such as Normal Saline or Ringer's Lactate to dengue patients, hypotension and tachycardia, or low urine output, were identified by 78.4% and 50.5% respectively. Surprisingly, only 27.8% correctly identified high haematocrit as an indication for initial fluid replacement. For a given scenario of a six-year-old boy with fever of a few days duration, distended painful abdomen and lethargy, 66.0% of the respondents reported that they would order a dengue test and admit the patient for 24 hours of observation (Table 4).
Surveillance and prevention of dengue (domain 4)
The majority of the respondents were knowledgeable about dengue being a nationally notifiable disease (80.4%). However, only 47.4% knew that suspected or confirmed cases have to be reported every week. More than 80.0% of respondents were able to identify both the variables (i.e., age and case type) included in surveillance reporting. About 95.0% of respondents were able to identify prevention messages such as using mosquito repellent and wearing long sleeve pants and shirts to prevent dengue infection ( Table 5).
Comparisons of domains by respondent's characteristics
The cumulative median score was 32 (IQR: 22-39) out of a possible score of 54 for all the four domains. Boxplots demonstrated higher median scores for domain 4 followed by domain 1. Domain 2 and 3 had the same median score with slightly lower upper quartile scores for domain 2 (Fig 4). Being a medical doctor and having diagnosed dengue were significantly associated with a higher median score across all domains (p-value<0.05). Those working in the hospital had a significantly better score for the first three domains (p-value<0.05). Performance of the respondents did not vary by age group, sex or medical experience for any of the domains (S1 Table).
Discussion
MPs play a crucial role in early detection and timely management of dengue patients, and notification of dengue cases to national authorities. Having a high level of accurate knowledge on dengue management amongst these professionals can save the lives of patients and prevent other healthy people from becoming infected. The current study revealed major gaps in the knowledge of MPs on transmission, clinical presentation, diagnosis and treatment, and prevention of dengue in Bhutan. Knowledge varied according to the type of profession, healthcare setting and past experience in managing dengue cases. To our knowledge, this is the first study of health practitioner's knowledge and practices with regards to dengue in the WHO-South East Asia Region (WHO-SEAR).
In this study, poor knowledge on transmission of dengue was evident from the respondent's inability to recognize the feeding time of dengue mosquitoes. Similar findings have been documented by Huang et al. in Taiwan, where, only 14.4% of respondents correctly identified the feeding behaviour of dengue mosquitoes [22]. Having accurate knowledge on the behaviour of dengue mosquitoes will help MPs to impart appropriate health education [22], which remains one of the cornerstones of preventing dengue. Respondents had low knowledge of the signs that lead to dengue shock and thrombocytopenia. Such findings were also reported in Puerto Rico, where only 29.0% of the respondents correctly identified early signs of shock, and 48.0% identified severe abdominal pain and persistent vomiting as warning signs of severe dengue [23]. This could be due to a lack of training on the recognition of warning signs and case classification of dengue as per the updated WHO guidelines. Identification of warning signs of dengue and indications that lead to shock is critical for managing dengue [1]. Yusuf and Ibrahim reported that 56.0% of respondents lacked adequate training to manage dengue patients, including identifying warning signs, and recommended to close this gap [24].
In the diagnosis and management domain, respondents failed to recognize the importance of NS1 antigen as an early marker of dengue infection. This might indicate a lack of training, experience of managing dengue patients or a lack of diagnostic test kits for detecting NS1 antigen in health facilities as has been reported in other countries [24]. IgM would be negative during the febrile phase (it may be positive at or after defervescence), while NS1 antigen becomes detectable as early as day one of onset of the disease [1]. Ruberto et al. [25] reported that less than half of the healthcare providers were choosing the right diagnostic tools in the U.S. Respondents also performed poorly on knowledge regarding the detection of plasma leakage using Chest X-rays. This could be in part due to unavailability of X-ray machines in many facilities like BHUs, where they depend on simple blood test parameters, noting that 47.7% of the respondents from hospitals reported using Chest X-ray, while only 11.5% from BHUs reported the use of Chest X-ray for detecting plasma leakage. The feasibility of cheaper, portable and a point-of-care ultrasound machines may be considered to detect plasma leakage and other lifesaving diagnoses in remote areas without X-ray facilities [26].
Respondents also overlooked the importance of recognizing co-morbidities that require admission for patients suspected of having dengue. A similar pattern was also observed in Ecuador, where only 22.0% of healthcare providers were reported to be monitoring dengue patients having co-morbidities for hospital admission [12]. Patients with significant co-morbidities should be closely monitored before they can be sent back home regardless of the severity of the disease [14]. Respondents also had a poor understanding of elevated haematocrit as an indication for fluid replacement during the course of dengue illness. A similar finding was reported in Puerto Rico, where 30.0% of respondents correctly identified elevated haematocrit after an initial trial of intravenous crystalloids [23]. Only 47.4% of participants knew that aggregate cases, whether suspected or confirmed (based on the WHO case definition) have to be reported weekly. This suggests the need to evaluate and reinforce training of physicians on surveillance and reporting. Training on national disease surveillance and reporting is conducted every year by the Ministry of Health to sensitize healthcare workers, including physicians. There is a need to review the existing strategy of training healthcare workers on the surveillance system in the country to improve reporting.
The high proportion of respondents who had poor knowledge of transmission and clinical course might be due to the lack of experience in dengue case management by the respondents. This is evident with 44% of the respondents having never seen dengue in their professional career. In Bhutan, dengue transmission only occurred in Chukha district (Fig 3) from 2004 to 2012 [27]. Eventually, it spread to eight new districts by the end of 2018, and a further 10 districts in 2019 [28]. Prior to 2019 dengue epidemic, training on dengue management was mostly conducted among clinicians and healthcare workers in the dengue-endemic districts. Since 2019, dengue is reported from 19 districts in Bhutan. Therefore, it is an opportune time to train and provide refresher courses to all clinicians and health works of Bhutan.
Overall, knowledge on transmission, clinical course, diagnosis and management, and surveillance and prevention of dengue was higher for medical doctors, physicians working in hospitals and healthcare workers who had experience in managing dengue cases in their clinical practice. In hospitals, most of the physicians are medical doctors, which might have led to increased performance as compared to those working in BHUs, which are mostly staffed by HAs/COs. Variation in performance between the type of profession may be related to their educational qualifications and responsibilities. A study in Ethiopia has shown that physicians were 39 times more likely to have a high level of knowledge on dengue prevention than nurses [24]. The same study also demonstrated that healthcare workers who worked in referral hospital settings were 75 times more likely to have a high level of knowledge on dengue prevention when compared to those who worked in health centres [24]. Additionally, physicians with past dengue clinical experience had better knowledge than dengue-inexperienced physicians [14], and findings of significant differences in knowledge of dengue among healthcare workers in different clinical settings have been reported in other regions [6,15]. This emphasizes the need to prioritize specific health centres and novice health professionals in dengue case management training and continuing medical education to reduce morbidity and mortality due to dengue.
This study has some limitations worth mentioning. The response rate was low despite our endeavours to increase it by extending the duration of the survey for collecting the data by more than two months. Firstly, respondents located in remote or hard-to-reach areas with limited internet connectivity may not have accessed the survey despite a follow-up reminder to participate in the survey. Secondly, due to limited health workforce, respondents might have other competing priorities like patient care and health promotion activities at the time of the survey and had to forego their participation. Thirdly, as the number of respondents were higher in the southern region, we speculate that respondents working in the dengue-endemic districts including Chukha, Samtse, Sarpang, Zhemgang, Pemagatshel and Samdrup Jongkhar were more likely to take part in a survey as compared to those working in non-endemic areas due to the familiarity with the disease. High response rates in Thimphu can be attributed to the national referral hospital, where severe dengue patients are referred from other districts. However, this survey does include respondents from all 20 districts, thus covering the whole country.
Conclusion
The study reveals a critical gap and an urgent need to strengthen the knowledge of dengue management and clinical practices among physicians in Bhutan. Emphasis has to be given to educating physicians on transmission, clinical manifestations, early diagnosis and appropriate management of dengue patients. In addition, strategies should be put in place to enhance the physician's awareness of surveillance and reporting requirements. Exploratory studies such as focus group discussions with healthcare workers in both hospitals and BHUs or PHCs is suggested to understand critical gaps in dengue management. | 2021-07-18T06:16:09.197Z | 2021-07-16T00:00:00.000 | {
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235909480 | pes2o/s2orc | v3-fos-license | BLOG NO ENSINO DA RESSUSCITAÇÃO CARDIOPULMONAR: UMA FERRAMENTA PARA A FORMAÇÃO DO ENFERMEIRO BLOG IN THE TEACHING OF CARDIOPULMONARY RESUSCITATION: A TOOL FOR THE NURSE’S EDUCATION BLOG EN LA ENSEÑANZA DE LA REANIMACIÓN CARDIOPULMONAR: UNA HERRAMIENTA PARA LA FORMACIÓN DE LA ENFERMERA
Objective: To develop and validate a blog for the teaching and learning of cardiopulmonary resuscitation of the adult focused on the nurse’s education. Method: Applied research, of technological production, which addressed the development of a blog about the cardiopulmonary resuscitation of adults in the intra and extra-hospital environment for nursing. The elaboration followed the stages of analysis, design, development, implementation and evaluation of the blog. The validation of this virtual learning environment involved 11 nurses from the area of urgency and emergency and three experts of informatics, totaling 14 participants. Results: A virtual educational tool was created for teaching and learning, called “Blog of Cardiopulmonary Resuscitation”. The evaluation of three experts in informatics approached the fields: response time, quality, interface and tools, and resources, covering 33 criteria, considered excellent by the majority. The experts in nursing evaluated 32 criteria among educational aspects, the interface of the virtual environment and the teaching resources, pointed out, predominantly, as excellent. Conclusion: In this study, a blog for the teaching of cardiopulmonary resuscitation of adults was elaborated and validated, representing a framework of updated scientific, reliable, interactive and technological evidence for nursing, which can be replicated in other learning environments. Descriptors: Cardiopulmonary Resuscitation; Education, Nursing; Strategies; Learning; Blog.
INTRODUCTION
The current days are permeated by a social technological revolution from the mass use of new resources to propagate information, especially through the internet in the daily life of individuals, the proliferation of social networks and educational blogs (1)(2) .
The blog is considered a virtual environment structured into small blocks, called "posts", which cover texts, images and media objects, as well as the presence of links for user navigation. 3 This digital tool can contemplate the process of teaching and learning, and represent an attractive and easy-access cross-sectional pedagogical axis for students and professionals (1)(2) .
Also emerging in health education, the blog is represented by a disruptive technological innovation model, i.e., an educational product simple, convenient and accessible for the user, which promises to accompany and overcome the books produced in mass and the traditional lectures for health education (4) .
Specifically in nursing, among the challenges for the rupture of the traditional paradigm of education still in force, there is the adaptation to the demands of a new generation of students, with expectations of differentiated learning, characterized as "Net generation", and the difficulty of appropriation, by the faculty, of technology involved in the teaching and learning process (5) .
In this perspective, adopting the use of blogs as a complementary tool for teaching in nursing can cause significant changes in how, where and when students have access to educational materials relevant to the development of clinical skills, mainly for elucidation of complex issues, such as the cardiopulmonary resuscitation (CPR) (6) .
In the process of teaching and learning of the CPR for nursing, the adoption of courses and training that do not address innovative strategies and technology is frequent, impacting negatively on the development of knowledge, skills and attitudes and retention of learning for nursing students and professionals (7) .
Despite the use of blogs for the teaching of the CPR be recommended by the literature and by the American Heart Association (AHA), main entity focused on the dissemination of knowledge about cardiovascular diseases and CPR, scientific studies, well prepared methodologically, intending to develop blogs are still incipient (8)(9)(10) .
Considering the potential of the blog as educational resource and innovative virtual tool to facilitate the learning of the CPR in nursing, this study aimed to develop and validate a blog for the teaching and learning of the CPR in adults focused on the nurse's education.
METHOD
This is an applied research, of technological production, which addressed the development of a blog about CPR in adults, in the intra and extrahospital environment, for the teaching and learning process of nursing undergraduate students.
The research was developed in the Nursing School of Ribeirão Preto of the University of São Paulo (USP), and its population was 11 nurse practitioners, experts in the area of urgency and emergency and/or nursing professors, who teach contents of this nature, for the evaluation of the blog virtual learning environment (VLE), and three experts in the area of informatics, totaling 14 participants.
The experts were identified by the Lattes Curriculum Platform, based on the criteria of Fehring (11) for selection, which consider: 4 points for Master's Degree in Nursing; 1 point for Master's Degree in Nursing, with a dissertation on the area of interest in the study; 2 points for a doctoral thesis in the area of study; 1 point for clinical practice for at least 1 year in the area of interest; 2 points for a certificate of clinical practice (specialization), in the area of interest; 2 points for publication of research relevant to the area of interest; 2 points for publication of article about the topic in a journal of reference. The inclusion criteria were: being a nurse, specialist in the area of urgency and emergency or professor of this theme, and/or professional specialist in the area of informatics with an emphasis on creation of VLE, and a minimum sum of 5 points, as the criteria of Fehring (11) .
Exclusion criteria were professionals who were on vacation or leave in the period defined by the researcher for the evaluation of instruments and those who did not attend the assessment within the specified period of 15 days after receiving the instruments and accepting the evaluation.
The VLE was produced by a team composed of a system programmer and web designer, and by the researcher. In this way, the following phases were accomplished: analysis, design, development, implementation and evaluation (12) . Batista In the first stage, of analysis, there was a review of the literature, in the period from March to June 2018, on databases PubMed®/MEDLINE®, Latin American and Caribbean Health Sciences Literature (LILACS), Scopus, Cumulative Index to Nursing and Allied Health Literature (CINAHL) and Web of Science, and in the documents relating to the international guidelines for CPR and emergency cardiovascular care (ECC) The American Heart Association (AHA) (8) to prepare de guide relevant to the content of the blog proposed, based on scientific evidence.
Then, the content was divided into sequential and complementary modules, settling the following structure: content of the module; references; links/material for directed study; forum/chat and e-mail. Subsequently, there was the creation of the educational goals, from Bloom's taxonomy, which considers the cognitive, affective and psychomotor domains for learning. (13) The following learning goals related to the cognitive domain were adopted: permanent update of CPR guidelines; understand the definition and the mechanisms of cardiorespiratory arrest (CRA); know the anatomy and the respiratory and cardiocirculatory physiology; know the risk factors for CPR; understand the rhythms of CPR; understand the intra and extra-hospital chain of survival; identify and understand the materials and equipment used in the CPR (emergency cart, devices for airway and defibrillator); and understand the science of realistic simulation.
The learning objectives that comprised the psychomotor domain were: educate to acquire skills and knowledge acquisition on CPR in adults; and cause changes in the behavior of the leaner, for the high quality care of CPR in adults. The affective domain addressed the following intentions: realize the importance of the study of the theme for the professional life; influence the favorable attitude to seek and acquire knowledge; share respect in interpersonal relationship, always responding to other participants when questioned and receiving contributions from other participants analytically; provide bi-or multidirectional communication with the interaction student-content, users-content and experts on the subject.
In the second phase, of design, there was the reformulation and expansion of all didactic material determined for the VLE, selecting the following modules: what is CPR; pathophysiology of CPR; risk factors for CPR; rhythms of CPR; chain of survival (intra and extra-hospital); defibrillator; materials and devices for the care of CPR (medicines, emergency cart and airway devices); registration of CPR; simulated environment care (intra and extra-hospital environment) and content folders.
In this stage, there was also the creation of a storyboard to define the primary elements of screen, which comprised the Blog of CPR, aiming to guide the development process by a professional web designer, considering the following elements to the layout: source ("Libre Franklin", "Helvetica Neue", "Helvetica", "Arial" and "Sans-Serif"); font size 14; color scheme of white, gray and red; device-adaptive screen size; average size of video files (videos allocated -on demand -on YouTube platform); size of image files (image files with resolution of 72dpi, in .JPG and .PNG -compressed -with a maximum size of 200 KB); elements of layout used (CMS Word Pressver are free, using free template Twenty Seventeen, version 2.2); location of the horizontal menu at the top of the page; vertical scroll bar.
In the third phase of development, the content for the teaching and learning process of CPR in adults was structured, associating images, videos of CPR intra and extra-hospital call, conducted by researchers from the postgraduate program, at the Nursing School of Ribeirão Preto, USP and photographs belonging to the researcher's personal file, in addition to the provision of useful links, in the intention of making the blog dynamic, interactive and based on reliable scientific evidence.
In the fourth phase, implementation, there was the development of the web-based VLE and the blog in Word Press version, configured as a platform used as a basis for the construction of blogs, with versions of hosting and free and paid upgrades. Finally, the last stage of preparation of the blog, the evaluation, was performed by specialists in computer technology and experts in nursing, using two validated instruments (14) .
The instrument intended for experts in informatics 14 comprises three areas analyzed in a Likert scale with a score from 1 to 3, meaning unsatisfactory (1 point), satisfactory (2 points) and excellent (3 points). In the topics assessed as unsatisfactory, they were guided to comment or justify in the field "Considerations", aiming to implement improvements and adaptations in the CPR VLE blog.
The first area of this instrument referred to the response time, used to assess issues of cyberworthiness, accessibility, system feedback to the user. The second area covered the quality of interface to evaluate the visual aspects or design of the VLE, such as colors, menu, buttons and other elements. The third area, tools and resources, evaluated the type, the presentation and the functioning of the system tools, such as forum, e-mail and links. The second instrument (14) was intended to nursing experts and divided into three domains. The first, called "educational aspects", aimed to evaluate the proposed theme; the coherence of educational goals, texts and hypertexts; the user's autonomy; and the teaching resources employed. The second domain, "interface of the environment", focused on evaluating items such as cyberworthiness, accessibility and design for the environment. The third domain, "teaching resources", evaluates the interactivity of the system, the functionality of didactic resources and screen resources. For online submission of forms of evaluation, the virtual application Google Docs was used.
The data obtained were organized by evaluator and issues in absolute numbers and percentages, exported as a spreadsheet to Microsoft Excel® 2013, with dual typing, by two different researchers, analyzing them through descriptive statistics on the program Statistical Package for Social Sciences (SPSS), version 23.
For compliance with ethical precepts, this study was submitted to and approved by the Research Ethics Committee at the Nursing School of Ribeirão Preto, USP, protocol CAAE: 99791118.4.0000.5393, following the Resolution 466/2012 of the National Health Council (NHC), as accepted by the participants' online Informed Consent Form.
RESULTS AND DISCUSSION
The VLE was as a blog, called CPR Blog (http://www.blogdarcp.com.br). After typing the VLE address, the user had access to the home page of the blog, as shown in Figure 1. The menu of the blog was exposed, horizontally, in the upper part of the screen, comprising the following buttons: Presentation, Highlights, Modules, Useful links, Tips for study, Courses and events, and Contact.
The proposal of the first screen, the presentation of the blog, was to allow for a visual call that corresponded to the identity of the subject. The button "Highlights" assured access to posts published by the coordinators of the blog, with themes and updates on the science of CPR.
In the contents present in "Modules", the user developed the knowledge on the call of CPR in adults, providing, in each unit, information based on the guidelines of the American Heart Association (8) and educational videos on the call of a CPR. At the end of the contents of each module, a list of updated references on the subject studied and external links were made available, to support Batista DFG, Nascimento JSG, Oliveira JLG, et al.
Revista da Enfermagem do Centro Oeste Mineiro 2020;10/3643 www.ufsj.edu.br/recom -5 and validate the information. The buttons "useful links" and "tips for study" gathered all the external links selected for study of CPR.
The button "courses and events" covered courses and events that happen along the year. National and international congresses of relevance in the area would be listed for that users could be in constant update. The button "Contact" set up a dynamic way for users to contact the authors of the blog, containing the name, e-mail address, subject and message content that would be sent to the e-mail blogrcpusp@gmail.com.
Regarding the evaluation and validation of the CPR VLE Blog for layout and content, firstly, the profile of selected experts was identified, according to their training area, length of professional experience in the field, greatest degree and main current activity.
The experts in informatics presented distinct training and current activity, namely: Bachelor's Degree in computing, system analysis and information technology, exercising, currently, consulting functions, systems analysis and development, and web development. The shortest time of experience was 8 years and the highest was 20 years, and the largest degree was specialist.
Concerning the profile of nursing experts, the shortest length of professional experience was 7 years and the longest, 36 years. The smallest degree was Masters and the highest, Post-Doctorate, with the majority PhD in the area of urgency and emergency, and the current main activity exercised was nursing teaching. Table 1 exposes the evaluation of the blog proposed by the experts in informatics, in relation to the domains "response time", "interface quality" and "tools and resources", pointing to the number and percentage of professionals who considered unsatisfactory, satisfactory or excellent each criterion approached. Source: data obtained from the study, 2020.
The experts in informatics assessed 33 criteria of the CPR VLE Blog. The domain of response time encompassed five criteria, considered excellent by most evaluators. No criterion was regarded unsatisfactory. The quality of the interface covered 25 criteria, and most evaluators considered excellent. In the tools and resources domain, composed of three criteria, most evaluators considered it excellent; however, the item "forum" was considered unsatisfactory and could be improved. The experts in informatics suggested, especially, the improvement of the cyberworthiness for internal links, the improvement of accessibility displaying the next screen and previous screen, and the incorporation of forum postings. The evaluation of the CPR Blog, by nursing experts, involved educational aspects, interface of the environment and didactic resources, shown in table2. The educational aspects domain presented unsatisfactory notes on the criteria "depth of approach" and "relevant updated references", making the appropriate adjustments, both in the field "teaching resources" and, more specifically, in the criterion "forums and chats", also signaled as unsatisfactory and revised for the excellence of the CPR blog.
The nurses performed considerations about the depth of the approach of the subject, relevance of vocabulary, updated references, figures and the use of the forum, which also generated new evaluation and suitability by the researcher.
The nurse's primary role is health education, which should be guided in attractive and innovative pedagogical approaches (15) In this perspective, the adoption of educational blogs is an option of easy employability and access, pleasant interface, didactic, which allows posting from a simple text until complex constructions (16) .
In the nursing area, technological strategies of teaching and learning are in a great development, although the production on the existing educational technologies, and the construction and validation of virtual learning objects became significant only from the year 2008 (16)(17) .
The quality presented in innovative educational tools for the teaching of the CPR enables self-directed learning, without time, place or personal restrictions, which are the main challenges of the current traditional nursing courses, which makes the blog a promising virtual learning object in the development of digital education (18) .
The experts in informatics responsible for evaluating the blog in question presented training and professional degree in line with the proposal and a sufficient experience length, demonstrating a broad knowledge and grasp of the subject.
The experience of experts in informatics to evaluate the layout and the content of web platforms and blogs of scientific dissemination, as virtual learning space, allow maintaining the quality of their drafting process, allowing for the construction of a trustworthy and reliable tool (18) .
With the same intent, the nursing experts selected also demonstrated length of professional experience consistent with the proposal, and the fact that most of them are nursing professors contributed to the ease and accuracy in the evaluation process of this educational tool.
The use of computational tools in the teaching and learning process occurs in an expressive manner in nursing undergraduate courses, opening the doors for use of technologies and indicating statistically significant results, supporting the adoption of this educational resource (19) . Quantitative study of exploratory and descriptive nature carried out with 14 students from the eighth semester of a nursing course, in a regional university, located in northwestern Rio Grande do Sul, aimed to identify the influence of a blog on the teaching of mechanical ventilation. The blog contributed positively to the teaching and learning construction and process for nursing, increasing students' knowledge and motivating them (20) .
Concerning the effectiveness and the motivation of the use of blogs to the nursing teaching of study, an experience report, performed in a public university in the state of Ceará, involving professors and students of Masters in Health, on the elaboration and implementation of an educational blog, in this context, evidenced the participants' satisfaction in the use of this tool, recommending it as current educational practice, contextualized and interdisciplinary (21) .
CRP is an event that demands from nursing professionals the scientific knowledge and technical ability to act in such a situation, being recommended constant updates and the scientific deepening on the subject, which can be facilitated by technological and innovative educational tools, such as the blog (8,22) .
A research conducted on the hybrid teaching of CPR with support of educational blog at University of Rio Grande do Norte, open to the population, identified the use of this pedagogic resource, used in the online modality, as important, comprehensive and impactful, before the reports identified by participants, considering the mechanism practical and attractive for the dissemination of knowledge (23) .
Thus, the highlight is the viability of blogs to the nursing teaching and learning process and in the teaching of the CPR towards the technological digital society, which demands subjects and knowledge that cooperate, mutually, to solve complex problems, in this world of almost immediate access to information (23)(24)(25) .
CONCLUSION
A virtual educational tool was obtained for teaching and learning called CPR Blog, about the Batista DFG, Nascimento JSG, Oliveira JLG, et al.
Revista da Enfermagem do Centro Oeste Mineiro 2020;10/3643 www.ufsj.edu.br/recom -8 science of cardiopulmonary resuscitation in adults, developing the stages of analysis, design, development, implementation and evaluation, in addition to the validation of layout and content of this contemporary learning object.
The main limitations were the lack of evaluation of the blog by Nursing students and the scarcity of studies aiming to develop and validate educational blogs for nursing, especially regarding the CPR.
More experimental researches should be developed, well delineated, intending to test the effectiveness of blogs on the teaching and learning process of cardiopulmonary resuscitation for nursing.
This research contributes to science, assistance and education in nursing, by presenting a tool for the teaching of CPR, constituting as a new and important aspect in this context, demonstrating the methodology of elaboration and validation, which can be replicated in other learning environments. | 2020-05-07T09:14:22.389Z | 2020-10-14T00:00:00.000 | {
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53572088 | pes2o/s2orc | v3-fos-license | A review of antibiotic prophylaxis for traveler’s diarrhea: past to present
As there is rapid increase in international travel to tropical and subtropical countries, there will likely be more people exposed to diarrheal pathogens in these moderate to high risk areas and subsequent increased concern for traveler’s diarrhea. The disease may appear as a mild clinical syndrome, yet a more debilitating presentation can lead to itinerary changes and hospitalization. As bacterial etiologies are the most common causative agents of TD, the use of antibiotic prophylaxis to prevent TD has been reported among travelers for several years. The most common type of antibiotic used for TD has changed over 50 years, depending on many influencing factors. The use of antibiotic prophylaxis for TD prevention in travelers is still controversial, mainly because of difficulties balancing the risks and benefits. Many factors, such as emerging drug resistance, side effects, cost and risk behavior need to be considered. This article aims to review antibiotic prophylaxis from the 1950s to 2000s, to describe the trend and reasons for different antibiotic use in each decade. We conclude that prophylactic antibiotics should be restricted to some high-risk travelers or short-term critical trips.
Introduction
International travel is rapidly increasing, with 1.2 billion travelers in 2016, and Asian and Pacific regions being particularly popular [1]. Traveler's diarrhea (TD) is one of the most common travel-related illnesses among short-term travelers to low-and middle-income countries [2,3]. Incidence of TD ranges from 2 to 57%, for different traveler characteristics and destinations [4][5][6][7][8][9][10]. High risk destinations for contracting TD include most countries in Asia, the Middle East, Africa, Mexico, and Central and South America [11,12]. Most TD usually results in mild symptoms and is self-limiting [13,14], however clinical symptoms can be severe and cause several issues, including disruption to travel, or long-term effects and hospitalization [15,16].
Etiology of traveler's diarrhea
The causative pathogens of TD vary in each region, but bacteria are the most common, followed by viruses and protozoa. In Latin America and the Caribbean, the most common pathogens causing TD are enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) [17,18]. In addition, ETEC has been documented as the most common pathogen in travelers returning from African countries [19]. As well as ETEC, Campylobacter, Giardia and Shigella have frequently been reported to cause TD in travelers in the Indian subcontinent [20,21]. Interestingly, Campylobacter is the most common pathogen isolated from travelers returning from Southeast Asia [17,22]. Increasing reports of norovirus in travelers returning from multiple regions of the world are of concern, as it is an important cause of TD [23].
Travelers to remote areas far from medical facilities are often advised to take antibiotics when symptoms of TD develop [24]. In contrast, some high-risk groups, such as immunocompromised travelers, might prefer to take antibiotics prophylactically to prevent TD. There have been many reports that bacterial etiology is the most common cause of TD, therefore antibiotic use might be the most effective method of prevention [17][18][19][20][21][22]. It has been reported previously that approximately 15% of travelers take antibiotics to prevent TD [25]. However, the use of antibiotic prophylaxis for TD prevention in travelers is still controversial, mainly because of the challenge of managing risks and benefits [26].
Antibiotic prophylaxis
Traditionally, standard pre-travel consultations include advice to "boil it, cook it, peel it, or forget it" to prevent TD [11]; nevertheless, studies have reported that even travelers who follow these rules may develop TD [27,28]. Therefore, the use of antibiotic prophylaxis for travelers could be considered, to decrease the pathogen burden and prevent long-term morbidity [29,30]. The prophylactic antibiotic of choice has been changing over the last few decades, as resistance patterns developed [31]. According to previously published randomized controlled trials (RCTs), antibiotic efficacy varies from 28% up to 72% [32][33][34][35][36], for different types of antibiotics and traveler's destinations.
Method
We searched the PubMed database for publications on the protective efficacy of antibiotics as chemoprophylaxis for TD. Search terms included antibiotics, travel, diarrhea, neomycin, furazolidone, doxycycline, trimethoprim/sulfamethoxazole, erythromycin, mecillinam, bicozamycin, ciprofloxacin, norfloxacin, azithromycin and rifaximin. All search articles yielded 616 in numbers. Inclusion criteria were papers written in English and related to the use of antibiotics as prophylaxis for TD. Overall, 27 studies were included in this review, to illustrate the trend of antibiotic use in the prevention of TD over the past 50 years (as shown in Table 1).
Enterovioform, neomycin, phthalylsulfathiazole and furazolidone
The first attempt to use antibiotics to prevent TD was in the late 1950s, using enterovioform and neomycin [37]. Since the use of enterovioform-an iodochlorhydroxyquin-was associated with myelo-optic neuropathy, the drug was withdrawn from the market [38]. In the early 1960s, a double-blind study was conducted in American college students in Mexico City, to compare a placebo with low doses of neomycin sulfate and a sulfonamide called phthalylsulfathiazole [39]. Low protective efficacy was observed in neomycin sulfate, yet phthalylsulfathiazole halved the incidence of TD. There were no adverse drug reactions reported during the 14-day study. Another trial using neomycin-trisulfamide, Streptotriad (a combination of streptomycin and triple sulphonamides), or a placebo was conducted in British Airways personnel and their families, traveling to different countries worldwide, for up to 3 weeks [40]. Both drugs resulted in a low protection rate against TD. Comparable low protective efficacy was reported in the use of a nitrofuran derivative furazolidone for TD prophylaxis in 223 Royal Air Force pilots [41].
Doxycycline
During the late 1970s, doxycycline was used initially. The role of doxycycline for the prevention of TD was supported by research that showed the drug was highly effective against ETEC as major cause of TD, had long-acting properties and minimal adverse events [42]. Daily administration of 100 mg of doxycycline for 3 weeks was up to 86% effective for TD prevention among American Peace Corps Volunteers, deployed to multiple regions of the world [43,44]. Unfortunately, lower protective efficacy was observed over the proceeding 5 years, because of high proportions of tetracycline-resistant ETEC [45][46][47]. Hence, a trial using a higher dose of doxycycline (200 mg per day) was conducted. The results showed a significant reduction in the incidence of TD, but 12% of cases reported gastric upset in the doxycycline group [48]. Today, because of the high risk of antimicrobial resistance and potential side effects from using doxycycline, no guidelines recommended its use.
Trimethoprim/sulfamethoxazole
During the late 1970s to early 1980s, some studies showed that the use of trimethoprim/sulfamethoxazole (TMP/SMX) prophylaxis resulted in a significantly higher reduction of TD incidence in college students traveling to Guadalajara, Mexico when compared with a placebo and TMP alone [49,50]. Administration of 160 mg TMP and 800 mg SMX twice daily for 3 weeks yielded a protective efficacy of 71% [49]. Another study showed that the same daily dosage of TMP/SMX for 14 days resulted in 95% protection against TD [50]. Emerging drug resistance was a concern, as a higher resistance pattern for E. coli had been observed since the early 1980s [50][51][52]. Dermatologic adverse reactions, especially skin rashes, were also reported.
Erythromycin, mecillinam and bicozamycin
Emerging drug resistance resulted in the need to find new regimens for the chemoprophylaxis of TD (Fig. 1). In early 1980s, trials in TD prevention using erythromycin, mecillinam and bicozamycin were conducted. This is because erythromycin had been reported to eliminate Enterobacteriaceae from human fecal flora without evidence of recolonization [53]. A placebo-controlled RCT on American travelers to Mexico, using 1 g erythromycin during travel showed a protective efficacy of 71% against TD [54]. Furthermore, a non-absorbable antibiotic, mecillinam, was also identified as having selective bactericidal activity against Enterobacteriaceae [55]. A trial using 200 mg of mecillinam for 25 days as prophylaxis for TD was conducted in adult travelers visiting Asia, Africa or Latin America, resulting in 75% protection against TD [56]. A similar protection rate was reported among Danish travelers to Mexico [57]. However, it should be noted that etiology of most TD cases remained unknown. Bicozamycin, or bicyclomycin, has been found to have similar activity against Enterobacteriaceae [58]. Administration of 500 mg of bicozamycin four times daily for 21 days, to adult US citizens traveling to Guadalajara, Mexico, showed high protection against TD [59]. As well as the inconvenience of frequent pill taking for travelers, resistance of bicozamycin had also been detected [59]. The drug was not further developed for human use.
Fluoroquinolones: ciprofloxacin and norfloxacin
Discovery of quinolone antibiotics gave researchers hope to prevent traveler's diarrhea, as this group of antibiotics is highly effective against gram-negative pathogens including Enterobacteriaceae [60]. The early clinical trials using ciprofloxacin and norfloxacin were conducted during the late 1980s and early 1990s [51,[61][62][63][64][65]. These two drugs have been shown to be highly effective in the prevention of TD, with less frequent adverse events. Administration of 500 mg of ciprofloxacin daily in two studies demonstrated protective efficacies of 84% and 94% [51,61]. To compare fluoroquinolones with [36] previous antibiotics, ciprofloxacin provided a higher protection rate, compared with TMP/SMX (84% versus 51%, respectively); however, the Asian region was not included in this study. A double-blind randomized trial using 250 mg of ciprofloxacin was conducted in a Himalayan expedition team in 1994, yet the protection rate for TD was not clearly reported [62]. Further studies using norfloxacin with a dose of either 400 mg daily or 200 mg twice daily showed 70-93% protection against TD [63][64][65]. Antibiotic resistance to fluoroquinolones, especially in Campylobacter species has been reported, particularly in Southeast Asia [66][67][68]. Broad use of fluoroquinolones in both humans and in animals along with chromosomal mutations in drug's target enzymes and efflux systems, were sufficient to cause important levels of clinical resistance [69,70]. In addition to its ease in drug resistance development, the risk of tendinitis/tendinopathy, QT prolongation, and delirium could limit the use of fluoroquinolones for TD prevention [2,71].
Azithromycin
The protective effect of azithromycin against dysenteric diarrhea has been accidentally discovered during malaria chemoprophylaxis efficacy trial in Kenya in 1995. A total of 231 volunteers were divided into 4 arms: azithromycin 250 mg daily, azithromycin 1000 mg weekly, doxycycline 100 mg daily, or placebo. Result showed that only 2.6% of volunteers in the daily or weekly azithromycin groups developed dysentery, while 13.9% of volunteer in doxycycline or placebo group developed dysentery in the same period, so the estimated protective efficacy of azithromycin was 81% [72]. Unfortunately, although azithromycin is very well known for its efficacy in treatment of TD; the study of its use as chemoprophylaxis against TD is rarely reported.
Rifaximin
Side effects and development of resistance among extraintestinal bacteria means the use of absorbable (systemic) antibiotics to prevent TD has been discouraged [73]. In the 1990s, an antibiotic called rifaximin was discovered and shown to be effective for the prevention of TD without causing significant adverse effects. Rifaximin is a rifamycin derivative, which is poorly absorbed and able to reach high concentrations in the intestinal lumen [74]. Rifaximin has also been shown to effectively prevent shigellosis [75].
Studies in short-term travelers to Mexico, using various regimens of rifaximin for 14 days, demonstrated protective efficacies ranging from 28 to 72% [32,33,35]. Another study showed that the use of a higher dose of 1100 mg rifaximin for 14 days resulted in 67% protection against TD in military personnel deployed to Turkey [34]. The latest trial using 200 mg of rifaximin, twice daily from the day of departure to 7 days post-return in adult travelers visiting South Asia and Southeast Asia, showed a protective efficacy of 48% [36].
Low to moderate efficacy of rifaximin has been observed in several studies, however the majority of the studies [33][34][35][36] did not report the resistance pattern of the causative pathogen. Therefore, it remains unclear whether the low protection level was because of rifaximin resistance. One study reported unusually high rifaximin resistant E. coli in several countries [76].
Factors to consider and recommendations
Several factors need consideration when prescribing prophylactic antibiotics for the prevention of TD. Individual risk assessment, including type of population (Table 2), travel purpose and itinerary will be helpful for making prescribing decisions [31]. All the RCTs reported here were conducted in travelers from high-income countries. Applying similar regimens to travelers from low-or middle-income countries may not yield similar efficacy. Furthermore, the incidence of TD in Asian travelers only ranges from 1.6 to 8%, therefore in general travelers, reducing the incidence of TD with antibiotic prophylaxis may not be beneficial [7][8][9]. Immunocompromised travelers, those taking important trips, or visiting remote locations with a lack of medical provision, may be targeted for antibiotic prophylaxis [26,31]. As up to 50% of immunocompromised travelers will experience a gastrointestinal illness including diarrhea [77], it is advisable for this group of travelers to take prophylactic antibiotics; otherwise they will be at risk of more severe illness and hospitalization during travel [78]. In addition, drug side effects should also be considered when making prescribing decisions [79]. Host microbiome may be an important factor involved in protecting travelers from TD. Alterations in native gut microbiome, known as dysbiosis, are known to be associated with travel [80]. Ingestion of antibiotics could affect gut microbiome by several mechanisms such as decrease the diversity, expansion of the resistant strain, loss of the keystone species that support normal ecology [81]. All of these factors can increase the susceptibility of TD. Unfortunately, even short term antibiotics exposure could disrupt the gut microbiome for a year or more and repeated exposure could delay its recovery [81]. Apart from the use of antibiotic, several factors including change of sleeping pattern, exposure to local diet and water or ingestion of antibiotics during the trip can disrupt the gut microbiome [80,82].
Other factors may affect the efficacy of prophylactic antibiotics, such as the predominant pathogens present in each geographical region and their pattern of antibiotic resistance [83]. Drug resistance has emerged over several years, from ETEC resistance to tetracyclines and TMP/SMX, followed by fluoroquinolone-resistant Campylobacter strains from Southeast Asia. Hence, fluoroquinolones should not be prescribed as prophylactic antibiotic for TD in travelers traveling to Southeast Asia. Unfortunately, data for antibiotic resistance are unavailable in some regions of the world, especially in low-and middle-income countries [84]. The current data for rifaximin suggest it could be a suitable prophylactic treatment for TD, as there is no evidence of resistance developing.
Although TD is perceived as a self-limiting disease, it represents a substantial socioeconomic burden from traveler's perspective [85]. This illness may affect not only a change in lifestyle induced by the illness itself, but also expenses for medication and medical services. Cost-benefit analysis, by comparing the cost of antibiotic prophylaxis and treatment involving loss of productivity, may also be a useful approach [79,86].
Current recommendations suggest that antibiotic prophylaxis for TD may be prescribed selectively in some travelers, especially in high-risk short-term travelers [26]. Rifaximin is preferred to other antibiotics because of its poor absorption, reducing the risk of development of resistance in extraintestinal bacteria [26]. However, it is important to note that not all TD is caused by bacteria, so even if we had ideal antibiotics, these would not prevent TD caused by viruses or protozoa.
Conclusions
Trends in antibiotic use for TD prevention have been changing over several decades. Prophylactic antibiotic prescribing for TD should always include an individual risk assessment, including type of traveler, their destination, travel purpose, itineraries, drug side effects, and cost-benefit analysis. The global increase in antibiotic resistance limits the choice of antibiotics. In the past, doxycycline, TMP/SMX or fluoroquinolones may have been effective for TD prevention; however currently, minimally-absorbed rifaximin is recommended for travelers. | 2018-11-11T01:10:52.644Z | 2018-11-07T00:00:00.000 | {
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15372800 | pes2o/s2orc | v3-fos-license | Epidemiology of Injuries in Belgium: Contribution of Hospital Data for Surveillance
Objectives. Investigating injuries in terms of occurrences and patient and hospital stay characteristics. Methods. 17370 stays, with at least one E code, were investigated based on data from 13 Belgian hospitals. Pearson's chi-square and Kruskal-Wallis tests were used to assess the variations between distributions of the investigated factors according to the injury's types. Results. Major injuries were accidental falls, transport injuries, and self-inflicted injuries. There were more men in the transport injuries group and the accidental falls group was older. For the transport injuries, there were more arrivals with the support of a mobile intensive care unit and/or a paramedic intervention team and a general practitioner was more implicated for the accidental falls. In three-quarters of cases, it was a primary diagnostic related to injury and poisoning which was made. The median length of stay was nearly equal to one week and for accidental falls, this value is three times higher. The median cost, from the social security point of view, for all injuries was equal to €1377 and there was a higher median cost within the falls group. Conclusion. This study based on hospitals data provides important information both on factors associated with and on hospital costs generated by injuries.
Introduction
In Belgium, as in other countries all over the world, injuries remain a public health problem. In 2009, in the European Union, mortality due to external causes was equal to 30 deaths for 100000 inhabitants, with higher values in Belgium than in its neighboring countries: 36.8/100000 versus 31.0/100000 in France, 29.9/100000 in Luxembourg, 19.8/100000 in Germany, and 16.4/100000 in the Netherlands [1]. Still in 2009, without considering the age groups, suicide and transport injuries were among the top ten causes of death in Belgium, with a proportionality mortality ratio (PMR) of 1.9% (7th place in the ranking) and 1.0% (10th place in the ranking), respectively. In the 15-24-year age group, suicide and transport injuries were the two first leading causes of death, with a PMR of 36.7% and 22.9%, respectively. The situation was nearly the same in the 25-44-year age group (suicide PMR equal to 22.9%, 1st place in the ranking, and 11.6% for the transport PMR, 3rd place in the ranking) [2]. Next to this high mortality, there is significant morbidity and also an important burden of these injuries. In the European region, according to the Global Burden of Disease report road traffic injuries are in the 6th place in the ranking of the leading causes of burden diseases with 3.7 million of disability-adjusted life year (DALY's), corresponding to 2.4% of total DALY's; selfinflicted injuries are in the 10th place with 3.1 million DALY's, corresponding to 2.0% percent of total DALY's [3]. Generally, mortality is well documented, especially for certain type of injuries (e.g., traffic injuries), but there is lack of information about the morbidity. However, several types of data sources are available for injury morbidity surveillance, for example, population based survey data and emergency department data or hospital data [4]. Within the European region, injuries represent an estimated number of 7200000 hospital admissions, 34800000 emergency department attendances, and 18600000 other medical treatments [5]. According to the summary of injury statistics for the years 2008-2010 published by the European Association for Injury Prevention and Safety Promotion (EuroSafe) [6], in Belgium, the estimated percentage of injury related to hospital discharge is equal to 10%. Although hospitalized injuries represent a small proportion of nonfatal injuries, they are generally more severe and are associated with higher medical and treatment cost than those who are not treated in hospital, and because patients admitted to hospital usually have longer stays than those treated in the emergency department, hospital inpatient records usually contain more detailed and accurate information about the diagnosis of injury than emergency department visit records [4,7,8].
In Belgium, the Minimal Clinical Dataset is a standardized and concise summary of the patient's medical record that general hospitals are required to register since 1990. The registration has the objectives to identify needs for hospital equipment, to define the standards of qualitative and quantitative recognition of hospitals and their services, to organize the funding of hospitals, to determine policy on the exercise of the art of healing, and to define epidemiological policy [9]. In the early 2000s, a published ministerial circular has strongly encouraged the registration of the codes E [10]. To our knowledge, no epidemiological studies have investigated specifically the distribution of E codes in the database, nor patient characteristics, and even less the cost associated with the hospitalization. So the objectives of our study were to investigate these E codes, based on the more available recent data, in terms of occurrences, patient characteristics, and hospital stay characteristics.
Cases' Selection.
These analyses were performed with the 2010 data from 13 Belgian hospitals, which are included in the PACHA project, a project focused on the analysis of stays and pathologies cost. These hospitals are either private or public. In 2010, the total of inpatient stays from this sample represented 11.4% of all Belgian inpatient stays. On the 473426 available stays (inpatient stays and day care stays), only hospital stays with at least one external cause (E codes) were selected. There were up to 3 E codes for some case files ( Figure 1). Data were coded according to the ninth revision of the International Classification of Diseases, clinical modification (ICD-9-CM), so the E codes' groups taken into account in this study were presented in Table 1 [11]. As in the EUROCOST project [12,13]-concerning the cost estimation of injury-related hospital admissions in European countries-and as in Meerding and colleagues [14] study on the costs of injuries in the Netherlands, we have not considered the following E codes: "misadventures to patients during surgical and medical care" [E870-E876], "surgical and medical procedures as the cause of abnormal reaction of patient or later complication, without mention of misadventure at the time of procedure" [E878-E879], and "drugs and medicinal and biological substances causing adverse effects in therapeutic use" [E930-E949].
In the ICD-9-CM, causes and place of occurrence were found in the same chapter, so we have first differentiated, on Accidents occurring in residential institution (E849. 8) Accidents occurring in another specified place (E849.9) Accidents occurring in unspecified place one hand, the causes and, on the other hand, the places of injuries. Because of this absence of differentiation between place and cause, all the patients have not both code for the cause and another one for the place (Figure 1). Besides that, for the management of the stays with more than one cause we have created, for the extraction of a "main" external cause, a decision algorithm based on the gravity's perception. and/or choice of the main "cause" only with "cause" only with "place" with "cause" and "place" 16406 "E codes" "cause" 8493 "E codes" "place" or stay after a one-day stay hospital stays that are "secondary" stays Extraction of the "place"
Associated Factors.
The other variables taken into account have been chosen to try to follow the patient from its arrival to its discharge. The demographic characteristics of the patients were gender and age and we also have information about the moment (month and day), the type and the initiative of the hospital's admissions, the primary diagnosis that justifies the hospitalization, the stays description (in terms of care units), the type of discharge, the patient's destination, the cause of death, the type and the length of stay, and finally the cost incurred from the social security point of view.
Type of Admission.
Based on the preexisting categories, we have regrouped the type of admissions in 4 categories that may already reflect certain gravity of the injury: 1: admissions through the emergency department (ED) without ambulance; 2: admissions through the ED with an ambulance; 3: admissions through the ED with an ambulance and also with the intervention of a MICU, mobile intensive care unit, and/or a PIT, paramedic intervention unit; and finally 4: unplanned admission, meaning emergency hospitalization not through the emergency department.
Primary Diagnosis.
It is defined as the affection which, upon medical examination, proved to be the main cause of patient's admission. For the description of this primary diagnosis, we have used the chapter headings of the ICD-9-CM classification ( Table 2) and we have detailed more the chapter concerning the injury and poisoning (Table 3).
Stays Description.
The different services were the emergency unit, the one-day unit, the intensive care unit, the burn unit, the diagnostic and surgical care unit, the diagnostic and medical care unit, the geriatric unit, the pediatric unit, the neuropsychiatric unit, the specialized unit for the treatment and rehabilitation (with cardiopulmonary affections, musculoskeletal affections, neurological diseases, palliative care, polychronic diseases, and psychogeriatric affections), and finally the obstetric and neonatal unit. During the stays, patients could change, one or more times, service according to the evolution of their health status. We therefore have investigated up to three consecutive services, with a focus on the passages by the intensive care unit or by the specialized units for the treatment and rehabilitation. Focus was made because a stay in one of these services may reflect certain gravity of the health status.
Type of Discharge, Patient's Destination, and
Cause of Death. Based on the preexisting categories, we have regrouped the type of discharge in 5 categories. Two categories were related to the hospital's discharge with or without medical advice and the 3 others were related to a transfer to another place: 1: for specialized care, 2: for reeducation, or 3: for logistic reasons (e.g., for financial problems). The patient's destination was taken into account because this variable contains relevant information in terms of patient's redirecting to psychiatric homes/hospitals or to elderly homes. The other variable's categories were the domicile, another hospital, or all Diseases of the blood and blood-forming organs (290-319) Mental disorders (320-389) Diseases of the nervous system and sense organs (390-459) Diseases of the circulatory system (460-519) Diseases of the respiratory system (520-579) Diseases of the digestive system (580-629) Diseases of the genitourinary system other possible destinations such as jail or a boarding school. By the way of these two variables it was also possible to know if the patient was dead during their stay. The cause of death was reported in one of the fields of the patient file. It is important to note that the reported cause of death was the one that appears on the death certificate and that this cause is, for the nonnatural deaths, the circumstances of the accident (or the place of injuries if no type of injuries was reported) unless there was a morbid condition underlying (e.g., the neoplasms and the circulatory diagnoses). This death can be natural or nonnatural, so for this variable, there was coexistence of codes related to diseases and codes related to external causes (Tables 2 and 3).
Type, Length, and Cost of Stays.
The type of stays can provide first information about the gravity, because a patient who only stays for a day hospitalization is probably less affected than a patient who stays as an inpatient. Furthermore, patients in the case of the day hospitalization, day surgery, or outpatient emergency have a length of stay equal to one day, so they are not taken into account when it is the question of the estimation of mean or median length of stay (reported in days). The estimation of the costs borne by social security (reported in euros) was also only based on the inpatient stays and the costs were broken down into costs resulting from medical procedures, from pharmaceutical products, and from day lump sums. The percentage of each of these 3 specific costs in relation to the whole cost was also calculated.
Statistical Analyses.
Categorical variables were described with both absolute and relative frequencies and the variations between these distributions according to the different groups Table 4 shows that the three major groups of injuries observed were accidental falls [E880-E888] (58.2%), transport injuries [E800-E848] (11.2%), and suicide and self-inflicted injuries [E950-E959] (10.6%). According to these observations the next results would be presented
Demographic Characteristics of the Patients.
There were significantly more men in the transport injuries group than in the two other groups (66.7% versus 39.3% and 37.0%, resp.) and the patients in the accidental falls were older than those in the two other groups (median age equal to 74 years versus 32 years and 41 years, resp.) ( For the month of admissions, the percentages were nearly the same for each month (from 7.4% in February to 9.3% in December) for all injuries, but the repartition was significantly ( < 0.001) different between traffic injuries, accidental falls, and self-inflicted injuries. The highest proportions of admissions for traffic injuries were observed between May (10.8%) and August (10.3%). For accidental falls, December (10.6%) was the most observed, and finally for self-inflicted injuries, higher proportions were observed in January (9.6%) and July (9.5%).
Regarding the type of admission, there were significantly ( < 0.001) more arrivals through the emergency department with the support of a mobile intensive care unit and/or a paramedic intervention team in the group of the transport injuries than in the accidental falls and suicide groups (36.4% versus 10.3% and 23.5%, resp.). Relating to the person at the initiative of the admission, the general practitioner was more implicated in the case of an accidental fall (21.9% versus 2.5% and 7.0%, resp.), while a third party was implicated in nearly half the situations of transport injuries and a little more than forty percent of the self-inflicted injuries (47.4% and 43.3% versus 27.9%, resp.). It was also observed that a not inconsiderable proportion of admissions were due to the initiative of the patient (Table 6). Table 7 showed that for all injuries, in three-quarters of cases (74.6%), it was a diagnostic related to injury and poisoning [800-999] which was made, and this proportion was significantly different ( < 0.001) between the three studied groups, with a lower proportion in the accidents' falls group relative to the transport injuries and the self-inflicted injuries (72.2% versus 93.1% and 90.0%, resp.). In the accidental falls group, there were diseases of the circulatory system and in the self-inflicted injuries group mental disorders were found in a higher proportion than in the other groups. If we look in more detail to the injury and poisoning diagnosis group, half of it consisted of fractures: 22 Figure 4 shows that two-thirds of the 17370 patients have stayed in a diagnostic and surgical care unit (C) (34.4%) or in a diagnostic and medical care unit (D) (36.7%) on their arrivals, and a little less than five percent (3.9%) were directly admitted to the intensive care unit. For 409 of the 17370 patients, the stay was extended by a passage in the intensive care unit. Therefore, 6% of all patients remained at least one day in an intensive care unit. After the stay in the intensive care unit, 28.6% patients have left the hospital. Among the patients who were returned to one other service, after the stay in the intensive care unit, 31.3% were returned to a C unit and 31.3% other were returned to a D unit. Figure 5 shows that, regarding the specialized units for the treatment and the rehabilitation, 616 patients have gone through these units after a first ward, with 412 for the musculoskeletal affections. Finally, 85.3% percent of these 616 patients were discharged from the hospital after their stays in one of the six specialized units. Data are n (%) and median (p25-p75). patients for whom we only have the place of occurrence, and the two major places reported were the residential institution [E849.7] for 45 cases (6.2%) and the home for 17 cases of death (2.3%) (data not shown). Therefore, the proportion of death was significantly higher in the group of accidental falls than in the other two groups (5.2% versus 2.5% and 1.3%, resp.) ( Table 9). About the type of discharge, the proportion of transfer for specialized care was higher in the self-inflicted group (5.8% versus 3.6% and 2.4%, resp.) and the proportion of discharge without medical consent was also higher in this group than the other two (8.0% versus 1.2% and 1.0%, resp.). Concerning the destination after the discharge, a large part of the patients returned to their home, but in the accidental falls group nearly 14% went to elderly homes and in the selfinflicted injuries groups 5.7% went to psychiatric structures (Table 9). If we look in more detail to the causes of death, we observe that 31.6% in the transport group, 17.5% in the accidental falls group, and 29.4% in the self-inflicted injuries group were directly related to the type of injuries which have led to the admission, and for another part it was the injury and poisoning cause which has been mentioned (23.7%, 12.5%, and 41.2% in the three groups, resp.). The other major cause of death observed was a disease of the circulatory system both for the transport injuries group and for the accidental falls group (36.8% and 27.6%, resp.) (Table 10).
Type, Length of Stay, and Cost Incurred.
Concerning the hospital stays, the inpatient stays were the most observed. According to the three groups investigated the proportion of inpatients was significantly higher in the suicide group (94.7% versus 91.3% and 92.4%, resp.). Overall, the median length of stay was nearly equal to one week (6 days) and in the accidental falls group, this value was at least three times higher than in the two other groups (9 days versus 3 days and 2 days, resp.). The median cost borne by social security for all injuries was equal to C1376.6, with little more than twenty-five percent of the total inpatient stays being with a cost higher than C2500. There was also a higher median cost within the accidental falls group compared to the transport injuries group and the self-inflicted injuries group (C1760.4 versus C112.6 and C669.5, resp.). On the whole cost, the medical procedures were significantly proportionally highest for the accidental falls group and for the transport injuries group than for the suicide group (68.0% and 67.3% versus 58%); and the costs of the pharmaceutical procedures were significantly proportionally highest for the suicide group than for the transport injuries group and the accidental falls group (19.5% versus 13.6% and 10.7%, resp.) (Table 11).
Discussion
To our knowledge, in Belgium, no epidemiological studies have recently investigated specifically the distribution of E codes in the Minimal Clinical Summary database, nor patient characteristics, and even less the cost associated with the hospitalization.
E Codes Observed and Demographic Characteristics of the Patients.
As for mortality, transport injuries and selfinflicted injuries were also within the major groups observed [13,16]. Regarding the gender, more men were present in the transport injuries group but there were more women in the self-inflicted injuries group. This observation is consistent with the known risk factors: women have more attempts on their lives than men [17]. Women were also more present in the accidental falls group. This group is also the one in which patients are older. These two demographic characteristics were equally found in other studies [18,19].
Moment, Type, and Initiative of the Hospital's Admissions.
Our study showed that there were on the whole-and it is the same for the accidental falls group-more admissions on Mondays and Tuesdays than on other weekdays, but there were more admissions for traffic injuries on Saturdays and Sundays and more admissions for suicide and self-inflicted injuries on Sundays and Mondays. It was the same trend as that reported not only by Hawton and van Heeringen [17] in their International Handbook of Suicide and Attempted Suicide, but also by the studies of Beauchamp and colleagues [20] or Colman and colleagues [21].
Regarding the type of admission, there were, for the transport injuries group, more arrivals through the emergency department with the support of a mobile intensive care unit and/or a paramedic intervention team than in the other groups, and relating to the person at the initiative of the admission, the general practitioner was more implicated in the case of an accidental fall. These observations might inform about the seriousness of the traffic injuries, as a mobile intensive care unit's intervention involves "intensive care, " while for accidental falls, the intervention of a general practitioner may be explained by the fact that the patients in this group are older.
Primary Diagnosis That Justifies the Hospitalization.
In three-quarters of cases, a diagnosis related to injury and poisoning was reported, with a lower proportion in the accidental falls in which there were more diseases of the circulatory system and in the self-inflicted injuries group, where more mental disorders were found. This difference, in the accidental falls group, must probably be explained by the fact that, in this group, these older patients have more preexisting chronic conditions; the observation in the self-inflicted group is consistent with the literature which reports that there is a significant link between the mental health and the suicidal behaviors [17,22,23]. Regarding the injury and poisoning diagnosis group, fractures were the most common diagnosis observed in the accidental falls group and in the traffic injuries group, with a nonnegligible part of intracranial injuries in this last group. These observations were also made in other studies [12,14,24]. Finally, in the self-inflicted injuries group, poisoning by drugs and medicinal and biological substances was the most frequent diagnosis observed. This is consistent with the fact that women, who are more represented in this group, were known to choose less violent methods than men, with a predilection for ingesting drugs [22].
Stays Description.
A little more than five percent of the whole patients were remaining at least one day in an intensive care unit. In absence of real information about the severity of the injury-as in other European countries, the data do not include, for example, the AIS (abbreviated injury scale)the seriously injured patients could not be distinguished even if a stay in the intensive care unit is an important indication of the gravity [12]. Regarding the specialized units for the treatment and the rehabilitation, 616 patients have gone through these units after a first ward, with 412 for the musculoskeletal affections. This observation is consistent with the fact that a large number of patients who have had a stay in the specialized units must be linked with the fact that there was an important proportion of limbs fracture. These stays in specialized units might give an idea about the future disabilities of these injured patients, but only a longitudinal follow-up could confirm these disabilities [13,16].
Type of Discharge, Patient's Destination, and Cause of
Death. The proportion of death was higher in the group of accidental falls, with a lower proportion of related death to this fall which has led to the admission. It is certainly correlated with the older age of the patients in this group and with the presence of other diseases as observed in other studies [23]. For the alive patients in this group, a lot of patients returned to their home, and for a little more than one out of ten, the return was toward an elderly home. According to our data we cannot know if the patients were already in this type of structure before their admissions. In a little less than half of the self-inflicted cases it was the injury and poisoning cause which has been mentioned as the cause of death and; for the alive patients in this group, the proportion of transfer for specialized care and to psychiatric structures as well as of discharge without medical consent was higher than in the other two groups. This need for specific psychiatric care and the problem of unauthorized discharge are clearly documented in suicidology theory [17,22]. 4.6. Type, Length of Stay, and Cost Incurred. The median length of stay was nearly equal to one week and was similar to that observed by McKenzie and colleagues [25] in their study. In the accidental falls group, this value is at least three times higher. Scuffham and colleagues [23] have also observed a longer stay for the older elderly patients. Concerning the cost, we have observed that a little more than a quarter of the total inpatient stays have been supported by the social security cost higher than C2500. In the accidental falls group, the median cost was higher than in the other groups; this observation is again correlated with the fact that these patients were older, have other diseases, and have longer hospital stays [12,13,23]. Therefore, that is why, on the whole cost, the medical procedures and the day lump sums were proportionally highest in this group. On the other hand, it was the drug cost that was significantly the most important part of the total cost in the suicide group.
Even if it is difficult to compare our costs' results with other studies because of the data's content in our dataset, our observations contributed to the better knowing of the burden of injuries in Belgium. But it is also important to note that taking only into account the hospital costs is (sometimes) only to consider the "tip of the iceberg" because injuries lead (sometimes) to long-term follow-up costs, as shown in the study of Meerding [14] and colleagues who have investigated the costs of injuries based not only on hospital care but also on nursing home care and rehabilitative services.
Quality of the Data.
Although there is a ministerial circular that strongly encourages the registration of the codes E when they are present, we have observed that there were shortcomings regarding the encoded information: some cases have only the injury's cause or only the place of occurrence. We have also observed that there is an important utilization of the ".9" code; codes corresponding to the "other accident"/"unspecified cause" and "other place"/"unspecified place" and these codes are generally not useful to researchers because they lack details. These observations were also made in other studies [26][27][28]. According to McKenzie and colleagues [25] it is essential that clinicians and coders alike be aware of the documentation and coding problems related to the capture of cause-injury data. Another problem is the existence of more than one code of cause. In our study we have elaborated a decision's algorithm, but without knowing the hierarchy of the event, we maybe have made some misinterpretations. In their study, Scuffham and colleagues [23] present the example of a fall occurring on the road which will be coded as traffic injuries. Lawrence and colleagues [27] complete with the fact that "a fall should be E coded only if it causes an injury that is medically treated. If a patient falls down as a result of an illness or poisoning, but does not sustain an injury from the fall, then the fall should not be coded in the patient's record. But we often found records E coded as falls where the only diagnoses are heart conditions. " In this paper, we have made the choice to not take into account the hospital readmissions. Other authors have made the same choice with the justification that, to consider those events, in a simple way, do not allow overestimation of the impact of injuries [12][13][14]18]. Nevertheless, the study of the patients with more than one admission during a year must be really interesting in terms of understanding the repetitive injuries, in terms of investigations of complications/disabilities correlated with the initial injury, and also in terms of incurred costs [18]. It will be also interesting to investigate the costs related to other used health services (e.g., outpatients visits, general practitioner visits, outpatient physical therapy, and home care), as Meerding and colleagues have done [14].
Conclusion
Our study, the first of such kind in Belgium, has documented the occurrences, the related diagnosis, and the nonnegligible cost for the social security of all types of hospitalized injuries, specifically for three majors groups: the traffic injuries, the suicide and self-inflicted injuries, and finally the accidental falls. In this last group, we have also shown that because these injuries affect the elderly, there is a significant comorbidity which must be also taken into consideration.
Finally, despite the fact that injuries remain an important public health, especially in Belgium, and despite the European Union initiative, our country is not in the list of members' states that have committed to participate to the Joint Action on Monitoring Injuries in Europe (JAMIE) initiative, even it is obvious that the hospital sector provides the best setting for collecting information as this piece of information is related to the more severe cases, and information can be obtained easily on a large number of cases at low cost [5]. The total hospital costs generated by injuries indicate the relative importance of injuries in the healthcare sector as a whole and may be useful in convincing politicians of the importance of preventing injuries and investing in trauma care, and ideally costs and burden of injury should be analyzed in a combined perspective [12,13]. | 2018-04-03T02:26:27.973Z | 2014-04-28T00:00:00.000 | {
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55113811 | pes2o/s2orc | v3-fos-license | Wild Golden Iris (Iris aurantica) in Syria
In Syria, Iris is considered as a wild perennial herbaceous plant that subjected to strict protection, though Iris grows naturally in many regions of Syria. It presents some 30 species grown in Syria [1]. There are five subgenus found in the world. Apogon, Pogonias, Xiphion, Guno and oncocyclus which includes most of the Syrian species, that are considered as rare endemic plants, characterized by special, beautiful forms that have a great importance in applied studies for genetic biodiversity, such as Iris aurantiaca Dinsm.
Current Trends in Biomedical Engineering & Biosciences
Iris aurantica has green falcate leaves, they can grow up to 15-25cm long and between 1-1.5cm wide ( Figure 2). Leaves can sheath up to haft of the stem after the iris has bloomed. At the summer sun with dry conditions, leaves fade and die. Golden Iris has a slender stem, which can grow up to 30-50cm tall. The stems hold terminal flowers (top of stem), blooming during May and Jun, they can flower for up of month long [4]. Golden Iris has a slender stem, which can grow up to 30-50cm tall. The stems hold terminal flowers (top of stem), blooming during May and Jun, they can flower for up of month long [4]. The flowers like other irises, it has two pairs of petals, three large sepals, known as the falls and three inner smaller petals or sepals, known as the standards. The standards are golden yellow to coppery-brown, 8-9cm long, 5-5.5 wide, with fine purple veins. The falls are oblong-shaped, 7cm long, and 4cm wide, abovate, with minute purplish red spots and very fine reddish veins. It has golden yellow with fine purple. Style branches (stigma) keeled, and have lobes tips that are a similar color like falls ( Figure 3). After the iris has flowered, it produces a seed capsule, 8cm long, rather narrow.
The golden iris flowered in Europe in June, a whole month later than in their habitat in Syria. The unexpectedly cold European weather in the spring of 1962 had nearly everywhere in Europe killed the flowers on the iris species. The only exception and a unique consolation, was the plants of Iris aurantica from Syria, which gave us much joy with their flowers [5].
Golden Iris Propagation
The propagation of Iris species is usually accomplished vegetatively through bulbs or splitting of rhizomes (rhizomatous Iris). In rhizomatous Iris, splitting the rhizomes gives a maximum of 10 plants per year per rhizome [8]. Furthermore, the propagation of Iris species through seedlings is known to be difficult due to a poor fruit set and a very low germination rate.
Plant tissue culture is a powerful alternative technique for propagation and conservation of plants, especially for those that are rare and difficult to propagate by conventional methods. Therefore, a new trend has evolved to propagate these species through tissue culture techniquein order to preserve it from deterioration and to study the possibility of using them as a medical or ornamental plant.
Micro propagation is the aseptic culture of cells, pieces of tissue, or organs. It is possible to regenerate new plants from small pieces of plant tissue identical to the plant from which it was derived.
The process of micro propagation can be divided into four stages: A. Initiation stage: The objective of this stage is to achieve an aseptic culture. An aseptic culture is one without contaminating bacteria or fungi. Base of leaves and shoot tips of rhizomes in Iris aurantica (after surface disinfection by chlorox 3%) were cultured on solidified MS medium containing 30g/l sucrose, and supplemented with 2mg/lBAP and 0.2mg/ lIBA (Abouzedan and Al-Batal 2015). Results showed, after one month of culture, that using shoot tips of rhizomes resulted in the highest growth percentage (35.76%) in initial stage [9].
B. Multiplication stage:
A growing ex plant can be induced to produce vegetative shoots by including a cytokinin in the medium and different media. In Iris aurantica, the highest average number of shoots per ex plant was found (3.43) under BAP at a concentration of 3.0mg/l (Figure 4), and MS media resulted in the highest multiplication rate and shoot length with significant difference compared with Heller media, Subculture of the plantlets on the same medium resulted in increasing multiplication rate and shoot length in Iris aurantica [9] the Current Trends in Biomedical Engineering & Biosciences treatment of high concentrations of cytokinins (5 and 10mg/ lBAP) consist of appearance of vitrification.
C.
Rooting stage: Growing shoots can be induced to produce adventitious roots by including an auxin in the medium. In iris aurantica, the highest root percentage (88.5%) was obtained on medium containing 3mg/lIBA ( Figure 5). The highest root number (4.25) was recorded when using the concentration 0.5mg/lIBA [9] (Table 1).
Other Compounds
Activated Carbon 3g dm -3 --D. Acclimatization: A growing, rooted shoot can be removed from tissue culture and placed in soil. When this is done, the humidity must be gradually reduced over time because tissue-cultured plants are extremely susceptible to wilting. The acclimatization in vivo was achieved easily with high percentage of success (86.95%) in iris aurantica ( Figure 6). Two months, later, plantlets were cultured in greenhouse and the average length of shoots were 23. 25 cm [10].
In Vitro Conservation
Conservation is a very simple in vitro technique that permits conservation plants material for periods ranging from 6 months to 5-7 years, depending on species [11]. This technique is based on reducing the growth rates of the tissue cultured plant and yet increasing the intervals between subcultures [12]. Research was conducted, to develop an in vitro technique for short-term conservation and relieve of growth and increase the period of time between transfers of Iris aurantica, The best osmotic agents for in vitro conservation was sucrose compared with (mannitol and sorbitol) and the best medium concentration was 1/10 MS [10] in iris aurantica the cultured stored at (3 ˚C) gave the highest survival (93.33%) and lengthening the time period between transfers up to 6 months [10]. ABA was found to regulate expression of many genes that are responsible for the syntheses of proteins needed for osmotic adjustment in the cell, such as, membrane stabilization proteins, and the LEA proteins that would modify water state in the cell to cope with osmotic stress [13]. Some researchers reported that ABA is responsible for low temperature tolerance capacity of plant tissues [14]. In our research Iris aurantica micro shoots were conserved for more than nine months at normal growth room conditions in the media supplemented with 0.5 to 3.0mg/lABA, also ABA significantly decreases growth of shoots in medium when compared with control.
Results showed that the best survival after three, six and nine month were obtained in media supplemented with 2 and 3mg/l ABA with significant difference compared to the control at 3 ˚C and normal growth room conditions. The best treatments on germ plasm conservation were in 3mg/l ABA at 3 ˚C, in these treatments the survival rate was 60%.
Long -Term Conservation (Cryopreservation)
The principle of cryopreservation is the storage of plant material at ultra low temperature (-196 ˚C) that takes a place in a cryogenic condition which is liquid nitrogen [15]. At this
Current Trends in Biomedical Engineering & Biosciences
temperature, all forms of cellular divisions and metabolic activities of plant cell are ceased and consequently plant material can be stored unaltered for an indefinite time scale [16,17]. Two techniques of cryopreservation were used in the case of Golden iris:
Vitrification
Vitrification is based on three major phases, the loading phase, dehydration with the highly concentrated vitrification solutions, and unloading phase [18]. In loading phase samples are exposed to cryo protectants or diluted vitrification solutions [19] the samples are dehydrated by a highly concentrated vitrification solution before being plugged in LN, [20]. In unloading phase vitrification solution is drained out of the cryovials after rapid thawing, and then replaced routinely with 1.2 M sucrose for 10-20 min [21]. Plant vitrification solution 2 (PVS2) is commonly used in most vitrification protocols [22].
In our research Shoot-tips in Iris aurantica were excised aseptically from in vitro grown plants and incubated for 3 days on solid (HF-MS) media supplemented with 0.3 M sucrose under complete darkness at 24 ± 1˚C.in vitrification, shoot-tips were loaded in 0.4 M sucrose and 2 M glycerol for 20 min followed by desiccation with different combinations and concentrations of PVS2, before immersion in LN. Results showed that Subjecting I. aurantica shoot-tips to gradual increase in PVS2 concentrations (20,40, 60, and 100%) before plunging them in LN gave the highest survival for the non-cryo preserved shoot-tips (50%), and cryo preserved shoot-tips ( 30%), respectively, Using of 2 M glycerol plus 0.4 M sucrose resulted in the highest survival (80%, 25%) for the non cryo preserved and cryo preserved Iris aurantia shoot-tips, respectively, The highest survival for the non cryo preserved I. aurantica shoot-tips was recorded after 30 min of loading.
Encapsulation-Dehydration
This method, developed [23] in which shoot tips, somatic embryos or callus cells are encapsulated within alginate beads and subsequent culture in a medium containing elevated concentrations (0.7-1.5M) of sucrose for 1 to 3 days [24,25]. The beads are then allowed to dehydrate using silica gel or by air under the laminar air flow until the moisture content drops to 20-30% before being immersed in LN [23].
In our study shoot-tips in Iris aurantica were encapsulated in 3% calcium alginate and dehydrated under laminar air flow cabinet for0, 2, 4, or 6h. the highest survival were obtained after pre-treating encapsulated non-cryo preserved shoot-tips for 3 days in 0.5M sucrose supplemented media with 4h dehydration.
Chemical Analysis
The medicinal parts of Iris (orris) species are the rhizomes with the roots. They contain volatile oil (α, β, γ, irons) giving the odor of violets triterpenes, isoflavonoids, flavonoids, xanthones and starch [26]. Some of Iris species were reported for their medical use. The isolated compounds from some species were demonstrated to have pesticide, anti neo plastic and anti tuberculosis properties [27]. The extract of I. germanica was found to have central anti serotonin activity [28].
Gas chromatography-mass spectrometry (GC-MS) analyses of the essential oil have indicated the presence of 23 compounds in Iris germanica, and 19 in Iris aurantica [29]. The major compound in these essential oils was Myristic acid (61.42%, 70.67%) in Iris germanica, and Iris aurantica respectively with no significant differences [29]. The findings here agreed with those obtained [30] which noted that the myristic acid was the major compound of the oil of the fresh and naturally aged rhizomes in Iris pallida which has antifungal properties [31]. The other sub major compounds obtained were Lauric acid, Decanoic acid (Capric acid), Palmitic acid methyl ester, Octadecanoic acid methyl ester, Elaidic acid methyl ester (9-Octadecenoic acid methyl ester (E) and Palmitic acid [29].
The highest percentage of Lauric acid was obtained (6.97%) in I. aurantica, with no significant differences comparing with Iris germanica (5.69%), these findings give us the possibility to investigate the use of iris aurantica for medical purposes [29].
Iris Cultivation
Wild Iris aurantica is very difficult to cultivate. It can withstand the cold and the heat as long as it is dry. Golden iris needs well drained soil and at least 6-8 hours sunlight. If the soils are heavy, sand or humus may be added to improve drainage. The ideal pH is less than 7, slightly acidic. Iris should be planted in September or October, when the weather starts to cool, it is preferably to be divided and planted at least six weeks before the first frost in any area.
The rhizomes produce more rhizomes, which in turn lead to more leaves and flowers. One rhizome of golden iris can give more than ten flowers. When the bloom production slows and it is necessity to divide the plants, removing and replanting the baby rhizomes in spacing 15-25 cm. Close planting results in immediate effect, faster clump formation, and makes dividing clumps a necessity in two to three years. New plantations of irises need moisture and fertilizer to help their root systems becomes established. Watering depends on the soil quality and the climatic conditions (Figure 7). It is preferable to give deep watering at long interval is better than shallow watering. | 2019-06-13T13:11:18.334Z | 2017-02-27T00:00:00.000 | {
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258615512 | pes2o/s2orc | v3-fos-license | Reliability Improvement of Circular k-out-of-n: G Balanced Systems through Center of Gravity
This paper considers a circular k-out-of-n: G balance system equipped with homogeneous and stationary units. Building on previous research by Endharta et al. (Reliability Engineering&System Safety, 2018), we propose a new balance definition in circular k-out-of-n: G balance systems based on the concept of center of gravity. According to this condition, a circular k-out-of-n: G balance system is considered balanced if its center of gravity is located at the origin. This new balance condition is not only simple but also advantageous as it covers the previous two balance conditions of symmetry and proportionality. To evaluate the system's reliability, we consider the minimum tie-sets, and extensive numerical studies verify the enhancement of system reliability resulting from the proposed balance definition.
Notation
• n: number of units in a system • k: minimum number of non-failed units for a functioning system • r: probability that a unit is functioning properly • U: index set of all units in the system; U = {1, 2, ..., i, ..., n} • U or U j : (j th ) subset of U denoting the set of non-failed units; U j = {u 1 , u 2 , ..., u l , ..., u |U j | } where each u l corresponds to a unit index i ∈ U such that u 1 < u 2 < · · · < u |U j | .
Introduction
This paper considers systems with spatially distributed units such as balanced engine systems in planetary descent vehicles and Unmanned Aerial Vehicles (UAVs), commonly called drones equipped with multiple rotary-wings as shown in Fig. 1. In the balanced engine system, if an engine in a pair fails during landing, system dynamics mandate that the other engine in the pair must be turned off in order to uphold the balance of the descent vehicle [9]. The k-out-of-n balanced system can be used to analyze the reliability in such a situation because there may be a minimum number of pairs of engines required to prevent rapid descent during landing. Similarly to the descent system, there may be a minimum number of operating rotary-wings to operate drones reliably. In fact, many systems with spatially distributed units have a circular configuration with evenly distributed units. The definition of balance varies depending on the context of the analysis, so reliability evaluation does not have a clear-cut solution. Additionally, considering the units' positions is the most significant factor that makes the problem difficult when defining the system's balance.
Inspired by drones and descent systems, Hua and Elsayed [10,11,12], along with a series of papers, defined this situation as a k-out-of-n pairs: G balanced system and conducted extensive reliability studies for this system. Unlike descent systems where each engine provides propulsion in a vertical direction to the ground, however, the rotors of a drone generate lift force while rotating, making the definition of balance more complicated. In the series of research studies regarding drone reliability, the balance condition was mainly defined based on system symmetry.
Meanwhile, Endharta et al. [5] proposed a new balance condition based on the proportionality of the system for a similar situation. That is, as long as the operating units are evenly distributed throughout the system, balance can be maintained. Furthermore, considering such balance conditions, the units of the system no longer need to necessarily form pairs, which can lead to a relaxation of the target system from a k-out-of-n pairs system to a k-out-of-n system. As a result, Endharta et al. [5] investigated a reliability analysis problem for circular k-out-of-n: G balanced systems. Note that a k-out-of-n pairs system can be seen as a special case of a k-out-of-n system. For example, if Fig. 1(b) is considered a k-out-of-4 pairs system, it can be regarded as a 2k-out-of-8 system with some additional conditions. Therefore, a balance condition based on symmetry can also be applied to examine the balance of k-out-of-n systems, which can lead to a direct comparison between two different balance conditions.
Building on the previous research study by Endharta et al. [5], this paper presents another new balance definition for circular k-out-of-n: G balanced systems. This new balance definition is advantageous due to its simplicity and generality, as it covers the two balance conditions previously investigated. In essence, the new balance definition directly takes into account the concept of center of gravity. The main contributions of our work are summarized as follows.
• We propose a new balance definition for circular k-out-of-n: G balanced systems that can enhance the system reliability.
• We investigate the inclusion relationships among the three different balance conditions discovered so far through mathematical proofs and numerical examples.
• We demonstrate the reliability improvement resulting from the new balance definition using extensive numerical analysis. We also discuss the effect of system parameters on the overall system reliability.
The remainder of this paper is organized as follows. Section 2 conducts a literature review of existing reliability studies on a variety of k-out-of-n: G balanced systems. Section 3 describes the target system and outline the key modeling assumptions. Section 4 presents three definitions of balance conditions, including the newly proposed one. This section also provides an in-depth investigation of the relationships among the three balance conditions. Section 5 explains the reliability evaluation method with a descriptive numerical example. Section 6 provides extensive numerical results showing the enhancement of reliability resulting from the proposed balance definition. Finally, Section 7 concludes and suggests possible extensions of the research study.
Literature Review
The k-out-of-n models can be largely divided into two types: k-out-of-n: F and k-outof-n: G. A k-out-of-n: F system fails when at least k units are failed whereas a k-out-of-n: G system functions when at least k units are non-failed. In a k-out-of-n: G system with homogeneous units of binary-states (failed and non-failed), the number of non-failed units follows the binomial distribution; the system reliability coincides with the probability that k or more units are non-failed [3].
The circular k-out-of-n: G balanced system, which is the target system of this paper, is a variant of k-out-of-n: G model in which the units are arranged circularly, and the reliable system requires to satisfy a certain balance condition. Among the variants of circular kout-of-n: G balanced models, the most relevant special case is the k-out-of-n pairs: G balanced system which has n pairs of units distributed evenly on a circular configuration. For example, the graphical model in Fig. 1(b) can be considered to have four pairs of units: (1,5), (2,6), (3,7), and (4,8). Sarper and Sauer [17] presented the first reliability study for such a system: balanced engine systems in planetary descent vehicles, e.g., four-engine (i.e., two pairs) and six-engine (i.e., three pairs) configurations. The authors provided a basic reliability evaluation framework based on simple stochastic models such as Bernoulli distributed unit state and exponentially distributed failure time. Applying the k-out-of-n pairs: G balanced model to drone systems, Hua and Elsayed [10,11] conducted the extensive reliability studies regarding degradation analysis and reliability estimation. Notably, Hua and Elsayed [11] were the first to define the balanced state of a drone system by symmetry, considering the concept of Moment Difference to measure the degree of symmetry. The authors considered two different scenarios (rebalancing is allowed or not) and develop a systematic approach for estimating the reliability of kout-of-n pairs: G balanced systems. Although the proposed approach was effective for reliability evaluation, the computational intractability when systems are large remained a limitation. In this regard, Hua and Elsayed [12] proposed a Monte Carlo simulationbased reliability approximation method which is fast as well as accurate hence applicable even for large systems. Considering more complex configurations of drone systems, Guo and Elsayed [6] presented a (k 1 , k 2 )-out-of-(n, m) pairs: G balanced system. The system modeled a rotary UAV with multi-level of rotors where m rotors are arranged vertically in parallel in the same position. Reliability estimations for two scenarios (forced-down rotors are considered as failed and forced down rotors are considered as standbys) have been obtained by enumerating operational states and calculating the probability of their occurrences.
Unlike the above literature that only considered system symmetry as a measure of balance in drone systems and conducted extensive reliability studies, Endharta et al. [5] proposed a new balance definition based on system proportionality that can be applied to drone systems. By considering the new balance definition, the target system can be relaxed from a k-out-of-n pairs to a k-out-of-n system. Endharta et al. [5] considered a system with homogeneous and stationary units, and the minimum tie-sets were enumerated to evaluate the system reliability. The results showed the reliability improvement compared to when considering only the symmetry-based balance definition. In the subsequent study by Endharta and Ko [4], a load-sharing system was considered where the amount of load is equally distributed among the working units. The failure time of each unit was assumed to follow an exponential distribution, and the load-sharing relationship between the amount of load and the failure rate is assumed to follow a power rule. System failure paths were arranged to evaluate reliability, and for larger systems, reliability was approximated using Monte Carlo simulation.
Regarding the technique of reliability evaluation, the Inclusion-Exclusion (IE) method is a well-known approach that can be used for the systems consisting of units with constant failure probability. Using the IE method, reliability expression of such systems can be derived based on the concepts of minimum tie-sets or cut-sets [3]. To improve the computational efficiency of the IE method, Heidtmann [8] proposed an improved version that eliminates certain terms in the expression. Another popular method called the sum-ofdisjoint-products (SDP) method, which is also related to minimum tie-sets or cut-sets, has been explored by McGrady [15]. For k-out-of-n: G systems with independent but nonidentical units, Rushdi [16] developed a pivotal decomposition approach for evaluating reliability. Recently, Hao et al. [7] proposed a new IE method called Quick Inclusion-Exclusion (QIE) to increase the efficiency of the IE method and reduce the amount of required memory.
There have been a body of recent reliability studies on a variant of balanced systems. Wang et al. [23] studied a multi-state k-out-of-n: F balanced system with a rebalancing mechanism, which is important in engineering fields like new energy storage and aeronautics. The components and system are assumed to have multiple states, and an age maintenance strategy and optimization model were investigated to obtain the optimal results. The proposed model was demonstrated through numerical examples based on a product line balancing problem. Dui et al. [2] proposed a model for studying the mission reliability and structure optimization of an UAV swarm based on importance measures. The mission reliability model was based on polygonal linear consecutive-k-out-of-n: F systems, and the structure optimization has been analyzed using conditional reliability, conditional failure rate, and remaining useful life. The proposed method was demonstrated through numerical examples of triangular and quadrilateral UAV swarms. Wang et al. [21] presented a condition-based preventive maintenance policy for balanced systems with identical components. The system state was evaluated based on the deterioration levels of all components, and PM activities are employed to avoid competing failures. The optimal preventive maintenance thresholds are determined by minimizing the sys-tem maintenance cost within the framework of semi-Markov decision process. Zhao and Wang [29] suggested a new maintenance policy optimization method for systems with two balanced components, assuming that the components degrade over time according to a bivariate Wiener process. The objective of the maintenance actions was to eliminate the differences of degradation levels of system components at the cost of aggravating the degradation, and the model was optimized using Markov decision process with both finite and infinite planning horizons. Wang et al. [22] proposed a maintenance policy optimization model for balanced systems composed of multiple functionally-exchangeable units, where a unit may fail due to self-failure or external stress. The objective was to find the optimal number of operating units to minimize the maintenance cost per unit time, and an illustrative example was used to demonstrate the effectiveness of the proposed policy. Wu et al. [26] studied a load-sharing consecutive-k-out-of-r-from-n subsystems: F balanced system with linear and circular structures that maintains balance by forcing working components to standby or resuming standby components to operate. The system fails if there are r consecutive subsystems in which at least k subsystems fail, and the system reliability was analyzed using the Markov processes and finite Markov chain imbedding approach. Tian et al. [19] presented a new reliability evaluation method for Performance-based balanced systems with common bus performance sharing (PBSs-CBPS), which considers the balance degree threshold, transmission loss, and transmission capacity limit. A continuous-time discrete-state Markov model was built to address the transition behaviors of components, and the universal generation function method combined with nonlinear programming was proposed to calculate system reliability. Wang et al. [25] investigated two reliability models for balanced systems with multi-state protective devices, considering rebalancing mechanisms and failure criteria of both dynamic and static balanced concepts. The models involved new triggering and variable protection mechanisms to reduce the impact of shocks and internal degradation on units, and the reliability was derived using the Markov process imbedding method with Monte-Carlo simulation.
In the next section, we describe the target system and introduce the key assumptions considered in this paper.
System Description
Consider a system with circularly arranged units such as descent systems equipped by n engines or a drone product equipped by n rotary-wings. Although the system starts up in the perfect state with n operational units, some of them can go wrong as time goes by. The system with some failed units (say a subsystem), however, still has possibility to be reliable if the number of non-failed units are enough to generate sufficient forces to remain the system in the air, for example, at least k units are non-failed then the drone can stay afloat in the air. In such situation, what we must consider together is the balance of the overall system. The set of non-failed units that can keep the balance will generate the total thrust in the same direction to the lifting forces so that the system stays in the air. The circular k-out-of-n: G balanced system is a reliability model that abstracts out such a situation. Fig. 1 shows a pair of such system (a drone product) and its graphical model.
Here, we explain some of the main notations to mathematically describe the target system. First of all, we consider a set of indexes of units U (U = {1, 2, ..., n}) that comprise Note that u l 's are in an ascending order: u 1 < u 2 < · · · < u |U | . We also introduce a collection of sets denoted by U k that consists of all the k-combinations of a unit set U, that is, A circular k-out-of-n: G balanced system is said to be operational if at least k among n units are non-failed as well as the system maintains a balance with the non-failed units. Since On-Off control is not a highly sophisticated control mechanism in modern electronic devices, it is reasonable to assume each non-failed unit can be forced up and down. This On-Off mechanism can be utilized to rebalance the system by forcing down some operational units that are harming the system balance. To evaluate the system-wise reliability of such systems, it is necessary to take the unit-wise reliability into account. In this regard, we assume that the system consists of independent and homogeneous units each of which has a stationary survival probability. In short, we assume a constant survival and failure probabilities r and 1 − r for each unit and for all the planning horizon.
Below, we summarize the key assumptions considered for the last of this paper.
• The system consists of independent and homogeneous units each of which has a constant survival and failure probabilities r and 1 − r.
• Each non-failed unit is subject to On-Off control. For example, any non-failed operating unit can be forced down for rebalancing the system.
In the next section, we formally define the balance conditions that can be considered regarding the reliability analysis of circular k-out-of-n: G balanced systems.
Balance Conditions
We consider three balance conditions for circular k-out-of-n: G balanced systems: Balance Condition I (symmetry, BC1 [11]), Balance Condition II (proportionality, BC2 [5]), and the newly proposed Balance Condition III (center of gravity at origin, BC3). Note that just satisfying one of the balance conditions does not necessarily mean that the system is functioning; for a k-out-of-n: G system to be operational, there should be at least k non-failed units as well. Taken together, a circular k-out-of-n: G balanced system is operational when it satisfies at least one of the balance conditions with at least k non-failed units. Although this section introduces all the three balance conditions for explanatory purpose, we want to emphasize that the proposition of BC3 is the contributing part of this paper. Hence, we refer readers to the previous literature [11,5,4] for in-depth explanation on BC1 and BC2.
BC1: System is symmetric
The first balance condition BC1 is related to the symmetry of the target system. That is, if the system is symmetric in terms of a certain definition, it is considered as balanced. BC1 has been originally suggested to evaluate the circular k-out-of-n pairs: G balanced systems. Since the system can be regarded as a special case of circular k-out-of-n: G balanced system in which an additional pair constraint is considered, BC1 can also be applied to any of the circular k-out-of-n: G balanced systems for evaluating its balance. The following definition states BC1 that is suggested by Hua and Elsayed [11].
Definition 1 (BC1). A circular k-out-of-n: G balanced system is said to be balanced if it is symmetric in the sense that all its operating units are symmetric w.r.t at least a pair of perpendicular axes as well as the number of pairs is an even number. Regarding all the graphical models that will be shown in this paper, white circles express the operating units whereas black-colored circles correspond to the failed or turned-off units. First, the graphical model in Fig. 2(a) describes a subsystem with U = {4, 5, 6, 10, 11, 12}. As shown in the figure, it forms a pair of perpendicular axes of symmetry so that is identified to satisfy BC1 hence balanced. In comparison, Fig. 2(b) provides a subsystem with U = {4, 8, 12} that forms three distinct axes of symmetry in which no pair of perpendicular axes is observed. According to definition 1, it is identified not to satisfy BC1 hence unbalanced. Later in Section 4.2, we will show that the subsystem in Fig. 2(b) also can be identified to be balanced by considering more generalized balance condition (see Fig. 3(a)).
To explain the quantitative way of judging whether or not the system satisfies BC1, we introduce some mathematical notations. First, let d l denote the distance between units u l+1 and u l defined by We also define D U be a tuple that collects all the distance enumeration: Note that an E (l) that satisfies E (l) = D U corresponds to an axis of symmetry. By collecting all such E (l) 's, we generate a set of reverse tuples denoted by E B U . Combined with definition 1, the following proposition states a quantitative way of figuring out if a system satisfies BC1. [5]). For a circular k-out-of-n: G balanced system with an index set of non-failed units U , the axis of symmetry is located between units u 1 and u l+1 for l such that E (l) = D U . Thus, |E B U | can represent the number of axes of symmetry in the system and the system balance is examined as follows.
(a) If |E B U | is an even number, the system is symmetric w.r.t. at least a pair of perpendicular axes and balanced.
U | is an odd number, the system is symmetric w.r.t. certain axes, but not balanced.
(c) If |E B U | is 0, the system is not symmetric, then it is not balanced. Simply put, the result of proposition 1.1 leads towards the following remark. Remark 1.1. A circular k-out-of-n: G balanced system with an index set of non-failed units U satisfies BC1 if |E B U | is a nonzero even number. In the next subsection, we will explain an expanded balance definition (BC2), which is the main result of the previous study by Endharta et al. [5].
BC2: System is spread proportionally
The second balance condition BC2 focuses on the proportionality of the operating units in a system. According to BC2, a circular k-out-of-n: G balanced system is considered as balanced if the system is spread proportionally by the operating units.
Definition 2 (BC2).
A circular k-out-of-n: G balanced system is said to be balanced if the operating units are spread proportionally within the system.
To quantify the definition 2, we investigate the angles between two non-consecutively neighboring operating units (say sector angle), and then examine the patterns observed from the sector angles. Fig. 3 shows some representative examples of the systems satisfying BC2 and the following remark states the quantitative way of evaluating BC2 proposed by Endharta et al. [4].
Remark 2.1. Let a s be the s th sector angle between the two non-consecutively neighboring operating units and N be the total number of such angles. Then, a circular k-out-of-n: G balanced system showing at least one of the following patterns is said to be balanced.
(a) All sector angles a 1 , a 2 , ..., a N are congruent angles; a 1 = a 2 = · · · = a N . When N is an even number, all sector angles a 1 , a 2 , ..., a N Regarding the method of examining BC2 given a subsystem U , Endharta and Ko [4] previously found that the existence of more than one reverse distance tuple E (l) such that E (l) = D U implies the spread proportionality of the system. Hence, the following remark can be used to check if a subsystem satisfies BC2.
Remark 2.2 (Endharta and Ko [4]).
A circular k-out-of-n: G balanced system with an index set of non-failed units U satisfies BC2 if |E B U | > 1.
Combining remark 2.2 with remark 1.1, we draw a conclusion that a subsystem satisfying BC1 also satisfies BC2 whereas the opposite is not always true. For example, among the graphical models that depicted in Fig. 3, only three of them also satisfies BC1 with at least a pair of perpendicular axes of symmetry (see Figs. 3(a), 3(c) and 3(d))). As such, BC2 is regarded as a more generalized balance condition than BC1. For another descriptive example, Fig. 4(a) shows a subsystem which is the same graphical model as previously shown in Fig. 2(b), which has been identified as unbalanced according to BC1. Now, we notice that it can be regarded as a balanced system by considering BC2.
In the next subsection, we will introduce the most generalized balance condition (to the best of our knowledge) BC3 that covers both BC1 and BC2. For example, although the subsystem in Fig. 4(b) cannot be identified as balanced even if we consider both BC1 and BC2, it will be regarded as balanced by taking BC3 into account (see Fig. 5(a)).
BC3: System has a center of gravity at origin
The motivation of BC3 stems from the following research question: What is the essence that BC1 and BC2 are trying to evaluate? In consequence, both BC1 and BC2 can be considered as the conditions representing the states in which a subsystem comprised of only a part of units can maintain the balance. Such condition may correspond to the states in which the center of gravity formed by the operating units is located at the geometric center of the system. To be specific, the center of gravity stands for the mean point of an object's or system's weight distribution. By the assumption that we have homogeneous units, the system's direction of total thrust should be in the same direction as the lift force given the center of gravity located at the origin. Based on this simple and intuitive concept, we state the following definition for the new as well as more generalized balance condition.
Definition 3 (BC3).
A circular k-out-of-n: G balanced system is said to be balanced if the system with operating units has a center of gravity at the origin.
For example, plots in Fig. 5 depict two graphical models describing the meaning of BC3. First, Fig. 5(a) corresponds the case where BC3 is satisfied because the operating units in a subsystem U = {1, 4, 5, 9, 10} forms the center of gravity at the origin; the blue-colored 'x'-marker indicates the location of the center of gravity. Note also that the subsystem is the same as the previous figure Fig. 4(b) which was examined as unbalanced by considering both BC1 and BC2. On the other hand, Fig. 5(b) shows another subsystem U = {3, 6, 8, 12} in which the center of gravity (the red-colored 'x'-marker) is not located at origin, which is identified as unbalanced although the most generalized condition BC3 is considered.
To quantitatively examine BC3 for a circular k-out-of-n: G balanced system, we consider a conventional two-dimensional coordinate system with x-axis and y-axis. Without loss of generality, we assume that each unit of the system is located on a unit circle with radius 1. That is, the distance between the origin (0, 0) and each unit is 1 for all units. Again, without loss of generality, we adjust the coordinate of unit 1 to (1, 0) and arrange all the other units counterclockwise in ascending order of unit index such that the x-y coordinate of unit i, (x i , y i ), becomes (cos ((i − 1) θ) , sin ((i − 1) θ)) for i = 1, ..., n where θ = 2π/n. Then, the coordinate of the center of gravity given a subsystem U = {u 1 , u 2 , ..., u |U | } can be simply calculated by u∈U x u /|U |, u∈U y u /|U | . In summary, the following remark states a quantitative way of examining whether or not a subsystem U satisfies BC3.
Relationship between the balance conditions
In this subsection, we discuss the relationship among the three balance conditions BC1, BC2, and BC3. The main purpose of this investigation is to show that BC3 is the most generalized balance condition. First, we start with the following proposition which shows that BC3 is a more generalized balance condition than BC1. Proof. See Appendix A.
Next, the following proposition states that BC3 is a more generalized balance condition that BC2. Recall that we have observed an example of this relationship graphically by comparing Fig. 4(b) with Fig. 5(a). In the next section, we explain how to evaluate the system reliability based on the well-known minimum tie-set method considering the balance conditions discussed so far.
Reliability Evaluation
The reliability of circular k-out-of-n: G balanced systems can be evaluated considering the balance conditions. We apply the minimum tie-set method (also known as minimal path set method) [3]. That is, we interpret the system as a parallel system consists of minimum tie-sets and find the probability that at least one minimum tie-set is operational. In the following two subsections, We will introduce the notion of minimum tie-sets in the context of circular k-out-of-n: G balanced systems and then explain how to evaluate the system reliability.
Minimum tie-sets
A tie-set (also known as path set) is a subset of units in the system which by operating ensures the system is functioning. Hence, a tie-set for a circular k-out-of-n: G balanced system should include at least k elements and satisfy one of the balance conditions. Among the ordinary tie-sets, the minimum tie-sets (also known as minimal path sets) are the tiesets that cannot be reduced without losing their property as a tie-set. The minimum tie-set plays an important role when we evaluate the system reliability since the system can be interpreted as a parallel structure of minimum tie-sets [3].
To examine the minimality of a tie-set, we need to check its inclusion relationship with other tie-sets; a tie-set which is a superset of another tie-set cannot be minimal. The flowchart in Fig. 6 summarizes the procedure to find all the minimum tie-sets in a circular k-out-of-n: G balanced system. Note that the procedure applies for all the balance definitions by changing the balance condition checking step.
System reliability
In this subsection, we explain how to evaluate the system reliability using the set of minimum tie-sets. Let X i be the binary state variable of unit i; X i = 1 if unit i is functioning, X i = 0 otherwise. Collecting the states of all units, we let X be the system state vector; X = [X 1 , X 2 , ..., X n ]. Then, we define the system structure function φ(·) of X as follows: By the above definition, φ(X) becomes an indicator random variable that is 1 if X corresponds to an operational system and 0 otherwise.
Using the system structure function, the system reliability R S can be directly derived by calculating P [φ(X) = 1] which is equal to E [φ(X)] due to the property of indicator random variable. Since the probability that a unit is functioning properly is a constant r and all units are assumed to be homogeneous, we have E[X i ] = r for all i. Together with the assumption of independent units, R S is obtained by From the last Eq. (1) for the system reliability, calculating the value of R S is straightforward once the set of minimum tie-sets T M is acquired by enumeration. Next, we provide a specific example of system reliability evaluation for descriptive purpose. Check the balance condition for each in and remove the unbalanced elements. Define an empty set which will contain non-minimum tie-sets.
Initialization: , , balance condition
Define an empty set that will contain all the tie-sets.
Check the minimality of each element in set and store the non-minimum tie-sets in set .
• For any two different tie-sets and ′ such that ⊂ ′ , ′ is not minimal.
Define an empty set and store the minimum tie-sets in there. That is, ← \ .
Termination: is the set of minimum tie-sets.
A descriptive numerical example
Consider a circular 4-out-of-12: G balanced system. Following the procedures in Fig. 6, we enumerate all the minimum-tie sets according to each balance definition and visualize them in Fig. 7. As shown in the figure, we have 15 minimum tie-sets considering only BC1, 19 minimum tie-sets considering BC2, and 31 minimum tie-sets considering BC3, for a circular 4-out-of-12: G balanced system. Since the Eq. (1) implies that the system reliability R S is proportional to the number of minimum tie-sets |T M |, the increment of the number of minimum tie-sets will result in the enhancement of the system reliability. To better understand the systems with different parameters, Table 1 compares the number of minimum tie-sets for various combinations of (k, n) for each balance condition. Although not all the systems show the difference in the number of minimum tie-sets between BC3 and BC2, we observe significant differences (larger than 10) for some systems such as 4-out-of-12 (diff = 12), 6-out-of-12 (diff = 21), 6-out-of-14 (diff = 14), and 8-out-of-14 (diff = 14).
In the next section, we provide extensive numerical studies on the reliability enhancement effect resulted from introducing the new balance condition BC3. Figure 7: Enumeration of all the graphical models of minimum tie-sets in a circular 4-out-of-12: G balanced system (each legend exhibits the balance conditions that the corresponding minimum tie-set satisfies)
Numerical Analysis
We investigate the reliability of circular k-out-of-n: G balanced systems with various system parameters k (minimum number of non-failed units for working system), n (number of units in the system), and r (unit reliability), for different balance condition consideration. Though the main purpose of this section is to show the reliability enhancement effect thanks to the new balance definition BC3 compared to BC2, we include the BC1's results together in order to provide richer experimental data that helps understanding the gradual improvement from BC1 to BC2 to BC3. Fig. 8 depicts the effect of the unit reliability on the system reliability for each balance condition (say R BC1 S , R BC2 S , and R BC3 S ) for k-out-of-12 (three plots in upper row) and k-out-of-14 (another three plots in lower row) systems where k = 4, 6, 8. The first important but anticipatory observation is that considering more generalized balance condition consistently improves the system reliability for all the systems: R BC3 1]. For some systems such as 6-out-of-12, the level of improvement is quite significant so that introducing the new balance condition seems to be promising in terms of operating circular k-out-of-n: G balanced systems. Second, the system reliability shows definite "S"-shaped curve regardless of the system parameters. Together with the first observation, it is implied that the effect of unit-wise reliability enhancement on the system reliability is stronger in the middle of r's range [0, 1] and goes weaker as it approaches extreme values. Fig . 9 shows the system reliability with varying k's and r's for the systems with n = 12 (upper row) and n = 14 (lower row) under each balance condition. Similar to the observations from the previous plots, the system reliability shows "S"-shaped curve in terms of unit reliability r with the increasing trends from BC1 to BC2 to BC3, for all the cases. In addition, we find the decreasing tendency of R S as k increases because the systems with larger k require more non-failed units for the system to be operational. Note that the degree of decrement looks more significant when k increases from an even number to an odd number (say k o ), for example, see the changing slopes of R S for k = 4, 5, 6 for each plot in Figs. 9(d)-9(f). This phenomenon aligns with our intuition because maintaining the physical balance with the odd number of units must be more difficult than with the even number of units. Hence, there must be only few additional minimum tie-sets for the system with k = k o compared to the system with k = k o + 1 (the smallest even number greater than k o ). 10 provides the system reliability plots with varying k and n for r = 0.5, 0.7, 0.9 and different balance conditions. It is worth mentioning that the reliability is only evaluated for the systems with k and n such that k ≤ n − 1 hence the plots have empty space for some pairs of (k, n). From the plots in Fig. 10, we understand the overall changing shape of the system reliability R S affected by changing k, n, and r. For example, we observe the decreasing trend of R S as k increases for a fixed n for all the cases. Similar to the observed phenomena in Fig. 9, the level of R S 's decrement looks significant when k is increased from an even number to an odd number. On the other hand, R S shows increasing trend as n increases for a fixed k for all the cases. The sensitivity of R S to (k, n) looks greater for smaller r's (see Figs. 10(a)-10(f)) whereas we mostly observe few changes except for the marginal area of the plots when r is larger (see Figs. 10(g)-10(i)).
Summary with interpretation
In this section, we summarize the experimental results followed by the corresponding interpretations. Firstly, we consistently observed R BC3 and it demonstrates that the introduction of BC3 can enhance the system reliability of circular k-outof-n: G balanced systems. The analysis in Section 4.4 provides the supporting theory, which shows that BC3 covers BC2 and BC1. Although BC3 is considered as the most generalized balance condition for circular k-out-of-n: G balanced systems to the best of our knowledge, the system reliability can be further improved if one can find another effective balance condition.
Secondly, R S shows definite "S"-shaped curve to the unit reliability r when all the other parameters fixed. That is, the unit-wise reliability improvement is more effective in terms of enhancing the system reliability when r is not around the extreme values 0 or 1. This tendency can be useful for the impact-effort analysis of unit-wise reliability improvement. Finally, it is trivial that the increment of k results in the decrement of R S . However, we consistently observed more significant R S 's reduction when k is increased from an even number to an odd number from Figs. 9-10. From this observation, we realize that determining k in a circular k-out-of-n: G balanced system as even as possible must be advantageous for most of the systems.
Conclusion
This article examines the reliability of circular k-out-of-n: G balanced systems based on several definitions of the balance condition. Motivated by two previously investigated conditions, BC1 (symmetry) and BC2 (proportionality), we propose a new balance condition BC3 based on the center of gravity concept. Among the three conditions, the newly proposed BC3 is advantageous in terms of its simplicity and generality because both BC1 and BC2 can be regarded as special cases of BC3. We investigate the inclusion relationships among the balance conditions through mathematical proofs and several numerical examples. For system reliability, we apply the minimum tie-set method in which the system is interpreted as a parallel system consists of minimum tie-sets. Since the system reliability is proportional to the number of minimum tie-sets, considering BC3 can discover more minimum tie-sets, thereby enhancing system reliability. Extensive numerical studies demonstrate the effect of considering BC3 and the changing trends of system reliability for varying system parameters.
There are several future research suggestions that extend this paper. First, the reliability evaluation method used in this paper requires complete enumeration of all working states, which can be computationally challenging when k and/or n are large. In this regard, a more efficient method that reasonably approximates system reliability in a shorter time can be explored. Second, assumptions considered throughout this paper can be relaxed. For example, we have assumed that survival and failure probabilities for each unit are constants at any time and that the unit is only of two states (failed and non-failed). The constant probability assumption can be relaxed by introducing a failure time distribution such as exponential and Weibull, etc. Also, considering multi-state units would relax the binary-state assumption, thereby opening up another interesting research direction regarding circular k-out-of-n: G balanced systems.
A Proof of Proposition 3.1
Consider a conventional two-dimensional coordinate system with x-axis and y-axis on which the units are circularly and evenly located. Assume that we have a subsystem U = {u 1 , u 2 , ..., u |U | } that satisfies BC1. Then, the subsystem U is symmetric with respect to at least a pair of perpendicular axes by the definition 1. That is, for any operating unit u ∈ U , we have another operating unit u ∈ U such that the angle between u and u is π. Letting (x u , y u ) be the x-y coordinate of u, we have where r is the Euclidean distance from the origin (0, 0) to the unit u and θ is the angle between x-axis and unit u. Since the angle between u and u is π, we have x u y u = r cos (θ + π) r sin (θ + π) .
B Proof of Proposition 3.2
Assume that we have a subsystem U = {u 1 , u 2 , ..., u |U | } that satisfies BC2. Then, the sector angles are either identical for all s = 1, 2, . . . , N , as described in remark 2.1(a), or the opposite angles of one another as described in remark 2.1(b). In this proof, we will only show that remark 2.1(a) implies BC3 because remark 2.1(b) is trivially considered as satisfying BC1 (symmetry) hence implies BC3 by proposition 3.1.
Given the identical sector angles, we can partition the subsystem U into N SG mutually exclusive and exhaustive subgroups U j 's each of which have the same number of units such that U = {u 1 , u 2 , . . . , u |U | } = N SG j=1 U j and |U 1 | = |U 2 | = · · · = |U N SG |.
Because all the subgroups are homogeneous and all the sector angles are congruent, we can interpret the subsystem U as an equivalent system V comprised of N SG virtually aggregated units, say units v j 's, such that V = {v 1 , ..., v N SG } where the angle between any two such units is θ = 2π/N SG . Considering the physical properties, we define the x-y coordinate of v j to be the center of gravity formed by the units in the associated subgroup U j : (x v j , y v j ) ≡ (x U j ,ȳ U j ). Then, the center of gravity formed by the subsystem U , (x U ,ȳ U ) can be expressed as follows: Let r be the Euclidean distance between the origin (0, 0) and a virtual unit v. Without loss of generality, we adjust the coordinate of unit v 1 to (r, 0) and arrange all the other units v j 's counterclockwise in ascending order such that the x-y coordinate of unit v j , (x v j , y v j ), becomes (cos ((j − 1) θ) , sin ((j − 1) θ)) for j = 1, ..., N SG where θ = 2π/N SG . Then, (x U ,ȳ U ) is To show that the RHS of Eq. (B.1) is indeed the origin (0, 0), we will use the following lemma. | 2023-05-12T01:15:48.668Z | 2023-05-10T00:00:00.000 | {
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55838578 | pes2o/s2orc | v3-fos-license | Surface-Enhanced Raman Scattering Substrate for Trace Analysis of Furazolidone in Fish Feeds
Nanoparticles (NPs) composed of ferromagnetic and noble metal materials show dual functions of magnetic activity and local surface plasmon response and have great potential as substrates for surface-enhanced Raman scattering (SERS) in trace analysis. Easy-to-prepare superparamagnetic Fe 3 O 4 /Ag hybrid NPs were synthesized and optimized by adjusting the ratio of silver particles aggregated with APTMS-modified Fe 3 O 4 NPs.The hybrid NPs were assembled under an external magnetic field before being used as substrate for SERS analysis.The SERS spectral features of furazolidone standard solution were clearly identified at concentrations as low as 40 ngmL, and furazolidone in fish feeds could be detected at 500 ng g. The results indicated that the Fe 3 O 4 /Ag hybrid NPs as SERS substrates had a great potential for detection of trace amount of furazolidone and other prohibited or restricted antibiotics in the animal and fish feeds.
Introduction
Surface-enhanced Raman scattering or surface-enhanced Raman spectroscopy (SERS) has emerged as an important tool for trace analysis in many fields, such as chemistry, biology, environment, and food due to its ultrahigh sensitivity and potential rapidity or nondestructivity [1][2][3].Raman enhancement effect is mainly attributed to the surface plasmon resonance (SPR) very close to the surface of nanostructured noble metal substrate [4,5].Enormous SPR is often present at the gaps between microstructures, resulting in the so-called hot spots.In general, nanoparticles suspended in a well-dispersed system containing target molecules lead to rather weak SERS signals, while aggregated NPs result in better enhancement effect since more interparticle narrow gaps could be provided on the surface of aggregated colloids [6].The use of magnetic oxides/noble metal hybrid NPs makes it possible to assemble NPs in a relative orderly aggregated pattern with the assistance of an external magnetic field and has shown great advantages as SERS substrates [7][8][9].For instance, Hu and Sun reported a robust strategy to construct Au coated multilayer magnetic nanoparticles with tunable hot spots by controlling the seed-mediated plating process, resulting in ultrasensitive SERS detection for rhodamine 6G as low as femtomolar [7].Bao et al. fabricated Ag-Fe 3 O 4 NPs and maximized SERS sensitivity through changing the size and density of Ag particles.The optimized NPs could be used to detect rhodamine 6G at a concentration as low as 10 −12 [8].Wheeler et al. demonstrated that magnetically induced aggregation of the Fe 3 O 4 -Au coreshell NPs enhanced SERS activity for ∼7 times compared to nonmagnetically aggregated Fe 3 O 4 -Au NPs [9].
SERS is considered as first layer effect, and the analyte molecules must be very close to or adsorbed onto substrates to achieve significant enhancement effect.The surface of a SERS substrate must be clean so that targeted molecules could be adsorbed onto the surface.However, precursors and reducing agents used during NPs synthesis are often present on the surface of NPs, interfering with effective adsorption of analytes molecules [10].The precursors and reducing agents used for the synthesis of noble metal-coated Fe 3 O 4 NPs could be washed off under an externally exerted magnetic force, resulting in clean substrate surfaces.Therefore, magnetic noble metal NPs often lead to higher enhancement effects than nonmagnetic NPs [11].
The objective of this study was to synthesize Fe 3 O 4 /Ag hybrid NPs suitable for analysis of trace amounts of furazolidone in animal feeds.Furazolidone is a nitrofuran antibiotic often applied illegally as animal or fish feeds, although it is prohibited by many countries due to its carcinogenicity and other adverse health effects [12].There are very few studies published on the application of SERS for analysis of nitrofuran antibiotics, and these studies mainly used standard solutions instead of actual samples [13][14][15].To the best of our knowledge, there is no report on applying SERS to analyze nitrofuran antibiotics in animal or fish feeds.Complex sample systems (such as fish feeds) contain numerous nontargeted compounds that may interfere with and sometimes even make it impossible for the SERS analysis of targeted molecules.In this study, easy-to-prepare superparamagnetic Fe 3 O 4 /Ag hybrid NPs were synthesized and applied for trace detection of furazolidone in fish feeds with pronounced SERS activity, which helps understand the matrix effects on the SERS analysis and serves as a basis for further studies on rapid SERS method development for nitrofuran antibiotics in complex animal/fish feed systems.
Synthesis of Fe
O 4 /Ag Hybrid NPs.The coprecipitation method was used to synthesize Fe 3 O 4 NPs, in which ferric and ferrous irons (molar ratio 2 : 1) were coprecipitated to Fe 3 O 4 in alkaline solutions at room temperature and the resultants were treated under hydrothermal conditions [16,17].The as-synthesized iron oxide particles were hydrophilic, which could be easily dispersed in AgNO 3 solutions for later Ag particles growing and aggregating with Fe 3 O 4 NPs.The reaction was performed under oxygen-free environment by passing N 2 gas.In brief, FeCl 3 ⋅6H 2 O (5.40 g) and FeCl 2 ⋅4H 2 O (1.99 g) were thoroughly mixed in 100 mL water.Next, NH 3 ⋅H 2 O (30%) was added with vigorous stirring until the pH was 10, and the mixture was continuously stirred for 10 minutes at room temperature.The mixture was then heated at 80 ∘ C for 30 min.Following this, the mixture was washed under magnetic field with ethanol and water three times for each and then dried in vacuum oven at 70 ∘ C for 2 hours.
To be able to attach Ag particles and Fe 3 O 4 NPs, APTMS was used as a linker between Fe 3 O 4 and Ag NPs [18,19].
To obtain APTMS-modified Fe 3 O 4 NPs, Fe 3 O 4 NPs (0.25 g) were added into ethanol (100 mL) and sonicated for 40 minutes.Next, APTMS (1 mL) was added to the mixture followed by stirring for 6 hours.Then the mixture was rinsed four times with ethanol under an external magnetic field and dried in a vacuum oven at 70 ∘ C for 2 hours.
Four different types of hybrid NPs were constructed by aggregation of APTMS-modified Fe 3 O 4 NPs and Ag particles through adjusting the molar ratio of AgNO 3 and Fe 3 O 4 NPs (2 : 1, 3 : 1, 4 : 1, 5 : 1).The amount of Fe 3 O 4 NPs was fixed (0.05 g, 0.22 mmol), and four different levels of AgNO 3 solutions (0.07 g, 0.44 mmol; 0.11 g, 0.66 mmol; 0.15 g, 0.88 mmol; 0.19 g, 1.10 mmol) were mixed with Fe 3 O 4 NPs and sonicated for 40 minutes, respectively.Then, freshly prepared reductant mixtures consisting of 50 mL 0.1 M NaOH and 45 mL 0.06 M hydroxylamine hydrochloride were added dropwise and mixed for 45 minutes.Finally, the NPs were washed with water under an external magnetic field until the supernatant was clear.The four Fe 3 O 4 /Ag hybrid NPs samples were labeled as FA-1, FA-2, FA-3, and FA-4, respectively.Figure 1 is a summary of the procedure for synthesis of Fe 3 O 4 /Ag hybrid NPs and later substrate preparation.
Characterization of Composite Nanoparticles.
General shapes and sizes of Fe 3 O 4 NPs and Fe 3 O 4 /Ag hybrid NPs were characterized by a JEM-2100F transmission electron microscopy (TEM; JEOL Ltd., Tokyo, Japan).The samples for TEM characteristics were prepared by making a carboncoated copper grid soaked in as-prepared NPs for 30 minutes before being dried at room temperature.Scanning TEM equipped with a high angle annular dark field detector (STEM-HAADF) and energy dispersive spectroscopy (EDS) elemental mapping was used to further understand the structure and component of particles.The magnetic properties were investigated by using a Quantum Design superconducting quantum interference device (SQUID) magnetometer (MPMS SQUID VSM, Quantum Design Ltd., USA) at room temperature.
Preparation of Fish Feed Samples. Fish feeds from the College of Fisheries and Life Science in Shanghai Ocean
University were spiked with furazolidone at a series of concentrations (200, 500, 1000, and 5000 ng mL −1 ).Furazolidone was then extracted and purified from fish feeds based upon the method of Hu et al. [20].In brief, furazolidone was extracted with dichloromethane three times and purified through anhydrous sodium sulfate and alumina column successively.The column was a syringe (10 mL) containing a filtration membrane (0.45 m), 4.0 g of anhydrous sodium sulfate, and 2.0 g of alumina.The eluate was evaporated to almost dryness at 50 ∘ C in water base with a rotary evaporator (R206B, Shanghai SENCO Technology Ltd., Shanghai, China), dissolved into 1.0 mL of acetonitrile/water (8 : 2) and 1.0 mL of hexane, and then centrifuged.The supernatant hexane was discarded, followed by adding 1.0 mL of hexane to defat the matrix.Finally, the subnatant was collected and used for SERS analysis.
SERS Analysis.
A series of furazolidone standard solutions (1000, 500, 100, 50, 40, and 20 ng mL −1 ) were prepared in acetonitrile.For SERS measurement, 10 L of Fe 3 O 4 /Ag NPs was first added onto a glass slide on top of the magnet, and furazolidone standard solution was added on top of NPs droplets.After the substrates were dried under ambient conditions, 20 spectra were collected at different positions of the substrates and the average of these spectra was used as the final spectrum for the sample.To compare the enhancement effect of four different Fe 3 O 4 /Ag hybrid NPs, SERS spectra of furazolidone standards (100 and 500 ng mL −1 ) were collected with each type of NPs, respectively, and the experiments were repeated five times.In addition, SERS enhancement effect without the use of magnetic field was examined by removing the magnetic field during Fe 3 O 4 /Ag NPs substrate preparation for the SERS measurement.
The SERS measurement of Fe 3 O 4 /Ag NPs with different amounts of silver was carried out on a Nicolet DXR microscopy Raman spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).A 780 nm HP He-Ne laser source with 20 mW laser power and a 20x objective was used for spectral collection.All spectra were recorded with a 2-second exposure time for each scan, and the final spectra were the sum of two scans.NPs, which allows for coordination and static binding of Ag particles with negatively charged surface [18].Figure 2(c) shows statistical histogram of diameters and relative standard deviation (RSD) of four Fe 3 O 4 /Ag hybrid NPs ( = 100).The particle sizes were 58 ± 7 nm, 63 ± 8 nm, 73 ± 9 nm, and 106 ± 14 nm, respectively, increasing gradually with an increase of molar concentration of Ag + ions used for hybrid NPs aggregation.The size distributions of four samples were all less than 15%, indicating relatively uniform particle size.Figure 4 exhibits the magnetization curves of dried Fe 3 O 4 NPs and two Fe 3 O 4 /Ag hybrid NPs (FA-3 and FA-4) measured at room temperature.The saturated magnetization (SM) value of Fe 3 O 4 NPs was 83.0 emu⋅g −1 , which was higher than 78.0 emu⋅g −1 reported by Kim et al. [22] and 53.7 emu⋅g −1 by Bao et al. [8].The negligible coercivity and reversible hysteresis behavior indicated the superparamagnetic nature of Fe 3 O 4 NPs, which is common for magnetite smaller than 30 nm in diameter [23].The SM values of FA-3 and FA-4 were 46.7, 36.5 emu⋅g −1 , respectively.The SM values decreased with an increase of Ag ratio in the hybrid NPs due to the diamagnetic contribution of Ag particles [22].The magnetic response for all four hybrid NPs is strong enough to make it easy to rinse the NPs and to conduct magnetically induced aggregation in a magnetic field.The excellent magnetic property of hybrid NPs also allows separating the particles over a short time (less than 30 seconds) in an external magnetic field (Figures 4(D) and 4(E)).
Effect of Magnetic Self-Assembly Method on SERS Sensitivity.
In order to understand the effect of the ratio of Ag particles on the SERS sensitivity, four different Fe 3 O 4 /Ag hybrid NPs were used as substrates to collect the SERS spectra of furazolidone standards 100 and 500 ng mL −1 , respectively.Figure 5(a) displayed the representative SERS spectra of 500 ng mL −1 furazolidone acquired with the use of four hybrid NPs substrates.All four spectra consist of characteristic peaks at 1561, 1490, 1348, and 1014 cm −1 associated with furan ring stretching vibrations.The peak at 1606 cm −1 is assigned to C=N stretching vibration in the plane, while medium bands at 1243, 960, and 807 cm −1 are attributed to C-H bending vibrations and C-C stretching vibrations [13].
Notably, the SERS signals from FA-3 were much stronger than the others.Similar trends were observed for SERS spectra of 100 ng mL −1 furazolidone.Using the characteristic peak at 1348 cm −1 as an example (Figure 5(b)), its Raman intensity increased with an increase in hybrid particle size until reaching 73 ± 9 nm but then decreased with a further increase of particle size.Controlling the amount of Ag particles aggregated with modified-Fe 3 O 4 NPs through adjusting the molar ratio of Ag + ions and Fe 3 O 4 NPs makes the SPR tunable and eventually optimizes Raman enhancement effect for furazolidone.In addition, although it is often a challenge to obtain reproducible SERS spectra, by using the average of spectra collected at 20 different positions of a substrate, we can overcome this problem.As shown in Figure 5(b) for the Raman intensity of 500 ng mL −1 and 100 ng mL −1 , with furazolidone at the characteristic peak of 1348 cm −1 , the error bars calculated from quintuplicate analyses indicated very small standard deviation for spectra collected independently, implying that repeatable spectra could be obtained with Fe 3 O 4 /Ag hybrid NPs.We also attempted to analyze furazolidone with Ag NPs varying in particle size (34-67 nm) synthesized using the method of Leopold and Lendl [24], but very weak SERS enhancement was obtained.This was probably due to the interference of remaining reductive agent on the surface of Ag NPs that affected the effective adsorption of furazolidone.On the contrary, the surface of Fe 3 O 4 /Ag hybrid NPs was clean because reductive agent and other nontargeted compounds could be removed by washing with copious amounts of water under external magnetic field before the NPs were used for SERS analysis.
Using a magnetically directed self-assembly method is a simple and effective way to condense NPs on a glass slide used as holder for substrate and analyte molecules to increase SERS signals [6]. Figure 6 shows the SERS spectra of furazolidone obtained using 73 ± 9 nm Fe 3 O 4 /Ag hybrid NPs assembled onto a glass slide with and without an external magnetic field.The spectra obtained with substrates assembled under magnetic field displayed stronger SERS signals, with about twice to fourfold increase in enhancement effect compared to those without magnetic field.NPs assembled in a more orderly arrangement by the magnetic force led to the formation of more relatively homogeneous SERS hot spots and therefore better enhancement effect [6].
The FA-3 Fe 3 O 4 /Ag hybrid NPs were used as SERS substrate to further acquire spectra of furazolidone with different concentrations, as shown in Figure 7(a), characteristic peaks of furazolidone were clearly visible at concentration as low as 40 ng mL −1 , and the intensity of SERS signals increased as the concentration changed from 40 to 1000 ng mL −1 .This indicated that Fe 3 O 4 /Ag hybrid NPs exhibited significant Raman enhancement effect as SERS substrate for furazolidone detection.Figure 6: SERS spectra of (a) 500 ng mL −1 and (b) 100 ng mL −1 furazolidone molecules adsorbed on the FA-3 substrates prepared by magnetically directed self-assembly method (curve A) and nonmagnetic force (curve B).(The inset was the mean Raman intensity at 1348 cm −1 corresponding to curves A and B.) hybrid NPs) of extracts from spiked fish feeds consist of main characteristic peaks of furazolidone consistent with those of the standards, although some extra bands (such as the ones at around 1050 and 1447 cm −1 ) are observed in the spectra of extracts (Figure 7(b)).These extra bands may be due to some residual compounds, such as amino acids and pigments naturally present in fish feeds.Due to the high sensitivity of SERS method, some trace amounts of nontargeted compounds from fish feed may produce SERS signals.In addition, nontargeted compounds in the extracts may interfere with effective adsorption of furazolidone molecules onto the NPs and reduce the sensitivity of SERS method.As shown in Figure 7(b), the intensity of furazolidone characteristic peaks for extracts was weaker than that for standards.The lowest concentration with discernible characteristic peaks of furazolidone was 500 ng g −1 for extracts, which is much higher than that of 40 ng mL −1 for standards.However, the sensitivity of the SERS method for furazolidone in fish feed is comparable to a reported HPLC-UV method (detection limit, 1 g g −1 ) [25] and a LC-MS method for animal feeds (detection limit, 100 ng g −1 ) [26].In addition, the sensitivity of the SERS method could meet the requirement for analysis of furazolidone in animal and fish feeds, which are often added with the antibiotics ranging from 8 g g −1 to 400 g g −1 [27].
Conclusions
Superparamagnetic Fe 3 O 4 NPs were synthesized and modified using APTMS to add NH 2 groups on their surfaces and then aggregated with Ag particles to form Fe 3 O 4 /Ag hybrid NPs.The Fe 3 O 4 /Ag hybrid NPs exhibited tunable plasmonic properties of SERS by adjusting the amount of Ag particles aggregated with Fe 3 O 4 NPs through modulating molar ratios of the Ag + and Fe 3 O 4 .The use of 73 ± 9 nm Fe 3 O 4 /Ag hybrid NPs assembled onto a glass slide under an external magnetic field as SERS substrate could detect furazolidone standards and furazolidone in fish feeds at the levels as low as 40 ng mL −1 and 500 ng g −1 , respectively.To improve the applicability of the SERS analysis method, simplifying sample preparation and development of selective SERS substrates are much needed for future research.
4 Fe 3 O 4 A 2 Fe 3 O 4 / 2 FeCl 3 A g +Figure 1 :
Figure 1: Schematic illustration of major steps for synthesis of Fe 3 O 4 /Ag hybrid NPs and preparation of SERS substrates.
Figure 2 (
b) shows TEM images of representative Fe 3 O 4 /Ag hybrid NPs (FA-3) synthesized in this study, in which the lighter small particles are Fe 3 O 4 , while the darker large particles are Fe 3 O 4 /Ag hybrid NPs (FA-3).The figure indicates that APTMS-modified Fe 3 O 4 NPs are successfully aggregated with Ag particles.Through the condensation reaction between -OH groups on the surface of Fe 3 O 4 NPs and APTMS, NH 2 groups are then decorated on the Fe 3 O 4
Figure 2 (
d) is the EDS spectrum of Fe 3 O 4 /Ag NPs, which shows the peaks corresponding to Ag and Fe elements.The presence of Cu elements was due to the carbon-coated copper grid used as the holder for nanoparticles.The STEM-HAADF image and element mapping images for Fe 3 O 4 /Ag NPs indicate that Fe 3 O 4 /Ag hybrid NPs are aggregates of Fe 3 O 4 and Ag particles and contain denser Ag particles than Fe 3 O 4 particles (Figures 3(a)-3(d)).
Figure 7 :
Figure 7: Respective SERS spectra collected with FA-3 NPs of (a) furazolidone standard solution and (b) furazolidone extracts from fish feeds. | 2018-12-09T09:12:50.091Z | 2014-01-01T00:00:00.000 | {
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9207746 | pes2o/s2orc | v3-fos-license | Structural and Biophysical Analysis of BST-2/Tetherin Ectodomains Reveals an Evolutionary Conserved Design to Inhibit Virus Release
BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-Å crystal structure of the complete mouse BST-2 ectodomain reveals an ∼145-Å parallel dimer in an extended α-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 Å for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.
Viruses must utilize host cell machinery to replicate and produce new virions. Efficient amplification, including virus release and infection of new cells, is obligatory for virus survival and disease pathogenesis. To subvert this process, host cells encode several interferon-inducible antiviral factors that constitute the first line of innate immune defense (1,2). These antiviral factors interfere with various steps in the viral replication pathway to aid in clearance.
BST-2 is a type II transmembrane protein with a unique topology consisting of a short N-terminal cytoplasmic tail; a single transmembrane region (TMR); 3 an ectodomain; and a second membrane anchor, a C-terminal glycosylphosphatidylinositol (GPI). It has been found to localize within lipid rafts on the cell surface and trans-Golgi network membrane (17). Because the activity of BST-2 requires co-localization with budding virions, many viruses encode countermeasures to remove it from the cell surface. HIV-1 Vpu interacts with the TMR and directs surface removal of BST-2, although the exact mechanism remains unclear (18 -23). Karposi sarcoma-□ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. The atomic coordinates and structure factors ( associated herpesvirus K5 interacts with the cytoplasmic tail and mediates a ubiquitin-dependent down-regulation of BST-2 (11,24). Interestingly, the envelope glycoproteins of HIV-2 (Env) (19,25) and Ebola (GP) (26,27) antagonize BST-2 directly through interaction with the ectodomain. The bulk of the literature suggests that BST-2 restricts enveloped virus release by directly bridging the host and viral membranes through simultaneous inclusion using its two opposing membrane anchors (28,29). Indeed, deletion of either the TMR or GPI renders BST-2 nonfunctional for HIV-1 restriction (7,28). BST-2 is surface-expressed as a disulfidelinked homodimer (30). Disulfide cross-linking through at least one of the three conserved cysteines is necessary for antiviral function (28,31). Recent structural studies of a human BST-2 ectodomain C-terminal fragment revealed an irregular coiled coil that exhibits conformational flexibility (32).
Here, we present the first high resolution crystal structure of the full-length mouse BST-2 ectodomain. The structure reveals an extended ␣-helix spanning nearly the entire ectodomain, predominantly forming a disulfide-linked coiled coil. Thermal denaturation dictates this structure to be unstable in the absence of the disulfides. This instability produces structural plasticity in solution as detected by small angle xray scattering. Our structural and biophysical analysis of mouse and human BST-2 ectodomains suggests an evolutionarily conserved design encoding an unstable coiled coil that requires intermolecular disulfides for stability. Such a design produces a conformationally labile dimer that can adapt during the dynamic events of viral assembly and budding while providing necessary separation for the membrane anchors.
EXPERIMENTAL PROCEDURES
DNA Constructs and Recombinant Proteins-The ectodomains of mouse (amino acids 53-151) and human (amino acids 45-160) BST-2 were cloned into pET23b with artificial start methionines as tagless constructs. The mouse BST-2 (mBST-2) ectodomain was generated by PCR amplification from full-length cDNA. A human BST-2 (hBST-2) ectodomain construct was codon-optimized for expression in Escherichia coli generated by PCR-based gene synthesis (33). Both constructs were confirmed by sequencing and transformed into Rosetta 2(DE3) cells (Novagen) for protein expression.
Protein Expression and Purification-BST-2 ectodomains were expressed in E. coli as insoluble proteins, which were recovered from inclusion bodies, denatured in guanidine hydrochloride, and then refolded under oxidative conditions by rapid dilution. The resulting soluble proteins were purified by gel filtration followed by ion exchange chromatography. The proteins were well folded as assessed by circular dichroism.
Antibody Binding Assay for the Refolded mBST-2 Ectodomain-The refolded mBST-2 ectodomain was assessed for proper folding using anti-mBST-2 mAb 927, which was raised against cell surface-expressed mBST-2. Direct binding was assessed by biolayer interferometry using a ForteBio Octet platform. Briefly, streptavidin-coated biosensors from ForteBio were used to capture biotinylated mAb 927 onto the surface of the sensor. After reaching base line, sensors were moved to the association step containing 900, 450, 225, 112.5, 56.3, 28.1, 14.1, or 7.03 nM mBST-2 ectodomain for 300 s and then dissociated for 300 s. A buffer-only reference was subtracted from all curves. The running buffer consisted of 10 mM Hepes (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, 1% BSA, 0.01% azide, 0.05% Tween, and 0.005% Triton X-100. Affinities were estimated from global kinetic analysis of the three lowest concentrations (K D ϭ 1.8 nM). No binding to the refolded hBST-2 ectodomain at 900 nM was observed under the same conditions.
Cell-based Competitive Binding Assay-The refolded mBST-2 ectodomain was additionally assessed for proper folding using a competitive binding assay. The mouse T cell hybridoma 14-3-1 (high BST-2-expressing) and human 293T cells (non-BST-2-expressing) were plated at 1-2 ϫ 10 5 cells/ well in 96-well round-bottom plates. The refolded mBST-2 ectodomain diluted in FACS buffer (2% BCS in PBS) was added to wells. Immediately after adding mBST-2, anti-mBST-2 mAb 927 (rat IgG2b) diluted in Fc block (1:100) was added to the wells (final dilution of 1:200). Cells were incubated for 30 min on ice and then washed three times with FACS buffer. Allophycocyanin-conjugated streptavidin (1:400 in FACS buffer) was added to the wells for 20 min on ice. Cells were then washed three times with FACS buffer, resuspended, and analyzed on a FACSCalibur using propidium iodide to gate out dead cells. Samples were run in duplicate and repeated. Fig. 1B shows the mean fluorescence intensity of mAb 927 staining using different concentrations of the mBST-2 ectodomain.
Crystallization, Data Collection, and Structure Determination-Attempts to determine the crystal structure of the hBST-2 ectodomain were hindered by poorly diffracting crystals and endogenous proteolytic degradation of the protein that made optimization difficult. A single degradation product was always observed at ϳ8 kDa. N-terminal sequencing and mass spectrometry of the degradation product revealed the cleavage site to be Phe-81.
Crystals of the mBST-2 ectodomain were obtained by mixing protein solution (at 10 mg/ml) with reservoir solution (100 mM phosphate/citrate (pH 4.6), 35% isopropyl alcohol, and 5% PEG 1000) at a 1:1 ratio. Crystals routinely formed over the course of 1 week. Crystals were cryoprotected in reservoir solution (with isopropyl alcohol reduced to 30%) supplemented with 25% 2-methyl-2,4-pentanediol and streamfrozen at 100 K. X-ray diffraction data were collected at Advanced Light Source beamline 4.2.2 at the Lawrence Berkeley National Laboratory. Crystals contained one dimer in the asymmetric unit with 42% solvent content. Data were processed using HKL-2000 (34). Data collection and refinement statistics are given in Table 1.
The phase problem was solved by molecular replacement in the program PHASER (35) by locating a single 40-residue polyalanine dimer fragment from the coiled-coil protein cortexillin I (Protein Data Bank code 1D7M) (36) followed by two copies of an ensemble consisting of 10 20-mer ␣-helices. The model was built by a combination of auto-rebuilding with PHENIX (37) using RESOLVE (38) and manual building with COOT (39). Refinement against data to 1.6 Å was carried out in PHENIX. Hydrogens were generated in the final stages of refinement and included as a riding model. The final model consisted of residues 57-151 (chain A) and residues 58 -144 (chain B). All residues are within the most favored (98.3%) and additionally allowed (1.7%) regions of a Ramachandran plot. Molecular graphics figures were created using PyMOL. Structures were superposed using the "user defined match" option in CCP4MG Version 2.4.0 (40). Knobs-into-holes packing analysis with SOCKET (41) determined coiled-coil boundaries. Variations in coiled-coil structure were analyzed using TWISTER (42). Sequence conservation was calculated using ALSCRIPT (43), and GPI anchor positions were predicted using the BIG-PI predictor (44). The coordinates and experimental structure factors of the mBST-2 ectodomain have been deposited in the Protein Data Bank (code 3NI0).
Circular Dichroism Studies-CD spectroscopy measurements were performed using a JASCO J-815 spectropolarimeter equipped with a Peltier temperature controller. Thermal denaturation experiments were carried out in 20 mM phosphate (pH 7.0) and 100 mM NaCl. For reducing conditions, the buffer contained 1 mM DTT. Ellipticity was measured at 222 nm in 1°C steps from 20 to 90°C at a rate of 1°C/min.
Small Angle X-ray Scattering-Solution x-ray scattering data were collected at the National Synchrotron Light Source (Brookhaven National Laboratory) on beamline X-9. Scattering intensity data for both hBST-2 and mBST-2 ectodomains were collected at 2, 5, and 10 mg/ml in buffer consisting of 20 mM Hepes (pH 7.5), 100 mM NaCl, and 5 mM EDTA. Scattering data for each concentration were collected in triplicate and averaged after normalization and buffer subtraction. All data analysis and calculations were performed using the AT-SAS software suite (45). Data were processed in PRIMUS and regularized using GNOM. Calculated scattering plots from CRYSOL using the mBST-2 ectodomain crystal structure dimer as well as tetramers produced by crystal packing did not fit the observed scattering data well. Mixtures using these three models in OLIGMER did not fit the data well either; thus, ab initio modeling was employed. Models were created using GASBOR (46) with the final models a result of 10 independent predictions averaged in DAMAVER.
Multi-angle Light Scattering-Purified BST-2 ectodomains were subjected to size exclusion chromatography/multi-angle light scattering (MALS) using a Waters HPLC system connected to a DAWN HELEOS II 18-angle light scattering detector (Wyatt Technology Corp.) combined with an Optilab rEX differential refractometer. Protein samples (300 l) were loaded onto a Superdex S-200 10/300 GL gel filtration column (GE Healthcare) at a concentration of ϳ2.0 mg/ml and run at 0.5 ml/min in buffer consisting of 20 mM Tris (pH 7.5), 150 mM NaCl, and 0.01% NaN 3 . Data were recorded and processed using ASTRA software (Wyatt Technology Corp.).
Biochemical and Functional Characterization of Refolded
BST-2 Ectodomains-Recombinant mBST-2 and hBST-2 ectodomains were produced by refolding from denatured inclusion bodies; thus, the proteins were characterized to verify that they were similar to the natively folded surface-expressed forms. Both hBST-2 and mBST-2 ectodomains displayed a shift to dimer molecular masses on SDS-PAGE under nonreducing conditions (Fig. 1A), indicating that intermolecular disulfide bonds had formed, as observed in the native surface-expressed protein (30). Additionally, the refolded mBST-2 ectodomain was assayed for binding to mAb raised against cell surface-expressed mBST-2 (47). The mBST-2 ectodomain bound with high affinity (K D ϭ 1.8 nM) (data not shown) and was able to compete for binding with BST-2-expressing cells (Fig. 1B). No antibody binding to the hBST-2 ectodomain was observed. Thus, the refolded BST-2 ectodomains displayed features consistent with properly surface-expressed proteins. Defined higher order oligomers were not observed by gel filtration chromatography coupled with MALS, as both mBST-2 and hBST-2 ectodomains eluted predominantly as a single species in solution with determined masses corresponding to the respective covalent dimers (Fig. 1C).
BST-2 Ectodomains Are Thermally Unstable in the Absence of Intermolecular Disulfide Bonds-Previous biophysical studies of the hBST-2 ectodomain indicated that the coiled-coil dimer is highly unstable upon reduction of the intermolecular disulfide bonds (32). We sought to evaluate whether or not this structural instability is a conserved feature of the BST-2 ectodomain. The hBST-2 and mBST-2 ectodomains display a sequence identity that is near the average for this molecule when compared across species (50% identical) (see Fig. 3, A and H). Both mBST-2 and hBST-2 ectodomains are highly ␣-helical in solution as assessed by circular dichroism, and helical content was slightly reduced by breaking the intermolecular disulfide bonds via the addition of reducing agent (Fig. 1D). Thermostability was greatly affected, however, as the denaturing temperatures dropped dramatically in the presence of reducing agent (for the mBST-2 ectodomain, T m(ox) ϭ 68°C and T m(red) ϭ 32°C; and for the hBST-2-ectodomain, T m(ox) ϭ 61°C and T m(red) ϭ 31°C) (Fig. 1E). Thus, the intermolecular disulfide bonds are required to provide stability. This suggests that the coiled-coil instability encoded in the BST-2 ectodomain may be an evolutionarily conserved feature that is required for function.
Crystal Structure of the Full-length mBST-2 Ectodomain-The crystal structure of the mBST-2 ectodomain was determined to a very high resolution (1.6 Å). The final model was very high quality ( Table 1) and consisted of one homodimer in the asymmetric unit ( Fig. 2A). Electron density was visible for nearly the entire ectodomain (Fig. 2B), with chain A missing only four N-terminal residues and chain B missing five N-terminal and seven C-terminal residues. The structure reveals an elongated parallel ␣-helical dimer bridged by three intermolecular disulfide bonds. The ectodomain is ␣-helical for nearly the entire length with the exception of the C-terminal seven-residue GPI-connecting loop and presumably the initial disordered five-residue TMR-connecting loop. The extended conformation has the approximate dimensions of 25 Å (width) ϫ 145 Å (length).
A majority of the ectodomain (amino acids 68 -138) conforms to coiled-coil packing. Deviations from coiled-coil packing occur in short ␣-helical regions on either side of the central coiled coil (HR1 and HR2). Near the N terminus, the ␣-helices are slightly splayed due to the presence of two destabilizing residues that occur at heptad interface positions, an ␣-helix-destabilizing glycine (Gly-61) followed by a hydrophilic residue (Gln-65) (see Fig. 3, A and B). The C-terminal end of the coiled coil is disrupted by Lys-142, which lies at a heptad interface position and splays apart the coiled coil. In contrast to the previously determined structure of a C-terminal fragment of the hBST-2 ectodomain (Fig. 2C) (32), the mBST-2 ectodomain is more tightly packed as gauged by the coiled-coil radius and pitch (Fig. 2D) and displays no stutters (discontinuities) in the coiled-coil heptad repeat. Overall, the two structures are quite divergent (root mean square deviation (r.m.s.d.) of 2.35 Å for C-␣ overlay of the dimers and r.m.s.d. of 1.15 and 0.94 Å for individual chains A and B, respectively). This is due mostly to four nearly consecutive conservative differences at heptad positions in a short section from Lys-110 to Ile-121. The comparative positions between mBST-2 and hBST-2 are Lys-110/Glu-105, Ala-114/Gly-109, Ile-121/Val-113, and Val-128/Ile-120. The largest variation in this stretch is the Glu-105 and Gly-109 combination in hBST-2, which causes a stutter (discontinuity) in the heptad repeat at Gly-109 (32). In comparison, the equivalent positions of mBST-2 actually pack tighter as evidenced by the decrease in coiled-coil radius in this section (Fig. 2D). The other major structural divergence occurs near the C-terminal end, where the mBST-2 ectodomain starts to splay apart due to Lys-142, whereas this section of hBST-2 becomes more tightly packed.
The interface residues in the region N-terminal to Cys-96 are highly conserved when comparing mouse and human. There are four differences in this region: Gln-65/ Val-60, Thr-82/Ala-77, Leu-86/Phe-81, and Ala-89/Val-84. Interestingly, the occurrence of Phe-81 (in the hBST-2 ectodomain) at the coiled-coil interface may explain the proteolytic susceptibly at this position compared with the mBST-2 ectodomain, as the large Phe side chain would upset regular coiled-coil packing. In summary, the mBST-2 coiled coil displays tighter packing, no coiled-coil discontinuities, and reduced endogenous proteolytic susceptibility compared with the hBST-2 ectodomain due largely to a handful of conservative substitutions at the dimer interface. However, despite these structural observations, the mBST-2 ectodomain is highly unstable in the absence of the intermolecular disulfide bonds.
Design of an Unstable Coiled Coil Revealed by Structurebased Sequence
Analysis-Elucidation of the full structure of the coiled-coil ectodomain allowed analysis of the heptad interface across all BST-2 sequences (Fig. 3, A and B). Much of the sequence conservation and invariance across species are found at the coiled-coil interface (Fig. 3, C and G). Coiled coils possess characteristic heptad sequence repeats in which the A and D positions are normally occupied by small hydrophobic residues (e.g. valine, isoleucine, and leucine). This creates a hydrophobic seam that zippers ␣-helices together with A and D positions interlocked in what is termed "knobs-into-holes" packing. Introduction of hydrophobic side chains that are too large (e.g. phenylalanine) or too small (alanine) or hydrophilic residues is not favored and can disrupt the interface. Sequence analysis of the BST-2 coiled-coil interface across species revealed a unique design composed of short stretches of invariant stabilizing residues alternating with stretches of destabilizing residues (Fig. 3B). Coiled-coil formation is a cooperative phenomenon, requiring a critical fragment length for stability. Thus, it appears that BST-2 has been evolutionarily designed with encoded instability and that this designed instability is required for optimal antiviral function. 53-151). A, the 1.6-Å crystal structure of the mBST-2 ectodomain reveals a parallel ␣-helical covalent dimer bridged by three intermolecular disulfide bonds. The approximate dimensions shown were calculated in MOLEMAN2. Chain A includes residues 57-151, whereas chain B includes residues 58 -144. Cysteine residues involved in intermolecular disulfides (blue boxes) are shown as sticks, as are the two N-linked glycosylation sites (green boxes). TM, transmembrane region. B, electron density after rigid body refinement of the molecular replacement solution. The model shown is the final coordinates. Grey mesh represents a 2F o Ϫ F c map contoured at 2.0 , whereas green mesh represents F o Ϫ F c contoured at 3.0 . Note the readily identifiable disulfide electron density. C, superposition of the mBST-2 ectodomain (cyan) and an hBST-2 ectodomain fragment (grey; Protein Data Bank code 2X7A) (32). Structures were superposed using the "user defined match" option in CCP4MG Version 2.4.0. The C␣ r.m.s.d. between superposed dimers is 2.35 Å (r.m.s.d. for individual chains are 1.15 and 1.10 Å, respectively). D, comparison of coiled-coil (CC) parameters for the mBST-2 (blue) and hBST-2 (grey) ectodomains. Values were measured using TWISTER (42). Ang., angstrom.
Surface Analysis of the BST-2 Ectodomain-Surface mapping of sequence conservation can highlight functional hot spots. So far, the antiviral activity is the only function of BST-2 that is conserved across all species studied. Thus, we would expect clusters of sequence-conserved surface residues to highlight regions important for the antiviral function. Two major patches of surface conservation occur in the areas near the two N-linked glycosylation sites, Asn-70 and Asn-97 (Fig. 3B). A third major patch occurs at the C-terminal half in a region from Arg-120 to Val-128. It is possible that this could be a binding site for a yet unidentified accessory protein. Interestingly, a cluster of surface mutations near the N terminus that were shown to disrupt BST-2 antiviral function against HIV-1 are partially coincident with the first patch of sequence conservation (Fig. 3F) (32). The electrostatic surface of mBST-2 shows some features, most notably a large patch of basic residues at the N terminus (Fig. 3E).
BST-2 Ectodomains Are Conformationally Labile in Solution-
The solution structures of the mBST-2 and hBST-2 ectodomains were evaluated by small angle x-ray scattering (SAXS). Guinier analysis revealed radii of gyration of 46.2 Å for the mBST-2 ectodomain and 51.2 Å for the hBST-2 ectodomain (supplemental Fig. S1). Calculation of the predicted scattering pattern using the mBST-2 ectodomain dimer did not fit the experimental data well, so ab initio modeling was employed. Maximal protein dimensions (D max ) calculated from the distance distribution function p(r) were 150 Å for the mBST-2 ectodomain and 180 Å for the hBST-2 ectodomain. The ab initio models fit experimental data well with discrepancies () of 1.7 and 2.6 for the mBST-2 and hBST-2 ectodomains, respectively (Fig. 4, A and B). Individual predictions were all irregular twisted rods, which averaged together to produce overall models of elongated bent rods for both ectodomains (Fig. 4, C and D). The distribution of dimensions for the mBST-2 ectodomain is 150 ϫ 60 ϫ 45 Å, whereas that for the hBST-2 ectodomain is 180 ϫ 85 ϫ 65 Å. The predicted structure for the hBST-2 ectodomain showed a much more diffuse distribution than that for the mBST-2 ectodomain, indicating a higher degree of conformational lability. Further analysis of scattering data revealed features at FIGURE 3. Sequence and surface analysis of the BST-2 ectodomain. A, alignment of BST-2 sequences. Invariant residues (except cysteines) are magenta, invariant cysteines are blue, and residues highlighted yellow denote a conservation index of 6 or higher as determined by ALSCRIPT (43). The secondary structure of the mBST-2 ectodomain is shown above the alignment. Numbering is from mBST-2 (mBST-2#). Green boxes denote the N-linked glycosylation (N-Glyc) sites, and blue boxes highlight trafficking motifs utilized by AP-1 and AP-2. GPI anchor positions predicted by the BIG-PI Predictor (44) are shown as purple boxes. The letters A and D indicate coiled-coil core heptad repeat positions as determined by TWISTER. Dots above the sequence indicate residues located at the interface of the two possible dimer-of-dimers assemblies in the crystal: assembly 1 (red dots), assembly 2 (blue dots), and both assemblies (violet dots). Suffixes in sequence names indicate species as follows: mouse, Mus musculus; human, Homo sapiens; rat, Rattus norvegicus; chimp, Pan troglodytes; rhesus, Macaca mulatta; African green monkey (agm), Chlorocebus tantalus; dog, Canis lupus familiaris; and pig, Sus scrofa. B, sequence logo frequency plot of coiled-coil interface residues. Blue, cysteine; green, favorable coiled-coil interface residues (ILVT); red, unfavorable coiled-coil interface residues (all others). Numbering of interface position is color-coded according to sequence conservation as shown above. C, sequence conservation and invariance at the covalent dimer interface. One chain of the dimer is shown as space-filled and colored according to the sequence alignment shown in A, whereas the other is shown as a cyan coil. D, sequence conservation mapped onto the solvent-accessible surface of the mBST-2 ectodomain. E, charge-smoothened electrostatic surface as calculated by PyMOL. Blue denotes positively charged surfaces, whereas red denotes negatively charged surfaces (gradient, ϩ70 to Ϫ70). very low angles indicative of oligomerization (45). In summary, SAXS analysis indicated that the BST-2 ectodomain is conformationally flexible in solution, that the hBST-2 ectodomain is more labile than the mBST-2 ectodomain, and that both appear to form higher order associations in solution.
DISCUSSION
The literature to date indicates that BST-2 restricts enveloped virus release through simultaneous inclusion in both host and viral membranes (28,29). The BST-2 ectodomain performs the crucial role of linking the two membrane-anchoring regions (TMR and GPI anchor). Although an artificial BST-2 mimic in which much of the native ectodomain is replaced by a generic coiled coil from dystrophia myotonica protein kinase is still able to inhibit virus release, activity against HIV-1 is ϳ10-fold lower compared with native BST-2 (28), implying that the unique features of the BST-2 ectodomain outlined in this study have optimized the protein for antiviral function. Here, we have shown for the first time that the entire BST-2 ectodomain can form an extended ␣-helical confirmation, much of which is a coiled coil that is similar in appearance to a two-stranded rope. This extended structure allows for maximal separation of the membrane anchors to preclude inclusion of both into a budding virion, which would thwart successful virus capture. In addition, the length of the BST-2 ectodomain (150 Å for mouse and 180 Å for human) is longer than that of many virus spikes (e.g. ϳ120 Å for HIV-1 (48)), which would also be required to bridge the separating membranes.
The BST-2 ectodomain displays conserved structural and physical properties that are crucial to function. The mBST-2 ectodomain is a parallel homodimer bridged by three disulfide bonds. The functional importance of the intermolecular disulfides has been demonstrated, as mutation of single cysteines has little effect on virus-tethering function, whereas mutation of all three destroys antiviral function against HIV-1 (28,31). Here, we have demonstrated that the coiled-coil ectodomain is unstable in the absence of the intermolecular disulfides, as both mBST-2 and hBST-2 ectodomains displayed greatly decreased thermostability under reducing conditions. This appears to be a evolutionarily conserved feature of the ectodomain, as sequence analysis suggested a design composed of alternating stretches of residues that stabilize and destabilize the coiled-coil interface. The importance of proper formation of the last two intermolecular disulfides is highlighted by the invariant stretch of coiled coil-stabilizing residues immediately preceding them. Interestingly, the first disulfide is preceded by an invariant ␣-helix-destabilizing glycine followed mostly by a hydrophilic residue. These residues disrupt regular coiled-coil packing, yet the disulfide is able to form. It should be noted that clusters of mutations designed to completely disrupt the coiled-coil interface ablate the antiviral function against HIV-1 (32). Thus, a fine balance between instability and functional design has been achieved.
These encoded instabilities also likely produce the conformational lability observed for the mBST-2 and hBST-2 ectodomains in solution. Although both are predicted to be bent, non-rigid structures in solution, hBST-2 appears to be more conformationally diffuse, likely due to the introduction of more coiled coil-destabilizing side chains and discontinuities compared with mBST-2. In terms of antiviral function, the assembly and budding of virions are structurally dynamic processes, and the conformational plasticity of the BST-2 ectodomain would be advantageous for viral trapping. Similar coiled coil-destabilizing designs are also observed in streptococcus M1 protein (49), tropomyosin (50), and myosin (51). In these proteins, the instabilities cause helical unwinding, which, for myosin, is necessary for function (51,52). BST-2 is unique in this regard, as the intermolecular disulfides prevent major separation of the helices, so the end result of the coiledcoil instabilities is a dynamic structure. It is also possible that BST-2 may have a yet unidentified conserved cellular function involving a cell-surface binding partner as suggested by the presence of large surface patches of sequence conservation. Indeed, hBST-2 has been shown to bind the plasmacytoid dendritic cell receptor ILT7 and signal to inhibit type I interferon secretion (53). However, this function may not be conserved between humans and mice, as there is no known murine ortholog for ILT7.
Analysis of crystal contacts can highlight possible oligomers that may have functional relevance. Such analysis for the mBST-2 ectodomain revealed two possible dimer-ofdimers assemblies that bury significant amounts of surface area (Fig. 5, A-C). These associations may be weak, as they were not observed under the flow conditions of size exclusion chromatography. However, extensive contacts are observed in the crystal, and features of SAXS in solution suggest that some self-association may occur. Interestingly, both bear a slight similarity to the BAR domain family (Fig. 5D). BAR domains are ␣-helical modules that dimerize and form crescentshaped structures that bind to and stabilize curved membranes during membrane trafficking events (54). Of the two assemblies, assembly 2 is more likely to occur, as the interface is larger, is composed of more non-polar residues, and is composed of residues near the C terminus, away from the N-linked glycosylation sites that would prevent dimerization. In addition, this assembly would place the GPI anchors of adjacent dimers near each other, which is also consistent with the propensity of GPI anchors to localize to lipid rafts. This assembly also contains patches of basic residues on the predicted membrane-facing side, consistent with a possible membrane-binding function. Because viral assembly and release from the cell surface involve membrane deformation and bending, it is reasonable to hypothesize that these curved assemblies may have some functional purpose in the antiviral function of BST-2.
High local concentrations would support the formation of such oligomers, and in support of this, recent immunogold cryo-EM studies demonstrate that BST-2 forms clusters at HIV-1 budding sites (55,56). Viral assembly is a dynamic process involving sorting and clustering of surface glycoproteins, membrane bending, and finally membrane fission. Thus, it is reasonable that such BAR-like curved shapes could have functional significance. For example, they could aid in targeting BST-2 to cluster around the neck of a budding virus for correct positioning to avoid full inclusion in the progeny virion, or higher order structures could form proteinaceous collars around the neck, such as observed for the CIP4 F-BAR module (57). Such collars could stall or inhibit viral budding.
On the basis of our structural analysis and the current literature, we propose the following model of BST-2-mediated restriction of virus release. BST-2 must first locate to regions of viral assembly. Most enveloped viruses are believed to begin their assembly processes in lipid rafts. Given that GPI anchors prefer these cholesterol-rich microdomains, this is the most likely mechanism of co-localization. Another possible mechanism could involve sorting via an intracellular partner, such as RICH2 (58). Given the current information, we prefer the former, with an orientation such as that observed in assembly 2 (Fig. 5C) being likely. This orientation allows clustering of the GPI anchors of a pair of BST-2 homodimers near a lipid raft viral assembly site (maximizing for probability of inclusion) and keeps the N-terminal TMRs at a safe distance to prevent their inclusion in the viral membrane. As the viral proteins assemble and the membrane begins to curve outward, the structural plasticity of the BST-2 ectodomain would come into play, as it would be able to change shape as needed while staying firmly anchored in the viral membrane. At this point, the curved structures of BST-2 assemblies could be utilized, allowing BST-2 to cluster and collar around the neck to keep it near the host membrane and prevent full inclusion in the viral membrane. Next, the virus buds off the surface with a membrane anchor of the BST-2 homodimer planted in the viral membrane. Thin-section EM images support a large distance between virions and the plasma membrane (7,28), and immunogold cryo-EM studies suggest a rod-like structure for BST-2 connecting the virus to the plasma membrane (56). This evidence points to a model with opposite ends of the homodimer embedded in the host and viral membranes, respectively. Taking into account steric considerations, we prefer an orientation with the GPI anchor stationed in the viral membrane, as this would place the narrower end of the molecule in the viral membrane with the larger N-terminal end of the dimer (containing four bulky glycosylation appendages) juxtaposed. Having the narrower end of the molecule in the viral membrane would allow for better avoidance of viral coat proteins during assembly and budding.
The physical features of the BST-2 ectodomain appear to be optimally developed for function as a virus tether. First, the extended structure provides separation of the membrane anchors so as to avoid full inclusion in the viral membrane. This structure is also longer than many enveloped virus spikes, so it can span the distance between the host and viral membranes. The conformational plasticity of the dimeric ectodo- FIGURE 5. Possible assemblies created by crystal packing. A, interface surface analysis for the two major assemblies created by crystal packing. Symmetry operations creating the assemblies were as follows: assembly 1, Ϫx ϩ 5/2, y, Ϫz ϩ 1; and assembly 2, Ϫx ϩ 1/2, y, Ϫz. ASA, accessible surface area; N HB , number of hydrogen bonds; N SB , number of salt bridges; N DS , number of disulfide bonds; Non-cons., non-conserved. B and C, assemblies 1 and 2, respectively, viewed from the perspective of a side view on an outwardly curving membrane (left) and the membrane-facing surface (right). Below the ribbon diagrams are the respective electrostatic surfaces as calculated by PyMOL. For B, the C-terminal GPI-connecting loop has been removed from chain A. D, comparison with the FBP17 F-BAR domain (Protein Data Bank code 2EFL), a member of the BAR domain superfamily that binds to curved membranes. main allows it keep up with dynamic membrane deformation and protein assembly events that occur during viral genesis. Finally, curved assemblies of BST-2 may bind curved membrane to correctly position BST-2 near the neck of the budding virus.
Although much mechanistic detail of BST-2 antiviral function has been elucidated in a short time, many questions remain. BST-2 appears to be effective against a number of enveloped viruses (in the absence of known antagonists); however, the full spectrum of BST-2 antiviral capability is unknown. Additionally, the contribution of intracellular binding interactions to BST-2 function has not been investigated. Also, in a broader context, the normal cellular function of BST-2 is largely unknown. Most importantly, the detailed mechanisms of viral antagonism of BST-2 are poorly understood. A full understanding of this host-pathogen relationship could lead to the development of broad-spectrum antiviral therapies against enveloped viruses that exploit BST-2 activity. | 2018-04-03T04:57:02.291Z | 2010-11-17T00:00:00.000 | {
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265020207 | pes2o/s2orc | v3-fos-license | The predicting role of serum tumor-specific growth factor for prognosis of esophageal squamous cell carcinoma
Objective Tumor-specific growth factor (TSGF) is an immune-related factor that offers good performance in the clinical management of human cancers. However, the role of serum TSGF in esophageal squamous cell carcinoma (ESCC) has not been fully clarified. Methods A total of 562 ESCC cases were collected in our study, with available information on preoperative serum levels of TSGF at diagnosis. Preoperative serum TSGF was detected using the rate method. We retrospectively analyzed its correlation with clinicopathological features of ESCC and survival. Results The cut-off value of serum TSGF was determined to be 60.5 U/mL by receiver operating characteristic (ROC) analysis. Serum TSGF was associated with gender (P < 0.001), tumor location (P = 0.022), tobacco use (P < 0.001), alcohol consumption (P < 0.001), lymph node involvement (P = 0.007), and TNM staging (P = 0.004). The survival analysis revealed that ESCC patients with high levels of serum TSGF had poorer prognosis than those with high TSGF (P = 0.006), especially for male ESCC cases (P = 0.001), under 60 year (P = 0.036), male middle location (P = 0.023), tobacco consumption (P = 0.004), G1 + G2 (P = 0.031), advanced T staging (P = 0.033), lymph node involvement (P = 0.003), TNM staging (P = 0.003). Univariate Cox regression analysis indicated that exposure to smoking and drinking, tumor grade, T staging, lymph node metastasis, TNM staging, and serum TSGF level were the prognosis-related factors of ESCC. Multivariate regression analysis revealed that smoking history, higher serum TSGF levels, and advanced T stage enhanced the risk of ESCC-related death. Conclusion In brief, serum TSGF levels had in relation to malignant features of ESCC. It was positively correlated with survival but was identified as an independent risk factor for ESCC.
Introduction
Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies involving the digestive system, and the leading cause of cancer-related deaths worldwide owing to its aggressiveness and adverse survival probability.In China, ESCC is the principal subtype of esophageal cancer and remains the sixth leading cause of cancer incidence and the fourth leading cause of cancer mortality [1].In recent years, the clinical approaches to overcoming ESCC have focused on the combination therapy comprising operative treatment, chemotherapy, and radiotherapy based on patient background and TNM staging by the American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) clinical stage [2].Currently, emerging immunotherapy is an additional treatment method [3].However, tumor development dominated by diverse tumor environments and complex heterogeneity.Despite the more extensive efforts to defeat ESCC and improve the prognosis, it is indispensable to find a reliable clinically beneficial biomarker to trace the progression of ESCC.
Tumor-specific growth factor (TSGF) is a collective name that includes multiple substances related to the malignant growth of tumors.Based on previous efforts, accumulating studies have revealed the importance of serum TSGF for the clinical identification and assessment of treatment effects in various malignancies, such as breast cancer, and digestive tract carcinoma [4][5][6][7][8][9][10][11][12][13][14][15][16][17].Of note, combining TSGF and conventional biomarkers improved essential clinical utility.The combination of color Doppler ultrasound with serum carcinoembryonic antigen (CEA), carbohydrate antigen 153 (CA15-3), and TSGF detection enhanced the diagnostic efficiency of breast cancer [4].TSGF and other tumor markers combined with 18 F-FDG-PET achieved approximately 97.3% of diagnostic accuracy, and yielded 100% specificity for discriminating prostate cancer [11].The joint detection of serum CEA, carbohydrate antigen 72-4 (CA72-4), carbohydrate antigen 19-9 (CA19-9), and TSGF has shown robust discriminatory power in gastric cancer (AUC = 0.913, sensitivity: 88.9%) [6].Similarly, the combined assay with serum TSGF, carbohydrate antigen 242 (CA242), and CA19-9 appeared to be able to identify pancreatic cancer [7].Meanwhile, three serum markers, consisting of AC007271.3,squamous cell carcinoma antigen (SCCA), and TSGF, resulted in an AUC of 0.917 with a sensitivity of 80.0% and specificity of 93.1% in oral squamous cell carcinoma [14].TSGF predicts the survival consequence of pancreatic cancer patients undergoing cryoablation treatment [8], utilizes to monitor the treatment response in colon cancer [9], and determines the survival of bladder cancer patients undergoing robotassisted radical cystectomy [10].For primary hepatocellular carcinoma, patients carrying increased TSGF levels obtained a reduced 3-year survival rate, showing its value for evaluating survival benefit of transcatheter arterial chemoembolization (TACE) [12].For osteosarcoma, higher TSGF levels decreased 5-year survival rate [15].Yang L et al. performed a study with 753 retecal carcinomas and demonstrated that decline of TSGF in 5th postoperative day (POD) less than 10 U/mL showed a positive correlation with anastomotic leakage (AL) after anterior resection for rectal cancer with a double stapling technique [16].Altogether, these results demonstrate that serum TSGF plays a crucial role in the screening, prognosis prediction, and treatment outcome of cancer.However, its role in ESCC is unclear.
Unexpectedly, only a few studies are currently available that have examined the role of TSGF in esophageal carcinoma.The combined detection of AgNOR, SCCA, CEA, and TSGF was favorable for screening esophageal carcinoma [18].Therefore, we retrospectively reviewed 562 ESCC cases to assess their clinicopathological relevance and potential role as prognostic indicators for ESCC.
Data collection
The clinical data were obtained from patient's medical records and included basic information such as age at diagnosis and sex; cancer-related information such as tumor location, tumor grade, pathologic T stage, lymph node status, and TNM stage; lifestyle information such as tobacco consumption and alcohol consumption; preoperative detection based on blood assays including serum TSGF level; and treatment options.Follow-up was performed via telephone and regular outpatient consultation according to National Comprehensive Cancer Network (NCCN) guidelines.Overall survival (OS) time was calculated from the date of surgery to the date of the most recent follow-up or death.And the follow-up period ended in December 2022.
Serum TSGF assay
A 3-mL sample of peripheral blood was withdrawn from ESCC patients and the serum was obtained for detection after centrifugation at 1000 × g for 10 min.Hemolysis, jaundice, and lipids were excluded from all samples.Importantly, if the serum TSGF was not detected immediately, it was stored at -20℃ or -80℃ and protected from repeated freezing and thawing.Serum TSGF levels were measured by BIOELAB ES-200 Automatic Biochemistry Analyzer(Changsha, China) according to the manufacturer's instructions(tumor-specific growth factor assay kit, Hunan Newland Biotechnology Co., Ltd, China).After the completion of the test, the results were obtained based on the standard curve.
Statistical analysis
All data were processed using SPSS 22.0 software (IBS SPSS, Armonk, NY, USA).The ideal cut-off values for TSGF levels were determined using receiver operating characteristic (ROC) analysis: the survival status of ESCC patients as the status variable and TSGF as the test variable.Its correlation with clinicopathological features of ESCC was investigated via the chi-square test.Overall survival and stratified analysis were examined using the Kaplan-Meier method and log-rank test.The univariate and multivariate analyses were executed following the Cox proportional hazards regression model.P values less than 0.05 were regarded as statistically significant.
The characteristics of ESCC patients
As shown in Tables 1, 562 ESCC patients were recruited including 372 males and 190 females.The age of the enrolled ESCC cases ranged from 40 years to 81 years old, with a median age of 61 years.Drinking and smoking habits were risk factors promoting ESCC.In our study, 286 cases with ESCC had exposure to tobacco use, and 181 patients harbored an alcohol-drinking habit.The dominant location was the middle esophagus in 417 cases, the lower segment in 116 cases, and the upper esophagus in only 29 cases.For tumor grade, moderately differentiated ESCC is the dominant subgroup, accounting for the majority of the total cases (62.46%), followed by poorly differentiated (35.77%) and well-differentiated ESCC (1.78%).Moreover, 66.01% of ESCC patients were at the advanced T stage at diagnosis, and 33.99% were diagnosed at the early T stage.In total, 42.53% of ESCC patients harbored advanced TNM stages at the time of diagnosis, and 43.77% had lymph node involvement.Of note, the patients at the II stage were the majority of all ESCC cases (266/562), followed by III stage (207/562).
Serum TSGF predicts survival for prognosis of ESCC patients
Given its predictive role for cancer-related prognosis, we further performed the survival analysis.We found that ESCC patients with low levels of serum TSGF had worse clinical outcomes than those with high levels of TSGF (P = 0.006) (Fig. 1).This result suggested that serum TSGF may be a prognostic indicator of ESCC.
To tailor the refined prediction, we further performed stratified analysis.The results indicated that increased serum TSGF was an excellent predictor for adverse outcomes in ESCC patients with younger age (< 60 years) (P = 0.036), male sex (P = 0.001), middle location (P = 0.023), tobacco consumption (P = 0.004), G1 + G2 (P = 0.031), advanced T staging (P = 0.033), lymph node involvement (P = 0.003), and TNM staging (P = 0.003).Of note, we found that higher serum TSGF levels in ESCC patients with or without a drinking history were prone to shorten the survival time.(Fig. 2).Additionally, we evaluated the predictive role of different levels of TSGF for ESCC-related deaths(Fig.3).The analysis showed that high TSGF increased 1.476-fold the risk of death for ESCC under 60 year group than level TSGF.Similarly, high TSGF was a risk factor for poor prognosis in the male ESCC group(HR = 1.576).In another subgroup, for the cases with middle ESCC, having smoking and drinking habits, well and moderate differentiation, positive lymph node, and advanced TNM staging, high TSGF acted as an unfavorable factor for survival of ESCC.
Discussion
In our present research, we investigated the predictive property of preoperative serum TSGF and explored its clinical utility in ESCC.We analyzed the correlation between serum TSGF and some clinical parameters, including the survival of ESCC.We found that it correlated with sex, tumor location, smoking and drinking history, TNM stage, and lymph node involvement.Epidemiologically, smoking and alcohol drinking enhanced the risk of ESCC [19].Even the studies depicted tobacco and alcohol may play a positive synergistic effect to increase 3-fold of the risk of suffering ESCC compared with the predicted risk of any single factor alone(only 20-30%) [20].In Non-small cell lung cancer, the median and positive rates of TSGF were 64(56-67)μ/mL and 10.14%(28/276).However, TSGF level showed no statistical difference between smokers(median: 63, range: 53-66) and non-smokers(median: 62, range: 51-67) (P > 0.05) [21].To date, there was no report about its relationship with drinking alcohol.We firstly indicated that TSGF was associated with smoking and drinking history.And higher TSGF in the smoking and alcoholdrinking groups predicted poor clinical outcome of ESCC.More importantly, habitually consuming tobacco, alcohol, and areca nuts can influence the age-onset of male ESCC [22].
Commonly, metastasis leads to an unfavorable clinical outcome of ESCC.For ESCC, lymph metastasis showed a remarkable correlation with malignant features such as tumor differentiation, perineural invasion, Note: Age(< years vs. ≥ years), gender: female vs. male, location: middle vs. upper, lower vs. upper, grade: G3 vs. G1 + G2, T stage: T3 + T4 vs. T1 + T2, smoking history: yes vs. no, dringking history: yes vs. no, lymph node status: positive vs. negative, TNM stage: III+IV vs. I+II, TSGF: high vs. low Fig. 3 Predicating role of preoperative serum TSGF level for risk of deaths of ESCC patients.LN: lymph node advanced-stage tumor, and venous invasion.It predicted a worse prognosis and could act as an independent prognostic factor for ESCC [23].In our cohort, 246 of 302(43.77%)ESCC cases harbored lymph node involvement, which was slightly higher than the previous report with 36.8% lymph node metastasis [23].Min BH et al. demonstrated that the different LNM rates may be associated with tumor invasion depth [24].The LNM rates of tumors invading the lamina propria, muscularis mucosa, and SM1 layers were 3.7%, 15.5%, and 40.7%, respectively.To date, there have been few available studies on the predictive role of the serum level of TSGF for lymph node involvement in cancer.TSGF is a polypeptide that triggers initiation and facilitates metastasis of cancer.TSGF levels have been related to tumor differentiation in pancreatic cancer [8] and lymph node involvement in colon cancer [9].Our previous study revealed that increased serum TSGF may enhance the emergence of multifocality and the development of PTC, suggesting that TSGF may be qualified as an excellent promising marker for predicting lymphatic involvement in PTC [25].Our results focused on the clinical utility of TSGF in cancer.Nevertheless, its role in ESCC for predicting prognosis was not uncovered.Our analysis supported that increased TSGF was likely to develop metastasis involving the lymph node in ESCC.This result suggested that serum TSGF may play a distinct role in cancer progression.
In general, the TNM stage represents the tumor burden in tumor-bearing patients.Advanced TNM stage not only results in increased metastasis probability, including more lymph node involvement, but also contributes to poor survival.In the past several years, the therapeutic benefit of ESCC patients was limited by the late diagnosis and asymptomatic early disease [1].In our present study, we identified that the cases were predominantly at stage II, and 239 of 562 (41.72%) had advanced disease.Moreover, the ESCC patients with high TSGF levels were prone to be at advanced TNM stages.And TNM stage was a predictor of lymph node involvement and poor prognosis [26].Our result was indicative of its pro-tumor effect in ESCC progression.In addition, there was no significant correlation with other traits, such as age, tumor grade, or pathologic T stage.
To date, whether serum TSGF has a connection with cancer-related deaths has been previously described in several types of cancer.It has been reported that serum TSGF exhibits a superior early detection capability [4-7, 11, 13], prognosis prediction role [12,15], and response to the therapeutic outcomes [8-10, 12, 14-16] of malignancies.Based on the previous exploration, combining serum TSGF with traditional biomarkers or other detection tools caused better diagnostic accuracy [5].Higher levels appeared to correlated with worse clinical outcomes of cancer [12,15].Our findings indicated that ESCC patients with decreased serum TSGF had a better prognosis than those with high TSGF.This finding seemed to be consistent with the predictive value of hepatocellular carcinoma [12] and osteosarcoma [15].Furthermore, we conducted the strata analysis to achieve the satisfactory survival prediction for the special ESCC patients.We found that younger-aged ESCC patients with elevated serum TSGF had worse survival.There were similar results in the ESCC subgroup with male cases, middle esophagus, tobacco and alcohol consumption, well and moderate differentiation, advanced TNM staging and pathologic T stage, and lymph node metastasis.These findings might aid clinicians in determining the population at risk for exposure for precise therapeutic intervention and clinical management.
Finally, the univariate analysis showed that higher serum TSGF levels, some malignant features including TNM stage, lymph node status, pathologic T stage, and ESCC lifestyle factors such as tobacco consumption and alcohol exposure could lead to unfavorable clinical outcomes in patients with ESCC.However, multivariate analysis showed that only advanced pathologic T stage, smoking habit, and increased serum TSGF increased the risk of ESCC-related prognosis.Our results suggested it's necessary to explore the clinical implications of serum TSGF in ESCC.
There were also some limitations in this study.Noticeably, the cases at stage II accounted for most of our cohorts, while the minority were in stage IV.In our cohort, not all subjects had received the same post-operation treatment, and due to the relatively small sample size, it was unfavorable to perform a subgroup analysis based on different treatment options.In addition, the sample size from the single center was limited, although it was larger than the previous cohort.Therefore, it's necessary to enlarge the sample from multiple centers to obtain a better performance of TSGF.
Conclusion
Collectively, serum TSGF was relevant to malignant features of ESCC.Increased TSGF predicted the adverse prognosis of ESCC, especially for male patients aged under 60 and with lymph node involvement, advanced disease stage, and smoking and drinking habits.Moreover, the serum TSGF was qualified as an independent predictor for the clinical outcome of ESCC by multivariate analysis.Since the assay of cytokine levels in ESCC patients is feasible and convenient in clinical practice, we will perform further observation to investigate its clinical utility and the molecular mechanisms underlying TSGF activity to shed light on its role in ESCC progression.
Table 1
The clinical characteristics of ESCC patients
Table 3
The univariate and multivariate analysis | 2023-11-06T14:36:43.541Z | 2023-11-06T00:00:00.000 | {
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7659613 | pes2o/s2orc | v3-fos-license | Ten years on ART – where to now ?
Next year marks the tenth anniversary since the rollout of antiretroviral therapy (ART) in South Africa (SA), the country with the largest number of people living with HIV in the world. SA also has the world's largest antiretroviral therapy (ART) programme, with approximately 1.8 million people estimated to have received ART by mid-2011.
Next year marks the tenth anniversary since the rollout of antiretroviral therapy (ART) in South Africa (SA), the country with the largest number of people living with HIV in the world.SA also has the world's largest antiretroviral therapy (ART) programme, with approximately 1.8 million people estimated to have received ART by mid-2011. [1]he massive investments in treatment and prevention have been accompanied by great successes.Increasing ART coverage has led to a decline in HIV-related adult mortality.During the first 3 years of ART rollout, although ART uptake averaged around 40% of those in need, it has been estimated that AIDS mortality dropped by approximately 25%. [2]ince the announcement of a comprehensive care, management and treatment programme by the Department of Health in late 2003, access to ART in SA has increased dramatically.Over time, revisions to the national treatment guidelines have meant that more patients than ever are eligible to initiate lifesaving therapy.The shift from an ART eligibility criterion of a CD4 count <350 cells/µl, from the previous threshold of <200 cells/µl, resulted in adult ART coverage dropping from 79% to 52%, although clearly more patients were eligible to start ART.Although HIV testing figures have increased in the country, mainly because of the National Testing Campaign launched by the Minister of Heath in 2010, they are not yet high enough.Over a quarter of the population has been tested for HIV, but this has not translated into all those with a positive result being enrolled into care.The 2012 SA Clinicians Guideline recommends starting ART at a CD4 count <350 cells/µl and argues for the use of ART in discordant couples.This again increases the number of patients deserving of being enrolled into care.
There remain significant obstacles, both to access to ART for those who need it and to sustaining those already on treatment.The disparity between rural and more highly resourced urban areas, significant operational problems, and lack of public healthcare infrastructure and adequately trained healthcare workers to provide long-term quality care and treatment for the masses of HIV-infected patients are just some of the challenges.These obstacles must be dealt with even in the current context of severe shortages in the healthcare workforce.Two of these issues are addressed by research presented in this issue of SAMJ. [3,4]n years on ART -where to now?
Task shifting has been widely promoted as a mechanism for expanding ART access.Changes in treatment guidelines calling for less toxic treatment regimens, higher CD4 count thresholds for initiation and simplified laboratory monitoring schedules should facilitate such task shifting to lower-level personnel. [5]In the early years following ART rollout, Médicins Sans Frontières used task shifting in their community-based HIV programme to integrate HIV treatment successfully into well-established primary healthcare services.Strategies to increase treatment access while conserving resources without compromising patient outcomes have shown promise.Early studies of nurse-provided ART from rural Rwanda, Ethiopia and Lesotho established successful implementation.Results from the CIPRA-SA study, an SA randomised trial, confirmed that doctor-initiated, nurse-managed HIV treatment was not inferior to doctor-initiated, doctor-managed care. [6]Long and colleagues demonstrated also that stable patients managed at primary health clinics had a similar, or even better, 12-month outcome compared with those maintained at a hospital-based ART clinic. [7]There was the additional advantage of an 11% cost saving per patient treated.While this was not full Nurse-Initiated Management of Antiretroviral Treatment (NIMART), it did demonstrate that nurses could play a substantive role in managing HIV treatment.
Large-scale NIMART also appears to be feasible and acceptable in the primary sector health services in SA.Evidence for NIMART in Africa is limited and little is known about the key barriers to implementing these programmes on a large scale. [5]Data from the STRETCH study (Streamlining Tasks and Roles to Expand Treatment and Care for HIV) suggest that expansion of the primary care nurse role to include ART initiation and maintenance can be done safely and will improve health outcomes and quality of care, although it may not reduce time to ART or impact on mortality.Time to death for those receiving ART from primary care nurses was similar to that in patients receiving ART from doctors (hazard ratio 0.94, 95% confidence interval 0.76 -1.15). [5,8]As Nyasulu and colleagues highlight in this issue, decentralisation of ART by primary healthcare workers increases ART uptake and reduces workload at referral facilities. [3]In their study, 45 professional nurses from 17 primary healthcare facilities around Johannesburg were trained to initiate patients on ART.The number of initiations at the PHC facility increased significantly by 99 patients immediately after NIMART rollout and continued to increase by a further 9 initiations every month.Their study showed that decentralisation of services strengthened retention in care and reduced the burden of managing complicated cases at referral hospitals.
Although the shift to nurse-initiated care has already begun, it does have its limitations, the main ones being adequate and sustainable training, support and salaries for staff in new roles, the integration of new members into healthcare teams, and compliance with regulatory bodies. [9]While NIMART is promising, its success will require a comprehensive approach with clinical guidelines tailored to nurses along with a strong monitoring and evaluation capacity.Accordingly, efforts to determine whether ART can be managed safely and effectively at even lower levels of the healthcare system must also be explored.We await treatment outcome data to determine whether NIMART implementation will mirror the CIPRA results.
Another problem that the SA HIV programme faces is how to deal with the twin epidemics of HIV and tuberculosis (TB).It is estimated that one-third of the people living with HIV and AIDS worldwide are co-infected with TB.SA has one of the highest estimated TB rates in the world, ranking 4th among the 22 high-burden countries.For many years, TB and HIV care were largely separate; however, efforts to integrate TB and HIV services are included in the National Strategic Plan and National TB Programme to ensure that co-infected patients receive appropriate care and treatment.As Chimbindi and colleagues demonstrate in this issue, an integrated service that includes HIV and TB testing and treatment is convenient for most patients and ensures timely treatment initiation and optimal care. [4]s highlighted in this article, patients with TB face several obstacles in accessing care.However, among those who did access TB care there was a high uptake of HIV testing and appropriate management of co-infected patients in this rural public programme.
Should SA follow Ethiopia, Malawi and Uganda [10] and consider community health worker (CHW) programmes for HIV care and treatment?CHWs could play an important role in scaling up ART by taking over a number of tasks from professional health workers.CHW programmes throughout sub-Saharan Africa have improved access to, and coverage of, communities with basic health services.There is some evidence that these programmes can improve outcomes; however, moving HIV care to CHWs has only just begun to be explored.Interestingly, findings from a community-based programme in Kenya demonstrated that task shifting of ART from healthcare workers to people living with AIDS in the community had similar outcomes in terms of detectable viral load, mean CD4 count, decline in Karnofsky score, change in ART regimen, new opportunistic infections and pregnancy rates compared with those receiving clinic-based HIV care. [11]Evidence in areas other than HIV suggests that CHW programmes are feasible and effective.In Pakistan, community case management with oral amoxicillin of children with severe pneumonia by female health workers was equivalent to the standard of care, [12] while in Zambia CHWs were found to provide prompt and effective malaria case management with high adherence. [13]A study from Bangladesh, where CHWs screened villagers, collected sputum samples and administered directly observed therapy for TB treatment, reported high rates of case detection and treatment compliance, with a cure rate of at least 85% and a drop-out rate of only 3.1%. [14]gain, the success of these programmes depends on continuous training, simple guidelines with standardised protocols, appropriate support and supervision, strong relationships with formal health services and, finally, adequate remuneration or professional development to retain CHWs in the programme.Inadequate remuneration and lack of quality supervision and continuous training are of particular concern and may lead to a decrease in the quality of the programme over time.
While results from ART programmes are promising, limited human resources continue to hamper rollout.Task shifting offers high-quality, cost-effective care to more patients than a physiciancentred model and should be considered for implementation where human resource shortages threaten rollout of programmes.Given the shortfall in healthcare workers there is emerging consensus that some form of NIMART or ART provision by non-physicians will be required to achieve optimal ART coverage in SA.An integrated care model, perhaps in the hands of nurses, which incorporates appropriate management of HIV and TB with non-communicable diseases such as hypertension, diabetes and cancer, could be instrumental in the success of the proposed National Health Insurance scheme.NIMART and other strategies of integrated HIV care may help to alleviate the burden at hospital-based HIV clinics.With approximately 383 nurses per 100 000 people in SA (2011 figures), an inclusive approach of community-based organisations, home-based care or even caregivers themselves being able to access treatment may be essential for longterm treatment programme sustainability.While these options are likely to be feasible for stable patients, there will always be a need for referral facilities or specialty clinics for particular groups such as children, adolescents, pregnant women or complicated or resistant medical cases.
D Evans
Health Economics and Epidemiology Research Office, Department of Internal Medicine, School of Clinical Medicine, University of the Witwatersrand, Johannesburg, South Africa Corresponding author: devans@witshealth.co.za | 2018-04-03T03:41:23.985Z | 2013-02-26T00:00:00.000 | {
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245501672 | pes2o/s2orc | v3-fos-license | Long‐term cannabidiol treatment for seizures in patients with tuberous sclerosis complex: An open‐label extension trial
Abstract Objective To evaluate the long‐term safety and efficacy of add‐on cannabidiol (CBD) in patients with seizures associated with tuberous sclerosis complex (TSC) in the open‐label extension (OLE) of the randomized, placebo‐controlled phase 3 trial GWPCARE6 (NCT02544763). Results of an interim (February 2019 data cut) analysis are reported. Methods Patients who completed the randomized trial enrolled to receive CBD (Epidiolex® in the United States; Epidyolex® in the EU; 100 mg/mL oral solution). The initial target dose was 25 mg/kg/day, which, based on response and tolerability, could be decreased or increased up to 50 mg/kg/day. The primary end point was safety. Key secondary end points included percentage reduction in TSC‐associated (countable focal and generalized) seizures, responder rates, and Subject/Caregiver Global Impression of Change (S/CGIC). Results Of 201 patients who completed the randomized phase, 199 (99%) entered the OLE. Mean age was 13 years (range, 1–57). At the time of analysis, 5% of patients had completed treatment, 20% had withdrawn, and 75% were ongoing. One‐year retention rate was 79%. Median treatment time was 267 days (range, 18–910) at a 27 mg/kg/day mean modal dose. Most patients (92%) had an adverse event (AE). Most common AEs were diarrhea (42%), seizure (22%), and decreased appetite (20%). AEs led to permanent discontinuation in 6% of patients. There was one death that was deemed treatment unrelated by the investigator. Elevated liver transaminases occurred in 17 patients (9%) patients; 12 were taking valproate. Median percentage reductions in seizure frequency (12‐week windows across 48 weeks) were 54%–68%. Seizure responder rates (≥50%, ≥75%, 100% reduction) were 53%–61%, 29%–45%, and 6%–11% across 12‐week windows for 48 weeks. Improvement on the S/CGIC scale was reported by 87% of patients/caregivers at 26 weeks. Significance In patients with TSC, long‐term add‐on CBD treatment was well tolerated and sustainably reduced seizures through 48 weeks, with most patients/caregivers reporting global improvement.
| INTRODUCTION
Tuberous sclerosis complex (TSC) is a highly variable genetic disorder characterized by benign hamartomas in multiple organ systems, most notably in brain, skin, kidneys, lungs, heart, and eyes. [1][2][3][4] It is caused primarily by mutations in tumor suppressor genes TSC1 or TSC2, resulting in an increased mechanistic target of rapamycin (mTOR) activation with subsequent excessive cell growth and proliferation. [1][2][3]5 The incidence of TSC is estimated at 1 in 6000 live births, affecting 40 000-80 000 people in the United States and 1-2 million people worldwide. 6,7 Approximately ~85% of patients with TSC experience epilepsy, with onset usually during the first 2 years of life; it can persist lifelong with multiple seizure types. 6,[8][9][10][11] Patients with TSC experience infantile spasms and focal seizures as infants and various other seizure types as the disease progresses. 9,12 Current treatments for seizures associated with TSC include antiseizure medications (ASMs), commonly referred to as antiepileptic drugs; the mTOR pathway inhibitor everolimus; surgical procedures; vagus nerve stimulation; and dietary therapy. 6,[13][14][15][16][17] Despite these treatment options, more than 60% of patients have treatmentresistant epilepsy, 12 which can be associated with various neurodevelopmental disorders. 10,18 Highly purified pharmaceutical formulation of cannabidiol (CBD; Epidiolex ® in the United States and Epidyolex ® in the United Kingdom, European Union, and Australia) has demonstrated efficacy, with an acceptable safety profile against seizures associated with Dravet syndrome and Lennox-Gastaut syndrome in four randomized, placebocontrolled phase 3 trials. [19][20][21][22] Results from an expandedaccess program demonstrated that CBD may also be an effective and well-tolerated treatment for TSC-associated seizures. 23 The effect of CBD on seizures associated with TSC was further evaluated in a 16-week, randomized, double-blind, placebo-controlled, multicenter phase 3 trial (GWPCARE6). In this trial, add-on CBD produced almost 50% reduction in TSC-associated seizures compared with an ≈30% reduction with placebo, and had an acceptable safety profile. 24 Based on these results, CBD was approved for treatment of seizures associated with TSC in patients aged 1 year and older in the United States 25 and at least 2 years of age in the United Kingdom and in the European Union. 26 Patients who completed treatment in the placebocontrolled, double-blind phase of GWPCARE6 were eligible to enroll in the open-label extension (OLE) phase under the same protocol for evaluation of the long-term safety and efficacy of CBD. Herein we present results of an interim analysis (data cutoff, February 26, 2019) of safety, efficacy, and patient/caregiver-and physician-reported outcomes in patients with TSC enrolled in the OLE phase of GWPCARE6.
| METHODS
This study was an OLE of the 16-week randomized, double-blind, placebo-controlled, multinational phase 3 trial GWPCARE6 (NCT02544763) and enrolled patients TSC 1 and treatment-resistant epilepsy were eligible if they were 1-65 years of age; had at least eight TSC-associated seizures during the 4-week baseline period, with at least one seizure occurring in at least 3 of the 4 weeks; and were taking at least one ASM at baseline of the randomized, controlled phase. Key exclusion criteria were a history of nonepileptic seizures, clinically significant illness other than epilepsy, surgery for epilepsy in the 6 months before screening, felbamate use for less than 1 year before screening, and a history of alcohol or substance abuse or recreational or medicinal use of cannabis or cannabinoidbased medications. Although not allowed during the randomized phase, on-label use of mTOR inhibitors for the treatment of seizures and tumors was permitted during the OLE. The trial protocol was approved by the relevant institutional review board or ethics committee at each participating site and was conducted in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Tripartite Guideline on Good Clinical Practice. All patients or their caregivers provided written informed consent before any trial procedure was carried out. Patients who were developmentally mature enough to understand the trial provided written assent. All patients received a pharmaceutical formulation of highly purified CBD derived from Cannabis sativa L. plant (100 mg/ml oral solution; Epidiolex ® in the United States and Epidyolex ® in the United Kingdom, European Union, and Australia; GW Research Ltd, Cambridge, United Kingdom). After completing treatment in the randomized phase, patients started a 2-week blinded transition period, during which the blinded medication (CBD 25 mg/kg/day, CBD 50 mg/kg/day, or placebo) from the randomized, controlled phase was tapered down to zero while simultaneously CBD was titrated up to 25 mg/ kg/day (Table S1). The dose could then be titrated up to 50 mg/kg/day during a 3-week titration period in increments of 2.5 mg/kg/day every 2 days. The daily dose of CBD was maintained throughout the trial; however, the investigator could decrease the dose if a patient experienced intolerance or could increase it up to the maximum dose of 50 mg/kg/day if required for better seizure control, until the optimal dose was found. CBD was taken twice daily in equally divided doses in addition to the patient's current ASM. The dose for concomitant ASMs could also be adjusted to manage side effects associated with their use. Patients could receive treatment for up to 1 year, except in the United States and Poland, where they could continue treatment beyond 1 year. At the end of treatment, patients (outside the United States and Poland) could continue using CBD outside the study. For patients who did not immediately continue using CBD outside the trial or upon decision to withdraw, CBD dose was tapered down by 10% per day for 10 days, unless continued dosing was not possible because of an adverse event (AE). A follow-up visit was performed 4 weeks after the last dose of CBD (including the final taper period dose) in patients completing or withdrawing from the trial. The study design is shown in Figure S1.
The primary objective of this OLE study was to evaluate the long-term safety and tolerability of add-on CBD based on the incidence, type, and severity of treatmentemergent AEs in patients with uncontrolled seizures associated with TSC. Patients used paper diaries for daily recording of any AEs, CBD intake, and the use of concomitant ASMs and rescue medications throughout the study. Blood and urine samples for clinical laboratory assessments were collected at all clinic visits (when possible).
The secondary objective was evaluation of efficacy through assessment of percentage change in the frequency of TSC-associated seizures; the number of patients considered treatment responders with ≥25%, ≥50%, ≥75%, and 100% reduction in TSC-associated seizures; change in overall condition of patients on the Subject/Caregiver Global Impression of Change (S/CGIC) scale and Physician Global Impression of Change (PGIC) scale; and percentage change in the frequency of total seizures. All changes were assessed relative to the pre-randomization baseline of the trial's blinded phase. TSC-associated seizures included countable focal motor seizures without impairment of awareness, focal seizures with impairment of awareness, focal seizures evolving to bilateral generalized convulsive seizures, and generalized seizures (tonicclonic, tonic, clonic, or atonic); they excluded absence, myoclonic, focal sensory, and infantile/epileptic spasms. This functional definition of TSC-associated seizures was approved by the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Epilepsy Study Consortium's independent committee of experts. Total seizures included TSC-associated seizures, infantile/ epileptic spasms, and absence, myoclonic, and focal sensory seizures. Patients or their caregivers used an interactive voice-response system to record the number and type of seizures, severity of focal seizures, and the number of status epilepticus episodes. The S/CGIC and PGIC scales are both 7-point scales that include three categories for improvement (slightly improved, much improved, and very much improved), three for worsening (slightly worse, much worse, and very much worse), and an option to indicate no change.
No formal sample size was calculated for this openlabel trial, and all patients who completed the blinded, placebo-controlled phase were eligible to enroll. All patients who received at least one dose of CBD during the OLE were included in the safety evaluation data set. Safety data for the complete OLE phase are reported here. Seizure frequency (average per 28 days) was calculated for each 12-week treatment window and expressed as median percentage reduction from pre-randomization baseline. Seizure outcomes for up to 48 weeks of treatment are presented here. Analyses of seizure frequency and treatment responder rates were repeated using the last observation carried forward (LOCF) imputation step, which is described in the Appendix S1. Seizure outcomes analyses were conducted for patients who completed weeks 37-48 of the treatment period. In addition, we conducted a post hoc analysis to evaluate the safety and efficacy of CBD by modal dose categories ≤25 and >25 mg/kg/day. The S/ CGIC and PGIC results at the 26-week visit are reported. There was no formal hypothesis testing and results are presented descriptively.
| Patients
Of the 201 patients who completed treatment in the placebo-controlled, double-blind phase of the trial, 199 enrolled in the OLE phase ( Figure 1 (Table S2).
The median patient age was 10.8 years; 46 (23%) were age 18 and older (Table 1). Patients had previously tried and discontinued a median of four ASMs and were taking a median of three ASMs at the start of the placebo-controlled phase. Valproate (41%), vigabatrin (36%), clobazam (32%), and levetiracetam (29%) were the most commonly used medications during the OLE. Although mTOR inhibitors were not allowed during the randomized phase, 9 patients received everolimus and 25 sirolimus during the OLE. Five patients (3%) were on concomitant ketogenic diet therapy, and 23 (12%) were using vagus nerve stimulation. The median number of TSC-associated seizures during the 4-week pre-randomization baseline was 56.9.
Overall median treatment duration was 267 days (range, 18-910 days) and patient-years on treatment was 152.9, and the mean modal (standard deviation [SD]) dose was 27 mg/kg/day (7.3). The mean modal (SD) dose was 26 mg/kg/day (6.1) for weeks 1-12 and 28 mg/kg/day (7.7-8.8) for the remaining 12-week treatment windows through week 48. Of the total 199 patients, 156 (78%) had a CBD modal dose ≤25 mg/kg/day with a mean (SD) of 24 mg/kg/day (3.3) and 43 (22%) had modal dose >25 mg/ kg/day with a mean (SD) of 38 mg/kg/day (7.4). The median treatment duration was 232 days (range, 18-876 days) and patient-years on treatment was 110.5 for patients with a modal dose ≤25 mg/kg/day. For patients with a modal dose >25 mg/kg/day, the median treatment duration was 318 days (range, 40-910 days) and patient-years on treatment was 42.4.
| Safety
Treatment-emergent AEs were reported in 92% of all patients, 91% of patients with modal dose ≤25 mg/kg/day, and 98% of patients with modal dose >25 mg/kg/day ( Table 2). Most AEs were of mild or moderate severity, with 36% of patients reporting the severity as mild, 47% as moderate, and 9% as severe. Diarrhea, seizures, and decreased appetite were the most frequently reported AEs, and most of these were of mild or moderate severity ( Table 2 and Table S3). Somnolence was reported more frequently in patients taking concomitant clobazam (16 of 66 patients [24%]) than in patients not on clobazam (15 of 133 patients [11%]); the overall incidence of AEs was similar between the two subgroups (94% of patients taking clobazam vs 92% of those not taking clobazam). An AE was listed as one of the reasons for permanent treatment discontinuation in 12 patients (6%). The most common AE leading to permanent treatment discontinuation in >1% of patients was seizures (four patients [2%]; Table S3). Fifty patients (25%) had permanent dose reduction because of an AE, primarily diarrhea (21 patients [11%]; Table S3). Serious AEs were reported by 15% of all patients, with seizures and status epilepticus as the most frequently reported serious AEs in >1% of patients (Table 2). There was one death due to cardiopulmonary failure during the study, which was deemed not treatment related by the investigator.
Laboratory testing showed elevations in serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels of more than 3 times the upper limit of normal (ULN) in 17 patients (9%), 12 of whom (71%) were taking concomitant valproate. No patient met the standard criteria for severe drug-induced liver injury (Hy's Law) with concurrent bilirubin levels more than 2 times ULN. One patient discontinued treatment because of elevated transaminase levels. Elevations occurred within 1 month of starting treatment in 9 of the 17 patients and between 1 and 3 months of starting treatment in 6 patients; 2 patients had elevations more than 3 months (100 days) after starting treatment. At the time of this analysis, increased ALT or AST levels had resolved in 14 of 17 patients-spontaneously in 6 patients, following treatment discontinuation in 1, and after CBD or ASM dose reduction in 7 (4 patients reduced valproate dose); transaminase levels had not resolved in 3 patients.
| Efficacy
During the first 12 weeks of the OLE, CBD treatment produced a median reduction of 54% from the randomized phase baseline in the monthly TSC-associated seizure frequency (Figure 2A). The effect was maintained throughout the treatment windows at weeks 13-24, 25-36, and 37-48, with 54%-68% reduction from baseline. Median reduction of 52%-73% was observed in patients with modal dose ≤25 mg/kg/day and 49%-61% in those with modal dose >25 mg/kg/day across the 12week treatment windows up to 48 weeks ( Figure S2A). In the LOCF analysis, a 54%-56% reduction from baseline in TSC-associated seizures was observed across the 12-week windows ( Figure S3A); reduction was 61%-68% F I G U R E 1 Patient disposition. a One patient taking cannabidiol (CBD) 25 mg/ kg/day in the randomized phase was withdrawn after completing the treatment because of vomiting and did not enter the open label extension (OLE); another patient taking CBD 50 mg/kg/day did not enter the OLE after completing treatment in the randomized phase because of logistical problems and on psychiatrist's recommendation. b Withdrawals are reported by the primary reason reported for each patient. c One patient met the withdrawal criterion of elevation in ALT/AST levels. d Of patients who had other reasons, nine withdrew due to lack of efficacy, one withdrew because of difficulty maintaining compliance, and one switched to a commercial product. among patients who completed the weeks 37-48 treatment window ( Figure S4A). More than 50% of patients had ≥50% reduction in TSCassociated seizure frequency across the 12-week treatment windows; ≥75% reduction in seizures was observed in >25% of patients ( Figure 3A). More than 5% of patients were seizure-free during each 12-week treatment window, with seven patients (4%) remaining seizure-free the entire treatment period. The proportion of patients who had ≥25%, ≥50%, ≥75%, and 100% reduction in TSCassociated seizures remained consistent across the treatment windows when LOCF was used to calculate responder rates ( Figure S5A), as well as among patients who completed the weeks 37-48 treatment window ( Figure S6A).
A median reduction from baseline of 51% in the monthly total seizure frequency was observed at the weeks 1-12 treatment window; reduction in seizures was maintained across the 12-week treatment windows through week 48 (54%-67% reduction from baseline; Figure 2B). Reduction in total seizures ranged from 53%-70% in patients with a modal dose ≤25 mg/kg/day and 48%-55% in those with modal dose >25 mg/kg/day ( Figure S2B). In the LOCF analysis, 50%-53% reduction in total seizure frequency was observed ( Figure S3B). Among patients who completed weeks 37-48 of treatment, 61%-67% reduction from baseline was observed ( Figure S4B). Overall, a ≥50% reduction in total seizure frequency was observed in 51%-60% of patients across the 12-week treatment windows for up to 48 weeks ( Figure 3B). Patients reporting ≥25%, ≥50%, ≥75%, and 100% reduction in seizures were consistent across the treatment windows in the LOCF analysis of responder rates ( Figure S5B), as well as among patients who completed the treatment window at weeks 37-48 ( Figure S6B).
At the 26-week visit, 87% of the 124 patients or caregivers who completed the S/CGIC evaluations reported an improvement from baseline in the overall condition of patients ( Figure 4). Improvements were reported by 80% of physicians on the PGIC scale.
| DISCUSSION
In this interim analysis of the OLE phase of trial GWPCARE6, add-on CBD treatment had an acceptable safety profile and resulted in a sustained reduction in seizures associated with TSC through 48 weeks of treatment. The safety profile of CBD in the OLE was consistent with that in the randomized, placebo-controlled phase of the trial, with more than 90% of patients on CBD reporting a treatment-emergent AE; most AEs were of mild or moderate severity. The safety profile was also consistent with that observed in the trials for Dravet and Lennox-Gastaut syndromes, and no new safety concerns were identified. 19,21,22,[27][28][29] The most frequently reported AEs were similar in the OLE and the randomized, controlled phase, with diarrhea being most common. Although the etiology is not known, diarrhea has been reported as the most frequent AE in other clinical trials of CBD; in most cases, however, the severity was mild or moderate and resolved before the end of treatment. 30,31 Permanent treatment discontinuation because of an AE was low (6%) despite some patients being on treatment for up to 130 weeks, supporting a favorable safety profile of CBD for long-term use in patients with TSC-associated seizures.
Elevations in serum ALT/AST levels (>3 times ULN) were more frequently reported in patients on concomitant valproate. This possible interaction between CBD and valproate leading to elevation in liver enzyme levels was also observed in the randomized phase of this trial, as well as in trials of patients with Dravet and Lennox-Gastaut syndromes. 19 that transaminase levels be monitored regularly in all patients taking CBD, especially those taking concomitant valproate. 25 In 53% of patients with ALT/AST elevations, onset occurred within 1 month of starting treatment, which is also consistent with the controlled phase. In most patients, elevations resolved spontaneously, after (14) 6 (14) 28 (14) Upper respiratory tract infection 19 (12) 7 (16) 26 (13) Alanine aminotransferase increased b 7 (4) 6 (14) 13 (7) Fall 8 (5) 6 (14) 14 (7) Cough 7 (4) 5 (12) 12 ( treatment discontinuation, or following CBD/ASM dose reductions. Thus discontinuation or reduction of CBD and/or concomitant valproate dose may be considered for management of liver enzyme elevations. Although the potential for interaction between CBD and the mTOR inhibitors everolimus and sirolimus was not evaluated in this study, CBD treatment has been shown to produce a clinically significant increase in the levels of mTOR inhibitors in some patients with TSC. 32 Therefore, in patients taking CBD in addition to everolimus or sirolimus, the doses of these mTOR inhibitors may need to be adjusted to avoid potentially significant toxicity. Treatment with CBD produced a sustained reduction in TSC-associated seizures, including countable focal and generalized seizures but excluding absence, myoclonic, focal sensory, and infantile/epileptic spasms, as well as in total seizures, which included all of the abovementioned seizures types. In the randomized, placebocontrolled phase of the trial, CBD produced an ~40% reduction in the frequency of seizures associated with F I G U R E 3 Responder rates for (A) TSC-associated seizures and (B) total seizures. Abbreviation: TSC, tuberous sclerosis complex TSC. Improvements during the OLE were higher and were sustained through 48 weeks of treatment. More than 50% reductions in TSC-associated and total seizures were observed during the 12-week treatment windows. Seizure responder rates (≥25%, ≥50%, and ≥75% reduction in seizures) were sustained throughout the study, with 29%-45% of patients experiencing ≥75% reduction in seizures and at least 5% of patients becoming seizurefree during each 12-week treatment window reported here. This effect on seizure reduction and responder rates over time was not due to patient withdrawal, as consistently robust response was also observed with LOCF analyses conducted to assess the effect of patient withdrawals on seizure reduction end points.
In addition to the substantial reduction from baseline in seizure frequency, more than 80% of patients or their caregivers and physicians reported improvements in the overall condition of patients, assessed using S/CGIC and PGIC scales, indicating that CBD treatment provided meaningful improvement in patient quality of life.
In an analysis by modal dose, >90% of patients with modal dose ≤25 mg/kg/day and those with modal dose >25 mg/kg/day had an AE. A similar reduction in monthly seizure frequency was observed in patients with modal dose ≤25 mg/kg/day and those with modal dose >25 mg/ kg/day. However, both the safety and efficacy results by modal dose must be interpreted with caution because the dose categories were based on patients' modal or most frequent dose, which could vary between the treatment windows; in addition, the dose could be titrated up or down, depending on tolerability and response, throughout the follow-up.
Retention rate in OLE studies has been suggested as a measure of long-term benefit of antiseizure medications. 33 The 1-year retention rate in the GWPCARE6 OLE was 79%, suggesting that most patients were receiving clinically meaningful benefit from CBD treatment. In addition, the mean modal dose did not vary considerably between the 12-week treatment windows, suggesting that patients did not develop tolerance to CBD, necessitating a dose increase to control seizure frequency.
The goal of an epilepsy treatment for patients with TSC is to prevent or control seizures as soon as possible after the diagnosis and with a minimum number of ASMs and AEs. 13 ASMs are frequently prescribed treatment for seizures associated with TSC 13 ; in a study of patients enrolled in the TSC Natural History Database, >99% of patients had received an ASM therapy. 34 TSC-associated seizures are difficult to manage and ~60% of patients taking ASMs have usually tried three or more distinct ASMs. 34 Currently, vigabatrin is recommended as the first-line monotherapy for infantile spasms and/or focal seizures associated with TSC in the first year of life 13,35 ; however, the risk of a visual field constriction associated with prolonged use of vigabatrin remains a concern. 13,36 Adrenocorticotropic hormone is recommended as the second-line treatment for infantile spasms. 13,36 Topiramate, carbamazepine, and oxcarbazepine are also used as second-line therapies for focal seizures. 37 The mTOR inhibitor everolimus has also shown efficacy as adjunctive treatment of TSC-associated focal-onset seizures. 38 Standard treatment guidelines are recommended for other TSC-associated seizures. 39 The results of our study show that add-on CBD can be an efficacious long-term treatment for TSC-associated seizures F I G U R E 4 Global impression of change in patients' overall condition on the S/CGIC and PGIC scales at the 26-week visit. Abbreviations: PGIC, Physician Global Impression of Change; S/CGIC, Subject/Caregiver Global Impression of Change with manageable side effects and has been approved in patients as young as 1 year of age in the United States. 25 Our study does have some limitations. As an OLE, it did not include a placebo comparator. Efficacy measures and patient-reported outcomes were evaluated relative to the pre-randomization baseline of the trial; therefore, CBD exposure varied among patients depending on whether they were randomized to placebo or CBD group. Efficacy outcomes could have been affected by a change in the patient's concomitant medications, especially by addition of a new ASM. Patient/caregiver's impression of the improvement in patient's overall condition could have been affected by close monitoring and the perception of increased attention and care associated with participating in a trial. Because this was an interim analysis of the OLE, not all patients had completed later treatment windows. Although classification of seizures in this trial was confirmed by the Epilepsy Study Consortium, videoelectroencephalographic confirmation of the individual seizure subtypes was not done.
Despite certain limitations, the results of this OLE of GWPCARE6 trial demonstrated that CBD had an acceptable safety profile as a long-term add-on treatment for seizures associated with TSC. Reduction in the seizure frequency observed during the randomized, placebo-controlled phase of the trial was sustained during long-term CBD treatment. Patients and their caregivers reported meaningful improvements in the patient's overall condition on the S/CGIC scale; improvements were also reported by most physicians on the PGIC scale. Thus our results support the long-term benefit of add-on CBD therapy for patients with treatment-resistant epilepsy associated with TSC. This trial was conducted with Epidiolex ® /Epidyolex ® and results do not apply to other CBD-containing products.
ACKNOWLEDGMENTS
The authors would like to thank the patients, their families, and the staff at sites that participated in this study. Medical writing support for the development of this manuscript, under the direction of the authors, was provided by Ritu Pathak, PhD, and editing support by Dena McWain, both of Ashfield MedComms, an Ashfield Health company, and funded by Greenwich Biosciences, Inc.
CONFLICT OF INTEREST
Elizabeth A. Thiele received support from GW Research Ltd as an investigator during the conduct of the trial; outside of the current work, she serves as a principal investigator on clinical trials for GW Research Ltd and Zogenix and serves as a consultant for Aquestive Therapeutics, Biocodex, West Therapeutics, Greenwich Biosciences, and Zogenix. E. Martina Bebin received support from GW Research Ltd as a site principal investigator during the conduct of the trial and serves as a consultant for Greenwich Biosciences and Biocodex. Francis Filloux and Patrick Kwan have nothing to disclose. Rachael Loftus is a full-time employee of GW Research Ltd. Farhad Sahebkar is a full-time employee of Greenwich Biosciences. Steven Sparagana has received personal compensation for serving as a consultant for Greenwich Biosciences and Nobelpharma. His institution has received research support from Greenwich Biosciences, Tuberous Sclerosis Complex Alliance, and Novartis. He has a noncompensated relationship as a professional advisory board member or committee member with Tuberous Sclerosis Complex Alliance. James Wheless has received personal compensation for serving as a consultant for Eisai, Supernus, Aquestive, GW Pharmaceuticals companies, and Neurelis. He has served on a speakers bureau for LivaNova, Eisai, Supernus, GW Pharmaceuticals companies, UCB, BioMarin, and Zogenix. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. | 2021-12-28T06:21:57.565Z | 2021-12-27T00:00:00.000 | {
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231753611 | pes2o/s2orc | v3-fos-license | A novel nasal co-loaded loratadine and sulpiride nanoemulsion with improved downregulation of TNF-α, TGF-β and IL-1 in rabbit models of ovalbumin-induced allergic rhinitis
Abstract Purpose The work aimed to develop a co-loaded loratadine and sulpiride nasal nanoemulsion for allergic rhinitis management. Methods Compatibility studies were conducted adopting differential scanning calorimetry and Fourier transform infrared spectroscopy. Nanoemulsion formulations were prepared using soybean lecithin, olive oil and tween 80. Sodium cholate and glycerol were employed as co-surfactants. Nanoemulsions were assessed for viscosity, pH, droplet size, polydispersity index, zeta potential, electrical conductivity, entrapment, In vitro drug release and corresponding kinetics. Stability of the selected formulation was investigated. The biological effectiveness was evaluated in rabbit models of ovalbumin-induced allergic rhinitis by measuring TNF-α, TGF-β and IL-1. Results Compatibility studies revealed absence of drug/drug interactions. Nanoemulsions exhibited > 90% entrapment efficiency. The selected nanoemulsion demonstrated small droplet size (85.2 ± 0.2 nm), low PDI (0.35 ± 0.0) and appropriate Zeta Potential (−23.3 ± 0.2) and stability. It also displayed enhanced in vitro drug release following the Higuashi Diffusion and Baker–Lonsdale models. The mean relative mRNA expression of TNF-α, IL-1 and TGF-β significantly decreased from 9.59 ± 1.06, 4.15 ± 0.02 and 4.15 ± 0.02 to 1.28 ± 0.02, 1.93 ± 0.06 and 1.56 ± 0.02 respectively after treatment with the selected nanoemulsion formulation. Conclusion The results reflected a promising potent effect of the combined loratadine and sulpiride nasal nanoemulsion in managing the symptoms of allergic rhinitis.
Introduction
Allergic rhinitis is a disease provoked by IgE-mediated immune response and demonstrates a long-lasting inflammation of nasal mucosa. IgE/allergen interaction on the exterior of basophils and mast cells results in the stimulation of these cells for the liberation of mediators comprising histamine, leukotrienes, prostaglandins, platelet activating factor (PAF) and cytokines. T-helper cell (Th2) cytokines are known to have a principal responsibility in the developing allergic sensitization and pathology of allergic inflammation (Maes et al., 2012). Furthermore. IL-1, TNF-a, and TGF-b cytokines are commonly exist in the inflamed sites in the body. These mediators can consequently recruit extra inflammatory cells, initiate the release of more inflammatory mediators and stimulate afferent nerves (Greiner et al., 2011). Moreover, it has been reported that this condition is accompanied by increase of lysophosphatidyl choline that in turn increases the cell permeability to sodium (Na þ ) and calcium (Ca 2þ ), induces membrane depolarization, enhances IgE response, supports phagocytic action, prevents adenylate cyclase, and stimulates phosphodiesterase and consequently reducing cAMP levels in the cells. These changes promote the liberation of mediators from mast cells generating airway inflammation (Agrawal et al., 1986). The released mediators in this allergic condition trigger allergic manifestations (Wang et al., 2016) including nasal symptoms such as rhinorrhoea, sneezing, nasal itching and nasal obstruction (Aria Workshop Group;World Health Organization, 2001). Eye redness, itching and tearing can also develop. As the related symptoms may disrupt sleep, cause lethargy, and affect patient concentration, it has been reported that this type of allergy affects the quality of patient life and adds loads on the health care systems (Kim et al., 2007). The usual pharmacological therapeutic protocol for the management of allergic rhinitis involves the use of topical and/or oral antihistamines, intranasal corticosteroids, anticholinergics or antihistaminedecongestant combinations, leukotriene receptor antagonists and mast cell stabilizers such as cromoglycic acid (Sur & Scandale, 2010). As histamine has the major role in the allergic reactions, this type of rhinitis is commonly treated with antihistamines that have been clinically used for several years. Antihistamines manage the baseline symptoms including sneezing and nasal secretions and inhibit the allergenprovoked liberation of mediators from mast cell in the nasal mucosa. As histamine release is responsible for all the pathological features of allergic rhinitis except the inflammatory reactions of the late phase (Iriyoshi et al., 1996), patients do not attain entire symptom management with a single drug therapy (Fabbri et al., 2014). Thus, combination treatment can be recommended for managing the entire symptoms of the condition.
The second-generation antihistamine, loratadine is known to be used in the treatment of allergic rhinitis. The drug has good absorption from the gastrointestinal tract when given orally reaching peak plasma concentration after 1 to 1.5 hour (Moffat et al., 2004). However, it has poor oral bioavailability (40%) as it experiences rapid first-pass hepatic metabolism (Borgaonkar et al., 2011). Therefore, in order to avoid the drug metabolism in the liver, an alternate route of delivery would be favored.
Sulpiride, an antipsychotic drug, is a selective dopamine receptor antagonist. It has high affinity to D2 and D4 receptors. It has been reported that sulpiride has an anti-inflammatory effect through increasing the intracellular cAMP level regardless the presence of dopamine signaling at D2 receptors (Brustolim et al., 2006). Based on this finding, it can be speculated that the drug can have a beneficial effect in the management of allergic rhinitis inflammatory manifestations. Sulpiride experiences a limited oral bioavailability not more than 27% due to its poor water solubility (Zidan et al., 2015).
The nasal route represents a promising noninvasive alternative route of drug delivery for treatment of different conditions. Rapid absorption of drugs into systemic circulation is permitted through the porous endothelial membrane of the lush vascular capillary layer beneath the nasal mucosa (Illum, 2002). Moreover, nasal delivery improves poor bioavailability and avoid hepatic first pass metabolism and drug degradation in gastrointestinal tract (Khan et al., 2018). This route is adopted also for local drug delivery to avoid the systemic exposure to certain drugs and reduce the related side effects (Djupesland et al., 2013).
Nanoemulsions are colloidal systems consisting of two immiscible phases. They are translucent having droplet size around 200 nm. These systems are kinetically stabilized by the aid of surfactants or a mixture of surfactants and co-surfactants (Gurpreet and Singh, 2018). Nanoemulsions have the advantages of controlling the drug release and the possibility of delivering a wide variety of therapeutic agents (Hoeller et al., 2009). Additionally, they are superior over macroemulsion in having larger surface area and free energy. They also avoid coalescence, flocculation, creaming and sedimentation. Nanoemulsions can be prepared adopting lower concentration of emulsifying agents and thus reducing surfactant-related toxicity (Liu et al., 2011). Furthermore, these systems have the ability to solubilize poorly water-soluble drugs and thus improve their permeation through mucosa and provide an advantageous formulation for delivering such drugs (Gaba et al., 2019).
The present work, represent for the first time the co-loading of loratadine and sulpiride into nanoemulsion for nasal delivery as a new approach with enhanced therapeutic effect for the management of allergic rhinitis. Lecithin based nanoemulsions of combined loratadine and sulpiride were formulated. The developed nanoemulsions were investigated for in vitro characterization. Biological studies have been conducted to evaluate the efficacy of the selected formulation in ovalbumin-induced allergic rhinitis rabbit models.
Fourier transform infrared spectroscopy
Fourier transform infrared (FTIR) spectra of loratadine, sulpiride and their physical mixture (1:1) were inspected using a FTIR spectrometer (Perkin Elmer, NY, USA). 5 mg samples was squashed into disks of potassium bromide. The spectra were attained at the wavelength range from 500 to 4000 cm À1 .
Differential scanning calorimetry study
Differential scanning calorimetry (DSC) examination of loratadine, sulpiride and their physical mixture (1:1) were performed using a DSC-131 Evo (Setaram Inc., France). 2-5 mg samples were weighed in crucible pans covered with pierced caps. The temperature was raised gradually from 25 to 500 C at a heating rate of 10 C/min under a nitrogen flow rate of 40 ml/min.
Preparation of the co-loaded loratadine and sulpiride nanoemulsions
Combined loratadine and sulpiride nanoemulsions (o/w) was developed adopting ultra-sonication technique. Different compositions of the developed formulations are shown in Table 1. Specified amounts of Tween-80 and soybean lecithin were mixed at 25 C using a magnetic stirrer (400 rpm for 30 minutes) to form the oil phase. The specified amounts of olive oil (3.5 g), loratadine (0.25 g) and sulpiride (0.05 g) were added and stirred till obtaining a uniform mixture. The calculated amounts of deionized water, sodium cholate and glycerol (co-surfactants)were mixed to form the aqueous phase. The oil phase was then added gradually to the aqueous phase using a suitable syringe under stirring for 10 minutes. The resultant mixture was further sonicated for 30 minutes adopting ultrasonic processor at 20 kHz (FB-110Q, Shanghai Litu Machinery and Equipment Engineering Co., LRD, Shanghai, China).
Appearance, viscosity and pH
The prepared nanoemulsions were visually examined by inspecting the transparency on light reflections. Their pH was determined using a pH meter (model 361, Systronics). Viscosity of the prepared nanoemulsions were investigated using a Brook Field Viscometer (LVF 69726) with a UL-adapter. All the investigations were accomplished in triplicate at 25 C.
Entrapment efficiency
Centrifugation method was employed to establish the entrapment efficiency of the co-loaded loratidine and sulpiride nanoemulsion (Prabhakar et al., 2013). Briefly, 2.0 mL sample was centrifuged at 3500 rpm for 15 minutes. The aqueous phase or the supernatant was separated, while the residue was washed two times with distilled water and recentrifuged to ensure entire separation of the free drugs. The separated supernatants were added together, mixed with equivalent volume of ethanol, and mixed for five minutes using a vortex mixer. The amount of free loratadine and sulpiride was established by measuring the spectrophotometric absorbance at 395 and 218 nm respectively using a-1900PC UV-Vis spectrophotometer (Shanghai Puyuan Instrument Co., LRD, China) and adopting ultraviolet spectroscopy chemometric technique (Gad et al., 2013;Sood et al., 2014). The drug amounts entrapped in the oil phase were estimated by calculating the difference between the total drug amount incorporated and the free drug amount detected in the aqueous phase. Entrapment efficiency (%EE) was computed using the subsequent equation: 2.4.3. Droplet size, polydispersity index, zeta potential and electrical conductivity The droplet size, polydispersity index (PDI) and zeta potential of the selected nanoemulsions were investigated through dynamic light scattering adopting N4Plus submicron particle size analyzer (Beckman Coulter, UK). Samples were appropriately diluted with distilled water and measured at room temperature. The average values of three measurements ± SD were recorded. A conductivity meter (CM 180) was used to determine the electrical conductivity of the selected nanoemulsions by direct dipping of electrode into the sample at 25 C.
In vitro release studies
To study the in vitro release of loratadine and sulpiride from the prepared nanoemulsions, a dialysis bag method was adopted (Sood et al., 2014). 4 mL sample was loaded into a dialysis bag of cellulose membrane (14 kDa molecular weight cutoff). The bag was dipped in a beaker containing 100 ml phosphate buffer solution, pH 6.8 ± 0.5 kept at 37 ± 0.1 C and stirred at 50 rpm for 12 hours. 2 ml samples were removed at appropriate time intervals. Loratadine and sulpiride amounts were determined by measuring spectrophotometric absorbance at 395 and 218 nm respectively. The resultant release profiles were compared to those obtained from the individual raw loratadine and sulpiride. Statistical analysis of the release data has been carried out using similarity factor, f2 whereas the release profiles were compared to a reference (Shah et al., 1998).
To define the release mechanism of loratadine and sulpiride from the selected nanoemulsions (F2, F3), data was fitted into different kinetic models: Chawla et al., 2000) Whereas R, Q or Mt/M1 refers to the fraction of drug released at time t, K or KH is the rate constant related to each model, UR is the unreleased fraction of the drug while n is the diffusional exponent that characterizes the type of release mechanism during the dissolution process.
Morphology analysis
The morphology of the selected nanoemulsion (F3) was investigated through transmission electron microscope (JEM-2100, JEOL, Japan). The selected nanoemulsion was suitably diluted with deionized water and dripped on a copper mesh. For enhancing the image quality, 2% w/v phosphotungstic acid solution was added as a negative stained standard (Mallick et al., 2020). The morphology was also further confirmed via scanning electron microscope. The samples were positioned on polycarbonate substrate while the excess water was removed by drying first at ambient temperature and then by carbon dioxide. Samples were coated with gold and examined under a scanning electron microscope (KYKY EM3200, China) running at an accelerating voltage of 20 kV.
Stability study
The selected nanoemulsion F3 was stored at 4 C and 25 C for one month. The stored nanoemulsion was investigated for changes in droplet size, PDI and % EE (Ghosh et al., 2013).
Statistical analysis
All investigations were conducted in triplicate and by freshly prepared samples. Statistical analysis of data was accomplished using graph pad Prism 8.3 computer software (Graph Pad Software San Diego, CA). All investigational data were conveyed as mean ± standard deviation (SD). One-way ANOVA was used for analysis of the results considering the difference was statistically significant when p value < .05 to be.
In vivo efficacy study in rabbit models of ovalbumin-induced allergic rhinitis
The study was permitted by the Committee of Animal Ethics in Minia University, Minia, Egypt, that guaranteed the of animals corresponded to the National Institutes of Health guide for the use and care of laboratory animals (NIH Publications No. 8023, revised 1978).
Induction of allergic rhinitis
Allergic rhinitis rabbit models were developed according to a modified reported protocol (Sagit et al., 2017). The rabbits were sensitized via intraperitoneal (IP) injections of 1 ml freshly prepared ovalbumin every other day over 7 days followed by intranasal administration of 1 ml physiological saline (PS) having 0.3 mg ovalbumin and 30 mg Al(OH) 3 for 14 days (Sagit et al., 2017;Senturk et al., 2018). Treatment was initiated after the 21 days of the induction and for 14 days. The studied formulations were administered as 70 ll dose "holding 17 lg of the loratadine and 7 lg of sulpiride" in every nostril by means of a micropipette with a low-density polyethylene tubing, having an internal diameter of 0.1 mm at the administration site.
Study groups and drug treatment
Twenty-five male New Zeeland rabbits weighing 1.5-2 kg were used. The rabbits were maintained at room temperature and had access to typical laboratory diet and water. The rabbits were allocated into five groups of five rabbits each: Group I: Negative control (normal) in which rabbits were injected with 1 ml intraperitoneal PS followed by intranasal administration of 1 ml PS.
Group II: Positive control group in which rabbits were sensitized with intraperitoneal ovalbumin and provoked with intranasal ovalbumin but did not receive treatment. Group III: placebo in which rabbits were sensitized and provoked with ovalbumin and treated with drug-free nanoemulsion. Group IV: In which rabbits were sensitized and provoked with ovalbumin and treated with intranasal co-loaded loratadine and sulpiride conventional emulsion. Group V: In which rabbits were sensitized and provoked with ovalbumin and treated with intranasal co-loaded loratadine and sulpiride nanoemulsion (F3).
Evaluation of allergic rhinitis symptoms
The early and late response of the rabbit models after rhinitis induction and intranasal treatment was assessed on the first and fourteenth day of treatment by otorhinolaryngology specialist. Allergic rhinitis was assessed concerning the severity of the classic clinical symptoms including nasal irritation, sneezing, nasal secretions and conjunctivitis and eye secretions. The rhinitis sign scores were classified into a four-point scale, ranged from 0 to 3. For motion of nasal itching: 0 ¼ no nose rubbing; 1 ¼ 2 nose rubbing/min; 2 ¼ 4-6 nose rubbing/min; while 3 ¼ > 6 nose rubbing/min. For sneezing: 0 ¼ none; 1 ¼ 1-3/10 min; 2 ¼ 4-9/ 10 min; while 3 ¼ more than 10 sneeze/10 min. For nasal mucus: 0 ¼ no mucus; 1 ¼ mucus inside a nostril; 2 ¼ mucus outside a nostril; while 3 ¼ overflowing. For eye secretions: 0 ¼ none; 1 ¼ inside eye; 2 ¼ outside eye; while 3 ¼ overflowing or change in color. For conjunctivitis: 0 ¼ none; 1 ¼ mild; 2 ¼ moderate; while 3 ¼ severe. On the last day of sensitization or day 1, the predominant signs including nasal rubbing, sneezing, and nasal secretion were scored to start treatment. The allergic rhinitis model succeeded when the total sign score was more than 7 while the treatment succeeded when the total sign score was less than 4 ( Zhou et al., 2009;Senturk et al., 2018).
Measurement of inflammatory parameters
Different inflammatory parameters were determined prior and post treatment to assess the efficacy of the selected nanoemulsion (F3). These parameters include TNF-a that is a powerful pro-inflammatory cytokine having fundamental responsibilities in stimulating leukocyte staffing to injured spots via provocation of expression of inflammatory chemokines and adhesion molecules. Another inflammatory parameter is TGF-b that is known as a very effective immunosuppressive and anti-inflammatory cytokine, conflicting TNF-a activities, stimulating the production of T regulatory cells, and facilitating the anti-inflammatory activities of these cells (Ohno et al., 1992;Hamaguchi et al., 1994;MULLOU et al., 1995). Interleukin-1 (IL-1) also is a cytokine that is essential for triggering the inherent immune response, facilitating the staffing, stimulation, and adherence of phagocytes (macrophages and neutrophils), and ending the inherent immune response is the same as TNF-a (Ott et al., 2007).
Statistical analysis
Codes were set to the data and inputted employing the Graph Pad prism version 7 software. Differences between groups were verified by means of the Chi-Square test (qualitative variables), independent sample T-test, and analysis of variance (ANOVA). Post hoc Bonferroni test was used for normally distributed quantitative variables. The difference was considered a statistically significant when P-values were equal to or less than .05.
Fourier transform infrared spectroscopy
The FTIR spectra of raw loratadine, sulpiride and their 1:1 physical mixture are displayed in Figure 1. Loratadine spectrum showed characteristic absorption bands in the range between 3,000 and 2,850 cm À1 corresponding to C-H stretch. A strong peak appeared at 1,702 cm À1 corresponding C ¼ O group of ester. Other peaks were detected at 1,474 and 1,227 cm À1 and 996 cm À1 related to benzene ring stretching vibrations, C-H stretching and aryl C-Cl stretching respectively (Akhgari et al., 2016). The typical peaks of sulpiride were spotted at 3385 cm À1 (N-H), 3211 cm À1 (NH2), 1643 cm À1 (C ¼ O), and 1322 cm À1 (SO2) (Zidan et al., 2015). The corresponding physical mixture spectrum displayed the presence of the typical peaks of both drugs. 3.1.2. Differential scanning calorimetry study DSC thermograms of raw loratadine, sulpiride and their 1:1 physical mixture are revealed Figure 2. Loratadine exhibited a sharp endotherm at 135 C ascribed to its melting temperature Akhgari et al., 2016(Akhgari et al., 2016. Thermogram of sulpiride displayed a sharp endotherm at a temperature of 175 C attributed to its melting transition (Zidan et al., 2015). The physical mixture DSC thermogram presented the melting endothermic peak of loratadine at 135 C while showed the absence of the melting endotherm of sulpiride.
Appearance, pH and viscosity
Appearance, pH and viscosity of different nanoemulsion formulations are displayed in Table 2. The resulting nanoemulsions were homogenous in appearance and did not display any indications for drug precipitation or phase separation. F1, F2 and F3 showed less cloudy appearance while F4, F5, F6 and F7 were cloudy. Viscosity of different nanoemulsion formulations ranged from 73.3 ± 3.7 (F2) to 203 ± 3.1 (F7). F6 and F7 had viscosity values that were significantly greater than those of the other formulations with no significant differences between their values (p > 0.05). pH values were recorded in the range from 6.2 ± 0.0 to 7.2 ± 0.02.
Entrapment efficiency
The EE% of the prepared nanoemulsions was investigated to determine the amount of loratadine and sulpiride loaded in the internal phase (oily phase) of these formulations and the results are displayed in Table 2. The EE% of the prepared nanoemulsions for both drugs exhibited high values more than 90% with insignificant differences between different formulations (p > .05). F3 displayed the highest values for loratadine (97.5 ± 0.00) and sulpiride (98 ± 0.1).
Droplet size, polydispersity index, zeta potential and electrical conductivity
Droplet size is an important characteristic for evaluation of the stability of nanoemulsion and improvement of drug bioavailability (Xi et al., 2009). It is an essential factor since it influences the drug release and biological absorption (Parul et al., 2013). Depending on the appearance, viscosity and entrapment results, F1, F2 and F3 were selected for this investigation. The mean droplet size, polydispersity index and zeta potential of these formulations are displayed in Table 3. F3 demonstrated the smallest droplet size (85.2 ± 0.2 nm) while F1 showed the largest one (149 ± 2 nm). F2 and F3 had the lowermost values of PDI (0.44 ± 0.02 and 0.35 ± 0.0 respectively) while F1 exhibited a PDI value of 0.78 ± 0.01. Zeta Potential ranged from À20.8 to À29.7 mV with F2 having the highest value. The electrical conductivity values ranged from 0.00 to 0.02 mS/cm. Thus, F2 and F3 were selected for following investigations.
In vitro release studies
This study was carried out to assess the release rate of loratadine and sulpiride from the selected nanoemulsions F2 and F3 in comparison to the raw drugs ( Figure 3). The release of Figure 2. DSC thermograms of a. raw loratadine, b. raw sulpiride, c. 1:1 loratadine/sulpiride physical mixture. 85.2 ± 0.2 0.35 ± 0.0 À23.3 ± 0.2 0.00 ± 0.00 84.7 ± 0.5 Figure 3. Release profiles of loratadine and sulpiride from the selected nanoemulsions, F2 and F3 compared to release profiles of the raw drugs (n ¼ 3).
both drugs was enhanced from the investigated nanoemulsions displaying significantly higher values relative to those of the raw drugs (F 2 <50). The percentage amounts loratadine and sulpiride released after half an hour were 52.3%±.16 and 30%±0.3 from F2 or 58%±3 and 30%±2 from F3 respectively. On the other hand, the corresponding raw drugs showed 7%±1 and 6%±0.5 respectively at the same time interval. At the end of 8 hours, the percentage amounts loratadine and sulpiride released were 98%±2 and 93%±0.1 from F2 or 95%±1.5 and 91%±2 from F3 while the raw drugs displayed 35%±0.1 and 30%±0.3 respectively. The difference between the release of loratadine and sulpiride from F2 and F3 was insignificant (F 2 >50). Table 4 shows the correlation coefficient (r) and release exponent (n) for the selected nanoemulsions (F2, F3). It is clear that the Higuchi's diffusion and Baker-Lonsdale models had the highest correlation coefficient (r). Therefore, the release data of loratadine and sulpiride from the selected formulations was best fit to both models.
Morphology analysis
The TEM and SEM images of the selected formulation, F3, were displayed in Figure 4 revealing spherical shape of the nanoemulsion droplets with the presence of some aggregations.
Stability studies
Stability studies for the optimized formulation F3 were conducted at 4 C and 25 C for one month, the results are illustrated in Table 5. Insignificant alterations were detected in the droplet size, PDI and entrapment efficiency of loratadine and sulpiride (p > .05). Additionally, the appearance of the studied nanoemulsion persisted with lower cloudiness and absence of phase separation till the end of the study time.
3.8. In vivo efficacy study 3.8.1. Allergic rhinitis symptoms Table 6 displays the symptoms in different animal groups before and after the start of treatment with different formulations. Allergic rhinitis symptoms including nasal irritation and secretions, sneezing, eye secretions and conjunctivitis were found to gradually increase following induction procedures. The total score of the symptoms ranged from 11-12 before the start of treatment (day 1). After 14 days of treatment with the co-loaded loratadine and sulpiride emulsion, these symptoms were significantly reduced (p < .05) displaying a total score of 6. Further reduction of the symptoms was detected after treatment with the co-loaded loratadine and sulpiride nanoemulsion with a total score of 3 (p < .001). On the other hand, Placebo group displayed non-significant reduction of the symptoms with a total score of 9 (p >.05). Figure 5 show the mRNA expression of TNF-a, IL-1 and TGF-b after induction of allergic rhinitis and treatment with different formulations. The Nasal mucosa relative mRNA expression of TNF-a, IL-1 and TGF-b was significantly increased in positive control group (II) after induction of nasal mucosa inflammation relative to the negative control (I) (p < .0001). However, after treatment by placebo (III), the relative expression at the end of experiment showed no relative increase or decrease in the marker's expression in comparison to the positive control (II). Instead, after treatment with the conventional emulsion co-loaded with loratadine and sulpiride (IV), the nasal mucosa relative mRNA expression of TNF-a, IL-1and TGF-b was significantly reduced relative to the positive control (II). However, nasal mucosa relative mRNA expression of TNF-a, IL-1and TGF-b in the group treated with the nanoemulsion formulation, F3 (V) displayed a further and highly significant decrease relative to the positive control (p < .0001).
Discussion
FTIR and DSC have been employed to investigate the possibility of incidence of interaction between loratadine and sulpiride. The existence of the characteristic FTIR absorption bands of both loratadine and sulpiride in the spectrum of the corresponding physical mixture (at the same positions compared to the spectra of the individual drugs) refers to the absence of drug/drug interaction. The presence of the melting endotherms of loratadine in the physical mixture DSC thermogram revealed the nonexistence of drug interaction supporting the FTIR results. The absence of melting endotherm of sulpiride could be attributed to the dissolution of the drug in the melt of loratadine as the two drugs have close melting temperatures. The less cloudy appearance of F1, F2 and F3 compared to other formulations could be related to diminished droplet size resulting in reasonably weak scattering rendering the nanoemulsion system optically translucent . The higher viscosity values of F6 and F7 relative to other formulations might be ascribed to the higher concentration of (ns), p < .05: mild significant ( Ã ), p < .01: significant ( ÃÃ ), p < .001: highly significant ( ÃÃÃ ), p < .0001: very highly significant ( ÃÃÃÃ ) and p <.0001: very highly significant (####) by T-test unpaired. lecithin or glycerol respectively. Similarly, Zhou et al., have revealed a rise in the viscosity of a lecithin nanoemulsion by the increase in the concentration of lecithin or glycerol (Zhou et al., 2009). The high entrapment efficiency of loratadine and sulpiride in the prepared nanoemulsions could be attributed to increased drug solubility in the crude olive which contains a combination of unsaturated fatty acids that provide a cosolvent effect (Balata et al., 2016). This result could also be ascribed to the high ester value of olive oil (190.86) indicating high percentage of ester groups (Zambiazi et al., 2007). This value is an indication for the proportion of glycerol existing in the oil that gives high solubilizing capacity for the oil (Azeem et al., 2009). In a recent study, olive oil has been reported to have the uppermost solubilizing capacity for the hydrophobic drug gliclazide relative to other screened oils (Balata, 2018). These results are also in accordance with a preceding work that revealed that olive oil has a good solubilizing ability for the hydrophobic drug resveratrol (Balata et al., 2016). In addition, the hydrophilic nonionic surfactant, Tween 80, having HLB 15 might maximize the solubilizing power, which is essential for affording a uniform emulsion. Furthermore, lecithin as a natural emulsifying agent might enhance the solubilization of both drugs in the oil and hence entrapment efficiency. The resulted small droplet size is important for drug bioavailability as it leads to greater surface area for drug absorption. The small droplet size of F3 compared to F1 and F2 could be ascribed to the higher total concentration of the used surfactants (Tween 80 and sodium cholate) that might jacket the surfaces of the new droplet produced throughout homogenization and lower the oil/water interfacial tensions (Samson et al., 2016). It has been reported that the average droplet size of nanoemulsion reduces with the increase in concentration of surfactant due to the formation of larger water-oil interface (Joung et al., 2016). Furthermore, combination of hydrophilic surfactant Tween 80 and lecithin (natural emulsifying agent) decreases the interfacial tension during emulsification process and consequently reduces the nanoemulsion droplet size (Guttoff et al., 2015). The small PDI values of F2 and F3 indicates narrow globule size distribution, which reflects uniformity in the size distribution and droplet diameter of both formulations (Balakumar et al., 2013). The measured zeta potentials reflect good stability of the selected systems with F2 having the highest stability. Generally, it has been reported that zeta potential of ± 30 mV was appropriate for nanoemulsion stability; (M€ uller et al., 2001;Balakumar et al., 2013). Formulations with high zeta potential have higher stability, as they resist coalescence of oil droplets through enhancing the electrostatic repulsion between the charged globules (Balakumar et al., 2013). The electrical conductivity of the selected nanoemulsions revealed good quality nanoemulsions and support stability (Sari et al., 2015). The enhanced release of loratadine and sulpiride from F2 and F3 compared to the corresponding raw rugs could be ascribed to the small droplet size of the investigated nanoemulsions which imparts large surface area for release (Alshehri et al., 2020). Thus, both drugs existed in solubilized micellar solution which greatly enhanced their release from the selected nanoemulsions (Balata, 2018). Kinetic of release of loratadine and sulpiride from F2 and F3 obeyed the Higuashi Diffusion model denoting that there was a direct proportional relationship between the amount of drug release and either the square root of the total amount of the drugs or the drug solubility in the nanoemulsion formulation (Sarpal et al., 2010). Also, the release kinetics followed the model of Baker-Lonsdale that was established from Higuchi model and described the release of drugs from the sphereshaped matrices. Analogous findings have been documented for the release of chlorehexidine HCL from a promising antibacterial root canal irrigant nanoemulsion (Abdelmonem et al., 2019). Stability study revealed that F3 maintained excellent physical stability at 4 C and 25 C for one month.
The high significant reduction of the allergic rhinitis symptoms that was observed after treatment with the co-loaded loratadine and sulpiride nanoemulsion revealed a promising effect that was superior to that of the corresponding emulsion formulation. In response to actuation by extraneous particles, macrophages discharge TNF-a, a motivator of the inherent immune response. In a paracrine way, TNF-a instigates adjoining cells to generate interleukin-8 (to provoke phagocyte staffing) (Yao et al., 2005) and E-selectin (to support adhesion of phagocytes to the adjacent endothelium) (Hermosilla et al., 2006). Autocrine activity by TN-Fa encourages further TNF-a production and initiate macrophages to produce and release IL-1. In a paracrine way, IL-1 provokes the localized formation of Interleukin-6 and the expression of Intercellular Adhesion Molecule-1 (ICAM-1). The overall products of protein produced by TNFa and IL-1 acting together to facilitate the inherent immune response and induce the adaptive immune response (Cottam et al., 2004;Ott et al., 2007). TGF-b has a responsibility in numerous processes involving regeneration of epithelial cells, inflammation and healing of tissue. TGF-b plays an essential role in the initial immune response as it acts as a chemoattractant and activator of inflammatory cells. It also fosters downregulation of inflammation across impediment of actuated cells and stimulation of apoptosis exerting anti-inflammatory effects (Otto & Wenzel, 2008).
The significant increase of the nasal mucosa relative mRNA expression of TNF-a, IL-1 and TGF-b in positive control group after induction of nasal mucosa inflammation indicated that the induction of inflammation was successful resulting in upregulation of this expression. The absence of change in the marker's expression after treatment by free drug nanoemulsion indicated that the treatment with the plain nanoemulsion had no effect on these markers. The significant decrease of the marker's expression after treatment with co-loaded loratadine and sulpiride conventional emulsion indicated that the treatment with the emulsion causes downregulation of these marker's expression. However, the further and highly significant decrease of these markers after treatment with the nanoemulsion formulation, F3, revealed a superior downregulation of the inflammatory markers. Thus, the co-loaded loratadine and sulpiride nanoemulsion had a more efficacious action in managing inflammation compared to the corresponding emulsion formulation.
Conclusion
In the present work, co-loaded loratadine and sulpiride nanoemulsions were developed using soybean lecithin and olive oil in addition to surfactants. F3 that displayed small droplet size, low PDI, applicable Zeta Potential and enhanced in vitro drug release was selected as the prime nanoemulsion. The loratadine and sulpiride release of from F3 followed the Higuashi Diffusion and Baker-Lonsdale models. F3 presented a good stability over a month upon storage at 4 C and 25 C. The biological study revealed enhanced downregulation of inflammatory parameters; TNF-a, TGF-b and IL-1 in rabbit models of ovalbumin-induced allergic rhinitis compared to corresponding emulsion formulation. These findings support that the co-loaded loratadine and sulpiride nasal nanoemulsion as a new approach can provide an encouraging effect in handling the symptoms of allergic rhinitis. | 2021-02-03T06:16:50.514Z | 2021-01-27T00:00:00.000 | {
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261318687 | pes2o/s2orc | v3-fos-license | Frequency of prehospital ambulance utilization by patients with chronic diseases: A retrospective study
Abstract
The increasing number of elderly people will coincide with the increasing number of chronic diseases, because every elderly person is said to have a predominant one or more chronic diseases [3]. The increase in numbers can have an impact on increasing the use of health services. Repeat ambulance use is frequent and has an association with chronic health problems such as chronic respiratory disorders, epilepsy, mental disorders with alcohol abuse, and comorbidities [4]. Another study also reported that chest pain was present in one in 10 ambulance calls and the causes of the cases varied, with substantial differences depending on age and sex [5]. The data was presented using graphs and frequency distribution tables.
Utilization in Denpasar City
Denpasar City has an ambulance that serves pre-hospital cases under the coordination of the Regional Disaster Management Agency.
The Integrated Emergency Management
System in Denpasar City is managed by the Table 1 The number of chronic diseases served by Denpasar City ambulance 2019 Table 3 The number of chronic diseases served by ambulances in Denpasar City 2021 The use of the pre-hospital ambulance has begun to be used by people with chronic diseases, but not optimized. This can be seen in the number of chronic disease ambulance utilizations that do not reach half of total ambulance services. Similar results were also presented on the low use of ambulances in the country of Ethiopia. In that country, victims experiencing emergency conditions are mostly delivered by Bajaj or taxi [11]. The results of this study also did not find any repetition of the use of the service by patients.
Chronic diseases
Other studies have reported that patients with complaints such as breathing difficulties, seizures, chest pain, and alcohol poisoning sometimes repeat the use of ambulance services [4]. Furthermore, lack of awareness of ambulance access and / or lack of an integrated EMS system are barriers that can contribute to low ambulance utilization [11]. pandemic [12]. The data in Table 3 showed that stroke, diabetes mellitus, and heart disease are the three main chronic diseases comorbid with COVID-19, which have contributed to the increase in the use of prehospital ambulance services due to chronic diseases. These three diseases also experience an increase in patients every year, as can be seen in Figure 5. An increase in the use of prehospital services was also suggested to be associated with an increase in COVID-19 infections.
LIMITATION OF STUDY
This study only summarizes the number of ambulance uses, and the researchers did not examine the types of services or actions provided to patients or accident victims.
RECOMMENDATION
The three highest cases of pre-hospital ambulance use in Denpasar City are accident, fire, and sick patient evacuation services. The use of ambulances by chronic diseases has not yet reached half of the ambulance services.
Stroke, diabetes mellitus, and heart disease are the three most common chronic diseases that use pre-hospital ambulance services.
It is necessary to increase the capacity of ambulance services, so that the utilization of pre-hospital ambulance services increases.
The government, through related agencies, should conduct socialization so that there is an increase in the use of pre-hospital ambulance services by the community.
ACKNOWLEDGMENT
The researcher would like to thank the Institute of Technology and Health Bali for funding assistance so that this research could be carried out. The researchers also thank the participation and support of the Denpasar City Regional Disaster Management Agency and the Denpasar City Health Office, especially the Damakesmas Ambulance, for the opportunity to collect research data.
CONFLICT OF INTEREST
Authors disclose no conflicts of interest related to the work in this manuscript. | 2023-08-30T15:09:38.084Z | 2023-07-31T00:00:00.000 | {
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1800398 | pes2o/s2orc | v3-fos-license | Editorial: Dialogues in Music Therapy and Music Neuroscience: Collaborative Understanding Driving Clinical Advances
Music is a complex, dynamic stimulus with an
un-paralleled ability to stimulate a global network
of neural activity involved in attention, emotion,
memory, communication, motor co-ordination and
cognition. As such, it provides neuroscience with
a highly effective tool to develop our understanding
of brain function, connectivity and plasticity.
Increasingly sophisticated neuroimaging technologies
have enabled the expanding field of music
neuroscience to reveal how musical experience,
perception and cognition may support neuroplasticity,
with important implications for the rehabilitation
and assessment of those with acquired brain
injuries and neurodegenerative conditions. Other
studies have indicated the potential for music to
support arousal, attention and emotional regulation,
suggesting therapeutic applications for conditions
including ADHD, PTSD, autism, learning
disorders and mood disorders.
In common with neuroscience, the music therapy profession has advanced significantly in the
past 20 years. Various interventions designed to address functional deficits and health care needs
have been developed, alongside standardised behavioural assessments. Historically, music therapy
has drawn its evidence base from a number of contrasting theoretical frameworks. Clinicians are
now turning to neuroscience, which offers a unifying knowledge base and frame of reference to
understand and measure therapeutic interventions from a biomedical perspective. Conversely,
neuroscience is becoming more enriched by learning about the neural effects of ‘real world’
clinical applications in music therapy. While neuroscientific imaging methods may provide
biomarking evidence for the efficacy of music therapy interventions it also offers important
tools to describe time-locked interactive therapy processes and feeds into the emerging field of
3 June 2017 | Dialogues in Music Therapy and Music Neuroscience
social neuroscience. Music therapy is bound to the process of creating and experiencing music
together in improvisation, listening and reflection. Thus the situated cognition and experience
of music developing over time and in differing contexts is of interest in time series data.
We encouraged researchers to submit papers illustrating the mutual benefits of dialogue between
music therapy and other disciplines important to this field, particularly neuroscience, neurophysiology,
and neuropsychology.
The current eBook consists of the peer reviewed responses to our call for papers.
Dialogues in Music Therapy and Music Neuroscience: Collaborative Understanding Driving Clinical Advances
Over 30 years of neuroscientific investigations of music perception and cognition have developed an understanding of music involving and supporting global brain processing in virtually every sphere of human activity (Levitin and Tirovolas, 2009;Särkämö et al., 2013). Increasingly sophisticated neuroimaging technology is able to provide objective biomedical evidence of musical activity supporting neuroplasticity (Münte et al., 2002;Pantev and Herholz, 2011), which has been shown as underpinning recovery and rehabilitation in stroke (Schlaug et al., 2009;Särkämö et al., 2014). Furthermore, the therapeutic potential of musical activity has been evidenced by neuroscience methods in relation to effects between common areas of processing between speech, memory, attention and motor activity (Schlaug et al., 2009;Besson et al., 2011;Patel, 2011), in how it influences arousal (Pelletier, 2004;O'Kelly et al., 2013), and through the modulation of wide ranging neurochemical activity involved in stress, immunity, social affiliation, and reward (Chanda and Levitin, 2013;Fancourt et al., 2014).
The effective use of music with clinical populations by credentialed music therapists is also an area of increasingly robust research activity, as evidenced by recent Cochrane reviews of music therapy with autism (Geretsegger et al., 2014), depression (Maratos et al., 2008) schizophrenia (Mössler et al., 2011), and acquired brain injury (Bradt et al., 2010). Various interventions designed to address functional deficits and health care needs have been developed (e.g., Thaut and Hoemberg, 2014), alongside valid and reliable scales sensitive to the effects of music in assessment and treatment with diverse clinical populations, including disorders of consciousness , parent-child relationships (Jacobsen and McKinney, 2015), and neurodegenerative conditions such as dementia (McDermott et al., 2014) and Huntington's disease (O'Kelly and Bodak, 2016). Historically, music therapy has drawn its evidence base from a number of contrasting theoretical frameworks, wherein an abundance of heterogeneous, sometimes contradictory theoretical approaches are hard to generalize to a wider multidisciplinary and international milieu (Hillecke et al., 2005). Clinicians are now turning to neuroscience, which offers a unifying knowledge base and frame of reference to understand and measure therapeutic interventions from a biomedical perspective.
Neuroscience methods offer exciting opportunities to understand the effects and mechanisms involved in music therapy practice through (i) in situ studies, where measures are used during music therapy sessions to explore underlying neurological processes, (ii) empirical comparisons where neuroimaging and neurological measures provide biomarkers of general changes in brain processes pre and post interventions, and (iii) approximations, where methods are focussed on the effects of specific musical features, and findings explored to identify brain based action mechanisms in the music therapy process (Fachner, 2016).
Whilst music therapy is benefiting from neuroscience collaborations, neuroscience is becoming more enriched by learning about the neural effects of "real world" clinical applications in music therapy. Not only do neuroscientific imaging methods provide biomarking evidence for the efficacy of music therapy interventions, they also offer important tools to describe time-locked interactive therapy processes, feeding into the emerging field of social neuroscience. Music therapy is bound to the process of creating and experiencing music together in improvisation, listening and reflection. Thus the situated cognition and experience of music developing over time and in differing contexts is of interest in time series data (Fachner, 2014(Fachner, , 2016. This research topic developed as a consequence of the editors shared commitment to promoting fruitful dialogues between music therapists, psychologists, neuroscientists, and other medical professionals, at a time where these professions are increasingly sharing the same platforms at conferences (e.g., Luck and Brabant, 2013;Bigand and Tillmann, 2015;O'Kelly et al., 2014), and in collaborative research topics such as this, and its predecessor of a similar theme (Tervaniemi, 2014, for music and brain plasticity; Särkämö et al., 2016 for music in neurorehabilitation). Our research topic features the work of 115 authors in 18 papers across the titles Frontiers in Human Neuroscience and Frontiers in Auditory Cognitive Neuroscience. Whilst the majority of papers detail music therapy and neuroscience collaborations with specific clinical populations, two further areas are covered (i) commentary on the challenges and opportunities of music therapy and neuroscience collaborations, and (ii) studies with healthy populations offering both insights into how we process music and transferrable lessons for music therapy.
A diverse range of clinical populations are covered by the topic, reflecting the many areas of health care where music therapy and neuroscience collaborations are providing important new understandings. Two areas of stoke rehabilitation are detailed in the topic. Street et al. outline feasibility, efficacy, and patient experience of a music therapy treatment protocol aimed at promoting measurable changes in upper limb function in hemiparetic stroke patients. The authors detail the use a neurologic music therapy (NMT) technique designed for this purpose: Therapeutic Instrumental Music Performance. Similarly, Cortese et al. explore the effectiveness of "Melodic-Rhythmic Therapy" in the treatment of aphasia with six stroke patients. Bukowska et al. combine several NMT techniques ("Therapeutic Instrumental Music Performance, " "Rhythmic Auditory Stimulation, " and "Pattern Sensory Enhancement") in a pilot study examining mobility and stability with Parkinsons patients. Using a range of novel methods including 3D Movement Analysis, the authors demonstrate significant improvement in the majority of the spatiotemporal gait parameters in the experimental group (n:30) compared to the control group (n:25).
Neurological populations feature in several other studies in the topic, such as Baker et al.'s exploration of flow and meaningfulness using songwriting with those with traumatic brain injuries or spinal cord injury. Whilst they found the intervention was positively associated with well-being outcome in the 10 participants, they also observed that those who found the songwriting process had strong personal meaning, experienced increased anxiety and depression in the process of accepting their emotions. Steinhoff et al. (2015) detail a pilot music therapy study with five individuals in an unresponsive wakefulness state (or "vegetative state") using Positron Emission Tomography to measure changes in brain activity, finding increases in tracer uptake across the frontal, hippocampal, and cerebellar region of the brain of four patients receiving music therapy for 5 weeks. Krick et al. (2015) use structural brain scanning (magnetic resonance imaging, MRI) methods to examine the effects of the Heidelberg model of music therapy on tinnitus at a cortical level. The authors found increases in gray matter volume of a range of brain areas dedicated to auditory processing concurrently with decreased symptoms. Finally, neuro-developmental issues in autistic spectrum disorder (ASD) are addressed in a study of the effect of sung speech on socio-communicative responsiveness by Paul et al. Using an adapted single subject design with three autistic children, the authors concluded sung directives may play a useful role in engaging children with ASD serving as an effective intervention for promoting socio-communicative responsiveness. Ramirez et al. (2015) detail a pilot study using a novel musical EEG neurofeedback system to treat depression in the elderly. The authors found an average of 17% improvements on depression scores (BDI), concurrent with significant decreases of relative alpha activity in their left frontal lobe (p = 0.00008) in subjects receiving the intervention. As with all the studies in the topic featuring pilot level data, the small sample (n:10) in the study suggests findings must be cautiously interpreted, but points to the potential benefits of this novel technology. Linnemann et al. detail a study of voluntary (not therapist initiated) music listening with 30 female participants with fibromyalgia syndrome, which is characterized by chronic pain. Whilst neuro-chemical measures of stress response (cortisol and alpha-amylase) did not indicate significant effects, significantly improved perceived control over pain was observed using VAS type Ecological Momentary Assessment Items. The final study with a clinical population was provided by Fritz et al., in their study of the psychological effects of listening to self-made music during a prior musical feedback intervention with 22 polydrug abusers. The study design compared scores range of scales (e.g., PANAS) completed after listening to pre-recorded drum and base music or listening back to music co-created with other participants on exercise machines ("Jymmin") capable of modulating musical sounds, producing a similar, but original co-created music. They found a positive effect of listening to the recording of joint music making on self-efficacy, mood, and a readiness to engage socially, proposing participants were influenced by "recapitulating intense pleasant social interactions during the Jymmin intervention" (p. 1).
As detailed, a range of review and commentary papers explore the challenges and opportunities afforded by neuroscience and music therapy collaborations. Magee and Stewart frame this discussion by exploring existing and potential collaborations, whilst highlighting the misconceptions from both parties that may impede further expansion of the field. In a similar vein, Hunt comments on the boundaries of research methods employed in the neurosciences with regard to capturing inter-subjective, holistic experiences in music therapy, highlighting the potential of emerging technologies providing methods for delivering clinically relevant information for music therapists. Further to providing an overview of our neuroscientific understanding of auditory processing in premature infants, Shoemark et al. build the case for music-based interventions, including a hypothetical vignette from their shared clinical experience. Similarly Moore and Hanson-Abromeit review the neuroscience of emotional regulation development in childhood to frame the rationale for "Musical Contour Regulation Facilitation, " an interactive intervention for emotional regulation. Finally Sachs et al. provide a systematic review of the "Pleasures of Sad Music, " concluding that such pleasures exist where music is perceived as nonthreatening, is aesthetically pleasing, and where it produces psychological benefits such as mood regulation. The authors continue by exploring the neural mechanisms involved in producing sadness which can also induce a positive affective states, with implications for informing music therapy work in this field.
A range of research papers explore the therapeutic potential of music through neuroscientific investigations with non-clinical participants. Here, the emotional effects of music are explored from a range of perspectives. Psychologists Sharman and Dingle explore the conception that listening to music from the extreme metal genre might have a causal relationship with anger behaviors. Thirty-nine extreme metal fans were tested for heart rate and positive/negative feelings of effect on the PANAS scale after an "anger induction" followed by listening to 10 min of either preferred music or silence. Contrary to expectations, participants reacted to their preferred extreme music with stabilized heart rate and positive emotions. The role of music listening in emotional regulation receives further attention from Carlson et al., who investigated relationships between music listening behaviors ("Discharge" or "Diversion") and gender, levels of depression, anxiety and neuroticism in a large nonclinical sample (n:123). Interestingly, in the context of Sharman and Dingles findings, Carlson et al. found on psychological scales (MADRS, BFQ, and HADS-A) that Discharge (using music to express negative emotions), was related to increased anxiety and Neuroticism, particularly in males. However, comparisons can only be made cautiously given the different samples, methods and range of genres involved in Carlson's study. Carlson et al. also present brain imaging (functional magnetic resonance imaging, fMRI) findings highlighting decreases in medial prefrontal cortex activity in high Discharge males, with increases for females preferring music listening for Diversion, exploring these findings in relation to the neurological and psychological impact of maladaptive listening practices.
Focussing on more active music making in non-clinical populations, the effects of improvised and pre-composed choral singing on experiences of flow, engagement, and neurobiological measures of social affiliation and arousal (oxytocin and adrenocorticotropic hormone/ACTH) were investigated by Keeler et al. Significant increases on a validated measure of flow for both singing conditions were observed concurrently with decreases in ACTH, significantly for pre-composed music. Whilst the small sample of one choral quartet with a limited range of material indicates caution, the authors propose group singing as effective in reducing stress and arousal, highlighting the importance of flow states in this process.
In setting up this research topic, we aimed to investigate the following question from different viewpoints: what can we understand about the musical, therapeutic, relational, or creative processes in music therapy from a neuroscience perspective, and how can this perspective advance music therapy practice? Moreover, we aimed at introducing various experimental approaches designed recently in order to investigate the efficacy and underlying principles of music therapy. As we hoped, authors working with those with acquired, developmental or neurodegenerative neurological and psychiatric conditions submitted empirical research, systematic reviews, and case studies adopting neuroscientific methods. Furthermore, the richness, challenges, and potentials of this field have been explored in commentary, position statement, and theoretical papers. Though, convergent thinking and research activity this volume illustrates how much music therapy and neuroscience have to learn from each other. The authors wish to thank all authors, peer reviewers, and participants in the research featured here for the important contribution to this evolving field they have made in this topic. The papers featured show the great potential for more important and synergistic collaborations to benefit the wellbeing of both clinical populations, and those interested in harnessing the therapeutic power of music in their everyday lives. | 2017-05-04T15:50:02.562Z | 2016-11-22T00:00:00.000 | {
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248986342 | pes2o/s2orc | v3-fos-license | Phase Shifts Measured in Evanescent Acoustic Waves above the Solar Photosphere and Their Possible Impacts to Local Helioseismology
A set of 464-min high-resolution high-cadence observations were acquired for a region near the Sun's disk center using the Interferometric BI-dimensional Spectrometer (IBIS) installed at the Dunn Solar Telescope. Ten sets of Dopplergrams are derived from the bisector of the spectral line corresponding approximately to different atmospheric heights, and two sets of Dopplergrams are derived using MDI-like algorithm and center-of-gravity method. These data are then filtered to keep only acoustic modes, and phase shifts are calculated between Doppler velocities of different atmospheric heights as a function of acoustic frequency. The analysis of the frequency- and height-dependent phase shifts shows that for evanescent acoustic waves, oscillations in the higher atmosphere lead those in the lower atmosphere by an order of 1 s when their frequencies are below about 3.0 mHz, and lags behind by about 1 s when their frequencies are above 3.0 mHz. Non-negligible phase shifts are also found in areas with systematic upward or downward flows. All these frequency-dependent phase shifts cannot be explained by vertical flows or convective blueshifts, but are likely due to complicated hydrodynamics and radiative transfer in the non-adiabatic atmosphere in and above the photosphere. These phase shifts in the evanescent waves pose great challenges to the interpretation of some local helioseismic measurements that involve data acquired at different atmospheric heights or in regions with systematic vertical flows. More quantitative characterization of these phase shifts is needed so that they can either be removed during measuring processes or be accounted for in helioseismic inversions.
INTRODUCTION
Helioseismology investigates the Sun's interior structure and dynamics through analyzing the oscillation signals observed in either Doppler velocities or intensities in or arXiv:2205.10410v1 [astro-ph.SR] 20 May 2022 above the Sun's photosphere (Christensen-Dalsgaard 2002). Despite great advances and breakthroughs obtained in the last few decades using both global and local helioseismological analysis methods, helioseismology still faces challenges of some systematic effects, whose presence in either data or analysis methods often complicates the interpretation of measurements or hinders a reliable inference of the Sun's interior properties. For instance, the helioseismic center-to-limb effect (Zhao et al. 2012) is an order of magnitude larger than the meridional-circulation-induced signals for certain travel distances (Zhao et al. 2013;Kholikov et al. 2014;Rajaguru & Antia 2015;Chen & Zhao 2018), posing a great challenge to our inferences of the Sun's deep meridional circulation. Another systematic effect occurs in and near magnetic regions. As demonstrated by Gizon et al. (2009), the inferences of subsurface sound-speed perturbations using different helioseismic analysis techniques gave sharply different results, implying a systematic effect in such regions. To reduce this effect, Liang & Chou (2015) and Chen & Zhao (2017) excluded all the magnetic regions with a strength above a certain threshold.
What causes the center-to-limb effect and the magnetic effect in the helioseismic analysis is not exactly known, but recently Zhao & Chen (2020) suggested that the center-to-limb effect and part of the magnetic effect might have a similar cause: the helioseismic signals used in the analysis, particularly those pairs of signals used to compute the cross-correlation functions in time-distance helioseismology, are not observed in the same atmospheric heights. This height difference may introduce unexpected phase shifts in acoustic waves. Actually, the possibility that the helioseismic center-to-limb effect may be related to different heights where oscillatory signals are taken has already been explored by various authors. In the photosphere, convective blueshifts in granules dominate in areas over redshifts, systematically shifting the phases of acoustic waves and causing the observed center-to-limb effect according to Baldner & Schou (2012); but the shift caused by this process is not expected to be frequency dependent and the values of shifts are expected be small. Through coupling the Doppler and intensity observations, Schou (2015) demonstrated how granulation can change the observed amplitude and phases of the oscillatory signals. However, how the phases of the oscillatory signals vary with atmospheric height and acoustic frequency has not been well studied, although a detailed analysis of the behaviors of these waves above the photosphere is expected to offer a key insight of the physical causes of the observed effect.
Meanwhile, we have to recognize that most of the helioseismic waves that we observe are evanescent waves. Although helioseismic waves are observed between approximately 2.0 and 8.0 mHz, the photospheric cutoff frequency of the waves is believed near about 5.0 mHz (Jiménez et al. 2011), meaning that waves with frequencies below 5.0 mHz, including the strongest oscillatory power near 3.0 mHz, are evanescent waves, and those with frequencies above 5.0 mHz are propagating waves. While it is often expected that phases of evanescent waves no longer change with height in the atmosphere unless perturbed by flows or other physical factors, whether this is exactly the case in observed intensities and Doppler velocities remains to be examined. Despite the early effort examining how phases change with height (Lites & Chipman 1979), similar efforts with a higher precision that meets the current helioseismic measurement requirements have not been done, likely due to the scarcity of high-quality simultaneous observations covering multiple atmospheric heights.
In this article, through analyzing a set of well-observed long-duration data with high spatial and spectral resolutions, we study how the phases of both evanescent and propagating waves change with atmospheric height. We believe that our results have a profound impact on the interpretation and inversion of many local and global helioseismic measurements, which may challenge us to design new methods removing these systematic phase shifts. This article is organized as follows: we introduce our data acquisition and reduction in Section 2, present our measurements of various types of phase shifts in Section 3. We then discuss our results in Section 4, and give conclusions in Section 5.
OBSERVATION AND DATA REDUCTION
The data used in this study were previously acquired and used for different studies (Rajaguru et al. 2010;Couvidat et al. 2012). These imaging spectropolarimetry data were obtained on 2007 June 8 using the Interferometric BI-dimensional Spectrometer (IBIS ;Cavallini 2006) installed at the Dunn Solar Telescope at Sacramento Peak, New Mexico. These observations have a spectral resolution of 25 mÅ, with all the Stokes parameters (I, Q, U, V ) taken at 23 line positions along the spectral line Fe I 6173.3Å. The spatial resolution of this set of observations is 0. 330 (or 0. 165 pixel −1 ), and the circular field of view has a diameter of 80 . The observed region is close to the disk center at S07W17, with a medium-size sunspot of NOAA AR 10960 near the center of the field (see Figure 1a). The temporal cadence of the observation is 47.5 s, and a total of 586 time steps were taken with a duration of approximately 464 min. The Fe I 6173.3Å line, which is also used by the Helioseismic and Magnetic Imager onboard Solar Dynamics Observatory (SDO/HMI; Scherrer et al. 2012;Schou et al. 2012) and by the Polarimetric and Helioseismic Imager onboard Solar Orbiter (Solanki et al. 2020), is believed to form between ∼100-270 km above the photosphere according to various authors (Norton et al. 2006;Fleck et al. 2011;Kitiashvili et al. 2015). The spectral images were then dark subtracted, flat-fielded, and re-registered to remove the atmospheric distortions that were derived from the white-light images recorded simultaneously (Rajaguru et al. 2010).
In this study, we first derive line-of-sight Doppler velocities from the spectral-line profiles by use of three methods: bisector, MDI-like algorithm, and center-of-gravity method, and then compare velocities from various heights and methods for their relative phase shifts in oscillations. For the bisector method, we extract Doppler velocities of plasma motions within the line-forming layers from the line bisectors, in a similar way used in previous studies (Rajaguru et al. 2007(Rajaguru et al. , 2010. For the spectral-line profile acquired at each spatial location, we use 10 bisector levels with equal spacing in line intensity to derive 10 Doppler velocities, corresponding to 0%, 10%, . . . , 90% intensity levels and relative to the line-core wavelength 6173.34Å in the rest frame of reference (see Figure 2a). The Doppler velocities derived from different intensity levels form at different optical depths, which correspond to, approximately, different atmospheric heights, with 90%-intensity level slightly above the line's continuum formation height of 100 km, and 0%-intensity level corresponding to line-core formation height at ∼270 km. The intensity levels in between are approximately formed evenly between these heights; however, one needs to be cautioned that the line-formation heights vary depending on solar structures. In this study, for the relative phase shifts measured in Section 3, we only use Doppler velocities derived for the 80%, 60%, 40%, and 20%-intensity levels, denoted hereafter from the lower to higher atmosphere as V 80 , V 60 , V 40 , and V 20 , respectively. Figure 1b displays a sample image of V 40 , and Figure 3a shows a comparison of these four velocity curves at a random location with a 100-min duration. The comparison (Figure 3a) shows that the amplitudes of the Doppler velocities derived for different atmospheric heights are quite different, larger in higher atmosphere and smaller in lower, but the oscillations at different heights show clear similarities with little visual difference.
The second Doppler-velocity derivation method, MDI-like algorithm, is the method that was previously used for the data taken by Michelson Doppler Imager onboard Solar and Heliospheric Observatory (SOHO/MDI; Scherrer et al. 1995) and is also currently used in the SDO/HMI's routine production pipeline of Doppler velocities. Because SDO/HMI only takes observations at 6 line positions while the IBIS observations cover the full line profile, we can determine Doppler velocities in the same way as done for SDO/HMI. As shown in Figure 2b, we first multiply the IBIS-observed line profiles with the six SDO/HMI filter transmission profiles to match SDO/HMI's 6-point intensities. We then follow the procedure prescribed by Couvidat et al. (2012) to derive the HMI-like Doppler velocities (hereafter, V HMI like ): where dV /dλ = 48, 562.4 m s −1Å−1 , T is the observed wavelength span equal to 412.8 mÅ in this case, and a 1 and b 1 are the first Fourier components: The third Doppler-velocity derivation method, center-of-gravity method (see Figure 2c), computes the Doppler shift corresponding to the intensity-weighted average of the whole line profile following the formula: where I j represents the intensity at the j-th line position with a wavelength of λ j , and λ 0 is the line-core wavelength in the rest frame of reference. The center-of-gravity Doppler velocity (V CofG ) is then derived from ∆λ. Figure 3b displays a comparison of V 40 , V HMI like , and V CofG at a random location for a 100-min duration. The comparison shows that the Doppler velocities derived from the MDI-like algorithm are similar to V 40 , and the velocities from the center-ofgravity method are similar to the V 60 from the bisector method. This implies that the atmospheric height corresponding to the SDO/HMI Doppler velocities is at about the 40%-intensity level of the Fe I line. Despite being obtained from very different methods, the oscillation patterns in these three velocities show remarkable similarities although the following analysis will disclose their small phase differences.
For all the above three Doppler-velocity derivation methods, at each location we first use both the left (I − V ) and right (I + V ) circular polarization profiles to derive two Doppler velocities separately, and then average the two as the final velocity to be used in the follow-up analyses. This is essentially consistent with the standard SDO/HMI Doppler-velocity derivation procedure, in which two sets of Doppler velocities from the left and right circular polarization are averaged. After all the Doppler velocities are derived at each location, we then rebin the data by 2×2 in space to enhance the signal-to-noise ratio before carrying out our phase measurements.
DATA ANALYSIS
One main objective of this study is to investigate the relative phase shifts between acoustic waves observed at different atmospheric heights, particularly for the waves with frequencies below the cutoff frequency, i.e., evanescent waves. It is understandable that in sunspot regions, due to the complicated interaction between helioseismic waves and magnetic field as well as the substantial reduction of the cutoff frequency, the results from sunspot regions will differ significantly from quiet-Sun regions (see Rajaguru et al. 2010). Therefore, in this study, we limit our analysis to the quiet-Sun region, delimited by the two dashed circles in Figure 1b, and focus only on the phase shifts in evanescent waves between different heights.
Cross Spectrum
We first examine the phase-shift diagrams between oscillatory signals observed at different atmospheric heights, i.e., phase shifts in higher atmosphere of V 20 , V 40 , and V 60 relative to the lower atmosphere of V 80 . The phase-shift diagram as a twodimensional function of harmonic degree and frequency ν can be calculated following where V 80 ( x , y , ν) represents three-dimensional Fourier transform of V 80 (x, y, t), x and y are horizontal components of ( = 2 x + 2 y ), † represents the conjugate of the Fourier transform, arg represents the argument of a complex numbers, i.e., relative phase, and n represents one of the three numbers: 20, 40, and 60. Figure 4a shows the power diagram of the cross-spectrum between V 20 and V 80 , so that the relative locations of acoustic modes (or p-modes), f -mode, convection, and internal gravity waves can be easily identified. Figure 4b-d show the phase diagrams of the three higher-atmosphere velocities relative to V 80 , calculated using only the quiet-Sun signals. As can be seen, positive phase shifts (seemingly downward propagating) dominate the areas beneath the f -mode ridge, between of 500 − 5000 and ν of 0 − 3 mHz. These are believed to mostly correspond to internal gravity waves, consistent with the results reported by Straus et al. (2008). For acoustic modes above the dashed curve, the phase shifts clearly show negative signs (seemingly upward propagating) in higher frequencies and positive signs (seemingly downward propagating) in lower frequencies. It is not very clear in these plots at what frequency the phase shifts switch the sign from positive to negative. Figure 1, and this explains why the area of < 500 is not shown. The color bar on the right represents the relative phases, with positive (negative) values indicating oscillations in the higher atmosphere leading (lagging behind) those in V 80 . The dashed curves in all panels represent the line separating the f -and p 1 -ridges, below which signals are not used in the phase-shift analyses that follow.
Relative Phase Shifts between Different Heights
To more accurately measure how phases change in both evanescent and propagating waves above the photosphere, we measure the phase shifts δφ at each spatial location between the Doppler oscillations obtained at higher atmospheric levels and those at V 80 following the formula: where V n (x, y, ν) represents the one-dimensional Fourier transform of V n (x, y, t), and n represents one of the three numbers: 20, 40, and 60. Because we are only interested in studying the behaviors of acoustic waves, all signals corresponding to the f -mode, convection, and internal gravity waves are filtered out, and only signals above the dashed curves in Figure 4 are kept. Then a two-dimensional Gaussian smoothing is applied on the x-y space to further enhance the signal-to-noise ratio. The FWHM of the Gaussian function is 1. 5 for the results presented in this article; however, different values of FWHM are tested and the analysis results remain largely unchanged. After the data are filtered and smoothed, for each of the three higher atmospheric levels, the δφ n (x, y, ν) is measured at each quiet-Sun location, and then all the δφ(x, y, ν) are averaged in x-y space for δφ(ν). Figure 5 shows the relative phase shifts δφ and travel-time shifts δτ measured for all atmospheric levels as functions of ν, averaged from the entire quiet-Sun region where the net vertical flow is presumed to be close to 0. The δτ is computed from δφ following δτ (ν) = δφ(ν)/2πν. It can be seen that the δφ, albeit very small between 1.5 and 4.0 mHz, show positive values below ∼3.0 mHz and negative values above ∼3.0 mHz, indicating that even for evanescent waves in a region with no or little net vertical flows, the measured phases continue to change with height. The positive values indicate that the phases in the higher atmosphere lead those in the lower atmosphere, as if the waves propagate downward. Negative phase shifts persist from 3.0 mHz to 7.0 mHz and continue to grow in values with frequency. It is not surprising for the phases of ν > 5.0 mHz waves to grow because these waves are generally believed to be propagating waves, but it is a bit surprising that the magnitude of δφ drops beyond ∼6.0 mHz. This may be due to the increased observational noises with the increase of acoustic frequency, or due to the acoustic halos (e.g., Rajaguru et al. 2013;Rijs et al. 2016) outside of the sunspot where the data are taken for these analyses. However, whether the measured values match the expected values above 5.0 mHz and how to explain the phase behaviors above 6.0 mHz are beyond the scope of this article. Figure 5b shows that even for the relatively small δφ's, the corresponding δτ 's can be around 1 s or larger, implying that these small phase changes can cause substantial errors for some local helioseismic measurements, particularly when travel-time shifts caused by very weak flows are measured, such as meridional circulation in the Sun's deep interior or vertical flows beneath supergranules (see discussions in Section 4.1).
Figure 5 also shows that δφ (δτ , too) are not just functions of ν, but also functions of height: while the positive δφ remain largely unchanged with height, the negative δφ grow substantially. Figure 6 shows both δφ and δτ as well as the change rate of δτ as functions of intensity level (or approximately, atmospheric height), obtained for a few 1.0-mHz-wide frequency bands with their middle frequencies marked in the figure.
In most atmospheric layers, the δφ around 2.0 mHz remain largely flat and positive, and the corresponding δτ are around 1 s. The δφ around 3.0 mHz also remain mostly flat, close to 0 across all layers. For all the other frequency bands above 3.0 mHz, the δφ are negative and have a trend of growth with height, and the δτ values are of an order of 1-2 s. Figure 6c shows the phase changing rate, i.e., how rapid phase changes with height, is mostly similar for a same frequency in different heights, except a few notable points.
To investigate behaviors of acoustic waves in areas with persistent upward and downward flows, we select areas with the top 10% of upflow speeds and the top 10% of downflow speeds for further analysis. After removing a uniform background velocity, i.e., caused by rotation, the selected upflow (downflow) region has an average speed of −190 (+190) m s −1 during the observation period. Figure 7 shows that the δφ and δτ , measured from V 60 relative to V 80 , exhibit a frequency-dependent variation trend similar to those measured in the entire quiet region ( Figure 5). It is also clear that the δτ measured in the upflow region is systematically above the δτ measured in the downflow region, with the differences around 0.5 s. Note that this difference between the two δτ can be seemingly explained by the upward (downward) flows that speed up (slow down) both evanescent and propagating waves, but as a matter of fact, the measured values are too large to be explained this way. Given that the sound speed is much greater than 10 km s −1 above the photosphere and the total distance between these two layers is <50 km, a flow speed difference of 380 m s −1 cannot cause a δτ that matches the measured values. Measurements between V 80 and other higher atmospheres give similar values below 3.0 mHz and considerably larger values above that. As noted in Section 2, in addition to the bisector method, the MDI-like algorithm and the center-of-gravity method are also used to derive Doppler velocities from the observed spectral-line profiles. It is also of interest to examine relative phase shifts in the Doppler velocities derived from these different methods. Figure 8 shows δφ of the four bisector Doppler velocities relative to V HMI like as functions of ν. It can be found that the phases of V HMI like are relatively close to the phases of the bisector velocities below ∼3.0 mHz, but beyond that their phases show substantial differences. The δφ of the bisector Doppler velocities relative to the V CofG (plots not shown in this article) give similar results.
Relative Phase Shifts between Doppler Velocities from Different Methods
As demonstrated in Figure 3b, both the V HMI like and V CofG show similar magnitudes and variation patterns to V 40 , indicating that these two Doppler velocities correspond best with the bisector velocity inferred for the 40%-intensity level. Figure 9 shows δφ and δτ between these three Doppler velocities. None of the pairs fully agree with each other in the frequency-dependent δφ, indicating that all the Doppler-deriving methods show a different amount of combination of information from various atmospheric heights, and these combinations seem to be frequency dependent as well.
Comparing IBIS and SDO/HMI Results
We have by now examined the relative phase shifts in Doppler velocities obtained at different atmospheric heights and from different velocity-derivation methods. It would be interesting to examine how the oscillatory phases observed in intensities change with height, and how the measured results from IBIS observations compare with those from SDO/HMI. Unfortunately, for both IBIS and SDO/HMI, intensities can only be reliably inferred at line core and continuum of the spectral line but not for other optical depths; IBIS data were acquired long before SDO was launched, thus simultaneous comparison of IBIS and SDO/HMI data is not possible; and, SDO/HMI does not have multiple Doppler velocities available like those used in Sections 3.2 and 3.3. Double-height Doppler-velocity proxies were attempted previously using SDO/HMI's 6-position data (Nagashima et al. 2014), but the accuracy of those data cannot meet the high-precision requirements of this study. Therefore, in this study Figure 10. (a) Phase shifts measured from line-core intensities relative to continuum intensities, for both the IBIS and SDO/HMI observations. (b) Phase shifts measured from Doppler velocities, which are derived using MDI-like algorithm, relative to line-core and continuum intensities, respectively, for both the IBIS and SDO/HMI observations. for both IBIS data and one selected dataset from SDO/HMI, we compute relative phase shifts δφ(I lc − I c ) between line-core intensity (I lc ) and continuum intensity (I c ), which are then compared. For both sets of data, we also compute the relative phase shifts, δφ(V − I lc ) and δφ(V − I c ), between Doppler velocities V , derived using the MDI-like algorithm for both IBIS and SDO/HMI data, and their corresponding I lc and I c , respectively. However, meanwhile, one needs to keep in mind that the velocity and intensity data show different senses of mode asymmetries (Duvall et al. 1993a), and discussions related to this are in Section 4.3.
In this study, I c , I lc , and V data are taken from 2011 January 1 SDO/HMI observations near the disk center, with a duration of 464 min (same as the IBIS data duration) and covering an area of 256 × 256 with a 45-sec cadence. The SDO/HMI spatial resolution is 1.0 (or 0.5 pixel −1 ), much coarser than the IBIS data; but the field of view of this selected dataset is nearly as 10 times large as the IBIS data. Figure 10a shows δφ(I lc − I c ) for both datasets, in which it can be seen that the trends of the curves are similar, with oscillations in the higher atmosphere lead those in the lower atmosphere substantially between about 2.0 − 5.5 mHz, although the sign-reversal frequency differs in these two curves. However, it is also clear that the δφ(I lc −I c ) values from SDO/HMI are substantially larger than (about twice of) those from IBIS. Figure 10b shows δφ(V − I lc ) and δφ(V − I c ) for both sets of observations, in which V leads both I c and I lc substantially in phases. Again, the variation trends for both δφ(V − I lc ) and δφ(V − I c ) curves show many similarities in the IBIS and SDO/HMI results; however, the δφ measured from the SDO/HMI data are about as twice large as those measured from the IBIS data.
To understand the differences in the IBIS and SDO/HMI results, we compute phase diagrams of the cross-spectra between I c and I lc for both datasets (Figure 11b and 11c). Understandably, because of its higher spatial resolution and smaller field-ofview, the IBIS data has poorer wavenumber (or harmonic degree ) resolution relative to the HMI data. Both of the phase diagrams show similar and clear frequency dependency that is also seen in Figure 10: large positive δφ in the range of about 2.0 − 5.5 mHz and small negative δφ above about 5.5 mHz. Figure 11b also shows at least two δφ-variation trends: (1) The δφ show alternating positive and negative signs on either side of the p-mode power ridges, with positive values overlapping more with the power ridges, which results in giving a net positive sign in phase-shift analysis shown in Figure 10a; (2) Despite the alternating signs of δφ, there is a general trend of decreasing positive values and increasing negative values with the increase of . The trend (2) explains, we tend to believe, why the values of δφ measured from IBIS and SDO/HMI differ substantially (Figure 10): the IBIS data are more sensitive to the higher-wave signals, getting more contributions from the low-δφ portion to the measured δφ than the SDO/HMI data.
Impacts to Local Helioseismology Studies
In this study, we have analyzed relative phase shifts, δφ, between oscillatory signals observed in Doppler velocities of different optical depths (approximately, different atmospheric heights), derived using a set of long-duration IBIS observations with full spectral-line profiles, high spatial and spectral resolutions, and a high temporal cadence. Due to the scarcity of high-quality simultaneous Doppler observations of multiple atmospheric heights, similar types of analysis were rare in the past. We believe that the δφ, as a function of frequency and atmospheric height, reported in this article have profound impacts to local helioseismology studies, including timedistance helioseismology (Duvall et al. 1993b) among others.
As introduced in Section 1, the Sun's acoustic cutoff frequency (ν ac ) in the photosphere is around 5.0 mHz (e.g., Deubner & Gough 1984;Jiménez et al. 2011), thus our observed waves consist of both evanescent and propagating waves. It is often presumed that the phases of evanescent waves remain unchanged with height unless the waves are perturbed by vertical flows or other dynamic parameters; or if perturbed, local helioseismic inversions are expected capable of recovering these perturbations. However, our analysis shows that for the acoustic-mode oscillations inferred from the Fe I 6173.3Å line in a quiet-Sun region, where the net vertical flow is expected to be close to 0 (for discussions of the role of convective blueshift, please refer to Section 4.3): relative to lower-atmosphere oscillations, higher-atmosphere oscillations lead in phases while ν < 3.0 mHz and lag behind while ν > 3.0 mHz (for discussions of evanescent waves, please refer to Section 4.2). These measured δφ are not consistent with the general presumption of the stationary phases in evanescent waves, nor can be simply explained by vertical flows. The long-term (around 7 hrs in this study) average of the vertical flows, if not exactly net 0, is not expected to exceed an order of 100 m s −1 , which, to the waves with a phase speed >10 km s −1 within a travel interval of <200 km, can hardly cause phase (or travel-time) shifts that match the measured values in Figures 5 & 6. Equally importantly, vertical flows cannot explain the measured frequency dependency of δτ , either.
What possibly causes these observed phase shifts will be discussed in Section 4.3, and here we focus on how these results impact local helioseismic studies. Let us take time-distance helioseismology as example, which measures (see Figure 12) travel time τ AB of acoustic waves traveling from one surface location A to another surface location B through the solar interior, and measures travel time τ BA along the opposite direction. Suppose the Doppler observation at location A forms at one atmospheric level, and the Doppler observation at location B forms at a slightly higher levellocation B . Based on our earlier analysis, there is a travel-time shift τ BB between positions B and B ; therefore, the presumed τ AB measurement becomes τ AB , which is equal to τ AB + τ BB , and the presumed τ BA measurement now becomes τ B A , which is equal to τ BA − τ BB . The travel-time difference, δτ AB ≡ τ AB − τ BA , which is often interpreted as caused by the plasma flows along the subsurface travel path, becomes δτ AB + 2τ BB . A measurement error of 2τ BB is thus introduced, which will be mistakenly interpreted as interior flows in the inversions. Based on our analysis shown in Section 3.2, this τ BB can be around +1 s between 1.5 -3.0 mHz and around −1 s between 3.0 -5.5 mHz, approximately the same order of magnitude as caused by the near-surface meridional flow, thus non-negligible. Actually, this model is not strictly accurate because oscillations at location B and B consist of all types of wave Figure 12. Schematic plot showing a subsurface ray path connecting two surface locations A and B, along which waves travel between these two locations. Signals presumably observed at location B are actually observed at a slightly higher location B . Plots are not to scale.
modes, while waves traveling from A to B consist of only a few modes. So the τ BB measured locally may not be identical to the τ BB in the waves traveling to here from A. Nevertheless, it is still reasonable to believe, although the values may slightly differ, using oscillatory signals observed at different atmospheric heights to measure δφ (or δτ ) can introduce non-negligible errors.
As a matter of fact, it is not rare that observed oscillatory signals form at different atmospheric heights on and above the Sun's photosphere, albeit most times with only small height differences. For instance, due to the limb-darkening effect, the formation heights of most spectral lines gradually increase with the distance to the disk center; and in fact, this is believed to be directly linked to the helioseismic center-to-limb effect (Chen & Zhao 2018). Due to Wilson Depression, the observed sunspot umbra, penumbra, and quiet-Sun regions can differ in heights by ∼500 km, likely causing a few seconds of δτ measured between them, which are not due to any flows but were mistakenly ascribed to flows in some past helioseismic inversions (e.g., Zhao & Kosovichev 2003). Another example is supergranules, which are often thought of as quiet-Sun regions but their central areas may be slightly warmer, thus spectral lines there form slightly higher, than their boundary areas. For a long-time (a few hours) analysis typically required by time-distance helioseismology, the δτ measured between supergranular centers and boundaries will likely carry a systematic shift that is unrelated to the supergranules' subsurface structure or flows, but such a systematic shift is not accounted for in the time-distance analysis (e.g., Duvall & Hanasoge 2013).
Another important implication to the time-distance helioseismic measurements is in the areas with systematic long-term (an order of a few hours) up-or down-flows. As illustrated in Figure 7, the measured travel-time shifts in the areas with up-or down-flows cannot be accounted for by the vertical flows inferred from Doppler shifts, and some other factors must play a role in causing these measured shifts. Therefore, if one end of the acoustic waves used for time-distance measurements (say, location A or B in Figure 12) is inside a region with systematic vertical flows, one needs to be cautious in interpreting the measured δτ . There is no shortage of such locations on the solar surface, e.g., again, the boundaries (central areas) of supergranules are usually associated with downward (upward) flows. This vertical-flow-related effect may either be just another manifestation of the effect caused by the different lineformation heights that is discussed above, because supergranular upflow (downflow) areas are often associated with hotter (cooler) areas, or it is an independent effect in addition to the line-formation height.
It is now clear that the unaccounted-for phase shifts in evanescent waves impact significantly how we interpret local helioseismic measurements, but it is not immediately clear how this observational fact impacts global helioseismology. Intuitively, the systematic change of oscillatory phases with distance to the disk center, i.e., the center-to-limb effect, albeit small, can systematically change the ridge location ( , ν) in the power spectrum, hence change the inversion for sound-speed structures inside the Sun. The effect on the inference of the internal rotation by global helioseismology should be negligible, because the effect from both sides of the central meridian can cancel each other. In addition, our observations can only identify non-zero phase shifts in evanescent waves in and above the photosphere, but unable to investigate how phases change beneath the photosphere. For example, let us consider 3.0 mHz waves, which become evanescent a few hundred km beneath the photosphere: are extra phase shifts introduced before they are observed at the surface? A tiny change of their phases in this depth interval that are unrelated to flows will greatly challenge our interpretation of both global and local helioseismic measurements.
Our analysis also shows that the Doppler velocities derived from the MDI-like algorithm and the center-of-gravity method both show small yet non-negligible phase shifts relative to the velocities that are derived from the bisector method and match those two methods best. This does not imply that any of these Doppler-velocity derivation methods introduce extra phase shifts, but caution us that it may not be appropriate to measure relative phase shifts between velocities derived using different methods.
Evanescent Waves
Our analysis on this specific set of IBIS observations shows that for most atmospheric heights, the δφ of the acoustic waves switches from positive to negative at around 3.0 mHz, where a mixture of both evanescent and propagating waves is expected. However, what frequency separates evanescent waves from propagating waves is not very clear in this set of observations.
Studying the helioseismic center-to-limb effect as a function of acoustic frequency, Chen & Zhao (2018) pointed out that the sign reversal of the center-to-limb effect occurs at about 5.5 mHz, which is close to the cutoff frequency, in most regions on the solar disk, and this sign-reversal frequency decreases with distance to the disk center. If the center-to-limb effect is indeed, most likely it is, connected to the spectral-line's formation height, we would expect the observed sign reversal in our present analysis to occur around 5.5 mHz instead of near 3.0 mHz, because the analyzed region is very close to the disk center and both this analysis and their analysis (Chen & Zhao 2018) used observations from the same spectral line Fe I 6173.3Å. A few factors may play a role in causing this apparent discrepancy: first, in this study the IBISobserved region is in the vicinity of a sunspot, with visible pores in our "quiet-Sun" region. The presence of magnetic regions, though sparse, and the systematic moat flows around the sunspot, will undoubtedly affect our results while the magnitude of the influence is difficult to assess. Second, the sign reversal near 3.0 mHz seen in this analysis is from the bisector-derived Doppler data, and it is difficult to estimate how the MDI-like algorithm-derived Doppler velocities would behave as a function of frequency and height, thus it is difficult to compare these results directly against the SDO/HMI results. Third, it is possible that different instruments with different spatial resolutions and modulation transfer functions, despite using the same spectral line, can cause systematic differences in the measured results. All these above factors are for Doppler velocities, but the results obtained from intensity data, presented in Section 3.4, may help shed light on understanding more of the sign-reversal frequency.
The comparison of measurements, made using IBIS and SDO/HMI line-core and continuum intensities, show similar frequency-dependent trends of δφ but with substantially different values. The phase diagrams of the cross-spectra between the two intensities from both datasets help to explain the discrepancies in the δφ values: the δφ are not just a function of frequency and height, but also a function of wavenumber. With a higher spatial resolution, the IBIS δφ measurements are constituted more of low phase shifts than the SDO/HMI observations, causing the discrepancies in the measured values. Although this conclusion is only for intensity data, we speculate the similar thing for the Doppler data. It would be reasonable to speculate that the δφ in SDO/HMI-observed Doppler data will show a frequency-and height-dependency similar to those measured from IBIS data, but likely with higher values and a higher sign-reversal frequency.
Overall, although it is unclear at what frequency the acoustic waves become evanescent and whether the phase-shift sign reversal is directly related to the cutoff frequency, it is quite clear that the phases of the evanescent waves continue to change, albeit rather small, with the atmospheric height. The sign and amount of the phase shifts seem to depend upon acoustic frequency, as well as spectral lines, telescopes, and instruments that are used for observations in addition to vertical flows.
Physical Causes of Phase Shifts
Our analysis has shown that above the Sun's photosphere, the measured phases of evanescent acoustic waves continue to shift, and this frequency-dependent shift cannot be explained by vertical flows in the region. What causes such phase shifts?
For an enclosed ideal and adiabatic gas that is experiencing a periodic variation, the velocity change is expected to lead the temperature change in phase; however, the Sun's atmosphere is neither enclosed nor adiabatic. For an evanescent wave, even if the phases of oscillatory signals in velocities do not change or only change subtly with atmospheric height, the temperature responses to them, thus the observed intensity changes, at different heights may not be simultaneous, but likely with a gradual height-dependent time delay due to the non-adiabaticity of the atmosphere. That is, intensities at different heights do not respond simultaneously to a same velocity perturbation. As clearly shown in Figure 5a and Figure 11a, oscillatory signals show small phase changes with height when observed in Doppler velocities, but show substantial, an order of magnitude larger, phase changes with height when observed in intensities. Now, we need to remind ourselves that all Doppler velocities are inferred from spectral-line intensities, and a small leakage from the large heightand frequency-dependent phase shifts in intensities can result in non-negligible phase shifts in Doppler velocities, even when local velocities do not carry such phase shifts intrinsically. Indeed, Fe I 6173.3Å line is not perfectly symmetric, and its asymmetry gets enhanced by convective blueshifts (e.g., Löhner-Böttcher et al. 2018;Stief et al. 2019). And, it is also well known that the red wing of spectral lines oscillates with larger amplitudes, thus carrying stronger oscillatory power, than the blue wing (Cavallini et al. 1985;Bertello & Caccin 1990). Therefore, the Doppler velocities derived from this asymmetric line profile, with unbalanced oscillatory power in both of its wings, may carry a height-dependent phase shift leaked in from intensities. That is, a combination of the atmosphere's non-adiabaticity and the spectral line's properties can cause height-and frequency-dependent phase shifts in Doppler velocities. This is a scenario that we speculate can explain most of the observed phase shifts while the rest can be explained by the scenarios discussed below; meanwhile, we also recognize that this scenario needs to be confirmed using numerical simulations (e.g., Kitiashvili et al. 2015), and perhaps, more disk-location-dependent observations with the full spectral-line profiles are needed for a more systematic analysis.
Other factors may also play a role in the measured phase shifts. To explain the helioseismic center-to-limb effect, which is probably a manifestation of the frequencyand height-dependent δφ reported in this work, Baldner & Schou (2012) tried to explain that the ubiquitous convective blueshift above the photosphere likely caused negative phase shifts in both the evanescent and propagating waves. (Note that the role of convective blueshift here is to shift phases of acoustic waves, different from its role in causing the spectral-line asymmetry mentioned above.) This effect undoubtedly plays a role in causing some phase shifts, but the amount of the shifts estimated from numerical models are about one order of magnitude smaller than the observed values, indicating that other factors may play a leading role. In addition, this mechanism cannot explain the observed frequency dependence of the phase shifts.
Another cause of the observed δφ may be related to the line-asymmetry (Duvall et al. 1993a) across the oscillation power ridges. (Note that the line asymmetry here is across acoustic power ridges, different from the optical spectral-line asymmetry mentioned above.) It appears (see Figure 11b) that the δφ observed in intensities show opposite signs on either side of the power spectrum ridges, and the line asymmetry can cause unequal contributions to the δφ in our δφ or δτ measurements. If the observed line asymmetry across the acoustic power ridges is caused by correlated noise in observations (Nigam et al. 1998), then it is possible that such correlated noise is an indirect cause of part of the observed phase shifts, although the magnitude and the detailed frequency dependence that this mechanism can incur remain to be investigated.
CONCLUSION
Through analyzing oscillatory signals observed at different optical depths, which roughly correspond to different atmospheric heights, we have found that the phases of evanescent acoustic waves continue to change with height, increasing for lowerfrequency waves and decreasing for higher-frequency waves, and the changes are not accounted for by vertical flows. We also found that in areas with systematic upward or downward flows, the phase shifts measured in the acoustic waves are more substantial than those expectantly caused by flows. These pose great challenges to some helioseismic analyses that involve measurements using observations obtained at different atmospheric heights or in areas with systematic vertical flows. The comparison of the phases measured from IBIS and SDO/HMI intensity data shows significant differences in magnitude, but these differences are likely owing to the different sensitivities of the instruments to high-waves. We speculate that our measured frequency-and heightdependent phase changes in acoustic waves are probably due to the non-adiabaticity of the Sun's atmosphere with a combination of the asymmetry in the spectral line used in observations. More investigations are needed to better quantify the phase changes as functions of height and frequency, so that this systematic effect can be either removed in helioseismic measuring processes or can be accounted for in helioseismic inversions. | 2022-05-24T01:16:13.534Z | 2022-05-20T00:00:00.000 | {
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245353293 | pes2o/s2orc | v3-fos-license | Cosmic adventures in stringland: Scattering of bosonic and fermionic fields in gravitating cosmic string spacetimes
We show that when considering a scalar field scattering in a gravitating cosmic string spacetime, the standard partial-wave approach's scattering amplitude is singular. In order to avoid the divergence caused by the spacetime asymptotically conical structure, we propose a modification of the asymptotic ansatz in the partial-wave formalism and find the corrections in the phase-shift and total scattering cross-section. We also developed a toy model for the spacetime metric of a cosmic string and showed how local interaction with the vortex gauge field affects the scalar field total cross-section. Then we apply this formalism to a Dirac field and show the explicit formula for the fermionic total cross-section. Finally, we study the scattering of bosonic and fermionic fields in the spacetime of an abelian and a nonabelian gravitating cosmic strings and show that the cross-sections have damped oscillations. In order to understand the origin of this behavior, we used the aforementioned toy model to show that the spacetime particular asymptotical structure causes the observed oscillations.
Introduction
In 1976, Thomas Kibble showed that cosmological phase transitions during the early universe should lead to the formation of topological defects, namely cosmic strings, domain walls and monopoles [1]. Among these, cosmic strings have been showed to be the most promising ones [2,3]. Along the years many attempts have been made in order to develop reasonable observational constraints on the parameters of real cosmic strings [4,5]. Some works suggested cosmic strings as possible sources for gravitational lensing phenomena [6]; and although some of these claims have been debunked [7], recent data from 12 years of observation of the NANOGrav collaboration [8] suggests that cosmic strings gravitational interaction might be affecting the detection of pulsar blinking [9,10]. It appears the subject of cosmic strings is about to be revived. However, the attention toward cosmic strings is not exclusively by cosmological reasons. These objects have strong condensed matter counterparts. The U(1) string solution, for instance, is also present in the theory of superconductors [11][12][13]. Furthermore, even the gravitational interaction of cosmic strings, in the wire approximation, have condensed matter analogues called wedge disclinations and edge dislocations. Therefore the interest in cosmic strings/vortex solutions, are not exclusive for the high-energy physicists.
Many cosmic string solutions have been found in many diverse models, specially during the 20th century, such as the Nielsen-Olesen vortex in 1973 [14], Semilocal strings in 1991 [15] and Eletroweak strings in 1993 [16]. Nonetheless their gravitational interaction with nearby matter have been most studied using the wire approximation. This is possibly because it is too hard to study the gravity-coupled system, since one usually has to solve 5 (or more) coupled partial differential equations. Hence one has to resort to numerical methods to have even a basic understanding of the field and metric solutions. Even the flat-spacetime cosmic string solutions have not been analytically solved completely. With a symmetry-breaking model in hands one frequently resorts to proving the existence of such a line-like object without actually finding the solution analytically. Only in some restricted situations the equations of motion are analytically solvable, such that it is of no surprise that the gravity-coupled system is only possible to be studied using numerical methods.
Although Garfinkle [17] made significant advances in understanding the asymptotical conical limit of an abelian string spacetime, there was still plenty of aspects to be understood. For instance, how do the vortex internal parameters affect the asymptotical metric limit or the curvature profile? Is the conical limit a general property of any cosmic string solution? Do the gravitational description changes significantly when we consider the vortex size to be non-negligible? These are all legitimate questions, but one needs to build a comprehensive understanding of gravitating cosmic string solutions to answer them. The works of Christensen, Larsen, and Verbin [18] and Brihaye and Lubo [19] on the abelian-Higgs model appeared as a light in this long cosmic journey. [18] established the default notation in studying gravitationally extended vortex solutions of the abelian-Higgs model and following them Brihaye and Lubo [19] showed that the metric of the U(1) vortex is asymptotically conical and how the conical parameters depend on the internal ones. Subsequently many gravitating vortex solutions have been found to also be asymptotically conical. This feature, which seems to be general to all gravitating cosmic string solutions, is a key point to the work presented in chapters 4 and 5 of this thesis.
This thesis is organized as follows. In Chapter 2 we present the essential theory of cosmic strings without taking into account their gravitational interaction. We study the Nielsen-Olesen vortex with a reasonable amount of details and present the numerical solution to the scalar and gauge fields as well as the energy density with two different winding numbers. In Chapter 3, we study the gravitational aspects of cosmic strings. We present the wire approximation and two of the local physical effects of a conical spacetime. In the second part of Chapter 3 we dive into the world of gravitationally extended vortices. We present the results from [18] and [19] on the gravitating U(1) string, and the results of Pádua Santos and Mello [20] on a non-abelian-Higgs model developed recently. We see that both of these models present conical structure far from the core. In Chapter 4 we study the scattering of scalar and fermionic fields in the spacetime of a gravitating cosmic string. We show that the partial-wave formalism presents inconsistencies when applied to the scalar field scattering in this class of spacetimes and propose a modification to solve them. We explicitly show the corrections in the phase-shift, scattering amplitude and total cross-section. In order to apply the formalism we develop a toy-model for the spacetime of a cosmic string and study the scattering of a scalar field in this toy-model. We also take into account the interaction with the gauge field that generates the vortex and compare the cross-section with and without the local gauge-field interaction. In the last part of 4 we apply the same formalism to the fermionic field and find the expression of the total scattering cross-section.
In Chapter 5 we apply the formalism to the spacetime found by [20]. In the first part we study the scattering of the scalar field and show how the mass of the field affects the total crosssection. We then turn our attention to analyze the reason behind the observed oscillations in the total cross-section. In order to do that we use the aforementioned toy-model to conclude that the oscillations are caused by the spacetime asymptotical conical structure. In the second part of Chapter 5 we study the fermionic scattering in the same spacetime. We show the dependence of the cross-section with the fermion mass and also observe oscillations in the total cross-section. Finally we present a crude estimation of why the cross-section is larger when the deficit angle of the asymptotical spacetime is bigger.
Throughout the text we use natural units where = c = 1. Hence mass is measured in Planck mass, m p , and length in Planck length, l p . For future reference, these values are approximately m p = 2.1 × 10 −8 kg and l p = 1.6 × 10 −35 m. Vicki Weisskopf [21] In field theory, a topological defect is a field configuration that only depends on the value of the field at the boundaries. Since, for a localized configuration, the boundary field values are also the vacuum ones we can also define topological defects by the non-trivial structure of the vacuum of the theory, which is commonly expressed as a disconnectedness in the vacuum manifold. This motivates a more general definition of topological defects which is also valid outside the field theory context: a topological defect is a discontinuity (defect) in the order parameter 1 that cannot be removed (topological).
Furthermore, if the field configuration cannot be continuously transformed to the vacuum keeping the energy finite then it is considered topologically stable since, as we shall see, it is possible to define a conserved charge that depends only on the field values at the boundaries.
The non-trivial topology of the vacuum is commonly connected to a form of symmetry breaking, which can be related to phase transitions in a variety of systems, ranging from condensed matter ones, as in liquid crystals, liquid helium, and superconductors, to the formation of structures in the early universe, such as domain walls and cosmic strings. In this chapter, we will see the basic topological defects from a field-theoretical point of view. In the last section, however, we encounter some interesting connections between condensed matter systems and the universe's structure.
The simplest model: φ 4
When confronted with a new subject, it is often illuminating to start with a straightforward problem. A simple model that admits a topological defect solution is the 1+1-dimensional φ 4 model, i.e. we are searching for static solutions in 1 spatial dimension.
The φ 4 Lagrangian describes a real scalar field with a quartic self-interaction potential. The quadratic term in the field, λη 2 φ 2 /2, means the mass of the field is √ λη. Also, the minimum of the potential, V (φ) = 0, is achieved when φ = ±η, hence we say φ = ±η are the vacuum states. The Euler-Lagrange equation yields the solutions which are called kink (positive sign) and anti-kink (negative sign). The kink is a one-dimensional, localized, time-independent, stable solution. We shall see it is also topologically stable, i.e. the boundary conditions enforce stability under field perturbations.
In Figure 2.1 we see that the kink interpolates between both vacuum states, ±η, which are at ±∞. Also, the order parameter is the vacuum expectation value (VEV), φ , the field's value at the minimum of the potential as it labels the different equilibrium field configurations. The φ 4 model has many applications. For example, it can represent domains walls [22], 2dimensional structures expected to have formed during the early universe, but also non-linear excitations in polyacetylene [23]. For a historical overview of the φ 4 model we suggest [24]. Now, if we look at the vacuum manifold (the space of vacuum states), we see that it is disconnected. It is impossible to go from one vacuum to the other passing only through vacuum states. This means that the order parameter, φ , has a discontinuity between the vacua; thus, the vacuum manifold is disconnected.
It was mentioned earlier that topological defects are usually connected with symmetry breaking, but which symmetry is broken by the quartic potential? The answer is Z 2 parity, which is equivalent to φ → −φ. If we make η → 0 we see that both vacua coalesce to the same value, namely φ = 0, and the configuration becomes Z 2 invariant. The degenerate minima and the disconnected vacuum manifold only appear because η = 0. Topological defects are topologically stable. In order to destroy the kink, one would have to take every point of the solution and move to zero, which would require an infinite amount of energy since the solution extends to infinity. This guarantees the stability of the kink. This argument can be tracked down to the disconnectedness of the vacuum, where the vacuum structure ensures stability.
We can see topological stability more clearly defining the current where µν is the two-dimensional Levi-Civita antisymmetric tensor. One can easily see that the current (2.3) is conserved, ∂ µ j µ = 0, which gives rise to a conserved charge Naturally, one might want to explore solutions of the φ 4 model in more spatial dimensions. However, it is not possible to find such stable static solutions. Derrick's theorem [25] states that there are no non-linear scalar field models with stable, time-independent, and localized solutions in more than one dimension which imposes a big restriction on higher-dimensional solutions to (2.1). Essentially, if one has a non-linear scalar model in more than one spatial dimension it is guaranteed no static and localized 2 solution is stable under field perturbations. Derrick's argument is based on the fact that any D-dimensional pure scalar field theory, with static solution is φ(x), has total energy given by where ∇ x is the gradient with respect to the x-coordinates. For further use we define the two quantities We can make the rescaling x → y = αx, α = 0, and define the deformed field φ α = φ(αx). The energy of the new configuration is given by Now we can analyze how the energy behaves with respect to the rescaling parameter α and the spatial dimensions. The main point here is that if the solution φ(x) is stable then α = 1 is a minimum of the energy. Extremizing the energy we find the condition for the solution to be at the equilibrium point: Now we need to know if this point is a minimum, δ 2 E > 0, or a maximum, δ 2 E < 0. 11) which is greater than zero only when D < 2. This means that for D ≥ 2 a deformation of φ(x) shall decrease the energy of the configuration, hence the solution is not stable.
The alternatives to construct stable non-linear scalar field solutions in higher dimensions are split into two categories: 1) extending the model by including more fields, like a gauge or spinor field, and b) relaxing the time-independence or localized condition. Higher-dimensional topological defects can be obtained following any of these two paths. Before diving into any of these routes, we need to understand a straightforward generalization of (2.1).
Spontaneous Symmetry Breaking
Come forth into the light of things, let nature be your teacher.
William Wordsworth
Knowing the symmetries of the Lagrangian is often regarded as the first step in getting a comprehensive description of a system. If a symmetry is continuous, Noether's theorem assures a related conserved quantity, making some calculations a lot easier. Nevertheless, sometimes a symmetry of the Lagrangian might not be the symmetry of the vacuum. In such cases, we say the symmetry is spontaneously broken.
A simple model: Goldstone
We can illustrate the main features of spontaneous symmetry breaking using the Goldstone model which looks similar to the φ 4 one, but now φ is a complex scalar field instead of a real one. Although, according to Derrick's theorem, model (2.12) in more than one dimension is unstable, it is also pedagogically useful to illustrate spontaneous symmetry breaking. We see that (2.12) is invariant under the action of the global U (1) group, i.e., (2.12) does not change if we multiply φ by a constant phase α, φ → e iα φ. The vacuum state, φ = ηe iθ , however, is not invariant under U (1). The global U (1) symmetry is spontaneously broken.
Source: the author (2021) Since the coupling constant λ is considered to be small, we can use the term vacuum expectation value referring to the ground state, since when λ → 0, the classical and quantum values become the same. For small η, the potential V (φ) can be approximated by which has a maximum V max = λη 2 at φ = 0 and a minimum V min = 0 at |φ| = η. In order to keep track of field excitations, we expand φ around the specific ground state φ = η, where φ 1 and φ 2 are treated as two independent real scalar fields. Each term of (2.12) becomes (2.15) Now plugging (2.15) into (2.12) yields where L exc refers to the excitation of the scalar field and L int includes all the interaction terms.
What is important to notice here is that the free part describes a massive scalar field φ 1 , with mass η λ/2, and a massless scalar field φ 2 . This suggests the symmetry breaking, which can be tracked down to the inclusion of a complex scalar field, generates a massless excitation of the Higgs field, φ 2 , called Goldstone boson. In 1961 Jeffrey Goldstone conjectured this to be a general feature of models with symmetry breaking. The Goldstone conjecture [26] states that "every spontaneous breaking of a continuous symmetry generates a massless scalar particle". It was demonstrated, becoming a theorem, in 1962 by Goldstone, Salam and Weinberg [27]. We can give meaning to the Goldstone boson by looking at Figure 2.3. In order to use the expansion (2.14) suppose that the system is at the vacuum with θ = 0, i.e. at Im(φ) = 0 and Re(φ) = η. In this situation, a change in φ 1 (the real part of the field) is energetically disfavoured, since the state would have to climb the potential hill, while a change in φ 2 takes the field to an energetically equivalent state, only changing the representative vacuum (value of θ). The Goldstone boson encodes the energetic symmetry of the vacua. Naturally, any interpretation to φ 1 or φ 2 , as defined in (2.14), have to consider them as perturbations around φ = η. There is, however, a loophole to the Goldstone theorem. If we consider a broken local symmetry, we can make the Goldstone boson disappear. One might take a moment and try to figure out why.
A hard one: abelian-Higgs
Now we are going to see what happens when we impose a local symmetry in the Goldstone model. First of all, local symmetry plus Lorentz-invariance forces the existence of a gauge field communicating the symmetry. Consider the abelian-Higgs model with the following Lagrangian where D µ = ∂ µ − ieA µ is the covariant derivative in field space, e is the coupling to the gauge field (usually referred to as charge), A µ is the gauge field, and F µν = ∂ µ A ν −∂ ν A µ is the Faraday tensor. Because we are dealing with a gauge field, there is a subtlety. The Lagrangian (2.17) have U (1) local symmetry, which means invariance under gauge transformations for any arbitrary coordinate dependent function α(x). Transformations (2.18) tell us that for every choice of the four-vector A µ , the vector A µ is equivalent. This is called gauge freedom.
It means that we have the freedom of choosing a function α, the gauge, that better suits our intentions. In this case we can choose a gauge that annihilates the imaginary part of φ, the Goldstone boson. Expanding the field around vacuum results in the Lagrangian (2.20) One might notice that now we do not have any massless scalar boson in (2.20), the gauge boson "ate" the Goldstone boson. In addition, the field A µ has a self-interaction term, e 2 η 2 A µ A µ , meaning that the gauge boson, initially massless, acquires a mass m A = √ 2eη. This result can be generalized to any number of non-abelian gauge fields. It can be shown that the corresponding gauge boson acquires a mass proportional to the vacuum energy for every broken symmetry.
Global strings
Now consider the Goldstone potential and assume the field φ is near the minima, φ = φ 0 e iθ , where θ is the phase in the field space. We can map circles, in field space, near the minima of the potential. which we denote by the letter L, to loops in physical space, which we denote by S, since both are periodic closed curves. Once we travel around a loop S, in physical space, we know the angular coordinate, ϕ, must change by 2π while the phase of L, the image of S in field space, θ, must change by an integer multiple of 2π, ∆θ = 2πn. Now imagine one continuously deform the loop S, in physical space, to a point p. Since the field has to be uniquely determined at p there are two options: φ 0 (p) = 0 or φ 0 (p) = 0. If φ 0 (p) = 0 then we know there is an energy excitation inside S.
If φ 0 (p) = 0, because φ is single-valued at p then ∆θ = 0 at some intermediate loop L , which is mapped to S between S and p in physical space, but since ∆θ has to be an integer multiple of 2π, ∆θ has to jump from 2π to 0 at L , which contradicts the hypothesis that φ is a smooth function of the coordinates. Hence φ 0 = 0 at least at one point of a loop L located between L and L . The excitation confined by the loop S , the image of L in physical space, is called a cosmic string. This reasoning can be better visualized in Figure 2.4 and works for any model that possesses a symmetry breaking potential of the form ((2.21)). We can show the phase difference around the loop L , or any other above it, in Figure 2.4b is indeed zero. The phase θ of any point in L is So, starting at point (Re(φ), Im(φ)) = (0, 0) and going anticlockwise on the loop L we see that θ increases in the first quarter of the circle, and so it does on the second quarter. However, on the third quarter, when Re(φ) < 0, the phase difference is negative, and so it is on the fourth quarter, yielding ∆θ = 0. The deformation argument outlined above assures that there must be an excitation of the Higgs field at some point of the surface bounded by L, and it is clear that this excitation should have cylindrical symmetry since L is on a bounded 2-surface. Moreover, the fact that ∆θ should be an integer multiple of 2π means that we can make the correspondence θ = nϕ, where n is called the winding number. Finally, the vacuum manifold is a circle, which is not simply connected because a loop on the circle cannot be continuously shrunk to a point on the circle [28]. Now we turn our attention to the simplest vortex solution, the global string. This solution arises from the Goldstone model (2.12), which breaks global U (1) symmetry. Consider a localized configuration, V (φ) r→∞ − −− → 0, which means that far from the core of the string the scalar field is approximately φ = ηe inϕ . The energy density is given by 3 which gives the total energy (excluding the contribution from the core) (2.24) In the above expression, δ is the width of the string 4 , R is the cutoff radius, and the winding number n labels the kinetic energy of the string. We see that (2.24) diverges as R → ∞, suggesting the isolated global string is unstable, which is expected from Derrick's theorem since this a static solution of a non-linear pure scalar model. We could anticipate the instability based on Derrick's theorem.
Local strings
A local string is a vortex solution with local gauge symmetry breaking. Here we consider the abelian-Higgs model (2.17), which is the Goldstone model coupled with the U (1) gauge field. Notice that, because we are coupling the scalar field with a gauge field, Derrick's theorem no longer predicts instability. The actual vacuum is the same as in the pure Goldstone case, so the scalar field cannot be affected by the gauge field far from the core, i.e., |φ| r→∞ − −− → η. The effect of the gauge field should be concentrated on the string core since we are searching for a localized solution.
The Euler-Lagrange equations for the abelian-Higgs model are Now plugging the field far from the core φ = ηe inϕ in (2.25b), we get which indicates a magnetic flux, φ B , on the string (2.27) The additional minus sign in A ϕ appears because the metric has signature -2. We have seen that the winding number n labels the kinetic energy of the string, and now we see that it also labels the magnetic flux, which is quantized! Denoting the field covariant derivative as D µ = ∂ µ − ieA µ , we know that far from the core which makes the energy depend purely on V (φ), H ≈ V (φ), which has finite integral. It can be shown that the mass per unit length µ of the local U (1) string is given by [29] µ We have dodged Derrick's theorem by employing a gauge-field coupling.
In 1973 Nielsen and Olesen [14] found explicitly the existence of a static cylindrically symmetric solution to the field equations (2.25). For this reason, the U (1) local string, or abelian-Higgs string, is usually called the Nielsen-Olesen vortex. In the next section, we show the derivation of the Nielsen-Olesen solution and discuss some of its properties. Also, from now on, we use the words string and vortex interchangeably.
Nielsen-Olesen vortex
The scientist does not study nature because it is useful to do so. He studies it because he takes pleasure in it, and he takes pleasure in it because it is beautiful. If nature were not beautiful it would not be worth knowing, and life would not be worth living. I am not speaking, of course, of the beauty which strikes the senses, of the beauty of qualities and appearances. I am far from despising this, but it has nothing to do with science. What I mean is that more intimate beauty which comes from the harmonious order of its parts, and which a pure intelligence can grasp.
Henri Poincaré
The equations of motion of the abelian-Higgs model can be expressed in the form (2.30c) For convenience we rescale the quantities, , and choose the Lorentz gauge ∂ µ A µ = 0. Besides that, we take the following ansatz where f (r) and α(r) are unknown functions that satisfy the boundary conditions Notice that far from the string we recover A ϕ = n er , φ = ηe inϕ . Also, the magnetic flux is in fact given by 2πn/e since α(r → ∞) = 1. Now we can determine f (r) and α(r) by plugging the ansatz (2.31) in the equations of motion (2.30). One of the terms is giving rise to for (2.30a). Moreover, we obtain (2.30c) and Finally, the equations of motion take the form An analitical solution to (2.37) can only be obtained in some restricted situations.
In the regime r → ∞ we have f (r → ∞) = 1, which simplifies (2.37b). By rescaling the radius, r = √ 2er, and changing the variable, α(r) − 1 =rξ(r), where ξ(r) is to be determined, we arrive at a new form of (2.37b) in the asymptotic regime, where prime denotes differentiation with respect tor. Equation (2.38) is the modified Bessel equation, and since we want the solution to be finite at large r, it shall be proportional to the modified Bessel function of the second kind of order 1, K 1 (r). The asymptotic behavior of α(r) is given by where the integration constant is set to ±1.
If we rescale the coordinates x → x/e in (2.30) we can see that the solution to the field equations are only dependent on the parameter β = (m φ /m A ) 2 , which measures the relative strength between scalar and gauge fields. In Figure 2.6 we show the numerical solution of f (r), α(r) and their dependence on β. We shall see that the parameter β measures the ratio between disruptive and confining forces. In Figure 2.7 we see how the vortex energy density is affected by the winding number. In general, the string has zero energy density at the axis and is surrounded by a cylindrical energy density that goes to zero at infinity. This feature will be important when coupling strings with gravity.
Moreover, we notice that the width of the vortex is proportional to the winding number n. In fact, if we consider the magnetic flux uniformly distributed over the vortex, the characteristic width is proportional to √ n. The reason for this is that the vortex width should increase with n to accommodate the increasing magnetic flux. Recently Alexander Penin and Quinten Weller, in [30] and [31], used an asymptotic expansion of the field functions in inverse powers of n to find a piece-wise perturbative analytical solution of (2.37) in the limit n → ∞.
Properties of the Nielsen-Olesen vortex
Equipped with the solution of the Nielsen-Olesen vortex, we can start analyzing some of its properties.
Energy-momentum tensor
Given an action S defined in a spacetime with metric g µν with signature -2, the energymomentum tensor T µν is given by where g is the determinant of the metric. For a Lagrangian L, (2.40) yields Now if we apply (2.41) to the Lagrangian (2.17) we get which for the ansatz (2.31) results in Equations (2.43) tell us that besides the energy density, ε = T t t , and the longitudinal tension, p z = T z z , there is also radial flux of momentum, p r = T r r , and azimutal momentum p ϕ = T ϕ ϕ . Although in some restricted situations we can neglect p r and p ϕ , they are essential to analyze the vortex internal dynamics which are non-vanishing in general.
Stability
One interesting feature of the Nielsen-Olesen solution is that if the winding number n is larger than 1, the vortex can unwind, i.e: the string decays into n strings each with magnetic flux equal to 2π/e. In 1976 Eugene Bogomol'nyi [32] showed that Nielsen-Olesen strings are unstable to such unwinding if β = m φ m A 2 > 1 and stable if β < 1. This can be explained by the following reasoning. The magnetic lines repel each other while the Higgs field act to confine the vortex. Since the field strength is inversely proportional to the mass, when m A > m φ (β < 1) the Higgs field is stronger than the repulsion of the magnetic lines, while when m A < m φ (β > 1) the repulsion overcomes the confinement and the string unwind. Hence we can see β as the ratio between disruptive and confining forces. At this point, we can see the connection between cosmic strings and vortex lines in superconductors. Type-II superconductors (β < 1) at certain values of the applied magnetic field present stable vortex lines with quantized magnetic flux, while in type-I superconductors (β > 1), the quantized vortex lines are unstable and immediately unwind due to Meissner effect. In 1976, Vega and Schaposnik [33] showed that in the critical coupling, β = 1, the equations of motion decouple, and it is possible to construct an analytical power-series solution to the field equations. In this situation T rr = 0 5 suggesting an equilibrium in the radial direction, i.e: there is no radial net flux.
Here we finish our discussion on the Nielsen-Olesen solution. In the next section, we will see a simple mechanism for the cosmological creation of cosmic strings.
Kibble and the cosmic tale
...so many out-of-way things had happened lately, that Alice begun to think very few things indeed were really impossible.
From "Alice's adventures in Wonderland", by Lewis Carrol.
The discussion of the Goldstone potential up to now has been simplified for pedagogical purposes. When the temperature is above 0K, the Goldstone potential has a different form caused by the interaction of the matter with the environment energy. Furthermore, according to the standard model of cosmology, the universe, with all its fields, started in a highly dense, hot state and cooled through a series of epochs, during which matter interaction drastically changed. In what follows, we will see how the temperature affects the Higgs field and how it is related to the formation of topological defects in the early universe.
The role of temperature
Before getting to the Kibble mechanism in the next section, we sketch the essential temperaturedependence of the Goldstone potential. The discussion of this section is summarized and for further details and calculations we refer the reader to [29] The Goldstone potential at non-zero temperature can be approximated by [29] For T > T c = √ 6η, m 2 (T ) is positive and the VEV is φ = 0. As the universe evolved, the temperature dropped and, for T < T c , m 2 (T ) < 0. When m 2 (T ) < 0 the state φ = 0 is not stable anymore and the scalar field acquires a non-zero VEV, leading to U (1) symmetry breaking.
which reduces to η by setting T = 0. In summary, decreasing temperature drives symmetry breaking.
Finally, when T ≈ T c the approximation is not valid anymore because this regime represents a second-order phase transition and we need to include further corrections to (2.44).
Kibble mechanism
Looking at a piece of iron at high temperatures, we see that the magnetization direction varies from point to point; magnetization does not have a preferred direction. This is the most symmetric state of the system since it possesses rotational symmetry, i.e., we can rotate the iron, and magnetization will look the same. Now suppose we cool it; the molecules have less kinetic energy and, if the cooling is uniform throughout the material, the magnetization in each region ends up in a common direction: the iron becomes a magnet! The magnetized metal is the less symmetric state of the system since we no longer have rotational symmetry. But if things happen this way, why do not all pieces of iron we encounter are magnets?
The loophole of the reasoning is the assumption of uniform cooling. In nature, such uniform cooling never happens. We certainly can achieve uniformity in a laboratory with ever-increasing levels of precision, but this is not favored spontaneously. In nature or most simple industrial processes, the non-uniform cooling leads to different regions choosing different magnetization directions, leading to a non-magnetic material. In the early universe, things were quite similar. As the universe cooled down, the Higgs field went from a symmetric state, φ = 0, to an asymmetric one φ = 0. Nevertheless, the phase of the field, i.e., the specific representative vacuum it will be in, is not a priori defined. Random fluctuations of φ will determine it. As one can anticipate, this settling process is not uniform through the universe. Causally disconnected regions may choose different vacuum states, and the way these regions organize themselves must give rise to topological defects.
Consider the φ 4 model. Two nearby regions in space might end up in opposite minima of the potential. Between them, there can emerge a kink interpolating both vacua. The emerging 2-dimensional (though the dynamics is restricted to 1 dimension) kink-like structure is called domain wall. Now consider the Higgs potential. We can imagine a region of space where the field is in the symmetric state φ = 0 surrounded by patches where the field is in the asymmetric state | φ | = η. This gives birth to 1-dimensional defects, called cosmic strings. This mechanism for the cosmological formation of topological defects is called Kibble mechanism, after Thomas Kibble, who proposed it in 1976 [1]. Source: The author (2021).
The ideal string
When it comes to the world around us, is there any choice but to explore?
In this section, we will see how a zero-thickness cosmic string affects the geometry of spacetime. To this end, we use the wire approximation, which treats the vortex as a thin pipe with negligible radius and constant energy density. This is a good starting point since some physical properties can be worked out analytically, and it gives us some valuable physical intuition when studying more complicated, non-analytical scenarios. We start with the consequences of the wire approximation on the energy-momentum tensor and then proceed to its gravitational effects.
Wire approximation
By treating the vortex as a thin wire we can average the energy-momentum (EM) tensor T µ ν over the string cross-section and consider all the energy to be localized at one point in the x-y plane,T This is called the wire approximation which is specially important when considering gravitational effects of cosmic strings. Because the string is supposed to be invariant under boosts along the z-axis we must have T 0 0 = T 3 3 , such that when we apply a Lorentz transformation to (3.1) it does not mix these components. In addition, we assume the string does not have any internal shear forces, T 3 i = 0 for i = 1,2 or energy flux in any direction T 0 i = 0 for i = 1, 2, 3. These assumptions guarantee the string does not have any considerable internal dynamics. In addition, because the divergence of the EM tensor vanishes, ∂ µ T µ ν = 0, we have where we used integration by parts. Assuming that the components of the EM tensor fall exponentially when r → ∞, one can say T x ν = 0, and, by a similar reasoning, also T y ν = 0 for ν = 1, 2. Hence T i j = 0 for i, j = 1, 2. The only non-vanishing components of T µ ν are T 0 0 and T 3 3 . Denoting the mass per unit length by µ and knowing T 0 0 = ε(r) is the energy density, we Notice that the only conditions we used were that the EM should fall exponentially at r → ∞ and that it is invariant under boosts along the z-axis, so these results are valid for every localized narrow 1 string configuration. The string solution with these simplifications is an ideal string. Alternatively, one could use the lorentz-invariance, localizability of the energy, the no-shear and no disruptive force conditions to show that any static infinite straight string should have an EM tensor proportional to diag(1, 0, 0, 1). We have seen that the Nielsen-Olesen vortex does have momentum in r and ϕ directions, so the wire approximation could only represent this solution when β = 1, which is also called supersymmetric limit. However, it could be useful when considering large-scale gravitational effects, when the internal dynamic of the string is not important.
Gravitating ideal string
In this section, we shall see how an infinite straight wire affects spacetime around itself. We will follow the approach independently done by Richard Gott [5] and William Hiscock [34]. The general procedure is straightforward. We consider a cylindrically symmetric energy density ε(r) with finite radius r 0 , find the metric in r < r 0 , and match it with the exterior, r > r 0 , vacuum solution of the Einstein field equations (EFEs). In the end, we take the limit r 0 → 0 in the exterior metric, which yields the metric of a zero-thickness infinite straight string. We need to solve EFEs with an energy-momentum tensor inside the tube given by where ε is considered to be any r-dependent function. Though Gott and Hiscock used constant energy density we will stick to a general approach and consider a constant energy density ε(r) = ε 0 , only when needed. The ansatz for the line-element proposed by Gott III [5] and Hiscock [34] is where χ(r), ψ(r), ω(r) are functions of r only. We use exponentials just to ensure the metric components are positive. Equation (3.6) yields the following non-zero components of the Einstein tensor G µ where prime denotes derivative with respect to r. Conservation of energy-momentum, ∇ µ T µ ν = 0, where ∇ denotes the covariant derivative, gives one more constraint on the metric functions Using (3.9) in (3.7c) we get (χ ) 2 e −2ψ = T ϕ ϕ = 0 which means χ = ψ = 0, hence χ and ψ are constants. From now on we denote e χ = χ 0 and e ψ = ψ 0 . With these results we notice that only the three last terms of (3.7d) do not vanish, resulting in the equation from which we can find ω once ε(r) is given. Now we consider the exterior metric. Taking the ansatz (3.6) and setting T µ ν = 0 in (3.4) results in the exterior solution where K, C i are constants to be determined. If we maker = r + K we get which results in the following exterior metric (3.14) Now we need to determine a, b, c based on the continuity of the metric functions and their derivatives on the boundary r 0 . Let us start with the continuity of derivatives. Inside the cylinder the functions χ and ψ are constants hence continuity of derivative yields m = 0. Now taking m = 0 on the continuity of the functions e χ and e ψ we see that c 2 = χ 2 0 and b 2 = ψ 2 0 , which means the scaling of the coordinates t and z are equal in the interior and exterior of the cylinder. Also invariance under boosts along the z-axis demands c = b. For these reasons we set c = b = 1. Therefore, the exterior metric takes the following form Since we have neither applied any condition on the size of the cylinder nor on the energy function ε(r), we know that the spacetime outside a hard-wall cylindrically symmetric energy density has to have the form (3.15). Nevertheless, notice that we still have to determine two constants, namely a and K. When r 0 → 0 we know, by regularity of the metric at the origin, the inside metric is Minkowskian, and continuity of metric at r = r 0 implies K = r 0 (1 − a), hence K vanishes when r 0 → 0. For this reason from now on we user = r. Now we intend to determine a. Imposing continuity of the derivative of e ω across the boundary yields e ω (r 0 )ω = a. (3.16) Integrating (3.11) gives and employing the condition that the metric at r = 0 is Minkowskian, e ω r→0 − − → r, we see that where (3.16) was used. By now we have already determined the exterior metric in terms of the matter content of the string, but we can go even further. The mass per unit length of the string (or linear energy density), µ, is given by where g is the determinant of the interior metric. This yields One interesting feature of this metric is that it does not depend on r 0 , so it seems there is no meaning in applying the zero-thickness limit; we will get back to this point shortly. When r 0 → 0 a reasonable approximation is that the energy distribution inside the tube is uniform, ε(r) = ε 0 . Consider Equation (3.11) with ε(r) = ε 0 . Employing regularity of the metric at the origin, the solution is e ω = r * sin(r/r * ), where r * = 1/ √ 8πε. With the metric functions in hand, we can calculate the mass per unit length explicitly µ = εr * sin(r/r * )drdϕ = 2πε 0 r 2 * 1 − cos Now we see that when we apply the limit r 0 → 0 we have to do it such that µ, hence r 0 /r * , is kept constant. It implies that we should also have ε 0 → ∞, turning the EM tensor into a delta-function. This procedure justifies that the metric (3.15) is valid for all values of r, i.e., we extended the validity of (3.15) to the whole space.
Consider the line-element outside the zero-thickness infinite straight string This line-element represents a locally flat spacetime where the angular variable range from 0 to 2π(1 − 4µ) or, equivalently, with a deficit angle ∆ϕ = 8πµ. Notice that ∆ϕ = 2π is not physical, hence µ is constrained, 0 < µ < 1 4 . Since we are using natural units, µ = 1 4 means roughly 3.2 × 10 26 kg/m. At this density, a string with the mass of the Jupiter planet, roughly 2 × 10 27 kg, would be approximately 10 meters long. If we consider the mass of the black hole at the center of the Milky way galaxy, Sagittarius A*, with mass of approximately 4 × 10 36 kg, the corresponding string would have 1.3 × 10 7 km.
One can think of this geometry as a flat spacetime where one has cut a slice and glued the edges, resulting in a conical structure. Imagine taking a flat sheet of paper, cutting a slice of it with a specific angle, and then gluing the edges. It is only possible if one turns the flat sheet into a cone. Now take another sheet on a table and draw a constant vector field (a set of arrows with the same size and same direction) all over the paper, cut a slice, and glue the edges again. One can see that the vector field on the two sides of the glued edge is not in the same direction. The vector field changes the direction when it is parallel transported around the tip of the cone, meaning that there is curvature somewhere. However, the curvature can not be extended since we have not folded the paper, so it must be located on the tip. We can formally demonstrate this idea. Taking the trace of EFE (3.4) 24) and using the averaged energy-momentum tensor T µ ν = µδ(x)δ(y)diag(1, 0, 0, 1). It results in which means the discontinuity generated by the curvature is located precisely at the string axis. The spacetime is locally flat, i.e., it is everywhere flat except a small region. Here we will not address the controversy of using distributions 2 as sources in EFE. A distributional formulation of the straight string can be found in [35,Chapter 7]. The "cut and glue" process described above is one of Volterra processes, which are used to visualize the formation of crystalline defects in materials. By geometrizing a crystalline lattice, we can describe crystalline defects by geometrical properties such as curvature and/or torsion. For instance, defects called disclinations cause non-vanishing curvature while dislocations are related to non-zero torsion [36][37][38]. The cosmic string metric shown here is equivalent to a linelike crystalline defect called wedge disclination, which is "created" 3 using the process outlined before. However, this is not the only way to create such a line-like defect. Some works propose that the cosmic string can also be described by a torsion singularity, instead of a curvature one [39][40][41]. In this case the cosmic string condensed matter counterpart is called an edge dislocation.
Another interesting property of (3.23), is that if we remove the z-axis from the conical line element (3.23) the resulting line-element describes the spacetime around a point-particle with mass µ in (2+1)-dimensional gravity [42,43].
Finally, a geometrical way of detecting a cosmic string is to enclose the string with a circular loop and measure its perimeter. In doing this one sees that the perimeter is 2πr(1 − 4µ) and not just 2πr. This is, however, a global measure. In the next section we will see that this global feature induces some important local physical effects.
Topology induces physics
In this terrifying world, all we have are the connections we make.
Bojack Horseman
In the last section, we have seen that the curvature outside a zero-thickness string vanishes everywhere. Considering only this fact, we could wrongfully conclude that the string is gravitationally sterile. Nevertheless, we shall see that the conical structure is phenomenologically quite rich.
We start with analyzing the geodesics in conical space. The line element (3.15) can be written in a Minkowski-form if we make θ = (1 − 4µ)ϕ which is more convenient since θ is what we measure outside the string. Besides that, we know the geodesics in Minkowski coordinates are straight lines. A displacement in the radial direction is given by (3.27) Now the displacement in the angular direction is where ω = dϕ dt . The final equations of motion are given by Now, suppose two light rays coming from the same star encounter a cosmic string on their paths, each ray traveling by one side of the string. It is important to find the difference, if there is any, in the angle between these two light rays when they reach a distant observer, like us here on earth. First, we adapt the formula for dθ shown before because we want it to reflect the fact that the light rays are coming from opposite sides of the string, so We can express the ϕ-coordinate of both light rays as (3.31) When t → ∞, they become (3.32) The angular difference between the two light rays are This is a rather interesting result. The measured angular difference between the two light rays, ∆θ, is precisely the deficit angle of the spacetime! Moreover, this is a purely geometrical effect that arises from the global conical structure, not from local spacetime curvature. Notice that we have not used any assumption of the particle's mass, so this result is also valid for massive particles. A careful study of the possible detection of cosmic strings via gravitational lensing can be found in [5]. Now imagine we are asked to find the Newtonian gravitational force on a massive point particle near a cosmic string. At first, it might seem there should be no such force since the space is flat, though the non-vanishing mass of the string could correctly anticipate that a tidal force does exist. Suppose the particle has mass m and is at rest at (r, θ, z) = (a, θ 0 , 0), where θ 0 is an arbitrary angle. The equation for the gravitational field Φ is which is to be solved under the boundary conditions . [44] solved this problem and found that the particle experiences an attractive self-force, F , proportional to m 2 where f (µ) is a monotonically increasing function of µ Four years before Gal'tsov, in 1986, Bernard Linet [45] solved the problem of a charged particle in conical space, which is mathematically equivalent to the gravitational one, but with a sign change in Poisson equation, and found a repulsive interaction proportional to the charge squared. An intuitive explanation for arising of an electrostatic (gravitational) force is that the field lines "refract" on the string, causing the illusion that there is another particle on the opposite side. Also, because the charged (massive) particle accelerates, it radiates electromagnetic (gravitational) waves. Both of them can, in principle, be measured.
The appearance of both gravitational and electrostatic forces sheds light on the common cause of both phenomena: the non-trivial boundary conditions both fields have to satisfy are caused by the non-vanishing deficit angle. This is commonly mentioned as a topological feature since it comes from a global property of space.
Extended gravitating vortices
La nature ne fait jamais des sauts Nature does not make jumps
Gottfried Leibniz
The zero-thickness cosmic string is a good approximation in a cosmological context, but the presence of a delta-function in the energy can be seen with distrust, especially when one realizes that the scalar curvature becomes a distribution in the limit r 0 → ∞. Here we use the expression "extended vortex" to denote vortex solutions with internal structure, i.e., we take into account all components of the energy-momentum tensor. In fact, by taking a look at the energy-density profile of the Nielsen-Olesen vortex, one could anticipate that its effect on spacetime has a finite length and is highly dependent on the vortex parameters.
However, the cosmic string zoo has many more characters. As we have seen in Chapter 2, any model with the Goldstone/Higgs potential V (φ) ∝ (|φ| 2 − η 2 ) 2 admits a vortex excitation, hence it is of no surprise that many cosmic string solutions have been found through the years. Abelian-Higgs strings, described by the Nielsen-Olesen solution, were found in 1973 [14], then Vachaspati and Achucarro, in 1991, showed the existence of semilocal strings 4 [15], and in 1993 Vachaspati found Eletroweak strings in the Weinberg-Salam model [16], to cite a few. This section aims to see how the coupling of an extended vortex with gravity affects the spacetime at the location of and far from the string.
Gravitating cosmic strings with non-negligible thickness can be generated by coupling the Lagrangian of the matter with gravity and then solving Euler-Lagrange and Einstein field equations simultaneously. These equations are usually too hard to be solved in a closed form, and frequently one resorts to approximations or asymptotical limits in order to get an idea of what happens with the system before solving the equations numerically.
Given an action S defined in Minkowski spacetime, we can minimally couple it with gravity by doing the following substitutions inside the action where η µν is the metric tensor in Minkowski spacetime, g µν is the metric tensor in a curved spacetime, g is the determinant of the curved metric and ∇ µ is the covariant derivative operator. When dealing with fermions we still need to substitute the gamma matrices, γ a with their curved-spacetime versions, γ µ = γ a e µ a , where e µ a is the a-th tetrad vector field, defined by e a µ e aν = η µν . For example, consider the action of the free scalar field φ defined in Minkowski spacetime Coupling this action with gravity results in which means the Lagrangian of the free scalar field in curved spacetime is As we are searching for cylindrically symmetric solutions that are invariant under boosts along the z-axis, the usual metric ansatz is 42) where N (r), L(r) are functions of the radial coordinate only, and shall be determined by the matter content. Additionally, we impose spacetime boundary conditions The metric should be regular at the origin, i.e: N (r → 0) = 1 and L(r → 0) = r. Spacetime should be asymptotically flat, R(r → ∞) = 0. (3.43) This is the basic prescription to find gravitating cosmic string solutions. In 1981 Vilenkin [3] found that, as we have seen in the last section, an infinite string using the wire approximation creates conical geometry outside it. This approach, however, neglects terms that might be present in the EM tensor, other than T zz and T tt , which accounts for internal dynamics. In 1985 Garfinkle tackled this issue by showing that, under some weak conditions, any cosmic string solution of the gravitating abelian-Higgs model has to be asymptotically conical [17]. Finally, Christensen, Larsen, and Verbin [18] showed the existence of at least four different classes of solutions to the gravitating abelian-Higgs model, including cosmic strings. The other three are Melvin, where the functions N (r), L(r) are asymptotically powers of x, Kasner, and inverted cone solutions; the last two being in the category of closed solutions since the metric is terminated at some finite radial coordinate, suggesting that they do not represent an isolated system. In what follows, we deal only with cosmic string solutions.
In [18] the authors used the following standard ansatz where n is the winding number and f (r), H(r) are functions to be determined. Notice that this notation is different from the one we used in Chapter 2. Considering (3.44), the boundary conditions are In particular, in [18] the authors used the parameters α, γ, given by to show that every solution in the cosmic string branch is related to a solution in the Melvin branch with the same position in the α − γ plane. Following the approach of [18], in 2000 Brihaye and Lubo [19] studied classical gravitating solutions of the abelian-Higgs model with n = 1 and explictly showed that the metric of cosmic string solutions become conical 5 far from the core, i.e., In particular the authors showed the dependence of the conical parameters a, b and the mass per unit length M in , on α and γ. In Figure 3.7 we can see that while M in does not change much, the parameter b decrease linearly with γ. This means the angular deficit is proportional to the scale of symmetry breaking of the vortex solution. There is however a critical value of γ, γ cr (α), for which b = 0 and the solution becomes non-physical, since the deficit angle becomes 2π. This means there is a region in the α − γ plane where the metric solution stops making sense. Moreover, in Figure 3.7 we can see that when α changes from 1.0 to 3.0, a changes from decreasing to increasing, which suggests that for some α = α 0 , 1 < α 0 < 3, we have a(α 0 ) = 1.
(3.48) Figure 3.7: Conical parameters generated by the Nielsen-Olesen vortex with n = 1. Notice that for α = 1.0, a decreases with γ, while it is increasing for α = 3.0. The parameter b seems to always decrease with γ.
In [20] the authors studied the following gravity-coupled non-abelian-Higgs model which describes two bosonic fields, φ and χ, interacting via a potential V (φ a , χ a ) given by in the presence of the SU (2) gauge field A a µ that generates the field strength In the above expression, e is the gauge coupling, and abc is the antissymetric Levi-Civita symbol. The gauge-covariant derivatives have the form and just for the sake of clarity (D µ φ a ) 2 = D µ φ a (D µ φ a ) * and (φ a ) 2 = φ a φ * a . Notice that each bosonic field has three complex components, and there is one vector field for each component of the scalar field. Although the situation seems much more complicated than the abelian-Higgs scenario, the prescription is the same, i.e., solve the E-L and EFEs that arise from (3.49) using the boundary conditions (3.45) and (3.43). The authors considered the following ansatz
54)
A a (r) = δ a, 3 1 − H(r) erφ , (3.55) which means the scalar fields are orthogonal in the field space, φ a χ a = 0, and only the third gauge field, A 3 , is non-vanishing. Notice that this model reduces to the abelian-Higgs one when λ 2 , λ 3 , and χ all vanish. For numerical analysis it is useful to define the following dimensionless functions which makes the Lagrangian depend only on the dimensionless parameters Their results for the field and metric functions can be seen in Figures 3.8 and 3.9. In Figure 3.10 one can compare the metric functions in the abelian and non-abelian scenarios. Source: [20] From Figures 3.8, 3.9, and 3.10, one can conclude that non-abelian cosmic strings also generate asymptotical conical geometry, and this effect is more pronounced compared with the abelian case. Moreover, the transition from Minkowski to conical is smooth in these spacetimes. They consist of a region with non-vanishing curvature, where the exact form is dependent on the vortex parameters (see Figure 3.11). Here we end our discussion on gravitating extended vortices. It is worth mentioning that we did not try to exhaust the literature. There have been many studies in this line of research, and we did not include everything here. Instead, our goal was to give an introduction with some guiding references. Now we turn our attention to how the asymptotical conical structure of cosmic string spacetimes presents a difficulty for studying bosonic and fermionic scattering and our approach to deal with the problem.
Vincent van Gogh
In Chapter 3 we have seen the general structure of the spacetime around a cosmic string. It consists of three distinct regions: at the center of the vortex, spacetime is flat, with Minkowskian coordinates, then at a finite distance from the center, it becomes curved and, far from the core, it approaches the conical limit.
In 1988 Deser and Jackiw [50] studied the quantum scattering in a pure conical background and found that the usual partial wave approach produces singularities in the scattering amplitude; they fixed this problem by changing the usual asymptotical ansatz of QM. In [51] we showed that although the spacetime of a cosmic string is much more complex than pure conical, the usual partial-wave approach also generates a divergent scattering amplitude. To avoid the singularity, we proposed a modification of the asymptotic ansatz in the partial-wave formalism and presented the corrections in the phase-shift, scattering amplitude, and cross-section. The first part of this chapter is devoted to these results.
After that, we apply the same formalism to the scattering of a massive fermionic field in the same background.
Scalar field scattering
The lagrangian of the free massive scalar field Φ in a curved spacetime is where M is the mass of the field. Using the relations we get the following E-L equation which is the Klein-Gordon equation minimally coupled with gravity. We can go further and add non-minimal terms inside (4.3), for example where = ∇ µ ∇ µ is the D'Alembertian operator, R is the Ricci scalar and ξ is the non-minimal coupling.
where prime denotes derivative with respect to r. Imposing conservation of energy, cylindrical symmetry and restricting the dynamics to the r − ϕ plane (invariance under boosts along the z-axis) we can take the following ansatz where E is the energy of the field, k the momentum in z-direction, m = ±1, ±2, ±3, ..., is the mode of the field, and a m is a mode-dependent constant to be defined by the initial condition. Notice m has to be integer for the solution to be single-valued in ϕ. The summation over m reflects the fact that for a linear equation, the sum of solutions is still a solution. Combining (4.6) with (4.5) and using (4.7) Now remember that near the origin we have N (r) = 1 and L(r) = r, hence R m (r → 0) ∝ J m (λr) where J m is the Bessel function of first kind of order m, and we absorb the proportionality constant in a m . Here we set a m = i m such that in the limit r → 0 the field is a plane-wave in thex-direction. The general solution in the limit r → ∞ is In the above expression β m = λ c b − α m + d m (λ), α m = π 2 (m + 1/2) while C m (λ) and d m (λ) are model-dependent constants to be determined, usually numerically. Also notice that the phase shift of the m-th mode is given by δ m (λ) = β m + α m = λ c b + mπ It is interesting to take a look at the effect of conical structure in other observables of the scalar field. For instance, a temporal element dt at the center of the vortex becomes a dt far from the core, hence the frequency of oscillations appears higher for a local observer outside the string than it is measured by someone at the core. Although the energy of the field does not change, the hamiltonian operator, which is the local measuring scale, does change. Explicitly we havê whereĤ in andĤ out are the hamiltonian near and far from the core, respectively. Equation (4.11) impliesĤ out Φ = E/aΦ, which makes sense if we compare the expression of λ and λ . Because g tt = −g zz the linear momentum in the z direction is perceived as larger far from the core. Finally, angular momentum is also affected by the conical structure. The angular momentum operator is given by whereL in andL out are the angular momentum operators near and far from the core, respectively. Equation (4.12) suggests the angular momentum outside the vortex is measured to be a noninteger value m = m/b. Thus, the angular deficit of spacetime affects the measured angular momentum of the field.
In addition, note that if the flat spacetime for r → ∞ is not conical, i.e. c = 0 and b = 1, the phase shift becomes equal to d m (λ) determined by the gravitational potential in the region 0 < r < ∞. Moreover, one might realize that any local interaction does not change the general form of the solution at infinity. The effect of any such interaction shall be felt only by the parameters C m and d m to be measured at infinity. For example, suppose the scalar field also interacts with the gauge field creating the vortex. In this situation, the constants C m and d m are certainly modified by the local interaction when compared with the situation without the gauge field. This illustrates the fact that the constants C m and d m store information about any local interaction in the region 0 < r < ∞. Although cosmic string models are usually too hard to solve in closed form, we later devise a toy model and show the effects of the gauge-field coupling in the total cross-section of a scalar test field. Now let us get back to the scattering. In usual partial wave approach we rewrite the cosine in (4.10) in terms of plane waves, resulting in (4.13) The usual QM ansatz is given by 14) where f (ϕ) is called scattering amplitude. This ansatz says the scattered wave is a cylindrical wave, regulated by f (ϕ), plus a plane-wave accounting for the part of the incident wave that does not interact with the potential. Usually in QM, the spacetime before and after the potential is the same. Hence the unscattered wave has momentum λ, the same as the incoming plane wave 1 . However, this is not possible in the spacetime of a cosmic string since a change in the metric component N (r) affects λ. Also, far from the core, the radial part of the solution to the Klein-Gordon equation, R m (r), is a Bessel function of non-integer order, making it impossible to take the pure plane-wave in the second part of the ansatz. Finally, it is clear that the regions before and after the scattering are not equivalent, which motivates a change in the canonical approach. Later we show another reason to do such modification. We take the asymptotic ansatz in the form The reason for writing the second term in this form is that when A m = 1, a = 1, b = 1 and c = 0 it takes the form of a plane wave with momentum λ travelling in the x-direction. Here, however, we left A m free to be determined by the form of the solution at infinity. Expressing the ansatz in terms of plane waves, we get Now comparing the coefficients of e −iλ w , we obtain Comparing the coefficients of e iλ w and considering Equation (4.17), results in where δϕ = π i.e., the scattering amplitude has a delta-contribution coming from the deficit angle of spacetime. In our approach, with A m free to be determined by the field solution at infinity, the scattering amplitude becomes which does not have any delta contribution due to the nontrivial mode dependent constants C m and d m . In conclusion, our formalism avoids the singularity of the scattering amplitude. Let us pause and connect our approach with the one in [50] for the particle scattering in a conical geometry. In their scenario, the whole space is conical and analytically determined. Because of a non-vanishing deficit angle, the scattering amplitude f (ϕ) is singular, taking the standard partial wave expansion ansatz in quantum mechanics. They circumvented this problem by modifying the second term in (4.15) to match the solution determined at r → ∞. Our reasoning is similar. Due to the conical structure at r → ∞, we need to leave an extra free parameter to be fixed with the asymptotic solution after the scattering. In contrast with the result in [50], one needs to find the parameters C m and d m numerically since our formalism deals with a class of spacetimes typically too complex to have a metric in a closed-form due to non-trivial matter-gravity interaction. Similar to [50], here the optical theorem is not satisfied, although there is no problem with unitarity. It can be shown that the probability current J is It means that the probability current on the plane is divergenceless, hence conserved. We conclude that in this class of spacetimes the optical theorem is no longer suitable to determine particle or probability conservation. Moreover, the appearance of an angular deficit in the scattering amplitude originates from the spacetime's deficit angle equal to δ = 2π(1 − b) [20]. In fact, we noticed that the extra factor δϕ is proportional to the angular difference between geodesics in the ideal cosmic string spacetime (3.33) Now we turn our attention to the phase shift. The phase shift of the scalar field scattered in this class of spacetimes is given by and notice the first term accounts for the rescaling of the radial coordinate, the second term matches the result found in [50] and comes from conical structure, and the last one is due to any interaction in the midway, e.g. curvature and/or gauge field for example. In the low-energy regime, the isotropic mode, m = 0, holds the largest contribution to scattering [53] since any other mode has some part of the flux in the azimutal direction, as seen in (4.21). In QM the scattering length is defined as such that, for instance, the potential of a hard sphere of radius R 0 yields l sc = R 0 . Knowing that for λ = 0 there is no scattering, which means d 0 (λ = 0) = 0, the scattering length of a cosmic string is given by Now we have all the ingredients to calculate the scattering cross-section. Ignoring the z-axis, due to the dynamics being restricted to the x-y plane, and knowing the incoming and outgoing momenta, λ and λ , are not the same in general, the differential cross-section is given by [54] One can see that if we include the z-direction, the total cross-section diverges. Sustituting eq. (4.18) into eq. (4.28) and then integrating (4.28) in ϕ results in This result is very similar to the standard expression from QM, except for the factor |C m | 2 . However, the extra factor becomes 1 in the limit where the asymptotical spacetime has the same parametrization as the origin. Clearly, the convergence of this formula depends on the convergence of the amplitudes C m , which, as we shall see, does converge for both a general toy model and a realistic scenario.
Toy model
To illustrate our formalism in a concrete example, we develop an analytical model similar to a cosmic string spacetime, although somewhat simplified, in order to calculate the factors C m and d m analytically. We have seen in Chapter 3 that the metric outside a hard-wall cylindrically symmetric energy density of radius r 0 is conical. We then use the following metric as a first approximation to the cosmic string spacetime r < r 0 : N (r) = 1, L(r) = r r > r 0 : N (r) = a, L(r) = br + c, (4.30) which has conical geometry outside, as expected. Continuity of L(r) at r = r 0 results in Imposing continuity of the gradient of φ at r = r 0 gives where ∆J m (x) = J m+1 (x) − J m−1 (x). Using (4.33) in (4.34) gives It is worth mentioning, however, that the cross-section that comes from (4.36) and (4.37) converges too slowly, which may happen because this toy model has serious limitations. The curvature scalar that comes from (4.30) 3 diverges at r = r 0 , so this is a singular spacetime.
To circumvent this problem, we design a smooth version of (4.30). The metric (4.30) can be expressed using the Heaviside step function Θ(x) One might have noticed that (4.38) presents no transition between the interior and the conical spacetime, which is precisely the reason why the delta-curvature appears. We avoid this problem by employing a smooth transition via an analytical approximation of the step function, H(x), defined by which imitates the spacetime around a vortex, as can be seen in Figure 4.1. One might realize that r 0 still defines a characteristic radius of the vortex since r 0 is the center of the transition between Minkowski and conical. In this situation the curvature is no longer divergent and size of the curvature well is controlled by the parameter p. With a faithful toy model in hand, all we need to do is to insert (4.40) in (4.7), extract constants C m , d m from the numerical solution of the equation of motion and then use (4.29). Now we consider the scenario where the scalar test field also interacts with the gauge field of the vortex. To simulate this situation, we take the gauge field solution of the abelian-Higgs vortex where α(r) is coming from the Nielsen-Olesen solution found in Chapter 2. For the abelian-Higgs vortex with n = 1 and β = 0.5, Pádua Santos and Mello [20] found conical parameters approximately a = 0.98, b = 0.64, c = 0.39. We take the same spacetime and field parameters, together with p = 3.0. In Figure 4.3, we show the scattering cross-section of the scalar field with and without the gauge field interaction. It is clear that the gauge field has most influence in large wavelength (small momentum) particles, while it becomes irrelevant for small wavelength (large momentum) particles. As the momentum increases, all local interactions tend to become irrelevant and the cross-section approaches zero.
Fermionic cross-section
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Samuel Taylor Coleridge
Now it is fairly straightforward to extend the formalism to analyze scattering of spin-1 2 fields. Each component of the spinorial field can be considered to be similar to a scalar field with its own associated differential cross-section. The average value of all 4 cross-sections is the total cross-section of the fermionic field [55].
We start with the Dirac equation in a curved spacetime where {γ µ } is the set of gamma matrices in a curved spacetime, M is the mass of the fermion field, and ∇ µ is the spinorial covariant derivative. In order to convert the known gamma matrices, defined in flat spacetime, to their curved counterparts we have to make use of tetrads, which are objects that parametrize the geometry of spacetime. The tetrad field in 4-dimensional spacetime {(e a ) µ } is a set of 4 vector fields labelled by the latin index a, each one with, in general, four components labelled by the index µ. These objects are related to the metric via g µν = e aµ e a ν = η ab e b µ e a ν , η ab = e aν e ν b = g µν e µ a e ν b , Inverting (4.44b) and using it in ds 2 = g µν dx µ dx ν yields which provides a straightforward prescription to construct matrices (e a ) µ . With a tetrad field in hands, the curved gamma matrices are calculated using The covariant derivative is defined as where Γ µ is called the spin coefficient, (ω µ ) ab the spin connection and the set Σ ab , here called Σ matrices, is the set of generators of Lorentz transformations for spinors. Spin connection can be expressed in terms of the tetrad field and Christofell symbols {Γ λ µν } [38,[56][57][58] via while the Lorentz generators Σ ab are constructed with flat gamma matrices Such a choice, however, does not reflect the symmetries of spacetime, which yields a set of equations of motion that are not separable. A more suitable choice of the tetrad field is which results in and one can verify that indeed (e a ) µ (e b ) µ = δ a b . From now on, when writing components explictly, latin indices are replaced by numbers, a, b, c... → 0, 1, 2, 3, and greek indices by letters representing the coordinates, µ, ν, λ... → t, r, ϕ, z. In addition notice that, when reading the tetrad matrices, lower index runs on the columns while upper index runs on the rows of the matrix, so, for example, e 2 ϕ = cos ϕ/L and e ϕ 2 = L cos ϕ. The curved gamma matrices read (4. 54) In what follows we use the following representation of the flat gamma matrices which gives the Σ matrices To calculate the spin connection we consider each term separately. So, we split (ω µ ) ab in part A and part B (4.58) The only non-vanishing components of the spin connection are which when put together with the Σ matrices inside the spin coefficients yield The covariant derivative contracted with the gamma matrices gives (4.61) Now, in order to make calculations easier we split the spinor Ψ in two parts We employ the following ansatz for the spinor field Ψ where j = ±1/2, ±3/2, ±5/2..., and For clarity, we have dropped the index j from the components of the spinor ψ j . The angular dependence of (4.65) may seem as an imposition, but one can perform the calculations with general angular phases and the separation of variables force them to appear this way. Substituting the ansatz (4.65) in (4.63) leads to the following equations of motion (4.66) We have denoted ξ(x) ≡ N /N + (1/2L)(L − 1) , that vanishes in Minkowski spacetime, but not in conical one. All the above ψ (i) are complex and only dependent on r. By cylindrical symmetry, the solution does not change if we make ϕ → −ϕ, and since we are summing over all modes from −∞ to +∞, we do not have to worry about the sign of j. Now in order to apply the partial wave approach, we need to know the solution of (4.66) near and far from the origin. In the limit r → 0 we have [59] Ψ j (t, r → 0, ϕ, z) = a j e −iEt e ikz e ijϕ where j = ±1/2, ±3/2, ..., j = sgn(j), s = ±1, β j = |j| − j /2 and λ 2 = E 2 − k 2 − M 2 as before. Here there is a sublety. The constant a j has to be set the same way for all components, which means we cannot make all of them plane waves. Here we choose a j = i j− 1 2 such that the component Ψ (0) is a plane wave in the x-direction near the origin. The field solution in the limit r → ∞ is given by 68) where k = k/a, E = E/a, λ 2 = E 2 −k 2 −M 2 and C j (λ), d i j (λ) are model-dependent constants. Notice that here we have four different phase-shifts, d i j , and only one global amplitude C j . We express the asymptotic ansatz as where the index i = 0, 1, 2, 3 labels the component of the spinor field. Here we need to define the second term of (4.69) such that in the limit r → ∞ it reduces to a term similar to the solution near the origin but with the free parameter A i j and j → j , r → w, λ → λ . The ansatz for the 0-th component becomes and notice that (4.71) Instead of summing over j, we can sum over n = j − 1/2. The ansatz becomes where n = j − 1/2, and β j = n . The asymptotic form of the 0-th component of the actual solution is given by which when compared with (4.72) gives with δϕ = π 2 n n − 1 . The cross-section for the 0-th fermionic component is Calculating σ 2 is completely analogous since the coefficients of the Bessel function and the angular exponential are the same. We can adapt this result for σ 2 by just making the change C n → C n k −is nλ E +M . Therefore, the cross-section of the second component is as follows The computation of σ 1 and σ 3 are tedious but straightforward. The ansatz for the first component is However, notice that which suggests that we can sum over m = n + 1 = j + 1/2, instead of j. The asymptotic ansatz for Ψ 1 becomes where m = n + 1 = j + 1/2 and β j + j = m . The asymptotic form of the actual solution is which when compared with (4.79) gives Now it is easy to compute the total cross-section of the fermionic field. The average of all which is analogous to the scalar case (4.29). In the same spirit, we expect the total cross-section to behave similarly to the scalar case, with damped oscillations caused by the spacetime's asymptotical structure. One might notice that as the energy of the field increases, E M , the contribution from the second and third components becomes just the sinusoidal terms, similar to the 0-th and first components.
Here we end our discussion on how to calculate the cross-section of fermionic and scalar fields in the spacetime of a cosmic string. It is worth mentioning that, although in the fermionic case, the computational work is considerably harder, the whole discussion about local non-minimal interactions we had in the scalar case is equivalent here, i.e., their effects shall be felt only by the constants C j , d i j . In the next chapter, we finally apply this formalism to a realistic gravitating cosmic string and analyze the results. 5. Bosonic and fermionic scattering in a gravitating cosmic string spacetime
Bosonic scattering
Acredito que se pense muito mais corretamente quando as ideias surgem do contato direto com as coisas, do que quando se olham as coisas com o objetivo de encontrar esta ou aquela ideia.
Vincent van Gogh [60] In the final part of this thesis, we apply the method developed in Chapter 4 to the gravitating cosmic string found by Pádua Santos and Mello [20]. Our goal is to find the dependence of the cross-section of the scalar field on the mass M and momentum λ of the field. To do that, we need to solve the Klein-Gordon equation in this spacetime, find the parameters C m , d m , and use formula (4.29) to compute the total cross-section. Later we apply the same procedure to the fermionic field. Remember that the model consists of two scalar fields φ and χ coupled with a SU (2) gauge field. The complete model is given by which becomes the abelian-Higgs model for λ 2 = λ 3 = 0 and χ = 0. The authors used dimensionless functions and the dimensionless parameters From now on we set the dimensionless parameters to be α = 1.0, γ = 0.6 for both abelian and non-abelian scenarios and q = 1.0, β 2 = 2.0, and β 3 = 1.0 in the non-abelian case. The For solving the equations of motion (4.7) numerically, we employed the Runge-Kutta method of eighth order. From each solution with specific mode m and momentum λ we extracted the parameters C m (λ) and d m (λ), which are needed to construct the cross-section. We also verified that the cross-section indeed converges for a finite number of modes. In Figure 5.2 we present |C m | and sin 2 (d m ) for three different values of m for a massive scalar field with M = 1.0. We noticed |C m |, unlike d m , is symmetric under m → −m and is always larger in the non-abelian scenario. It originates from the larger deviation of the conical parameters from the Minkowski counterparts. We also noticed that larger mass delays sin 2 (d m ).
In Figure 5.3 and 5.4, we show the total cross-section in the abelian and non-abelian scenarios, respectively, for three values of mass. We can see that in both cases, the mass dampens the cross-section in the small-momentum regime, but as λ increases, the difference between the cross-sections becomes smaller. Eventually, the mass becomes irrelevant in the regime λ → ∞.
In Figure 5.5 we compare the massless cross-section in abelian and non-abelian cases and show, in the zoomed region, how mass affects the region of large momentum in the non-abelian scenario. For large momentum, we see that the presence of mass dampens σ but also delays the signal. This was expected since sin 2 (d m ) is also delayed.
We can see that the cross-section diverges as the incident momentum approaches zero and tends to zero as λ → ∞, which is expected. However, in both scenarios, the total cross-section presents an unusual oscillatory behavior that becomes more evident as λ increases. Since we have not detected any oscillatory pattern in the values of |C m |, we are led to think the oscillation comes from an interference between the sinusoidal terms of equation (4.29). As we shall see, this particular interference pattern seems to be related to the asymptotical form of the spacetime metric.
The high-energy tail oscillation is not entirely new. Burt and Sebhatu [61] already showed that non-linear persistent self-interactions lead to damped oscillations in the total cross-section of baryon-antibaryion scattering. In their case, the non-linear interaction appears as a potential in the lagrangian density, while in our case, it manifests itself as the asymptotical conical configuration of spacetime, which affects the lagrangian through the covariant derivative. In fact, we can use the toy model developed in Chapter 4 to investigate this hypothesis. We start considering the metric toy model (4.40) with a = 1, r 0 = 2.5, p = 3.0 and analyze the scalar field total cross-section with respect to the conical parameter b. For consistency c is always calculated according to c = r 0 (1 − b). The curvature profile for the three chosen values of b is shown in Figure 5.6. We then calculated the total cross-section in these spacetimes. In Figure 5.7 we see that the frequency of oscillations in the total cross-section is proportional to the deficit angle of the asymptotic spacetime and it tends to zero as b → 1. However, that is not the end of the story. We set b = 0.85, r 0 = 2.5, p = 3.0 and analyzed how the cross-section changes with the parameter a, which represents a blue-shift of the time coordinate 1 . The curvature profile for the three chosen values of a is shown in Figure 5.8. The corresponding total cross-section is shown in Figure 5.9. One can see that the parameter a also has a strong influence on the frequency of oscillations and the average amplitude of σ. It is worth mentioning that the oscillations tend to disappear as the metric coefficients tend to pure Minkowski ones, namely a = 1, b = 1, c = 0. Therefore, we conclude that the damped oscillations in the total cross-section are caused by the persistent interaction of the field with the spacetime geometry, represented by the asymptotical structure of spacetime or, equivalently, by the parameters a, b and c.
Fermionic scattering
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Nemo Nobody
In this section, we analyze the scattering of a fermionic field in the same spacetime. In Chapter 4 We have shown that the total scattering cross-section of the fermionic field is given by the formula (4.83). Again we need to extract the parameters C j (λ), d i j (λ), and for doing so, we used the Runge-Kutta method of eighth order to solve the equations (4.66) numerically. The numerical procedure is similar to the one in the last section for the bosonic case.
In Figure 5.10 we show the amplitude C j and the phase d 1 j of the component ψ 1 . Unlike the phase of the scalar field, here sin 2 (d i j ) is symmetric under j → −j. We noticed the presence of some plateaus in sin 2 (d 2 j ), which means that for each mode, there are windows, in λ, for which that mode has no contribution to the total cross-section. Though these windows are seen in both scenarios, in the abelian case, they are wider and were detected only for large momentum, i.e., λ > 15. In addition, the amplitude |C m | behaves similarly to the scalar counterpart. It is symmetric under j → −j and vanishes after a finite number of modes. In Figures 5.11 and 5.12, we show the influence of mass on the total cross-section in both abelian and non-abelian scenarios, respectively. We see that the effect is similar to the scalar case, i.e., larger mass reduces the average value of σ. Comparing the fermionic and bosonic results, one can conclude that the mass effect in the fermionic case is larger than the bosonic one. Finally, it is clear that the fermionic cross-section also presents damped oscillations, which can be seen as evidence that damped oscillations in the total cross-section are a fundamental property of scattering in asymptotically conical spacetimes. In Figure 5.13 we show the crosssection for a massive fermionic field in both abelian and non-abelian scenarios. One might have noticed that the cross-section of both scalar and fermionic fields is more significant in the non-abelian scenario compared with the abelian one. A crude estimation could have anticipated this result. Remember that the cross-section is proportional to the ratio outgoing flux/incident flux (5.4) and that for a slice of a cylinder of height ∆z, the effective area for the outgoing particles is proportional to ab∆z, hence when the deficit angle is larger (b is smaller), the area of the outgoing particles is smaller, hence the flux is larger. Of course, there is also the contribution from the increase in momentum/velocity of the particle since λ > λ.
Conclusion
This thesis has studied the general theory of cosmic strings and their gravitational interaction with nearby matter fields. In Chapter 2, we developed the basic theory of cosmic strings, discussed the two general types, global and local strings, and their topological stability. Then we presented the Nielsen-Olesen vortex, the first local string solution, and discussed some of its properties. We have seen that for some region of the Bogomolny'i parameter β the Nielsen-Olesen vortex is unstable to unwinding, i.e. every string with winding number n > 1 eventually decays to n strings each with n = 1. At the end of Chapter 2, we outlined how cosmic strings are expected to have formed in the early universe via the Kibble mechanism.
In Chapter 3 we proceeded to study gravitational properties of cosmic strings in the wire approximation and have seen that the conical structure presents non-trivial phenomenology. It was observed that gravitational (eletromagnetic) interaction is changed due to the non-trivial boundary conditions the conical spacetime imposes on the gravitational (eletromagnetic) potential. Still, in Chapter 3 we investigated the literature on extended cosmic strings, i.e., vortex solutions with non-negligible internal structure, and have seen that the spacetime generated by a gravitating extended vortex presents a conical structure far from the core. Garfinkle [17] showed this feature to be quite general in the abelian-Higgs model, and we presented a recent non-abelian model that possesses this feature as well.
In Chapter 4 we showed that the asymptotically conical structure of gravitating cosmic string spacetimes creates a divergent scattering amplitude if we follow the standard ansatz of the partial-wave formalism. Deser and Jackiw [50] had already investigated the simpler case of scalar field scattering on a cone and found a similar divergence. We avoided the singularity by modifying the asymptotical ansatz in the partial-wave approach, which leads to corrections in the phase-shift and total cross-section. The correction on the phase-shift is the addition of two terms induced by the conical structure, while the correction on the total cross-section is a multiplicative constant on each term of the cross-section series. This correction accounts for the idea that we need information about the field at infinity in order to construct the asymptotical ansatz, which is exactly what removes the singularity on the scattering amplitude. This happens because the spacetime before and after the scattering are not equivalent and it is impossible to construct the usual plane-wave solution at infinity. The essential conclusion is that we also need information about the amplitude, besides the phase, of the scattered field to be able to construct the total cross-section. We then developed an analytical toy model for the spacetime metric of an extended vortex, which is based on an analytical approximation of the Heaviside step function, and showed how the cross-section changes when considering the interaction with the string gauge field. In the second part of Chapter 4, we applied the same formalism to a Dirac field and found the explicit formula for the fermionic total cross-section. We have seen that defining the asymptotical ansatz for the fermionic field is tricky since we cannot make all components plane-waves before the scattering. The cross-section formula, however, is not dependent on how you define the initial condition, as expected. In addition, it was shown that in the high-energy limit all components of the fermionic field contribute to the cross-section in a symmetric way.
In Chapter 5, we applied this formalism to an abelian and a nonabelian gravitating cosmic string model found by Pádua Santos and Mello [20] and compared the cross-sections of both fields for two sets of the vortex parameters. We have seen that the spacetime with an asymptotically larger deficit angle has a larger cross-section for both fields which is related to a smaller effective area for the outgoing particles. In addition, all cross-sections present damped oscillations that, with the aid of our toy model, were shown to be caused by the particular spacetime structure, including the conicity, far from the core of the string. We also showed how each parameter of the asymptotical spacetime contributes to the curvature profile and oscillations in the total cross-section. We have found that both parameters, the conicicty b and the blue-shift a, contribute to the cross-section oscillations.
The natural next step of our work is to apply this formalism to the scattering of gauge fields and see if it presents the same damped oscillations seen in the scalar and fermionic cases. We also look forward to explore our toy model and see if it could reveal new features of this class of spacetimes without the hard computational work usually required to obtain the metric components. Moreover, in the near future, we plan to study a cosmic string model from scratch in order to have control over all parameters and see how the scattering of classical and quantum particles is affected when we change the string configuration and when we consider interaction with the fields generating the vortex. Besides that, we plan to study other types of defects, topological and nontopological, and see if their interaction with nearby matter fields reveals novel or similar features. | 2021-12-22T02:15:35.215Z | 2021-12-21T00:00:00.000 | {
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260146954 | pes2o/s2orc | v3-fos-license | Avena sterilis ssp. ludoviciana (Durieu) Control in Wheat Through Integration of Tillage, Seeding Rate, and Herbicide Application
Avena sterilis ssp. ludoviciana (Durieu) is a problematic weed in the wheat crop of Australia. Pinoxaden is an effective herbicide for the control of this weed. However, late cohorts of A. ludoviciana escape from the early application of pinoxaden and produce seeds. This research investigated the integrated effect of tillage systems (no-tillage and conventional tillage), seeding rates (100 and 200 seeds m−2), and weed control treatments (nontreated control and pinoxaden application at Z12 and Z33 stages of wheat) on Avena ludoviciana control and wheat yield. The wheat yield remained similar in no-tillage and conventional tillage systems; however, the no-tillage system helped in reducing A. ludoviciana seed production by 28% compared with the conventional tillage system. In the nontreated control, the increased seeding rate of wheat reduced A. ludoviciana biomass and seed production by 33 and 66%, respectively, compared with the low seeding rate. These results suggest that a high seeding rate could be useful in the organic production of wheat. Application of pinoxaden at Z12 and Z33 stages of wheat resulted in an improvement in grain yield by 170 and 150%, respectively, compared with the nontreated control. At both seeding rates, the application of pinoxaden at the Z33 stage of wheat reduced weed seed production by 99% compared with the nontreated control. These results implied that the delayed application of pinoxaden at the Z33 stage of wheat effectively reduced weed biomass and seed production of A. ludoviciana without compromising grain yield as the yield in this treatment was similar to the pinoxaden application at the Z12 stage.
Introduction
Weeds in crops are an issue for improved crop productivity as they compete with crops for water and nutrients, caus-doviciana is important in improving wheat productivity in Australia.
In eastern Australia, the no-till wheat production system is common, especially in dryland regions, and has been adopted at a large scale (Llewellyn et al. 2012).The no-till production system helps in residual soil moisture utilization and improves nutrient uptake, as well as improve the soil structure through crop residue retention (Llewellyn et al. 2012).However, weeds remain a problem in no-till production systems due to weed emergence prior to crop emergence, and the low efficacy of pre-emergence herbicides as a result of residue crop cover (Chauhan et al. 2006;Walsh and Powles 2007;Triplett andDick 2008, Nord et al. 2012).The expansion of the cotton (Gossypium hirsutum L.) industry in eastern Australia has increased the area of conventional tilled irrigated wheat in some pockets as it helps in breaking disease and pest cycles in cotton-based cropping systems (Sykes 2012).Weeds behave differently in response to varied tillage systems (Chauhan et al. 2006(Chauhan et al. , 2012)).
Avena ludoviciana could behave differently in no-till and conventional-till production systems due to varied emergence patterns in response to burial depth.It has been found that A. ludoviciana emergence was greater from 2 and 5-cm soil depths compared with that on the surface (Mahajan and Chauhan 2021b).These results suggest that the emergence of A. ludoviciana could be greater in conventional systems compared with the no-till system.Information on the growth behavior and seed reproduction potential of A. ludoviciana in response to the tillage system is limited in Australia.The ability of A. ludoviciana to produce seeds in a water-stress environment could increase the persistence of this weed in response to varied tillage systems (Sahil et al. 2020).The plants of A. ludoviciana are prolific in seed production.It was estimated that early cohorts (May) of A. ludoviciana could produce up to 4000 seeds plant -1 (Mahajan and Chauhan 2021c).The peak emergence time of A. ludoviciana coincides with the optimum sowing time of wheat (first fortnight of May) in Australia (Mahajan and Chauhan 2021b).These observations suggest that it is essential to control A. ludoviciana before it produces seeds to reduce the weed seed bank in the soil and for improving wheat yield.
In Australia, A. ludoviciana biotypes have evolved resistance to Group 1 and 2 herbicides (Heap 2023).Herbicides belonging to Groups 1 and 2, in general, are applied at a young seedling stage (2-3 leaf stage) for high efficacy and effective control (Beckie et al. 2002).However, growers wait for a greater number of weeds to emerge to obtain maximum weed control in the field.However, by that time weeds become large and difficult to control (Chauhan et al. 2021).This type of situation is very frequent when weeds have multiple cohorts and late emerging weeds es-cape from timely spray applications (personnel, communication).In Australia, multiple cohorts of A. ludoviciana appeared from March to October (Mahajan and Chauhan 2021b).The staggered emergence pattern of A. ludoviciana could lead to a decision-making process difficult for spraying post-emergent herbicide application.
Pinoxaden is a selective post-emergence herbicide used for controlling annual weeds in winter cereals (Anonymous 2022).It kills weeds by inhibiting acetyl-CoA carboxylase (ACCase) (Hofer 2006, Bitarafan andAndreasen 2020).Many weeds have evolved resistance to pinoxaden in more than 15 countries (Heap 2023).Large weed plants may build up resistance to herbicides with time, even when applied at an optimal rate, due to poor weed control (Botterman and Leemans 1988).Therefore, the stage of the plant is very critical for effective control during the spray application of herbicides.We hypothesized that late application of pinoxaden in wheat (Z33 stage: third node formation) may provide complete control of early and late cohorts of A. ludoviciana.
Exploring crop competition for weed suppression is found to be an effective cultural strategy for weed control in Australian cropping regions (Lemerle et al. 1996;Scott et al. 2013).Crop competitiveness against weeds can be increased by using a high seeding rate (Mahajan and Chauhan 2022).We hypothesized that the competitiveness of a crop in response to a tillage system can be increased by using a high seeding rate and that competitiveness can also be exploited for reducing the seed production of A. ludoviciana without using any herbicides.Increased crop competition using a high seeding rate in wheat may suppress weeds without using any herbicides and promote organic wheat.
This research aimed to answer two questions: (i) can an increase in seed rate of wheat in different tillage systems suppress A. ludoviciana, reduce its seed number, and improve wheat productivity; and (ii) can a late application of pinoxaden provide effective control of A. ludoviciana without compromising grain yield.
Materials and Methods
A 2-yr field study was conducted at the Research Farm of the University of Queensland, Gatton (27.5514°S and 152.3428°E),Australia.In each year (2020 and 2021), the experiment was conducted in the winter season (May-October).The experimental location belongs to the subtropical climatic region of Australia and has an average annual rainfall of 728 mm.The soil in the experimental field was clay with a pH of 6.9 and 1.4% organic matter.The experimental location was the same in both years.Experimental plots in conventional tillage treatments were tilled (12-15 cm deep) K with a disc-harrow (two times) followed by a rotovator operation.The field was kept fallow after the first wheat crop.
Experiment Design and Herbicide Treatments
The experiment was arranged in a split-split plot design with two tillage regimes (no and conventional tillage) in main plots, two seeding rates (100 and 200 seeds m -2 ) in sub-plots, and three weed control treatments (nontreated control, pinoxaden application at Z12 (two-leaf stage), and Z33 stage (third node formation) of wheat) in sub-sub plots.Wheat (variety Spitfire) was sown using a cone planter (six rows) at a row spacing of 35 cm in both years.The crop was sown on 7th May in both years.
All treatments were replicated thrice each year, and the size of each plot was 4.0 m (length) × 1.4 m (width).Avena ludoviciana seeds at a rate of 40 kg ha -1 were mixed with sand and broadcasted before wheat planting and tillage operation for ensuring uniform weed infestation across the field each year.Pinoxaden spray was done with a CO2 backpack sprayer equipped with four flat-fan nozzles (Airmix 110015 TeeJet nozzles, Model 25611) spaced at 50 cm and using a volume of 100 L of water ha -1 at 200 kPa.Pinoxaden 100 EC (Axial = active constituent; 100 g L -1 pinoxaden +25 g L -1 cloquintocet-mexyl; Syngenta, Australia) was applied at a rate of 20 g ai ha -1 .To create the herbicide solution, pinoxaden was mixed with Adigor adjuvant at 500 ml per 100 L water.
The crop was harvested on 14th and 7th October in 2020 and 2021, respectively.At crop harvest, A. ludoviciana density, biomass, and seed number m -2 were determined using a quadrat (50 cm × 50 cm) placed randomly in each plot at two places.Weeds from the quadrat area in each plot were counted and converted into plants m -2 .For biomass sampling, weeds were removed from the base level in each plot, placed in paper bags, and then dried in an oven at 70 °C for 72 h.After oven drying, weed samples were weighed for biomass.For weed seed count, florets (empty and nonempty) of each A. ludoviciana's panicle were counted that occurred in the quadrat area.In both years, the crop was harvested when it attained maturity using a combine harvester.For effective tillers, tillers bearing earheads were counted from two places in each plot by placing a ruler of 1 m length randomly and converted into plants m -2 .Grain yield was recorded from a net area of 4.2 m 2 (3.0 m × 1.4 m) per plot and converted to t ha -1 at a 12% moisture content.
Statistical Analyses
The results of the analysis of variance (ANOVA) for each parameter presented an overall picture of the relative effects of years, tillage, seeding rates, weed control treatments, and all possible interactions on A. ludoviciana and wheat (Table 1).In a combined analysis of data, parameters where the interactions of years × treatments were nonsignificant, data were pooled over the years.
Data were analyzed using the software CPCS1 developed by Punjab Agricultural University (www.pau.edu),Ludhiana, India (Mahajan and Chauhan 2022) and verified with GenStat 21st edition (VSN International, Hemel Hempstead, UK).Before ANOVA, data were also validated for meeting the assumptions of normality and equal variance.Treatment means were separated with the use of Fisher's Protected LSD test.Unless indicated otherwise, after ANOVA, means were separated using LSD at P = 0.05.
Weather Conditions
The crop received a higher amount of rainfall in May and July of 2021 (May: 90 mm and June: 82.6 mm) compared with 2020 (Fig. 1; Mahajan and Chauhan 2022,).In both years, July had the lowest mean monthly maximum temperature (21.7 °C for 2020 and 20.5 °C for 2021).The mean monthly minimum temperature for June and July 2021 was lower than in 2020.In 2020, the mean monthly minimum temperature was lowest in August (6.8°C); however, in 2021, it was lowest in July (7.1 °C).These observations suggest that the early season of 2021 (July) was more congenial for the emergence of A. ludoviciana than in 2020 due to the occurrence of higher rainfall and more favorable temperatures, as supported by a recent study conducted in Australia revealed that those conditions in the winter season proved to be a catalyst for the emergence of A. ludoviciana (Mahajan and Chauhan 2021b).
Weed Biomass and Weed Seed Production
For weed (A.ludoviciana) biomass, the interaction between the seeding rate and the weed control treatment was found to be significant and all other interactions were found to be nonsignificant (Table 1).In the nontreated control, the high seeding rate reduced the biomass of A. ludoviciana by 33% compared with the low seeding rate (Table 2).In herbicide-treated plots, the seeding rate did not influence weed biomass.Averaged over tillage treatments, at the low seeding rate, weed biomass in the nontreated plot was 508 g m -2 , and this biomass was reduced by 89 and 99% with pinoxaden application at Z12 and Z33 stages, respectively (Table 2).Averaged over tillage treatments, at the high seeding rate, weed biomass in the nontreated plot was 340 g m -2 , and this weed biomass was reduced by 95 and 99% with pinoxaden application at Z12 and Z33 stages, respectively (Table 2).
Seed production of A. ludoviciana followed a similar trend to biomass as the interaction between seeding rate and weed control treatment was significant (Table 2).In the nontreated control, the use of the high seeding rate reduced weed seed production by 66% compared with the low seeding rate (Table 2).Application of pinoxaden at Z12 and Z33 stages of wheat grown at the low seeding rate reduced weed seed production by 89 and 99%, respectively, compared with nontreated control (3500 seeds m -2 ) (Table 2).Similarly, at the high seeding rate, application of pinoxaden at Z12 and Z33 stages of wheat reduced weed seed production by 88 and 98%, respectively, compared with the nontreated control (1200 seeds m -2 ) (Table 2).
Averaged over seeding rate and weed control treatments, weed biomass was similar in both tillage systems.However, seed production of A. ludoviciana was reduced by 28% in K Fig. 2 Avena ludoviciana seed production (number m -2 ) in response to tillage treatments (Averaged over seeding rate and weed control treatments).Error bars indicate the least significant differences (LSD) at a 5% level of significance the no-tillage system compared with the conventional tillage system (Fig. 2).
Grain Yield
All interactions (two-way and three-way) between tillage, seeding rate, and weed control treatments for grain yield were found to be non-significant.Averaged over seeding rate and weed control treatments, grain yield was similar (4.7-5.1 t ha -1 ) in both tillage systems (data not shown).
Averaged over tillage and weed control treatments, the grain yield at both seeding rates was also similar (4.7-5.1 t ha -1 , data not shown).Averaged over tillage and seeding rate, wheat yield in the nontreated control plot was 2.1 t ha -1 and it increased by 171 and 148% with pinoxaden application at Z12 and Z33 stages, respectively, compared with nontreated control (Fig. 3).A similar trend was also observed for effective tillers m -2 (Fig. 3).Averaged over tillage and seeding rate, effective tillers m -2 in the nontreated control plot were found to be 131 and it increased by 232 and 227% with pinoxaden application at Z12 and Z33 stages, respectively, compared with nontreated control (Fig. 3).
Discussion
This research demonstrated that wheat yield did not increase with an increased seeding rate, irrespective of tillage systems.However, an increased seeding rate helped in reducing weed biomass and weed seed production, especially in the nontreated control treatment.This information could be useful in making strategies for weed control in the organic production of wheat.Effective weed control through increasing seeding rate is a proven cultural weed management practice and is exploited in many crops (Gill and Holmes 1997;Lemerle et al. 2004).The concept behind the increased seeding rate in different crops is to increase the competitive ability of crops against weeds by attaining an early canopy closure (Lemerle et al. 1995;Mahajan and Chauhan 2022).The manipulation of this cultural practice is very useful where weeds look phenotypically similar (Lemerle et al. 2001, Cousens 1996).Thickening of a crop canopy through increased seeding rates was traditionally practiced to control weeds when high tillering varieties and herbicides were not available (Downing 1921).
Previous studies in Australia also reported the effect of increased wheat planting density on the suppression of A. ludoviciana (Radford et al. 1980).The authors found a negative correlation between wheat density and A. ludoviciana biomass (Radford et al. 1980).Reduced biomass of Lolium rigidum Gaudin (rigid ryegrass) was observed when the density of lupin was increased (Lupinus spp.), which resulted in an increase in yield (Allen 1977;Medd et al. 1985).Similarly, the effect of increased wheat density on L. rigidum suppression and improved wheat yield was also observed in Western Australia (Hashem et al. 1998).
In the current study, weed seed production decreased with an increased seeding rate.Likewise, previous workers also reported that increasing the seeding rate of wheat from 50 to 200 kg ha -1 helped in reducing the seed production of L. rigidum (Fee and Anderson 1997).
The current study also demonstrated that yield remained similar when pinoxaden was applied at the Z12 and Z33 stages of wheat.However, the reduction in magnitude for weed biomass and weed seed production was higher when pinoxaden was applied at the Z33 stage compared with the nontreated control.These observations suggest that pinoxaden may be applied at a late stage (Z33) of the crop and could effectively reduce weed seed reduction without compromising grain yield.Late application of pinoxaden could help in controlling early and late cohorts of weeds as A. ludoviciana emerges in multiple cohorts.In addition to this, a delayed application of pinoxaden in wheat (ZCK31-32) caused a biomass reduction (60%) of Alopecurus myosuroides Huds (Pintar et al. 2021).
It is well-known fact that younger weeds are more sensitive to herbicides than those in more advanced stages (Kudsk 2017).However, this phenomenon depends on many factors, such as weed growth rate, type of weeds, herbicides, and environmental conditions.Pinoxaden causes inhibition of ACCase enzyme and results in chlorosis of leaves within a week after application followed by necrosis and death of rapidly growing meristematic tissue.Pinoxaden kills the susceptible plants (Avena spp., Phalaris spp., Lolium spp., etc.) completely within two to three weeks after application (Hofer 2006;Bitarafan and Andreasen 2020;Anonymous 2022).
Weed control strategies that prevent flowering and reduce the seed production potential of weeds may help in reducing the soil weed seed bank and enable fewer weed problems in the future.Avena ludoviciana plants have high seed-shattering ability (Mahajan and Chauhan 2021a), therefore, late application of pinoxaden may provide effective control of early and late cohorts before producing seeds.The current study also suggested that the no-till system helps in reducing weed seeds per unit area.A previous study revealed that A. ludoviciana seeds decayed fast on the soil surface and its emergence was lower on the soil surface (Mahajan and Chauhan 2021b).This could be the reason for reduced weed infestation in no-till plots compared with the conventional tilled plots, and ultimately lower weed seed production per unit area.Several studies reported a faster decline in the seed bank of Avena spp. in no-till compared with the conventional till system (McGillion and Storrie 2006;Nugent et al. 1999;Osten et al. 2007).It was postulated that the germination of Avena spp. was promoted with tillage operations (Chancellor 1976).
Conclusions
An increased seeding rate of wheat under weedy situations helped in reducing A. ludoviciana biomass and weed seed production, which resulted in a significant improvement in the yield.This information could be useful in the organic production of wheat.Wheat yield under no-till and conventional tilled systems remained similar; however, the notill production system helped in reducing weed seed production.This information suggests that the no-till system had an added advantage of weed control.The application of pinoxaden at the Z33 stage (third node formation) helped in controlling early and late cohorts of A. ludoviciana without compromising the grain yield.Radford BJ, Wilson BJ, Cartledge O, Watkins FB (1980)
Fig. 1
Fig. 1 Weather parameters (mean monthly maximum and minimum temperature, and rainfall) recorded at Gatton, Queensland, in 2020 (a) and 2021 (b) during the growth duration of wheat crops (Mahajan and Chauhan 2022)
Fig. 3 a
Fig.3a Grain yield (t ha -1 ) and b effective tillers (number m -2 ) and of wheat in response to weed control treatments (Averaged over tillage treatments).Error bars indicate the least significant (LSD) differences at a 5% level of significance.Z12 (two-leaf stage), and Z33 stage (third node formation) of wheat
Table 1
Analysis of variance (ANOVA) for different parameters of Avena ludoviciana and wheat yield *indicates significance at a 5% level of significance
Table 2
Interaction effect of seeding rate and weed control treatments on biomass and seed production of Avena ludoviciana (Averaged over Effect of wheat seeding rate on wild oat competition.Aust J Exp Agric 20:77-81 Sahil, Mahajan G, Loura D, Raymont K, Chauhan BS (2020) Influence of soil moisture levels on the growth and reproductive behaviour of Avena fatua and Avena ludoviciana.PLoS ONE 15(7):e234648 Scott BJ, Martin P, Riethmuller GP (2013) Row spacing of winter crops in broad scale agriculture in Southern Australia.Graham Centre Monograph, vol 3. NSW Department of Primary Industries, Orange Storrie A (2007) 'Wild oat resistance options', Grains Research Update-Northern region, Grains Research & Development Corpo-ration.http://users.tpg.com.au/icanadsl/newsletters/NL37V4.pdf.Accessed 10 Jan 2023 Sykes J (2012) Irrigated wheat-Best practice guidelines in cotton farming systems.Cotton Catchment Communities CRC and Grains Research & Development Corporation Triplett GB Jr, Dick WA (2008) No-tillage crop production: A revolution in agriculture!Agron J 100:153 Walsh MJ, Powles SB (2007) Management strategies for herbicideresistant weed populations in Australian dryland crop production systems.Weed Technol 21:332-338 | 2023-07-26T15:04:25.793Z | 2023-07-24T00:00:00.000 | {
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236944933 | pes2o/s2orc | v3-fos-license | High BMI and Low Muscular Fitness Predict Low Motor Competence in School-Aged Children Living in Low-Resourced Areas
Childhood obesity is a relatively new problem for Sub-Saharan developing countries. Especially in children with a low socioeconomic background, the link between motor competence, muscular fitness, and body mass index (BMI) remains poorly investigated. Due to the interrelatedness of BMI and physical fitness, the aim of this study is to determine the predictive value of these factors in relation to low motor competence in school-aged children living in low-resourced areas. Motor competence and physical fitness were assessed in 1037 school-aged Ghanaian and South African children using the Performance and Fitness test battery (PERF-FIT). “Low motor competence” was predicted using odds ratios calculated from backward logistic regression analyses. Low motor competence was less prevalent in Ghanaian children (3.7–11.1%) compared to the South African children (21.9–24.2%). Increased BMI and decreased muscular fitness predicted low motor competence in both Ghanaian and South African children. For example, the chance for a Ghanaian child to have low static balance increased by 22.8% (OR = 1.228, p < 0.001) with a 1-point increase in BMI, whereas this decreased by 30.0% (OR = 0.970, p < 0.001) with a 10-cm increase on the standing long jump. In the case of the South African children, if their BMI increased by 1 point, the chance for those children of having low static balance increased by 7.9%, and if their SLJ performance decreased by 10 cm, their chance of low performance increased by 13%. Clearly, motor competence is associated with both BMI and muscular fitness. Policy makers can use this information to counteract the establishment of childhood obesity by promoting weight control through physical activity and stimulating motor competence at school.
Introduction
Overweight and obesity were previously perceived as typical problems of developed countries, but currently in low-and middle-income countries, a relatively rapid transition from underweight to overweight and obesity is occurring [1]. The increasing prevalence of overweight and obesity in western and southern Africa is remarkable [1]. For example, between 2000 and 2013, overweight and obesity increased from 11 to 19% in southern African countries, whereas the prevalence of stunted children remained almost the same [2]. In 2020, approximately 17% of the children between age 5 and 19 in South Africa presented with overweight and obesity [3], which was estimated at 19% for all children under age 19 in Ghana [4]. In general, it is often reported that more girls tend to have overweight or obesity compared to boys [5].
To reduce obesity, good nutrition and physical health are vital [6]. Recent systematic reviews and meta-analyses showed that physical health is associated with physical activity
Participants
Children aged 6 to 12 years were recruited from four primary schools in South Africa, (two in the Western Cape and two in North West Province) and from five primary schools in Ghana (four near Accra and one in the Eastern region of Ghana). Twelve hundred children were invited to participate in the study and were recruited through stratified sampling, spread over nine schools (located in different SES areas: four schools low SES; three schools low-middle SES; one school middle SES; and one school located in high-middle SES). The participating schools were situated in low SES areas and were recruited through an indirect network of the researchers. Once the schools gave consent to participate, the children were randomly selected. The children participated in this cross-sectional study after their parents provided written informed consent. Data collection took place between January and September 2019. The study protocol was approved by the local ethical committees (NWU-00491-19-A1, HREC Ref 598/2019, and GHS-ERC 084/04/19).
The parent(s) filled in the child physical activity readiness questionnaire [27]. Children were excluded from the sample if they had: (i) a formal diagnosis that would significantly impede motor performance as reported by the parents, (ii) refused testing, or (iii) incomplete test results due to absence from school during test administration. Neither children nor legal guardians received financial compensation for their participation.
Measurements
Weight (BF 511 Omron, measured to nearest 0.1 kg) and height (a wall-mounted tape measure, measured to the nearest 0.1 cm) were recorded to calculate BMI (kg/m 2 ). Next, the PERF-FIT, a valid and reliable test to measure movement skills, musculoskeletal fitness, and agility in children this age in low resourced communities [24][25][26]28], was administered. The test consists of two subscales. The power and agility subscale comprises three agility items (running, stepping, and side jumping) and two explosive power items (overhand throw and Standing Long Jump (SLJ)). Although the SLJ is considered a fundamental movement skill, due to its strong relationship with other measures for lower extremity power [33,34], it represents a general index of muscular fitness in youth [29,30]. The motor skills performance subscale contains five series of tasks (referred to as skill item series (SIS)) with increasing difficulty: (1) throw and catch, (2) bounce and catch, (3) static balance and dynamic balance, (4) jumping, and (5) hopping. For studying the relation with body composition, muscular fitness, and motor competence, the SLJ, static balance, dynamic balance, jumping, and hopping items are reported in this study and are therefore described in more detail in Table 1. Table 1. Description of PERF-FIT standing long jump and motor skill item series.
Standing Long jump 1
The child is asked to stand with his/her toes behind a starting line and to jump as far as possible without falling [35]. One practice trial with submaximal force is given, followed by two test trials with 15 s rest between trials. The distance (cm) between the starting line and the heel of the foot that landed closest to the starting line is measured. The best performance is the final result.
Static balance SIS 4
Four items: (1) standing and hugging the knee for maximum 15 s (left and right), (2) standing and grasping the foot for maximum 15 s (left and right) [35]. Timing started when the knee was hugged or the foot was grasped and stopped if the raised leg touched the standing leg, corrective hops were made, or the child lost balance or fell. Per item, a second trial was allowed if the child performed submaximal during the first trial. The best trial was the final result.
The scores of each item were summed, resulting in scores between 0-60 s.
Skill Item Series Items Description
Standing Long jump 1 The child is asked to stand with his/her toes behind a starting line and to jump as far as possible without falling [35]. One practice trial with submaximal force is given, followed by two test trials with 15 s rest between trials. The distance (cm) between the starting line and the heel of the foot that landed closest to the starting line is measured. The best performance is the final result.
Dynamic balance SIS 6
(1) walking while hugging a knee, (2) walking while grasping a foot, (3) standing on one leg while moving cans from close to far (left and right), and (4) standing on one leg while moving cans from far to close (left and right) [35].
First, the child was asked to walk slowly in an agility ladder (max 8 steps) while hugging a knee or grasping a foot without touching the borders, stepping outside the borders, or losing balance. For each correctly placed step, a point was awarded. For both items, a second trial was allowed if the child performed submaximal during the first trial. The best trial was the final result.
With these two items, a maximum of 16 points could be earned [35]. Afterwards, the child stood on one leg while picking up 4 cans consecutively and moving them from close to far (or the other way around) without moving the stance foot, losing balance, or placing the raised leg on the ground. One point was awarded for each correctly placed can (max 4 points). The children performed this for both legs and in both directions (i.e., 4 items) and could earn a maximum of 16 points. The scores of the dynamic balance items were summed, with scores between 0 and 32 points.
Jumping and hopping SIS 12
Four jumping items and four hopping items (left and right) [35]. The jumping and hopping SIS consists of four levels of difficulty: jump/hop in each square, in every other square, in every other square over a five-centimeter foam pad, and in every other square over a ten-centimeter foam pad. One point was awarded for each correct jump/hop. For the jumping items a total of 20 points could be earned and for the hopping items 40 points, resulting in a total score varying between 0 and 60 points.
Statistical Analysis
Statistical analyses were performed with SPSS 27.0 for windows. The sample was described using demographic data (age, sex), anthropometric data (weight, height, BMI), and muscular fitness data from the PERF-FIT. The Shapiro-Wilk test was used to check for normality. The prevalence of overweight and obesity was determined using the BMI-for-age-and-sex percentiles defined by Cole et al. (2012) [36] and expressed as a percentage of the entire sample. To compare the children's age, BMI, and muscular fitness per country (Ghana versus South Africa) in the entire sample and within the age groups, a one-way analysis of variance (ANOVA) was used and a two-tailed Chi-squared test for the distribution of the sexes.
Age and sex may or may not have an influence on motor competence [7], depending on the applied outcome. These factors were therefore considered in the analyses. To explore age-related differences in the motor competence outcome variables, the Kruskal-Wallis test was applied.
The distribution of the dependent variables was skewed, such that the use of cut-off values, to identify low performance, were clinically more relevant as opposed to using median values or interquartile ranges (IQR). Therefore, to predict low motor competence, the dependent variables were dichotomized (0 = normal performance, 1 = low performance) using percentile 15 (P15) as a cut-off value. This cut-off value was chosen as motor scales usually apply the 15th percentile as a cut-off value to decide whether a motor performance is below average for age because this represents performances 1SD away from the mean. p < 0.001), age-specific percentile values were used for dichotomization, hereafter referred to as the motor competence groups (normal versus low motor competence).
Subsequently, a two-tailed Chi-square test revealed that the distribution of Ghanaian and South African children across the motor competence groups was significantly different for all dependent variables (static balance SIS: X 2 = 89.893, p < 0.001; dynamic balance: X 2 = 28.637, p < 0.001; jumping and hopping SIS: X 2 = 36.459, p < 0.001). Therefore, the next analyses were performed separately per country (Ghana versus South Africa). As such, a two-tailed Chi-square test was used to establish whether the sex and SES distribution and the percentage of children living in urban or rural areas were similar between each motor competence group. The Mann-Whitney-U test was used to compare the BMI and SLJ performance in the normal (>P15) versus the low (≤P15) motor competence groups. Next, backward conditional logistic regression analysis was applied to calculate odds ratios. The predicted value was "low motor competence" for the static balance, dynamic balance, and jumping and hopping, with BMI (continuous) and SLJ performance (continuous) as predictors. Sex was not added to the model because the Chi-square test was not significant for the static and dynamic balance SIS. There were differences between boys and girls regarding jumping and hopping performance (see Figure S1 and Table S1), and their distribution was also significantly different across the motor competence groups. Furthermore, for the jumping and hopping SIS, the residential area was also significantly differently distributed across the groups. Hence, sex and residential area were added to the model in addition to BMI and SLJ. However, sex was not a significant predictor in the model. Odds ratios (ORs) were derived from the exponential β value and the corresponding 95% CI. Significance was set at p < 0.05.
Participants
Approximately eighty-seven percent (1040/1200) of the invited children participated in this study. Three 5-year-olds were excluded because they were too young, leaving 1037 children for data analysis. Table 2 contains a detailed description of the entire sample and the different age groups. An equal number of boys and girls participated in both countries (X 2 = 0.046, p = 0.830). The Ghanaian children were significantly older (F 1,1035 = 121.146, p < 0.001), had a lower BMI (F 1,1035 = 23.149, p < 0.001), and higher muscular fitness (F 1,1033 = 227.268, p < 0.001) ( Table 2). The combined prevalence over the age groups of overweight and obesity was 12.9% in the Ghanaian sample, of which 47% lived in urban area, and the prevalence of overweight and obesity was 22.6% in the South African sample, of which 71% lived in urban area. The prevalence of underweight was 21.5% and 7.8%, for Ghana and South Africa, respectively ( Figure 1). model. Odds ratios (ORs) were derived from the exponential β value and the corresponding 95% CI. Significance was set at p < 0.05.
Participants
Approximately eighty-seven percent (1040/1200) of the invited children participated in this study. Three 5-year-olds were excluded because they were too young, leaving 1037 children for data analysis. Table 2 contains a detailed description of the entire sample and the different age groups. An equal number of boys and girls participated in both countries (X 2 = 0.046, p = 0.830). The Ghanaian children were significantly older (F1,1035 = 121.146, p < 0.001), had a lower BMI (F1,1035 = 23.149, p < 0.001), and higher muscular fitness (F1,1033 = 227.268, p < 0.001) ( Table 2). The combined prevalence over the age groups of overweight and obesity was 12.9% in the Ghanaian sample, of which 47% lived in urban area, and the prevalence of overweight and obesity was 22.6% in the South African sample, of which 71% lived in urban area. The prevalence of underweight was 21.5% and 7.8%, for Ghana and South Africa, respectively (Figure 1).
PERF-FIT Performance
The prevalence of low motor competence was significantly lower in the Ghanaian sample compared to the South African children for all motor competencies. Table 3 shows the distribution of the children in motor competence groups and the medians and IQR for age, BMI, and SLJ performance in each motor competence group. For each skill, fewer Ghanaian children showed low motor competence compared to the South African children and, in both countries, the motor competence groups consisted of a similar number of boys and girls (Ghana: X 2 = 0.504, p = 0.495; South Africa: X 2 = 0.378, p = 0.543). For both the Ghanaian and South African children, BMI and SLJ performance were significant predictors for low motor competence in static balance, dynamic balance, and jumping and hopping. In both the Ghanaian and South African sample, the group with a low static balance performance had a significantly higher median BMI (Ghanaian group: U = 6606, z = 3.059, p = 0.002; South African group: U = 28,519, z = 2.228, p = 0.026) and lower median SLJ performance (Ghanaian group: U = 2916, z = −2.784, p = 0.005; South African group: U = 20,181.5, z = −3.232, p = 0.001) compared to those with a normal static balance (Table 3; Figure 2). Table 4 shows the probability of low static balance based on changed BMI and SLJ for both groups in terms of odds ratios. In both the Ghanaian and South African sample, the group with a low static balance performance had a significantly higher median BMI (Ghanaian group: U = 6606, z = 3.059, p = 0.002; South African group: U = 28,519, z = 2.228, p = 0.026) and lower median SLJ performance (Ghanaian group: U = 2916, z = −2.784, p = 0.005; South African group: U = 20,181.5, z = −3.232, p = 0.001) compared to those with a normal static balance (Table 3; Figure 2). Table 4 shows the probability of low static balance based on changed BMI and SLJ for both groups in terms of odds ratios.
Dynamic Balance SIS
In both the Ghanaian and South African sample, the group with a low dynamic balance performance had a significantly higher median BMI (Ghanaian group: U = 15,173, z = 2.126, p = 0.033; South African group: U = 30,029, z = 3.397, p < 0.001) and lower median SLJ performance (Ghanaian group: U = 8556.5, z = −4.171, p < 0.001; South African group: U = 19,364.5, z = −3.656, p < 0.001) compared to those with a normal dynamic balance (Table 3; Figure 2). Table 4 shows the probability of low dynamic balance based on changed BMI and SLJ for both groups in terms of odds ratios. Figure 2). Table 4 shows the probability of low dynamic balance based on changed BMI and SLJ for both groups in terms of odds ratios.
Jumping and Hopping SIS
In the Ghanaian sample, the group with a low jumping and hopping competence had a lower median SLJ performance (U = 5403.5, z = −5.028, p < 0.001) but similar BMI compared to normal motor competence group (Table 3; Figure 2). The South African children with a low jumping and hopping performance had both a higher median BMI (U = 30,855, z = 5.064, p < 0.001) and a lower median SLJ performance (U = 18,019.5, z = −3.801, p < 0.001) compared to those with a normal jumping and hopping performance (Table 3; Figure 2). Table 4 shows the probability of low jumping and hopping performance based on living in an urban area or changed BMI and SLJ for both groups in terms of odds ratios.
Discussion
The aim of this study was to investigate the prevalence of low motor competence in Ghanaian and South African school-aged children living in low-resourced areas and subsequently to establish the extent to which body composition and muscular fitness predict low gross motor competence. Our study showed that: (1) Significantly fewer Ghanaian children had a low motor competence compared to South African children; (2) In both samples, low motor competence in static and dynamic balance as well as jumping and hopping can be predicted by both body composition (BMI, ranging between 7.9-22.8% per increasing BMI unit) and muscular fitness (SLJ, ranging between 13-35% per decreasing 10 cm); and (3) Children living in urban areas are more likely to have low jumping and hopping performance compared to those living in rural areas.
In line with the literature, BMI was related to all motor skills tested. In general, except for the 6-year-olds where the mean is below P50, the children in the current sample have a slightly higher BMI than those in the growth curves of the World Health Organization [32]. First, Ghanaian children have a lower BMI compared to the South African children, and there is a smaller portion with overweight and obesity (Ghana: 12.9%; South Africa: 22.6%). Additionally, overweight and obesity tended to be more prevalent in the older age groups, especially in the South African sample. Nevertheless, for both samples the chance of belonging to the low motor competence group increased significantly if their BMI increased by 1 point (ranging from 7.9% to 22.8%). These findings confirm the inverse relationship between BMI and motor skill competence [7]. Previous research has shown that older children with high BMI levels exhibit lower motor competence compared to younger children, stressing the enlarging impact of growing older on the reciprocal association between motor competence and overweight [14,15,19]. Interestingly, the Ghanaian children were older than the South African children and also showed less overweight and obesity. Hence, increasing BMI has a much larger impact on motor competence, as shown by higher odds ratios in this specific group. This may suggest that the negative spiral between motor competence, BMI, and muscular fitness has not reached a similar level in both countries.
Furthermore, we found a link between muscular fitness and motor competence. Although the impact of the individual factors is important, both BMI and SLJ performance additively predicted low performance on all assessed motor skills. This is not surprising as physical fitness or strength and weight are associated with each other [33]. A heavier body is more difficult to move and control during functional movements as it requires relatively more power [7], a feature limited in these children, especially the South African sample. Similar to a global trend of children becoming less active and overweight, physical fitness in our African samples is lower compared to previous studies [37]. Especially, the Ghanaian sample showed that when children have lower muscular fitness, the impact on their motor competence is detrimental. Similar to what the Stodden model [13] suggests, our results indicate that higher levels of muscular fitness may protect a child from presenting with low motor competence, which is why attention should be given to this factor as well [33,37]. Indeed, strength training (combined with anaerobic and aerobic exercises) positively influences BMI and adiposity in children with overweigh and obesity [38,39].
Investigating these specific groups of children provided a unique opportunity to map the impact of children living in different environmental circumstances. Interestingly, in both countries, living in urban areas predicts low motor competence for jumping and hopping. This is not surprising as such skills require room for movement. South African children living in informal settlements in urban areas do not have many opportunities to participate in organized sports activities and play outside because of the increased crime and unsafe environment [40,41]. Subsequently, for safety purposes, approximately 40% of the South African children do not walk to school, but go by taxi busses, decreasing their daily physical activities [40]. Although many Ghanaian children also live in low resourced areas, they do not face such unsafe environments, allowing them to play outside, and most children in our sample walk to school. Hence, it is not surprising that a significantly larger number of South African children have low motor skill related fitness compared to their Ghanaian peers. The current results indicate that these physical activity restrictions also have large implications for the children's body composition and muscular fitness. Nevertheless, even for the Ghanaian children, living in urban areas may have a negative impact on motor competence, indicating that both our samples would benefit from more opportunities that allow practicing movement skills. However, this would require safe environments, such as public parks, playgrounds, community centers, and walking trails/hiking areas, where the children can be active and keep practicing.
Studies performed in developed countries, which have been dealing with the impact of overweight and obesity on child development for several decades, have already shown that high BMI at a young age contributes to declines in motor competence at a later age [17,18], but also the reverse impact has been shown [18]. Therefore, early childhood interventions targeting weight control can positively impact motor competence [17]. To ensure long-term effects, weight control needs to be targeted, not only through diet, but also by increasing physical activity and physical fitness. Because motor competence in our sample was related to muscular fitness and previous research showed that a certain level of motor competence is required to enjoy and maintain long-term physical activity [12], promoting physical activity and fitness is much needed. Especially children with low balance, such as in our sample, benefit from weight loss and training [21]. These results have important implications for policy makers in both countries targeting overweight. For South African children, the vicious circle of physical inactivity, overweight and obesity, and low motor competence needs to be broken, e.g., more physical activity at school. Although physical education forms part of the life skills curriculum in South Africa, its actual delivery in the schools may be insufficient [40]. For Ghanaian children, the current results show that discontinuation of the gradually increasing prevalence of overweight and obesity in children is imperative and that prevention is in order, which also requires implementation of regular physical education at schools and organized sports activities, next to attention regarding nutrition. Interventions focusing on improving motor skills in children with overweight and obesity have proven to be successful on the body composition, physical fitness, and gross motor competence [42].
Study Strengths and Limitations
Although several systematic reviews and meta-analyses have been performed on factors that influence motor competence, literature on this topic in children living in low and middle-income regions is scarce. The included sample is large and was recruited in different regions in two African countries, which improves generalizability of the results. A limitation of this study is that the sample was not entirely randomly selected. However, the specific group of children targeted did not allow such a sampling method, and the main aim was to recruit a representative sample. More background on sedentary behavior in our sample would have helped to interpret the data better. Physical fitness and motor competence were both measured using fundamental motor skills, for which some authors [43] argue that a general motor ability could explain the interrelatedness. Nevertheless, motor abilities are task-dependent as the tasks dictate which aspects of motor competence and their underlying body functions, such as muscle strength, coordination, or balance, are being tapped into. Thus, although the standing long jump, used for assessing muscular fitness also requires coordination and balance, this test has been proven to correlate strongly to other measures for lower extremity power [33].
Conclusions
The present study showed that low motor competence measured with the PERF-FIT can be predicted by increased BMI and decreased muscular fitness. These findings provide policy makers with important knowledge on the current state of overweight and physical fitness in children living in low-resourced Sub-Saharan areas, which can be used to counteract the establishment of childhood obesity by promoting physical activity, preferably at school. Evaluation using the PERF-FIT can therefore be used to plan additional support to stimulate motor competence through physical education and organized sports activities at school in children living in low and middle-income countries.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijerph18157878/s1, Table S1: Differences in motor competences between boys and girls across different age groups. Figure S1: Median jumping and hopping SIS for boys and girls across age groups. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
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 privacy. | 2021-08-08T05:23:18.482Z | 2021-07-25T00:00:00.000 | {
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224911040 | pes2o/s2orc | v3-fos-license | Music Education in Krasnoyarsk as a Resource Factor for Building the Civic Solidarity: The Situation by the Outbreak of the War
The purpose of the study is a comprehensive analytical review of various sources that reveal the role of music in Krasnoyarsk by the outbreak of the war. A hypothesis: interdependence of cultural and social events and their mutual influence during a period of increased social tension could be observed. Methods. During the study the following research methods were applied: cultural-historical, axiological, continuous sampling, descriptive methods and elements of statistical research. Materials published on the pages of the Krasnoyarsk periodicals for 1937–1941, as well as scientific works about music education and music culture by various outstanding scientists were used. Results. It was proved that music education by the outbreak of the war is considered a foundation for the realization of ideological and educational purposes. And it is also outlined that The Great Patriotic War set new challenges in the cultural life of the Krasnoyarsk Territory in general and Krasnoyarsk in particular. It is concluded that music education is considered a resource for the national solidarity building. Due to such a strong foundation, music education at the beginning of the war was able to fulfil an ideological function while concert musical activities were used as a tool for creating of civic identity.
I. Introduction
The study was carried out to understand the role of art in the regions during the war, in particular in the Krasnoyarsk Territory. This article is devoted to Krasnoyarsk. Previously, the art of the Krasnoyarsk Territory was studied within the framework of art history, through the cataloging of material, analysis of artworks whereas this study starts a series of articles devoted to the problems of the civic identity building in the Krasnoyarsk Territory in 1941-1945 through musical and concert activities. It gives characteristics to the situation with music education as a resource for the cultural environment formation in the city of Krasnoyarsk, which could make it possible to use art as a tool for creating a national community. The purpose of this study is a comprehensive analytical review of various sources that reveal the role of music in the Krasnoyarsk Territory during the war. A hypothesis is put forward about the interdependence of cultural and social events and their mutual influence during a period of increased social tension.
III. Results
It should be noted that the subject of music education in Krasnoyarsk has been deeply studied by several scientists. For instance, the monograph "To Teach Creativity! Concerning the history of professional education in the field of art in Krasnoyarsk" is worth mentioning [6]. According to this work, before the outbreak of the Great Patriotic War, the musical cultural life in Krasnoyarsk was quite active. E.V. Prygun gives an overall picture of the cultural musical life development of the city and distinguishes four periods of its formation [5]. By 1941, the National Conservatoire had been functioning for about 20 years. Pianists, singers, string and brass players and future voice teachers graduated from the first professional music institution on a regular basis. Before the conservatoire was established, pupils at Krasnoyarsk primary and secondary educational institutions had had the opportunity to study music at singing and music classes. They learned to play musical instruments, sang in the choir, and played in the orchestra. The students also took part in concerts. Fortunately, in Krasnoyarsk by that time there were venues necessary for the functioning of creative teams. It was this fact that largely determined the further development of cultural events in Krasnoyarsk, and the city turned into a Siberian musical centre. The purpose of the conservatoire was to teach all those wishing the basics of general music education for three age categories. A couple of years later, the conservatoire was renamed into a music technical school, and in 1936 into a music college. The first separate music school was opened in Krasnoyarsk in 1930. Before 1930, the primary and the secondary levels of professional music education were united at the conservatoire. Besides the National Conservatoire, by the outbreak of the war there were the Philharmonic Society (whose slogan on the posters was "Music to the masses"), the Regional Concert and Variety Bureau, the Pushkin Theatre with drama, opera, and operetta companies, orchestras (symphonic, Great Russian and mandolin-symphonic orchestras), the County Exemplary Choir headed by S.F. Abayantsev. Due to the Slovtsovs, an opera group was created, and opera performances were a great success with the public. Lack of funding at the National Conservatoire led to teachers' various part-time jobs in different musical groups and orchestras. Also, teachers had to give private lessons. Apart from professional activities, there were hobby clubs for choir and musical instrument practice. The situation of that time is described in detail in the second volume of "Musical culture of Krasnoyarsk" [4].
Refugees, prisoners of the First World War (some of them had academic music education) as well as White Guards contributed to Krasnoyarsk music culture too. For instance, local authorities organized a three-month training course for music specialists to transfer knowledge from Europeans to Siberians. E.S. Tsareva gives much interesting information on the prisoners-of-war symphony orchestra that entertained Krasnoyarsk citizens with both classical masterpieces and popular dance and folk music [6].
The composer, teacher, founder of Krasnoyarsk professional music education P.I. Ivanov-Radkevitch created the first mixed choir consisting of Krasnoyarsk students and organized a symphony orchestra of city music lovers and students. He also staged opera scenes and a children's opera with the participation of the pupils.
There was also some theoretical background. For example, K.N. Sementovsky lectured in musical arts at the University of Culture for the Krasnoyarsk party, Komsomol and Union Activists. He also made opening remarks before concerts and performances, wrote reviews and articles for the periodical "Krasnoyarsky Rabotchy", broadcast on the radio, combining these activities with pedagogic work at schools and colleges. Moreover, a musical lecture institution for senior pupils and students was created. In the Regional House of Folk Art, refresher training for leaders of amateur art hobby clubs took place. As for non-professional activities, plant employees lived an amateur musical life. There was also a choir and a brass band at the pedagogical university.
Despite the fact that the Great Russian Orchestra at the Red October club, the best Krasnoyarsk amateur group in 1925 (piccolo, domra prima I, domra prima II, domra alto I, domra alto II, domra bass I, domra bass II, balalaika prima, balalaika second, balalaika alto, balalaika bass), ceased to exist four years before the outbreak of the war, musicians playing the folk instruments continued to participate in Krasnoyarsk musical life. Before the disbandment, the repertoire of the orchestra included transcriptions of works by Russian and foreign classics (Georges Bizet, Camille Saint-Saëns, Vittorio Monti, Frédéric Chopin, etc.) as well as fantasies based on Russian folk songs ("Barynia /a mistress/1", "Vo piru byla /I was having a feast/", etc.). In addition to the fact that the head of the orchestra G.I. Troshin conducted and sometimes performed himself, he often carried out the instrumentation of the pieces. The orchestra sometimes played the music written by its conductor too.
In the 1930s, the tour poster was diverse: Leningrad Ballet, Novosibirsk and Irkutsk opera companies, vocal quartets (the Moscow Art Theatre School-Studio Quartet and Lysenko Quartet), the Travelling Opera Ensemble, the Auer Quartet, the Glazunov Quartet, the David Oistrakh and Yuri Bryushkov's Duet, the Nathan Perelman (piano) и Hertz Tsomyk's (cello) Ensemble, soloists Miron Poliakin (violin), Grigory Ginzburg (piano), Vera Dulova (harp) and many others. In 1940, the citizens has opportunity to attend the performances of the State Song and Dance Ensemble of the North Ossetian Autonomous Soviet Socialist Republic and the Leningrad State Academic Capella under the direction of A.V. Sveshnikov, the jazz orchestra of the composer Dmitry Pokrass (the program included new songs "May Moscow", "If tomorrow will be war", "Rifle", "Farewell Komsomol Song", etc.).
Thus, on the eve of the war, both art education and amateur performing were largely developing. Also cultural life was very active: musicians were working, educational institutions were teaching students and organizing concerts and musical and literary evenings as well. Even on the last pre-war day of preparing the layout, the newspaper "Krasnoyarsk Rabochy" reported about the concert of the Lviv State Theatrical Jazz under the direction of I. Gert and about One Farewell Tour of the A.M. Gorky Central Park of Culture and Leisure (Green Theatre) [1, p. 4].
At the beginning of the war, Krasnoyarsk turned into one of the defense centres with factories operating at that time to help the front. Both the preparation of reserves for the front and the formation of military units took place. Nevertheless, musical life in the city continued to intensify: in addition to concerts given by the symphony orchestra, opera theatre, chamber music soloists and groups, there were also performances in hospitals, military units, industrial enterprises and collective farms. Many musicians and professors from the state universities as well as Dnepropetrovsk and Odessa opera and ballet theatre artists were evacuated to Krasnoyarsk. This fact also played a great role. Evidently, there was not only patriotic theme, anthems and military songs. On the one hand, people were preparing to defend the country; on the other hand, people were trying to create an illusion of peace and to believe that life went on. Musical comedies staged at the Pushkin Theatre prove it. It is not surprising, as they are laughter, light music, an interesting plot, the illusion that everything is fine, that life goes on as usual. This is an opportunity to go to the theatre, to get away, to smile, to listen to well-known motives familiar to people even before the war. As for the folk songs performed at the concerts, they are also people's culture, because everyone can sing along to them. It is also a kind of identity, civic solidarity consciousness and national folklore awareness. Folklore can represent slow long melodies, love and dance songs. This is a part of the culture; it also forms the patriotic spirit. Therefore, victorious patriotic creativity arose precisely at the moment. At the same time, the war did not cancel the achievements of previous years, but brought something new to the concert activity. Undoubtedly, events taking place on the international political scene and news from the front presented by the media influenced the cultural development of the city and mobilized all the forces. Not only scientists, engineers, technicians, but also writers, poets and artists were inspired to hard work and dedication. The newspaper "Krasnoyarsk Rabotchy" also encouraged people to this: «Может ли быть что-либо выше, жизненнее, священнее для советского патриота, как отдать все силы на защиту советского отечества, если надо, то и саму жизнь, чтобы он мог сказать вместе с Владимиром Маяковским: «Я с теми, Кто вышел Строить И месть В сплошной лихорадке Буден. Отечество Славлю, Которое есть, Но трижды -Которое будет2» [2, p. 2].
Thus, the hypothesis is confirmed. Music education by the outbreak of the war can be considered as a foundation for the realization of ideological and educational purposes. The Great Patriotic War set new challenges in the cultural life of the Krasnoyarsk Territory in general and Krasnoyarsk in particular.
IV. Discussion
The study strengthens the considerable work on music education and musical culture in Krasnoyarsk done by the scientists and deepens it going into the civic identity aspect. Due to such a resource as long-term music education (both in the direct academic sense, and in terms of transferring knowledge from experienced masters to amateur students), musical art was able to fulfil an ideological function for the formation of the civic solidarity among the population. They managed to create a fairly dense cultural environment where there were plenty of opportunities to use musical activities as a tool for bringing people together. The way of using these opportunities will be described in the following works. The research will be continued within the framework of the war period in the Krasnoyarsk Territory. 1) Hereinafter the translation is made by the authors. 2) Can there be anything higher, more vital, and more sacred for a Soviet patriot, than to devote all the efforts to defend the Soviet homeland, and if necessary, then life itself, so that one could say together with Vladimir Mayakovsky: "I am with those who went out to build and sweep in a continuous fever of the weekdays. I am glorifying the Homeland, which exists, but I am glorifying three times -the Homeland that will be". | 2020-10-16T02:23:33.941Z | 2020-09-28T00:00:00.000 | {
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269612949 | pes2o/s2orc | v3-fos-license | Berberine inhibits the progression of breast cancer by regulating METTL3‐mediated m6A modification of FGF7 mRNA
Abstract Background Berberine (BBR), an isoquinoline alkaloid from Coptidis rhizoma, has been found to have powerful activities against various human malignancies, including breast cancer. However, the underlying antitumor mechanisms of BBR in breast cancer remain poorly understood. Methods Breast cancer cells were cultured and treated with different doses (0, 20, 40, and 60 μM) of BBR for 48 h. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3‐(4, 5‐dimethyl‐2‐thiazolyl)‐2, 5‐diphenyl‐2H‐tetrazolium bromide (MTT), 5‐ethynyl‐2′‐deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. Fibroblast growth factor 7 (FGF7), methyltransferase‐like 3 (METTL3), and insulin‐like growth factor‐2 mRNA‐binding protein 3 (IGF2BP3) mRNA levels and protein levels were measured using real‐time quantitative polymerase chain reaction (RT‐qPCR) and western blot. Interaction between METTL3 and FGF7 m6A was assessed using methylated RNA immunoprecipitation (MeRIP)‐qPCR and RNA immunoprecipitation (RIP) assay. Binding ability between IGF2BP3 and FGF7 mRNA was analyzed using RIP assay. Results BBR treatment hindered breast cancer cell proliferation, invasion, migration, and induced apoptosis. FGF7 expression was upregulated in breast cancer tissues, while its level was reduced in BBR‐treated tumor cells. FGF7 upregulation relieved the repression of BBR on breast cancer cell malignant behaviors. In mechanism, METTL3 stabilized FGF7 mRNA through the m6A‐IGF2BP3‐dependent mechanism and naturally improved FGF7 expression. BBR treatment inhibited breast cancer growth in vivo. Conclusion BBR treatment blocked breast cancer cell growth and metastasis partly by regulating METTL3‐mediated m6A modification of FGF7 mRNA, providing a promising therapeutic target for breast cancer treatment.
INTRODUCTION
As the most common occurring malignancy in women, breast cancer has been considered an increasing health and economic burden all over the world, with about 3 million new cases and 1 million deaths by 2040. 1,2In recent years, breast cancer incidence rates have been slowly increasing, especially in younger women associated with poorer clinical outcomes. 3Despite the fact that 70%-80% of individuals with early-stage breast cancer are considered curable, thanks to the advancement of multimodal therapies, including mammography screening, surgical resection, adjuvant chemotherapy, and immunotherapy, outcomes for subjects with advanced/metastatic disease remain dismal. 4,5Therefore, it is imperative to identify more effective therapies for breast cancer to achieve better treatment outcomes.In recent years, traditional Chinese medicine (TCM) and some extracted monomers have become more and more popular against different diseases due to their unique advantages such as multitargeting and low side effects. 6,7As a nature-driven phytochemical isoquinoline alkaloid from Chinese herbs and used in TCM, BBR has been reported to possess multiple protective properties, such as antibacterial, antiapoptosis, anti-inflammatory, and anticancer. 8,9Mounting evidence has indicated that BBR is a multitarget drug that prevents and treats many cancers by inhibiting proliferation and inducing apoptosis. 10,11 Yet, the detailed underlying mechanism of the anticancer ability of BBR is far from being addressed in breast cancer.
As a potent mitogen for different types of epithelial and cancer cells, fibroblast growth factor 7 (FGF7, also known as keratinocyte growth factor [KGF]) has been reported to modulate the migration and differentiation of these cells and defend them against various injuries under stress conditions. 14Several studies have suggested that FGF7 is expressed at a high level in most human cancers, including breast cancer. 15Further reports verified that the inhibition of FGF7 signaling might reduce breast cancer cell growth, migration, and invasion. 16,17Beyond that, a recent study indicated that a TCM extract (aqueous extract of eucommia leaves) might significantly hinder the proliferation of osteoblasts by decreasing FGF7 expression. 18However, whether FGF7 participates in BBR-mediated antitumor effects in breast cancer remains unknown.
Numerous RNA modifications have been identified to possess the potential to change RNA function.As the most prevalent internal mRNA modification, N6-methyadenosine (m6A) has received increasing attention as an important regulator of gene expression. 19Indeed, some articles have presented that m6A methylation exerts a vital role in modulating gene expression in many human cancers, 20 including breast cancer. 21As a dynamic and reversible process, it can be installed by methyltransferase complexes ("writers") consisting of methyltransferase-like 3 (METTL3), METTL14, and WTAP, and eliminated by demethylases ("erasers"), containing FTO and ALKBH5. 22,23Moreover, the regulatory function of m6A is required for the recognition and binding of m6A sites via m6A binding proteins ("readers"), like insulin-like growth factor-2 mRNA-binding protein 1/2/3 (IGF2BP1/2/3) and YTHDF1/2/3. 24,25It has been reported that m6A modifications are involved in regulating mRNA stability, degradation, and translation efficiency. 24Of note, the silencing of METTL3 might block breast cancer progression via decreasing PD-L1 mRNA stability in an m6A-IGF2BP3-dependent way. 26In addition, METTL3 has been reported to partake in BBR-mediated neuroprotection in ischemic stroke. 27In the present study, SRAMP software was used and it was discovered that FGF7 possessed the underlying m6A sites.Apart from that, FGF7 was identified as a possible target gene of METTL3.Accordingly, this study aimed to explore whether METTL3 might affect the antibreast cancer of BBR by modulating FGF7 expression.
Breast cancer and matched adjacent normal tissues were provided by 29 patients at People's Hospital of Dongxihu District.No patient had received chemotherapy or radiation therapy prior to surgery.Subsequently, these excised specimens during the surgery were quickly frozen in liquid nitrogen and stored at À80 C.This research was approved by the Ethics Committee of People's Hospital of Dongxihu District with written informed consent from each participant.
5-diphenyl-2H-tetrazolium bromide (MTT) assay
MCF-7 and MDA-MB-231 cell viability were evaluated in this experiment.After being treated with or without BBR for 48 h at 37 C, 20 μL of 5 mg/mL MTT reaction solution (Sigma-Aldrich) was added to 3 Â 10 4 tumor cells in 96-well plates.After 4 h, the precipitates generated were dissolved by the addition of 150 μL DMSO.Finally, the absorbance was measured based on a microplate reader.
Flow cytometry for cell apoptosis
After treatment with or without BBR for 48 h, collected breast cancer cells were washed with phosphate buffered saline (PBS) and resuspended in binding buffer.Subsequently, 5 μL annexin V-FITC and 10 μL PI (Solarbio) in the dark were used to stain the cells, which then were analyzed using the flow cytometer and FlowJo software version 10.5.0.
Matrigel invasion assay
In brief, 1 Â 10 5 breast cancer cells in 200 μL serum-free medium were introduced into the upper chamber (BD Biosciences) with coated Matrigel (BD Biosciences).Meanwhile, 600 μL medium with 10% FBS was added to the lower chambers.After incubation at 37 C for 24 h, cells invaded into the lower side were immobilized by 4% paraformaldehyde and stained with 0.1% crystal violet solution.Finally, the penetrated cells were counted using a microscope in five randomly selected fields.
Wound healing assay
The activity of tumor cell migration was measured in this experiment.Briefly, cells in 24-well plates were cultured until they reached approximately 90% confluency.After that, 200 mL sterile pipette tips were used to scratch across the surface of the cell monolayer (record 0 h).After removing the nonadherent cells, the tumor cells were cultured in serumfree medium for 24 h.Finally, wound closure was captured under a microscope and analyzed using Image J software.
Real-time quantitative polymerase chain reaction (RT-qPCR)
In general, total RNAs from clinical samples and cell lines were extracted using TRIzol reagent (Invitrogen).Then, 2 μg of total RNAs were utilized to synthesize cDNA based on HiScript 1st strand cDNA synthesis kit (Vazyme).On ABI7900HT fast real-time PCR system (Applied Biosystems), amplification reaction was conducted with a real-time fluorescent quantitative PCR kit (Vazyme).The PCR conditions were as follows: 30 s at 95 C, followed by 40 cycles at 95 C for 5 s, 45 C for 30 s, and 72 C for 30 s.After being normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), RNA expression fold changes were calculated by the 2 ÀΔΔCt method.The primers used are shown in Table 1.
Methylated RNA immunoprecipitation (MeRIP)
In order to examine m6A modification of FGF7 mRNA, total RNAs from MCF-7 and MDA-MB-231 cells transfected with si-NC, si-METTL3 or without were isolated using TRIzol (Invitrogen).After being fragmented, one-ninth of the mRNA was used as an "input" control.Meanwhile, the A/G immunomagnetic beads and anti-m6A (Abcam) or IgG (Abcam) were premixed and then incubated with the rest mRNA.After being digested, m6A enrichment was subjected to RT-qPCR analysis and input normalization.
RNA immunoprecipitation (RIP)
To further prove the binding ability between FGF7 mRNA and METTL3 or IGF2BP3, this assay was performed in MCF-7 and MDA-MB-231 cells transfected with si-NC or si-METTL3.In summary, complete RIP lysis buffer (Millipore) was used to obtain sufficient cell lysates, which were incubated with magnetic beads coupled with anti-IGF2BP3 (Abcam) or anti-IgG (Millipore) at 4 C overnight.
After being digested and purified, RNAs from the RNAprotein complex were determined using RT-qPCR assay.
Measurement of mRNA stability
In brief, MCF-7 cells with si-NC or si-METTL3 were harvested at 0, 3, 6, and 9 h after treatment with actinomycin D (act D, a transcriptional inhibitor, Sigma-Aldrich).After being extracted, the half-life of FGF7 mRNA was assessed using RT-qPCR.
Tumor xenograft assay
In this animal experiment, 100 μL MCF-7 cells (1 Â 10 6 cells) were subcutaneously injected into female nude mice (5-week-old, Slaike Jingda Laboratory, Hunan, China).When the tumors attained volumes of about 40 mm 3 , the mice were randomly assigned to two groups (n = 5 each group): (1) the control group, mice were treated with saline; (2) the BBR group, mice were treated with 10 mg/kg BBR by gavage administration.During tumor growth, size was examined and calculated every 3 days.Then, 22 days later, tumor weight was measured after mice were euthanized by cervical dislocation after deep anesthesia with 2% isoflurane (Baxter Healthcare Corporation).In addition, dissected tumors were subjected to western blot and immunohistochemical (IHC) staining analysis.All processes involving this experiment were authorized by the Animal Ethics Committee of People's Hospital of Dongxihu District.
Statistical analysis
Data were analyzed using GraphPad Prism7 software and expressed in the form of mean ± standard deviation (SD).The statistical significance of the difference was assessed by student's t-test or one-way analysis of variance (ANOVA) with Tukey's tests.Statistical significance was accepted for p < 0.05.
BBR repressed breast cancer cell proliferation and metastasis
To investigate the effect of BBR in breast cancer cells, MCF-7 and MDA-MB-231 cells were exposed to different concentrations of BBR (0, 20, 40, or 60 μM) for 48 h.After that, cell viability was assessed using MTT assay.Data exhibited that BBR treatment might decrease MCF-7 and MDA-MB-231 cell viability in a dose-dependent way (Figure 1a).Considering that no significant changes in suppression of tumor cell viability after 40 μM or 60 μM BBR treatment, we selected 40 μM for subsequent experiments.As shown in Figure 1b, BBR treatment might apparently block the number of EdU-positive cells in MCF-7 and MDA-MB-231 cells relative to the control group.On the contrary, an obvious enhancement in apoptosis rate was observed after BBR exposure in MCF-7 and MDA-MB-231 cells (Figure 1c).Furthermore, transwell results showed that the number of MCF-7 and MDA-MB-231 cell invasions was clearly hindered after BBR exposure (Figure 1d, e).Meanwhile, the wound healing assay also presented the inhibitory role of BBR treatment on MCF-7 and MDA-MB-231 cell migratory ability (Figure 1f).Collectively, these results suggested that BBR treatment might constrain breast cancer cell growth and metastasis.
FGF7 expression was reduced in BBR-treated breast cancer cells
Furthermore, the GSE85871 database microarray data set from the Gene Expression Omnibus (GEO) database was applied in an optimized analysis to identify differentially expressed genes in breast cancer treated with BBR.As a result, we found that FGF7 expression was significantly downregulated in the BBR group (Figure 2a), implying that FGF7 might partake in the BBR-mediated breast cancer process.Moreover, we further validated that the mRNA level and protein level of FGF7 were remarkably hindered in BBR-induced MCF-7 and MDA-MB-231 cells compared with the control group (Figure 2b, c).In addition, FGF7 mRNA level and protein level were highly expressed in 29 breast cancer samples versus 29 normal tissues (Figure 2d, e).Simultaneously, the significant downregulation of FGF7 was found in breast cancer cell lines (MCF-7 and MDA-MB-231) in comparison with MCF-10A cells (Figure 2f).Together, these data highlighted that dysregulated FGF7 might be associated with BBR-mediated breast cancer progression.
BBR treatment suppressed breast cancer cell malignant phenotypes by modulating FGF7
Then, to check the impact of FGF7 on BBR-mediated malignant phenotype inhibition of breast cancer cells, in vitro gain-function analysis was conducted in MCF-7 and MDA-MB-231 cells.At first, FGF7 protein level were markedly increased in pcDNA-FGF7-transfected cells compared with the pcDNA group (Figure 3a), suggesting that the overexpression efficiency is available.Under BBR treatment, MTT and EdU assays displayed that the overexpression of FGF7 suppressive role of BBR exposure on MCF-7 and MDA-MB-231 cell invasion and migration were evidently abrogated after pcDNA-FGF7 introduction (Figure 3f, g).In addition, our data also confirmed that upregulation of FGF7 might partly relieve the repression of BBR exposure on the growth and metastasis of BT-474 and BT-549 cells (Figure S1).Collectively, these findings implied that FGF7 overexpression partially attenuated the inhibitory role of BBR treatment on breast cancer cell growth and metastasis in vitro.
METTL3 might improve the stability and expression of FGF7 mRNA by regulating m6A methylation
Based on MeRIP analysis, we found that FGF7 contained abundant m6A methylation (Figure 4a).Subsequently, SRAMP (an online tool to predict m6A sites) database discovered that FGF7 mRNA possessed the potential m6A sites (Figure 4b).In addition, the knockdown efficiency of METTL3 in MCF-7 and MDA-MB-231 cells was detected and shown in Figure 4c.After that, RT-qPCR results showed that the FGF7 mRNA level was apparently blocked by METTL3 knockdown in MCF-7 and MDA-MB-231 cells (Figure 4d).Moreover, MeRIP-qPCR analysis presented that the downregulation of METTL3 might apparently decrease the m6A level of FGF7 mRNA in MCF-7 and MDA-MB-231 cells (Figure 4e).To further validate their interaction, RIP assay was carried out.Data showed that FGF7 RNA enrichment was significantly reduced in the si-METTL3 group relative to the si-NC group (Figure 4f).In addition, the actinomycin D experiment showed that METTL3 knockdown might enhance the degradation rate of mRNA in MCF-7 cells (Figure 4g).Beyond that, western blot assay presented that METTL3 content remarkably declined in BBR-treated MCF-7 and MDA-MB-231 cells compared with the control group (Figure 4h).Collectively, these results indicated that METTL3 might sustain FGF7 mRNA stability through m6A modification.
IGF2BP3 recognized the m6A modification of FGF7 mRNA and enhanced its expression Previous studies have shown that the effects of m6A modifications on mRNA transcripts require that these modifications be recognized by m6A binding proteins called m6A readers.Therefore, we knocked down IGF2BP1, IGF2BP2, and IGF2BP3 in MCF-7 cells.Among these reader proteins, our data displayed that the downregulation of IGF2BP3 might obviously decrease FGF7 mRNA levels in breast cancer cells (Figure 5a).Beyond that, the GSE85871 database determined that BBR treatment might remarkably decrease the IGF2BP3 mRNA level (Figure 5b).Moreover, we further validated that IGF2BP3 protein level was clearly repressed in BBR-treated MCF-7 and MDA-MB-231 cells versus the control group (Figure 5c).Apart from that, FGF7 RNA enrichment was significantly increased in the IGF2BP3 group compared with the IgG group when METTL3 knockdown in MCF-7 cells (Figure 5d), suggesting that silencing METTL3 inhibited the binding of FGF7 RNA to IGF2BP3.In addition, the overexpression efficiency of IGF2BP3 in MCF-7 and MDA-MB-231 cells was measured and displayed in Figure 5e.Then, western blot assay presented that the cotransfection of pcDNA-IGF2BP3 might significantly abolish the repression of METTL3 deficiency on FGF7 protein level in MCF-7 and MDA-MB-231 cells (Figure 5f).Overall, these results suggested that METTL3 might increase FGF7 expression by regulating the RNA m6A modification, which might be recognized by IGF2BP3 and is essential for mRNA stability.
Knockdown of METTL3 might block breast cancer cell malignant phenotypes by targeting FGF7
Next, to further explore whether the influence of METTL3 on breast cancer cell development was mediated by regulating FGF7, rescue experiments were performed in MCF-7 and MDA-MB-231 cells.Western blot results exhibited that the lack of METTL3 might strikingly dampen FGF7 protein level in MCF-7 and MDA-MB-231 cells, which were partly overturned after pcDNA-FGF7 co-transfection (Figure 6a).Functionally, reduced cell viability and proliferation were observed due to the downregulation of METTL3, whereas these effects were ameliorated by FGF7 overexpression (Figure 6b, c).Beyond that, METTL3 silencing-mediated apoptosis rate promotion was partly mitigated through the upregulation of FGF7 in MCF-7 and MDA-MB-231 cells (Figure 6d, e).In addition, the forced expression of FGF7 might markedly abrogate the repression of METTL3
BBR treatment might inhibit FGF7 expression by regulating METTL3
As previously mentioned, we speculated that BBR treatment might repress breast cancer progression partly by the METTL3/FGF7 regulation pathway.To prove the assumption, we further investigated whether BBR exposure might affect the expression of FGF7 by METTL3.First, western blot results exhibited that METTL3 protein level was clearly improved in pcDNA-METTL3-transfected MCF-7 and MDA-MB-231 cells compared with the pcDNA group (Figure 7a), indicating that the overexpression efficiency is successful.After that, BBR treatment might significantly dwindle FGF7 protein level in MCF-7 and MDA-MB-231 cells, while these effects were partly abolished by METTL3 overexpression (Figure 7b, c).Collectively, these findings implied that BBR might exert its function by regulating the METTL3/FGF7 pathway.
BBR might hinder breast cancer cell growth in vivo
Additionally, mice xenograft models of breast cancer were established to explore the influence of BBR treatment on tumor growth.As shown in Figure 8a, b, BBR treatment significantly blocked tumor volume and weight, implying the repression of BBR exposure on tumor growth.Beyond that, western blot results presented that METTL3 and FGF7 protein levels were obviously lower in tumor tissues from the BBR group than in the control group (Figure 8c).In addition, IHC staining also uncovered that the positive expression rate of METTL3 and FGF7 was apparently decreased in the BBR group compared with the control group (Figure 8d).Collectively, these data illuminated that BBR treatment diminished breast cancer xenograft tumor growth in vivo.
DISCUSSION
In recent years, natural herbal extracts from TCM have attracted extensive attention in the field of tumor research for their distinctive benefits of multitargeting and minimal side impacts. 28,29As a natural bioactive alkaloid mainly found in the Chinese herb Coptis chinensis, BBR possesses a broad range of pharmacological effects, containing antiapoptosis, antioxidative, antimicrobial, and anticancer activities. 9,30Lately, it has become evident that BBR displays anticancer effects on various cancers, including breast cancer. 31Herein, our data verified that BBR treatment might suppress breast cancer cell proliferation, invasion, migration, and induce apoptosis in vitro, in agreement with previous studies. 32,33Consistently, the repression of BBR exposure on breast cancer growth has been validated in vivo.In total, the above findings demonstrated the anticancer activity of BBR in breast cancer.
Regarding the molecular mechanism, numerous studies have validated the antitumor effects of BBR occur in breast cancer via different biochemical mechanisms, containing regulating gene expression and signal pathways. 13,34,35erein, we used the GSE85871 database to screen FGF7, which clearly differs in BBR-treated breast cancer.Beyond that, our data further verified that FGF7 content was obviously reduced in BBR-triggered breast cancer cells.Of interest, FGF7 level was identified to be upregulated in breast cancer, consistent with a former study. 15As a mesenchymespecific heparin-binding growth factor, FGF7 was reported to bind FGF receptor 2 to affect various cellular processes. 36,37It has been confirmed that the knockdown of FGF7 might hinder breast cancer cell growth and metastasis. 16,17Therefore, we inferred that FGF7 might be implicated in BBR-mediated antitumor activity.In the present study, our data confirmed that BBR-triggered breast cancer growth and metastasis inhibition were partly overturned by FGF7 upregulation.These observations provide first-hand evidence of the antitumor effects of BBR through the regulation of FGF7.
As a novel layer of epigenetic modulation, m6A is considered an abundant cotranscriptional modification in mRNA and participates in diverse aspects of posttranscriptional mRNA metabolism. 38,39Herein, our data identify for the first time an important role for METTL3 in mediating the m6A modification of FGF7 mRNA.Moreover, it has been confirmed that IGF2BPs might affect target mRNA expression by regulating the stability of target mRNA. 40Meanwhile, IGF2BP3 might facilitate breast cancer cell proliferation through destabilizing NF1 mRNA via an m6A-dependent way. 41In the current study, this modification is recognized by IGF2BP3, which is responsible for maintaining the mRNA stability and expression of FGF7.In addition, METTL3 might increase PD-L1 mRNA stability and its protein expression in an m6A-IGF2BP3-dependent manner, thereby repressing tumor immune surveillance in breast cancer. 26Herein, our study showed that FGF7 overexpression might partially abolish the repression of METTL3 deficiency in breast cancer development.Some studies have indicated that METTL3 might be implicated in BBR-or allocryptopine (an isoquinoline alkaloid extracted from Macleaya cordata)-mediated antiischemia-reperfusion injury or antitumor activity. 27,42As expected, our data verified that METTL3 upregulation might partly abrogate BBR-mediated FGF7 reduction in breast cancer cells, further supporting the regulatory mechanism of BBR/METTL3/FGF7 in breast cancer.In addition, previous studies have indicated that aberrant activation of the Wnt/βcatenin signaling pathway is one of the main reasons for breast tumorigenesis and metastasis. 43,44Meanwhile, it has been reported that BBR might suppress tumor growth in various human cancers by repressing Wnt/β-catenin signaling, 45,46 containing breast cancer. 47 FGF7 might induce the accumulation of cytoplasmic β-catenin. 48Therefore, we will explore whether BBR treatment impedes the growth and metastasis of breast cancer cells through the METTL3/FGF7-mediated Wnt/β-catenin signaling pathway in further studies.In addition, the subcutaneous xenograft model used in this study may not fully mimic the natural breast cancer microenvironment.Hence, some of our findings may not be reproducible in the natural disease state, and the patient-derived xenograft (PDX) model needs to be performed in future studies.
In conclusion, our results provided compelling evidence that BBR might hinder breast cancer cell growth and metastasis by regulating the METTL3/FGF7 axis, which provides novel strategies for the design of new METTL3/FGF7-based antitumor agents and drug combinations.
might effectively abolish BBR-mediated cell viability and proliferation inhibition in MCF-7 and MDA-MB-231 cells (Figure3b, c).Furthermore, BBR-induced MCF-7 and MDA-MB-231 cell apoptosis rates were distinctly relieved through FGF7 upregulation (Figure3d, e).In addition, transwell and wound healing assays showed that the F I G U R E 1 Effects of berberine (BBR) on breast cancer cell proliferation and metastasis.(a) A 5-diphenyl-2H-tetrazolium bromide (MTT) assay was applied to measure cell viability in MCF-7 and MDA-MB-231 cells exposed to different concentrations of BBR (0, 20, 40, and 60 μM).(b-f) MCF-7 and MDA-MB-231 cells were treated with BBR (40 μM) or without (control).(b) A 5-ethynyl-2 0 -deoxyuridine (EdU) assay was used to assess cell proliferation in treated MCF-7 and MDA-MB-231 cells.(c) The cell apoptosis rate was examined using flow cytometry assay in treated MCF-7 and MDA-MB-231 cells.(d and e) A transwell assay was performed to measure cell invasion in treated MCF-7 and MDA-MB-231 cells.(f) Migration ability was detected using wound healing assay in treated MCF-7 and MDA-MB-231 cells.*p < 0.05, **p < 0.01, ***p < 0.001.F I G U R E 2 Berberine (BBR) might decrease the levels of fibroblast growth factor 7 (FGF7) in breast cancer cells.(a) The GSE85871 database was used to analyze the expression level of FGF7 in the control and BBR groups.(b and c) FGF7 mRNA level and protein level were determined in MCF-7 and MDA-MB-231 cells treated with 40 μM BBR or control using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot.(d and e) RT-qPCR and western blot analysis of FGF7 mRNA level and protein level in 29 breast cancer tissues and 29 normal tissues.(f) FGF7 protein level was assessed in MCF-10A cells and breast cancer cell lines (MCF-7 and MDA-MB-231).*p < 0.05, ***p < 0.001.
F I G U R E 3
Overexpression of fibroblast growth factor 7 (FGF7) might overturn the effects of berberine (BBR) on breast cancer cell malignant phenotypes.(a) FGF7 protein level was determined in MCF-7 and MDA-MB-231 cells transfected with pcDNA or FGF7 using western blot.(b-g) MCF-7 and MDA-MB-231 cells were treated with control, BBR, BBR + pcDNA, and BBR + FGF7.(b) Cell viability was assessed using a 5-diphenyl-2H-tetrazolium bromide (MTT) assay in treated MCF-7 and MDA-MB-231 cells.(c) A 5-ethynyl-2 0 -deoxyuridine (EdU) assay was used to measure cell proliferation in treated MCF-7 and MDA-MB-231 cells.(d and e) Flow cytometry assay was applied to analyze cell apoptosis rate in treated MCF-7 and MDA-MB-231 cells.(f and g) Transwell and wound healing assays were employed to examine cell invasion and migration in treated MCF-7 and MDA-MB-231 cells.**p < 0.01, ***p < 0.001.
F I G U R E 4
Fibroblast growth factor 7 (FGF7) was regulated by methyltransferase-like 3 (METTL3)-mediated m6A methylation.(a) Methylated RNA immunoprecipitation (MeRIP) assay was used to assess the m6A methylation level of FGF7 in MCF-7 and MDA-MB-231 cells.(b) SRAMP software predicted that FGF7 mRNA had an m6A site.(c and d) METTL3 protein level and FGF7 mRNA level were examined using MCF-7 and MDA-MB-231 cells transfected with si-NC or si-METTL3 using real-time quantitative polymerase chain reaction (RT-qPCR) or western blot.(e) Changes in the m6A methylation level of FGF7 after METTL3 knockdown were assessed using MeRIP-qPCR assay.(f) Their binding was verified using RNA immunoprecipitation (RIP) assay in MCF-7 and MDA-MB-231 cells transfected with si-NC or si-METTL3.(g) Influences of METTL3 downregulation on FGF7 mRNA stability after actinomycin D treatment was assessed using RT-qPCR in MCF-7 cells.(h) FGF7 protein levels were examined in MCF-7 and MDA-MB-231 cells treated with berberine (BBR) or control using western blot.**p < 0.01, ***p < 0.001.knockdown on MCF-7 and MDA-MB-231 cell invasion and migration (Figure 6f, g).Altogether, the above-mentioned results indicated that the METTL3 downregulation might impede breast cancer cell malignancy by modulating FGF7 in vitro.
Furthermore, F I G U R E 8 Berberine (BBR) blocked tumor growth in a xenograft model.MCF-7 cells were inoculated subcutaneously into the nude mice.Then, mice with tumor formation were given 10 mg/kg BBR.(a) Growth curve of xenografted tumors was presented.(b) Weight of resected tumor masses was measured.(c) Methyltransferase-like 3 (METTL3) and fibroblast growth factor 7 (FGF7) protein levels were assessed using western blot in the xenografts.(d) The positive expression rate of METTL3 and FGF7 was gauged in xenografts using immunohistochemical (IHC) staining.*p < 0.05, **p < 0.01, ***p < 0.001. | 2024-05-08T06:17:05.095Z | 2024-05-06T00:00:00.000 | {
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54840456 | pes2o/s2orc | v3-fos-license | TLC of selected sesquiterpenoids of the Asteraceae family
Visual chromatography has been employed for a preliminary identification of natural compound of a group of sesquiterpene lactones – Centaurea bella Trautv., C. crocodylium L., C. lusitanica Boiss. et Reuter, Helenium hoopesii = hoopesii (A. Gray) Rydb., Stizolophus balsamita (Lam.) Cass., baldschuanica – Santolina pinnata subsp. neapolitana (Jord. et Fourr.) Guinea = S. neapolitana Jord. et Fourr. A dependence of the colour of the spots, induced anisaldehyde reagent, on the presence of several substituents in the germacranolide ring – derivatives of partnenolide and salonitenolide – has been identified. The structures of the skeleton in the ring of sesquiterpene lactones in Helenium hoopesii can also be established by thin layer chromatography (TLC). The analysis of the chromatograms of extracts from dry and fresh Centaurea crocodylium herb has shown significant differences as for the chemical composition. Two sorts of germacranes of Santolina pinnata subsp. neapolitana display a characteristic colour of the spots.
Introduction
Among natural compounds in the species of family Asteraceae, sesquiterpenoids play an important role.They constitute a valuable chemotaxonomic material and might be also decisive for the medicinal value of the raw material, as in Chamomillae anthodium, Cnici benedicti herba, Arnicae anthodium, Millefolii herba, Chrysanthemi partheni herba, Cichorii radix et herba.A recent study has revealed antibacterial and cytotoxic activities of sesquiterpene lactones [1].These compounds can be detected through visual chromatography and isolated by simple column chromatography, with silica gel as the adsorbent.
The possibility of identification of several substituents at guaianolides by TLC with concentrated sulphuric acid as the developer had been mentioned earlier [2,3].Now, visual chromatography of compounds of four other types of sesquiterpenoids has been presented: germacranolides, germacranes, seco-pseudoguaianolides and pseudoguaianolides.
To develop the spots, the sole anisaldehyde reagent, which proved selective enough to rate the colours of the spots of the compounds in question, was used.
Plant material
The plant materials used for the studies employed aerial parts of Centaurea bella, C. crocodylium, C. lusitanica, Helenium hoopesii, Santolina pinnata subsp.neapolitana, Stizolophus balsamita, Zoegea baldschuanica, plants cultivated and identified in the garden of Department of Medicinal and Cosmetic Natural Products, University of Medical Sciences in Poznań (Poland), where their voucher specimens are deposited.
Isolation and identification of compounds
The herbs, collected right before blooming (about 450 g each) were extracted with methanol three times.The methanol extracts were inundated with distilled water (ca.600 cm 3 ), after the evaporation of the solvent.The water phase was extracted with chloroform.The chloroform extracts, in turn, having been dried with anhydrous sodium sulphate, were used for thin layer chromatography and isolation of compounds.Furthermore, some methanol/water/chloroform extracts were prepared from Centaurea crocodylium herb, which had been collected at the same time and from the same plot as the herbs designed to be dried.
The extracts were separated by column chromatography on silica gel (Merck art.7733).The fractions were subjected to repeated column chromatography on silica gel (Merck art.7729) and eluted by right mobile phases.The structures of the isolated compounds (Fig. 1) were identified on the basis of 1H NMR, IR and EI mass spectroscopy and by comparing the obtained data with those of the reference compounds or reported data [4][5][6][7][8].
TLC analysis
Thin-layer chromatrography was performed at room temperature on aluminium-backed silica gel plates DC Alufolien Kieselgel 60 (Merck art.5553).15-20 µg of each isolated compounds were applied per plate.Developed and dried chromatograms were sprayed by anisaldehyde reagent and heated at 100 o C by 3 minutes.Spots of isolated compounds exhibited, mauve, violet, red-brown, black-blue, orange, yellow, cherryred and gray colours when examined 3-15 minutes from the time of spraying (Tab.1).
Biosynthesis of cnicin during the drying of Centaurea crocodylium herb
While an attempt was being made to obtain compounds of C. crocodylium by column chromatography [silica gel, CH 2 Cl 2 -CO(CH 3 ) 2 8:1], from the extract from the fresh herb on large quantity (665 mg), salonitenolide (Fig. 1e) was isolated.Another compound, also in large quantity (845 mg), was obtained [silica gel, CH 2 Cl 2 -CO(CH 3 ) 2 4:1] from the dry herb of the plant.To our astonishment, rather than salonitenolide (Fig. 1e), it turned out to be its ester derivative: cnicin (Fig. 1f).It can be stated, thus, that at the time of drying, the ester had been connected to the hydroxyl group at C8 of e on Fig. 1.It is also worth noting that with the methods used so far in this work, the attempts to isolate either cnicin (Fig. 1f) from the fresh herb, or salonitenolide (Fig. 1e) from the dried herb have been unsuccessful.
Germacranes
Two out of the five germacranes of Santolina neapolitana [7], with the 4,5 epoxide and a substituent at C2 (Fig. 1i) exhibit dark-blue colour of their spots on the chromatograms (Fig. 3a,c).The spots of compounds j, k, l on Fig. 1 have a different, namely mauve, colour (Fig. 3b,d,e).They lack of the 4,5-epoxide ring and are characterised by the presence of the OH group at C5 and the =CH 2 substituent at C4.The 1,10 epoxide of l on Fig. 1 has no influence on the colour of the spot.
Seco-pseudoguaianolides
Another type of sesquiterpene lactones occurs in the herb of Helenium hoopesii [8], with seco-pseudoguaianolides (Fig. 1m-p) as the dominant feature.Hymenovin -mixture of C3 and/or C4 diastereoisomers (Fig. 1m) and hymenoratin B (Fig. 1n) with pyran skeleton with at least one OH group at C2 or C3 and C4 exhibited orange colour on the chromatograms.Hymenoratin B 2-O-β-D-(6'-O-acetyl)-glucopyranoside (Fig. 1o), on the other hand, with a pyran ring and blocked hydroxyl groups at C2 and C4, changes its colour to yellow on the chromatograms, while floribundin (Fig. 1p), without a substituent at these places, is invisible in the anisaldehyde reagent (but fast crystallizes as white, spectrally clear needles).This compound was presented in this paper for the first time as one of compounds isolated from H. hoopesii.
Guaianolides and pseudoguaianolides
The other compounds of this plant: guaianolides (Fig. 1r,s) and pseudoguaianolides (Fig. 1t,u) with a five-part ring exhibited cherry-red colour.The fading of the cherry-red colour can be observed in acetylhymenograndin (Fig. 1v).Three acetyl groups in this compound are responsible for the grey colour of the spot.
Conclusions
Sesquiterpenoids constitute an important group of natural compounds occurring in species of family Asteraceae.By means of the simple TLC method, it is possible to estimate several details of the structure within guaianolides, germacranolides, pseudoguaianolides, seco-pseudoguaianolides and germacranes.
The simple thin layer chromatography may surely rank among methods of comparative analysis of natural compounds.It is often easier to differentiate and classify species based on TLC than through the traditional botanical analysis.One might assume that with high probability.Zoegea species will biosynthesize "violet" germacranolides with substituent at C9, and that Stizolophus species will biosynthesize "mauve" germacranolides with substituents at C8, and some Centaurea species will biosynthesize "brown-red" ones, with the characteristic CH 2 OH group at C4. | 2018-12-05T20:57:22.385Z | 2011-07-25T00:00:00.000 | {
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236320583 | pes2o/s2orc | v3-fos-license | Antiproliferative and Antimicrobial Potentials of a Lectin from Aplysia kurodai (Sea Hare) Eggs
In recent years, there has been considerable interest in lectins from marine invertebrates. In this study, the biological activities of a lectin protein isolated from the eggs of Sea hare (Aplysia kurodai) were evaluated. The 40 kDa Aplysia kurodai egg lectin (or AKL-40) binds to D-galacturonic acid and D-galactose sugars similar to previously purified isotypes with various molecular weights (32/30 and 16 kDa). The N-terminal sequence of AKL-40 was similar to other sea hare egg lectins. The lectin was shown to be moderately toxic to brine shrimp nauplii, with an LC50 value of 63.63 µg/mL. It agglutinated Ehrlich ascites carcinoma cells and reduced their growth, up to 58.3% in vivo when injected into Swiss albino mice at a rate of 2 mg/kg/day. The morphology of these cells apparently changed due to AKL-40, while the expression of apoptosis-related genes (p53, Bax, and Bcl-XL) suggested a possible apoptotic pathway of cell death. AKL-40 also inhibited the growth of human erythroleukemia cells, probably via activating the MAPK/ERK pathway, but did not affect human B-lymphoma cells (Raji) or rat basophilic leukemia cells (RBL-1). In vitro, lectin suppressed the growth of Ehrlich ascites carcinoma and U937 cells by 37.9% and 31.8%, respectively. Along with strong antifungal activity against Talaromyces verruculosus, AKL showed antibacterial activity against Staphylococcus aureus, Shigella sonnei, and Bacillus cereus whereas the growth of Escherichia coli was not affected by the lectin. This study explores the antiproliferative and antimicrobial potentials of AKL as well as its involvement in embryo defense of sea hare.
Introduction
Lectins are carbohydrate-binding proteins, omnipresent in almost all life forms, which specially recognize carbohydrate structures of glycoproteins and glycolipids present on the cell surface [1]. Marine invertebrates possess diverse classes of lectins with various protein foldings and different carbohydrate-binding specificities [1][2][3][4][5]. Many of these lectins show affinity towards galactose-related carbohydrates and perform biological functions including antitumor and antimicrobial activities.
Endogenous lectins interact with cell-surface glycans to mediate numerous biological functions in cells [6]. Compared to normal cells, glycosylation pattern of cancer cells are altered, which affects multiple cellular mechanisms performed by lectins through their association with corresponding glycans [7]. As a result, this alteration supports their neoplastic progression [8,9]. For example, galactose-binding lectins or galectins aid the binding of tumor cells through the interaction with galactose-containing carbohydrate ligands on tumor cells [9]. Many marine invertebrate lectins have been reported to bind with glycans on the tumor cell surface and killed those inducing apoptosis [3,[10][11][12]. On the other hand, lectins can also modulate the entry and subcellular targeting of drugs into cancer cells [13,14]. Both these phenomena exhibit considerable influence of lectin-glycan interaction on the proliferation and regulation of tumor cells [5,[10][11][12]14].
As a component of innate immunity, lectins present in marine invertebrates agglutinate both Gram-negative and Gram-positive bacteria through the interaction with lipopolysaccharides and peptidoglycans on their cell walls [4,5,15,16]. Galactose residues present as terminal sugars in lipopolysaccharides (LPS) of bacteria impact the intracellular composition of bacteria and maintain the synthesis of UDP-galactose for LPS [17,18]. Bacterial endotoxins also possess galactose as a constituent [19]. On the contrary, antifungal activity of lectins is not well reported yet.
Sea hares are marine gastropod mollusks found in common coastal areas. Various lectins have been reported to be present in eggs and gonads of sea hares due to their possible roles in early development and body defense. D-galacturonic acid and D-galactosebinding lectins with molecular masses 16-34 kDa have been obtained from the eggs of Aplysia kurodai [11,[20][21][22] and other Aplysia species (A. depilans, A. dactylomela, and A. californica) [23][24][25]. Primary structures of all these previously purified egg lectins possess a novel triple tandem repeating sequence consisting of 210-230 amino acids and show striking similarities to domain DUF3011 of some uncharacterized bacterial proteins [26].
These Aplysia egg lectins showed cytotoxic activities against certain tumor cells and were involved with organogenesis in early developmental stages. They might have played protective roles in marine organisms as well. Hasan et al. reported an α-galactose-binding 40 kDa lectin from eggs of AKL in 2014 that inhibited streptolysin O-induced hemolysis and growth of Streptococcus pyogenes [22].
The 40 kDa lectin from Aplysia kurodai eggs will be designated here as AKL-40, to differentiate it from other AKLs. We characterized this egg lectin and determined its N-terminal sequence. Previously, in vitro antiproliferative activity of two AKLs was reported [11,21]. In this study, antiproliferative activities of this particular lectin were determined for the first time in vivo, using Swiss albino mice. In addition, other biological properties, such as in vitro anticancer activity against different cancer cell lines and antibacterial potential of the lectin, was further evaluated along with its antifungal activity.
Purification, Confirmation of the Molecular Mass and Hemagglutination Activity of Aplysia kurodai Egg Lectin (AKL-40)
Affinity chromatography showed different molecular masses of AKLs found in the eggs of Aplysia kurodai (Figure 1). After applying the crude protein sample, the melibosylagarose affinity chromatography column was washed with TBS and bound lectins were eluted with 10 mM D-galacturonic acid or D-galactose-containing TBS. Each polypeptide was separated with gel filtration chromatography by using Sephacryl S-200HR, (Cytiva, Marlborough, MA, USA) (data not shown). In the reducing condition, polypeptides with molecular weights of 40 kDa, 32/30 kDa, and 16 kDa were denoted as AKL-40, AKL, and AKL-2, respectively, in this study ( Figure 1: white triangle: AKL-40; black triangles: AKL and AKL-2). The minimum concentration of AKL-40 to agglutinate human and mice erythrocytes was found to be 24 and 32 µg/mL, respectively. and AKL-2). The minimum concentration of AKL-40 to agglutinate human and mice erythrocytes was found to be 24 and 32 µg/mL, respectively. (14 kDa). All samples are separated in the reducing condition and 15% polyacrylamide gels are used except AKL-2, which was separated by a 12% gel.
N-Terminal Amino Acid Sequence of AKL-40
The N-terminal region of AKL-40, including the first 35 amino acids of the polypeptide, was determined by Edman degradation with a repetitive yield of 84.19% (Supplementary Figure S1). This partial N-terminal sequence of AKL-40 fitted with those of AKLa to -d and ADEL, showing similarities ( Figure 2). Unlike others, AKL-40 possessed a forward extension comprising four additional amino acids. Thirty-five amino acids of the N-terminal sequence of AKL-40 were indicated by the singleletter amino acid code. Identical amino acids in the N-terminal sequences of 32-30 kDa Aplysia egg lectins isolated as AKL-a to -d [25] and ADEL [23] were shown as bold letters. X: unidentified, Gray box: skipped.
Toxicity of AKL-40 against Brine Shrimp Artemia nauplii
A dose-dependent graph showed that 70% of shrimp nauplii died at a concentration of 160 µg/mL of AKL-40 ( Figure 3). The LC50 value was determined to be 63.63 µg/mL indicating AKL-40 as a moderately toxic protein. (14 kDa). All samples are separated in the reducing condition and 15% polyacrylamide gels are used except AKL-2, which was separated by a 12% gel.
N-Terminal Amino Acid Sequence of AKL-40
The N-terminal region of AKL-40, including the first 35 amino acids of the polypeptide, was determined by Edman degradation with a repetitive yield of 84.19% (Supplementary Figure S1). This partial N-terminal sequence of AKL-40 fitted with those of AKL-a to -d and ADEL, showing similarities ( Figure 2). Unlike others, AKL-40 possessed a forward extension comprising four additional amino acids. and AKL-2). The minimum concentration of AKL-40 to agglutinate human and mice erythrocytes was found to be 24 and 32 µg/mL, respectively. (14 kDa). All samples are separated in the reducing condition and 15% polyacrylamide gels are used except AKL-2, which was separated by a 12% gel.
N-Terminal Amino Acid Sequence of AKL-40
The N-terminal region of AKL-40, including the first 35 amino acids of the polypeptide, was determined by Edman degradation with a repetitive yield of 84.19% (Supplementary Figure S1). This partial N-terminal sequence of AKL-40 fitted with those of AKLa to -d and ADEL, showing similarities ( Figure 2). Unlike others, AKL-40 possessed a forward extension comprising four additional amino acids. [25] and ADEL [23] were shown as bold letters. X: unidentified, Gray box: skipped.
Toxicity of AKL-40 against Brine Shrimp Artemia nauplii
A dose-dependent graph showed that 70% of shrimp nauplii died at a concentration of 160 µg/mL of AKL-40 ( Figure 3). The LC50 value was determined to be 63.63 µg/mL indicating AKL-40 as a moderately toxic protein. Thirty-five amino acids of the N-terminal sequence of AKL-40 were indicated by the single-letter amino acid code. Identical amino acids in the N-terminal sequences of 32-30 kDa Aplysia egg lectins isolated as AKL-a to -d [25] and ADEL [23] were shown as bold letters. X: unidentified, Gray box: skipped.
Toxicity of AKL-40 against Brine Shrimp Artemia nauplii
A dose-dependent graph showed that 70% of shrimp nauplii died at a concentration of 160 µg/mL of AKL-40 ( Figure 3). The LC 50 value was determined to be 63.63 µg/mL indicating AKL-40 as a moderately toxic protein.
In Vivo Anticancer Activity of AKL-40 against Ehrlich Ascites Carcinoma Cells, Their Morphological Changes, and Expression of Apoptosis-Related Genes
After administering 1 mg/kg/day and 2 mg/kg/day of AKL-40 for five days, growth of EAC-cells in the lectin-treated mice (Groups B and C) was inhibited to 28.7% and 58.3%, compared to the untreated (or control) mice from group A ( Figure 4A). Weight of the treated mice reduced significantly compared to the untreated mice (data not shown). EAC cells from untreated mice were spherical and regular-sized ( Figure 4B, column 0). Irregular-shaped kidney-bean-like EAC cells from the treated mice groups ( Figure 4B, column 1 and 2). Upregulation of p53 gene expression was observed in EAC cells from group C, though it was a weak one. Expression of Bax gene was there in EAC cells from lectin-treated mice whereas no expression was found in EAC cells from untreated mice. Cells from AKL-40-treated mice showed no expression of the Bcl-X L gene whereas, in EAC cells from control mice, it was visible. Expression of GAPDH gene in EAC cells from both untreated and lectin-treated mice confirmed the quality of mRNA isolated ( Figure 4C). The results were statistically significant (* p < 0.05, when mortality percentage of lectin-treated shrimps were compared to untreated shrimps).
In Vivo Anticancer Activity of AKL-40 against Ehrlich Ascites Carcinoma Cells, Their Morphological Changes, and Expression of Apoptosis-Related Genes
After administering 1 mg/kg/day and 2 mg/kg/day of AKL-40 for five days, growth of EAC-cells in the lectin-treated mice (Groups B and C) was inhibited to 28.7% and 58.3%, compared to the untreated (or control) mice from group A ( Figure 4A). Weight of the treated mice reduced significantly compared to the untreated mice (data not shown). EAC cells from untreated mice were spherical and regular-sized ( Figure 4B, column 0). Irregular-shaped kidney-bean-like EAC cells from the treated mice groups ( Figure 4B, column 1 and 2). Upregulation of p53 gene expression was observed in EAC cells from group C, though it was a weak one. Expression of Bax gene was there in EAC cells from lectintreated mice whereas no expression was found in EAC cells from untreated mice. Cells from AKL-40-treated mice showed no expression of the Bcl-XL gene whereas, in EAC cells from control mice, it was visible. Expression of GAPDH gene in EAC cells from both untreated and lectin-treated mice confirmed the quality of mRNA isolated ( Figure 4C).
In Vitro Anticancer Activity of AKL-40 against Different Cancer Cells and Activation of Signal Transduction Molecules
AKL-40 significantly inhibited the growth of K562 cells at a concentration of 100 µ g/mL or more ( Figure 5A, orange bar). On the other hand, the lectin could not influence the growth of human B-lymphoma cells (Raji) and rat basophilic leukemia cells (RBL-1) ( Figure 5A, blue and gray bars). During a period of 24 h, extracellular signal-regulated kinase (ERK)1/2 and p38kinase molecules became phosphorylated in AKL-40 treated K562 cells, as shown by western blotting ( Figure 5B).
Minimum agglutination concentrations of AKL-40 for EAC and U937 cells were 20 and 24 µ g/mL, respectively. The dose-dependent effect of AKL-40 against U937 and EAC cells was observed by MTT assay. At lower concentrations (100 µ g/mL), growth inhibition of both cell types was similar, but at higher concentrations, AKL-40 showed slightly higher activity against EAC cells. At the concentration of 250 µ g/mL, the percentage of . Growth inhibition in AKL-40 treated mice compared to the untreated mice. The lectin was administrated to mice at doses 0 (untreated), 1 and 2 mg/kg/day (treated). Data are expressed in mean ± SD (n = 6). The results were statistically significant (* p < 0.05, when cells from lectin-treated mice were compared to cells from untreated mice). (B). Change of morphology in Hoechst-stained EAC cells harvested from AKL-40 treated mice. EAC cells from untreated and treated (with 1 mg/kg/day and 2 mg/kg/day of AKL-40, for five days) mice were observed by a fluorescence microscope. White arrows show changes in the morphology of cells. Scale bar: 25 µm. (C). Expression of apoptosis-related genes (Bcl-X L , p53 and Bax) and GAPDH. L, 1000 bp DNA ladder; T, RNA of EAC cells from AKL-40 treated mice; C, RNA of EAC cells from untreated (control) mice.
In Vitro Anticancer Activity of AKL-40 against Different Cancer Cells and Activation of Signal Transduction Molecules
AKL-40 significantly inhibited the growth of K562 cells at a concentration of 100 µg/mL or more ( Figure 5A, orange bar). On the other hand, the lectin could not influence the growth of human B-lymphoma cells (Raji) and rat basophilic leukemia cells (RBL-1) ( Figure 5A, blue and gray bars). During a period of 24 h, extracellular signal-regulated kinase (ERK) 1/2 and p38kinase molecules became phosphorylated in AKL-40 treated K562 cells, as shown by western blotting ( Figure 5B). . Anticancer activity of AKL-40 against EAC and U937 cells. After treating with 100-250 µg/mL of AKL-40, percentages of growth inhibition were determined by MTT assay (n = 3, mean ± SD). Gray and black bars indicate EAC and U937 cells, respectively. The results were statistically significant (* p < 0.05, when lectintreated cells were compared to untreated cells). The results were statistically significant (** p < 0.01 and * p < 0.05, when lectin-treated cells were compared to untreated cells).
Antimicrobial Activity of AKL-40
AKL-40 showed antibacterial activity against Staphylococcus aureus, Bacillus cereus, and Shigella sonnei. No zone of inhibition was there for Escherichia coli. Slightly larger zones were observed around the disks soaked with higher doses (100 µg/disc) of AKL-40 in case of every susceptible bacterial species ( Figure 6A). Maximum activity of AKL-40 was found against Staphylococcus aureus in terms of the inhibition zones formed. Growth of Talaromyces verruculosus was also very efficiently inhibited by AKL-40. The fungi rapidly grew and after two weeks, nearly covered the whole petri dish. However, when discs soaked with AKL-40 were placed in the fungal media, almost no growth of the mold was observed ( Figure 6B). . Activation of MAPK pathway in AKL-40 treated K562 cells was observed during a period of 24 h. Phosphorylation of P38 was observed from a 3 to 12 h treatment and it diminished at 24 h (surrounded in red). Phosphorylation of Erk1/2 was also shown after the treatment for 12 to 24 h (surrounded in blue). No phosphorylation of JNK was observed. (C). Anticancer activity of AKL-40 against EAC and U937 cells. After treating with 100-250 µg/mL of AKL-40, percentages of growth inhibition were determined by MTT assay (n = 3, mean ± SD). Gray and black bars indicate EAC and U937 cells, respectively. The results were statistically significant (* p < 0.05, when lectin-treated cells were compared to untreated cells). The results were statistically significant (** p < 0.01 and * p < 0.05, when lectin-treated cells were compared to untreated cells).
Minimum agglutination concentrations of AKL-40 for EAC and U937 cells were 20 and 24 µg/mL, respectively. The dose-dependent effect of AKL-40 against U937 and EAC cells was observed by MTT assay. At lower concentrations (100 µg/mL), growth inhibition of both cell types was similar, but at higher concentrations, AKL-40 showed slightly higher activity against EAC cells. At the concentration of 250 µg/mL, the percentage of growth inhibition for EAC cells was 37.9%, compared to 31.8% for U937 cells ( Figure 5C).
Antimicrobial Activity of AKL-40
AKL-40 showed antibacterial activity against Staphylococcus aureus, Bacillus cereus, and Shigella sonnei. No zone of inhibition was there for Escherichia coli. Slightly larger zones were observed around the disks soaked with higher doses (100 µg/disc) of AKL-40 in case of every susceptible bacterial species ( Figure 6A). Maximum activity of AKL-40 was found against Staphylococcus aureus in terms of the inhibition zones formed. Growth of Talaromyces verruculosus was also very efficiently inhibited by AKL-40. The fungi rapidly grew and after two weeks, nearly covered the whole petri dish. However, when discs soaked with AKL-40 were placed in the fungal media, almost no growth of the mold was observed ( Figure 6B).
Discussion
Following the purification of two lectins AKL and AKL-2 from Aplysia kurodai eggs, a third lectin with different molecular mass (40 kDa) had been purified [11,21,22]. This study focused on the biological activities of AKL-40 and the comparison of those activities to other marine lectins, specially isolated from mollusks. A number of lectin families have been reported in the phylum Mollusca whereas the Aplysia egg lectin family is structurally unique consisting of various isotypes. Galacturonic acid has been found in polysaccharides present in mollusks, such as cuttlefish [27]. Eggs of Aplysia kurodai also contained galactose/galacturonic acid-binding lectins as a mixture of 40, 32/30, and 16 kDa polypeptides ( Figure 1). Such diversity of galactose/galacturonic acid-binding lectins in Aplysia eggs perhaps is a result of their response to changing marine environments. It might be interesting to find out the function of each molecular species of Aplysia egg lectins during each developmental stage of the embryo. The primary structure of the sea hare egg lectin family is different from other lectins because of the presence of homolog sequences that are also found in organisms, such as bacteria and brachiopods [26]. A partial but novel Nterminal sequence of AKL-40, consisting of 35 amino acids, indicated that this polypeptide was different from other variants (such as AKL-a to -d) by having a four-amino acid forward extension (Figure 2). This result also showed low sequence similarity of AKL-40 to ADEL.
Determining the level of toxicity of lectins could be important to study and predict their structures, physiological functions, and biological applications. With an LC50 value of 63.63, AKL-40 was moderately toxic to brine shrimp nauplii (Figure 3) whereas AKL-2, the lactose-binding counterpart, was more than three times toxic [28]. However, toxicity of lectins is not always related to cell regulatory effects as their interactions with glycans play vital roles in cell signaling. Despite having much lower LC50 value (384.53 µg/mL), show the petri dishes containing three discs soaked with 400 µg/mL of AKL-40 after one week and two weeks of fungal inoculation, respectively). C and D are the petri dishes with no AKL-40 (control) after one week and two weeks of inoculation, respectively. Orange and black arrows indicate the pigment produced by the fungus and its subsequent growth, respectively.
Discussion
Following the purification of two lectins AKL and AKL-2 from Aplysia kurodai eggs, a third lectin with different molecular mass (40 kDa) had been purified [11,21,22]. This study focused on the biological activities of AKL-40 and the comparison of those activities to other marine lectins, specially isolated from mollusks. A number of lectin families have been reported in the phylum Mollusca whereas the Aplysia egg lectin family is structurally unique consisting of various isotypes. Galacturonic acid has been found in polysaccharides present in mollusks, such as cuttlefish [27]. Eggs of Aplysia kurodai also contained galactose/galacturonic acid-binding lectins as a mixture of 40, 32/30, and 16 kDa polypeptides (Figure 1). Such diversity of galactose/galacturonic acid-binding lectins in Aplysia eggs perhaps is a result of their response to changing marine environments. It might be interesting to find out the function of each molecular species of Aplysia egg lectins during each developmental stage of the embryo. The primary structure of the sea hare egg lectin family is different from other lectins because of the presence of homolog sequences that are also found in organisms, such as bacteria and brachiopods [26]. A partial but novel N-terminal sequence of AKL-40, consisting of 35 amino acids, indicated that this polypeptide was different from other variants (such as AKL-a to -d) by having a four-amino acid forward extension (Figure 2). This result also showed low sequence similarity of AKL-40 to ADEL.
Determining the level of toxicity of lectins could be important to study and predict their structures, physiological functions, and biological applications. With an LC 50 value of 63.63, AKL-40 was moderately toxic to brine shrimp nauplii (Figure 3) whereas AKL-2, the lactose-binding counterpart, was more than three times toxic [28]. However, toxicity of lectins is not always related to cell regulatory effects as their interactions with glycans play vital roles in cell signaling. Despite having much lower LC 50 value (384.53 µg/mL), MytiLec-1, a lectin from another marine mussel, could kill Burkitt lymphoma and U937 cells in vitro, as well as Ehrlich ascites carcinoma cells in vivo [3,29]. A variable level of toxicity with different LC 50 values (850.1, 142.1, 9.5, and 6.4 µg/mL) was also observed in lectins purified from marine sponges and a sea cucumber [30,31].
Ehrlich ascites carcinoma cells originated from mammary tissues, are spontaneous, differentiated, transplantable, and aggressive in nature. These cells grow in certain mice strains and are approved worldwide as a standard mice model [32]. AKL-40 agglutinated EAC cells via galactosyl ligands present in their cell membrane [33,34]. In a previous report, another Molluscan D-Gal-binding lectin, MytiLec-1 strongly agglutinated EAC cells at a minimum concentration of 16 µg/mL and inhibited 28 and 49% of their growth at doses of 1 and 2 mg/kg/day, respectively [29]. In the present study, minimum agglutination concentration of AKL-40 for EAC cells was 20 µg/mL and, compared to MytiLec-1, similar growth inhibition activities (28.70% and 58.32% at doses of 1 and 2 mg/kg/day) have been observed ( Figure 4A). Lectin-treated cancer cells presented Irregular shapes and nuclear condensation compared to the control (or untreated) cells ( Figure 4B).
Appearance of a faint band of p53 indicated to the transcription of a pro-apoptotic gene like Bax in AKL-40 treated Ehrlich ascites carcinoma cells. Expression of Bcl-X L , an anti-apoptotic gene had also become downregulated in the treated cells ( Figure 4C). Similar expressions of p53, Bax, and Bcl-X L genes have been found when MytiLec-1 was applied on EAC cells [29]. A previous study reported a lactose-binding lectin from the marine sponge Cinachyrella apion to induce the apoptotic death of HeLa cells through activation of Bax protein. The anti-apoptotic Bcl-2 protein showed no significant change in expression, compared to the control [35]. Evaluation of gene expression of MCF-7 cells revealed that marine red alga Solieria filiformis triggered caspase-dependent apoptosis. The anti-apoptotic gene Bcl-2 became down expressed, whereas the pro-apoptotic Bax gene underwent over-expression [36]. Downregulation of the anti-apoptotic factor Bcl-2 was determined also in the case of two other lectins from Sea bass (Dicentrarchus labrax) and sea urchin (Strongylocentrotus purpuratus) [37]. Therefore, it can be suggested that AKL-40 possibly calling the Bcl-2 apoptotic protein family into play marks its ability to promote apoptosis.
Marine invertebrate lectins have already been reported to exhibit antiproliferative properties, promote apoptosis, and block angiogenesis. In contrast, tumor-derived galactosebinding lectins, especially galectins, could compromise the anti-tumor immune response of CD8+ T cells to escape from the host immune surveillance [38,39]. AKL-40 significantly reduced the growth of erythroleukemia K562 cells compared to Raji and RBL-1 cells ( Figure 5A). Phosphorylation of two major mitogen-activated protein kinases, p38 and ERK 1/2 became significantly upregulated by the administration of AKL-40, whereas no phosphorylation was observed for and c-Jun N-terminal kinase (JNK) ( Figure 5B). It can be suggested that like other lectins previously reported, the lectin might have participated in a signaling cascade controlling cellular responses, probably leading to apoptosis [3,5,14]. In another in vitro study, AKL-40 inhibited the growth of two other cell lines, EAC and U937 ( Figure 5B). U937 is a monocytic human myeloid leukemia cell line frequently used in biomedical research [40]. Compared to AKL-40, MytiLec-1 and Ricin inhibited growth of these cells in vitro at much lower concentrations, which also indicates the difference of glycan recognition for these three lectins [30,41,42].
In marine organisms, lectins recognize and bind to the surface polysaccharides of a variety of bacteria. They have ability to kill bacteria or inhibit their growth and thereby contribute to defense against infection as a part of the innate immune system [43,44]. Similar to AKL, AKL-40 also exhibited growth inhibitory activity against both Grampositive and Gram-negative organisms ( Figure 6A) [45]. Staphylococcus aureus were the most susceptible bacteria to both lectins. Unlike AKL, another egg lectin from Aplysia dactylomela (ADEL) agglutinated Staphylococcus aureus cells, but could not inhibit their growth [24]. A mannose/galactose-binding C-type lectin isolated from bay scallop Argopectenirradians also displayed this property against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and Vibrio anguillarum [46]. However, due to unknown reasons, AKL, AKL-40, and ADEL could not affect the growth of Escherichia coli [24]. On the other hand, CvL from marine sponge Cliona varians showed cytotoxic effect on Gram-positive bacteria, such as Bacillus subtilis and Staphylococcus aureus, but could not inhibit the growth of Gramnegative bacteria like Escherichia coli and Pseudomonas aeruginosa [15]. These results suggest that the bactericidal activity of lectins depend not only on glycan-binding specificities, but also on multiple systems, such as multivalency or binding constancy.
Antifungal properties are not very common in lectins isolated from marine invertebrates [47]. Aplysia depilans gonad lectin was previously used to study the distribution of galacturonic acids in the cell walls of some pathogenic fungi [48]. In this study, high concentration (400 µg/mL) of AKL-40 totally inhibited the growth of Talaromyces verruculosus ( Figure 6B). A study by Ruperez et al. reported the presence of galactose sugars in the cell wall of Talaromyces verruculosus, which justified our findings [49]. Another antifungal protein (Aplysianin E) from Aplysia kurodai eggs completely suppressed the growth of Saccharomyces cerevisiae and Candida albicans at a concentration of 16 µg/mL [50]. AKL also repressed the mycelial growth of Curvularia lunata at 100 µg/mL whereas growth inhibitory effects of two other lectins from Japanese black sponge (Halichondria okadai) and mussel (Crenomytilus grayanus) were found against Aspergillus niger and Pichia pastoris, respectively [5,45,51]. It can be postulated that certain polysaccharides present in fungal cell walls interacted with these lectins and inhibited their growth by disturbing spore germination, growth of mycelium, and synthesis of chitin, to alter the fungal cell wall [52]. Like eggs from other organisms, sea hare eggs are found lying exposed in the seashore, vulnerable to the attack of predators and microbes [53]. Therefore, egg lectins might have a role to serve as protective molecules.
Preparation of the Crude Extract
The eggs of sea hare were gathered from the Zushi coast, Kanagawa, Japan. Eggs were crushed in a mortar, solubilized with Tris-buffered saline or TBS (10 mM Tris(hydroxymethyl) aminomethane-HCl, pH 7.4, with 150 mM NaCl). Then, crushed eggs were blended with 10 volumes of TBS containing a protease inhibitor (10 mM, Protease Inhibitor Cocktail 100X, Wako Pure Chemical Corp, Osaka, Japan).The homogenized sample was filled up in 500-mL centrifuge bottles and centrifuged at 14,720× g for 1 h at 4 • C. A Suprema 21 centrifuge equipped with an NA-18HS rotor (TOMY Co. Ltd., Tokyo, Japan) was used for this step. After repeated centrifugation of the supernatant at the same speed for the same duration, a clear solution was prepared and stored as the crude extract.
Purification of the Lectin
The lectin was purified according to a previously described procedure [22]. The crude extract was twice centrifuged at 27,500× g for 1 h at 4 • C and was administered to a 5 mL melibiose-agarose affinity column (J-Oil Mills Inc., Tokyo, Japan). This column was connected to a 5 mL Sephadex G-75 pre-column. After loading on the crude protein sample, the column was washed well with TBS. Lectins bound to the column were eluted with 10 mM D-galacturonic acid or D-galactose-containing TBS. The mixture of AKLs was separated by using a gel filtration chromatography of Sephacryl S-200 (Cytiva, Marlborough, MA, USA) connected to a fraction collector (FRC-10A, Shimadzu Corporation, Tokyo, Japan). Molecular weights of the lectins were confirmed by SDS-PAGE using standard marker proteins [54]. The 40 kDa polypeptide species was denoted as 'AKL-40 in this study.
Determination of N-Terminal Partial Amino Acid Sequence of AKL-40
The N-terminal sequence of the 35 amino acids of AKL-40 was determined with automated Edman degradation by using a protein/peptide sequencer PPSQ (Shimadzu Corporation, Kyoto, Japan) [55].
Determination of the Toxicity of AKL-40 by Brine Shrimp Nauplii Lethality Assay
The assay was performed according to a method reported earlier [29]; 4 mL of artificial seawater was taken in test tubes. Ten brine shrimp nauplii were taken in each vial and AKL-40 was added to these vials to adjust its concentrations from 10 to 160 µg/mL. There were control vials containing only seawater and nauplii, but no lectin. The experiment was repeated thrice at 30 • C for 24 h with 6 h of light exposure. Percentages of mortality of the nauplii were determined for each concentration and the LC 50 value of AKL-40 was also determined according to the method of Finney [56].
In Vivo Anticancer Activity of AKL-40 against Ehrlich Ascites Carcinoma Cells Grown in Swiss Albino Mice
The in vivo experiment was approved by the Institutional Animal, Medical Ethics, Biosafety and Bio-security Committee (IAMEBBC) for experimentations on animals, human, microbes, and living natural sources, Institute of Biological Sciences (IBSc), University of Rajshahi, Bangladesh (memo no. 102(6)/320-IAMEBBC/IBSc). Four to six weeks old Swiss albino mice (weight range 25-30 g) of both genders were collected from ICDDR'B (International Center for Diarrheal Diseases Research, Bangladesh) and EAC cells were propagated into these mice by a bi-weekly intraperitoneal transformation. Cells in ascitic fluid were drawn from a donor mouse bearing 6-7 days old tumor cells and with the help of a hemocytometer, adjusted to 2 × 10 6 cells/mL. Normal saline was used for the dilution. Viability of tumor cells was checked by 0.4% trypan blue assay.
The mice were randomly distributed into three groups, consisting of six mice in each group. These groups were denoted as 'A' or control, 'B' or lectin-treated with lower dose (1 mg/kg/day)' and 'C' or lectin-treated with higher dose (2 mg/kg/day). Moreover, 0.1 mL of cellular suspension containing viable EAC cells was injected intraperitoneally to each Swiss albino mouse. After 24 h, both lectin-treated groups (B and C) were treated for five days with an intraperitoneal injection of AKL-40 at doses of 1.0 and 2.0 mg/kg/day. All mice in groups A, B, and C were weighed to check the rate of tumor growth. Mice were sacrificed on the sixth day and EAC cells were collected from the ascitic fluid. The total number of viable EAC cells in every mouse of the treated groups (B and C) was compared to those of the control group (A) using the following formula: Percentage of inhibition = 100 − {(cells from AKL-40 treated mice/cells from control mice) × 100}
Morphological Observation of AKL-40 Treated EAC Cells by Fluorescence Microscope
Morphological changes of control (or untreated) and AKL-40 treated EAC cells were observed using fluorescence microscopy (Olympus iX71, Seoul, Korea). EAC cells from mice treated with and without AKL-40 for five consecutive days were collected. After washing with phosphate buffer saline (PBS), the cells were stained with 0.1 µg/mL of Hoechst-33342 at 37 • C for 20 min in dark, washed again with PBS, and observed in the microscope.
RNA Isolation and Checking the Expression of Apoptosis-Related Genes from Ehrlich Ascites Carcinoma Cells
EAC cells from mice treated with and without AKL-40 were collected and the total RNA was isolated using a reagent kit (Tiangen Biotech Co., Beijing, China). Concentration and purity of the isolated RNA were checked at 260 and 280 nm using a spectrophotometer. cDNA samples were prepared following the protocol of Applied Biosystems, Waltham, MA, USA, and bands for all PCR reactions were visualized in 1.4% agarose gel with a gel documentation system (Cleaver Scientific Ltd., Rugby, UK). Ten µg/mL of ethidium bromide solution was used to stain the gel. GAPDH was used as a housekeeping gene to compare with the standard. GeneRuler 1000 bp DNA ladder (Fermentas, Waltham, MA, USA) was used as marker. Specific oligonucleotides (Integrated DNA Technologies or IDT, Singapore) like p53, Bax, Bcl-X L and GAPDH generated 458 bp, 477 bp, 780 bp, and 475 bp amplification products, respectively. Primer sequences of these genes under study are provided in Table 1. Table 1. Primer constructions for apoptosis related and housekeeping genes.
Primer
Forward Reverse For gene amplification, a program was set in a thermal cycler (Gene, Atlas 482, Tokyo, Japan) at 95 • C for 3 min, followed by 35 cycles of 95 • C for 30 s, 55 • C for 30 s, 72 • C for 50 s, finally at 72 • C for 10 min, and then was eventually held at 20 • C. In case of Bax and Bcl-XL, the annealing temperature was 54 • C instead of 55 • C.
Determination of Cytotoxic Activity of AKL-40 against Cancer Cell Lines and Detection of Activated Signal Transduction Molecules
Cytotoxic activity of AKL-40 against cancer cell lines was evaluated according to a previous report [5]. Three leukemia cell lines K562, Raji, and RBL-1 (2 × 10 5 cells) were seeded into a 96-well titer plate and treated with different concentrations of AKL-40 for 24 h at 37 • C. Ten micro liter of WST-8 solution was added to each well and incubated for 4 h at the same temperature. The absorbance was measured at 450 nm to assay the reduction in proportion of living cells by a GloMax Multi Detection System (Promega, Madison, WI, USA).
Determination of the Minimum Agglutination Concentration of EAC and U937 Cells by AKL-40
U937 cells (ATCC CRL-3253) were collected from Yokohama City University, Japan. Fifty µL of 20 mM Tris-HCl buffer saline containing 10 mM CaCl2 (pH 7.8) was taken in each well of two U-bottomed 96-well microtiter plates (one for EAC and the other for U937 cells). AKL-40 (50 µL) was added to the titer plates through serial dilution. The number of EAC and U937 cells in RPMI-1640 media was counted using a hemocytometer (Hirschmann EM Techcolor, Eberstadt, Germany) and around 5 × 10 5 cells were seeded in each well of the two plates. The plates were agitated for 5 min in a microshaker, kept at room temperature for 30 min, and agglutination titers for both cell types were recorded.
Anticancer Activity of AKL-40 in Vitro against U937 and Ehrlich Ascites Carcinoma Cells
One hundred µL of RPMI-1640 media was taken in two 96-well flat bottom titer plates (one for EAC and the other for U937 cells). AKL-40 was added to the wells at final concentrations of 0, 100, 200, and 250 µg/mL. 100 µL of EAC cells (collected from mice) and U937 cells were added to each well (5 × 10 5 cells/well). There were three control wells containing only cancer cells. After an incubation period of 24 h in 5% CO 2 incubator at 37 • C, the clear supernatant was carefully removed from each well. Then 180 µL of PBS and 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide or MTT (5 mg/mL) were added and both plates were kept in the incubator for 8 h at 37 • C. The supernatant was removed again from each well, 200 µL of acidic isopropanol was added, and absorbance of each well was recorded by a titer plate reader at 570 nm. Percentages of cell proliferation inhibition were calculated by the following equation: Proliferation inhibition ratio (%) = (A − B) × 100/A where A is the OD 570 nm of the cellular homogenate from control wells and B is the OD 570 nm of the cellular homogenate from wells treated with AKL-40.
Bactericidal Activity of AKL against Pathogenic Bacteria
Antibacterial activity of AKL-40 was determined by agar disc diffusion method against four bacteria-two Gram-positive (Staphylococcus aureus and Bacillus cereus) and two Gramnegative (Shigella sonnei and Escherichia coli). Petri dishes containing lysogeny broth (LB) media for each bacterial species were prepared and four paper discs (control, high dose, low dose, and antibiotic) were placed on each petri dish. Moreover, two hundred and one hundred µg of AKL-40 were used as high and low doses, respectively, whereas the antibiotic disc contained 15 µg of ampicillin. Control disc was soaked only with LB media. After keeping at 37 • C for 24 h in an incubator, zones of inhibition around the discs were observed in each petri dish.
Antifungal Activity of AKL-40 against Talaromyces verruculosus
Talaromyces verruculosus, a fungus from the division ascomycota, was cultured in petri dishes with potato dextrose agar (HiMedia Laboratories Pvt. Ltd., Mumbai, India) as a medium following the standard procedure. In the 'Test' petri dish, three discs soaked with 400 µg/mL of AKL-40 were placed around whereas the 'Control' petri dish had only one disc, soaked only with media. After keeping at 25 • C for 1 week in an incubator, growth of the fungi in these petri dishes was observed. After another week, there was a second observation to find any growth inhibition.
Statistical Analysis
All experiments of this research work were performed in three replicates. Experimental data were expressed as mean ± SD. To test differences between experimental conditions, one way ANOVA and Dunnett's post-test correction was used. The results were statistically significant when ** p < 0.01 and * p < 0.05.
Conclusions
This study might be the start of isolating and sequencing amino acids in the specific peptides/proteins accountable for the observed antiproliferative effects of AKL-40. Abolition of biological activities by AKL-40 was not checked in the presence of inhibiting sugars, such as D-galacturonic acid or D-galactose, which is a limitation of this study. Determination of MIC and MBC values for the bacteria and fungi could also be performed. Additional investigations are required to investigate the association of lectin in infection and pathogenesis of different bacteria, as well as to recognize the molecular mechanism of signaling pathways activated during infection, which could help in the development of new therapeutic approaches. It is quite interesting to predict that a protein domain from bacteria was evolved in the course of time to exert antimicrobial effects in marine organisms. Such predictions could be evaluated in the future via a combined study of functional genomics, transcriptomics, and glycobiology of Aplysia lectins. | 2021-07-26T05:21:23.492Z | 2021-07-01T00:00:00.000 | {
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226528930 | pes2o/s2orc | v3-fos-license | Ultra-Compact Bandpass Filter with Super Wide Upper Stopband
An S-band bandpass filter based on quarter wavelength stepped-impedance resonators (SIRs) is presented in this paper. Two SIRs are loaded to the BPF to obtain wide stopband suppression. The center frequency f0 of the bandpass filter is located at 2.105 GHz with 3-dB fraction bandwidth (FBW) of 11.9%. It shows that the spurs free upper stopband with 15 dB rejection level can extend to 40 GHz (19f0). The circuit size of this filter is extremely compact, which occupies only 7.35mm×7.5 mm.
INTRODUCTION
Recently, microwave bandpass filters (BPFs) with compact size, high selectivity, and harmonic suppression have aroused wide concern. The inherent spurs and harmonics in out-of-band due to the periodic characteristic of transmission line make the filters hard to be applied in RF front-end. Various practical and effective approaches focused on harmonic suppression are presented [1][2][3][4][5][6][7][8][9][10][11][12]. It is convenient to design a BPF with wide stopband by cascading a lowpass filter and a highpass filter [1], but it usually occupies larger circuit area. Defected ground structure (DGS) technology is widely used to suppress the harmonic responses due to the good lowpass characteristics [2,3]. Generally, DGS can provide harmonic suppression in upper stopband and reduce the circuit area. However, the fabrication of a DGS filter is complicated, and it is difficult to integrate with planar circuits. The method of using stepped-impedance resonators (SIRs) to push the harmonic frequencies to higher frequencies by adjusting the ratio of impedance has been demonstrated for the design of wide upper stopband BPFs [4][5][6]10]. Besides, harmonics-staggered filters [7,8], defected microstrip structures [9,10], and infused spur-lines [11,12] are popularly used to suppress the unwanted harmonic passbands. Although the merits of these filters have been fully demonstrated, the spurs-like responses in out-of-band should be further improved.
In this letter, we present an ultra-compact S-band BPF with super wide spurs free upper stopband. The filter consists of two cascaded quarter wavelength SIRs. By loading SIRs, transmission zeros are generated to suppress the out-of-band spurs. The method for spurious suppression is very simple and effective, while it does not increase the complexity of fabrication and circuit size at all. For experimental validation, the filter is designed, fabricated, and tested. The measurement results show that the center frequency f 0 is located at 2.105 GHz with 3-dB fraction bandwidth (FBW) of 11.9%. Its upper stopband with 15 dB rejection level can extend to 40 GHz (19f 0 ). The fabricated filter occupies very compact size of 7.35 mm × 7.5 mm.
BPF ANALYSIS AND DESIGN
The configuration and coupling topology of the proposed S-band BPF, respectively, are depicted in Figs. 1(a) and 1(b). It is shown that the BPF consists of two cascaded quarter wavelength SIRs. As shown in Fig. 1(a), L k and W k (k = 1, 2, 3, 4) denote the physical length and width of the resonator, respectively. S 1 and S 2 , respectively, are the gaps of two shorted coupling lines and two open coupling lines. R represents the diameter of the via holes. As shown in Fig. 1(b), mixed electric and magnetic coupling is introduced, in which the shorted coupling lines provide magnetic coupling, and the open coupling lines provide electric coupling. Figure 2(a) shows the configuration of a quarter wavelength SIR. According to transmission line theory: where, Here, Z 2 and θ 2 denote the characteristic impedance and electrical length of low-impedance transmission line. Z 1 and θ 1 represent the characteristic impedance and electrical length of highimpedance transmission line. Therefore, the input impedance Z in of quarter wavelength SIR can be expressed as: Here, Z L = 0. According to the resonant condition of Y in = 0 [13][14][15], we have where Y in denotes the input admittance of quarter wavelength SIR, and R Z is the impedance ratio. Therefore, the resonant modes f 1 and f 2 (the fundamental resonant frequency and the first harmonic resonant frequency) of the quarter wavelength SIR can be derived by solving Eq. (6).
Here, we define the ratio of electrical length α as under the condition of θ 1 + θ 2 = π/2. Figure 2(b) shows the relationship between f 2 /f 1 and α under different R Z . It can be found that quarter wavelength SIR is of harmonic suppression when R Z is less than 1. Fig. 3(a) plots the diagram of the normalized first and second harmonic resonant frequencies (f 2 /f 1 and f 3 /f 1 ) against α under different R Z . The ratio of normalized second and first harmonic resonant frequencies is depicted in Fig. 3(b). According to [16], the coupling coefficient can be extracted as: Figure 4(a) plots the extracted coupling coefficient M 12 versus varied L 1 and S 2 . As can be seen, the coupling coefficient decreases as S 2 increases from 0.07 mm to 0.27 mm, when L 1 is fixed. The coupling coefficient decreases slightly as L 1 gets larger, when S 2 is fixed. The extracted coupling coefficient M 12 versus varied S 1 and S 2 is depicted in Fig. 4(b). It shows that the coupling coefficient decreases as S 2 increases from 0.05 mm to 0.45 mm, when S 1 is fixed. However, the coupling coefficient keeps almost unchanged as S 1 becomes larger, when S 2 is fixed. Next, the external quality factor Q e can be derived by [16]: where ω 0 denotes the resonant frequency, and τ S 11 (ω 0 ) is the group delay of S 11 at ω 0 . Figure 5 shows the relationship between the extracted external quality factor Q e and the tap position t. As expected, with the increase of t, the coupling strength becomes larger. Due to the periodic characteristic of transmission line, the inherent spurs and harmonics in out-ofband usually make the filters hard to be applied in RF front-end. Therefore, the harmonic frequencies in upper stopband should be further suppressed. In this letter, two SIRs are loaded to the original BPF to suppress the harmonics. Fig. 6(a) plots the layout of the modified BPF. Figure 6(b) shows the frequency responses of BPF with and without SIRs loaded. It can be found that the out-of-band performance has been improved with SIRs loaded, whereas the passband keeps almost unchanged.
EXPERIMENTAL RESULTS AND DISCUSSION
In order to validate the analysis above, BPF with SIRs loaded is finally printed on a substrate of Rogers 4003C, with thickness of h = 0.508 mm and relative dielectric constant of ε r = 3.55. The circuit . This corresponds to 0.086λ g × 0.088λ g , where λ g is the guided wavelength of a 50 Ω microstrip line at f 0 . Agilent vector network analyzer N5244A is used to characterize the frequency responses of BPF. As shown in Fig. 7, the measurement results are in good agreement with the prediction ones. It can be observed from Figs. 7(a) and 7(b) that the center frequency f 0 is located at 2.105 GHz with 3-dB FBW of 11.9%. The measured return loss (RL) within the passband is greater than 35.8 dB. As can be seen, the lower stopband suppression can reach 45 dB from DC to 1.48 GHz, while the upper stopband with rejection level of 15 dB can extend to 40 GHz. Table 1 summarizes the comparison between this work and other reported BPFs, which shows that the proposed BPF is of very compact size, super wide upper stopband, and good in-band RL.
CONCLUSION
An S-band BPF with compact size, wide upper stopband, and good in-band RL based on quarter wavelength stepped-impedance resonators (SIRs) is presented in this paper. Mixed electric and magnetic coupling is introduced in this design. To suppress the spurs in out-of-band, two SIRs are loaded to the original BPF. It shows that the modified BPF is of super wide upper stopband, whose 15 dB rejection level can extend to 40 GHz (19f 0 ). The circuit size of the filter is extremely compact, which occupies only 7.35 mm × 7.5 mm. | 2020-08-20T10:03:54.485Z | 2020-01-01T00:00:00.000 | {
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209263275 | pes2o/s2orc | v3-fos-license | Using Mobile Health Tools to Assess Physical Activity Guideline Adherence and Smoking Urges: Secondary Analysis of mActive-Smoke
Background Rates of cigarette smoking are decreasing because of public health initiatives, pharmacological aids, and clinician focus on smoking cessation. However, a sedentary lifestyle increases cardiovascular risk, and therefore, inactive smokers have a particularly enhanced risk of cardiovascular disease. Objective In this secondary analysis of mActive-Smoke, a 12-week observational study, we investigated adherence to guideline-recommended moderate-to-vigorous physical activity (MVPA) in smokers and its association with the urge to smoke. Methods We enrolled 60 active smokers (≥3 cigarettes per day) and recorded continuous step counts with the Fitbit Charge HR. MVPA was defined as a cadence of greater than or equal to 100 steps per minute. Participants were prompted to report instantaneous smoking urges via text message 3 times a day on a Likert scale from 1 to 9. We used a mixed effects linear model for repeated measures, controlling for demographics and baseline activity level, to investigate the association between MVPA and urge. Results A total of 53 participants (mean age 40 [SD 12] years, 57% [30/53] women, 49% [26/53] nonwhite, and 38% [20/53] obese) recorded 6 to 12 weeks of data. Data from 3633 person-days were analyzed, with a mean of 69 days per participant. Among all participants, median daily MVPA was 6 min (IQR 2-13), which differed by sex (12 min [IQR 3-20] for men vs 3.5 min [IQR 1-9] for women; P=.004) and BMI (2.5 min [IQR 1-8.3] for obese vs 10 min [IQR 3-15] for nonobese; P=.04). The median total MVPA minutes per week was 80 (IQR 31-162). Only 10% (5/51; 95% CI 4% to 22%) of participants met national guidelines of 150 min per week of MVPA on at least 50% of weeks. Adjusted models showed no association between the number of MVPA minutes per day and mean daily smoking urge (P=.72). Conclusions The prevalence of MVPA was low in adult smokers who rarely met national guidelines for MVPA. Given the poor physical activity attainment in smokers, more work is required to enhance physical activity in this population.
Background
Smoking cessation and physical activity both lead to significant improvements in health [1]. Although smoking rates are decreasing because of regulation and taxation, behavioral counseling, and pharmacotherapy [1], an individual's attempts to quit smoking are still challenging [2]. Furthermore, because a sedentary lifestyle increases cardiovascular risk [3], inactive smokers have a particularly enhanced risk of cardiovascular disease. The 2018 US Physical Activity Guidelines recommend greater than or equal to 150 min per week moderate activity or greater than or equal to 75 min per week vigorous physical activity (VPA), accumulated over bouts of any duration [4]. However, the prevalence of meeting these activity guidelines in the general adult population is unsatisfactory, with half of the US adults attaining fewer than 150 min of moderate-to-vigorous physical activity (MVPA) during leisure time per week, by self-report [4]. Moreover, 2 studies in young adults [5] and youth aged 14 to 18 years [6] found that self-reported attainment of physical activity guidelines was positively associated with noncigarette forms of tobacco use (eg, electronic cigarettes) but inversely associated with cigarette smoking, suggesting that physical inactivity and cigarette smoking may be compounding risk factors.
Physical activity has been suggested as an aid for smoking cessation, potentially through moderation of cravings and prevention of weight gain [7,8], but evidence is conflicting. Although there is insufficient evidence to recommend exercise as an aid for smoking cessation [7,9], previous meta-analyses suggested that acute bouts of exercise decrease urges, with activities at moderate to vigorous intensity having the greatest effect [10,11]. In addition, a study on active smokers found that a higher level of habitual MVPA was significantly associated with lower smoking urges [12]. However, this study used 7-day physical activity recall for assessing the levels of MVPA, leading to potential recall bias.
Methodological limitations such as recall bias and poor ecological validity are common in prior studies of exercise and smoking. Self-report for physical activity has been shown to poorly correlate with accelerometer data in accurately measuring MVPA and sedentary time, whereas ecological momentary assessment using mobile health (mHealth) tools, designed to sample real-time behaviors and experiences in the natural environment, performed better [13].
Objectives
The goal of mActive-Smoke, a 12-week prospective observational study, was to assess the day-level association between objectively measured physical activity and concurrent smoking urges. Previously reported primary results [14] demonstrate that acute bouts of physical activity (ie, number of steps accumulated in a 30-min period before urge reporting), but not total daily steps, were associated with a modest decrease in smoking urges. Although we previously found a temporal association between acute bouts of activity and decreased urge, it is unclear whether the intensity of daily activity is associated with daily urge. In addition, despite well-established contributions of physical inactivity [3] and smoking on cardiovascular risk, there is limited research describing adherence to physical activity guidelines in adult smokers, with prior research mostly reliant on self-reported data. Thus, in this secondary analysis, we used prospective, objective measures to investigate adherence to guideline-recommended MVPA and the association between the intensity of physical activity and the urge to smoke.
Study Aim and Design
The aims of this secondary analysis were to report adherence to physical activity guidelines among smokers in the mActive-Smoke study population and to investigate the relationship between the intensity of physical activity and the urge to smoke. The methods for this 12-week prospective observational study have been previously reported [14], and a summary is provided below (Recruitment and Measurement of Baseline Variables and Data Collection). This study was approved by the Johns Hopkins School of Medicine Institutional Review Board.
Recruitment and Measurement of Baseline Variables
We recruited 60 participants from April 7, 2016, to September 2, 2016, using on-site advertisements, social media, and physician referrals. Participants met inclusion criteria if they were aged 18 years or older, smoked at least 3 cigarettes per day on average, owned a smartphone, and were able to perform normal physical activity. Participants were screened for eligibility via email. At an initial meeting with a study coordinator, participants completed an enrollment questionnaire to record demographic characteristics, self-reported BMI (weight [kg]/height [m 2 ]), physical activity, and smoking behavior. Baseline physical activity was assessed by the International Physical Activity Questionnaire (IPAQ)-short form, a questionnaire assessing walking time, sedentary time, and MVPA time in the past 7 days [15]. A high IPAQ score is defined as the equivalent of either VPA on 3 days or more per week at greater than or equal to 1500 metabolic equivalent of task (MET) minutes per week or 5 days or more per week of any combination of MVPA meeting greater than or equal to 3000 MET minutes per week [16]. Baseline smoking behavior was assessed with the Arizona Smoking Assessment Questionnaire [17].
Data Collection
For the measurement of physical activity, participants used the Fitbit Charge HR (Fitbit Inc), a wrist-worn triaxial digital accelerometer allowing continuous monitoring of activity and heart rate. Patients were not instructed to alter their physical activity, but they could access step counts via the Fitbit mobile app (Fitbit Inc). Data from the Fitbit, including steps and the Fitbit-generated intensity level, were compiled in Fitabase, a secure research platform that collects real-time data from activity tracking devices [18]. Day-level and minute-level data were downloaded from Fitabase for each participant.
To measure smoking urges, an automated messaging service sent SMS text messages to participants 3 times per day, requesting that they respond with their instantaneous urge to smoke on a 9-point Likert scale from low to high. These messages were sent at participant-defined times, corresponding roughly to waking up, lunchtime, and returning home at the end of a day.
Participants were asked to complete a Web-based end-of-study survey regarding the study experience and their perceptions on physical activity and smoking urges. Survey questions and results were previously reported [14].
Statistical Analysis
Baseline characteristics were summarized using descriptive statistics, frequency for categorical data and mean (SD) and median (IQR) for continuous data. Spearman and Pearson correlation coefficients were used for associations between variables.
As participants were not required to wear Fitbits during sleep, we defined nonwear time as 90 consecutive minutes of missing heart rate data between the hours of 10 am and 10 pm. Days with 2 or more 90-min nonwear periods and wear time of fewer than 6 hours within the target time window were excluded [19]. At least 6 total weeks of recorded data were required for inclusion. For the calculation of the prevalence of meeting weekly MVPA goals, we included weeks for which participants contributed 4 or more days of complete data [20].
Fitbit assigns minute-level activity into 4 intensity levels (0: sedentary, 1: light, 2: moderate, and 3: vigorous) [18]. We eschewed this measure of intensity given the proprietary algorithms and concern about accuracy [21,22] and used cadence as a surrogate measure of intensity. We elected to not include heart rate because of concerns about the accuracy of Fitbit's heart rate measurement, particularly at vigorous intensities [23]. However, to explore the nature of Fitbit's intensity variable, we compare daily MVPA minutes by Fitbit intensity levels (number of minutes spent at intensity level 2 or 3) with daily MVPA minutes by cadence threshold.
Cadence (steps per minute) is associated with objectively measured speed and intensity under controlled experimental conditions [24]. A threshold of 100 steps per minute is an evidence-based value generally associated with moderate intensity or greater than or equal to 3 METs and is best described as brisk walking, whereas a threshold of 130 steps per minute is associated with vigorous intensity or greater than or equal to 6 METs [25]. We created 4 cadence categories defined as 0 steps per minute (no movement), 1 to 59 steps per minute (incidental movement to purposeful steps), 60 to 99 steps per minute (slow to medium walking), and greater than or equal to 100 steps per minute (brisk walking and faster), whereas VPA was defined as cadence of greater than or equal to 130 steps per minute [25]. Daily minutes of MVPA were calculated by summing the minutes spent at cadence of greater than or equal to 100 steps per minute, and daily minutes of VPA was calculated by summing the minutes spent at cadence of greater than or equal to 130 steps per minute. We also calculated moderate+2×VPA, which weights moderate activity as 1 min and vigorous activity as 2 min, in accordance with physical activity guidelines [20], but as the results remained similar, we reported only MVPA and VPA.
Given the positively skewed data, we described MVPA minutes using median (IQR) and used the Wilcoxon rank sum test for comparing between-group differences. We reported the within-person and between-person prevalence of physical activity guideline adherence. We also estimated the prevalence of adherence with obtaining at least 150 min of MVPA or 75 min of VPA per week on greater than or equal to 50% of the study weeks. Although the physical activity guidelines were developed based on self-report, we opted to apply the physical activity guidelines to cadence measurements to provide a clinical context for the objective data. Daily urge to smoke was described using mean (SD) of the 3 to 4 urge messages sent each day. The mean daily urge was normally distributed and was treated as a continuous variable.
A repeated measures multivariable mixed effects linear model, accounting for autoregression and heteroscedasticity, was used to evaluate the relationship between daily MVPA minutes and daily urge. We adjusted for age, sex, race, education, BMI, baseline cigarettes per day, and baseline physical activity, which were selected a priori. Baseline physical activity was defined as a high level of activity or not by IPAQ. We explored interactions between age, sex, obesity status, baseline physical activity, and baseline cigarettes per day with daily MVPA minutes, with P<.10 considered evidence of interaction. The analysis was conducted using Stata (version 15·1; StataCorp).
Study Flow and Baseline Characteristics
The study flow diagram, baseline characteristics, and survey results have been previously reported [14]. In brief, 60 participants were enrolled, and 53 participants recorded at least 6 weeks of data and were thus included in this analysis. In addition, 45 participants recorded 12 weeks of data, and 8 participants recorded 6 to 12 weeks of data. Of all participant-weeks, 80.1% (144/723) of weeks included at least 4 days of complete data. Participants sent a mean of 290 (SD 62) SMS text messages quantifying the urge to smoke. Moreover, 49 participants completed the Web-based exit survey. After excluding days using nonwear criteria, data from 3633 days were analyzed, with a mean of 69 days of data contributed by each participant. The mean age was 40 (SD 12) years, with 57% (30/53) women, 49% (26/53) nonwhite participants, and 30% (16/53) with a bachelor's degree or higher. In addition, 40% (21/53) of participants were overweight, 38% (20/53) were obese, and 53% (28/53) had a high level of baseline activity as assessed by IPAQ (
Patterns of Physical Activity
Participants accumulated a median of 7807 steps per day (IQR 5383-10,824). Of 53 participants, 31 (58%) met the recommended 30 min per day of MVPA on 1 or more days over the study duration. Of these 31 participants, the 30 min per day MVPA goal was met on a mean of 19% of days. Prevalence of adherence to national physical activity guidelines, defined as the proportion of participants who obtained at least 150 min per week of MVPA on at least 50% of total weeks, was 10% (5/51; 95% CI 4% to 21%). No participants attained at least 75 min per week of VPA on at least 50% of total weeks. Of the 53 participants, only 15 (28%) met 150 min per week of MVPA at least once throughout the study, and those 15 participants met that goal on a mean of 36% of weeks.
Association Between Day-Level Intensity and Urge
The number of daily MVPA minutes was positively skewed, with no clear association with urge upon inspection ( Figure 1). Furthermore, logarithmic transformation of the MVPA variable did not reveal any clear relation with mean daily urge. There was no significant association between daily MVPA minutes and mean daily urge to smoke in either the unadjusted model (P=.74) or the adjusted model (P=.72).
We explored the interaction of daily MVPA minutes with binary demographic factors, defined as age greater than or equal to 40 years, sex, BMI greater than or equal to 30 kg/m 2 , and baseline high activity based on IPAQ. The P values for interaction are as follows: .41 for age, .15 for sex, .90 for BMI, and .32 for high activity. Thus, no interaction terms were included in the adjusted models.
Sensitivity Analysis
Given that our prior work validating the urge to smoke revealed a positive association between mean urge over the course of the study and the number of cigarettes per day reported at the end of the study [14], we explored associations by baseline cigarette consumption. When stratifying by baseline cigarettes per day, those who smoked more than 10 cigarettes per day (n=19; 1333 person-days) had 0.293 lower daily urge per 30 min per day of MVPA (P=.03; 95% CI −0.563 to −0.023; Figure 1), but this was not significant on stratifying by greater than equal to 15 (n=16; 1100 person-days; P=.80) or greater than equal to 20 cigarettes per day (n=11; 809 person-days; P=.88).
Comparison of Cadence Versus Fitbit's Intensity Levels
To elucidate the nature of Fitbit's intensity variable, we compared the distribution of daily MVPA minutes calculated using the definition of cadence greater than or equal to 100 steps per minute with the distribution of daily MVPA minutes as defined by the number of minutes spent at moderate-or vigorous-intensity levels as defined by Fitbit's algorithm ( Figure JMIR
Principal Findings
In this secondary analysis of data from mActive-Smoke, we described intensity of physical activity in free-living adult smokers and found that the prevalence of MVPA was low, with 10% (5/51) of participants attaining greater than or equal to 150 min of MVPA on at least 50% of study weeks and no participants attaining greater than or equal to 75 min of VPA on at least 50% of study weeks. Overall, the median daily MVPA was 6 min, and this differed by sex and BMI. Most participants achieved at least 30 min per day of light-intensity activity (60-99 steps per minute) over the study duration. In regression analyses, there was no association between daily MVPA minutes and mean daily smoking urges among all participants. In addition, this study provides exploratory insights on using Fitbit's intensity level to determine MVPA, compared with accepted cadence thresholds, which is a simpler marker of intensity available across measurement devices.
Comparison With Prior Work
This study highlights the low prevalence of MVPA in adult smokers in a free-living environment and poor adherence to the 2018 US Physical Activity Guidelines of greater than or equal to 150 min per week of MVPA. Our results corroborate observations by prior analyses of accelerometer data from the National Health and Nutrition Examination Survey (NHANES), which are representative of the general US population. Using 2005 to 2006 NHANES data, Tucker et al [20] found that 59.6% of adults met the 2008 US Physical Activity Guidelines by self-report, whereas 8.6% of adults met the guidelines by accelerometer measurement, using the goal of greater than or equal to 150 min per week of MVPA in the 10-min bout. In the 2018 US Physical Activity Guidelines, the 10-min bout requirement was removed; thus, we did not include bouts in the calculation of MVPA minutes. Doing so would likely further reduce the estimated attainment of recommended physical activity levels and, as such, would not impact the conclusion of low levels of physical activity guideline adherence.
Another analysis of 2005 to 2006 NHANES data found that US adults accumulated only about 7 min per day of self-selected activity at a cadence of greater than or equal to 100 steps per minute (generally defined as MVPA), but the participants did accumulate, on average, about 30 min per day of activity at cadences of more than 60 steps per minute [26]. The NHANES dataset from these prior studies included accelerometer data over 1 to 7 days, whereas our data were obtained over 6 to 12 weeks [20,26], thus suggesting that low levels of physical activity may persist over time. These results highlight a need for further work in promoting physical activity in smokers to mitigate the compounding cardiovascular risk factors of inactivity and smoking.
Although there was no intervention to increase physical activity, 82% (40/49) of participants reported in the exit survey that they believed the study helped increase their physical activity [14], affirming the potential of mHealth tools and self-monitoring in promoting behavioral change. Furthermore, the 2018 Physical Activity Guidelines recommend information technology and mHealth interventions as a future direction for tracking and promoting physical activity [4].
This study also addresses the recall bias in prior studies on physical activity and smoking urges. For example, Haasova et al [12] showed that more habitual MVPA minutes based on 7-day activity recall significantly correlated with decreased urge over the past 7 days in 98 smokers. The difference between results from this 7-day recall study and our analysis suggests that the prevalence of MVPA and granularity of data are important factors to consider in studies on physical activity and smoking urge. Specifically, median daily MVPA based on 7-day recall was 45 min (IQR 17-77) in the study by Hassova et al [12], whereas we measured median daily MVPA over 12 weeks to be 6 min (IQR 2-13) using minute-level and day-level granularity of data. This is unsurprising given that this analysis and prior studies [13] found poor correlation between self-reported intensity via IPAQ and accelerometer-measured intensity.
This study analyzed intensity on a day level by quantifying the total daily minutes of MVPA for each person-day, which builds on prior studies showing that short bouts of MVPA acutely decrease cigarette cravings in a controlled laboratory setting [11]. In addition, we build on our primary analysis of mActive-Smoke, which showed that increased rate of step accumulation within 15-, 30-, or 60-min time windows before urge reporting was associated with decreased urge. When comparing our results with these prior studies on acute effects, we conclude that although MVPA may modestly decrease the urge to smoke immediately after physical activity, the effect of MVPA on the urge to smoke does not appear to persist throughout the day.
Limitations
We acknowledge that this was a relatively small, single-center study, not generalizable to smokers everywhere. Despite the low prevalence of MVPA, the participants in this study had fairly high total daily step counts, suggesting that these participants may be active throughout the day at lighter intensities. This confers cardiovascular health benefits over a more sedentary lifestyle but may not be enough to affect smoking urges [4]. In addition, there is inherent selection bias, as participants who enrolled in this study were more likely to have an interest in behavior change, and step counts and urge reporting may be subject to the Hawthorne effect. However, it is important to note that even as part of a research study, the physical activity observed was low and similar to prior studies of the general population.
As this was a post hoc analysis, the sample size was not powered to test correlations between intensity and smoking urge. Although the total number of observations was high, the number of participants in this pilot study was relatively small, especially in the stratified models. However, we did account for repeated measures and autoregression in the model, and our smallest stratified group contained 809 person-days.
In addition, mActive-Smoke participants were lighter smokers, with 36% (19/53) reporting more than 10 cigarettes per day at baseline. Prior studies generally used a minimum of 10 cigarettes per day as the threshold for study inclusion [9,12]. We did not collect data on the time since the last cigarette to avoid overburdening participants with text messages; thus, we could not adjust for the potential confounding effect of recent smoking on urge. These factors may have generated a flooring effect, as physical activity may be less able to further reduce smoking urge when the urge is already low, either from a recent cigarette or because of lighter smokers having lower urges. We previously validated the urge scale and found that self-reported urges correlated well with daily cigarette consumption.
Cadence is an imprecise marker of intensity, correlating well with caloric expenditure, but does not account for types of activity other than walking or running, leading to possible underestimation of MVPA. In addition, the cadence thresholds used in these analyses were not adjusted for stride length variation among participants. Bias from lack of stride length adjustment is likely to be minimal, as overestimation of MVPA in participants with shorter stride is partially offset by underestimation of MVPA in participants with longer stride. Although the Fitbit Charge HR reports minute-level intensity levels, METs, and heart rate, we opted for cadence as the measure of intensity, as it is less dependent on other factors such as resting heart rate, comorbidities, and medications. Furthermore, validation studies have raised concerns about the accuracy of Fitbit's reporting of heart rate [23], intensity, and energy expenditure, although step count was generally accurate [21,27].
Despite the advantages of objective activity measures, it is important to note that physical activity guidelines were developed based on self-reported data, and there are currently no guidelines based on accelerometer data. Linking objective measures with physical activity guideline attainment to provide clinical context has been done previously [20], but this method requires further validation in future studies. More work is needed to develop guidelines based on objective metrics of physical activity. Future directions include devising the optimal method of incorporating heart rate data into measurement of MVPA while accounting for medications and clinical characteristics.
Finally, Fitbit provides other information about health behaviors, such as sleep, which could impact both physical activity and smoking urge and warrants further exploration.
Conclusions
In this 12-week observational study of adult smokers using mHealth tools for real-time assessment of physical activity and smoking urge, the prevalence of MVPA was low, and participants rarely met national guidelines for physical activity. We found no day-averaged association between intensity of activity and smoking urges. On the basis of the known benefits of physical activity and the low levels observed in this study, more work is needed to address physical activity promotion in smokers. | 2019-09-15T03:25:06.080Z | 2019-06-11T00:00:00.000 | {
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119139825 | pes2o/s2orc | v3-fos-license | A Maximal Extension of the Best-Known Bounds for the Furstenberg-S\'ark\"ozy Theorem
We show that if $h\in \mathbb{Z}[x]$ is a polynomial of degree $k \geq 2$ such that $h(\mathbb{N})$ contains a multiple of $q$ for every $q\in \mathbb{N}$, known as an $\textit{intersective polynomial}$, then any subset of $\{1,2,\dots,N\}$ with no nonzero differences of the form $h(n)$ for $n\in\mathbb{N}$ has density at most a constant depending on $h$ and $c$ times $(\log N)^{-c\log\log\log\log N}$, for any $c<(\log((k^2+k)/2))^{-1}$. Bounds of this type were previously known only for monomials and intersective quadratics, and this is currently the best-known bound for the original Furstenberg-S\'ark\"ozy Theorem, i.e. $h(n)=n^2$. The intersective condition is necessary to force any density decay for polynomial difference-free sets, and in that sense our result is the maximal extension of this particular quantitative estimate. Further, we show that if $g,h\in \mathbb{Z}[x]$ are intersective, then any set lacking nonzero differences of the form $g(m)+h(n)$ for $m,n\in \mathbb{N}$ has density at most $\exp(-c(\log N)^{\mu})$, where $c=c(g,h)>0$, $\mu=\mu(\text{deg}(g),\text{deg}(h))>0$, and $\mu(2,2)=1/2$. We also include a brief discussion of sums of three or more polynomials in the final section.
Here and throughout we use [1, N ] to denote {1, 2, . . . , N }, and we use |X| to denote the size of a finite set X. Furstenberg [5] answered this question in the affirmative via ergodic theory, specifically his correspondence principle, but obtained no quantitative information on the rate at which the density must decay. Independently, Sárközy [24] showed via Fourier analysis, specifically a density increment argument driven by the Hardy-Littlewood circle method, that if A ⊆ [1, N ] contains no nonzero square differences, then . Throughout the paper we use log to denote the natural logarithm. We use "≪" to denote "less than a constant times", with subscripts indicating on what parameters, if any, the implied constant depends.
A natural generalization of Lovász's question is to extend from perfect squares to the image of more general polynomials. Balog, Pelikán, Pintz, and Szemerédi [1] extended (2) to sets with no k-th power differences for a fixed k ∈ N, with c = 1/4 and the implied constant depending on k.
More generally, to hope for such a result for a given nonzero polynomial h ∈ Z[x], it is clearly necessary that h(N) contains a multiple of q for every q ∈ N, as otherwise there is a set qN with positive density and no differences in the image of h. It follows from a theorem of Kamae and Mendès France [10] that this condition is also sufficient, in a qualitative sense, and in this case we say that h is an intersective polynomial.
Equivalently, a nonzero polynomial is intersective if it has a p-adic integer root for every prime p. Examples of intersective polynomials include any nonzero polynomial with an integer root or two rational roots with coprime denominators. However, there are also intersective polynomials with no rational roots, such as (x 3 − 19)(x 2 + x + 1).
It is a theorem of Lucier [15], with minor improvements exhibited by Lyall and Magyar [17] and the author [20], that if h ∈ Z[x] is an intersective polynomial of degree k ≥ 2 and A ⊆ [1, N ] has no nonzero differences in the image of h, then |A| N ≪ h log log N log N 1/(k−1) .
Further, Hamel, Lyall, and the author [8] extended (2) to all intersective polynomials of degree two, for any c < 1/ log(3) and the implied constant depending on c and the polynomial.
Main results.
Here we adapt the Fourier analytic double iteration strategy first developed in [19] and extend (2) to the full collection of intersective polynomials. By more closely mimicking details of [1] and [8], one may be able to take the constant c in Theorem 1.1 to be independent of k, and perhaps arbitrarily close to 1/ log 3, a natural limit of the method. For our exposition, however, this range of values for c is optimal. We discuss this further at the end of Section 3.5.
Our crucial new ingredients are motivated and discussed in Sections 2.4 and 2.5, respectively, with the necessary exponential sum estimates established in Section 4. In addition, for the interested reader, a summary of our new exponential sum estimates is provided as a stand alone theorem in Section 2.6.
Further, we apply the exponential sum estimates established in Section 4 in a more straightforward L 2 density increment, exhibiting stronger density bounds on sets free of nonzero differences that are the sum of two polynomial images. where c = c(g, h) > 0, µ = µ(deg(g), deg(h)) > 0, and µ(2, 2) = 1/2.
Remark on generality of Theorem 1.2. We note that the necessary intersective condition makes perfect sense in a multivariable setting, and analogous results should hold for every intersective integral polynomial in several variables, not just diagonal forms. Further, there do exist intersective binary diagonal forms not covered in this theorem. For example, if p is a prime congruent to 1 modulo 90090 that is not the sum of two integer cubes (of which there are plenty), then, since p is a sum of two cubes modulo q for every q ∈ N, x 3 + y 3 − p is an intersective polynomial in two variables that cannot be expressed as the sum of two single-variable intersective polynomials.
Lower bounds and conjectures.
For k, N ∈ N, by fixing a prime N 1/k /2 ≤ p ≤ N 1/k and letting we see that A ⊆ [1, N ] has no nonzero k-th power differences. More generally, for any polynomial h ∈ Z[x] of degree k, the greedy algorithm produces a set A ⊆ [1, N ] satisfying |A| ≫ h N 1−1/k with no nonzero differences in the image of h.
Ruzsa [22] showed that if q ∈ N is squarefree and B ⊆ Z/qZ has no nonzero differences that are k-th powers modulo q, then there exists A ⊆ [1, N ] with no nonzero k-th power differences satisfying |A| ≫ N c , where c = (k − 1 + log |B|/ log q)/k, which is larger than the trivial construction with which we began this section. For k = 2, Lewko [12] utilized an extensive computer search and found an example with q = 205 and |B| = 12, yielding c ≈ 0.7334, which is currently the best-known lower bound for the original square-difference question.
As Ruzsa remarks, if k = 2, then log |B|/ log q cannot exceed 1/2 if q is prime, and he conjectured this to be the case for all squarefree q. This indicates that N 3/4 is a limitation of Ruzsa's construction for square difference-free sets. Further, an easy Fourier analytic argument yields that if p is prime and A ⊆ (Z/pZ) 2 = G has no nonzero differences of the form (t, t 2 ), a set of forbidden differences similar in density and structure to the squares in [1, N ], then |A| ≪ p 3/2 = |G| 3/4 . These observations could potentially be viewed as evidence toward N 3/4 as roughly the true threshold for avoiding square differences, but these heuristics are rather tenuous. In particular, the same Fourier analytic argument applied to G = (Z/pZ) k gives an upper bound of about |G| 1− 1 2k for sets without differences of the form (t, t 2 , . . . , t k ), while Ruzsa's construction can beat this exponent for certain values of k. All of these questions are still massively open, and many believe these thresholds grow faster than N 1−ǫ for any ǫ > 0.
Preliminaries
In this section we make some preliminary definitions and observations required to execute the Fourier analytic double iteration strategy utilized to prove Theorem 1.1, as well as the more straightforward density increment utilized to prove Theorem 1.2. We also provide context and motivation in Section 2.4 for our most notable new ingredient, a polynomial specific sieve that is defined and discussed rigorously in Section 2.5, and we summarize our sieved exponential sum estimates in a stand alone theorem in Section 2.6.
2.1. Fourier analysis and the circle method on Z/N Z. We identify subsets of the interval [1, N ] with subsets of the finite group Z N = Z/N Z, on which we utilize a normalized discrete Fourier transform. Specifically, for a function F : Z N → C, we define F : Z N → C by When considering a set A ⊆ Z N , we employ a common abuse of notation by letting A(x) denote the characteristic function of A. We analyze the Fourier analytic behavior of A using the Hardy-Littlewood circle method, decomposing the nonzero frequencies into two pieces: the points t ∈ Z N such that t/N is close to a rational with small denominator, and the complement.
Definition 2.1. Given N ∈ N and K, Q > 0, we define, for each q ∈ N and a ∈ [1, q], It is important to note that as long as 2KQ 2 < N , we have that M a,q (N, K) ∩ M b,r (N, K) = ∅ whenever a/q = b/r, q, r ≤ Q.
Auxiliary polynomials. Suppose h ∈ Z[x]
is an intersective polynomial. For each prime p, we fix a p-adic integer z p with h(z p ) = 0. The objects defined below certainly depend on h, as well as on the choice of p-adic integer roots, though any choice works equally well for our purposes, and we suppress all of this dependence in the coming notation.
By reducing modulo prime powers and applying the Chinese Remainder Theorem, the choices of z p determine, for each natural number d, a unique integer r d ∈ (−d, 0], which consequently satisfies d | h(r d ).
We define the function λ on N by letting λ(p) = p m for each prime p, where m is the multiplicity of z p as a root of h, and then extending it to be completely multiplicative. For each d ∈ N, we define the auxiliary polynomial, h d , by If p j | d for p prime and j ∈ N, then since r d ≡ z p mod p j , we see by factoring h over the p-adic integers that all the coefficients of h(r d + dx) are divisible by p jm , hence each auxiliary polynomial has integer coefficients.
It is important to note that the leading coefficients of the auxiliary polynomials grow at least as quickly, up to a constant depending only on h, as the other coefficients.
, while the magnitude of every coefficient is (quite brutally) bounded by 2 k d k (max 0≤i≤k |a i |)/λ(d). This fact combines usefully with the following proposition, which carefully keeps track of the extent to which a polynomial behaves like its leading term with regard to preimages.
where ∆ denotes the symmetric difference.
Proof. Fixing h ∈ Z[x] and x > 0 as in the proposition, and letting y = (x/a k ) 1/k , we see that elements of the symmetric difference at hand arise in three ways: n ∈ N such that h(n) ≤ 0, y + n ∈ N with n > 0 such that h(y + n) < x, and y − n ∈ N with 0 ≤ n < y such that h(y − n) ≥ x. Defining R as in the proposition, it suffices to show that these sets have size at most ⌊R⌋, ⌊R⌋ + 1, and ⌊R⌋ + 1, respectively. To this end, we observe that for n ∈ N we have a k n k − Ra k n k−1 ≤ h(n) ≤ a k n k + Ra k n k−1 . In particular, h(n) > 0 if n > R. Carefully expanding a k (y + n) k − Ra k (y + n) k−1 , yields which is at least x provided that n ≥ R. Analogously, expanding a k (y − n) k + Ra k (y − n) k−1 yields We see that for 0 < n < y, both summations are alternating such that each negative term can be paired off with a positive term that is at least as large. Further, the latter summation is at least as large in magnitude as the former, hence h(y − n) < x for R < n < y, and the proposition follows.
In particular, if deg(h) = k and b d > 0 is the leading coefficient of h d , then for any x > 0 we have which will serve as a convenient observation for our exposition.
2.3. Inheritance proposition. We define these auxiliary polynomials to keep track of an inherited lack of prescribed differences at each step of a density increment iteration.
Proof. Suppose that A ⊆ N, A ′ ⊆ {a : x + λ(q)a ∈ A}, and a − a ′ = h qd (n) = h(r qd + qdn)/λ(qd) > 0 for some n ∈ N, a, a ′ ∈ A ′ . By construction we see that r qd ≡ r d mod d, so there exists s ∈ Z such that r qd = r d + ds, and therefore e 2πih(n)α is much smaller than the trivial bound M , unless α is well-approximated by a rational number with small denominator.
On a coarse scale, this principle is captured by combining the pigeonhole principle with Weyl's inequality (see Lemma 4.4), but for a more refined treatment, we must address the following question: If α is close to a rational with a small denominator, for example a denominator smaller than a tiny power of M , can we beat the trivial bound at all?
This question turns out to be quite straightforward, as under these conditions (4) has a convenient asymptotic formula, and the gain from the trivial bound resides in a local version of the sum. Specifically, if α is close to a/q with q small, then, up to a small error, the magnitude of (4) is at most M times In general, the best decay we can hope for from (5) is q −1/k , where k = deg(h) (see [3] for example). Unfortunately, it is completely vital to the double iteration method developed in [19] to establish (2) that one has decay at or near q −1/2 (what we refer to as square root cancellation) for small denominators q. Of course, this is not an obstacle if k = 2, a fact exploited in [8] to establish (2) for all intersective quadratics.
In [1], Balog, Pelikán, Pintz, and Szemerédi observed that by sieving the initial set of inputs, letting W equal a product of small primes and considering only inputs coprime to W , the gain from the trivial bound for α near a/q with small q is given roughly by In the case of h(n) = n k , one then exploits that, for all but the primes dividing k, units modulo p j for j ≥ 2 are k-th power residues if and only if they reduce to k-th power residues modulo p. This allows one to use orthogonality of characters and reduce to the case of squarefree q, where there is always square root cancellation.
However, a generic intersective polynomial need not have the root-lifting properties necessary to establish square root cancellation in (6), and so sieving away small prime factors is not a winning strategy for maximally extending (2). Alternatively, given an intersective polynomial h ∈ Z[x], we utilize a non-traditional sieve dependent on h, essentially defined by removing the zeros of the derivative h ′ modulo small primes, in order to mimic the aforementioned lifting observation using Hensel's Lemma.
2.5. Sieve definitions and observations. For an intersective polynomial h ∈ Z[x] and each prime p and d ∈ N, we define γ d (p) to be the smallest power such that the derivative h ′ d is not identically zero modulo p γ d (p) , and we let j d (p) denote the number of roots of h ′ d modulo p γ d (p) . Then, for d ∈ N and Y > 0 we define In the absence of a subscript d in the usage of γ(p), j(p), and W (Y ), we assume d = 1, in which case the definitions make sense even for non-intersective polynomials. Further, for any g ∈ Z[x] and q ∈ N, we define The size of W (Y ) can be estimated with a standard Brun sieve calculation, which for completeness we include below.
where c > 0 depends only on deg(g) and the collection of moduli for which g ′ is identically zero.
Proof. We fix g ∈ Z[x] and X > 0, and for each collection p 1 < p 2 < · · · < p s of primes, we let Fixing Y > 0 and letting r denote the number of primes that are at most Y , we have by the Chinese Remainder Theorem and the inclusion-exclusion principle that and moreover the true size lies between any two consecutive truncated alternating sums in s. Further, we know that We now observe that j(p) ≤ k − 1 if γ(p) = 1, and trivially j(p) ≤ p γ(p) . In particular, j(p) can be bounded above in terms of only deg(g) and the collection of moduli for which g ′ is identically zero, which allows us to apply the Chinese Remainder Theorem again and extend (9) to For this and the remainder of the proof we let C denote a positive constant, which may change from line to line but depends only on deg(g) and the collection of moduli for which g ′ is identically zero.
For any even 0 ≤ t ≤ r, we have by (8) and (10) that and we obtain analogous lower bounds by choosing odd 0 ≤ t ≤ r. To control E 1 , we observe that Using the standard fact that p≤Y 1/p ≪ log log Y , we then have If t > 2C log log Y , then each term of this series is at most half the previous, so the full tail is at most twice the first term. We then use the bound t! ≥ (t/e) t to establish To control E 2 , we use the trivial bound r s ≤ r s /s! to see that Since t ≤ r, each term of this series is at least twice the previous, so the full sum is bounded by double the final term. Since r ≪ Y / log Y by the Prime Number Theorem, and again t! ≥ (t/e) t , we have We now finish the proof by making a good choice for t. If Y ≤ log X, then we can choose t = r, in which case E 1 = 0 and E 2 ≤ C r ≤ exp(O(log X/ log log X)), which satisfies the proposition with room to spare. Finally, if Y > log X, then we choose t = log X C log Y . By our hypotheses on X and Y , this choice of t satisfies the lower bound needed for (11), and substituting this choice into (11) and (12) yields the desired error bounds.
As it applies to auxiliary polynomials, the conclusion of Proposition 2.4, as well as other steps in our future arguments, depends on the collection of moduli for which h ′ d is identically zero. The following observations assure that this collection of moduli remains under control.
Proof. We first note that g is identically zero modulo q if and only if the polynomial g/q is integer-valued. In this case, since the binomial coefficients form a Z-basis for integer-valued polynomials, we can write In particular, by clearing denominators we see that the coefficients of k!g are all divisible by q, and the proposition follows. For noting that any common factor of the coefficients of h ′ divides k!cont(h).
The last hurdle in controlling the set of "bad moduli" in our sieve, as well as a major issue in our future exponential sum estimates, is the possibility that the coefficients of the auxiliary polynomials h d gain larger and larger common factors as d grows, but the following lemma due to Lucier asserts that this is not case. Lemma 2.6 (Lemma 28, [15]).
we have now established control over not only the error term in the size of W d (Y ), but also the main term, since Proposition 2.5, Lemma 2.6, and the fact that h ′ d has at most k − 1 roots modulo every prime at which it is not identically zero, give 2.6. Summary of new exponential sum estimates. In Section 4, we combine new and old techniques to establish the sieved exponential sum estimates necessary to prove Theorem 1.1 and additional results. These estimates are obtained through a sequence of lemmas presented in the context of the larger proof, so we separately present a summary here in case the estimates are of independent interest to the reader.
For the following theorem, we utilize all the sieve-related notation and definitions from Section 2.5.
(ii) Square root cancellation: If (a, q) = 1 and Y > 0, then where C = C(k) and ω(q) is the number of distinct prime factors of q.
(iii) Minor arc estimate: If a k > 0, X, Y, Z ≥ 2, (a, q) = 1 and |α − a/q| < q −2 , then These estimates are obtained mostly by combining elementary sieve methods with traditional circle method techniques. The main novelty, the exponent 1/2 in estimate (ii), is established using Hensel's Lemma.
3. The Double Iteration Method: Proof of Theorem 1.1 In this section we execute an adapted version of the double iteration method originally developed in [19] and prove Theorem 1.1. We begin with an outline.
3.1.
Overview of the argument. We initially observe that if h ∈ Z[x] is an intersective polynomial, which by symmetry of difference sets we can assume has positive leading coefficient, and , then we can apply the circle method to show that this unexpected behavior implies substantial L 2 mass of A over nonzero frequencies near rationals with small denominator.
At this point, the traditional method, which we employ in Section 5 to prove Theorem 1.2, is to use the pigeonhole principle to conclude that there is one single denominator q such that A has L 2 concentration around rationals with denominator q. From this information, one can conclude that A has increased density on a long arithmetic progression with step size an appropriate multiple of q, leading to a new denser set with an inherited lack of polynomial differences and continued iteration.
Pintz, Steiger, and Szemerédi [19] observed that pigeonholing to obtain a single denominator q is a potentially wasteful step. We follow their approach, observing the following dichotomy: There is a single denominator q such that A has extremely high L 2 concentration, greater than yielded by the pigeonhole principle, around rationals with denominator q. This leads to a very large density increment on a long arithmetic progression.
Case 2. The L 2 mass of A on the major arcs is spread over many denominators. In this case, an iteration procedure using the "combinatorics of rational numbers" can be employed to build a large collection of frequencies at which A is large, then Plancherel's identity is applied to bound the density of A.
Philosophically, Case 1 provides more structural information about the original set A than Case 2 does. The downside is that the density increment procedure yields a new set and potentially a new polynomial, while the iteration in Case 2 leaves these objects fixed. With these cases in mind, we can now outline the argument, separated into two distinct phases. If the set A falls into Case 1, then the density increment procedure yields a small q ∈ N and a new subset A 1 of a slightly smaller interval with significantly greater density, and We can then iterate this process as long as the resulting interval is not too small, and the dichotomy holds as long as the coefficients of the auxiliary polynomial are not too large. We show that if the resulting sets remain in Case 1 until the interval shrinks down or the coefficients grow to the limit, then the density of the original set A must have satisfied a bound stronger than the one purported in Theorem 1.1. Contrapositively, we assume that the original density does not satisfy this stricter bound, and we conclude that one of the sets yielded by the density increment procedure must lie in a large interval, have no differences in I(h d ) for reasonably small d, and fall into Case 2. We call that set B ⊆ [1, L].
We now have a set B ⊆ [1, L] with (B − B) ∩ I(h d ) = ∅ which falls into Case 2, so we can adapt the strategy of [19], [1], and [8]. It is in this phase that we use the sieve outlined in Section 2.5, as the method breaks down without square root cancellation on the major arcs.
Phase 2 (The Inner Iteration): We prove that given a frequency s ∈ Z L with s/L close to a rational a/q such that B(s) is large, there are lots of nonzero frequencies t ∈ Z L with t/L close to rationals b/r such that B(s + t) is almost as large. This intuitively indicates that a set P of frequencies associated with large Fourier coefficients can be blown up to a much larger set P ′ of frequencies associated with nearly as large Fourier coefficients. The only obstruction to this intuition is the possibility that there are many pairs (a/q, b/r) and [19] and [1] on the combinatorics of rational numbers demonstrate that this potentially harmful phenomenon can not occur terribly often.
Starting with the trivially large Fourier coefficient at 0, this process is applied as long as certain parameters are not too large, and the number of iterations is ultimately limited by the growth of the divisor function. Once the iteration is exhausted, we use the resulting set of large Fourier coefficients and Plancherel's Identity to get the upper bound on the density of B, which is by construction larger than the density of the original set A, claimed in Theorem 1.1.
3.2.
Reduction to two key lemmas. For the remainder of Section 3, we fix an intersective polynomial h ∈ Z[x] with positive leading coefficient and deg(h) = k ≥ 2, and we let We fix ǫ > 0 and let m = k 2 + k + 2ǫ, with the goal of establishing Theorem 1.1 with c = (1 − 17ǫ)/ log(m/2). We note that any dependence of implied constants on m or is actually just dependence on k and ǫ.
We also fix a natural number N , and we let We preemptively assume that N is sufficiently large at all junctures with respect to h and ǫ, an assumption that is permitted because the implied constant in Theorem 1.1 is allowed to depend on those parameters. We deduce Theorem 1.1 from two key lemmas, corresponding to the two phases outlined in the overview in Section 3.1, the first of which yields a set with substantial Fourier L 2 mass distributed over rationals with many small denominators.
The second lemma corresponds to the iteration scheme in which a set of large Fourier coefficients from distinct major arcs is blown up in such a way that the relative growth of the size of the set is much greater than the relative loss of pointwise mass.
3.3. Proof of Theorem 1.1. In order to establish Theorem 1.1, we can assume that δ ≥ (log N ) − log log log log N .
Therefore, Lemma 3.1 produces a set B of density σ ≥ δ with the stipulated properties, and we set P 0 = {0}, U 0 = 3, and V 0 = K 0 = 1. Then, Lemma 3.2 yields, for each n, a set P n with parameters U n , V n , K n such that max{U n , V n , K n } ≤ (C log N ) (m/2) n+1 log log log log N , where the left-hand inequality comes from Plancherel's Identity, as long as max{U n , V n , K n } ≤ Q 1/m . This holds with n = (1 − ǫ)(log log log log N )/ log(m/2), as (m/2) n+1 ≤ (log log log N ) 1−ǫ/2 , and the theorem follows.
3.4. The Outer Iteration. We begin the first phase with the following standard L 2 density increment lemma, and we provide a proof for the sake of completeness.
then there exists an arithmetic progression Proof. Suppose B ⊆ [1, L] with |B| = σL. Suppose further that (20) t∈M ′ q (L,K) | B(t)| 2 ≥ θσ 2 and let P = {q, 2q, . . . , Xq} ⊆ Z L with X = ⌊min{θ, K −1 }L/16q⌋. We will show that either all or a portion of some translate of P , lifted to the integers, satisfies the conclusion of Lemma 3.3, with either L ′ = X or L ′ = ⌈σX/2⌉. We note that for t ∈ Z L , where · denotes the distance to the nearest integer. Further, if t ∈ M ′ q (L, K), then (22) qt/L ≤ qK/L ≤ 1/4πX. Therefore, by (21) and (22) we have To investigate the bias of B toward translations of P , we introduce the balanced function f B : Z L → R defined by f B (x) = B(x) − σ, noting by orthogonality of characters that By (20) and (23) we see Then, by (24), (25), Plancherel's Identity, and the fact that the Fourier transform takes convolutions to products (which in this finite context follows easily from orthogonality of characters), we have where P (n) = P (−n), and P + x = {x + ℓq : 1 ≤ ℓ ≤ X}. We now take advantage of the fact that f B , and consequently f B * P , has mean value zero. In other words, n∈Z f B * P (n) = 0.
As with any real valued function, we can write For a moment, let us suppose that f B * P (x) > σX/L for some x ∈ Z L . We note that P + x either lifts to a genuine arithmetic progression of integers, or the disjoint union of two such progressions. In the former case, P + x satisfies the conclusion of the lemma. In the latter case, if we call the two progressions P 1 and P 2 and their lengths X 1 and X 2 , then we have (|B ∩ P 1 | − σX 1 ) + (|B ∩ P 2 | − σX 2 ) ≥ σX. Consequently, at least one of the two progressions has length at least σX/2 and satisfies the required relative density condition.
With this observation made, we can now assume that f B * P (x) ≤ σX/L for all x ∈ Z L , which combined with the trivial bound of f B * P (x) ≥ −σX/L yields Then, by (26), (27), (28), and (29), we have We now let E denote the reduced residues x ∈ Z L such that x + Xq, as an integer, is less than L. Crucially, this means that if x ∈ E, then P + x does not "wrap around" and in fact lifts to a genuine arithmetic progression of integers. We see that |Z L \ E| ≤ qX, we have assumed f B * P is bounded above by σX/L, and by our choice of X we have qX ≤ θL/16, hence Noting that |E| ≤ L and recalling that L f B * P (x) = |B ∩ (P + x)| − σX, we have that there exists x ∈ Z such that the genuine arithmetic progression P + x ⊆ Z satisfies |B ∩ (P + x)| ≥ σ(1 + θ/16)X, as required.
Noting that the progression P in the conclusion of Lemma 3.3 can be partitioned into subprogressions of step size λ(q) ≤ q k , Lemma 3.3 and Proposition 2.3 immediately combine to yield the following iteration lemma, corresponding to Case 1 discussed in the overview, from which we deduce Lemma 3.1.
Proof of Lemma 3.1. Setting A 0 =A, N 0 = N , δ 0 = δ, and d 0 = 1, we iteratively apply Lemma 3.4. This yields, for each j, a set A j ⊆ [1, N j ] with |A j | = δ j N j and where C is an absolute constant, as long as either as the latter condition implies A j has density at least 3δ j /2 on the interval (N j /2, N j ], in which case we can take A j+1 to be a shift of A j ∩ (N j /2, N j ]. We see that by (14) and (32), the density δ j will exceed 1 after steps, hence (33) fails and |A j ∩ [1, N j /2]| ≥ δ j N j /3 for some However, we see that (14), (32), and (34) imply so we set B = A j , L = N j , σ = δ j , and d = d j , and we see further that Our task for Section 3 is now completely reduced to a proof of Lemma 3.2. and where we also use (13) to absorb the error term.
Remark. As discussed in Section 2.4, the inability to establish the exponent of −1/2 in (38), which we obtain thanks to our careful sieving of inputs and the application of Hensel's Lemma, had previously been the fundamental obstacle in extending this method. We highlight the need for this exponent at the end of the proof and discuss these estimates in detail in Section 4.
We have by (37), Cauchy-Schwarz, and Plancherel's Identity that which together with (36) yields We now wish to assert that we can ignore those frequencies in the major arcs at which the transform of B or B 1 is particularly small. In order to make this precise, we first need to invoke a sieved, weighted version of known estimates on the higher moments of Weyl sums.
Specifically, we have that where C = C(m).
Remark on (40) and the choice of m > k 2 + k. Without any sieving of inputs, Theorem 1.1 of [2] and the proof of Proposition 3.3 in [18] give the desired high moment estimate for m = k 2 + k, with a small power loss. As shown in the "ǫ removal argument" in Section 5 of [2], one can apply major and minor arc estimates and eliminate the small power loss by raising the exponent by any amount. The only additional insight required here is that this same trick can be used, with our major and minor arc estimates established in Section 4, to also eliminate any potential loss caused by the sieving of inputs.
Choosing a constant 0 < c 1 < (40C 1/m ) −m/2 , where C comes from (40), we define Using Hölder's Inequality to exploit the higher moment estimate on S, followed by Plancherel's Identity, we see that and hence by (39) we have For i, j, ℓ ∈ N, we define where the maximums are taken over nonzero frequencies t ∈ M a/q (L, η −1 ). We see that we have
|S(t)|.
Examining (38) more closely, we see that the bound of 1 inside the minimum will be used for at most two values of t, and for the remaining values of t that minimum can be bounded sequentially by 1/2, 1/3, . . . , η.
In particular, if we sum over all t ∈ M a/q (L, η −1 ), that minimum contributes a total of ≪ log(η −1 ). Therefore, it follows from (38), the bound U, σ −1 ≤ Q 1/m , and the standard estimate ω(q) ≪ log q/ log log q, that if (a, q) = 1 and q ≤ η −(2+ǫ) , then t∈M a/q (L,η −1 ) Therefore, by (43) we have By our definitions, the sets R i,j,ℓ exhaust Y by taking 1 ≤ 2 i ≤ η −(2+ǫ) and 1 ≤ 2 j , 2 ℓ ≤ U m/2 /c 1 σ (m−1)/2 , a total search space of size ≪ (log Q) 3 . Therefore, by (42) and (44) there exist i, j, ℓ in the above range with In other words, we can set V s = 2 i , W s = 2 j , and U s = 2 ℓ and take an appropriate nonzero frequency from each of the pairwise disjoint major arcs specified by R i,j,ℓ to form a set noting by disjointness that a/q ∈ R i,j,ℓ whenever q ≤ V s and M a/q (L, η −1 ) ∩ P s = ∅.
We now observe that by pigeonholing there is a subsetP ⊆ P with |P | ≫ |P |/(log Q) 3 , and hence for which the triple U s , W s , V s is the same. We call those common parametersŨ ,W andṼ , respectively, and we can now foreshadow by asserting that the claimed parameters in the conclusion of Lemma 3.2 will be U ′ =Ũ , V ′ =Ṽ V , and K ′ = K + η −1 , which do satisfy the purported bound.
We let By taking one frequency s + t associated to each element in R, we form our set P ′ , which immediately satisfies conditions (17) and (18) from the conclusion of Lemma 3.2. However, the crucial condition (19) on |P ′ |, which by construction is equal to |R|, remains to be shown. To this end, we invoke the work on the combinatorics of rational numbers found in [19] and [1]. It is a well-known fact of the divisor function that τ (n) ≤ n 1/ log log n for large n, and since η −1 , VṼ ≤ Q, we have that τ ≤ (log N ) ǫ . We also have from (15) that where the last inequality follows from (46), and hence E ≤W 2 (log N ) −1+ǫ .
Combining the estimates on τ and E with (45), (47), and Lemma 3.5, we have Recalling that we set U ′ =Ũ , we see that for sufficiently large N we have as claimed.
Remark on the necessity of square root cancellation. We now retrospectively observe that the exponent −1/2 in (38) directly leads to the exponent 1/2 onṼ in (45). That factor ofṼ 1/2 is then squared via Lemma 3.5, which then cancels the factor ofṼ in the denominator in (49). As the logarithmic gain in |P ′ |/(U ′ ) 2 is the crux of the method, the argument completely breaks down without this full cancellation ofṼ .
Remark on the hypothesis max{U, V, K, σ −1 } ≤ Q 1/m in Lemma 3.2. We make this assumption for convenience, as it ensures that not only the original parameters U, V, K, but also the new parameters U ′ , V ′ , K ′ , are bounded above by Q. This precise upper bound is only strictly crucial for the intermediate parameter V , as it is required to apply (15) in (48). This hypothesis can be carefully softened, though doing so does not lead to a stronger final result.
Remark on possible values of c in Theorem 1.1. In examining the proof of Theorem 1.1, we see that the value of c in that theorem is determined by the exponent m/2 on max{U, V, K} in the conclusion of Lemma 3.2. In examining the proof of Lemma 3.2, we see that this particular exponent is necessitated by the application of the high moment estimate (40), which leads to the definition of X in (41). The quoted result from [2] makes our choice of m > k 2 + k sharp, so for our chosen proof technique, our range for c is optimal. However, in [1], which restricts to h(n) = n k , the constant c = 1/4 is obtained independent of k, and with slight modification can be made arbitrarily close to 1/ log 3, as is done in [8]. However, this seems to be a hard limit of the method, as even if the exponent on max{U, V, K} in Lemma 3.2 could be taken independent of the high moment estimate, we would still be limited by the upper bound V ′ ≪ V η −(2+ǫ) ≪ V U 2+ǫ σ −(2+ǫ) seen in the proof, which requires that exponent to be at least 3.
Exponential Sum Estimates
In this section, we either invoke or prove all exponential sum estimates necessary to establish the crucial major and minor arc upper bounds in Section 3.5, namely (37), and (38). Throughout this section, given a polynomial g ∈ Z[x], we let γ(p), j(p), and W (Y ) be defined in terms of g as in Section 2.5. The first lemma provides asymptotic formulae for the relevant sifted Weyl sums near rationals with small denominator. Lemma 4.1. Suppose k ∈ N, g(x) = a 0 + a 1 x + · · · + a k x k ∈ Z[x], and let J = |a 0 | + |a 1 | + · · · + |a k |. If X, Y > 0, a, q ∈ N, α = a/q + β, and c log(X/q) ≥ log Y log log Y , then where c = c(k, cont(g)) > 0.
Proof. We begin by noting that for any a, q ∈ N and 0 ≤ x ≤ X, since for s ∈ W q (Y ) we have by the same calculation as Proposition 2.4 and partial summation that whereas for s / ∈ W q (Y ) the sum is zero. We note that c > 0 depends only on k and cont(g) by Prop. 2.5.
Then, by partial summation we have that if α = a/q + β, then Finally, noting that the lemma follows.
To establish the required square root cancellation for the restricted exponential sums that arise in the conclusion of Lemma 4.1, we use the following standard fact about lifting roots of polynomials, the details of which motivate the sieve outlined in Section 2.5. Here we focus only on the existence of the lifts, ignoring the extent of uniqueness or "closeness", and this statement follows, for example, from Proposition 2 in Chapter II, Section 2 of [11].
Armed with Lemma 4.2, we exhibit the restricted exponential sum estimate that was previously the missing ingredient to proving Theorem 1.1.
with deg(g) = k ≥ 2, a, q ∈ N with (a, q) = 1, and Y > 0, then where C = C(k) and ω(q) is the number of distinct prime factors of q.
Proof. Factor q = q 1 · · · q 4 , where q 1 , . . . , q 4 are pairwise coprime, q 1 houses the prime power factors p j of q satisfying p ≤ Y , γ(p) > 1, and j < 2γ(p), q 2 is a product of distinct primes p ≤ Y satisfying γ(p) = 1, q 3 is the product of p j satisfying p ≤ Y , j ≥ 2γ(p), and all prime factors of q 4 are greater than Y .
By the Chinese Remainder Theorem, we have where a 1 , . . . , a 4 are the unique residues satisfying a q ≡ a 1 q 1 + · · · + a 4 q 4 mod 1.
In other words, the map F on Z/p j−ℓ Z defined by g(p ℓ s 2 + s 1 ) ≡ p ℓ F (s 2 ) + g(s 1 ) mod p j is a bijection. In particular, if p ∤ b, then where the last equality is the fact that the sum in s 2 runs over the full collection of p j−ℓ -th roots of unity. Therefore, we have that Finally, noting that W q4 (Y ) = N, we utilize the standard complete sum estimate (see [3] for example) q4−1 s=0 e 2πig(s)a4/q4 ≪ k gcd(cont(g), q 4 ) 1/k q 1−1/k 4 , and the lemma follows.
We now invoke a variation of the most traditional minor arc estimate, Weyl's Inequality.
Lemma 4.4 (Lemma 3 in [4]
). Suppose k ∈ N, g(x) = a 0 + a 1 x + · · · + a k x k with a 1 . . . , a k ∈ R and a k ∈ N. If X > 0, a, q ∈ N with (a, q) = 1, and |α − a/q| < q −2 , then Finally, we carefully adapt Lemma 4.4 to our particular sieve to get the desired estimates far from rationals with small denominator.
Lemma 4.5. Suppose k ∈ N, g(x) = a 0 + a 1 x + · · · + a k x k ∈ Z[x] with a k > 0. Suppose further that X, Y, Z ≥ 2 and a, q ∈ N with (a, q) = 1. If |α − a/q| < q −2 , then Proof. Let r be the number of primes that are at most Y , and let P be the set of products p γ(p1) 1 · · · p γ(ps) s with p 1 < · · · < p s ≤ Y . By inclusion-exclusion and Proposition 2.5, While the first inequality above is simply the triangle inequality and the combination of the outer two summations, the second inequality uses a few things. Specifically, the inner range of summation in the second line is a disjoint union of arithmetic progressions modulo D, where the number of progressions is the product of the number of roots of g ′ modulo p γ(pi) i . We then use that if γ(p) = 1, then g ′ has fewer than k roots modulo p, while the cont(g) 2 term accounts for the primes p for which γ(p) > 1, as Proposition 2.5 guarantees that p γ(p) ≤ p 2(γ(p)−1) ≪ k cont(g) 2 .
Further, we see from Lemma 4.4 that where the last inequality uses that if C > 0, then If D ∈ P with D > Z, then, since D ≪ k cont(g) 2 Y ω(D) and Y ≥ 2, we know that Finally, by trivially bounding the inner sum and applying (51) and (50), we have and the estimate follows.
4.1.
Proof of (37) and (38). We return to the setting of the proof of Lemma 3.2 in Section 3.5, recalling all assumptions, notation, and fixed parameters. Further, we let Fixing t ∈ Z L , the pigeonhole principle guarantees the existence of 1 ≤ q ≤ L/Z 2k and (a, q) = 1 with Letting β = t/L − a/q, we have by Lemma 4.1 and (13) that We note that w q ≪ h 2 ω(q) by Proposition 2.5 and Lemma 2.6.
Finally, recalling that the leading coefficient b d of h d satisfies b d ≪ h d k ≤ L kρ , we have by Lemma 4.5, Lemma 2.6, (52), and partial summation that if L 2kρ ≤ q ≤ L/Z 2k , then and in particular (37) holds.
Single Iteration Method: Proof of Theorem 1.2
In this section, we further exploit the estimates established in Section 4 and apply a more traditional L 2 density increment, essentially an improved, streamlined version of Sárközy's [24] original method, in order to prove Theorem 1.2. The core of this method has been utilized in [15], [17], [23], and [21], among others.
We also provide a brief discussion on sums of three or more polynomials in Section 5.7. We begin with another preliminary discussion of the circle method, this time with a continuous frequency domain.
5.1. Fourier analysis and the circle method on Z. For this argument, rather than identify an interval of integers with a cyclic group, we embed our finite sets in Z, on which we utilize an unnormalized discrete Fourier transform. Specifically, for a function F : Z → C with finite support, we define F : T → C, where T denotes the circle parameterized by the interval [0, 1] with 0 and 1 identified, by Given N ∈ N and a set A ⊆ [1, N ] with |A| = δN , rather than singling out the zero frequency, we examine the Fourier analytic behavior of A by considering the balanced function, f A , defined by We then define the major and minor arcs on T, analogous to our definitions from Section 2.1.
is a collection of intersective polynomials, d 1 , . . . , d ℓ ∈ N, and A ⊆ N. If x ∈ Z, q ∈ N, for some n 1 , . . . , n ℓ ∈ N, a, a ′ ∈ A ′ , with all polynomial terms having the same sign as the corresponding leading coefficient.
By construction we know that r We deduce Theorem 1.2 from the following iteration lemma, which states that a set deficient in the desired pattern spawns a new, significantly denser subset of a slightly smaller interval with an inherited deficiency in the pattern associated to appropriate auxiliary polynomials. and for some c = c(g, h) > 0 and C = C(k, ℓ).
Proof of Theorem 1.2. Throughout this proof, we let C and c denote sufficiently large or small positive constants, respectively, which we allow to change from line to line, but can depend only on g and h. We use C ′ and c ′ similarly, but these constants can depend only on k and ℓ.
1 , d Further, we have that as long as However, we see that the density δ m will exceed 1, and hence (59) must fail for m = C log C ′ (δ −1 ), which by (56) and (58) yields (cδ) −C log C ′ (δ −1 ) ≥ e √ log N , and hence This establishes Theorem 1.2 outside of the claim that we can take c ′ = 1/2 if deg(g) = deg(h) = 2, which we discuss in Section 5.7.
5.4. Deducing Lemma 5.3 from L 2 Fourier concentration. The philosophy behind the proof of Lemma 5.3 is that a deficiency in polynomial differences in a set A represents nonrandom behavior, which should be detected in the Fourier analytic behavior of A. Specifically, we locate one small denominator q such that f A has L 2 concentration around rationals with denominator q, then use that information to find a long arithmetic progression on which A has increased density. | f A (α)| 2 dα ≫ g,h δ 2 log −C (δ −1 )N for some C = C(k, ℓ).
Lemma 5.3 follows from Lemma 5.4 and the following standard L 2 density increment lemma, the continuous analog of Lemma 3.3.
Partitioning P into subprogressions of step size Λ(q) = λ 1 (λ 2 (q)), the pigeonhole principle yields a progression Our task for this section is now completely reduced to a proof of Lemma 5.4. 5.5. Preliminary notation for proof of Lemma 5.4. Before delving into the proof of Lemma 5.4, we take the opportunity to define some relevant sets and quantities, depending on our intersective polynomials g, h ∈ Z[x], scaling parameters d 1 , d 2 , a parameter Y > 0, and the size of the ambient interval N . In all the notation defined below, we suppress all of the aforementioned dependence, as the relevant objects will be fixed in context.
d1 , and j (1) d1 in terms of g as in Section 2.5. We then define H 1 to be the collection of natural number inputs m ∈ W (1) d1 (Y ) such that g d1 (m) is strictly between 0 and ±N/18, where the sign is the sign of the leading coefficient of g. We let M 1 = N 18|b| 1/k , where b is the leading coefficient of g d1 , and we let . | 2018-11-14T15:34:41.000Z | 2016-12-06T00:00:00.000 | {
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245133991 | pes2o/s2orc | v3-fos-license | Early Transcriptional Changes in Rabies Virus-Infected Neurons and Their Impact on Neuronal Functions
Rabies is a zoonotic disease caused by rabies virus (RABV). As rabies advances, patients develop a variety of severe neurological symptoms that inevitably lead to coma and death. Unlike other neurotropic viruses that can induce symptoms of a similar range, RABV-infected post-mortem brains do not show significant signs of inflammation nor the structural damages on neurons. This suggests that the observed neurological symptoms possibly originate from dysfunctions of neurons. However, many aspects of neuronal dysfunctions in the context of RABV infection are only partially understood, and therefore require further investigation. In this study, we used differentiated neurons to characterize the RABV-induced transcriptomic changes at the early time-points of infection. We found that the genes modulated in response to the infection are particularly involved in cell cycle, gene expression, immune response, and neuronal function-associated processes. Comparing a wild-type RABV to a mutant virus harboring altered matrix proteins, we found that the RABV matrix protein plays an important role in the early down-regulation of host genes, of which a significant number is involved in neuronal functions. The kinetics of differentially expressed genes (DEGs) are also different between the wild type and mutant virus datasets. The number of modulated genes remained constant upon wild-type RABV infection up to 24 h post-infection, but dramatically increased in the mutant condition. This result suggests that the intact viral matrix protein is important to control the size of host gene modulation. We then examined the signaling pathways previously studied in relation to the innate immune responses against RABV, and found that these pathways contribute to the changes in neuronal function-associated processes. We further examined a set of regulated genes that could impact neuronal functions collectively, and demonstrated in calcium imaging that indeed the spontaneous activity of neurons is influenced by RABV infection. Overall, our findings suggest that neuronal function-associated genes are modulated by RABV early on, potentially through the viral matrix protein-interacting signaling molecules and their downstream pathways.
INTRODUCTION
Rabies is a fatal disease that occurs through the transmission of lyssavirus, mainly by one of the species called rabies virus (RABV) (Fooks et al., 2014(Fooks et al., , 2017Hampson et al., 2015). The virus is transmitted from infected animals to humans through broken skin caused by bites or scratches, or on rarer occasions, licking on the mucosa. RABV transmission from dog-mediated exposures is responsible for more than 98% of human rabies cases, leading to approximately 59,000 deaths every year. Rabies is hard to diagnose without a history of exposure since at the initial stage of the disease the symptoms are very similar to that of many other diseases (Hemachudha et al., 2013). However, as rabies advances, more specific symptoms appear which may include insomnia, anxiety, confusion, paralysis, excitation, hallucinations, agitation, difficulty in swallowing, hydrophobia and aerophobia (Jackson, 2014). The fatality rate is close to 100% once these neurological symptoms have manifested, and as to this date, there are no known cures or treatments that can effectively block the advancement of RABV (Dacheux et al., 2011;Rogée et al., 2019).
Human rabies cases do not show prominent pathological features such as neuron degeneration, adaptive immune cell infiltration, aggravated inflammation, or apoptosis during routine histopathological analysis (Adle-Biassette et al., 1996;Jackson, 2014;Davis et al., 2015;Fisher et al., 2018). This is due to the fact that RABV is capable of evading or delaying immune responses and cell death for the duration of replication and spread in the infected body. RABV encodes five viral proteins in its genome, Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Glycoprotein (G), and Large RNA polymerase protein (L), all of which actively mediate signaling pathways to subvert innate immune responses. Three of the viral proteins (P, M, and G) have been more extensively studied. The C-terminal domain of P protein is known to bind to STAT1/2, therefore blocks the translocation of the STAT protein complex (Vidy et al., 2005;Wiltzer et al., 2014) and suppresses the expression of antiviral effectors. M protein has been demonstrated to interact with RelAp43, a splicing analog of RelA (Luco et al., 2012;Khalifa et al., 2016;Besson et al., 2017) and intervenes with the activation of IFN-β and TNF-α. Conversely, a mutant of RABV, in which four critical residues were altered on M protein gene to hinder the interaction with RelAp43, showed to induce a stronger inflammatory cytokine response and a lower pathogenesis in vivo than the wild type virus (Sonthonnax et al., 2019). G protein also contributes to the replication of the virus by associating with MAST2 and PTPN4, which alters the balance of PI3K-AKT pathway and enables the infected neuron to evade apoptosis (Préhaud et al., 2010;Caillet-Saguy et al., 2015). The two other viral proteins, N (Masatani et al., 2013) and L (Tian et al., 2016) proteins have shown to be involved in the modulation of the host immune response but their mechanisms of action have been less studied. Even though these modulations by RABV may partially explain the lack of major inflammation and neuronal death, and how the network of neurons is relatively well preserved up until the demise of the host, the mechanism of neuronal dysfunction, which is the probable cause of neurological symptoms, remains elusive.
Different studies showed that the virus or virus infectionassociated changes in host gene expression could affect neuron activity. Inflammatory cytokines, such as TNF-α, IFN-γ or IL-1β were demonstrated to desensitize neurons to GABA stimulation and subsequently cause hyperexcitability (Clarkson et al., 2017). Increase of inflammatory cytokines in the brain could result in cognitive impairment and seizure as demonstrated in animal models (Nimmervoll et al., 2013;Habbas et al., 2015). Phosphoprotein of Borna disease virus competes with substrate proteins of PKC and decreases the phosphorylation level of PKC downstream proteins, such as SNAP25 and MARCKS. This action directly affects the synaptic vesicle recycling and structural integrity of synapse, and therefore disrupting synaptic plasticity (Bétourné et al., 2018). In addition, there are studies showing hyperpolarization and disturbance of neuronal network synchronicity as a result of RABV infection (Gourmelon et al., 1991;Ceccaldi et al., 1993;Iwata et al., 1999;Fu and Jackson, 2005); This was shown to be partly due to the lower membrane conductance of Na + and possibly reduced functional expression of the corresponding ion channels. However, the changes in membrane potential and the associated neuronal dysfunction could originate from multiple other mechanisms that remain unexplored, and more importantly, their connections to the RABV infection and the altered signaling pathways in neurons have not yet been properly studied.
RNA sequencing has been a crucial tool to understand gene expression kinetics in comparative studies. Here, we analyze the RNA transcriptome of RABV-infected human neurons in the early hours of infection and attempt to identify the origin of RABV-induced neuronal dysfunction, tracking the signaling pathways that are directly affected by the viral modulation.
Viruses and Reverse Genetics
Two recombinant rabies viruses were generated through reverse genetics from the rabies strain 8743THA (EVAg collection, Ref-SKU: 014V-02106), an isolate collected from the brain of a man who died of rabies after a dog bite in Thailand. In both viruses, E2-Crimson fluorescence gene (Clontech, 632556) was inserted between M and G protein genes of RABV to obtain, the wild-type virus, Tha-E2-Crimson (Tha) and an M protein mutant virus, Th4M-E2-Crimson (Th4M). The matrix protein of Th4M virus differs from Tha's at 4 amino acid positions: R77K, D100A, A104S and M110L. Both viruses were constructed and rescued as already described (Khalifa et al., 2016;Besson et al., 2019). Full-length viral cDNA (2.5 µg) and plasmids N-pTIT (2.5 µg), P-pTIT (1.25 µg), and L-pTIT (1.25 µg) were transfected in 106 BSR T7/5 cells (Buchholz et al., 1999) in 6-well plates. Cells were then passaged every 3 days until 100% of the cells were infected. The supernatant was harvested and titrated on BSR cells to determine the multiplicity of infection (MOI), following a previously described titration method (Hellert et al., 2020).
Differentiation and Infection of Neurons
hiPSC-derived NPCs were maintained only up to passage number 10 for the usage of neuronal differentiation, in DMEM/F12 medium (DMEM/F12/Glutamax (Gibco 10565-018), supplemented with N-2 1X (Gibco 17502-048), B27 1X (without vitamin A, Gibco 12587010), Penicillin/Streptomycin 1% (Gibco, 15140-122), HEPES 20 mM (Gibco, 15630080), MEM Non-essential amino acid 1X (Gibco, 11140050), bFGF 20 ng/ml (CHAMEDITECH) to the final concentration) and on Poly-L-Ornithine (Sigma P4957) and Laminin (Sigma L2020)-coated T25 flasks. Medium for maintenance was refreshed every second day. On day 0 of differentiation, the NPCs were dissociated by incubation with Accutase (StemPro, A1110501) for 30 min at 37 • C, counted and seeded onto PLO/Laminin-coated 96-well plates or 24-well plates for RNA-Seq and RT-qPCR, or 384-well plates for calcium imaging with 10 µM ROCK inhibitor (Y27632; Tocris 1254). The ROCK inhibitor was withdrawn within 2 h after seeding, and fresh Neurobasal medium for differentiation (Neurobasal (Gibco 21103-049), supplemented with B27 2%, and Glutamax 1X to the final concentration) was added onto the cells. The differentiation medium was 50% refreshed every 2∼3 days for 2 weeks. On day 14 of differentiation, the hiPSCderived neurons were subjected to RABV infection with a MOI 3 and media was changed after 2 h. Two days post-infection hiPSC-derived neurons were subjected to RNA preparation for RNA-Seq or RT-qPCR, and 5 days post-infection were fixed with 4% paraformaldehyde (Thermo Fisher Scientific, AAJ19943K2) and analyzed by immunocytochemistry or were processed for calcium imaging. H9-NSCs were maintained and differentiated in N2B27 medium on PLO/Laminin-coated flasks. N2B27 medium was 1:1 mixture of N2 and B27 media: N2 medium was composed of DMEM/F12/Glutamax, N-2 1X, MEM Non-essential amino acids 1X, Insulin 5 µg/ml (Sigma I9278-5ML), 2-Mercapoethanol (Sigma M7522) and Pen/Strep 1% to the final concentration. B27 medium was composed of Neurobasal, B27 1X, Glutamax 1X, Pen/Strep 1% to the final concentration. EGF 20 ng/ml (Millipore GF144) and bFGF 20 ng/ml were added to N2B27 medium to complete NSC maintenance medium. For differentiation medium, B27 with vitamin A (Gibco, 17504044) was used instead and the growth factors were withdrawn. BDNF (Peprotech, 450-02) and GDNF (Peprotech, were added to the differentiation medium to aid neuron survival at a final concentration of 10 ng/ml each. On day 0 of H9-NSC differentiation, cells were detached with Accutase and seeded in T25 flasks in 20% confluence. Within 2 h after seeding, the medium was replaced by N2B27 differentiation medium and once more the next day. The medium was refreshed every second day until day 12. On day 12, the post-mitotic H9 neurons were gently dissociated from T25 flasks and seeded onto 24-well plates for RT-qPCR or 384well plates for calcium imaging with ROCK inhibitor. The inhibitor was withdrawn within 2 h, and the differentiation medium was 50% refreshed every 2∼3 days up until day 27. The neurons were subjected to RABV infection as above and subsequently used for calcium imaging and RT-qPCR at 2 days post-infection.
RNA Preparation, RNA-Seq and Data Processing
Total RNAs from collected cell pellets were prepared using Trizol (Invitrogen, Cat. No. 15596026) and ethanol precipitation. For RNA-Seq, non-infected/infected neurons were collected in biological triplicates at 0, 8, 24, and 40 h post-infection using Accutase and kept as pellets at -80 • C until the RNA extraction. RNA preparation followed the Trizol manufacturer's protocol, with addition of Glycogen 10 µg per sample to increase RNA yield. After that, RNAs were treated with DNase I (Promega, Cat. No. M6101) for 30 min at 37 • C followed by second Trizol extraction. The acquired RNAs were mixed with 3 volumes of ethanol and 0.1 volumes of 3M sodium acetate, and kept at -20 • C for overnight. Next day, the samples were centrifuged and washed with 75% ethanol twice to achieve RNAs in high purity. Sequencing libraries were constructed from the polyA RNA fraction using Illumina TruSeq Stranded mRNA library kit and were sequenced on the Illumina NovaSeq 6000 targeting 12 giga-base reads per sample. After that, raw data was subjected to data cleaning to remove adapter sequences and low-quality sequences using cutadapt version 1.11 (Martin, 2011). Only the sequences greater than 25 nucleotides (nt) in length were considered for further analysis. Mappings to reference genomes were conducted using STAR version 2.5.0a (Dobin et al., 2013) for human DNAs (reference genome: Human (GRCh38) from ENSEMBL version 98) and Bowtie version 1.2.2 (Langmead et al., 2009) for viral DNAs (reference genome: rabies virus genome from NCBI) with default parameters. Following this, genes were counted using featureCounts version 1.4.6-p3 (Liao et al., 2014) from the Subreads package (parameters: -t gene -g gene_id -s 2 -p). An estimated expression level (or read count) for each annotated gene was achieved, and statistical analyses followed using R packages, including DESeq2 (Love et al., 2014). The statistical analysis process includes data normalization, graphical exploration of raw and normalized data, test for differential expression for each feature between the conditions, raw P-value adjustment and export of lists of features with significant differential expression between the conditions. The genes with adjusted P-value less than 0.05 were considered as differentially expressed compared to non-infected condition. The datasets generated and analyzed for this study can be found in NCBI's Gene Expression Omnibus (accession number GSE178583).
Geneset Enrichment Analysis With Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Databases, Keywords Filtering, and Upstream Analysis
Over-Representation Analysis was performed to generate lists of GO/Kyoto Encyclopedia of Genes and Genomes (KEGG) terms that are over-represented by the differentially expressed genes (DEGs): Enrichment scores for genesets were calculated based on log 2 fold-change rankings of DEGs. P-value was used to calculate false discovery rate (FDR; adjusted P-value) in a given geneset, that is a probability of the geneset with an enrichment score to be falsely overpresented. Odds-ratio of each geneset was calculated by the following formula: Odds-ratio = (DEG in geneset/non-DEG in geneset)/(DEG outside of geneset/non-DEG outside of geneset). In order to highlight the relevant ontologies and pathways to the study, subsets of Biological Process (GO) or KEGG Pathways were created by detecting the same set of keywords (stringr package, R Studio). For instance, the keywords we used to produce "GO_interest" or "KEGG_interest" included "signal, " "cascade, " "cytokine, " "receptor, " "transport, " "neuron" and 24 more, and the produced lists of subsets were provided on Supplementary
RT-qPCR
For the selected and grouped DEGs, we confirmed the gene regulation at 40 h post-infection (hiPSC-derived neurons) or 48 h post-infection (H9-NSC neurons) by RT-qPCR. Total RNAs were prepared from Trizol extraction and ethanol precipitation, and RT-qPCRs were carried out using SuperScript IV VILO master mix with ezDNase (Invitrogen, Cat. No. 11766050) and QuantiTect SYBR Green PCR kit (Qiagen, Cat. No. 204143) following manufacturer's protocols. Specific primers for each DEG were selected from QuantiTect Primer Assay (Qiagen, Cat. No. 249900), and listed in Supplementary Table 3. Viral RNA level was assessed by N protein-specific primers (N1130: 5 -CTGACGTAGCACTGGCAGAC-3 ; N1247: 5 -AGTCGACCTCCGTTCATCAT-3 ), and human GAPDH (forward 5 -GAAGGTGAAGGTCGGAGT-3 ; reverse 5 -GGTCATGAGTCTTCCACGAT-3 ) was measured as a reference gene for each sample. One-way ANOVA was used to validate the significant differences between conditions.
Spontaneous Activity Recording With Calcium Imaging
At the end-point of RABV infection, differentiated human neurons were incubated with 3 µM Fluo-4 AM calcium indicator (Invitrogen, Cat. No. F14201) at 37 • C for 40 min in 50% BrainPhys Neuronal Medium (STEMCELL, Cat. No. 05790) and 50% of differentiation medium. BrainPhys medium (Bardy and Gage, 2015) was supplemented with 2% B27, and used as loading and reading medium in this study. Subsequently, the cells were gently washed by 70% medium change for 4 times to remove excessive Fluo-4 probe and subjected to recording at 1.72 Hz for 4.5 min with 10X Air objective lens at 37 • C (Operetta system, PerkinElmer). From the collected TIFF images, information on the calcium oscillations of each detected soma (cell body of a neuron) was extracted, using FIJI macro and CalBlitz-incorporated Python scripts (open source) 1 : briefly, motion correction was applied on each image to find the common area between frames of the time-lapse acquisition (Giovannucci et al., 2019). Only the area commonly detected on all frames was subjected to the next analysis step. From the motion-corrected images, regions for the individual somas were segregated by applying binary definition (cell/background) and watershed algorithm. Subsequently, the relative intensity variation F/F for each soma was calculated from Fluo-4 intensity information of the images, by dividing intensity of the individual soma by the average intensity of all frames. After that, the raw F/F trace was transformed from nonlinear to linear trend by correlating with a mathematical model of neuron impulse. From the transformed trace of F/F, we extracted the frequency and amplitude information of calcium peaks, by setting the threshold to 2% of F/F. The number of calcium peaks was presented in individual value graphs (Prism 9). Further description of calcium imaging analysis and exemplary intensity trace can be found in "Results" section and Supplementary Materials.
Immunocytochemistry and Image Analysis
Non-infected or infected human neurons differentiated from neural progenitor cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and washed with PBS 3 times. Neurons were subsequently incubated with a primary antibody against TUBB3 (1:1,000; BioLegend, Cat. No. MMS-435P), diluted in PBS with 5% normal goat serum (Gibco, PCN5000) for 2 h at 37 • C, followed by incubation with the appropriate secondary antibody conjugated to AlexaFluor 488 (Thermo Fisher Scientific, Cat. No. A28175) for at least 1 h at room temperature. Nuclei were counterstained with Hoechst (1:1,000). For RABV N protein detection, Anti-Rabies Nucleocapsid Conjugate (Bio-Rad, Cat. No. 3572112) was used according to the manufacturer's protocol. Following the staining, images were Table 4. Biological Process was further filtered into 4 groups, Cell Cycle-related GO terms (E), Gene Expression (F), Immune Response (G), Neuronal Function (H) and the number of up or down-regulated genes indicated in pie charts. The GO terms that have Odds-ratios higher than 10, or FDR smaller than 0.05 on at least one time-point are displayed in panels, and the full list the specified GO terms is provided in Supplementary Table 2. The numbers in parentheses indicate the size of geneset for each GO term.
Frontiers in Microbiology | www.frontiersin.org obtained from Operetta or Opera Phenix (PerkinElmer) with 20X Air objective lens and analyzed in Columbus Image Data Management System (PerkinElmer).
Temporal Dynamic Expression of Significantly Selected Genes in Wild-Type Rabies Virus-Infected Neurons
To understand the transcriptional changes at early time points of RABV infection and distinguish the modulated processes or pathways that are potentially involved in neuronal dysfunction, we infected 2 weeks-differentiated neurons with wild-type RABV (Tha virus) and performed RNA-Seq: at 8, 24, 40 h post-infection (Figure 1 and Supplementary Figures 1A,B) in biological triplicates. At each time-point, infected samples were compared to the non-infected one to detect the DEGs.
Overall, the datasets showed strong correlations between experimental conditions and gene expression as described in Supplementary Figure 1C. The replicates are clustered according to non-infected/infected conditions and to the time-points, which demonstrates the genuine effects of RABV infection on the transcriptome changes. As the post-infection time-pointdependent changes in gene expression was an intriguing aspect of the datasets, we ran the DEG dataset that were obtained from comparison between Tha virus-infected and non-infected conditions (Tha dataset) through Gene Set Enrichment Analysis (GSEA) pipeline. In total, 610 genes are significantly modulated in Tha dataset ( Figure 1A), with 101 and 96 genes at 8 and 24 h, respectively, and then a larger group of genes (516) at 40 h post-infection (Table 1) Table 4). The GO terms that are associated with each time-point also varies. Between 8 and 24 h GO terms, there are only 237 terms in common, in contrast to the comparison of 24 and 40 h which shows 585 common terms in Biological Process (Supplementary Table 4). Combined together, these facts strongly suggest that wild-type RABV impacts the RNA transcription of the infected neurons differently at the earlier stage of infection compared to the later stages.
Neuronal Function-Related Processes Are Regulated Before Immune Response Processes
To understand which processes are regulated early on during the RABV infection and how they evolve over time, further analysis focusing on GO terms in Biological Process was performed. We filtered the GO terms using a keyword detection function in R dplyr package, and assembled 4 groups of GO terms that are relevant to the virus propagation and neuronal dysfunction: Cell Cycle (cell division, DNA damage and repair, apoptosis), Gene Expression (RNA transcription, protein translation and folding, vesicle transportation), Immune Response (cytokines and interferon, innate immune-responserelated pathways), and Neuronal Function (neuron projection and synapse formation, extracellular matrix, ion balance, membrane potential) (Figures 1E-H). Up-and down-regulated genes were distributed differently in each group, for instance in the group Cell Cycle (Figure 1E), the down-regulated genes compose 75% of DEGs of the group. DEGs from this group are connected to 120 GO terms (Supplementary Tables 2, 4). Further, the Odds-ratios of Cell Cycle GO terms gradually increase over time until 40 h post-infection, differently from the GO terms of other three groups, and most of these Cell Cycle GO terms have FDR smaller than 0.05 at the last time-point. Illustrating these points, Cell Cycle GO terms with the most significant Odds-ratios such as "G1/S transition of mitotic cell cycle" (27 out of 30 DEGs down-regulated), "mitotic spindle organization" (16 out of 17) and "mitotic cytokinesis" (16 out of 16) showed a higher ratio of downregulated DEGs at 40 h. In comparison, the group Gene Expression ( Figure 1F) presents GO terms with higher Oddsratios at 24 h compared to 40 h post-infection, implicating that the relevant biological processes are significantly regulated by RABV at 24 h. It shows 198 DEG involved in 87 GO terms (Supplementary Tables 2, 4), related to mRNA stabilization, protein folding, or sequestering transcription factor with significant Odds-ratio. Similar kinetics have been observed on the group Immune Response ( Figure 1G). Innate immune response-associated 133 genes are involved in GO terms related to cytokine production, regulation of NF-kB signaling, or response to TNF among others. Close to 70% of DEGs are up-regulated in this group, including NFKB1, NFKB2, RELB, and NFKBIA, all detected from 24 to 40 h. On the other hand, the genes involved in the group Neuronal Function ( Figure 1F) are detected from earlier on, among 212 GO terms 87 of them (41%) contain DEGs that are regulated at 8 h post-infection. Many of genes in this group such as CCL2, TNFAIP3, NFKB2, MEF2C, and others are implicated in both major signaling pathways involved in immune response and also in important processes for neuronal functions, which include dendritic morphology, synaptic membrane adhesion, neuronal differentiation, neurotransmitter secretion and more (Supplementary Table 4). Since the failure of autonomic nerve system is postulated as the main cause of RABV-associated death, we used a mutant RABV (Th4M virus; four point mutations on M protein at amino acid positions 77, 100, 104, and 110) that showed better longevities and delays of neurological symptoms in infected mice (Khalifa et al., 2016;Sonthonnax et al., 2019) to infect differentiated neurons and performed a RNA-Seq analysis. As we mentioned above, M protein from Th4M virus lacks the affinity for the NF-κB signaling protein RelAp43 and therefore allows the induction of IFN-β, TNF-α and other pro-inflammatory cytokines upon infection (Khalifa et al., 2016;Besson et al., 2017). We then undertook a comparative ontology analysis on the RNA-Seq datasets, of Tha and Th4M viruses (Figure 2 and Supplementary Figure 2). As expected, DEGs are regulated differently by two viruses at early time-points post-infection. As described on Tables 4, 5). This could mean that even though the transcriptome profiles of Tha and Th4M datasets are distinct, the DEGs of both datasets are involved in similar biological processes, at earlier time-points in the case of Tha compared to Th4M.
For the GO terms detected at 24 h post-infection, 596 terms are associated with Tha dataset and 1413 terms with Th4M ( Figure 2B), which is 2.4 times more than that of Tha dataset at the same time-point. We also found that amongst the terms detected with Th4M DEGs at 24 h, 507 terms are shared with Tha 24 h and then an even higher number of terms, 1006, with Tha 40 h dataset. This involves GO terms such as regulation of NF-kappaB signaling (GO:1901222), intrinsic apoptotic signaling pathway in response to ER stress (GO:0070059), cytokine-mediated signaling pathway (GO:0019221), and KEGG terms including TNF signaling (hsa04668), JAK-STAT signaling (hsa04630), MAPK signaling (hsa04010) and PI3K-AKT signaling (hsa04151), as further detailed on Figures 2C,D. Interestingly, most of the immune response-related processes from Th4M dataset overlap with that of Tha dataset, since the numbers of DEGs that are involved in the relevant pathways are very close between the two datasets. At 40 h post-infection, the two datasets show a relatively similar effect on biological processes, as 1,335 GO terms (87% of Tha and 79% of Th4M terms) ( Figure 2B) are commonly detected in both datasets and 372 genes are shared between them ( Table 1).
To conclude, Tha and Th4M datasets display different dynamics of gene regulation in differentiated neurons especially at early time-points, but they become similar at 40 h postinfection when the feedback response from host cells becomes more prominent. Overall, Tha virus secures more control over the gene modulation than Th4M at the early hours of infection, which emphasizes the role of M protein in the early regulation of host gene expression.
Rabies Virus-Associated Pathways Are Involved in Neuronal Differentiation, Synaptic Activity and Morphological Changes in Tha and Th4M Datasets
To understand which genes in the aforementioned RABVassociated pathways could directly impact neuronal functions or related processes, we investigated relevant DEGs identified in the KEGG pathways. At 24 h post-infection, TNF, IL-17 and NF-kB pathways scored relatively high Odds-Ratios compared to other gene sets ( Figure 3A). The DEGs detected in these pathways are mainly up-regulated in both Tha and Th4M datasets (Figures 3B-D), and many of these genes are induced at 24 h post-infection and maintained a high expression level until FIGURE 2 | Comparison of Tha and Th4M dataset. (A) From 0 to 40 hpi, DEGs (in red) were progressively selected more at the later time points. The common DEGs between two datasets were marked with a larger dot in red, and DEGs unique to one dataset with a smaller dot in red. The exact number of DEGs can be referred to Table 1. (B) Th4M dataset shares the majority of associated GO terms (Biological Process) with Tha. As the number of DEGs increases over time, the shared GO terms take the majority of ontology proportion at each dataset. At 8 hpi, Th4M shares 85% of its GO terms with Tha, and at 24 hpi, Tha shares 85% of GO terms with Th4M due to their lower number of DEGs compare to the opponent. At 40 hpi, both datasets share over 79% of GO terms with the other. Figure 3E shows more down-regulated DEGs than the previously mentioned pathways, notably IL1RAP (8 h on Tha; involved in inflammatory response, regulation of pre-/post-synaptic density assembly, positive regulation of NF-kB activity), CACNB3 (24 h on Th4M; involved in membrane depolarization), TEK (40 h on Tha and Th4M; involved in negative regulation of apoptotic process, negative regulation of inflammatory response, positive regulation of PI3K activity) as indicated on Supplementary Tables 4, 5. MAPK pathway is also associated through the subset genes to neuronal function-related biological processes such as synapse assembly (GO:007416), neuron differentiation (GO:0045664), and Wnt calcium modulating pathway (GO:0007223). As presented in the heatmaps, the overall DEG expression ranges from -1.2 to 3 log 2 -fold changes compared to non-infected conditions at each time point. This highlights the stealthy nature of RABV-induced modulation over gene expression in differentiated neurons and relatively mild feedback from the host defense mechanism during the first replication cycle of RABV.
The up-or down-regulated DEGs of RABV-associated pathways impact neuronal function-related processes through signaling transduction network. We have identified several direct and indirect connections from the signaling transductionrelated DEGs to the neuronal function-associated DEGs through upstream search using KEGG pathway database, TRANSFEC (geneXpression), GeneCards (GeneHancer Regulatory Elements), STRING and bibliography search. The examples of MAPK, NF-kB, and JAK-STAT pathway DEGs that are associated with neuronal functions were validated with RT-qPCR and assembled on Figure 4. DUSP5 on Figure 4A was previously studied for its contribution to the RABV replication , and plays an important role in de-phosphorylation of active ERK and controlling neurite outgrowth and postsynaptic density (Kim et al., 2015;Lacar et al., 2016;Kidger et al., 2017), along with other DUSP proteins. In our results, DUSP5 is detected as a DEG in Th4M dataset, but shows increased fold-changes in both Tha and Th4M RT-qPCR validations (Figure 4A and Supplementary Figure 3). Other MAPK pathway downstream elements FOS, FOSB and JUNB are up-regulated to varying degrees under Tha and T4M infection, which could then directly act as transcription factors for GDF15 [increases Glutamate release through upregulation of voltage-gated calcium channel (Liu et al., 2016)], SLC3A2 [forms cystine/glutamate transporter with SLC7A11 and regulates glutamate uptake (Bhutia and Ganapathy, 2016)], NR4A1 [NGF nuclear receptor, associated with dendritic spine attrition and cognitive impairment (Jeanneteau et al., 2018)] and GABRQ [extrasynaptic GABA receptor subunit theta (Fatemi et al., 2013)]. As part of GABAA receptor, GABRQ localizes on extrasynaptic membrane of neuron and contributes to GABA-mediated chloride ion fluxes and inhibitory regulation of action potential. All these DEGs have been confirmed to increase under Tha and Th4M infections in both RNA-Seq and RT-qPCR validation. WNT mediates neuronal circuit formation and could be up-regulated by MAPK pathway modulation through the upstream transcription factor CDX2 (De, 2011), which is validated by RT-qPCR. DYNLL1 (LC8) is an important protein involved in the translocation of RABV particles, but also in DNA damage response pathway and anti-apoptotic activity by interacting with TP53BP1, BIM and BMF (Fooks et al., 2017;He et al., 2018;West et al., 2019). DYNLL1 is up-regulated in our RNA-Seq and RT-qPCR experiments, for both datasets (Figure 4A and Supplementary Figure 3). In Figure 4B, selected DEGs from canonical and non-canonical NF-kB pathways and the possible downstream genes that are up-regulated are presented. NFKB1, NFKB2, RELB, NFKBIA are involved in host immunity and oncogenesis, as well as in synaptic plasticity, communication between synapse and nucleus, response to membrane potential changes and neurite outgrowth through downstream regulation (Kaltschmidt and Kaltschmidt, 2009;Engelmann and Haenold, 2016;Dresselhaus and Meffert, 2019). ICAM1 and VCAM1 could receive signals from both MAPK and NF-kB pathways for expression (Taniguchi and Karin, 2018), and contribute to neuron adhesion, stability of extracellular matrix, and permeability of brain barriers. TNFAIP3 (A20) is involved in negative regulation of inflammatory response and apoptosis, and heterozygosity of this gene causes an exacerbated cognitive impairment under inflammatory conditions (Daems et al., 2020;Martín-Vicente et al., 2020). The primary function of CCL2 and CXCL10 is chemokines for monocytes and macrophages, but also could indirectly cause neuronal dysfunction through the recruited immune cells (Lehmann et al., 2019). The chemokines could also increase intracellular calcium level within neurons and cause hyperpolarization and excitotoxicity (Kodangattil et al., 2012). In Figure 4C, STAT3 increases under Tha/Th4M infectedcondition, and so do the downstream effectors SLC7A11 and HSP90AA1. As mentioned above, SLC7A11 forms Xccystine/glutamate anti-transporter, and HSP90AA1 functions as scaffold protein and maintains the integrity of cytoskeletal proteins in neurons as well as induces clearance of abnormally aggregated proteins (Ou et al., 2014).
Rabies Virus Infection Hinders Spontaneous Activity of Neurons
To understand the impact of RABV infection on neuronal activity, we performed calcium imaging on the NPC-derived neuron cultures. Calcium imaging with Ca 2+ indicators can reflect the genuine neuronal activity, number of action potentials and the amplitude, correlating with the observed calcium traces (Kirwan et al., 2015). In our experimental setting, neurons expressed a neuronal marker TUBB3 after 2 weeks of differentiation and formed complex network over the desirable size of field of view (Supplementary Figure 5; over 500 somas detectable under 10X lens). The differentiated neurons were then infected with Tha or Th4M virus at MOI 3, same as in the RNA-Seq experiment, and subjected to Fluo-4 staining and calcium imaging on 5 days post-infection (more than 3 days after the last time point of the RNA Seq analysis). The acquired images were processed through Calblitz workflow (Supplementary Figure 6) to extract information on calcium peak frequencies and amplitudes from individual somas. Active neurons, which has more than one calcium peak over the course of imaging, were present in all three experimental conditions (Figure 5A and Supplementary Figure 6). However, Tha-and Th4M-infected neurons exhibited lower neuronal activities compared to the noninfected condition (Figure 5B). The frequency of calcium peaks was significantly higher on non-infected condition than on the Tha-infected condition (right panel), as well as for the ratio of active neurons (left panel). Comparison between Tha-and Th4M-infected neurons was not statistically significant on either activity ratio or the peak rate on 5 days post-infection. Average of amplitude of calcium peaks showed similar heights between non-infected and infected neurons, as presented in exemplary intensity traces in Supplementary Figure 6D. As summarized in graphs on Supplementary Figure 5, the results of calcium imaging did not originate from structural damages such as axonal/dendrite degeneration (neurite count/length) or apoptosis (bleb count). Interestingly, when we reduced the duration of infection to 2 days to observe the effect of Tha virus infection in similar timeline as the RNA-Seq experiment (Figure 5C), the difference of peak rates was less significant between non-infected and infected neurons in comparison to 5 days post-infection. However, the Tha-infected neurons exhibited a sensitivity to the inhibitory neurotransmitter GABA, with the frequency of calcium peak reduced further by the combination of infection and GABA treatment than on other conditions. This may indicate how the neuronal function is affected by the regulation of neurotransmitter-associated genes, such as the GABA receptor subunit GABRQ, under RABV infection.
DISCUSSION
As rabies advances, signs of autonomic and cognitive neuronal dysfunction become more prominent (Fooks et al., 2017). Many viral encephalitis studies attributed similar neurological symptoms to the strong immune responses from the macrophage/monocytes, neutrophils and adaptive immune cells, infiltrating central nervous system (CNS) or recruited FIGURE 4 | Validation of neuronal function-related DEGs with RT-qPCR at Tha-and Th4M-infected condition and grouping in the associated pathways. (A) DEGs associated with MAPK signaling. DUSP5, FOS, FOSB, JUNB are detected within KEGG MAPK signaling geneset, and the association of the remaining group was identified using different tools for upstream search (refer to text). (B) DEGs detected directly in KEGG NF-kB signaling geneset are NFKB1, NFKB2, RELB, NFKBIA, TNFAIP3, ICAM1 and VCAM1. (C) STAT3 was directly detected in JAK-STAT KEGG geneset, and the associations of SLC7A11 and HSP90AA1 were identified through upstream search. (D) RABV RNA level was detected in RT-qPCR using N protein primers. All comparisons between experimental conditions showed statistical significance (one-way ANOVA, P-value < 0.05 or lower).
through disrupted blood-brain barriers (Denizot et al., 2012;Ludlow et al., 2016;Carod-Artal, 2018). Excessive immune responses against viruses in the CNS result in neurodegenerative pathologies, such as neuronal death, axon degeneration or demyelination (Tsunoda, 2008;Ramesh et al., 2013;Kennedy, 2015). In contrast, rabies virus has developed strategies to evade immune responses at the site of initial entry as well as in the CNS, and widely spread across the brain even before the onset of symptoms (Jackson, 2006;Hemachudha et al., 2013). In order to understand the early modulations that occur in RABV-infected neurons, we have analyzed the dynamic transcriptome changes of the infected neurons around the first cycle of virus replication in absence of secondary inputs from glial cells, and provided neuronal function-associated genes that could directly be affected by RABV modulation of signaling pathways. The originality of this work lies on the fact that the transcriptome kinetic analysis has been performed from very early time-points of RABV infection up to 40 h post-infection, and that the role of M protein in transcriptome modulation and neuronal dysfunction has been investigated in differentiated human neurons.
M Protein Regulates Neuronal Function-Associated Genes From Early Time-Points of Infection
At 8 h post-infection, Tha dataset showed faster regulation of host gene expression than Th4M dataset, with the majority of these DEGs being down-regulated (81%). We showed that in Tha dataset, the group Neuronal Function particularly stood out at 8 h compared to the group Cell Cycle, Gene Expression or Supplementary Figure 6. (B) The active/inactive neuron ratios were counted from 12 imaging sessions of non-infected/infected conditions, and are presented as individual value graph on the left panel. The neurons that had more than one calcium peak detected during the imaging were considered as active. Bartlett's test evaluated the significant difference between the conditions. On the right panel, the graph represents frequencies of calcium peaks collected from over 5,000 individual somas at each condition. Student's t-test was performed to show the statistically significant difference between conditions. (C) H9-NSC-differentiated neurons and the decreased spontaneous activity on 2 days post-infection. The H9-NSCs were differentiated into neurons for 4 weeks and then infected with Tha virus for 2 days at MOI 1. The frequencies of calcium peaks were calculated from over 1,500 individual somas and presented in the graph. The non-infected and infected conditions show a statistical difference in Student's t-test, while the non-infected and infected conditions with GABA treatment (two graphs in the middle) presented stronger difference. GABA A receptor antagonist Bicuculline (Bic) partially reversed the effect of GABA and Tha infection, as shown in comparison of GABA/GABA + Bic treatment in infected conditions. Asterisks represent statistical significance; * for P-value < 0.05, * * for < 0.005, * * * for < 0.0005, and * * * * for < 0.00005. GABA, Gamma-aminobutric acid.
Immune Response, since 44% of DEGs at 8 h post-infection is associated with neuronal functions. Some of the high Odds-ratios we observe might occur due to the slightly smaller size of gene sets in the group Neuronal Function, or also because the specific gene ontologies are highly influenced by Tha virus infection at 8 h compared to the others. Also, the virus infection may have direct contribution onto this regulation, through immediate early genes (IEGs). IEGs regulation in host cells can be triggered by early event of virus infection-attachment, internalization, intracellular trafficking and uncoating-and activate series of transcription factors and DNA-binding proteins that are bestcharacterized in Ras/Erk/MAPK-associated signaling pathways (Tullai et al., 2007;Gallo et al., 2018). These genes are induced to maximum level within 1-4 h after stimulation and activate or suppress secondary transcription within few hours. Many of the group Neuronal Function-associated DEGs observed at 8 h (GRHL3, PDPN, TNFRSF11B, SPP1, COL11A1, PTX3, CRISPLD2, CDH2, EZR, NT5E, SMAD7, MEF2C, CLDN1, RGS4, ANXA1, CEMIP, ARNTL, LEF1, HBEGF, TGFA, WEE1) have transcription factor binding sites associated with Ras/Erk/MAPK or related pathways upstream (identified by TRANSFEC search), so it is possible that M protein of RABV may interact with Ras/Erk/MAPK signaling pathway proteins and affects the downstream transcription. Another point being addressed regarding the regulation of neuronal function in early timepoints is the proportion of down-regulated genes in DEGs. There are 86 processes in the group Neuronal Function at 8 h postinfection, and 64 of them are influenced by down-regulated genes. Compared to the 26 processes of Th4M dataset in the same criteria and the sudden expansion of DEGs at 24 h, the results show how important the role of M protein is in the early repression of host gene expression. Vesicular stomatitis virus (VSV), another rhabdovirus belonging to the Vesiculovirus genus (a genus closely related to that of the lyssavirus genus), also globally suppresses the transcription and translation of the host cells by 4-6 h post-infection, and the M protein of VSV is primarily responsible for this inhibition (Lyles et al., 2013). The scale of host gene expression inhibition induced by RABV M protein seems to be much smaller than that of VSV, as demonstrated in this study. However, if our results suggest that RABV is a milder inducer of host gene expression than VSV, RABV has developed more specific ways to control signaling pathways than general inhibition. This is in line with previous studies showing the crucial role of RABV M protein in the regulation of signaling proteins in vitro and in vivo (Luco et al., 2012;Khalifa et al., 2016;Besson et al., 2017).
Size of Differential Expression During the First Round of Virus Replication Is Controlled by M Protein
In Tha dataset, we observed that the control of M protein over the host gene expression was maintained at 24 h postinfection, in contrast to the delayed but drastically increasing number of DEGs on Th4M dataset. It seems that during the first round of virus life cycle (∼24 h post-infection), RABV has the competition between host response and virus replication under its control due to the presence of functional M protein, which implies that the suppression of host gene expression by M protein at early time-points of virus replication maybe significant for keeping the host cells relatively "quiet." Modulating host gene expression by the virus is not necessarily bound to immune responses here, since Tha and Th4M datasets display similar number of DEGs detected directly in immune responseassociated pathways at 24 h (according to KEGG pathway definition) and also similar number of GO terms related to innate immune responses at that time-point (Tha 92 terms, Th4M 105 terms by keyword filtering in Biological Process). This is to a certain degree a discrepancy from the previously reported studies showing stronger immune responses elicited by attenuated RABV (Wang et al., 2005;Koraka et al., 2018). Considering the genetic differences between the virulent RABV strain Tha and its mutant virus Th4M are 4 amino acids, the results we obtained only highlight the specific characters of M protein-derived regulation. Additionally, in our datasets, Th4M dataset has more DEGs that are directly or indirectly involved in gene expression, metabolism, or immune response processes from 24 h post-infection onward, which may affect the kinetics of virus replication as presented in Supplementary Figure 1 and partly cause the delayed neurological symptoms in Th4Minfected mice (Sonthonnax et al., 2019). In the current study, we were able to provide clearer perspectives on the kinetics of the progression of neuronal dysfunction in RABV infection, since we used neuron culture and shorter incubation time. In comparison, Zhang et al. (2016) used intranasally inoculated mice model to study CVS-11 and HEP-Flury transcriptome modification in the brain, but the earliest time point for RNA harvest was 10 days post-infection and the CVS-11 dataset showed over 2,800 DEGs ranging from -2 to 11 log 2 fold-changes. The analysis showed immune responses and cell death-oriented GO distribution for the CVS-11 dataset, which most likely comes from combination of cell types in the brain and therefore is difficult to interpret regarding the neuronal dysfunction. A study published in 2016 (Jamalkandi et al., 2016) took a systemic approach by meta-analyzing the existing datasets and extracted 7 relevant signaling pathways to RABV pathogenesis (WNT, MAPK, RAS, PI3K/AKT, TLR, JAK-STAT, and NOTCH), which are similar to the KEGG pathways filtered on p.i. 24 h based on higher Odd-Ratios in Figure 3. However, the datasets in this study as well were produced from mouse brain, spinal cord, and microglial cells with varying microarray systems and virus strains, and the time points for the sample collection were unclear. As the primary target for RABV replication, neurons are expressing receptor molecules such as nicotinic acetylcholine receptor (nAChR), neuronal cell adhesion molecule (NCAM), p75 neurotrophin receptor (p75NTR) or metabotropic glutamate receptor subtype 2 (mGluR2) (Zhang et al., 2014;Guo et al., 2019). Neurons are therefore exposed to host transcriptome modulation of RABV earlier than the other types of cells present in the nervous system, astrocyte, microglia or oligodendrocyte. From our study, the changes in neurons at early hours of infection seem crucial to maintain the control of RABV on preexisting host defense system and in particular on neuronal function regulation, as 137 out of 610 DEGs in Tha dataset are involved in neuronal function regulation, and 316 genes out of 969 DEGs for Th4M. Thus, this study provides an important insight on the sceneries of RABV's first replication cycle in its target domain and how the temporal regulation shifts to eventually affect neuronal functions.
Immune Response-Associated Pathways That Interact With Rabies Virus Impact Neuronal Functions
Regulation of many neuronal function-associated genes roots from the signaling pathways that are directly affected by RABV infection. One of such pathways is NF-kB signaling pathway. NF-kB pathway is intensively studied for its involvement in host immune responses, but also in relation to neuronal functions as it is localized in synapse for fast signaling transmission. NF-kB is constitutively activated in glutamatergic neurons (e.g., granule cells and pyramidial neurons in hippocampus CA1 and CA3) and responds to the increase of calcium level at the postsynaptic dendritic spine and translocate to nucleus (Kaltschmidt and Kaltschmidt, 2009;Engelmann and Haenold, 2016). Matrix protein of RABV interacts with RelA and more specifically its splicing variant RelAp43. Consequently, this interaction hinders the translocation and re-balances the downstream transcription by NF-kB pathway, including of IFN and TNF (Luco et al., 2012;Khalifa et al., 2016;Besson et al., 2017). RelA mainly partners with NF-kB p50, but also can interact with p52 and translocate to nucleus to act as transcription factor. With external stimulation such as TNF-α, IL1-β, CXCL10 or TGF-β1, or internal stimulation like viral RNA detection by RIG-I or TLRs, the RelA-p50 dimer transcribes inflammatory cytokines and inducible nitric oxide synthases (iNOS), and hinders the virus replication. With RelA stalled by M protein interaction, the downstream of NF-kB pathway weighs more on other transcription factors/co-activators/repressors, such as RelB, c-Rel, p50 and p52, frequently STATs, AP-1, p53 and IRFs in combinations (Taniguchi and Karin, 2018). The shifted transcription spectrum affects wide range of biological processes including neuronal functions, as exemplified by modulated transcription of TNFAIP3, ICAM1 or VCAM1. Also, the TPL2 (MAP3K8)-dependent interaction of M protein to p105 would prevent the positive regulation of MEK1/2 by TPL2 (Besson et al., 2017) and subsequently ERK1/2 or p38 signaling path, which as a result may suppress the expression of inflammatory receptors (Xu et al., 2018). This possibly leads to increasing the survival of neurons, but also alters the signaling pathways that interact with TPL2, such as release and destabilization of A20 binding inhibitor of NF-kB (ABIN2), which subsequently influence A20 (TNFAIP3)-mediated ubiquitination/de-ubiquitination of NF-kB signaling proteins (Webb et al., 2019;Martín-Vicente et al., 2020). Therefore, it is not surprising that Th4M, harboring 4 amino acids changes in the M protein impairing the M protein interaction with RelAp43, is not able to suppress host gene expression at early time-points of virus replication as efficiently as observed with the native virus Tha.
As mentioned before, modification of ERK/MAPK signaling plays important roles in both inflammation-and neuronal function-associated transcriptions. In our study, we have confirmed that several DEGs related to neurotransmitter uptake (SLC3A11, SLC2A11) and postsynaptic activity (GABRQ) are indeed regulated by RABV infection and this could result from the direct interaction between RABV proteins and MAPK pathway components. Of note, host genes of MAPK pathway that are important for virus replication are not necessarily modulated during the RABV infection, since the virus replication-assisting/inhibiting MAPK pathway genes observed in a previous study is not seen in our Tha dataset. JAK-STAT pathway could also be significantly modified by RABV infection, as STAT1/2 is efficiently sequestered by interaction with P protein. To a lesser degree in human neurons, P protein also binds to STAT3 (Harrison et al., 2020) and interferes with Gp130 receptor-dependent signaling that are involved in neuroprotection, neurite outgrowth and synapse formation (Lieu et al., 2013;Chen et al., 2016).
CONCLUSION
In conclusion, our study shows that the gene expression in neurons is significantly regulated by RABV infection from the early point of virus replication cycle, and M protein of RABV plays important roles in keeping the host gene expressions at bay during this time. RABV infection in neurons especially alters the expression of genes associated with neurotransmittergated ion channels/neurotransmitter uptake/morphology and extracellular matrix through RABV-interacting pathways at early hours of infection, which contribute to the eventual decrease of spontaneous activity in the infected neurons.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repository. Details of information is indicated in Materials and Methods.
AUTHOR CONTRIBUTIONS
SK, FL, RG, and HB contributed to the design of the study. HV, RL, and GD established analysis pipelines. SK, FL, and LF performed the experiments. SK, FL, HV, and RL analyzed the data. RM and GK provided the materials. SK, FL, and HB wrote the manuscript. All authors contributed to the revision of the manuscript and approved the submitted version. | 2021-12-15T14:22:54.636Z | 2021-12-13T00:00:00.000 | {
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44076493 | pes2o/s2orc | v3-fos-license | Extensive Bilateral Adrenal Rest Testicular Tumors in a Patient With 3β-Hydroxysteroid Dehydrogenase Type 2 Deficiency
Abstract Testicular adrenal rest tumors (TARTs) are presumably derived from ectopic adrenocortical tissue in the testis, affecting up to 49% to 94% of males with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Few reports have described TARTs in rarer forms of CAH such as 3β-hydroxysteroid dehydrogenase type 2 deficiency (3βHSD2D). A man with 3βHSD2D presented with massive bilateral testicular tumors. He had been treated with glucocorticoids and mineralocorticoids since infancy, with difficulties in suppressing dehydroepiandrosterone sulfate. At the age of 13 years, bilateral testicular lumps were found, and a radiologic diagnosis of TARTs was proposed. Subsequent sonographic examinations showed progression, despite intensifying his glucocorticoid therapy with metabolic complications. Following an open testicular biopsy, concerns of a Leydig cell tumor and risk of malignant transformation were raised, and because the patient also had local symptoms and azoospermia, he underwent bilateral orchiectomy at age 33 years. Histopathology was consistent with bilateral TARTs, exhibiting widespread immunoreactivity for adrenocortical markers, whereas no histological features of Leydig cell tumors were seen. The distinction between TARTs and Leydig cell tumors is important but can be challenging, and in our case, orchiectomy was needed to rule out the latter diagnosis. TART should be considered a differential diagnosis also in patients with 3βHSD2D who have testicular lumps.
periods of suboptimal hormonal control, inducing hypertrophy and hyperplasia of adrenallike cells within the testis (6,7). Recently, mixed testicular and adrenal characteristics of TARTs have been reported, challenging the sole adrenal origin of TARTs (8). TARTs have been claimed to be the main reason for the impaired fertility seen in males with CAH (3,6,9). Few reports have been published about males with 3bHSD2D and only single case reports regarding TARTs and/or fertility in this group of patients (7). We describe a male with 3bHSD2D with large, bilateral TARTs and infertility.
Case
The patient presented in the neonatal period with severe penoscrotal hypospadias, micropenis, cryptorchidism, and bifid scrotum. The 17-hydroxyprogesterone concentrations were elevated (15 nmol/L; normal, ,5 nmol/L) in infancy. After an acute adrenal crisis at 1 month of age, he was diagnosed with SW 3bHSD2D and initiated on regular doses of sodium chloride, cortisone acetate, and fludrocortisone. There was no consanguinity family history, the patient was of Swedish ethnicity, and the karyotype was 46XY. Mutation analysis of HSD3B2 revealed a rare mutation (Cys-72-Arg in homozygote form or as compound heterozygous with deletion). He was repeatedly operated on for hypospadias and retentio testis between ages 2 and 9 years.
Bone maturity was advanced during childhood and adolescence [+2 standard deviations (SDS)], and his final height was 174.5 cm (21 SDS compared with Swedish population, 22 SDS compared with his target height). The glucocorticoid and mineralocorticoid doses were successively increased over the years to control the elevated dehydroepiandrosterone sulfate (DHEAS) (Fig. 1) and renin (highest level 442 mIU/L; normal, 2.4 to 41 mIU/L), especially after the spontaneous onset of puberty. As a result, Cushing stigmata (obesity, osteoporosis, and striae) developed. During Figure 1. Long-term follow up in a male with 3bHSD2D deficiency. The TARTs were revealed by age 13 years, when the patient's height began to flatten (progressed bone age), and they gradually enlarged. Despite the depicted intensification in treatment (glucocorticoids and fludrocortisone), minimal response to DHEAS levels was observed. The increasing weight reflected the negative metabolic control. Finally, a bilateral orchiectomy was performed when the patient was 33 years old. The DHEAS concentrations were still markedly elevated at the last follow-up at 34 years of age. adulthood, he was treated with prednisolone (5 to 7.5 mg/d) and fludrocortisone (0.15 mg/d) with poor effects on DHEAS levels (Fig. 1). The paradoxically normal to high testosterone (30 nmol/L; normal, 10 to 30 nmol/L), androstenedione (12 nmol/L; normal, 1.2 to 5.0 nmol/L), and estradiol (164 pmol/L; normal, ,130 pmol/L) concentrations were attributed to peripheral conversion of DHEAS via 3b-hydroxysteroid dehydrogenase type 1 and 17b-hydroxysteroid dehydrogenase 5 enzymes. Eventually, testosterone substitution was initiated due to successive decline in testosterone (from 30 nmol/L to 11 nmol/L in 6 years) and elevated gonadotropins [LH 15 IU/L (normal, 1.2 to 9.6 IU/L); FSH 18 IU/L (normal, 1 to 12.5 IU/L)], at age 28 years.
At age 13 years, the testicular ultrasound revealed bilateral tumors (1.2 cm in diameter on the right side and 2 cm on the left side). They were described as hypoechogenic, well delimited, and normovascular. The tumors were considered TARTs and subsequently, over a 21-year follow up-period, advanced to larger, lobulated lesions, with blurred margins and hypervascularity, measuring finally 4 cm in diameter bilaterally. The normal testicular tissue was flattened to a minimal area in the periphery of the testes. A semen analysis performed at the age of 19 years to evaluate the fertility potential showed azoospermia.
Fine-needle testis biopsy with sperm aspiration detected no testis-specific tissue and no spermatogenesis, but ;100 immotile sperm with abnormal morphology could be found and were cryopreserved for future fertility treatment. Due to the high risk of permanent testicular damage, an open testis biopsy was postponed. At age 25 years, an open biopsy with enucleation of a 2.4-cm testicular tumor on the right side showed a presumed benign Leydig cell tumor with possible risk for malignant transformation. The sonographic control revealed progression, and because the patient was infertile and had discomfort, he underwent bilateral orchiectomy at age 33 years.
Histopathology
Macroscopically, the tumors were 4 cm in diameter, with a yellow to gray, lobulated to compact cut surface bilaterally. Microscopic examination showed the testicular parenchyma replaced by tumor cells with abundant, eosinophilic, slightly granulated cytoplasm and small, round to elliptical nuclei, with a loose chromatin and distinct nucleoli ( Fig. 2A). No Reinke crystals were observed. The tumor cells were admixed with a hyaline stroma with local lymphocytic infiltrates and adipose tissue metaplasia ( Fig. 2B and 2C), and tumor was also seen within the rete testis (the believed origin of TART; Fig. 2D). Less than one mitosis per 10 high-power field was seen, and the proliferation index (Ki-67) was 1%. Immunohistochemical stains revealed that the tumor cells were diffusely positive for adrenal markers inhibin A, Melan A, and calretinin and focally for synaptophysin (Fig. 2E). Partial immunoreactivity was seen for the androgen receptor. Negative staining was observed for EMA and OCT-3/4. Additional immunohistochemical markers (widespread immunoreactivity for CD56, focal for CYP11B1 and CYP11B2) supported the diagnosis of TARTs, stadium V (6) (Fig. 2F).
Discussion
To our knowledge, this is the first detailed report of a male with 3bHSD2D, extensive bilateral TARTs, infertility, and subsequent orchidectomy. TARTs can be found in children with CAH at a frequency of~20%. Testicular ultrasounds have been recommended as the method of choice for detection and follow-up of these lesions (2). Clinical control is less sensitive to detect lesions smaller than 2 cm (3). Five stages in the development of TARTs have been proposed (6), and in our case, the regular ultrasound control showed similar morphological progress.
Autopsy data have indicated TARTs in CAH boys of a few week of age, and rest tumors may already be stimulated in utero when the ACTH levels are elevated (6). Increased glucocorticoids doses can shrink the TART volume in the early stages, but continued growth can be seen when ACTH levels are suppressed. It is unknown if this is related to angiotensin II receptor stimulation, LH rise in adolescence, or other mechanisms (6). It is known that angiotensin II has a strong trophic effect on the adrenal gland, especially on the zona glomerulosa (7,10).
The impaired fertility is mainly related to the occurrence of TARTs (9). Glucocorticoid undertreatment leading to gonadotropin suppression due to increased adrenal androgen secretion and overtreatment also leading to gonadotropin suppression are additional reasons (3,5). The balance between under-and overtreatment has always been a challenge in patients with CAH. Semen quality has been reported to be very poor in CAH, with 100% being pathological, if all the World Health Organization criteria are considered (3).
Although testis-sparing surgery can be considered in advanced symptomatic TARTs when the conservative therapy is ineffective, 6% of males with CAH have been found to undergo unnecessary testicular surgery (3,6). The histological distinction between TART and Leydig cell tumor is difficult, although TARTs present bilaterally in 80% of cases, whereas Leydig cell tumors are bilateral in only 3% of cases. TARTs regularly display positivity for various adrenocortical immunohistochemical markers, which Leydig cell tumors do not. In addition, Reinke crystals are usually not seen in TARTs.
In conclusion, the clinical distinction between TARTs and Leydig cell tumors can be challenging, and it led to bilateral orchiectomy in this patient. Moreover, we show here that TARTs can be problematic in men with 3bHSD2D, a phenomenon that the practicing clinician should be aware of. | 2018-06-05T00:47:58.435Z | 2018-05-01T00:00:00.000 | {
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39930144 | pes2o/s2orc | v3-fos-license | Moreiba gen. n., a new Canarian genus in Laparocerini (Coleoptera, Curculionidae)
Abstract A new genus Moreiba is described for Strophosoma canariense Franz, 1995 (type species) and placed in Laparocerini. It differs from Laparocerus Schoenherr, 1834 by the small size, the strongly transverse rostrum, the dense longitudinal strigosity on head and rostrum, the body covered by dense, adpressed scales and short, semierect subspatulate to parallel setae, the slender antennae with bisinuate scape and short oval club, the granulate pronotum and all tibiae lacking a mucro in both sexes. Moreiba canariensis (Franz, 1995), comb. n., is the only described species, distributed in El Hierro and Gran Canaria. The tribal placement of the genera Aphyonotus Faust, 1895, Asmaratrox Heller, 1909, Straticus Pascoe, 1886 and Cyrtozemia Pascoe, 1872 is discussed.
Introduction
Herbert Franz (1995) described a new species of Canarian weevil as Strophosoma canariense (type locality: El Hierro, Las Playas) based on a holotype and 81 paratypes, coming from three localities on El Hierro (Las Playas, La Dehesa, El Sabinal) and one in Gran Canaria (Isleta). Reading the description, I noticed I had some similar specimens from Lanzarote sent by Gunnar Israelson and Lothar Dieckmann several years before for identification. I requested some specimens from Herbert Franz for study and he kindly sent me a dozen as a present for my collection. My materials and those from Franz belong to two different species of the same genus. Later some colleagues have found more populations in different islands, so I have decided to describe the genus as new (the original placement in Strophosoma Billberg, 1820 is incorrect), to allow them to publish their own discoveries under a legitimate generic name.
Material and methods
The specimens were studied under a binocular Leica Wild MZ8 microscope and photographed with an Olympus C7070WZ camera mounted on the same microscope. Microscope slides were studied and photographed with the same camera mounted on a Leitz Diaplan microscope, and some details were drawn by using a drawing tube. Extended focus images were generated using Alan Hadley's software CombineZP. The programs Adobe Illustrator CS5.0 and Adobe Photoshop CS5.0 were used for image postproduction and mounting. The description follows the usual terminology in Curculionidae, especially that in use in Machado (2010). Dissection methods follow Alonso- Zarazaga (1990). Genitalia and terminalia have been placed in a drop of DMHF on an acetate card accompanying the specimen for long-term conservation (Steedman 1958;Bameul 1990). Body length is measured from the midpoint of front margin of pronotum to the most apical point of the elytra (the apex is hidden under the overhanging declivity) in dorsal view, width is measured at the widest point of the elytra in dorsal view. In other structures, length and width are measured at the maximum points, unless otherwise stated. Franz, 1995, by present designation. Diagnosis. Small apterous Laparocerini with very short and wide rostrum; head and rostrum with a dense longitudinal strigosity; body covered by dense, adpressed scales; antennae distinctly slender with bisinuate scape and short oval club; pronotum granulate; elytra elliptical, weakly convex with declivity overhanging apex; legs with all femora edentate and all tibiae lacking mucro in both sexes, and endophallus devoid of visible sclerites. (Figs 1-2) densely covered by scales, completely covering integument, but not overlapping. Pronotal adpressed scales placed transversely with tips directed to midline, those on elytra pointing apicad. Elytral striae with very short, piliform setae. Antennal club densely tomentose. Tibiae without grooming patch. Ventral surface of body with sparse, adpressed to semierect piliform setae, integument clearly visible. Trochanteral setae present.
Description. Body
Rostrum in dorsal view ( Fig. 3) short, transverse; epistome transverse, medially notched apically, more or less V-shaped, delimited behind by fine raised line, naked, shiny, with one row of long parepistomal setae on each side; frons undelimited; epifrons flat, at the same level as head, medially sulcate, epifrons and head with a dense longitudinal strigosity from anterior border of pronotum to epistomal margin, covered by scales, sides of epifrons concave, tapering apicad in basal half, subparallel in apical half, lateral margin moderately projecting, extended above eyes in a supraocular ridge. Antennal scrobes in dorsal view inconspicuous, visible only in short apical part as narrow furrows, pterygia weakly prominent, in lateral view (Fig. 4) deep, short, curved in front of eyes, not reaching lower margin of rostrum, with ventral edge slightly longer than dorsal one, separated from eye about half width of scrobe.
Pronotum moderately wider than long, with rounded sides, anterior border distinctly narrower than posterior one, disc weakly curved, in the same curve as elytra, weakly depressed behind apical margin, without postocular lobes or setae, pronotal surface densely granulate. Base curved towards scutellum. Procoxae tangent, subglobular, situated at midlength of the pronotum.
Meso-and metaventrite. Mesoventrite transversally depressed, in a more dorsal plane than metaventrite. Mesocoxae subglobular, mesoventral process narrow, about as wide as 1/5 of diameter of mesocoxa. Metaventrite between coxae about as long as mesocoxa. Metanepisternal suture complete, metanepisternum narrow, its base oblique, projecting over outer angle of metacoxae. Metacoxae oval, transverse, not visibly touching costal margin of elytra; abdominal process distinctly narrower than largest diameter of metacoxa, subtruncate. Metendosternite very short, with long furcal arms in a flat angle, hemiducts weakly developed, anterior tendons well separated. Metathoracic wings absent.
Legs. Femora of all legs edentate, medially swollen. Protibia (Fig 7) in both sexes straight, apex slightly enlarged internally and externally, rounded, with a fringe of fine, yellow setae. Mucro of all legs not developed. Metatibial talus glabrous, slightly oblique, not ascending, without corbel or bevel. Tarsi slender, tarsomere 3 transverse, wider than the others, deeply bilobed, onychium strongly projecting beyond the lobes. Claws 2, equal, connate in basal half.
Note. Sexual dimorphism not apparent, except for slight differences in fifth ventrite. Distribution. This genus is presently known from two islands of the Canaries: Gran Canaria and El Hierro, but some samples are known as well from Tenerife, La Palma, Fuerteventura, Lanzarote and Montaña Clara. These are in study and may represent different species.
Etymology. Moreiba was the goddess of women and fertility among the ancient inhabitants of El Hierro (the 'bimbaches'). Gender feminine.
Integument reddish brown to pitch brown. Body densely covered by subtriangular scales, these apically awned (with a projecting bristle), adpressed to slightly raised. Head vestiture mostly creamy or whitish, that of pronotum different shades of brown, with one lateral band and a median thin band (sometimes incomplete anteriorly) creamy or whitish, elytra watery chequered in shades of brown, whitish and cream, 4 th interstria paler on declivity, 7 th and 8 th at base. Scutellum and legs with whitish scales. Semierect sublanceolate setae present among scales, on head forming a peculiar supraocular row, on pronotum denser, more perpendicular on sides, on elytra forming a regular row on interstriae. Semierect setae also present on antennae and legs.
Rostrum ca. 0.57 × as long as wide at base, as wide at pterygia as at base. Eyes asymmetrical, large-faceted, ommatidia separately convex, giving a blackberry aspect.
Elytra ca. 1.30 × as long as wide, ca. 2.5 × as long as pronotum, interstrial setae longer on sides than on disc, apically rounded to subtruncate, with sides subparallel, on disc ca. half the width of an interstria, separated in the row by a distance about twice their length, interstrial adpressed scales in 4-5 irregular rows. Interstriae flat, smooth, minutely punctate. Striae with shortly subrectangular punctures separated a distance similar to their length, punctures bearing small, piliform scales not surpassing margins of puncture. Upper part of declivity slightly overhanging elytral apex.
Wings. Franz (1995) stated that the species was brachypterous. In the three specimens I have dissected into pieces, I have been unable to find any trace of a wing, the insects being totally apterous.
Penis in dorsal view with pedon ca. 4.5 × as long as wide, sides slightly converging towards ostium, apical plate subogival, apex slightly projecting, in side view slightly curved ventrally.
Styli of ovipositor ca. 4 × as long as wide.
Spermatheca with ramus curved opposite to cornu. Material studied. 12 specimens, labelled El Hierro / Las Playas, ex coll. H. Franz, in coll. Alonso-Zarazaga (MNCN). The specimens have no date. They come from the type locality.
Biological notes. Franz (1995) mentions the presence of the species in xerophytic habitats in the islands, mostly under Periploca laevigata Aiton (Apocynaceae). A. Machado (pers. comm.) reports specimens under the basal leaves of Asphodelus sp. (Xanthorrhoeaceae). This is probably another polyphagous genus.
Discussion
The original placement of this species in the genus Strophosoma Billberg, 1820 is untenable, but easily understandable by the fact that Franz was not a specialist in Curculionoidea. In fact, the overall appearance and the size are more reminiscent of a Trachyphloeus Germar, 1817.
Of the characters stated by Machado (2010) to define the Laparocerini in the adult state, Moreiba matches each one, except the presence of mucronate tibiae. The character that Machado considered to be fundamental in the definition of the tribe (the presence of a spiculum relictum on the male VIII sternite) is present in Moreiba. Based on this definition, Machado (l.c.) excluded several genera from the tribe, and left the question open for three other genera: Aphyonotus Faust, 1895, Asmaratrox Heller, 1909and Straticus Pascoe, 1886. The study of some specimens of Asmaratrox intrusus Heller, 1909 andof A. coxalis Heller, 1909 in the collections of the NHM (London) and of the MNHN (Paris), and of specimens of Teripelus brachyderoides (Fairmaire, 1882), T. phoeostictus (Fairmaire, 1882) and Aseneciobius raffrayi (Fairmaire, 1882) (Perrin and Alonso-Zarazaga 2012) has convinced me that they are closely related. Alonso-Zarazaga and Lyal (1999), following the current opinions, placed the genus Asmaratrox in Laparocerini, Teripelus Heller, 1909 in Omiini andAseneciobius Hustache, 1939 in the Peritelini, reflecting thus the present chaos in Entiminae systematics. These three genera (and the Asian Cyrtozemia Pascoe, 1872) are related to Systates Gerstäcker, 1871 and belong to the so called 'African' Peritelini, characterized by the very short abdominal ventrite 2, about as long as the 3 rd , and the straight suture 1. I have not studied Straticus, but it could also belong here. The placement of Moreiba in Laparocerini is also supported by molecular data (A. Machado, pers. comm.). As here restricted, Laparocerini includes only the genera Laparocerus and Moreiba, that can be separated by using the following key: The presence of a spiculum relictum in Laparocerini is probably of the highest interest, as Machado (l.c.) has already pointed out, since it is absent in some Entiminae tribes (like Sciaphilini, Trachyphloeini, etc.). There is another peculiar feature that could be of some interest in characterizing the tribe Laparocerini, and that has been observed both in Laparocerus and in Moreiba. The basal margin of the metanepisternum protrudes obliquely over the outer angle of the metacoxa, hiding it in perpendicular view, so that the metacoxa does seem not to reach the elytral margin. In most Entiminae, the metanepisternal base ends transversely, not hiding the outer apex of the metacoxa, and the metacoxa is clearly seen touching the costal margin of the elytron. A survey of this character is needed to evaluate its phylogenetic significance.
The Laparocerini may be an evolutionary relict of former faunas that has been displaced by either climatological or competitive forces to a refuge in the Atlantic islands (Canaries and Madeira). There, at least Laparocerus has radiated into some two hundred species. The study of more Moreiba populations may help to cast light into the evolutionary history of this interesting taxon. a present of these 12 locotypic specimens and some other material of Curculionidae. Drs. Massimo Meregalli (Torino, Italy), Roman Borovec (Sloupno, Czech Republic) and Antonio Machado (La Laguna, Spain) are also thanked by their support during the preparation of this article. Dr Christopher H.C. Lyal (London, UK) checked the English language and is here warmly thanked. | 2016-05-04T20:20:58.661Z | 2013-09-20T00:00:00.000 | {
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243973772 | pes2o/s2orc | v3-fos-license | Promoting Junior School Students’ Anti-bullying Beliefs with the CATZ Cross-age Teaching Zone Intervention
In tackling the widespread problem of bullying victimisation, researchers have acknowledged the value of focusing on changing bullying-related beliefs and using peer-based interventions. In three studies (N = 419, 237 intervention and 182 controls), we tested the effectiveness of the CATZ cross-age teaching programme by inviting small groups of 11-year-olds to incorporate information supporting positive beliefs (concerning non-physical forms of bullying, the value of disclosing being bullied to adults, and helping victims) into a lesson they devised for themselves and to deliver that to small groups of 9-year-olds. Specifically, we examined if the intervention would promote that (i) non-physical forms of bullying are unacceptable (study 1), (ii) disclosing bullying to adults and getting the right kind of help have value and importance (study 2), and (iii) victims can be assisted in safe ways (study 3). Self-reports of nine specific aspects of these beliefs were collected from CATZ tutors and age-matched controls prior to and following the intervention, and at five-week follow-up in one study, using both open and closed questions. Results indicated significant positive effects of CATZ on all nine outcome variables, with mostly medium and high effect sizes. These findings support the use of CATZ to foster positive anti-bullying beliefs, and issues related to its wider uptake are discussed.
Introduction
Bullying is a sub-class of aggression that may take many different forms and involves intentional and repeated attempts to distress or harm a less powerful victim (Olweus, 1993(Olweus, , 2013. Common types include physical bullying (e.g. pushing, hitting), verbal bullying (e.g. name-calling, verbal threats), and relational bullying (e.g. rumour circulation; manipulation, social exclusion) (Baldry & Farrington, 2004). Like bullying, cyberbullying is another sub-type of aggression and despite sharing similar characteristics to bullying (i.e. intent to cause harm; repetition, power imbalance), it is also characterised by unique differences (Smith, 2016;Macaulay et al., 2020). Unlike bullying, cyberbullying is perpetrated online, and features of anonymity and publicity play a bigger role in the online domain Smith, 2016;Steer et al., 2020). It is common among school students (Menesini & Salmivalli, 2017;Olweus & Limber, 2010;Salmivalli, 2010) and is and continues to be a threat to their well-being, with teachers often struggling to address the issue within the school (Macaulay et al., 2018).
One recent large-scale survey in the UK of 9150 12-20-year-olds revealed that 51% were bullied at least once a month, with at least 34% being bullied each week (Ditch the Label, 2018). In addition, Przybylski and Bowes (2017) in their survey of 120,115 UK adolescents found that 27% had experienced being bullied on a regular basis. Despite scholars recognising variations in reported prevalence due to differing definitions and assessment methods used within research (see Volk et al., 2017), it is clear that bullying is a problematic issue that calls for intervention in the school environment. It can have diverse negative effects on the health and well-being of victims (Arseneault, 2017;Boulton et al., 2008;Hawker & Boulton, 2000;Reijntes et al., 2010), bullies (Copeland et al., 2013;Cowie & Myers, 2017), and onlookers (Midgett & Doumas, 2019;Rivers, 2012). Previous cross-sectional studies have reported how involvement in bullying can lead to serious adverse outcomes, such as increased suicidal ideation (Hinduja & Patchin, 2019), higher levels of social anxiety (Hawker & Boulton, 2000), and depression (Foody et al., 2020). These outcomes of school bullying not only occur within childhood experiences, but also through into adulthood (Zych et al., 2015), suggesting that it may precipitate adverse later life outcomes (Arseneault et al., 2010). Thus, bullying is a serious public health concern and it is imperative that effective anti-bullying interventions are put in place to address the issue.
While progress has been made in implementing interventions to tackle bullying, meta-analyses reveal residual rates that are usually far from zero (Merrell et al., 2008;Ttofi & Farrington, 2011). However, meta-analyses and systematic reviews on anti-bullying intervention strategies, in general, report some positive outcomes for anti-bullying prevention efforts, reducing bullying victimisation and perpetration (e.g. Evans et al., 2014;Gaffney et al., 2019). For example, Gaffney et al. (2019) found in their meta-analysis on the effectiveness of school-bullying prevention programs that there was a reduction of victimisation by 8-12% from evaluations conducted in the UK, with positive outcomes at reducing victimisation and perpetration globally. Despite this, anti-bullying interventions have not always been regarded as effective (Cunningham et al., 2016). One reason might be because students are not always receptive to antibullying initiatives delivered by teachers and other adults. Rigby and Bradshaw (2003) and Boulton and Boulton (2012) reported that many students believed teachers were not usually interested in tackling bullying and expressed little or no desire to collaborate with them in this regard. More recently, qualitative focus groups with young people suggested that anti-bullying interventions did not engage students, were delivered in a repetitive manner, and students felt that teachers were not the best group to deliver these anti-bullying messages (Cunningham et al., 2016). In addition, there is a belief among students, particularly victims of bullying, that teacher involvement may make the situation worse, and so students do not expect teachers to get involved (Newman & Murray, 2005;Smith & Shu, 2000). However, in order to solicit help, it is crucial that students do report bullying victimisation to teachers, and so the current study investigated how that might be encouraged. Partly for these reasons, student-led interventions, often a peer support service or buddy system (Boulton, 2005;Cowie, 2011;Tzani-Pepelasi et al., 2019), have also been implemented and evaluated. While these may help victims who use this service feel better, they do not come close to being a "proven" anti-bullying strategy because they are not directed at variables that are likely to mitigate bullying or its negative effects in the wider community of students (Gaffney et al., 2019;Houlston & Smith, 2009;Thompson & Smith, 2011). Nevertheless, students are now regarded as central to anti-bullying work, and efforts to find alternative ways of involving them are warranted, especially in community samples (Salmivalli, 2010). Studies have revealed significant, albeit modest, associations between beliefs about bullying and actual behaviour, suggesting that changing the former could lead to reductions in the latter Boulton et al., 2002). We now consider some specific beliefs that research suggests may do so.
Some sub-types of bullying, notably social exclusion and verbal bullying, are not perceived as serious as others, or even not as bullying per se, and this may be a reason why some students engage in them (Newman & Murray, 2005). For example, bullies may regard verbal bullying as a form of joking around with their peers (Shute et al., 2008). Some research has also reported that many young people and adults fail to recognise verbal bullying as something serious, at least relative to physical bullying (Jacobsen & Bauman, 2007;Maunder et al., 2010). Such beliefs may encourage the notion that incidents of verbal and social exclusion bullying are acceptable in the school environment or at least less likely to be "picked up" by the teachers. However, both forms can be considered a harmful form of peer relationships with adverse consequences (Coleman & Byrd, 2003;Hawker & Boulton, 2001). Given that verbal and relational bullying are seen as less serious than other forms of bullying, it is important that further work explores how harmful young people view verbal and relational bullying and if they regard these types of bullying as acceptable. In a sub-sample of approximately 6400 young people that reported experiencing bullying victimisation, verbal bullying and relational bullying were identified as the most prevalent types of bullying, with 88% having experienced verbal and 53% relational (Waasdorp & Bradshaw, 2015). So, while these forms of bullying are often not only considered less serious than other forms, they are also a more common experience for young people.
Considering gender differences, more girls than boys have been found to be victims of verbal or relational bullying (Waasdorp & Bradshaw, 2015) and, not surprisingly, to be more likely to view these types of bullying as more serious (Maunder et al., 2010;Shute et al., 2008). For example, Shute et al. reported that boys perceive verbal bullying as less serious and deemed such acts as harmless with no concern on the impact bullying could have. Despite this, one other study suggested that boys and girls are equally 1 3 likely to engage in verbal or relational bullying, with no gender differences on the perceived severity of the bullying (Newman & Murray, 2005).
Despite the distress it often arouses, bullying often goes un-reported by victims (Boulton, 2005;Cowie, 2000;Hunter et al., 2004). Encouraging more disclosure is a necessary condition for mobilising social support and is clearly warranted to help eliminate bullying in the school community and beyond. The bullying literature has noted that many students choose not to disclose bullying victimisation to teachers because they do not believe that teachers can or will provide help (Bradshaw et al., 2007;Unnever & Cornell, 2004). More recently, one study found that students were less likely to report their victimisation experiences if they did not trust the teachers and school staff (Berger et al., 2019). This consideration highlights the promising role of student-led interventions not only to combat bullying in general, but also to support students that may not trust the support of teachers in the school environment. Indeed, teachers can play an important role when providing social support to victims of bullying to reduce any negative outcomes. From a theoretical perspective, the stress buffering hypothesis suggests that increased perceived social support can reduce the relationship between a stressor and a negative outcome (Cohen & Wills, 1985). Boys have been found to be less inclined to partake in services that focus on social support for bullying, and girls are often more inclined to seek help generally (Boulton, 2005;Cowie, 2000). In addition, girls are more likely to view seeking help as the best strategy to overcome bullying and make them feel better (Hunter et al., 2004). In general, when it comes to the disclosure of bullying and seeking help, girls compared to boys are more likely seek social support to help cope with victimisation (Eliot et al., 2010;Oliver & Candappa, 2007).
One reason why boy's may be less inclined to seek help is that such help seeking behaviours may compromise boys' sense of masculinity, and boys may perceive it is less socially acceptable for them to ask for help (Cowie, 2000;Nadler, 1998). By encouraging young people to disclose victimisation to teachers, social support can be mobilised which will provide more options to the victim on how to cope with their victimisation, potentially reducing the negative outcomes. Teachers are important sources of support to help victims of bullying (Beckman & Svensson, 2015;Boulton et al., 2013), so it is important to encourage disclosure for young people so they can ask for the right kind of help and support and also understand when it is important to tell a teacher they have been bullied and would benefit from support.
Encouraging bystanders, those who witness bullying, to take responsibility to do something positive is also important since they are often reluctant to do so (Caravita et al., 2009;Gini et al., 2008;Thornberg et al., 2020). Young people are known to make evaluations about the likely impact of different types of bullying on victims (Chen et al., 2015) and so can respond in a negative or positive manner as they weigh up the risks and benefits according to different courses of action. Recent research with 868 11-13-year-olds in the UK found that bystanders reported they would provide emotional support to the victim and intervene to address the bully when they evaluated the incident to be severe, characterised by the intensity of the bullying, frequency of the victimisation, and extent the victim was upset (Macaulay et al., 2019).
As with other aspects of bullying, the moderating role of gender on bystander intervention is inconsistent. Boys showed more positive defending behaviour in some studies (Caravita et al., 2009), but in others, girls did so (Gini et al., 2008;Macaulay et al., 2019). It has been suggested that girls view bullying in general as more serious than boys and so may feel a greater responsibility to do something positive to help the victim (Maunder et al., 2010;Molluzzo & Lawler, 2012). Nevertheless, so many young people are often reluctant to intervene, leading Salmivalli et al., (1996, p. 117) to suggest that "bystanders were trapped in a social dilemma". It would appear that many young people recognise bullying as inappropriate but are afraid to intervene to support victims because of the perceived impact on their social status and safety (Boulton, 2013). Rock and Baird (2012) also suggested that students do not know what to do, and that teaching them safe intervention strategies would be helpful. Indeed, a recent meta-analysis showed that such bystander-targeted interventions led to more positive bystander behaviour such as supporting victims and dissuading perpetrators (Polanin et al., 2012). However, effect sizes (ESs) are not always large (e.g. Kärnä et al., 2011aKärnä et al., , 2011b and so different approaches to mobilising bystanders need to be developed and tested, including changing underlying beliefs about how to do it and why it is a helpful thing to do.
The Cross-age Teaching Zone Intervention
Co-operative group work (CGW) has been shown to assist students' learning in academic (Baines et al., 2007;Veldman et al., 2020) and social/behavioural domains (Blatchford et al., 2006;Cowie et al., 1994), including anti-bullying learning (Boulton et al., 2016;Ttofi & Farrington, 2011). Similarly, cross-age teaching (CAT) approaches have been shown to benefit tutors' academic development (Karcher, 2009;McDaniel & Besnoy, 2019;Robinson et al., 2005;Topping et al., 2011) and social/behavioural development (Robinson et al., 2005;Watts et al., 2019). For example, Watts et al. reported from a synthesis of the literature that the use of CAT approaches provides an effective platform in promoting academic, social, and behavioural skills for young people. Given these positive but separate results for CGW and CAT across such a wide variety of domains and variables, the first author developed an approach that combined them to target social outcomes, referred to here as the cross-age teaching zone (CATZ), and in this paper, we consider if it can be used to promote anti-bullying beliefs among students.
There are good theoretical and empirical reasons why a focus on the effect of CATZ on tutors rather than tutees is appropriate. Working with the lesson content (LC) facilitates cognitive restructuring and elaboration as it is incorporated into existing schemas (O'Donnell & O'Kelly, 1994;Slavin, 1996;Thurston et al., 2007;Topping & Ehly, 1998), which suggest that CATZ provides opportunities for learning as tutors work with the LC, make links with knowledge they already hold, and go on to develop more advanced cognitive structures and schemas. Applying Vygotsky's sociocultural theory (1978), the scaffolding of this type of learning provided by the adult facilitators of CATZ interventions, and the fact that tutors are required to re-work the LC into a viable lesson, means that tutors are in the zone of proximal development, that is, just outside what they can do or know unaided. In our case, that means CATZ tutors will likely be "thinking about bullying" in novel ways. The fact that CATZ tutors are working co-operatively to develop and deliver their lesson further optimises the likelihood that they will learn the LC. Slavin (1996) argued that such co-operative activities provide "implicit" reward and incentive structures, and so CATZ tutors are likely to see that they have a responsibility to their group that can be met if they themselves master/learn the LC. Role theory also suggests that acting as a teacher promotes that feeling of responsibility even more because it engenders a sense of care towards tutees (Biddle, 1986;Robinson et al., 2005). This implies that the tutors teaching the material to younger students (i.e. the tutees) would take their role seriously, hence facilitating an effective learning environment for both the tutors and tutees.
Moreover, it is now apparent that intervention approaches that only indirectly and subtly "challenge and change" existing thought patterns can be effective (Longmore & Worrell, 2007). This is especially important given that many students are resistant to direct attempts to change their bullying-related beliefs and actions (Boulton & Boulton, 2012). Thus, having CATZ tutors work on material about bullying in a general sense to help their tutees, in the absence of direct attempts to change their beliefs, could be sufficient for them to "take ownership" of this new information about bullying and internalise it. While attempts to stop students engaging in bullying are clearly warranted, it is now clear that this is very difficult to bring about, and researchers are beginning to explore interventions that target underlying beliefs, attitudes, and knowledge (Evans et al., 2014;Gaffney et al., 2019;Kärnä et al., 2011aKärnä et al., , 2011b. It is important to do this among the wider community of school students and not just those who currently act as bullies (Salmivalli, 2010). Partly for this reason, and partly because interventions that have the most positive effects on the most students are more likely to be taken up by school staff, we are currently looking at the effect of CATZ on tutees' attitudes/beliefs, feelings, and behaviour. The psychological mechanisms that might be responsible for any such positive effects are likely to be different to those outlined above for tutors, and so we will report them in detail in the near future.
In terms of anti-bullying practices, the acceptability of community interventions with students in schools is important because it can influence levels of engagement, treatment integrity, and ultimately treatment outcome (Cowan & Sheridan, 2003;Cunningham et al., 2016;Kazdin, 1980). Reports of the social validity of anti-bullying interventions are rare, and no study has so far reported it for CATZ with its co-operative and cross-age teaching characteristics. Considerable evidence suggests that girls are more open to acting as "agents of change" across a range of peer-led interventions (Boulton, 2005;Cowie et al., 2002), plausibly because acting in a somewhat formal helping capacity may compromise boys' sense of masculinity or macho self-image (Cowie, 2000;Petersen & Rigby, 1999).
Summarising the above, there is a clear need for studentled community-based interventions that help promote antibullying beliefs. As we have seen, there are good empirical and theoretical reasons to expect that CATZ may engender these beliefs among tutors. Our primary aim, therefore, was to conduct three linked studies that examined the effect of CATZ on beliefs that (i) non-physical forms of bullying are unacceptable (study 1), (ii) disclosing bullying to adults and getting the right kind of help have value and importance (study 2), and (iii) victims can be assisted in safe ways (study 3). Each intervention was delivered by different researchers in semi-autonomous ways. Given that the magnitude of positive effects of interventions delivered by their creators in one context are often attenuated when they are delivered by other people in another context (see Yeager & Walton, 2011), this would allow us to assess the likelihood that CATZ could be rolled out more widely by diverse groups of facilitators.
Related to the above, our second aim was to test if the effect of CATZ differed as a function of gender. As noted above, the literature has reported some, but not always consistent, gender differences in the kinds of beliefs that we measured and also in how they respond to peer-led interventions.
Finally, our third aim, also with relevance to the implications of our findings for anti-bullying practice, we assessed the social validity of CATZ by examining how acceptable it was to participants. Simply put, to be optimally effective, student-led interventions have to be well-received by students themselves.
Participants, Measures, Design, and Data Collection
Participants (N = 419) were drawn from five junior schools in the UK. Students' consent, and that of parents or head teacher in their loco parentis role, was solicited, and the response rate was 91% for taking part in our data collection procedures and 100% for taking part as a CATZ tutor. To explain the discrepancy, some parents requested that their children not be presented with questionnaires but none requested that their children did not take part in CATZ if the rest of their classmates would be doing so. At the request of the schools, and partly for logistical reasons, randomisation was at the class level such that a whole class was either randomly assigned to be CATZ or control. In each school, there were at least two classes and at least one class was a CATZ class and at least one class was a control class. In none of the participating schools were the classes streamed, meaning that the children in different classes were likely to be similar to each other. Overall, across the three studies, 237 and 182 students were randomly assigned in this way to act as CATZ tutors or controls, respectively. Teachers asked us to work with the oldest students in the school (mean age = 11.5 years in UK) because they deemed them most appropriate to deliver anti-bullying learning to their school mates.
In each study, data were collected on a whole class basis. Participants received a questionnaire, and a researcher read out instructions followed by each question. To encourage considered responses, students were informed, "This is not a test so there are no right or wrong answers. We just want to know what each of you think and so there is no need to copy what somebody else has put. Is that OK?" Preintervention (T1) data were collected immediately prior to the implementation of the intervention/control experience, and post-test (T2) data were collected about a week after it ended. Follow-up (T3) data were also collected five weeks later in study 2.
In each of the three studies, different outcome measures were employed (italicised below, nine in total) and different students in different schools took part. Details of each study are provided next, and then, the general approach used to deliver CATZ in all three studies will be described.
Study 1
Ninety-nine participants took part, 55 in the CATZ group (27 girls and 28 boys) and 44 acted as business as usual controls continuing with their normal lessons (28 girls and 16 boys). The four dependent variables were beliefs about non-physical forms of bullying, specifically Harmful Exclusion measured with the closed question "How harmful do you think social exclusion is?", Harmful Verbal measured with the closed question "How harmful do you think verbal bullying is?", Acceptable Exclusion measured with the closed question "How acceptable do you think social exclusion is?", and Acceptable Verbal measured with the closed question "How acceptable do you think verbal bullying is?" Each of these four questions had a 5-point response option initially anchored with "not at all" and "a lot", and they were subsequently scored from one to five so that low values were more desirable (i.e. participants saw the behaviour as less acceptable and more harmful).
A sub-set of 35 non-CATZ participants from the two control classes who were present at the time was asked these questions again one week later to assess test-retest reliability. It was good (all p < 0.001) for Harmful Exclusion (r = 0.59), Harmful Verbal (r = 0.75), Acceptable Exclusion (r = 0.72), and Acceptable Verbal (r = 0.44).
Study 2
This study involved 197 participants, 106 CATZ (58 girls and 48 boys), and 91 business as usual controls continuing with their normal lessons (50 girls and 41 boys). The two dependent variables were beliefs concerning getting help when one is bullied, specifically When to Tell measured with the open question "If you were bullied, how would you know when it would be a good idea to tell a teacher?", and Wanted Help measured with the open question "If you had been bullied and told a teacher, what could you do to help make sure you get the right kind of help?" For each open question, a researcher developed a coding scheme to identify common categories of responses, and two independent raters then used it to code all of the responses collected. High levels of inter-coder agreement were obtained, Cohen's kappa ≥ 0.89. Most cases of disagreement were resolved by a discussion among coders and in a few instances by a third coder. Once coded, the number of responses that represented "desirable and appropriate" knowledge was tallied for each participant and this value was used in the statistical analyses. For example, two examples of such responses for Wanted Help were "I could tell the teacher what I wanted them to do to help me" and "I would ask the teacher not to tell the bully that I had reported them".
A sub-set of (non-CATZ) participants was asked these two questions again one week later to assess test-retest reliability. Using the number of responses that indicated desirable and appropriate knowledge, reliability was good (p < 0.001) for Wanted Help (r = 0.66) and When to Tell (r = 0.70).
Study 3
There were 123 participants in study 3, 76 CATZ (39 girls and 37 boys), and 47 controls (18 girls and 29 boys). Unlike in studies 1 and 2, researchers delivered a 40-min presentation to control participants focused on the material that CATZ participants had been asked to include in their lessons (i.e. the CATZ LC). This allowed us to examine the effectiveness of CATZ against a form of direct instruction, something that is deemed another appropriate way -alongside a business as usual control group -to test an educational intervention (Yeager & Walton, 2011).
The three dependent variables were beliefs about supporting victims, specifically Victim Support -Emotional, Victim Support -Address Bully, and Victim Support -Other. The three DVs were derived from three open questions, "If you saw another child being bullied, what would you do?", "How could you try to stop a bully being nasty to someone without making them pick on you?", and "How could you help someone if they were bullied?" The coding procedure was similar to that employed in study 2. The number of "desirable and appropriate" responses across these three questions was tallied for each DV. Example responses meeting this criterion were "I would try to help the person feel better" (Victim Support -Emotional), "I would tell the bullying to stop doing it" (Victim Support -Address Bully), and "I would go and tell a teacher" (Victim Support -Other).
A sub-set of (non-CATZ) participants was asked these questions again one week later to assess test-retest reliability. Using the number of responses that indicated desirable and appropriate knowledge, reliability was good (all p < 0.001) for Victim Support -Emotional (r = 0.52), Victim Support -Address Bully (r = 0.66), and Victim Support -Other r = 0.67.
Social validity was assessed among a sub-set of CATZ tutors selected from across the three studies (N = 188), about a week after they had delivered their CATZ lesson, with four items: "How much would you like to design and give another CATZ lesson on something else about bullying?", "How much do you think designing and giving a CATZ lesson is a good way to help students your age learn about bullying?", "How much do you think CATZ is a good way to teach younger students about bullying?", and "How much do you think other students of your age in other schools would like to try CATZ?". They tap key aspects of acceptability, notably willingness to take part and perceived value (Elliott, 1986). A 1 ("not at all") to 10 ("a lot") response scale was employed. Cronbach's alpha was 0.88, and so a mean Social Validity score was computed across the four items. High scores indicate high social validity. A sub-set of participants (n = 74) were asked these questions again one week later to assess test-retest reliability and this was found to be high, r = 0.89, p < 0.001.
The CATZ Interventions and Training CATZ Tutors
CATZ was developed by the first author. The first author developed a semi-standardised protocol on the basis of considerable pilot work and previous evaluation studies of CATZ and used this to train co-authors to deliver CATZ in the three studies ("Facilitators" henceforward). The aim was to ensure relative -but not necessarily homogeneousconsistency in the way CATZ was delivered to students by Facilitators across the three studies, as this would mimic the kind of variation likely to result if CATZ was to be implemented more widely and delivered by teachers themselves in school communities (Boulton, 2014;Cowie et al., 1994). This was also made likely because each co-author of this paper acted as facilitator in only one of the three studies reported here.
Facilitators encouraged "buy-in" by explaining to CATZ tutors that taking part was voluntary, they could stop at any time (and re-join) without giving a reason, and they were being invited to work in small groups of about five students to design a (roughly) 30-min anti-bullying lesson and to deliver it to a small group of students who were two years younger than themselves. Facilitators stressed that this was an important task because the LC could help the younger students learn important things. Facilitators emphasised that, notwithstanding their responsibility to "educate" the younger students, tutors might actually enjoy taking part and themselves learn useful things. Indeed, given that students are often resistant to adult-implemented initiatives to tackle bullying partly because they are perceived as "boring" (Boulton & Bouloton, 2012), facilitators were asked to engender a sense of fun and ownership of the lesson among tutors that complemented their sense of responsibility. Tutors were informed that facilitators would provide them with the required lesson content (LC), offer suggestions about how to plan, test and deliver a lesson on it, but that the details would be left to them and they could augment that content. Facilitators aimed to strike a balance between being suitably supportive on one hand and leaving tutors to take ownership of their lesson on the other. While the final decision on the lesson itself was left to each group of tutors, facilitators ensured that as a minimum, they all designed a poster that contained the LC and prepared a script of what was to be said and done by each group member during their lesson. This ensured that the LC was addressed in each of the groups' lesson. Importantly, at no point did facilitators state or even imply that they "wanted" the tutors to learn this information or that tutors needed to change their bullying-related knowledge or behaviour. Rather, tutors were reminded that this was the information they would help the younger tutees learn via CATZ.
Across the three studies, CATZ tutors received similar guidance from facilitators and had similar time -about four 60-min sessions -to prepare their lesson, spread over 2-3 weeks. Then, within a few days, they delivered their lesson. Facilitators and class teachers observed these but did not take an active role. Facilitator reports confirmed the integrity, and hence relative consistency, with which they delivered CATZ to tutors, and in how CATZ tutors delivered their lesson to tutees. All groups of tutors were seen to take the task seriously and were judged to have done a good job.
Plan of Analysis
Because CATZ tutors were nested into groups, multilevel modelling was considered. However, we could not use these analyses because there was some changing of group membership that violates the assumption of independence across the different groups (Tabachnick & Fidell, 2014). Hence, to determine if CATZ did or did not have statistically significant effects on each variable, and to assess if gender was a moderator, a 2 (Condition) × 2 (or 3 where appropriate) (Time, repeated measures) × 2 (Gender) mixed analysis of variance (ANOVA) test was employed, and post hoc tests were used to identify sub-group differences, with Bonferroni corrections to control for family-wise inflation of type I errors.
Unlike tests of statistical significance, measures of effect size (ES) provide information about the practical significance of findings and the relative size of an experimental/ intervention effect and allow comparisons across studies and interventions (Thalheimer & Cook, 2002). Partial eta squared (η 2 ) was used as the index of ES in the ANOVAs, but we also calculated the much more widely used (and possibly understood) Cohen's d, with 95% confidence intervals. Cohen (1988) suggested effect sizes 0.20 to.49 be deemed small, those between 0.50 and 0.79 deemed medium, and those ≥ 0.80 deemed large. We also report the common language ES (McGraw & Wong, 1992) because it expresses an ES in an easy-to-understand format of a percentage. The latter represents the probability that any randomly selected person from one group will have a higher (or lower) value than any person selected at random from the other group after the intervention (Grissom & Kim, 2005).
Initial Equivalence of the CATZ and Control Groups
Independent group t-tests confirmed that on seven out of the nine outcome measures, CATZ and control groups did not differ at T1 (t's < 2.0, all p > 0.05). We now describe the two exceptions. For Acceptable Verbal, the CATZ group initially had significantly less desirable scores than controls (means = 1.53 and 1.20, respectively, t (97) = 2.93, p = 0.004).
Because there was more scope for CATZ participants to change in a desirable direction, this means it would be easier to detect a positive effect of CATZ on this measure. However, the CATZ group initially had significantly more desirable scores than controls on Victim Support -Emotional (means = 0.38 and 0.04, respectively, t (121) = 3.56, p = 0.001), and this means it would be more difficult here to detect a positive effect of CATZ. Thus, for all but one variable (Acceptable Verbal), there was scope for a "fair/conservative test" of the effects of CATZ.
Effects of CATZ on Individual Measures and Tests of Gender as a Moderator
The Condition x Time x Gender interaction was non-significant for all nine outcome variables, indicating that gender did not moderate any effects of CATZ. Hence, gender did not feature in any results we now go on to report concerning these nine outcome variables.
Descriptive data, summaries of Condition x Time interaction effects, and comparisons between CATZ and control participants at each assessment are given in Table 1. Across time comparisons (repeated measures t-tests) for CATZ participants are shown in Table 2. For all outcome measures, the Condition x Time interaction was significant. Using Cohen's (1988) scheme, partial η 2 ESs were low (< 0.06) for Acceptable Exclusion; medium (0.06 to 0.138) on four measures, Harmful Exclusion, Victim Support -Emotional, Victim Support -Address Bully, and Victim Support -Other; and high (> 0.138) on four measures, Harmful Verbal, Acceptable Verbal, Wanted Help, and When to Tell. With only one exception, involving Acceptable Exclusion at T2, CATZ participants had significantly more desirable scores than controls at T2 and at T3 on all variables. On none of the nine measures did control participants evidence a significant change in a positive direction from T1 to T2 or T1 to T3, but this was the case for all measures among the CATZ groups (Table 2). On the two study 2 measures with follow-up data (Wanted Help and When to Tell), T3 scores were significantly less desirable than at T2 among CATZ participants. Table 3 contains ESs for the nine outcome measures. In all cases, confidence intervals for Cohen's d did not contain zero, mirroring the ANOVA results reported above that indicated an effect of CATZ. For T1 to T2 changes, values of d were between "low" (0.2) and "medium" (0.5) for Harmful Exclusion (0.44) and Acceptable Exclusion (0.49); between "medium" and "high" (0.8) for Victim Support -Other (0.64), Victim Support -Emotional (0.68), and Victim Support 1 3 -Address Bully (0.79); and "high" plus for Harmful Verbal (0.83), Acceptable Verbal (0.95), When to Tell (1.51), and Wanted Help (1.57).
Effect Sizes
The common language ESs for the nine outcome measures indicated that there was between a 62% and an 87% (mean = 72.4%) probability that any randomly selected CATZ participant would have a more desirable T1 to T2 change score than any randomly selected control participant.
For T1 to T3 changes (study 2), values of d were "high" plus for Wanted Help (1.13) and When to Tell (1.14). The common language ESs indicated that there was a 79% probability that any randomly selected CATZ participant would have a more desirable T1 to T3 change score than any randomly selected control participant for both of these variables.
Social Validity
Overall, the mean social validity score was 8.6 on a 1-10 scale, with relatively little variability (standard deviation = 0.9), and 68.1% of participants scored 8 or above, a reasonable criterion for "high". The minimum score was 6.25. Social validity did not differ significantly between girls and boys, t (186) = 0.03.
Discussion
The main aim of this study was to investigate the effect of the relatively new CATZ co-operative cross-age teaching intervention on diverse bullying-related beliefs variables measured in three separate studies. With very few exceptions, results indicated that CATZ did have a positive effect. On eight out of nine measures, not Acceptable Exclusion, CATZ participants had significantly more desirable scores than controls at T2, despite this not being the case at T1. Moreover, CATZ participants showed significant improvements from T1 to T2 on all measures, whereas controls did not change for the better on any measure. On the two measures with follow-up data in study 2, Wanted Help and When to Tell, the T3 scores were significantly more desirable than at T1 among CATZ participants, but did not change in that direction among controls.
Cohen's d ESs for the effects of CATZ were between "low" and "medium" for Harmful Exclusion and Acceptable Exclusion; between "medium" and "high" for Victim Support -Other, Victim Support -Emotional, and Victim Support -Address Bully; and "high" plus for Harmful Verbal, Acceptable Verbal, When to Tell, and Wanted Help. The common language ESs for the nine outcome measures indicated that there was between a 62% and an 87% (mean = 72.4%) probability that any randomly selected Table 1 Mean (and standard deviation) scores of the individual studies, results of t-tests to compare CATZ and controls at each time, and condition × time interaction effects More desirable scores are low in study 1 and high in studies 2 and 3 *p < .05; **p < .01; ***p < . CATZ participant would have a more desirable T1 to T2 change score than any randomly selected control participant. Summarising this aspect of our findings, the mostly medium or high ESs indicate that CATZ is likely to have noteworthy practical benefits for those who experience it. The latter contention is endorsed by our other finding that the positive effect of CATZ was equally evident among girls and boys, and so the practical recommendations we offer below appear to be relevant to both. Positive effects of CATZ on boys are especially encouraging given that they have been found to score in a less desirable manner on a range of bullying related variables than girls; boys tend to disclose being bullied less than girls (Boulton, 2005;Hunter et al., 2004), are less likely to intervene in bullying and support the victim (Gini et al., 2008;Macaulay et al., 2019), and often see bullying as less serious (Maunder et al., 2010;Molluzzo & Lawler, 2012). We have shown that all of these beliefs can be addressed via CATZ and hence suggest that a wider implementation of the intervention would be beneficial within school. Benefits may extend beyond the students who hold more positive beliefs. For example, the covert nature of relational bullying introduces challenges for teachers to identify and support recipients of it and CATZ offers useful strategies that they can use to ask adults for help.
After experiencing CATZ, a sub-set of our participants (N = 188) were asked to rate it for acceptability and perceived value, and over two-thirds had very high scores, 8 or above on a 1-10 scale. Again, gender differences were not evident. It is encouraging that boys were as open as girls to engaging in CATZ, given that the former tend to be less enthusiastic towards other forms of peer support, broadly defined (Boulton, 2005;Cowie, 2000;Cowie et al., 2002;Peterson & Rigby, 1999). What it is about CATZ that appeals to students, especially boys, is worthy of study. Collectively, our findings of effectiveness and social validity provide strong support for CATZ as an anti-bullying intervention targeted at "improving" beliefs. Schools have many issues to deal with besides bullying, and "short but effective" interventions will likely be taken up more widely (Boulton, 2014). CATZ appears to meet this criterion, and future studies could explore how schools might incorporate CATZ into their wider antibullying efforts. Importantly, positive effects of CATZ Victim Support -Emotional 5.94*** Victim Support -Address Bully 6.80*** Victim Support -Other 3.98*** were found across the three studies, each of which were delivered by different facilitators. This is encouraging as for a more widespread school-level implementation of CATZ; different teachers will be acting as facilitators for the CATZ intervention and providing the learning content for the tutors to rework into a viable lesson to be delivered to younger tutees. One important aspect of any intervention implementation is the fiscal stability of resources needed for an effective outcome (Forman et al., 2009). Furthermore, to avoid replication failures, the training of implementers to run an intervention scheme needs to be feasible (Kumpfer et al., 2020). A helpful thing about CATZ is that teachers could easily be trained to act as facilitators for the CATZ sessions, which would in turn reduce the costs involved running the intervention. As schools have many different budgetary constraints which impact their decision to participate in an intervention scheme (Boulton, 2014), CATZ offers them a cost-effective way to enhance male and female students' anti-bullying beliefs. An important caveat for any enthusiasm for CATZ is that our follow-up data in study 2 showed that some of the gains were lost from T2 to T3. Nevertheless, T3 scores among CATZ participants were still significantly more desirable than T1 scores on the two relevant variables. Whether these "losses" can be eliminated with more CATZ experiences is a worthy issue for future studies. That this is a realistic possibility is suggested by research on memory and consolidation of learning which highlights the benefits of "extra" time and experience with learning material (McGaugh, 2000).
Cross-national research has shown variations in bullyingrelated variables (Boulton et al., 1999;Menesini et al., 1997) that could influence how receptive children are to CATZ and how much of a dose might be required. Researchers are beginning to test if CATZ can have similarly positive effects on students in different countries, and though early results are encouraging (Marx, 2018), more studies are clearly warranted. The same is true for age since we only studied upper primary school age students here. While CATZ has been shown to improve bullying-related beliefs among high school students (Boulton & Boulton, 2017), researchers would do well to examine the effectiveness and social validity as a function of age.
In evaluating this work, our three studies met four out of six criteria identified as important in intervention evaluations by Durlak et al. (1991); sample size exceeded 30 in each group, random assignment (at the class level) and intent-to-treat design were employed, and all pre-test posttest comparisons are reported. However, no blinded outcomes were recorded in any study, and an attention-only control condition was employed only in studies 1 and 2. Future studies should strive to meet the latter two criteria, but they are difficult to achieve in practice; many of our participants made spontaneous comments about their experiences of CATZ during data collection that would compromise blind testing, and teachers were reluctant to "waste time" on an attention-only placebo. Indeed, most teachers only agreed to take part in our studies if there was a "proper" intervention. Wait-list control methods offer a solution, but they are more disruptive and require extra time that schools are often unable to provide. Our finding from study 3 that CATZ had positive effects on beliefs that were not evident among children who experienced a direct attempt to change those beliefs via a lesson may help convince more schools that CATZ is a "proper" intervention. It must also be noted that different variables were assessed in the three studies, and that some of the effects found are therefore based from a relatively small sample size.
While seeking to explain the oft-found discrepancy between attitudes and behaviour, Ajzen (1991) proposed the theory of planned behaviour which is the modified version of an earlier model, the theory of reasoned action (Ajzen & Fishbein, 1980). The theory has been employed by researchers largely to investigate the impact of motivational factors on intentions to act and behaviour per se. Theorists believe that actors' behavioural intentions are the most immediate predictors of behaviour (Ajzen & Fishbein, 1980) and that attitude towards the behaviour, perceived subjective norm, and perceived control over the behaviour also play a role (Ajzen, 1991). While a strong case can be made for studying the kinds of beliefs/ knowledge variables included in our three studies, not least because they are thought to influence actual bullying-related behaviour (Boulton et al., 2002;Boulton et al., 2001;Boulton et al., 2002;Salmivalli & Voeten, 2004), the fact that we did not assess the effects of CATZ on any behavioural measure per se can be considered a limitation. As such, future research should endeavour to explore the components of theory of planned behaviour in the context of the CATZ intervention. For example, in the context of bystander behaviour, if bystanders do not know what do to when they witness bullying, CATZ can be used to promote knowledge on what strategies they can use. In other words, if we can promote positive attitudes on acting as a positive bystander, via CATZ, we can work to promote intentions to act the behaviour to combat bullying. In addition, interesting questions about the role of changes in the types of cognitions we studied as mediators of the effects of CATZ on actual behaviour arise out of our work and provide fruitful avenues to address in the future.
Our study is also limited by its focus on what might be described as rather "unintentional" beliefs about bullying as opposed to "intentional" beliefs more directly related to perpetrating bullying and/or intervening in a supportive way, such as "I believe I have a duty to help someone being bullied and I will do so if I see it taking place". Hence, future studies would do well to extend the range of beliefs examined that might improve following experience of CATZ.
That our dependent variables were (i) all single items, (ii) not from established measures, and (iii) high in social validity mean that it is possible that some of our results could be attributable to social desirability effects. Future studies would do well to address these limitations. However, that it would be a mistake to dismiss the entire set of results reported here as mere artifacts of social desirability is suggested by the facts that scores were not uniformly high at T1 and that at T2 and T3 scores improved among CATZ but not control participants.
Another aspect of our study that could limit the extent to which our results can be generalised is the fact that the CATZ facilitators, although different in each of the three studies, were nevertheless all trained by the same person. Hence, it remains possible that some of the positive effects found could be attributable to them and how they worked with the children rather than to CATZ itself. Future studies that involved diverse facilitators trained by other people are clearly warranted to rule out this possibility and at the very least the current study provides an empirical rationale for such a body of work.
In sum, with very few exceptions, the evidence from three separate studies suggests that CATZ can help students acquire important and diverse bullying-related knowledge. Unlike some other teacher-led interventions, students appear very receptive to it. While we need more research to understand how its effects may be maximised, and how effective it might be with other groups and with other facets of bullying-related beliefs, our findings support the wider take-up of CATZ as part of schools' efforts to tackle the pervasive problem of bullying.
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/. | 2021-11-11T16:11:19.632Z | 2021-11-09T00:00:00.000 | {
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16173957 | pes2o/s2orc | v3-fos-license | The line parameters and ratios as the physical probe of the line emitting regions in AGN
Here we discuss the physical conditions in the emission line regions (ELR) of active galactic nuclei (AGN), with the special emphasize on the unresolved problems, e.g. the stratification of the Broad Line Region (BLR) or the failure of the photoionization to explain the strong observed optical Fe II emission. We use here different line fluxes in order to probe the properties of the ELR, such as the hydrogen Balmer lines (Ha to He), the helium lines from two subsequent ionization levels (He II 4686 and He I 5876) and the strongest Fe II lines in the wavelength interval 4400-5400 \AA. We found that the hydrogen Balmer and helium lines can be used for the estimates of the physical parameters of the BLR, and we show that the Fe II emission is mostly emitted from an intermediate line region (ILR), that is located further away from the central continuum source than the BLR.
Introduction
Active galactic nuclei (AGN) are a ubiquitous phenomena, in a sense that most galaxies experience some sort of activity in their nucleus during their evolution. The most accepted scenario of the AGN structure is that they are powered by the accretion of matter onto a supermassive black hole (SMBH). One of the ways to study the inner emitting regions of an AGN, is by analyzing its emission lines, i.e. the broad (BELs) and narrow emission lines (NELs). So far many papers and textbooks are devoted to the physical properties of the emission line regions (ELR) (see e.g. Boroson & Green, 1992;Sulentic et al., 2000;Osterbrock & Ferland, 2006, and references therein), but, there are still many open issues.
Spectroscopy, in general, offers different methods for diagnostics of the emitting plasma (see e.g. Griem, 1997;Osterbrock & Ferland, 2006), but these methods could not be properly used to probe the physical conditions in some ELR of AGN, as e.g. in the Broad Line Region (BLR), since the forbidden lines, that are usually employed in plasma diagnostics of the Narrow Line Region (NLR) or HII regions, are not present in the BELs spectrum. Particularly, it is difficult to find a direct method which would only use the observed BELs to determine the temperature and density in the BLR. On the other hand, the optical Fe II (λλ 4400-5400 Å) lines are one of the most interesting features in the AGN spectrum. The origin of the optical Fe II extreme emission and the geometrical place of the Fe II emission region in AGN, are still open questions. Also, there are many correlations of the Fe II lines and other AGN spectral properties which need physical explanation such as: EW Fe II vs. EW [OIII] EWHβ , EW Fe II vs. peak [O III], EW Fe II and FWHM of Hβ, etc. (Boroson & Green, 1992).
Email address: dilic@matf.bg.ac.rs (D. Ilić) In order to estimate the physical conditions (such as the temperature and hydrogen density) of the BLR we use the Balmer and helium line ratios obtained in two ways: (i) using the photoionization code CLOUDY, a spectral synthesis code designed to simulate conditions within a plasma and model the resulting spectrum, and (ii) extracting a sample of AGN from the Sloan Digital Sky Survey (SDSS) database. We investigate these line ratios in order to find conditions in the BLR where socalled Boltzmann-plot (BP) method is applicable (Griem, 1997;Popović, 2003Popović, , 2006a. For these special cases, we study the correlations between the average temperature, hydrogen density and He II/He I line ratio. Moreover, we present an investigation of the optical Fe II emission in AGN, for which we have used an additional sample of 111 AGN from the SDSS database. The strongest Fe II lines are identified and classified into four groups according to the lower level of the transition: 4 F, 6 S, 4 G and 2 D1. In this progress report, we report our recent investigations of the physical and kinematical properties of the BLR and Fe II emitting region. This report is organized as follows: in §2 we describe the numerical simulations of the BLR and briefly introduce the BP method, and give the analysis of the simulated BELs; in §3 we study the SDSS sample of hydrogen Balmer and helium lines, while in §4 the selection and analysis of the SDSS sample of Fe II lines are given; in §5 we discuss some results and finally, in §6 our conclusions are given.
The BEL simulations
In order to study the BELs, we simulated the BLR emission line spectrum from different grids of the BLR photoionization models using the CLOUDY code (version C07.02.01: Ferland et al., 1998;Ferland, 2006). Input parameters for the simulations are chosen to match the standard conditions in the BLR (Ferland, 2006;Korista & Goad, 2000, i.e. the solar chemical abundances, the constant hydrogen density, the code's AGN template for the incident continuum shape. We compute an emission-line spectrum for the coordinate pair of hydrogen gas density n H [cm −3 ] and hydrogen-ionizing photon flux Φ H [cm −2 s −1 ]. The grid dimensions spanned 4 orders of magnitude in each direction, and with an origin of log n H = 8, log Φ H = 17 was stepped in 0.2 dex increments. A column density N H [cm −2 ] was kept constant in producing one grid of simulations. Even though many authors claim that the most probable value of the BLR column density is N H = 10 23 cm −2 (Dumont, Collin-Souffrin & Nazarova, 1998;Korista & Goad, 2000, we produce here 5 different grids of models changing the column density in the range N H = 10 21 − 10 25 cm −2 . We further analyze the BEL fluxes 1 from the CLOUDY grids of models. We consider in our analysis the hydrogen Balmer lines (Hα to Hε) and the flux ratio R of the helium lines He II λ4686 and He I λ5876, defined as R = F(HeIIλ4686)/F(HeIλ5876), where F is the line flux. We particularly consider these helium lines, since they are the lines of the same element but in two different ionization states, thus their ratio He II λ4686/He I λ5876 is sensitive to the change in the temperature and density (see e.g. Griem, 1997). Besides, these lines are in the same spectral range as Balmer lines. Additionally, from the grids of models we consider in our analysis an averaged temperature, which is the electron temperature averaged over the BLR radius (T av further in the text).
The Boltzmann-plot method for excitation temperature di-
agnostics For the plasma of the length ℓ that emits along the line of sight, assuming that the temperature and emitter density does not vary too much, the flux F ul of a transition from the upper to the lower level (u → l) can be calculated as (Griem, 1997;Popović, 2003Popović, , 2006aPopović et al., 2008) where λ is the transition wavelength, g u is the statistical weight of the upper level, A ul is transition probability, N 0 is the averaged total number density of radiating species which effectively contribute to the line flux (which are not absorbed), Z is the partition function, E u is the energy of the upper level, T is the averaged excitation temperature, and h, c and k are the Planck constant, the speed of light, and the Boltzmann constant, respectively. The additional assumption made here is that the population of the upper level in the transition follows the Saha-Boltzmann distribution (for more detailed derivation check Popović et al., 2008). From the above equation comes the so-called Boltzmann plot (BP) that can be used to estimate the excitation temperature T exc in the BLR log 10 (F n ) = log 10 F ul · λ g u A ul = B − AE u 1 The CLOUDY code gives all line fluxes normalized to the Hβ flux. Since it has no influence in our analysis, we have used these values. where B and A are BP parameters, and A = log 10 (e)/kT exc ≈ 5040/T exc is the temperature indicator. Therefore, for one line series (as e.g. Balmer line series) if the population of the upper energy states (n ≥ 3) 2 can be described with the Saha-Boltzmann distribution, then by applying the last equation to that line series and obtaining the value of the parameter A, we can estimate the excitation temperature of the region where these lines are originating. We should emphasize that the additional assumption in the BP method is that the Balmer lines are originating in the same emitting region.
In spite of the fact that the BP method has some obvious advantages, i.e. it is only using the measured Balmer line fluxes, that are easily observed, to estimate the excitation temperature, one should take into account some possible problems that could occur when using the emission line to determine the BLR physical properties: (i) the line profiles are usually very complex and indicate that more than one component contribute to the total line flux, therefore one should be aware of the multi-component BLR structure when estimating the BEL fluxes; (ii) the Balmer lines do not have to necessarily originate in the same region, e.g. there some indications that the Hα and Hβ line are forming in two distinct regions since it has been shown that the Hβ line is systematically broader than Hα ; (iii) there are different mechanisms contribution to the line formation. The photoionization seems to be working well, but other 2 We note here that since the emission deexcitation goes as u → l it is not necessary that level l has the Saha-Boltzmann distribution. heating mechanisms should be taken into account.
The analysis of the simulated BELs
The first step in analyzing the BEL ratios is to apply the BP method on the Balmer line ratios given by CLOUDY to estimate the parameter A, from which we then calculate the BP temperature, i.e. the excitation temperature (T BP further in the text) of the region where Balmer lines are formed. Also, from the best-fitting of the normalized line ratios, we obtain the error of the BP fit ( f further in the text). A few examples of the BP applied on the Balmer lines simulated with CLOUDY for column density of N H = 10 23 cm −2 are presented in Fig. 1. In many cases a satisfactory BP fit is not obtained, i.e. f has pretty large values. This is more noticeable in the case of higher values of the hydrogen density and ionizing-photon flux, hence we plot the error of the BP fit f in the hydrogen density vs. ionizing flux plane for all 5 grids of models of different column density in Fig. 2 (only the contours inside which f is less than 10%, 20% and 30% are given). From Fig. 2 can be seen that if the BP method could be considered valid if f is less than 10% (eventually 20% in the measured spectra) the parameter space where this is valid is pretty constrained for all column densities N H .
Out of all photoionization models, we select just those that follow these constraints: (i) the error of the BP fit f less than 10%, (ii) the average temperature T av less than 20,000 K, as for larger temperatures the Balmer line ratios are not sensitive to the temperature changes and the BP method cannot be applied Popović (2006b), and (iii) the ratio of helium lines R less than 2. Following this criteria, we constructed 5 samples of simulated BELs, of single column density, labeled as e.g. CD21 for column density N H = 10 21 cm −2 , etc. The models that satisfy these 3 constraints are represented with asterisks in Figure 2.
The correlations between the BEL ratios and the BLR physical properties
We investigate in more details the five sets of simulated spectra defined above. First, we plot the average temperature T av of the emitting region, one of the outputs of the model, with respect to the ratio R of the helium lines for all 5 sets (Fig. 3, upper). We fit data sets with the linear function T av = A + B · R and the best fitting results are given in Table 1. Another possible connection of the BLR physical parameters and BELs, can be obtained from the relation between the hydrogen density and the helium line ratio R. We plot the logarithm of the hydrogen density and the ratio R (Fig. 3, bottom), and we fitted it with the function: log n H = D/(C + R), where n H is given in the units of 10 7 cm −3 . It can be seen from Fig. 3 (bottom) that even when the column density changes, the same dependance of the hydrogen density on the helium line ratio R remains. The best fitting results are given in Table 1.
The hydrogen Balmer and helium line sample
We compare our results of the numerical simulations with the observed data. For that we consider our measurements of well defined sample of 90 AGN taken from the SDSS spectral database (La Mura et al., 2007), for which the Blamer line ratios have been precisely measured and the temperature parameter A has been estimated using the BP method applied on the Balmer line series (see for details La Mura et al., 2007). We found that for this sample in approx 50% of cases, the BP method can be applied. In this analysis we consider objects that have error of the BP fit smaller than 20% 3 and the BP temperature smaller than 20 000 K. This reduced our sample to 48 objects.
For that sample, we measure the fluxes of the helium lines He II λ4686 and He I λ5876, being particularly careful in the case of He II λ4686 line, where there is contamination from the Fe II 3 Even though in numerical simulations the error was taken to be smaller than 10%, here in real measurements, we take larger range of fitting errors due to the additional observational and line flux measurement errors. For the He II λ4686 line measurements, we perform Gaussian decomposition (for details see e.g. Popović et al., 2004) in order to subtract the contribution of the Fe II multiplet, Hβ and narrow He II line (as an example see Fig. 4). A standard deviation is taken as an error of the line flux measurements. The cases when helium lines could not be measured are not considered (e.g. when lines were too noisy or the contribution of Fe II was too strong and could not be properly subtracted), thus our sample is reduced to 20 objects. Using the above relations between the physical properties of the BLR and helium line ratio R (Table 1), we estimate the average temperature and the hydrogen density of the BLR of these AGN assuming different column densities. We obtain the following ranges of physical parameters in the BLR: average temperature T av = 5500 − 17600 K and hydrogen density n H = 10 8.3 − 10 11.6 cm −3 .
Moreover, in Fig. 5 we plot the ratio of the helium lines as a function of the BP temperature T BP for the SDSS sample. As can be seen in Fig. 5 there is a weak correlation (correlation coefficient r = 0.50, p 0 = 0.02321) between BP temperature and the He line ratio. This is also in agreement with the BLR physics, where for higher temperatures one should expect to have stronger He II than He I lines. We should note here that if we exclude the point that is clearly high above the best-fitting line (see Fig. 5), the correlation coefficient is slightly better r = 0.62 (p 0 = 0.00473).
The Fe II line emitting region
We selected a sample of 111 AGN from Sloan Digital Sky Survey (SDSS) according to the following criteria: high signal to noise ratio (S/N > 20), good quality of the pixels, negligible contribution of the stellar component and a good coverage (near uniform) of redshifts from 0 to 0.7. In order to investigate the geometrical place of the Fe II emission region in AGN, we analyzed kinematical connection between the Fe II and Balmer emission region by comparing the line shifts and widths, obtained from the best-fitting. We assume that the broadening of the lines is caused by the Doppler effect from the random velocities of emission clouds, while the shift of the line is caused by the systemic motion of the emission gas. The results are presented in Fig. 8-9.
The widths of the Fe II and Hβ (upper) and Hα (bottom) lines are compared in Fig. 8. The X-axis gives the Fe II line widths, while the Y-axis gives the parameters of the widths of the Hβ (upper) and Hα (bottom) components: the NLR (triangles), ILR (circles) and VBLR (squares). Dotted lines show the averaged values of widths: Fe II (vertical line) and Hβ and Hα line components (horizontal lines). On the sample of the 111 AGN that contain the Hβ line (upper), the average value of Fe II widths is 1770 ± 580 km/s, while the average values for Hβ components are: 310 ± 160 km/s (NLR), 1850 ± 660 km/s (ILR) and 4690 ± 1210 km/s (VBLR). Considering the selected sample of 58 AGN which have the Hα line (bottom) the averaged value of the Fe II width is 1770 ± 570 km/s and for the Hα components: 270 ± 130 km/s (NLR), 1470 ± 620 km/s (ILR) and 4070 ± 1080 km/s (VBLR). It is obvious that the averaged Fe II width (1770 km/s) is very close to the averaged widths of the ILR component of both the Hβ and Hα line (1850 km/s and 1470 km/s, respectively), while the averaged widths of the NLR and VBLR components are significantly different. Fig. 9 shows the correlation between the Fe II widths and the Hβ ILR ones (r = 0.67, p 0 < 0.0001). Considering the Hα ILR component, correlation is stronger, and it is r = 0.72, p 0 < 0.0001. We have also found that a weaker correlation between widths of the Fe II and Hα VBLR component (r = 0.55, p 0 < 0.0001) is present. Relations between the shifts of Fe II and Hα and Hβ (NLR, ILR and VBLR components) are also investigated. It is noticed a positive correlation with the shift of the Hα ILR component (r = 0.51, p 0 < 0.0001). There is no a significant correlation between the Fe II shift and other Hα and Hβ components. Boroson & Green (1992) Then, we divided our initial sample of 111 AGN (0<z<0.7) in 6 subsamples, which are made by gradually removing the objects with low redshift in 6 steps. Subsamples contain object with redshifts in intervals: 0.1<z<0.7, 0.2<z<0.7, 0.3<z<0.7, 0.4<z<0.7, 0.5<z<0.7 and 0.6<z<0.7 Table 2). We found that negative trend (r = -0.37, P<0.0001) observed between EW Fe II and EW [O III]/EW Hβ in initial sample (0<z<0.7), progress
Results and discussion
In this progress report we give some results of our recent investigations of different broad line parameters (the flux ratios, EW, FWHM, etc.) in order to probe the physics of the ELR, i.e. the hydrogen Balmer lines (Hα to Hε), the helium lines from two subsequent ionization levels (He II λ4686 and He I λ5876) and the strongest Fe II lines in the wavelength interval λλ4400 − 5400Å.
From the analysis of the hydrogen Balmer lines, we can say that the BP method gives valid results if the error of the BP fit f is less than 10% (eventually 20% in the measured spectra).
The parameter space of CLOUDY simulations where f < 10% is well constrained and in a similar range for different column densities N H (Fig. 2). This area of parameters contains lower ionizing fluxes and higher hydrogen densities, but depending on the column density the area increases keeping the same trend between the n H and Φ H , Therefore, for those parameters the BP method could be applied for the estimation of the excitation temperature. This indicates that for some physical conditions, even if we have the photoionization as the main heating process in the BLR, the hydrogen Balmer lines are produce in such way that they obey the Saha-Boltzmann equation, i.e. the BLR plasma is at least in the Partial Local Thermodynamical Equilibrium.
The physical parameters of the simulations for which the BP method could be applied ( f < 10%) follow some relations even when the column density changes. There is a linear relation between the average temperature T av of the emitting region and helium lines ratio R: T av = A + B · R (Fig. 4, Table 1). Also, the hydrogen density depends on the helium line ratio R as: log n H = D/(C + R), where n H is given in the units of 10 7 cm −3 (Figure 4, Table 1). For lower column densities (N H = 10 21 − 10 22 cm −2 ) this linear trend for T av seems to be broken after some value of R (Fig. 4, upper), therefore the results obtained for this column densities should be taken with caution. The ranges of the average temperature and hydrogen density, obtained when these relations are applied to the observed sample of AGN taken from the SDSS database, are in good agreement with the previous estimates of the physical conditions in the BLR (Osterbrock & Ferland, 2006). Having in mind problems given in §2.1 (i.e. different emitting regions of helium and Balmer lines or the multicomponent origin of BELs) the relations given in Table 1 could be use as a rough estimate of the BLR physical parameters from direct measurements.
On the other hand, from the analysis of the Fe II emission, we found the significant correlation between the Fe II and Hα, Hβ ILR widths which indicate the ILR origin of the Fe II emission. Also, the Fe II emission region is mainly characterized with random velocity of ∼1800 km/s, that corresponds to the ILR origin. This result is in favor to the results obtained by Popović et al. (2004), and with the recent findings that the optical Fe II line forming region seems to not be the same as the BLR (Kuehn et al., 2008;Hu et al., 2008;Popović et al., 2009
Conclusions
We used here the emission lines to estimate the physical parameters of the BLR and properties of the Fe II line forming region. From our investigations we can outline the following conclusions: (i) In the case of the pure photoionized plasma, there can exist the conditions that the excitation of the hydrogen Balmer lines is sensitive to the temperature. In this case, the He II/He I line ratio can be used for the estimates of the averaged temperature and hydrogen density. This can be very useful for determining the physical properties of the BLR, and for a small sample of AGN we found that the BLR temperatures are ranging from ∼ 5000 K to 18000 K, and hydrogen densities from 10 8 cm −3 to 10 12 cm −3 . These values of the temperature and density are expected in the BLR plasma (Osterbrock & Ferland, 2006). (ii) The optical Fe II emission is likely coming from a region that corresponds to the ILR. Also, there is a weak correlation with the VBLR component of Balmer lines indicating that a fraction emitted in the Fe II lines can originate also in the BLR. On the other hand, the correlation between the EW FeII and EW [OIII] (EW[OIII]/EW Hβ) discovered by Boroson & Green (1992) seems to be sensitive on redshift (or luminosity) of AGN.
Finally, the BLR physics (and geometry) still remains an open question, and we hope that with this work we approached some conclusions which will help us in understanding the BLR physics. | 2009-08-19T15:42:39.000Z | 2009-07-01T00:00:00.000 | {
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259499614 | pes2o/s2orc | v3-fos-license | Meningococcal B Immunisation in Adults and Potential Broader Immunisation Strategies: A Narrative Review
Recombinant vaccines against invasive meningococcal disease due to Neisseria meningitidis serogroup B (MenB) have shown substantial impact in reducing MenB disease in targeted populations. 4CMenB targets four key N. meningitidis protein antigens; human factor H binding protein (fHbp), Neisserial heparin binding antigen (NHBA), Neisseria adhesin A (NadA) and the porin A protein (PorA P1.4), with one or more of these expressed by most pathogenic MenB strains, while MenB-FHbp targets two distinct fHbp variants. While many countries recommend MenB immunisation in adults considered at high risk due to underlying medical conditions or immunosuppression, there are no recommendations for routine use in the general adult population. We reviewed the burden of MenB in adults, where, while incidence rates remain low (and far lower than in young children < 5 years of age at greatest risk), a substantial proportion of MenB cases (20% or more) is now observed in the adult population; evident in Europe, Australia, and in the United States. We also reviewed immunogenicity data in adults from clinical studies conducted during MenB vaccine development and subsequent post-licensure studies. A 2-dose schedule of 4CMenB generates hSBA titres ≥ 1:4 towards all four key vaccine target antigens in up to 98–100% of subjects. For MenB-FHbp, a ≥ fourfold rise in hSBA titres against the four primary representative test strains was observed in 70–95% of recipients following a 3-dose schedule. While this suggests potential benefits for MenB immunisation if used in adult populations, data are limited (especially for adults > 50 years) and key aspects relating to duration of protection remain unclear. Although a broader adult MenB immunisation policy could provide greater protection of the adult population, additional data are required to support policy decision-making. Graphical Abstract
countries recommend MenB immunisation in adults considered at high risk due to underlying medical conditions or immunosuppression, there are no recommendations for routine use in the general adult population.We reviewed the burden of MenB in adults, where, while incidence rates remain low (and far lower than in young children\5 years of age at greatest risk), a substantial proportion of MenB cases (20% or more) is now observed in the adult population; evident in Europe, Australia, and in the United States.We also reviewed immunogenicity data in adults from clinical studies conducted during MenB vaccine development and subsequent post-licensure studies.A 2-dose schedule of 4CMenB generates hSBA titres C 1:4 towards all four key vaccine target antigens in up to 98-100% of subjects.For MenB-FHbp, a C fourfold rise in hSBA titres against the four primary representative test strains was observed in 70-95% of recipients following a 3-dose schedule.While this suggests potential benefits for MenB immunisation if used in adult populations, data are limited (especially for adults[50 years) and key aspects relating to duration of protection remain unclear.Although a broader adult MenB immunisation policy could provide greater protection of the adult population, additional data are required to support policy decision-making.
INTRODUCTION
Invasive meningococcal disease (IMD) due to Neisseria meningitidis remains an important global public health concern [1,2].Mortality is high, with reported case fatality rates (CFRs) ranging from 4% to 20% (and up to 30% in older adults) [3].Serious sequelae, including a broad range of neurological complications (seizures, hearing loss, visual upset and language impairment), are reported in up to 20% of survivors [4][5][6], resulting in long-term physical disability, social impairment and reduced quality of life (QoL) [6,7].Such sequelae contribute to the reduced lifetime expectancy and premature death observed in those recovering from initial infection [6,8].
Recognised risk factors for IMD span a range of individual and social elements.The greatest risk (as evident from greatest incidence rates) is seen in infants and young children, and then in adolescents and young adults [2,9,10].IMD risk is higher in individuals with congenital or acquired immunosuppressive conditions (asplenia/splenic dysfunction, complement deficiency) or receiving specific immunosuppressive medications (e.g.complement component inhibitors) [11][12][13], and in specific groups such as men who have sex with men (MSM) [14].In addition, social aspects such as overcrowding, including dormitory accommodation (e.g. in higher education facilities and for military personnel) and that associated with lower socioeconomic status are also associated with greater risk (and disease outbreaks) [1,11,[15][16][17].Mass gatherings and religious events such as the Hajj and Umrah pilgrimages are also well-recognized risk factors for meningococcal transmission and subsequent outbreaks [18,19].Risk is also greater in people experiencing homelessness [20], and in immigrant and refugee populations [21].Tobacco smoking and passive exposure may also increase IMD risk [22].
As we detail later in this article, adoption and implementation of MenB vaccines in routine immunisation programmes continues to evolve, with the current focus in most countries towards younger children or in adolescents (and also younger adults in the United States); at present, there are no recommendations for routine use in the general adult population [57][58][59][60].Evidence for vaccine impact derived from such routine use (or when used in focused vaccination campaigns) have shown substantial impact in reducing MenB disease in targeted populations [61].However, in contrast to conjugated MenC and MenACWY vaccines, studies evaluating the impact of MenB vaccines on N. meningitidis carriage have shown no evidence for any significant effect [61][62][63][64][65], underlining the importance of MenB vaccination for direct protection of all those at risk of IMD due to MenB.
One impact of the introduction of MenB immunisation targeted towards children and adolescents (with subsequent substantial declines in IMD in these age groups) is that significant proportion of overall MenB cases in many countries (approximately 20%) now occur in adults and the elderly; a feature seen in Europe [28, 29,66], the United States [30,31], and Australia [33][34][35][36].Despite approval for use in adult populations in most countries where MenB vaccines are available (except North America), the current focus on childhood or adolescent MenB immunisation is such that the increasing burden of MenB disease in adults is likely to remain unchanged.
In the present review, we consider the burden of MenB in adults and describe the development and implementation of MenB vaccines.While data are limited, we review the immunogenicity and safety data for 4CMenB and MenB-FHbp when given to adult populations.While these are supportive of broader use of MenB immunisation in adult populations, an approach in alignment with growing support for a lifetime immunisation approach to vaccination policy [67,68], we also highlight the need for additional data to inform such decision-making.
METHODS
To inform this review, we initially searched PubMed electronic databases using search terms relating to meningitis and meningococcal disease to identify relevant publications reporting IMD epidemiology, meningococcal immunization policy, and clinical studies on 4CMenB and MenB-FHbp.The search strategy included a range of free-text and MeSH search terms, e.g.''meningococcal infection'', ''N.meningitidis'', ''IMD'', ''4CMenB'', ''MenB-FHbp'' ''Bexsero'', ''Trumenba'' in various combinations.Identified articles were supplemented by snowball sampling of relevant citations from the consulted papers.In addition, we reviewed relevant immunisation policy documents from a broad range of countries and publicly available reports on 4CMenB and MenB-FHbp approval and licensed indications.While comprehensive, our approach was not formally systematic.Searches were focused primarily on English language publications, although with no specific selection criteria or specific publication date limitations.Epidemiological data for Europe were also identified from the European Centre for Disease Prevention and Control (ECDC) Surveillance Atlas of Infectious Diseases tool [29] (last accessed 26 April 2023).In this, data for confirmed MenB case numbers were extracted for each year from 2011 onwards.As this tool presents data stratified in the following age groups (\1 year; 1-4 years; 15-24 years; and C 50 years), this format was retained in our reporting.For all other epidemiologic data, a broader range of age strata was used.
This article is based on previously conducted studies and does not contain any new studies with human participants or animals.
Despite this relatively lower incidence a substantial number of MenB cases occur in adults.This is illustrated by appraising data for confirmed MenB cases in regions/countries with robust surveillance reporting.In Europe, where a decline in MenB incidence between 2008 and 2017 has been observed across all age groups, this decline was greatest in younger children, less so in adolescents, and with the lowest relative decline observed in adults C 50 years (where rates have remained relatively stable) [28].This decline, and the recent implementation of routine infant 4CMenB immunisation in some countries, has led to changing patterns in the overall MenB case burden, with a high proportion of the overall case burden now observed in the adult population (Fig. 2).Surveillance data from the ECDC indicates that this pattern is seen across Europe, and in countries with established MenB infant immunisation such as the United Kingdom, and also in those countries (e.g.France) where the impact of more recent implementation of infant MenB immunisation (in 2022) remains to be established [29].In 2019, approximately 20% of all MenB cases involved individuals[50 years of age (Fig. 2).In the United States, data indicate that, of the 698 MenB cases reported by the CDC between 2015 and 2020, those involving individuals C 24 years accounted for 43% of the cases, and with 26% of all MenB cases occurring in adults aged C 45 years [30,31,[69][70][71][72] (Fig. 3).In Australia, of the 313 MenB cases reported across 2018-2021, 27% occurred in adults C 25 years of age (and 16% in those aged C 45 years) [33][34][35][36].These data highlight the increasing contribution of adult MenB disease to the overall burden within populations, and the potential value of adult MenB immunisation in reducing adult MenB disease.Although not the focus of this article, it should be recognised that, as well as the proportionately greater burden of MenB disease in adults, substantial increases in the incidence of IMD due to MenW and MenY are also apparent globally in recent years in adults (especially in adults[50 years) [2,73,74].
Initially, development and subsequent regulatory approval of 4CMenB and MenB-FHbp was on the basis of responses towards specific test MenB strains, chosen for their expression of one or more of these four main vaccine target antigens, and to accommodate meningococcal genetic diversity and global distribution of key pathogenic strains [45,81].In 4CMenB development, these included strain 44/76-SL (fHbp), strain 5/99 (NadA), strain NZ 98/254 (PorA P1.4), with the later inclusion of strain M10713 as a specific test strain for NHBA [45,81,82].For MenB-FHbp, four core strains were identified on the basis of fHbp subfamily A and B peptides (strains PMB80, PMB2001, PMB2948 and PMB2707) with ten additional strains expressing diverse fHbp peptides also used in selected studies [83].
Immunogenicity against these strains was assessed using specific bactericidal antibody assays with human complement (hSBA), with protective antibody responses against vaccine antigens considered as hSBA titres C 4 or C 5 observed for both 4CMenB and MenB-FHbp vaccines [43,81,83].As circulating MenB strains expressing target antigens show substantial diversity within different populations, novel platforms to define the extent of predicted coverage of 4CMenB and MenB-FHbp against MenB strains are now in established use [44,83].For 4CMenB, the meningococcal antigen typing system (MATS) is used in isolates from culture confirmed cases, and the complementary genomic system (gMATS) can predict coverage in non-culture PCR-confirmed cases, while an alternative antigen sequence type (BAST) platform can predict coverage for both culture-confirmed and PCR-confirmed cases [84,85].For MenB-FHbp, the Meningococcal Antigen Surface Expression (MEASURE) assay can quantify fHbp expression on isolates from culture-confirmed cases [43,45,84].Global studies utilising these platforms against extensive IMD isolate panels indicate relatively high predicted strain coverages for both 4CMenB and MenB-FHbp [75,84,[86][87][88], and both 4CMenB and MenB-FHbp induce robust responses towards MenB outbreak strains [43,[89][90][91][92][93].In addition, as these vaccine target antigens are capsule-independent, with antigens conserved across different capsular serogroups, 4CMenB and MenB-FHbp offer potential protection against non-B serogroups, with studies reporting high predicted coverage for both vaccines against MenC, MenW and MenY isolates from infants and adolescents [77][78][79], while predicted activity against other Neisseria species such as N. gonorrhoeae has also been reported [94,95].
MenB Vaccine Clinical Trial Programmes
Licensure of 4CMenB followed an extensive clinical trial programme evaluating immunogenicity and safety/reactogenicity in more than 7000 infants, toddlers, adolescents and adults [44,81,96].Most studies have focused on those age groups at greatest risk (infants, toddlers, and adolescents) with studies demonstrating robust immune responses to each of the three subcapsular target antigens, NadA, fHbp and NHBA, and also towards the OMV PorA1.4 protein [44,81,96].As in other meningococcal vaccine development, as IMD is relatively uncommon, the initial licensure was on the basis of immunogenicity, with an accepted surrogate of protection (hSBA C 1:4), with subsequent post-licensure studies required to demonstrate effectiveness in targeted populations [81].
Immunisation in the first year of life generates protective titres (hSBA C 1:4) against test strains in 84-100% of infants after three doses, with marked increases in hSBA geometric mean titres (GMTs) and in the percentage of children with hSBA titres C 4 observed after a subsequent booster dose [43,61,[97][98][99].While waning of immunogenicity is observed, persistence of protective titres for up to 36 months has been reported [98].A 2 ? 1 primary vaccination schedule with vaccinations at the age of 2, 4 and 12 months is now the preferred schedule in b Fig. 1 [43].Protective titres against MenB test strains were observed in 72-99% of individuals after a 3-dose schedule (and in 69-98% after a 2-dose schedule) [43,45].Persistence for up to 4 years is reported in over 50% of subjects, with booster vaccination eliciting robust responses [105,106].A 2-dose schedule is preferred for routine adolescent MenB-FHbp vaccination [43].Studies of MenB-FHbp and 4CMenB in adult populations are described below.
MenB Vaccine Approval
Both 4CMenB and MenB-FHbp are now widely available, although approval for use in specific age groups varies within different countries, with more restricted use in North America (Table 1).4CMenB was first licensed in the European Union (EU) in 2013 [47] (and such approval continues within the United Kingdom following departure from the EU) [48], and also in Australia [49] and Canada [55] the same year, and then in the United States in 2015 [53].4CMenB is currently approved in 50 countries worldwide, including Argentina, Brazil, Chile, Hong Kong, Israel, Saudi Arabia, New Zealand, Switzerland, Turkey, the United Arab Emirates and Uruguay [57].Recommended dosing in infants and toddlers is 2 or 3 doses given 1 or 2 months apart, and with a booster dose at 12--23 months of age [43,57].In older children, adolescents and adults, a 2-dose schedule is recommended [43,57].
MenB-FHbp was initially approved in the United States in 2014 [54], and in the EU [50, 51], Australia [52] and Canada [56] in 2017.MenB-FHbp is currently approved in over 50 countries worldwide, including Argentina, Brazil, Chile, Colombia, Hong Kong, Israel, Kazakhstan, Kuwait and Singapore [57].Recommended dosing in individuals aged from 10 through 25 years is 2 doses given at least 6 months apart, or where more rapid immunity is desired, e.g. in outbreak situations, as a 3-dose schedule with C 1 month between the first and second doses and the third dose given C 4 months after the second dose [43,57].Of some importance is that, unlike other meningococcal vaccines (e.g.quadrivalent MenACWY), 4CMenB and MenB-FHbp are not interchangeable, and so the same vaccine must be used for the complete vaccination series [43,57].
Implementation of MenB Vaccines
Inclusion of MenB vaccines in National Immunisation Programmes (NIPs) continues to evolve, in response to both shifting epidemiology and the introduction and availability of MenB vaccines at a national level [57][58][59].While most countries have a single national policy, implementation of MenB vaccine use may also be devolved on a regional/state basis, with individuals in some regions eligible for MenB vaccines out of national recommendations (e.g. in Australia) [107,108].At present, despite widespread approval and availability, inclusion of routine MenB immunisation within NIPs remains relatively restricted, and generally focused on infant/toddler immunisation (in Europe) and in adolescents and younger adults in the US [43,57,59].In Europe, fully funded infant/toddler MenB immunisation (using 4CMenB) is included in the NIPs of the United Kingdom (since 2015), and then Italy, Portugal, Ireland, Lithuania, Malta, and more recently in the Czech Republic [57][58][59].In France, following expert recommendations in 2021 [59], infant 4CMenB was formally included within the NIP in 2022 [109].In Spain, while infant/toddler 4CMenB immunisation was previously only implemented in some regions (Castilla y Leon, the Canary Islands, Catalonia and Andalusia), this policy will be implemented nationwide from 2023 [110].Other countries such as Austria and Hungary include infant 4CMenB immunisation in their NIP, but these are not funded [57].In most of these settings, infant/toddler dosing is usually in a 2 ? 1 schedule (although in some countries, e.g.Italy and Hungary, a 3 ? 1 schedule is used) [57,59].Adolescent MenB immunisation is also included in the Czech Republic [15,59], while vaccination of those aged 11-12 years is funded in the Puglia region of Italy (since 2018), where adolescent and adult vaccination has also been offered (chiefly through MenB-FHbp) [111].
Outside Europe, MenB vaccines have a more limited place in NIPs.In the United States, MenB immunisation (via either 4CMenB or MenB-FHbp) is recommended for those aged 16-23 years (on the basis of shared clinical decision-making), where the preferred age for vaccination is 16-18 years [13].In Canada, routine MenB immunisation is not included in either national or regional immunisation programmes [57,112].In Australia, while all infants and toddlers in at-risk ethnic populations (Aboriginal or Torres Strait Islander children) should be offered 4CMenB [107,108], broader routine infant 4CMenB vaccination is restricted to those residing in South Australia state (since 2018), where a funded school-based 4CMenB vaccination programme targeting adolescents aged 15-16 years also exists, and where catch-up immunisation for children aged up to 3 years and for those aged 17-20 years are also in place [57,113].While New Zealand has previously had no routine MenB vaccination policy, this will be implemented in 2023 for all infants up to 12 months of age, and also for people aged 13-25 years who are entering into or in their first year of specific close-living situations (boarding school hostel, tertiary education halls of residence, military barracks or prison) [114].In Brazil, although a MenB immunisation policy for infants, toddlers and adolescents is recommended by the Brazilian Pediatric Society, this is not publicly funded and remains out with the NIP [57,115].
Beyond these age-based recommendations, MenB is also offered to individuals considered 'at risk' within NIPs (and indeed were usually in place before the introduction of any age-based recommendations) [57][58][59].This spans a wide range of at-risk medical conditions or situational risk, and applies across all age groups, including adults, although what constitutes at risk varies widely across countries (reviewed in [57]).For example, in some countries, HIV infection is not a specific indication for MenB vaccination [57].In the United States and in the United Kingdom, persons considered at high risk due to pre-existing medical conditions include individuals with asplenia or splenic dysfunction (including sickle-cell disease) and those with persistent complement component deficiencies or receiving a complement component inhibitor (e.g.eculizumab) [13,116].Other individuals considered at situational risk include those with occupational health exposure (e.g.laboratory personnel) or living in specific high-risk environments (e.g.college dormitory students, military recruits) [13].In addition, immunisation of close contacts of MenB cases is also recommended.In these circumstances, either 4CMenB or MenB-FHbp can be used, depending on age [13].In Canada (where no routine MenB immunisation is recommended), a similarly broad range of at-risk categories where MenB immunisation should be considered also exist [117].While in North America, these recommendations would include adults out with the licensed age-based indication, such off-label use is considered appropriate based on expert opinion [13,117].
In Australia and New Zealand, national recommendations include a similar broad range of at-risk categories or situations where MenB immunisation should be considered [107,108,118].Within Europe, while individuals with underlying medical conditions associated with increased risk are included in most NIPs, specific eligibility criteria differ across countries [57].While some countries recommend immunisation of close contacts of affected MenB IMD cases, such policies also vary in different countries [57,59].
Effectiveness of MenB Vaccines in the Real-World
Post-licensure studies of 4CMenB evaluating effectiveness following its introduction in NIPs have demonstrated substantial reductions in MenB disease in vaccine-eligible populations (chiefly those\5 years of age) [61].In the United Kingdom, after 3 years of use within the NIP, a surveillance-based study across 2015-2018 in England reported a 75% reduction in the incidence of IMD due to MenB in children fully eligible for vaccination [119], with evidence of continued impact and with further reductions in MenB disease in subsequent years in children aged\5 years [120].Case-control and surveillance studies conducted in Australia, Portugal, Italy and Spain also report robust VE against MenB in chil-dren\5 years of age (ranging from 71.0% to 94.2%) [113,[121][122][123]. Data are limited for older populations.In South Australia, a cluster randomised trial reported that, following 4CMenB immunisation at 15-16 years of age, a 71% (95% CI 15-90%) overall reduction in the incidence of MenB in vaccinated subjects (aged 16-19 years) in the subsequent two years (2018-2019) compared with historical incidence (between 2003 and 2016) [124].
In addition to impact on MenB disease, some data indicate an impact of 4CMenB on other meningococcal serogroups (and also on other important Neisseria pathogens such as N. gonorrhoeae) [95].Cohort and case-control studies conducted in Australia and the United States have reported significant reductions in incident gonorrhoea (of 32.7% and 46%, respectively) in adolescents and young adults receiving 4CMenB compared to controls [113,125].These findings, allied with previous data indicating the potential impact of 4CMenB on gonorrhoea are now the subject of ongoing clinical studies [95].
At present, there are no data for the effectiveness of MenB vaccines in adults in reducing IMD due to MenB, and this remains a barrier to decision-making regarding broader recommendations for more routine use in older adults.
MenB Immunisation in Outbreak Control
Both 4CMenB or MenB-FHbp have been successfully used in outbreak control in a range of different settings and populations [17,61,126].Following outbreaks of MenB disease in university campuses in both the east and west coasts of the US (Princeton, New Jersey and UC Santa Barbara, California) in 2013/2014, 4CMenB was administered to more than 14,000 adults aged 18-65 years (comprising students, staff and adult family members), given in a 2-dose (0, 1 month) schedule [127][128][129].In both settings, no safety issues were identified, and no MenB cases occurred in those individuals who received at least one dose of 4CMenB [127][128][129].While this is reassuring, it should be noted that immunogenicity responses towards MenB strains were highly variable.An immunogenicity study evaluating protective serum antibody responses (hSBA C 4) in 499 individuals from Princeton (mean age 20.5 ± 1.3 years) receiving two 4CMenB doses reported that only 66.1% (95% CI 61.8-70.3) of individuals were seropositive for the outbreak strain [89].Geometric mean titres (GMTs) were also low (7.6, 95% CI 6.7-8.5)[89].In contrast, 86.9% were seropositive for the 44/76-SL strain (with 96% genetic similarity to the outbreak strain) and 100% for the 5/99 strain, and GMTs were higher (17.4 and 256.3, respectively, against the 44/76-SL and 5/99 strains) [89].
Focused use of 4CMenB in outbreak control has also been observed in response to a local school outbreak in France (in Brittany) in 2016/2017, where mass vaccination of students and local individuals aged 11-19 years was implemented, and with no further cases reported [130].In Canada, following a prolonged increase in the incidence of MenB disease in the Saguenay-Lac-Saint-Jean region of Quebec province between 2006 and 2013, 4CMenB vaccination of individuals aged between 2 months and 20 years was implemented in 2014 [131].In this campaign, 82% of this targeted age group received at least one 4CMenB dose [131].Between June 2014 and July 2018, five MenB cases were reported, one in a vaccinated child and four in unvaccinated adults (including three elderly adults).The estimated overall impact of the vaccination campaign was an 86% (95% CI -2 to 98%) decrease in risk of MenB disease [132].
MenB-FHbp has also been effective in outbreak control, notably following a college campus outbreak in Rhode Island in 2015 [133,134].
Supportive Immunogenicity and Safety Data for the use of MenB Vaccination in Adults
Licensure of 4CMenB followed an extensive clinical trial programme evaluating immunogenicity and safety/reactogenicity in more than 7,000 infants, toddlers, adolescents and adults [44,81,96].While most studies focused on those age groups at greatest risk (infants, toddlers, and adolescents), a number of studies within the 4CMenB development programme specifically evaluated immunogenicity in healthy adults [81,96,100,[102][103][104] (Table 2 and Fig. 4).
A phase 3b extension study (NCT02446743) conducted in Australia, Canada and Chile assessed persistence of bactericidal activity at 4-7.5 years after a 2-dose primary series of 4CMenB and the response to a third (booster) dose in adolescents and young adults [100,101].This was a follow-up to earlier studies evaluating immunogenicity and safety of a 2-dose schedule in adolescents aged 11-17 years (reported in [135,136]).In this extension study, as well as reporting on immunogenicity persistence and on booster responses at 4-7.5 years in previously primed individuals (now aged from 15-24 years), immunogenicity and safety of a 2-dose schedule (at 0, 1 months) were also evaluated in 255 vaccine-naı ¨ve subjects of a similar age, thus providing data on immunogenicity and safety of a 2-dose schedule in younger adults from these three countries [100].Another phase 3 study conducted in the United Kingdom (NCT01214850) evaluated the effect of 2 doses of 4CMenB given 1 month apart in young adults aged 18-24 years (additional responses to MenACWY conjugate vaccine were also assessed [137].While the primary outcome was the impact on N. meningitidis oropharyngeal carriage [133], an immunogenicity subset (n = 193) was included, in which antibody responses to 4CMenB and antibody persistence at 11 months after the 2-dose primary series were evaluated and subsequently reported [102].An earlier phase 1 study (conducted in Switzerland) evaluating the safety and immunogenicity of 3 doses of 4CMenB (and alternative vaccine formulations with different antigenic content) given at 0, 1 and 2 months in adults aged 18-40 years (with 28 subjects receiving the 4CMenB formulation) [103].A subsequent phase 2 study conducted in Germany and Italy assessed the safety and immunogenicity of 3 doses of 4CMenB (at 0, 2 and 6 months) in at-risk adults aged from 18 to 50 years (n = 46 laboratory workers exposed to a variety of meningococcal strains) [104].Across these studies, 2 doses of 4CMenB administered either 1 or 2 months apart resulted in robust immune responses, with hSBA titres C 4 (or hSBA titres C 1:4) towards fHBP and NadA observed in 98-100% of subjects 1 month after the second dose, and with hSBA titres C 4 (or C 1:4) against PorA P1.4 and NHBA observed in 79-96% and 91-95%, respectively (Table 2; Fig. 4).Prior to any vaccination, up to 78% of participants had baseline hSBA titres C 4 (or C 1:4) towards one or more of the reference strains, indicating some level of antigenic priming.Other studies in adults have also reported immunogenicity data [138,139].An early phase 2 study (NCT00962624) was conducted in the United Kingdom which evaluated co-administration of 4CMenB with a quadrivalent MenACWY vaccine in adult laboratory workers aged between 18 and 65 years (mean age, 34 years, range 23-55 years), with 32 subjects receiving two doses of 4CMenB and with available immunogenicity data [138].Immunogenicity was assessed against the same indicator strains and target antigens as previously described; H44/76 (fHbp), 5/99 (NadA) and NZ98/254 (PorA P1.4).While baseline antibody titres were high (with hSBA titres C 4 against indicator strains apparent in more than 60% of subjects), after the second dose the proportion of subjects with hSBA titres C 4 increased to 100%, with robust increases in GMTs also observed.Furthermore, co-administration with MenACWY vaccine was safe and well tolerated [138].A subsequent post-licensure study conducted in Canada (NCT02583412) evaluated immunogenicity in adolescents and young adults aged 17-26 years of age (mean age 20.6 ± 2.9 years) randomised to receive conventional (0, 2 months) and accelerated (with dose 2 given at 21 days) 4CMenB vaccination schedules in 121 healthy volunteers [139].This study found protective hSBA titres C 1:4 to fHbp, NadA and PorA P1.4 antigens in 98-100% of all participants 21 days after receiving the first 4CMenB dose, providing supportive evidence for use of a 1-dose schedule in outbreak control [139] (Table 2 and Fig. 4).
4CMenB was generally safe and well tolerated in adults in these studies (and broadly comparable to those observed in younger patients).After any 4CMenB dose, most subjects experienced reactogenicity, with the most common reaction being pain at the injection site.Most reported solicited local adverse events (AEs) i.e. local pain, erythema, induration, were mild to moderate in intensity with onset within 3 days after vaccination (after either dose), and the majority resolved within 7 days.The most common solicited systemic AEs included fatigue, headache, myalgia, and malaise [81,96,100,[102][103][104]139].Unsolicited AEs were collected for 1 month after each vaccination.Again, most unsolicited AEs were mild to moderate in intensity, with comparable rates reported in vaccinated and unvaccinated subjects, and most resolved before study completion.No serious AEs considered to be related to 4CMenB vaccination were reported [81,96,100,[102][103][104]139].
As well as these studies, a number of additional smaller studies have evaluated immunogenicity and safety of two doses of 4CMenB in adult populations.One conducted in Germany (NCT01911221) assessed the immune response and safety to two doses in 13 adults aged 18--65 years (mean age, 38.5 ± 12.2 years), all laboratory workers at increased risk due to occupational exposure to MenB samples.At 1 month after the second dose, there were protective hSBA titres of C 5 against all four vaccine target antigens, and with a robust increase in GMTs from baseline [140].Another study (NCT01478347) evaluated safety in 18 adults (mean age, 34.5 ± 5.7 years) with similar occupational risk conducted in Italy [141].In addition, a safety study (NCT02305446) assessed safety in 55 adults aged 18-50 years in Poland (mean age, 27.2 ± 6.9 years) [142].Across these studies, 2 doses of 4CMenB were found to be well tolerated, with a safety profile in line with that seen in other trials in adult populations.
For MenB-FHbp, data in adults are chiefly derived from a single large phase 3 study (NCT01352845) evaluating immunogenicity and safety of a 3-dose schedule (0, 2 and 6 months) in 2471 adults aged 18-25 years [143].Subjects were recruited from the United States, Canada and mainland Europe (Denmark, Finland, Poland and Spain [143].After dose 2, a C fourfold rise in hSBA titres against the four primary representative test strains was observed in 54.6-85.6% of subjects, and in 78.9-89.7%after the third dose.In addition, antibody titres against other strains were supportive of a broader protection against diverse MenB strains [45,143]. Earlier studies provide some data supportive of the use of MenB-FHbp in a slightly broader adult population.One phase 1 study (NCT00879814) evaluated different MenB-FHbp doses in 48 adults aged 18-40 years, again via a 3-dose schedule (0, 2 and 6 months), with 12 subjects (mean age 31.0 ± 7.6 years) receiving the subsequently licensed dose [144].In this study, which evaluated serum immunoglobulin G (IgG) titres against fHbp subfamilies A and B proteins, high IgG titres were observed after the second and third doses [144].A subsequent phase 2 study in Australia (NCT00780806) evaluated the licensed dose in 60 adults aged 18-40 years [145].After 3 doses, hSBA titres C 1:4 against the target stain expressing the vaccine-homologous fHbp subfamily A variant was observed in 94.3% of subjects, with hSBA titres C 1:4 against strains expressing other putative target fHbp variants achieved in 70.0-94.7%[145].
Both 4CMenB and MenB-FHbp are components of pentavalent MenABCWY meningococcal vaccines under development [146,147].While clinical studies evaluate immunogenicity and safety in younger adults (up to 25 years), these are reported as pooled data incorporating data from adolescents [148,149].As such, at the present time, no additional insight into specific adult immunogenicity data can be drawn from these programmes.It should also be recognised that, while the immunogenicity data for 4CMenB and MenB-FHbp we describe above are informative, some caution is warranted and limitations exist.The data we describe are chiefly from younger adults; indeed, product monographs make clear that there are no immunogenicity data for adults[50 years of age for 4CMenB [47], and that data are limited for MenB-FHbp in 40-to 65-year-olds with no data in those [65 years [50].There remains a need for additional data in older adults to inform decision-making for greater use in older adults.
One aspect of responses to MenB vaccines in adults that remains largely unknown is the duration of protection that may be achieved, and the need for subsequent boosting.In the persistence/booster study conducted in Australia, Canada and Chile reported by Nolan et al. (described above) there was evidence of waning of protection induced by 4CMenB, although, following a booster dose, protective hSBA titres C 4 were then observed in 93-100% of subjects [100].Evidence of waning may also be derived from follow-up data from the use of 4CMenB in outbreak control in Canada, where VE against MenB disease declined from 100% during the first 2 post-campaign years to 50% (95% CI -453, to 95) over an adjusted 5-year period [61,131,132].This would indicate that periodic boosting is necessary to maintain protection.Given the paucity of data in older adults[50 years of age, the immunogenicity and persistence of protective responses in older adults is unclear.In addition, as studies evaluating immunogenicity involved healthy individuals, the impact of comorbidity or frailty on vaccine responses is also unknown, and it may be anticipated that these are poorer.In some countries recommending MenB vaccine for atrisk adults (including those with immune dysfunction and laboratory workers), booster immunisation every 5 years is recommended [13,57].While data are required to define the duration of protection in older adults (and in those with comorbidity or frailty), it would seem that a similar booster vaccination approach would be required.Inevitably, this would increase programmatic costs associated with any broader adult immunisation policy.
TOWARDS A BROADER ADULT MENB VACCINATION APPROACH
These data in adults receiving either 4CMenB or MenB-FHbp indicate that a robust immune response to key antigens expressed by most pathogenic MenB serogroups are generated in adults across a diverse range of geographies and populations (and broadly comparable to that seen in children and adolescents).While most of the data were derived from use in younger adults (aged 18-26 years), equally robust hSBA responses were evident in those studies evaluating use in older subjects.As indicated earlier, it should be recognised that there remain important knowledge gaps as to immunogenicity in adults[50 years of age, persistence of protective responses, and impact of comorbidity/frailty in older adults.Further studies specifically examining this are welcome.
As we describe, present surveillance data indicate that there exists a substantial burden of MenB disease in many adult populations.It could be anticipated that direct protection through greater routine use of MenB vaccination in adult immunisation strategies would also have a clinical impact on IMD due to MenB, and help to reduce the case burden and adverse clinical outcomes (associated mortality and long-term sequelae in survivors) observed in adults.The current focus on predominantly age-based MenB immunisation recommendations towards younger at-risk populations (infants, toddlers and adolescents) is such that most adults remain at continued risk.
Greater efforts are needed to ensure access to and uptake of MenB vaccines in those adults at greater risk due to medical comorbidity.While data for vaccine uptake in at-risk individuals are limited, available data from the United States indicate relatively low coverage in at-risk populations, with uptake in individuals with complement deficiencies or with asplenia of 2.2% and 9.7%, respectively [150,151].In Australia, between 2014 and 2019 the reported MenB vaccine coverage in adults[50 years with at-risk medical comorbidities ranged from 12% to 14%, and was lower in those with lower socioeconomic status [152].Efforts to increase vaccine uptake in at-risk adults where MenB immunisation is recommended should be a priority.
It can be argued that a broader, more comprehensive MenB immunisation policy for adults could also be considered.This is in keeping with a lifelong approach to immunisation in which equitable access to vaccines throughout the lifespan is an essential element of healthy aging [67,[153][154][155].Although the focus in this approach is on immunisation against other vaccine-preventable disease (notably influenza, pneumococcal infection, pertussis and herpes zoster in older adults), this could also encompass a broader approach with adult meningococcal MenB immunisation (in those countries with broad age-based approval for use across adults of all ages).A broader adult immunisation approach would also be supportive of the aims of the World Health Organization's 'Defeating meningitis by 2030 global road map', with a target of the nearelimination of bacterial meningitis (including IMD due to MenB) by this date [156,157].However, there exist significant challenges to this.Immunisation recommendations by public health agencies must also accommodate other priorities and are subject to financial constraints, and any broader adult MenB immunisation policy would have substantial impact on healthcare budgets.While MenB in adults constitutes as much as 20% of the overall disease burden in a number of countries, the low incidence rate in adults is such that any MenB vaccine policy would not be considered cost-effective under existing criteria.These aspects pose considerable challenges when trying to balance healthcare financial resources with a more equitable approach to access to MenB vaccination.There remains a need to pursue additional studies to support policy decision-making.
Disclosures.Victoria Abbing-Karahagopian and Selim Badur are employed by GSK.Angelika Banzhoff, Serdar Ozturk and Mansour Khalaf are employed by and hold shares in GSK.George Kassianos is the Royal College of General Practitioners National Immunisation Lead, and British Global and Travel Health Association President; and participated in advisory boards or lectured at meetings organized by vaccine manufacturers (GSK, MSD, Sanofi Pasteur, AstraZeneca, Pfizer, Janssen, Valneva, Moderna and CSL Seqirus).George Kassianos, Victoria Abbing-Karahagopian, Angelika Banzhoff, Selim Badur, Serdar Ozturk and Mansour Khalaf declare no other financial and non-financial relationships and activities.Osamah Barasheed declares no financial and non-financial relationships and activities and no conflicts of interest.
Compliance with Ethics Guidelines.This article is based on previously conducted studies and does not contain any studies performed by any of the authors with human participants or animals.
Data Availability.All data generated or analysed are included in this published article or in the original data sources.
Trademark Statement.Bexsero is a trademark owned by or licensed to GSK.Trumenba is a trademark of Pfizer.
Open Access.This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial 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://creativecommons.org/licenses/bync/4.0/.vaccine against serogroups A, C, W-135, and Y in adults who are at increased risk for occupational exposure to meningococcal isolates.Clin Vaccine Immunol.2011;18:483-6.https://doi.org/10.1128/CVI.00304-10.
Fig. 2 a
Fig. 2 a Confirmed case numbers for IMD due to MenB across Europe (EU/EEA), and in the United Kingdom and France across 2011-2020 stratified by age [29].For the United Kingdom, data for 2020 are not available.
Fig. 3
Fig. 3 Confirmed case numbers for IMD due to MenB in Australia (2018-2021) [33-36] and the United States (2015-2020) [30, 31, 69-72] stratified by age.IMD invasive meningococcal disease, MenB N. meningitidis serogroup B , fHbp factor H binding protein, hSBA human serum bactericidal assay, NadA Neisseria adhesin A, ND not determined, NHBA Neisserial heparin binding antigen, PorA P1.4 Porin A; SD Standard deviation a Data represent the percentage of participants with hSBA C 4 (95% CI) against test strains and target antigens b Data represent the percentage of participants with hSBA C 1:4 (95% CI) against test strains and target antigens c Study evaluated two different vaccination schedules d Number enrolled in immunogenicity evaluation; not every subject had available data for evaluation.Percentages represent proportion of subjects with available data
Table 1
Licensed vaccines against meningococcal serogroup B
Table 2
Immunogenicity against reference indicator strains/target antigens in studies evaluating 4CMenB in adults | 2023-07-11T06:16:19.555Z | 2023-07-06T00:00:00.000 | {
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190699 | pes2o/s2orc | v3-fos-license | Candidate Luminal B Breast Cancer Genes Identified by Genome, Gene Expression and DNA Methylation Profiling
Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.
Luminal B BCs have a poor prognosis [32]. Although they express hormone receptors, their metastatic risk and resistance to hormone therapy and to conventional chemotherapy demand to develop appropriate therapies. Some proteins (e.g. CITED2, NCOR2) or molecular networks associated with BCAR (breast cancer anti-estrogen resistance) genes are involved in the resistance to anti-estrogen therapy and in the progression of these cancers [32][33][34][35]. Luminal B cancers exhibit various mutated genes, TP53 and PIK3CA being the most frequent (29% each) [28].
To further define molecular alterations associated with the luminal B subtype we studied DNA copy number aberrations (CNAs), DNA promoter methylation alterations (DPMAs), gene expression deregulation (EXP), and selected gene mutations in 188 primary BC samples. These analyses identified luminal B-specific candidate genes.
Ethics statement
The study was approved by our institutional review board: the ''Comité d'Orientation Stratégique'' of the Institut Paoli Calmettes (IPC) (Marseille, France). Each patient gave a written informed consent for research use.
Breast cancer samples
Pre-treatment tumor tissues were collected from 188 patients with invasive adenocarcinomas. Patients underwent surgical biopsies or initial surgery at the Institut Paoli-Calmettes between 1987 and 2007. The main histoclinical, biological and subtype characteristics were established for the 188 BCs as described [12,[17][18][19]. They are listed in Table S1A and illustrated in Figure S1.
Gene profiling and data analysis DNA and RNA were extracted as previously described [12,[17][18][19] and controled on Agilent Bioanalyzer (Agilent Technologies, Massy, France). Genomic profiles of the 188 BCs were established by using array-comparative genomic hybridization (aCGH) onto high-resolution 244K CGH microarrays (Hu-244A, Agilent Technologies, Massy, France). A pool of 13 normal male DNA was used as reference. Gene expression data from the same 188 BCs and 4 normal breast (NB) samples, which represented 1 pool of 4 samples from 4 women, and 3 commercial pools of respectively 1, 2 and 4 normal breast RNA (Clontech, Palo Alto, CA), were obtained using whole-genome DNA microarrays (HG-U133 Plus 2.0, Affymetrix). Both approaches and analysis methods have been used in our previous studies [12,17,18]. All probes for aCGH, gene expression and DNA promoters methylation analyses were mapped according to the hg18/NCBI human genome mapping database (build 36) to homogeneously integrate the data. The aCGH, gene expression, methylation data, as well as the integrated CNA/gene expression and DPMA/gene expression analyses are illustrated by pipelines ( Figures S2 and S3, respectively).
Validation and prognostic impact of candidate genes were evaluated in a large public series of BC samples. Thirty-six data sets, including a total of 5,765 non-redundant samples, were collected from public database i.e. Gene Expression Omnibus (GEO/NCBI), Array Express (EBI) and authors' websites (Table S1B). Raw data from each study were normalized using quantile normalization, and log2-transformed. The intrinsic molecular subtypes of each tumor sets were defined using Hu single sample predictor (SSP) [4]. To be comparable across data sets, each gene expression levels were standardized within each data set using luminal A population as reference.
The data (experiment called ''Candidate luminal B breast cancer genes identified by genome, gene expression and DNA methylation profiling'') are publicly available (ArrayExpress repository ref ID: E-MTAB-1861).
DNA promoter methylation profiling and data analysis
We captured the methylated DNA of 117 (109 tumors+8 NB) samples by using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). Genome-wide DNA-methylation analysis was done on custom [A-MEXP-2178 (arrayexpress)] human promoter arrays 26400K (Agilent Technologies, Massy, France) using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) [36]. Over 414,000 probes cover promoter regions approximately 2 3 kb to +3 kb relative to transcription start sites (TSSs) with a resolution of 280 bp in average. Scanning was done with Agilent Autofocus Dynamic Scanner (G2565C, Agilent Technologies). Raw data were obtained from Feature extraction 10.7.3 software (Agilent Technologies). Probes not mapped to exact positions in the genome as well as those under the background signal were removed with the control probes. The final dataset contained 326,350 unique probes covering 18,297 promoter regions according to the hg18/NCBI human genome mapping database (build 36). The M (Log 2 Red-Log 2 Green) values of each probe on the array were then obtained and normalized according to their GC content. Then, inter-array quantile normalization was done for the correction of distribution differences among experiments.
To estimate the global methylation level for a given gene in one sample, we computed a methylation score based on the sum of frequency of probes with a M value greater than zero combined with its amplitude and frequency of probes with a M value less than zero combined with its amplitude. Clustering was done with the Cluster program using Pearson correlation as similarity metrics and average linkage clustering. Results were displayed using TreeView program.
Gene mutations
Polymerase chain reaction (PCR) and direct sequencing were done using standard conditions with gene-specific primers designed to amplify coding sequence of ARID1A, ASXL1, FOXO3, L3MBTL4, MAP2K4, PIK3CA, RUNX1, RUNX3 (Table S1C). Most PCR amplifications were done in a total volume of 25 ml PCR mix containing at least 10 ng template DNA, Taq buffer, 200 mmol of each deoxynucleotide triphosphate, 20 pmol of each primer and 1 unit of appropriated Taq polymerase (Table S1C). PCR products were purified using Millipore plate MSNU030. The purified PCR products (2 ml) were used for sequencing using the Big Dye terminator v1.1 kit (Applied Biosystems, Courtaboeuf, France). After G50 purification, sequences were loaded on an ABI 3130XL automat (Applied Biosystems). The sequence data files were analyzed using the Seqscape software and all mutations were confirmed on an independent PCR product.
Statistical analyses
Correlations between sample groups and histoclinical factors were calculated with the Fisher's exact test for qualitative variables with discrete categories, and the Mann-Whitney test for continuous variables. Metastasis-free survival (MFS) was calculated from the date of diagnosis until the date of first metastatic relapse. Survivals were calculated using the Kaplan-Meier method and curves were compared with the log-rank test. Stratification into high-risk and low-risk groups was based on relative risk defined by Cox model using the natural threshold of 1. Univariate and multivariate survival analyses were done using the Cox regression model (Wald test). Variables tested in univariate analyses included patients' age at time of diagnosis (#50 years vs .50), pathological tumor size (pT: pT1 vs pT2-3), pathological axillary lymph node status (pN: negative vs positive), pathological grade (I vs 2-3), histological type, delivery of hormone therapy and chemotherapy, immunohistochemical (IHC) ERBB2 status (negative vs positive), molecular subtypes and RECQL4 expression (continuous value). Variables with a p-value,0.05 in univariate analysis were tested in multivariate analysis. All statistical tests were two-sided at the 5% level of significance. Analyses were done using R software (2.14.2) and associated packages. We followed the reporting REcommendations for tumor MARKer prognostic studies (REMARK criteria) [39].
Luminal B CNAs landscape was drawn with the Circos software [40] and significant mutual exclusive and co-occurring CNAs (FDR,0.05) were identified by DRP analysis [41].
In the meta-analysis of six recent NGS studies including 875 breast tumors, co-occurring and mutually exclusive gene mutations were identified by using a method previously reported [42].
Genomic characterization of 188 BC samples
We first describe the results on the whole set of 188 tumors before addressing the specific question of the luminal B cases. The 188 samples were first profiled using whole-genome gene expression microarrays. Figure S1 shows the hierarchical clustering of samples based on the expression of 13,031 probe sets. Samples were sorted into four major clusters, which strongly correlated with histoclinical features (grade, IHC data) and molecular subtypes. The 188 cases (Table S1A) (Table S1D).
Copy number aberrations in luminal B tumors
We next focused on the CNAs found in luminal B tumors. The luminal B subtype shows both common and specific alterations [7,9,16,19]. To identify the latter we did a supervised analysis comparing CNAs observed in luminal B to those found in each of the other subtypes ( Fig. S5 and Tables S2A-F).
The 33 and 6q, 9p22-p24, 13q34, and 18p11.31-11.32 regional gains and losses with a frequency $30%, respectively) are more frequent in luminal B samples (Fisher's exact test; FDR, 0.05) (Fig. S5, Tables S2C,D). Gains and losses targeted 1,091 and 3,339 genes, respectively ( Table S2D). The comparison with the ERBB2 subtype did not distinguish regions with a different CNA frequency, perhaps because of the low number of samples.
Integrated comparative analysis and luminal B candidate genes
We compared the degree of CNA-driven RNA up or downregulation in 32 luminal B (i) vs 64 luminal A, (ii) vs 54 basal, and (iii) vs 156 pooled non-luminal B tumors, by analyzing the 13,127 genes common to the two platforms (aCGH Agilent On the left is shown a hierarchical clustering of genome copy number profiles measured by aCGH on 24,907 probes or groups of probes (without X and Y). Red indicates increased copy number and green indicates decreased copy number. To the left are indicated chromosome locations with chromosome 1pter to the top and 22qter to the bottom. Next on the right, significant copy number amplifications (dark red), gains (red) and losses (green) observed in luminal B compared to non-luminal B tumors (Fisher's exact test), are plotted as a function of chromosome location. Only amplification, gains, and losses associated with luminal B tumors are shown (Fisher's exact test; FDR,0.05) for each chromosome. In addition to the previously reported 8p11-12, 11q13 amplifications, 17q, 20q gains and 18p losses we previously reported (16,20) and Hu233 2.0 plus Affymetrix) and retained after filtering based on the expression level. From these supervised analyses (Tables S2A-F), 160, 148 and 307 genes had an expression level that varied according to CNA (Mann-Whitney; FDR,0,05) after luminal B/luminal A (Table S3A), luminal B/basal (Table S3C), and luminal B/non-luminal B (Table S3E) comparison, respectively. Of these, 138, 132, and 251 genes had a deregulated gene expression in relation with CNA specifically associated with the luminal B subtype (t test, FDR,0.05) after luminal B/luminal A (Table S3B), luminal B/basal (Table S3D), and luminal B/nonluminal B (Table S3F) comparison, respectively. These specific luminal B candidate genes were qualified as potential oncogenes or TSGs if they presented up or downregulated expression in relation with amplification/gain or losses, respectively, as previously described [12,17,18]. Overall, out of the 337 candidates identified, 66% (221) were found in the TCGA data [28], deregulated in relation with their copy number alterations. (Tables S3B, 3D and S3F, respectively).
A total of 34, 47 and 2 candidate genes were identified as specific of luminal B tumors when compared to luminal A, basal, and normal-like tumors (Tables S3H-J), respectively. These different repertoires of candidate genes could help identify pathways and mechanisms either specifically affected in luminal B tumors or shared by the major subtypes.
RECQL4 was the most significant. Corresponding Kaplan-Meier MFS curves are shown in Figure 3A (p = 3.80610 29 ). Interestingly, concordant results were obtained with another referent molecular classifier i.e. PAM50 SSP [50] (Table S3M). Indeed, the definition of luminal B tumors is highly variable due to their molecular heterogeneity, making their assignment in the luminal B subtype non-reproducible by different gene expression signatures [51].This suggests that the robustness of our candidates is not impacted by the choice of the classifier [4,50]. A high RECQL4 gene expression level has recently been reported to confer proliferation advantage and survival to breast cancer cells [50]. We observed that RECQL4 overexpression was associated with MDM2 overexpression (t-test p = 2.75610 22 ) as well as with mutated TP53 or/and overexpressed MDM2 gene status (t-test p = 1,73610 220 ) in the independent public TCGA data set [28] (data not shown). This might highlight high genomic instability and DNA repair perturbations in such tumors.
In multivariate analysis including the other histoclinical prognostic features, RECQL4 expression remained significant for MFS (p = 3.4610 22 ), with pT and molecular subtypes ( Table 1). The clinical response study in regard to RECQL4 gene expression in 924 BCs (included in the large public series, see Table S1B) showed as expected that luminal B tumors have a better pathological response to chemotherapy (Fisher, p = 3.04610 24 ) than luminal A tumors. Moreover, RECQL4 overexpression was associated with a better pathological response in the group including both luminal A and B tumors (Fisher, p = 0.013) ( Figure 3B) but not within separated luminal subgroups (data not shown).
DNA methylation profiles of 109 BCs
The hierarchical clustering established with the most variant methylation scores observed in 5,492 promoters (SD.0.3) classified the 109 BC and 8 NB samples into clusters associated with different DNA methylation patterns (Fig. 4A) variably associated with ER and PR expression (p = 5.6610 27 and p = 2.26610 27 ), SBR grade, molecular subtype (p = 5610 24 ), and TP53 status (p = 1.2610 23 ) ( Table S4A). The 8 NB samples and most of the ER-tumors were included in cluster I while ER+ tumors were mainly distributed in cluster II. Most of the basal tumors were included in cluster I whereas cluster II contained most of the luminal A and luminal B tumors. ERBB2 tumors were distributed in the two clusters probably because ERBB2 tumors are heterogeneous for ER status [17]. These different profiles were consistent with recently reported BC methylation patterns [21][22][23], which pointed to a possible relationship between DNA methylation and ER status.
Validation of methylation results
The supervised analysis comparing methylation score data of ER+ and ER-tumors identified 3,484 gene promoters with methylation differences between the two groups (Table S4B) (t test, FDR,0.05). Among them, 1,753 gene promoters (including those of APC, CAV1, CCND2, CDCA7, CDH3, CDKN2A, CDKN2B, HEY2, RASSF1, RECK) had a DNA methylation level higher in the ER+ group (t-test, FDR,0.05). Conversely, 1,731 gene promoters (including those of BCL2, ESR1, HSD17B4, PISD, WNK4) had a higher level of methylation in the ER-group (FDR,0.05). These . For each region, heatmaps for genome copy number and gene expression profiles are consecutively drawn. Genome copy number was measured by aCGH on probes or groups of probes spanning each of these regions. Red indicates increased copy number and green indicates decreased copy number. In the heatmap tumors are organized from the tumor that presented the most copy number losses to the tumor that exhibited the most copy number gains. The next heatmap was established with the expression of the independent genes located on the corresponding 6q region and profiled in the same 188 tumors similarly organized. For gene copy number and gene expression heatmaps, we used color scale limits from 23 to +3 and 22 to +2, respectively. Next to the right, are plotted genes successively selected by steps I, II and III of the integrated analysis ''aCGH & mRNA expression'' as defined by the work pipeline ( Figure S2). Grey and green lines correspond to rejected and selected genes, respectively. Among genes with an expression level that varied according to CNAs, we retained genes showed significant differences (vertical line) in copy number loss correlated with downregulated expression in luminal B compared to non-luminal B data are in agreement with those reported on IlluminaH platform [20,21,23,24]. The strong correlation between DNA methylation data of RASSF1 gene promoter established by EpiTyper and promoter array approaches further validated our method (Results and References S1, Fig. S10, S11).
DNA methylation associated with breast cancer molecular subtypes
Using the ANOVA method, we identified 4,545 gene promoters with a DNA methylation level different in at least one subtype among the five major ones (Table S4C). They included several genes previously reported differentially methylated in BC subtypes and particularly in luminal and basal (or ER+ and ER2) tumors ( Table S4C). The median methylation levels of these 4,545 subtype-associated genes were highest in the luminal and ERBB2 subtypes and lowest in the basal subtype (p = 6.24610 212 ) (Fig. 4B).
Tukey's range test integrating ten supervised analyses was applied to distinguish gene promoters exhibiting a DNA methylation level different in only one subtype as compared to the others (FDR#0.05) ( Table S4C). A total of 375 and 431 promoters had a methylation level higher and lower in the luminal B tumors than in the other subtypes, respectively. Among them, 265 and 295 had a methylation level higher and lower than in NB tissues (t test, FDR,0.05). This analysis was extended to the other subtypes (Results and References S1).
Molecular subtypes and specific deregulated gene expression in relation with the DNA methylation level variation
Among the 4,545 promoters with a DNA methylation level different in at least one subtype (Table S4C), only 459 genes presented an associated mRNA deregulation (correlation,20.40) ( Table S4D). These genes are listed with their CNA status in Table S4E. The presence of CpG islands was observed in 62.5% of them. In the luminal B cases, among the 560 promoters associated with a significant DNA methylation variation, 46 corresponding genes presented a deregulated expression (24 and 22 were down and upregulated, respectively) (correlation,20.40) ( Table S4E). 52% (24 genes) were found significantly deregulated in relation with their level of DNA methylation in the TCGA data [28] (Table S4E).
Subtype-specific candidates presenting gene expression deregulation in relation with CNA and with DNA methylation aberrations
We integrated genomic, gene expression and DNA methylation profiles to identify subtype-specific candidate genes presenting expression deregulation in relation with CNA and with DNA promoter methylation aberrations (Table S4F).
In the luminal B cases, no gene was downregulated in relation with both copy number loss and with high methylation (Table S4F). Conversely, 8 genes were upregulated in relation with copy number gain or amplification and with low DNA methylation (Table S4F). C12ORF60 and two previously reported oncogenes, H2AFJ and RAB11FIP1/RCP were the most significantly upregulated (t-test, p,0.05) (Fig. S12A). Except for TPD52 and C12orf60, 75% were upregulated in relation with copy number gain or amplification and with low DNA methylation in the TCGA data [28]. The other subtypes were also analyzed (Results and References S1, Table S4F, Fig. S12B-S12D1-D4).
tumors. They were qualified as potential TSGs. For each region, only the first five most significant are listed. PNRC1, NCOA7 and TNFAIP3 genes were the most significant candidate TSGs for the 6q14.1-q22. 31 (Table S1B). Stratification into high-risk (red curve) and low-risk (black curve) groups were based on relative risk defined by the Cox model using the natural threshold of 1. RECQL4 gene expression is associated with MFS (p = 3.8010 29 ). B -From the same large public series, 924 BCs were informed for the pathological response ( Table S1B). The RECQL4 overexpression was associated with a better pathological response within the group including both luminal A and B tumors (N = 435) (Fisher, p = 0.013). doi:10.1371/journal.pone.0081843.g003
In these NGS studies, only 602 samples were subtyped ( Table S6A) including 248 luminal A, 134 luminal B, 132 basal, 71 ERBB2 and 17 normal-like BCs. A total of 517 genes exhibited more than 5 mutations in the 602 tumors. For each of them, the frequency of mutation was established and we identified gene mutations associated with a specific subtype by comparing the number of mutations with those observed in the others (Fisher, FDR,0.25 with odds ratio.1 dark grey colored in Table S6D). The first 15 most frequently mutated genes are reported in In an NGS subset [29], TP53 mutations were enriched in luminal B tumors (p = 0.04) and in high grade tumors (p = 0.02). In our meta-analysis of all NGS samples, TP53 mutations were enriched in only basal (FDR = 4610 218 ) and ERBB2 (FDR = 5610 26 ) tumors. The proportion of analyzed tumors and their heterogeneities could influence the results.
To identify co-occurring and mutually exclusive subtype-specific gene mutations, we retained in the 602 samples, the genes targeted by more than 3 mutations in each subtype. To identify key luminal B genes that could be altered by several mechanisms, we crossed in Table S6I information between genes found in the meta-analysis as mutated in luminal B cases and those identified in our study as significantly altered in luminal B tumors (Tables S2A-S2F) or as potential luminal B candidates (Table S3G). KCNB2 mutations were associated with luminal B tumors (Fisher, FDR,0.25 with odds ratio.1), and the gene was more frequently gained in luminal B than in the other subtypes (Fisher's exact test; FDR,0.05) (Tables S2A-S2F). Among luminal B candidate genes, only CIT (12q24) (3%), CHD6 (20q12) (2%), MDN1 (6q15), SRGAP1 (12q14.2) (1.5%), UTRN (6q24), BRCA1 (17q21) and EVPL (17q25) (less than 1%) were also targeted by mutations. Red indicates increased DNA methylation score and green indicates decreased DNA methylation score. The dendrogram of samples (above matrixes) represents overall similarities in DNA methylation profiles and is zoomed to the right. Three groups of tumor samples (I, IIa and IIb) are associated with various DNA methylation patterns and delimited by orange vertical lines. Below the dendrogram are some histoclinical and molecular features of the samples: from top to bottom, intrinsic molecular subtypes 4 , IHC ER status and SBR grade. Color legends for the various features are illustrated below. B -The median methylation levels of the 4,545 subtype-associated genes were highest in the luminal and ERRB2 subtypes and lowest in the basal subtype (p = 6.24 10 212 ). C -Compared to the other molecular subtypes, the DNA methylation levels (three top panels) of ASS1, C6ORF145 and ZFP36L2 gene promoters and their mRNA expression (three bottom panels) were higher (
Specific gains and amplifications target oncogene candidates in luminal B tumors
The high proportion of complex ''sawtooth and firestorm'' genomic profiles observed in luminal B tumors suggests a high level of genomic instability. Expectedly, among the most common specific alterations in luminal B tumors ($30%) were the amplified 8p11-p12 and 11q13.1-q13 regions. In these regions ZNF703, FGFR1 and CCND1 have already been found associated with luminal B BCs [9,19,46,51]. The comparison between luminal B and luminal A genomic profiles showed only differences in the frequency of these, which may contribute to phenotypic differences. ZNF703 interacts with WDR68/DCAF7, PHB2 and HSP60 and induces transcriptional repression [19]. Here, we found that WDR68/DCAF7 (17q23.3) is indeed a candidate luminal B oncogene. Genes of the 8p11 region were coamplified with ZNF703, supporting the idea that the 8p amplicon carries multiple genes, such as RAB11FIP1/RCP [47], FGFR1 [54] and PPAPDC1B [45], which contribute to the luminal B phenotype.
In the 8p12/11q13 coamplified regions [55], EIF4EBP1 and RPS6KB2 could be co-targeted and play a synergistic role associated with the development and cancer progression as AKT/MTOR activators [56]. The recurrent 8p12/11q13 coamplification was often accompanied by gain of 17q25.1 containing RPS6KB1, a paralog of RPS6KB2, suggesting again the involvement of the AKT/MTOR pathway in luminal B oncogenesis.
Specific losses target TSG candidates in luminal B tumors
Chromosome arm 6q showed frequent deletions in luminal B tumors. Several 6q regions were lost and rare homozygous or focal deletions of ARID1B, ASCC3, FOXO3, PARK2, MLLT4/Afadin and *Gene mutation associated with the corresponding molecular subtype. doi:10.1371/journal.pone.0081843.t002 UFL1/NLBP genes were observed. Deletion of 6q is also frequent in a variety of cancers [57]. Several other chromosomal regions were targeted by losses suggesting the existence of several potential TSGs in luminal B cancers (Table S7). We identified luminal B candidate TSGs PNRC1 (6q15), PTPRK (6q22.33) and L3MBTL4 (18p11.31). We previously reported L3MBTL4 as a potential BC TSG. PNRC1 is a coactivator of nuclear receptors such as ER. PTPRK negatively regulates the transcriptional activity of ßcatenin in cancer cells.
Gene expression deregulation of luminal B candidate genes could perturb epigenetic regulation Eleven of the luminal B candidate genes we found have been associated with endocrine resistance [33,34,44]. Upregulation of FOXM1 [58] could explain in part the DNA methylated landscape characterizing luminal B tumors. The deregulation of ARID1B, ASHL2, CHD6, KDM4C/JMJD2C, L3MBTL4 and ZFP161 suggests an important perturbation of the epigenetic regulation in luminal B tumors.
BC subtypes have specific methylation profiles [20,21]. Luminal B and basal BCs were reported as the most and least frequently methylated, respectively [21]. Our data are in agreement with these observations; the median methylation level of the 4,545 subtype-associated promoters was the highest in the luminal and ERRB2 subtypes and the lowest in the basal subtype. High DNA methylation level associated with the luminal B subtype targeted CITED4, SP100, SAMD9L, DCR1, FBXO32, ASS1, FAM78A and STAT5A genes previously reported as TSGs or associated with tumor progression ( Table S7). None of them is located in 6q. The two specific luminal B candidates, ASS1 and ZFP36L2 downregulated in relation with DNA methylation have been reported targeted in cancers. DNA methylation of the ASS1 promoter leading to its downregulation was associated with poor prognosis and chemoresistance in various cancers. Several phase I/II clinical trials of the arginine-lowering drug, pegylated arginine deiminase, showed encouraging evidence of clinical benefit and low toxicity in patients with ASS1-negative tumors that might be extended to luminal B tumors.
Finally, we further noted that multiple alternatively spliced transcript variants have been described for candidate genes ASB13, CIT, CPSF1, EPN2, LSM1, PDCD4, RNF146, TAF4, VTCN1. Transcripts of CIT, CPSF1, PDCD4 and TAF4 are overlapped by MIR1178, MIR939 and MIR1234 (both), MIR4680 and MIR1257, respectively [59]. Changes in MIR expression may also contribute to luminal B tumorigenesis by modulating the functional expression of critical genes involved in cancer growth and metastasis.
Mutation status
As expected, TP53 and PIK3CA were among the most frequently mutated genes in our luminal B series. FOXO3 and RUNX3 genes were lost and mutated. A possible role for RUNX3 as a tumor suppressor in ER+ BCs has been suggested [60,61]. FOXO3 functions as a tumor suppressor in both ER+ and ER2 BCs [62,63]. Its nuclear localization and subsequent transcriptional activity is a marker of good prognosis among breast cancer patients [64].
Our meta-analysis of NGS samples showed the frequent mutations of PIK3CA, TP53 and GATA3 in luminal B tumors but also that KCNB2 gene mutations were, for an unexplained reason, associated with this aggressive subtype. A total of 91 cooccurring mutations and GATA3/PIK3CA gene mutual exclusive mutations completed the landscape associated with luminal B tumors. Surprisingly, RUNX3 and FOXO3 mutations were not found in the NGS studies. This may be due the limited coverage and low depth of these early analyses, or to BC heterogeneity and the large number of alterations that could lead to similar deregulations of particular pathways.
Potential targeted therapy in luminal B breast cancer
Hormonal therapy is the preferred treatment for about twothirds of all BC patients whose tumor expresses ER. ER+ BCs are commonly treated with anti-estrogens or aromatase inhibitors [65]. Luminal B tumors respond less than luminal A tumors to such treatments. Seven of the luminal B candidate oncogenes we have identified could be targeted [43]. Several phase I/II clinical trials targeting IGF, FGF and PI3K/AKT pathways in luminal B BC have been reported [32]. Except for the IGF pathway, our data are in agreement with the potential activation of the FGF, PI3K/AKT, PIK3/MTOR pathways subsequent to the overexpression of candidate oncogenes such as RPS6KB1, RPS6KB2, EIF4EBP1, FGFR1, FRS2, and RAB11FIP1 and the high frequency of PIK3CA mutations. Interestingly, targeting the PI3K/AKT/ mTOR pathway is one of the most-promising therapeutic approaches to reversing endocrine resistance for ER positive breast cancer (for review, [66]). Therapeutic strategies combining endocrine treatment and signaling pathways inhibition (such as targeting growth factor receptors (ERBB2) PI3K/AKT/mTOR, CDK4 and CDK6, MDM2-TP53 interaction, FGFR pathway aberrations and histone deacetylases) might be pertinent [66]. However, the luminal B molecular heterogeneity depicted by its complex genomic and epigenomic landscape strongly suggests that other potential targets could also exist and should be identified for each patient. The candidate oncogene RECQL4, which might be involved in endocrine resistance [44], could be also a potential therapeutic target [43] as recently shown in breast cancer cells [52].
The targeting of candidates such as YWHAZ and its coregulated proteins such as FOXM1, or NHERF1, could also restore endocrine sensitivity and reduce the risk of BC recurrence [32,34,44,67,68]. FOXM1 has a significant role in endocrine therapy resistance [67]. In endocrine therapy-resistant BCs, high FOXM1 expression could result from the loss of FOXO function as transcriptional repressor.
In conclusion, this refined molecular dissection of luminal B BCs has pointed to both new and well-known specific candidates. Some code for proteins that participate to the same signaling pathways including those known to cross-talk with ER signaling pathways. Many of the candidate genes have not been previously reported in breast cancer, and deserve further functional validation. Similarly, further characterization of the 6q TSGs is an important goal. This should help better understand pathways and mechanisms affected, and find new therapeutic targets. 4 , GC (genome complexity) (green for simplex, orange for complex saw tooth and brown for complex firestorm), SBR grade (white for grade I, grey for II, and black for III), and IHC ER, ERBB2, and P53 status (white for negative, and black for positive). Crossed white boxes mean not assigned samples. 1-q13.4 (bottom) regions. For each region, heatmaps for genome copy number and gene expression profiles are consecutively drawn. Genome copy number was measured by aCGH on probes or groups of probes spanning each of these regions. Red indicates increased copy number and green indicates decreased copy number. In the heatmap tumors are organized from the tumor that presented the highest copy number gains and amplification to the tumor that exhibited the most copy number losses. The next heatmap was established with the expression of the independent genes located on the corresponding region and profiled in the same 188 tumors similarly organized. For gene copy number and gene expression heatmaps, we used colour scale limits from 23 to +3 and 22 to +2, respectively. Next to the right, are plotted genes successively selected by steps I, II and III of the integrated analysis ''aCGH & mRNA expression'' as defined by the work pipeline ( Figure S2). Grey and red lines correspond to rejected and selected genes, respectively. Among genes with an expression level that varied according to CNAs, we retained genes showed significant differences (vertical line) in copy number gains correlated with upregulated expression in luminal B compared to non-luminal B tumors. They were qualified as potential oncogenes.. For each region, only the first five most significant are listed. ZNF703 and CCND1 genes were the most significant candidate oncogenes for the 8p11.1-p12 (top) and 11q13.1-q13.4 (bottom) regions, respectively. (TIFF) Figure S9 Luminal B candidates and gene CNAs landscape. The Circos diagram presents from outside to inside, luminal B candidate genes, luminal B altered chromosomes, luminal B regional CNAs colored in red and green for significant gains and losses, respectively. Oranges and grey arcs indicate respectively genes/regions that present significant mutually exclusive and cooccurring luminal B CNAs (FDR,0.05) as identified in Table S3N. (no apparent CNA), T9345, T8056, T50115, T9934, T11568, T15120, and T9207. Arrow shows RUNX1 location on each genomic profile. Results show that RUNX1 is targeted by potential breaks in T8056, T9207 and T15120, but also by regional deletions (samples T50115, T9934, T11568, T15120, and T9207). The genomic profiles of T11568 (B), T15120 (C), and T9207 (D) BCs presenting the smallest regional deletions were established with CGH analyticsH software (Agilent Technologies), from centromere to telomere, within the genomic intervals [34.0-36.4 Mb] of the long arm of the chromosome 21. The common smallest deleted region observed in T9207 (D) involves RUNX1.
(TIF)
Results and References S1 In this section, results about: validation of our methylation approach; DNA methylation level associated with the other breast cancer molecular subtypes; and specific deregulated gene expression in relation with the DNA methylation level variation associated with the other breast cancer molecular subtypes; are presented with supplementary references. (DOCX) [28] was done. C: Comparison of expression levels according to CNAs (Mann-Whitney ; FDR,0.05) in luminal B vs basal BCs. D: Identification of specific luminal B candidate genes from comparison between luminal B and basal BCs. A comparison with TCGA data [28] was done. E: Comparison of expression levels according to CNAs (Mann-Whitney ; FDR, 0.05) in luminal B vs non luminal B BCs. F: Identification of specific luminal B candidate genes from comparison between luminal B and non-luminal B BCs. A comparison with TCGA data [28] Table S6 A: References of the six recent NGS studies used in the meta-analysis (dataset Meta-analysis). B: Frequency of somatic mutations identified in the 875 NGS breast tumors. C: Cooccurring and mutually exclusive mutations in the 875 NGS BCs (without molecular subtype distinction). D: Frequency of somatic mutations and association with molecular subtypes. E: Cooccurring and mutually exclusive gene mutations associated with luminal A subtype. F: Co-occurring and mutually exclusive gene mutations associated with luminal B subtype. G: Co-occurring and mutually exclusive gene mutations associated with basal subtype. H: Co-occurring and mutually exclusive gene mutations associated with ERBB2 subtype. I: genes targeted by several alterations mechanisms in luminal B subtype. (XLSX)
Table S7
Genes commented in discussion with supplemental references. (XLSX) | 2016-05-12T22:15:10.714Z | 2014-01-09T00:00:00.000 | {
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257162228 | pes2o/s2orc | v3-fos-license | Proliferation of household waste irregular dumpsites in Niger Delta region (Nigeria): unsustainable public health monitoring and future restitution
Inadequate collection and improper disposal of municipal waste have a direct negative impact on cities. Disease occurrence in Obio-Akpor (Port Harcourt metropolis, Nigeria) was suspected and linked to the proliferation of dumpsites and proximity to residential households. Evidence showed frequent incidence of diseases outbreak coupled with the topographic coastal nature and the morphological propelling dynamics of sediments transport in the area assisting the situation. The main objective of this study was to assess how the spatial distribution of irregular dumpsites is linked to the disease occurrence (cholera, diarrhoea and malaria) in the community. The data used for the study was obtained through questionnaires administrated at the hospitals, use of GPS for locating disease incidences and waste dumps, interviews and observations. Point pattern analysis using the G-function and the K-function was employed in analyzing the spatial distribution of dumpsites and disease incidences. Correlation tests were performed to test for the relationship between disease incidences and presence of dumpsites. The results showed that there was a significant relationship (p < 0.05) between disease incidences and presence of dumpsites. It was also observed that diseases could occur in areas where dumpsites were not present as cholera and diarrhoea are contagious but malaria is not, though it spreads. The study will be beneficial to governmental agencies, waste managers, institutions, environmentalists, health, social workers and future researchers.
Introduction
Irregular dumpsites serve as a receptor of heterogonous materials. Unsanitary landfilling or open dumping is common in developing countries [1][2][3][4][5][6][7] and known as the major threat to groundwater and other environmental resources [8,9]. Inefficient management of municipal waste produced; especially in Nigeria is increasing nuisance, environmental burdens and schematically linking disposal problems to public health concerns. Almost 95% of the municipal solid waste generated goes straight into a poorly managed hole called dumpsite, which decomposes and underflows to the land resources through infiltration from the precipitation processes in a very unsatisfactory manner thereby subjecting ground and surface water, environmental and public health to possible risks [10,11]. Since January 2009, Austria has given a red card to landfilling of waste materials and only allows pre-treated waste with very low organic matter to be legally landfilled through legislative actions. Since 2008, such an act defines waste management as a sustainable practice [12]. Inappropriate waste disposal can be very harmful [13]; it presents media for disease spreading and creates an overall negative impact on the environment because the released toxic heavy metals contaminate the soil [10], produce greenhouse gas [6], volatile organic compounds [14], and pollute water bodies [15,16].
Dumpsites in the Niger Delta are alternatively used as landfilling because it is a cheap method for waste disposal and no treatment goes on there. The leachate produced at these landfills causes contamination by organic, inorganic and microbial pollutants [13,14]. Locations where such indiscriminate dumping takes place become vulnerable to diseases since they become a harbor for mosquitoes and flies breeding, emission of methane gas, unpleasant odors, and serve as an invitation for all sorts of animalsdomestic and wild which can, in turn, serve as vectors transmitting diseases through food-chain to humans and environs. The objective of the study was to assess how the spatial distribution of the dumpsites is linked to the disease occurrence of malaria and acute enteric infections such as diarrhoea and cholera in the Obio-Akpor area of the Niger Delta. Holistic examinations of the risks posed by dumpsites and landfills were researched to determine the pollutants' migration. Adelopo et al. [16] reported heavy metal contents of landfills, Kumarathilaka et al. [14] focused on the volatile organic compounds, while Aboyeji and Eigbokhan [15] applied geospatial methods and GIS to represent the pollutants' spatial characteristics. Adamcova et al. [17] studied types of the waste (hazardous content and percentage of organic matter) present at the sites and their physicochemical reactions, but the diseases' occurrence and their clustering have not been established in the area. However, Yeukai and Kharlamova [18] have looked at the spatial distribution of some diseases in relation to the dumpsites. Figure 1 represents the study area. For the dry season, the daily relative humidity is 56%, and for the rainy season, it is 96% while the average annual rainfall in the Niger Delta is 2500 mm. Two identifiable patterns of rainfall occur, the longer one from April to October and the relatively shorter one from November to March. However, Uko and Tamunobereton-Ari [20] in their variability of climatic parameters of Port Harcourt cited the meteorological data of Port Harcourt airport, which provided an average 31.1-33.1°C of maximum temperature (during the dry season, November-February). Okpara et al. [19] have conducted a study of the heavy metal pollution from the dumpsite's leachate, soil and water. Due to the topography, sediment and solid waste transport enhances the propelling of debris [21]. People living in riverine areas frequently experience all kinds of illnesses and report mosquito's bite that leads to malaria. Water pollution and poor environmental sanitation encourage the incidence of cholera, dysentery, and diarrhoea.
Hospital survey and random sampling
We employed two-stage sampling techniques to select a representative sample. For the residential areas, hospital survey was conducted at five different hospitals within the communities living near the dumpsites to ascertain the level of disease cases diagnosed or treated in the area in recent time (10 months); and their reports were processed and analyzed. The selection of clusters (enumeration areas) was performed based on the households per person. According to Deaton [22], a sample size of 10% is adequate to draw statistical inferences. Therefore, 159 clusters were randomly selected from the study area. Six qualitative questionnaires were administered in selected hospitals in the community to seek information from the health experts (doctors, laboratory technicians, and matrons). From their file record, we configured our data to ascertain disease incidence and cases reported/treated. We also formed our objective based on the respondent's information and opinion expressed in the questionnaire delivered for the study. Based on the unstructured interviews' response, field surveys were conducted to observe and record the locations of irregular dumpsites and disease incidence using the Garmin GPS receiver for spatial distribution. The cases as represented in the clusters are residential areas or households within the surrounding dumpsite environment.
This study was carried out at Obio-Akpor local government area (LGA), created in 1989, located in the Niger Delta, Rivers State, southern Nigeria. The area is 260 km 2 with densely residential area of 2498 persons per km 2 . Based on the last census conducted in Nigeria in 2006, the projected population growth was estimated at 2000 per annum in Obio-Akpor LGA. Hence, as of 2016, we have about 649,600 officially reported, which means the additional population will amount to 8000 by 2020 [23]. A study by Ogwueleka [24] showed that more than 25 Mt of solid waste was generated in Nigeria annually with the average rate of generation ranging from 0.44 kg cap − 1 d − 1 in rural areas to 0.66 kg cap − 1 d − 1 in urban areas. On average, as reported by Okpara et al. [19], a total of 63.9 kt are being generated monthly in Rivers state of which Obio-Akpor is one of the main contributing figures. However, Rivers State where the present study was undertaken is home to the country's wealth. It is the oil-producing state and Port Harcourt is the capital city that has witnessed a high influx of migrants as a result of its rapid urbanization. The major urban center and their waste generation rate were estimated at 0.45, 0.98 and 1.16 kg cap − 1 d − 1 for Emougha, Obio-Akpor and Port Harcourt, respectively according to Babatunde et al. [25].
Population projection assumes the same rate of growth for all LGAs within a state according to the National Population Commission of Nigeria (https://www. nationalpopulation.gov.ng/) and National Bureau of Statistics (https://nigerianstat.gov.ng/); hence, there is a possibility of high error rates. We chose Eliozu Rumuokoro and Obiri Ikwere Rumuagholu communities for this study because they were located around dumpsites, in riverine areas with high disease incidence and low level of waste collection, and characterized by the presence of irregular dumpsites.
Analysis of the data used
We performed exploratory data analysis to view the pattern of the data generally. Data on the disease occurrence and distance of household's cases reported at the selected hospitals from the nearest dumpsite were tested for normality using the Kolmogorov-Smirnov test at 95% significance level. Correlations were used to test whether there is a significant relationship between disease occurrence and presence of selected dumpsites. Confirmatory data analysis involved the use of the K-function, which was used to further analyze the data and to reveal the patterns of progression. Calculation of distances between dumpsites and reported cases with disease incidence was done in SPSS (Statistical Package for the Social Sciences) using the following Pythagoras formula: , where D is the distance between disease cases reported and dumpsites, and (x; y) are the location coordinates of reported-households cases and dumpsites.
Point pattern analysis; G-function and K-function
Arc-Mag 10.3 was used to generate point maps for dumpsites and disease incidence. The G-function was used as an exploratory data analysis technique to determine the inter-event distribution of diseases. The Kfunction was used to determine the significance and scale of clusters that were explored using the Gfunction. The G-function and the K-function were used to determine if the occurrence of diseases and the presence of dumpsites show any clustering or regularity as opposed to being randomly distributed.
Results and discussion
Our findings showed that solid waste generated in Obio-Akpor LGA is influenced by a number of factors. These factors include the services rendered by the municipal government such as the provision of waste bins, irregular collection, transport, residence approach to wastes and disposal practices employed. The waste management plans and policies represent an inefficient waste management system. All wastes are disposed at dumpsites without any protection of the environment and population. Waste disposal by incineration and burning was common for the purpose of the waste volume reduction but had health implications. In our study, we looked at three possible diseases that could affect human beings through this source. Diarrhoea, cholera and malaria were conspicuous from other diseases. Prerequisite knowledge of the effects of poor waste management on human health was accessed through a qualitative interview conducted in the hospitals within the study area. Without periodic health surveillance to detect early signs of diseases, monitoring of disease clustering is difficult. Therefore, this study offers help to reveal the impact of the unregulated dumpsites' disease occurrence and their locations. It shows how clustering is going on in the area. The results in Fig. 2 suggest that waste dumping on the roadside or undesignated sites is visible due to irregular collection of waste by the management agency.
The spatial distributions have established the type of disease spreading across the residential areas from the dumpsites. Three health centres in the New Airport Road area reported disease frequently treated as malaria, diarrhoea and cholera. Previous studies conducted in the same area also revealed presence of heavy metal concentration found in leachate from dumpsites, soil and water [19].
K-function was used as confirmatory data analysis to evaluate the evidence of our assumptions in this study by challenging the data input. In this case, we conducted the test to ascertain the relationship between the presence of irregular dumpsites and disease occurrence and to determine whether there was evidence of disease occurrence in relation to the dumpsites in Eliozu Rumuokoro and Obiri Ikwere Rumuagholu communities of Obio-Akpor LGA. The results are displayed in Fig. 3.
The result disclosed that households, residential or industries closer to dumpsites have a high risk of getting diseases than those that are further away from the source. We observed that the dispersed distance had not reached the expected straight line as shown in the confidence envelope. However, clustering of diseases at 300 m is an assertion that there is high disease incidence at short distances from the nearest dumpsite. The confidence envelope review graph showed that. From 1800 to 4000 m, there is evidence of regularity, which further ascertains a decrease in the disease incidence. An observed behavior from the results suggests that diseases are also occurring in clusters; hence the clustering of diseases after 3000 m from the nearest dumpsite. All these are associated with unsanitary dumpsites multiplication and inadequate waste collection in the area.
In Fig. 4, general observation showed significant level, the p-values indicating lower or higher clusters in the random sample, while the z-score representing critical value. With p-values < 0.05 and z-score > 2.50 there is less than 1% likelihood that this high-clustered pattern could be the result of random chance.
However, the simulation envelopes were used to show the significance of the clusters at the 95% significance level. If the observed phenomenon K(d) lies above the upper envelope, the clustering will be significant, if K(d) lies below the upper envelope, the clustering will not be statistically significant. We observe from Fig. 5a for the New Airport Road that there is statistically significant clustering of diseases at 250, 350, and 400 m because K(d) lies below the upper envelope. The clustering continues significantly at 700 to 800 m because K(d) is above the upper envelope. While in Fig. 5b clustering of diseases is statistically significant at 1100 m and continues in 240 m away in Eliozu. This could be because of the terrain and Eliozu's older dumpsites. It further suggests that there is a significant number of diseases occurring at a short distance from the dumpsite as compared to the number of diseases occurring in the cluster at 800 and 2400 m.
We used colour differences in Fig. 6 to differentiate diseases clustering spots and to separate them.
In the study area, two general, two private and one health centre (community) hospitals provided confidential data on the reported cases for their patients living close to the major dumpsites. By percentage calculations, we tabled the diseases, and results showed that among all other cases, cholera reported cases were minimal while malaria and diarrhoea had a higher share. Frequent reports of mosquito bites, which lead to malaria, by residents could be confirmed in Table 1, which showed the highest occurrence and spread from 60 to 95%. This is followed by diarrhoea 5 to 30%.
The impact of monitoring irregular dumpsites
The Niger Delta region has more factors playing the key roles in supporting disease spread: the pattern of settlement, availability of wetland, and nature of the terrain. Rural settlements lack essential amenities like good infrastructures, drainage systems, and accessibility of potable water. Hence, the region is a prone area and deserves better access to solid waste collection and disposal options [25,26]. Monitoring, therefore, is important due to the lack of access to controlled waste treatment and disposal facility, which is very conspicuous. A larger settlement is found in the interior scale of the communities and they are generally facing a fragile ecosystem, delicate ecological sustenance and some containable disease spread. Most people in Eliozu Rumuokoro and Obiri Ikwere Rumuagholu communities live around dumpsites and riverine areas as seen in Fig. 2. Dumpsite impact monitoring will positively influence both environmental and ecologically sensitive areas. To effectively understand its impact, it is important to understand the dimensions and dynamics of anthological consequences on agriculture [27]. Moreover, a conscious effort is required to understand the present condition of dumpsite monitoring and the extent of ecological degradation, which affects both public health and natural ecosystems. However, in Table 1, the situation has not only provided rethinking strategies at the top management levels but has created a compromise requiring superabundant measures embracing all humankind.
Improve management approach for restitution
Strategic planning is important as it helps to know the actual extent of the ecological and public health disaster.
In [28]. Budget proposal of the same year revealed that health was on the 11th position and was offered just NGN 46 billion (approx. USD 120 million), by a factor of six less than works and housing that was on the first position and engulfed NGN 262 billion (approx. USD 683 million). This means that excluding malaria, diarrhoea, dysentery, maternal mortality, etc., Nigeria had 287 deaths caused by these three Figure 7 displays steps that can be employed in the planning process to tackle these problems.
Oil pollution led to the degradation and destruction of 10% of mangroves in Nigeria while there is no estimate to prove the contribution of pollution from dumpsites to the situation in the settlement areas. More so, there is total neglect on reports of the effects of heavy metal pollutants (eco-toxicants) in leachate and their transport pattern onto the plants, soil and water. Hence, the mangrove belt of the Niger Delta is "caught in the middle" between heavy metal pollution from leachates, brought by downstream flow from wetland area, and oil, spilt offshore and brought by the wave and tidal action to the mangrove estuarine ecosystem. Predominately, a typical eco-zone in the Niger delta has a floristic composition of vegetation such as Alstonia boonei, Cleistopholis patens, Mitragyna among others. More so, delta's eco-zone is rich in its floral biodiversity with over 219 plants species, which represent 66 families. The floral life forms are dominated by trees with 42%, followed by herbs 32.0%, shrubs 16.4%, and climbers/lianas 9.6% [29] but there is an on-going unaddressed threat to the Niger delta biodiversity.
Conclusions
This study established the fact that there is a correlation between the disease occurrence and irregular dumpsites in Obio-Akpor communities and additional actions must be taken to ascertain the effects of dumpsites proximity to residential apartments for public health promotion. As shown from clustering, diseases such as cholera and diarrhoea, with an exception of malaria, can occur in areas where dumpsites are not present because they are highly contagious. Therefore, appropriate siting of landfills, dumpsites or burrow pits is very significant and ones in charge must take cognisance of coastal proximity as well as other ecological importance into consideration. Total closure of dumpsites remains the best option for sustainable practice.
In conclusion, dumps are aiding acute infectious disease to spread, and malaria mosquitoes breeding spreads at every dumpsite as it has the highest percentage occurrence. Diarrhoea and cholera were found clustering around the dumpsites in the residential areas. From the interviewers' responds, unreported cases of diseases in question are more than reported cases. Observation also shows that residents often do not approach hospitals but choose local diagnosis of self-medication. Opinions of the health professionals and some documented cases used in the field studies revealed the occurrence of diseases (cholera, malaria and diarrhoea) spatially distributing from the Obio-Akpor LGA dumpsites' surroundings. | 2023-02-25T14:56:14.708Z | 2021-01-07T00:00:00.000 | {
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250034004 | pes2o/s2orc | v3-fos-license | Rapid Identification of Constituents in Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. Using UHPLC-Q-Exactive Orbitrap Mass Spectrometry
Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. (CT) belongs to the Rubiaceae family. Its dried leaves are widely used in traditional Chinese medicine to treat enteritis, dysentery, toothache, furuncles, swelling, traumatic injury, fracture, bleeding, and scalding. In order to further clarify the unknown chemical composition of CT, a rapid strategy based on UHPLC-Q-exactive orbitrap was established for this analysis using a Thermo Scientific Hypersil GOLDTM aQ (100 mm × 2.1 mm, 1.9 µm) chromatographic column. The mobile phase was 0.1% formic acid water–acetonitrile, with a flow rate of 0.3 mL/min and injection volume of 2 µL; for mass spectrometry, an ESI ion source in positive and negative ion monitoring modes was adopted. A total of 135 chemicals comprising 67 chlorogenic acid derivatives, 48 flavonoids, and 20 anthocyanin derivatives were identified by comparing the mass spectrum information with standard substances, public databases, and the literature, which were all discovered for the first time in this plant. This result broadly expands the chemical composition of CT, which will contribute to understanding of its effectiveness and enable quality control.
Introduction
Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. (CT), known as Ma Yanshu or Water Yangmei in traditional Chinese medicine (TCM) and 'Bagua Maple' in Dong medicine, belongs to the Rubiaceae family. The leaves have the effects of clearing away heat and toxic materials, as well as dispelling blood stasis and reducing swelling, and they are used for the treatment of enteritis, dysentery, toothache caused by acute gingivitis and acute pulpitis, furuncles, swelling, traumatic injury, fracture, bleeding, and scalding [1]. This plant is mainly distributed in Hunan, Guangdong, Hainan, and Taiwan provinces. However, no detailed studies on the material basis of its medicinal effects have been reported. To date, most studies on CT have focused on its ornamental value. A previous study reported on its pharmacognosy [2]. Therefore, it is necessary to clarify the unknown chemical composition of CT.
In order to further analyze and discover its chemical composition and pharmacological effects, it is necessary to find a suitable analytical technique to analyze the complex chemical composition in CT. There are many methods for analyzing herbal medicine, including Likewise, compounds 13 and 32 were confirmed as procyanidin B1 and procyanidin B2. [12,13], they were identified as trans-3-O-p-coumaroylquinic acid (pCoQA), trans-4-pCoQA, cis-4-pCoQA, and cis-5-pCoQA, respectively, according to their retention time and base peak ion in the MS 2 spectrum. were identified as trans-3-SQA, cis-3-SQA, trans-5-SQA, and cis-5-SQA through comparison with the literature data [14].
Identification of Speculative Anthocyanins
The [26]. was stored at the School of Pharmaceutical Sciences, Hunan University of Medicine, Changsha, China.
Reference Standards and Sample Preparation
The dried powder of CT (5 g) was extracted under reflux in 100 mL of 70% aqueous ethanol for 1 h, and then the extracted solution was filtrated and dried under reduced pressure to yield a brown residue, which was dissolved in methanol. The sample was centrifuged at 12,000 rpm for 20 min. A volume of 2 µL was injected into an UHPLC-Qexactive orbitrap MS for analysis. All reference standards were accurately weighed and dissolved in methanol before storing in a refrigerator at 4 • C until further analysis.
Instruments and Conditions
The instruments used for this study included a Thermo Q-exactive focus orbitrap MS connected to a Thermo Scientific Dionex Ultimate 3000 RS (Thermo Fisher Scientific, Carlsbad, CA, USA). Separation was performed on a Thermo Scientific Hypersil GOLD TM aQ (100 mm × 2.1 mm, 1.9 µm). The column temperature was kept at 35 • C, and the sample was maintained at 10 • C. The mobile phase was water with 0.1% formic acid (A) and acetonitrile (B). The gradient program was as follows: 0 min, 5% B; 2 min, 10% B; 5 min, 20% B; 10 min, 25% B; 12 min, 55% B; 20 min, 80% B; 25 min, 95% B; 26 min, 5% B; and 30 min, 5% B. MS analysis was performed in both positive and negative ionization modes using electrospray ionization (ESI) in the scan range of m/z 120-1000 at a resolution of 35,000. The source conditions were as follows: sheath gas, 30; auxiliary gas, 10; spray voltage, 3.0 kV for (−)-ESI and 3.5 kV for (+)-ESI; capillary temperature, 320 • C; auxiliary gas heater temperature, 350 • C. The MS 1 spectra were acquired in full MS mode at a resolution of 35,000, whereas MS 2 spectra were obtained by ddMS 2 or parallel reaction monitoring (PRM) mode triggered by inclusion ions [12]. The NEC (normalized collision energy) was set as 30%, with 5.0 × e 5 of the automatic gain control (AGC) target. Data were processed using Xcalibur™ version 4.1 and Compound Discoverer 3.0 (Thermo Fisher Scientific, Carlsbad, CA, USA).
Prediction of Expected Compounds
It is widely known that chemical constituents in the same category possess an identical carbon skeleton and homologous biosynthetic pathways. CGA analogues constitute a large family of esters formed between quinic acid or shikimic acid and one to four special residues, most commonly p-coumaric acid, caffeic acid, sinapic acid, and ferulic acid. Therefore, the molecular structure of the CGA derivatives can be predicted [11,26]. Likewise, flavonoids and anthocyanins can also be predicted. Quercetin and kaempferol are the carbon skeletons of flavonoids connected by hydroxyl (OH) and glycoside bonds; their structures differ in terms of the type and number of sugar units, e.g., glucose (C 6
Conclusions
In this study, a rapid and effective method for identifying the chemical constituents of CT was developed using UHPLC-Q-exactive orbitrap combined with PRM; the compounds were predicted using DFI and NL techniques. A total of 135 compounds were identified, comprising 67 chlorogenic acid derivatives, 48 flavonoids, and 20 anthocyanins, all of which are reported for the first time in CT. These results expand the knowledge on the chemical composition of CT and provide a scientific basis for the subsequent elucidation of the medicinal substances present and their activities, enabling further development and utilization of this plant. Overall, the results lay the foundation for in-depth research on the pharmacodynamic basis of CT. Furthermore, this research strategy can be used for the characterization of various samples.
Conclusions
In this study, a rapid and effective method for identifying the chemical constituents of CT was developed using UHPLC-Q-exactive orbitrap combined with PRM; the compounds were predicted using DFI and NL techniques. A total of 135 compounds were identified, comprising 67 chlorogenic acid derivatives, 48 flavonoids, and 20 anthocyanins, all of which are reported for the first time in CT. These results expand the knowledge on the chemical composition of CT and provide a scientific basis for the subsequent elucidation of the medicinal substances present and their activities, enabling further development and utilization of this plant. Overall, the results lay the foundation for in-depth research on the pharmacodynamic basis of CT. Furthermore, this research strategy can be used for the characterization of various samples. Data Availability Statement: Data will be provided upon request.
Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors. | 2022-06-26T15:07:15.149Z | 2022-06-23T00:00:00.000 | {
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221763269 | pes2o/s2orc | v3-fos-license | Brain-derived neurotrophic factor gene polymorphism in post-ST-elevationmyocardial infarction patients undergoing primary percutaneous intervention
Use your smartphone to scan this QR code and download this article ABSTRACT Introduction:The goal of this study was to elucidate a link of brain-derived neurotrophic factor (BDNF) Val66Met genewith combined 6-month clinical endpoints in post-myocardial infarctionpatients. Methods: 256 post-myocardial infarction patients who underwent primary percutaneous coronary intervention were enrolled in the study. Variants of Val66Met gene BDNF were identified by real-time polymerase chain reaction at baseline. Results: The combined clinical end points (major cardiovascular events and hospitalization) were determined in 61 (23.8%) post-STEMI patients; consequently, 195 (76.2%) patients did not meet the events. Univariate linear regression revealed that predictors for combined clinical end points were peak TnI levels, NT-proBNP, SYNTAX score, TIMI score, obesity, left ventricular ejection fraction, and genotype 66ValMet+66MetMet in BDNF gene. The cumulative clinical outcomes (major adverse cardiac events and admission) were determined in 61 (23.8%) patients. Kaplan-Meier curves demonstrated that 66ValVal genotype of BDNF gene was significantly associated with the low number of combined end points. Conclusion: The Val66Met polymorphism in BDNF gene independently predicted 6-month combined clinical end points in post-myocardial infarction patients.
INTRODUCTION
Recurrent major adverse cardiac events (MACEs) and heart failure (HF) remain the most common causes of premature cardiovascular (CV) mortality amid post-ST-segment elevation myocardial infarction (STEMI) patients, regardless of wide implementation of early re-vascularization strategies including primary percutaneous coronary intervention (PPCI) 1,2 . In fact, conventional pure approaches based on clinical assessment, transthoracic echocardiography and biomarkers of myocardial necrosis are insufficient today to catch vulnerable post-STEMI patients after successful PPCI 3 . Although there are several biomarkers (natriuretic peptides, high sensitive cardiac troponins, soluble ST2, etc.) that are designed for risk stratification among patients with STEMI 4-6 , the risk assessment and prediction of poor clinical outcomes after successful competed PPCI in post-STEMI patients are uncertain 7 . There are several limitations that could decrease the predictive value of conventional biomarkers in post-STEMI individuals, such as older age, morbid obesity, diabetes mellitus (DM), preserved or normal left ventricular (LV) ejection fraction (EF), co-existing kidney insufficiency and atrial fibrillation [8][9][10] . Neurotrophins are a superfamily of regulatory proteins which regulate proliferation, differentiation, survival and plasticity of neurons 11 . Brain-derived neurotropic factor (BDNF) was previously found as one of the neurotrophins with proliferative, cholinergic, serotoninergic and dopaminergic activities, and is predominantly synthesized in central and peripheral neurons 12 . Later, an expression of mRNA BDNF was found in myocardium, vessel vasculature, skeletal muscles, parenchymal organs (including lung, spleen and kidney), visceral epithelial cells, and mature and progenitor endothelial cells [13][14][15] . Previous animal and clinical studies have revealed that BDNF through an activation of nuclear factor kB receptors mediates endothelial cell survival and neoangiogenesis, reduces p75-mediated apoptosis of cardiac myocytes, enhances endothelial function, regulates blood flow in ischemic myocardium, and improves LV function after ischemic injury, thereby providing cardioprotective effects 16,17 . Observational and clinical investigations have shown strong inverted associations between BNDF levels in peripheral blood and cognitive dysfunction in HF patients, and CV risk in individuals with known DM, hypertension, cardiomyopathy, stable coronary artery disease and STEMI [18][19][20][21] . However, circulating levels of BDNF exhibited a close correlation with single nucleotide polymorphism (SNP) in BDNF gene that was associated with a replacement of valine to methionine in the 66 position of codon (Val66Met). It has been suggested that this SNP Val66Met affects the intracellular processing of the peptide and leads to a decline in the secretion of BDNF, resulting in lowered susceptibility to ischemia and myocardial injury 22,23 . The aim of the study was to elucidate a link of BDNF Val66Met gene with cumulative 6-month clinical end points in post-myocardial infarction patients.
Patient population
From 2016 August to 2019 February, 320 patients underwent selective coronary angiography due to suspected acute STEMI. Acute STEMI was diagnosed in accordance to the European Society of Cardiology (ECS) recommendation (2017) 24 . The study flow chart is shown in Figure 1. We identified 256 acute STEMI individuals who underwent PPCI with an implantation of bare-metal stent "COMMANDER" manufactured by "Alvimedica" (Turkey) in culprit artery (1 to 4 stents onto ischemic-related arteries) and discharged with post-PPCI TIMI III score from the hospital. All patients were treated with current adjuvant care 24 .
Coronary angiography
We used radial or femoral vascular access and twoto-three orthogonal projections to receive angiograms at baseline. A digital X-ray system "Integris Allura-9" (Philips Healthcare, Eindhoven, Netherlands) was used to visualize coronary anatomyto visualize coronary anatomy. Ultravist-370 (Baier Pharma, GmbH, Germany) was the contrast giving automatically through the injector.
Ethical declaration
All patients enrolled in the study gave voluntary informed consent to participate. The study was approved by the local ethics committee (Protocol №8, 29.08.2016). The study had been carried out in accordance with the 1964 Helsinki declaration.
Sample size calculation
We calculated sample size by taking into consideration the effect size estimation (0.99), type II error (0.2), type I error (0.05), expected mortality rate of 7%, and one-year mortality rate of 14% 25 . The sample size was 250 individuals.
Determination of risk factors and comorbidities
DM was diagnosed as serum level of fasting glucose of > 7.0 mmol/L, 2-h postprandial glucose serum level of > 11.1 mmol/L, or current therapy of anti-diabetic drugs 26 . Hypercholesterolemia (HCE) hypertension was diagnosed in accordance with current recommendation 27,28 . HF was diagnosed according to current ECS clinical guidelines 29 . Information of other diseases and medical history were obtained by investigators through a review of medical history or direct contact with the patient's general practitioner.
Echocardiography examination
Transthoracic B-mode echocardiography and Tissue Doppler Imaging were carried out using 3.5 MHz phase probe with "Aplio 500" (TUS-A500, Toshiba Inc., Japan) at baseline. LVEF were measured by Simpson's method. LV global longitudinal strain (e') and early transmitral velocity (E) were measured by tissue Doppler imaging technique and impulse transmitral Doppler regime, respectively.
Determination of STEMI prognosis
We used the TIMI score and the GRACE score to validate prognostic capacity after STEMI 30,31 .
SYNTAX score determination
SYNTAX score (SS) was calculated by an experienced interventional cardiologist accordingly 32 .
Determination of endpoints
The primary endpoint was MACEs in combination with hospitalization that occurred during 6-month period post-discharge period. MACEs included CV death, recurrent angina, and newly diagnosed HF. The endpoint was ascertained by personal or phone contact with a general practitioner or staff of the hospital in which the patient had been admitted.
Calculation of glomerular filtration rate
The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to calculate glomerular filtration rate (GFR) 33 .
Collection of blood samples
Venous blood samples were collected from the patients using vacutainers at baseline and at the end of the study. After 30-minute centrifugation, the serum was collected and then stored at -70 0 C until transfer to the laboratory of GI "L.T.Malaya TNI NAMSU". Hematology and biochemistry, including lipid profiles and fasting glucose, were provided for each patient. Serum levels of troponin I (TnI) were measured by chemo-luminescent immunoassay method on the biochemical analysator (Humalyser 2000, Mannheim, Germany). The average of the TnI level was 0.5-50 ng/mL. N-terminal fragment of brain natriuretic peptide (NT-proBNP) was evaluated by use of a commercially available kit manufactured by R&D Systems GmbH (Wiesbaden-Nordenstadt, Germany). The average of the NT-proBNP level was 10-12000 pg/mL.
SNP Val66Met (rs6265) in BDNF gene determination
The extraction of DNA was executed per the conventional protocol for a commercial set (TacMan TMSNP Genotyping Assays; Thermo Fisher Scientific Assay IDC_11592758_1). The BDNF genetic variants were evaluated by real-time polymerase chain reaction (PCR). Primers used in the BDNF Val66Met (rs6265) polymorphism assay were as follows: CCTACAGTTCCACCAGGTGAGAAGAGTG (forward), TCATGGACATGTTTGCAGCATCTAG-GTA (reverse).
Statistics
Baseline characteristics are presented as mean ± standard deviation (SD) or median (IQR) for continuous variables and proportions for categorical variables. Chi-square test or Mann-Whitney U test and T-test were used to compare the categorical or normallydistributed data, respectively. Hardy-Weinberg Equilibrium was conducted to estimate allele frequencies. Spearman correlation analysis was performed to investigate the relationship between circulating levels of cardiac biomarkers, co-morbidities, and BDNF gene SNP. Univariate and multiple variate linear regression analyses were done; factors that predict the combined endpoint were determined. The beta coefficient, standard error (SE), odds ratio (OR), and 95% confidence interval (CI) for predictors were evaluated. We also checked whether five common subject-level gene models (recessive, multiplicative, additive, dominant, and over-dominant models) were related to the explanatory variables 34 . We also used area under curve (AUC), integrated discrimination indices (IDI), and net-reclassification improvement (NRI) for prediction performance analyses. Significant differences between variables were set at p-value < 0.05.
RESULTS
The combined clinical endpoints (MACEs and hospitalization) were determined in 61 (23.8%) post-STEMI patients; consequently, 195 (76.2%) patients did not meet the events. General clinical characteristics of the entire patient population enrolled in the study and both cohorts (with MACEs and free MACEs) are reported in Table 1. The entire population consisted of participants with mean age of 58.76 years old, with male/female proportion of 77.3%/22.7%, with CV risk factors including hypertension (52%), type 2 DM (T2DM) (19.5%), hypercholesterolemia (63.7%), obesity (37.5%), and smoking (34.9%). We did not find significant differences between both cohorts of the post-STEMI patients in terms of demographics, glomerular filtration rate, and CV risk factor presentation, except in the incidence of T2DM and hypertension, which were more frequently seen in patients who met MACEs than in individuals who were free of MACEs (P = 0.032 and P = 0.0008, respectively). Therefore, participants with MACEs were older that those who did not meet MACEs (P = 0.027). The patients who met MACEs exhibited higher peak serum levels of troponin I and circulating levels of NT-proBNP than those who had no MACEs. Significant differences in lipid profiles among both patient cohorts were not found. The observed frequency of Val66Met BDNF genotype in post-STEMI patients was as follows: 66ValVal -74.2% and 66ValMet+66MetMet -25.8%. Genotype frequency matched to Hardy-Weinberg equilibrium (χ 2 = 0.17, P > 0.05). There were not differences between both cohorts in terms of phenotype of BDNF (P = 0.272). All post-STEMI patients were treated according to contemporary clinical protocol and there were not significant differences in concomitant medications, except for metformin which was prescribed frequently in the patient cohort with MACEs (P = 0.046). We found that TIMI risk in patients with MACEs was significantly higher in comparison with individuals who had no combined endpoint (P = 0.046) ( Table 2). Nevertheless, the number of participants in both cohorts having 22 -32 SYNTAX score points and ≤ 22 SYNTAX score points did not differ, but the proportion of the patients with MACEs having > 32 SYN-TAX score points occurred frequently when compared with those who did not meet the endpoint. Additionally, total GRACE score points were similar in both patient cohorts (P = 0.294).
Significant differences between both cohorts in terms of localization of STEMI were not found, even though one vessel injury frequently occurred in MACEs-free patients. In contrast, patients having MACEs represented frequent circumflex artery injury (p = 0.021). Hemodynamic characteristics in post-STEMI patients at baseline are reported in Table 3. The patients having MACEs had lower LVEF (P = 0.005), higher left atrium volume (P = 0.021), and E/e' ratio (P = 0.042) than those who had no MACEs.
Comparison of the predictive abilities of the models
We compared both models (genotype 66Val-Met+66MetMet in BDNF gene and NT-proBNP) for STEMI individuals ( Table 5). The genotype 66ValMet+66MetMet in BDNF gene was better than NT-proBNP and standard model, while the only adjusted models were entered in the analysis.
Determination of genetic model variants
We found that the model based on the genotype 66ValMet+66MetMet in BDNF gene carried out an additive value because it was defined by the following equation: OR2 = 2 − 1 / OR1 ( Table 6). This means that the regression analysis was the best fitted to the model by applying the variables which predicted cumulative clinical endpoint.
Kaplan-Meyer analysis for combined endpoint accumulation trends in post-STEMI patients
Kaplan-Meier curves demonstrated that post-STEMI patients having 66ValVal genotype of BDNF gene had the lowest accumulation of combined endpoint when compared with those who had the combination of 66ValMet and 66MetMet genotypes (Cox-criterion, P = 0.019; log-rank criterion, P = 0.03) (Figure 2).
DISCUSSION
The results of our study showed negative associations between the 66ValMet + 66MetMet polymorphisms in the BDNF gene and 6-month endpoints in post-STEMI patients. The prognostic significance of BDNF 66ValMet polymorphism was investigated in post-STEMI patients first, while these relations were previously determined in individuals with acute coronary syndrome 35 . Therefore, there was an association between the BDNF levels and severity of HF NYHA functional class among individuals with HF with reduced LVEF (HFrEF) 21 , atherosclerosis severity in stable CAD patients 36 , and patients with unstable angina 37 . The exact causes for the decline in circulating BDNF levels among patients with known CV diseases (including unstable angina, acute coronary syndrome, STEMI and HF) are uncertain. Probably, BDNF can be released from skeletal muscle, and skeletal muscle dysfunction (rather than loss of skeletal muscle mass) in HF patients may explain lowered levels of circulating BDNF in HFrEF. However, it does not determine declining levels of the peptide in post-STEMI patients with adverse cardiac remodeling, HF with preserved EF, nor recurrent CV events [38][39][40][41] . Whether BDNF production is under control by myocardial function and exercise is not well-understood. In this context, lowered levels of BDNF have been ensured by SNP in the 66ValMet BDNF gene, which controls synthesis of the peptide 42 . Previous animals and clinical trials have revealed that the BDNF levels were modulated by BDNF gene polymorphism and that CV actions of circulating BDNF likely correspond to this SNP 43,44 . Moreover, low circulating levels of BDNF were associated with adverse cardiac remodeling after STEMI and higher levels of NTproBNP 45 . Nevertheless, there are investigations that have yielded no close association between the genetic variants of BDNF and the serum levels of this peptide 43,45 . There is a hypothesis that the BDNF Val66Met genotype can affect cardiac function and CV risk through other mechanisms. We found an association between circulating levels of NT-proBNP and BDNF gene polymorphism; however, there is no plausible evidence for the interrelation between BDNF gene polymorphism and cardiac function, severity of atherosclerosis, and CV risk factors (not including T2DM). Importantly, previous clinical studies have shown associations between metabolic disease (such as morbid obesity and T2DM) and circulating levels of the BDNF, but not with the BDNF gene polymorphism 46,47 . Some researchers have proposed that adipocytokines can be triggers for the decline of BDNF levels, but BDNF Val66Met polymorphism has shown no significant association with circulating levels of leptin 47 . However, in our investigations, patients who were enrolled in these studies were not qualified as those having stable CAD or STEMI. We suggest that BDNF gene polymorphism is an independent factor contributing to MACEs and readmission to the hospital through altered cardioprotective effect. Since BDNF specifically binds to the tropomyosin-related kinase receptor B, it acts as modulator of various intracellular signaling pathways (e.g. Akt pathway, transforming growth factor-beta/ Smad pathway), which suppress inflammation and oxidative stress, upregulate the viability of ischemic tis-sue, promote differentiation of endothelial progenitor cells, and initiate neovascularization and angiogenesis in response to hypoxic and ischemia stimuli [48][49][50][51] . Interestingly, the predictive values for both the BDNF Val66Met gene polymorphism and serum levels of NT-proBNP were almost similar after adjustment for severity of atherosclerosis and STEMI. Although the results of the study demonstrated that the BDNF Val66Met gene polymorphism predicted the combined endpoint in post-STEMI patients, there is no explanation for the exact molecular mechanisms which support this effect. We believe this fact is required to be investigated in the future to clearly understand primary ways that BDNF Val66Met gene polymorphism affects the clinical outcomes of post-STEMI patients after successful PPCI. Our study had several limitations. The first limitation was associated with the small sample size, it is important to calculate sample size such that the sta-tistical difference between both cohorts can be discerned. However, a large clinical study is required to obtain more information regarding the BDNF gene polymorphism as a predictor for poor clinical outcomes in post-STEMI patients. The second limitation is the lack of optical coherent tomography to rule in correct expanding of stents. The third limitation relates to respectively lowered number of secondary endpoints including hospitalization due to HF, recurrent unstable angina, and STEMI and PPCI. We did not perform analysis of secondary endpoint accumulation.
CONCLUSION
We found that 66ValMet + 66MetMet BDNF gene polymorphisms independently predicts the combined 6-month endpoint in post-STEMI patients after treatment with PPCI. | 2020-09-03T09:04:37.292Z | 2020-08-31T00:00:00.000 | {
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248369385 | pes2o/s2orc | v3-fos-license | Effects of Changes in Osmolarity on the Biological Activity of Human Normal Nucleus Pulposus Mesenchymal Stem Cells
The expansion and maintenance of the NPMSC (nucleus pulposus mesenchymal stem cell) phenotype are considered as potential therapeutic tools for clinical applications in intervertebral disc tissue engineering and regenerative medicine. However, the harsh microenvironment within the intervertebral disc is the main limitation of its regeneration. The osmolarity of the intervertebral disc is higher than that of other tissues, which has an important influence on the biological characteristics of NPMSCs. In this study, we observed the effect of different osmolarities on the biological characteristics of human normal NPMSCs cultured in vitro and explored the role of osmolarity in intervertebral disc degeneration. Our data demonstrated that the change in osmotic pressure has an important effect on the biological activity of NPMSCs, and this effect may occur through the P16INK4A/Rb pathway. This study provides a theoretical basis for the future treatment of intervertebral disc degeneration.
Introduction
Low back pain is common, and its prevalence is increasing due to the aging of the population, seriously affecting the lives and health of patients. This condition is a major burden on society because of its high medical and financial costs [1,2]. Studies have shown that intervertebral disc degeneration (IVDD) is an important pathological factor in back pain [3,4]. IVDD is thought to be caused by a number of factors, including aging, biological factors, mechanical loading, smoking, and decreased cellular nutrition [5]. Degeneration of the intervertebral disc is characterized by damage to the nucleus pulposus (NP) cell function and reduced cell number, including degeneration of the annulus fibrosus, resulting in a disturbed ECM structure [6,7]. The intervertebral disc connects two adjacent vertebral bodies, and no blood vessels directly supply nutrients. This structure is composed of three parts: annulus fibrosus, NP, and cartilage endplate [8]. The NP is located in the center of the intervertebral disc and has a unique biological environment, which is characterized by hyperosmolarity, nutrient deficiency, acidity, and hypoxia [9]. Normal intervertebral discs lack blood vessels and a nutrient supply, and metabolites are mainly eliminated through the diffusion of the cartilage endplate pathway [10] because in a hypoxic environment, cells primarily provide energy through glycolytic metabolism [11,12]. As the degree of IVDD increases, the microenvironment of the cells in the intervertebral disc will change, the glucose supply will decrease, the oxygen supply will change, acidity will increase, and osmotic pressure will also change. These changes further aggravate IVDD and form a vicious cycle [13,14].
Studies have shown that NP cell damage is an important cause of IVDD [15]. Aggrecan and collagen are the most important extracellular matrix in the nucleus pulposus: aggrecan provides the swelling pressure required to increase water content and the collagen provides resistance to swelling. The nucleus pulposus possesses mostly collagen II [16]. NP cells play an important role in maintaining the metabolic balance of the extracellular matrix. NP cell damage can cause disorder of the matrix metalloproteinase (MMP) secretion, leading to the degradation of the extracellular matrix, and the water content of nucleus pulposus will decrease, resulting in IVDD [17]. Therefore, current research on the treatment of IVDD is mainly focused on preventing the degradation of the extracellular matrix and increasing the number of NP cells [18]. Blanco et al. [19] identified nucleus pulposus mesenchymal stem cells (NPMSCs) in human degenerated NP tissues; these cells could replicate and undergo adipogenesis, osteogenesis, and chondrogenesis, indicating they are potential candidates for cell therapy in IVDD. Stimulating the proliferation and maintenance of the endogenous NPMSC phenotype may be a new strategy for the treatment of IVDD. However, due to the complex microenvironment of the intervertebral disc, regeneration and repair of NPMSCs are difficult.
The NP tissue of the intervertebral disc contains a high level of hydrophilic extracellular matrix, and the main component is proteoglycan [20]. Aggrecan increases intervertebral disc tissue permeability and resistance to pressure, and degradation of aggrecan can lead to damage to intervertebral disc function and the occurrence of IVDD. Aggrecan plays an important role in maintaining the hydration state and osmolarity of intervertebral discs [21]. Sulfated glycosaminoglycan is highly negatively charged and adsorbs cations and water molecules, thereby increasing the osmolarity of the tissue, promoting water entry in the intervertebral disc tissue, and then increasing the expansion pressure of the tissue to bear the compressive stress load of the spine [22,23]. The various movements and loads of the spine throughout the day lead to dynamic changes in the osmolarity of the NP tissue. When the load stress exceeds the osmolarity of the NP tissue, water is squeezed out of the NP, and the osmolarity increases. When the stress is reduced, water is reabsorbed in the NP tissue to reduce osmolarity; thus, the osmolarity in the NP tissue is dynamically changing [24,25].
Aging is one of the factors of nucleus pulposus cell damage. The P16 INK4a /Rb signaling pathway is an important cellular senescence pathway and also an important pathway of cell proliferation. During normal cell proliferation, Cyclin D1 forms a complex with CDK4/6 to phosphorylate the Rb protein, and the phosphorylated Rb protein is dissociated from the transcription factor E2F, which promotes cell transition from G1 to S phase and enhances cell proliferation [26]. The P16 INK4A protein is an inhibitor of CDK4/6, which can compete with Cyclin D1 to bind CDK4/6, inhibit CDK4/ 6-mediated phosphorylation of Rb protein, prevent cells from entering the S phase from the G1 phase, and inhibit cell proliferation [26,27]. Messmer et al. [28] found that an increase in tear osmotic pressure is the main cause of ocular inflammation and corneal stem cell apoptosis and aging. Many studies have shown that as cells age, the level of P16 INK4A increases [29][30][31].
Studies have determined that the osmolarity of NP tissue is 430-496 mOsm/kg H 2 O [32,33], which is much higher than the normal osmotic pressure level of the human body. As an important factor for survival in the intervertebral disc tissue, osmolarity has a major effect on the activity of NPMSCs. Current research on the effect of osmotic pressure on NPMSCs mostly focuses on the intervertebral discs of cattle, mice, dogs, etc. In this study, for the first time, human normal NPMSCs were used to research the effect of changes in osmolarity on their biological activities and to explore the role of osmolarity in IVDD.
Materials and Methods
2.1. Cell Isolation and Culture. All experiments in this study were approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University. In the experiment, intervertebral disc samples (n = 6, males = 3 and females = 3, aged 18-40 years, disc level T12-L3, and Grade I or II) were collected from patients undergoing lumbar fracture surgery at the First Affiliated Hospital of Anhui Medical University. According to the Pfirrmann disc degeneration grading system, the patients with disc degeneration were classified as Grade I or Grade II. We refer to relevant references to isolate and expand NPMSC cells [19,[34][35][36]. Under sterile conditions, the NP (nucleus pulposus) is separated from the AF (annulus fibrosus), making sure there is no any visible contamination by the AF or any other tissue. Then, scissors were used to cut the NP tissue into pieces, and the tissue was placed in a cell incubator and digested with 0.2 mg/ml collagenase II (Gibco, USA) for 4 to 6 hours. The cells were centrifuged, and the supernatant was discarded. The partially digested tissue, along with liberated cells, was cultured in the MSC expansion medium, which consisted of Dulbecco's modified Eagle's medium-low glucose (HyClone, USA), 10% fetal calf serum (Gibco, USA), and 1% penicillin/streptomycin (Gibco, USA) at 37°C in 5% CO 2 . After 24-48 hours, the cells began to adhere to the bottle and underwent the first fluid change, followed by a change of medium every 3 days. When the cell confluence reached approximately 90%, the cells were subcultured at 1 : 3, and the third generation of cells was selected for the experiments.
2.2. Immunophenotype of NPMSCs. After trypsin (Biosharp, USA) digestion of NPMSCs, the cells were washed with phosphate-buffered saline (PBS, Sigma, USA) and centrifuged to generate a 100 μl cell suspension. CD105-PE, CD90-PE, CD73-PE, CD45-PE, CD34-PE, and HLA-DR-PE (eBioscience, USA) monoclonal antibodies were added to each tube. The samples were incubated in the dark for 30 min. After the cells were washed with PBS, they were resuspended in 400 μl of PBS, and then, flow cytometry (BD, USA) was used to detect the percentage of positive cells and the fluorescence intensity. An isotype control (eBioscience, USA) was used for each tube. assays were used to detect the proliferation of the NPMSCs cultured under different osmotic pressure conditions. NPMSCs were seeded in 96-well plates at a density of 5 × 10 3 cells/ml. These cells were cultured for 1, 3, 5, 7, 9, and 11 days in the four groups (1, 2, 3, and 4). The cells were incubated with 10 μl of CCK-8 reagent (Dojindo, Japan) at 37°C in the dark for 2.5 hours. No cells were added to the isotype group. A SpectraMax microplate reader was used to measure the absorbance of different groups at 450 nm.
2.6. Apoptosis Assay. The isolated cells were planted in a 6well plate. These cells were cultured in four groups (1, 2, 3, and 4) for 5 days in different osmotic media. The cells were digested with trypsin without EDTA (Biosharp, USA) and washed twice with PBS. The harvested cells were incubated with 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI; KeyGEN BioTECH, China) for 5 min at 37°C in the dark. Flow cytometry (BD, USA) was used to detect the percentage of apoptotic cells.
Cell Cycle
Analysis. PI (Beyotime, China) staining was used to analyze the cell cycle. Briefly, after NP cells were treated with medium with different osmolarities for 5 days, they were digested with 0.25% trypsin and fixed with cold ethanol overnight. The cells were washed with PBS to remove ethanol. The prepared 500 μl PI/RNase A staining working solution was added to the tube and incubated for 30 min in the dark at room temperature. After the cells were washed with PBS, the fractions of each phase of the cell cycle were detected by flow cytometry.
2.8. SA-β-Gal Staining Assay. The cells were seeded in a 6well plate and then cultured in the above four different osmotic pressure groups for 5 days. SA-β-Gal staining was performed according to the manufacturer's instructions (Beyotime, China). Then, the staining results were observed under an inverted microscope. Senescent cells were stained. The percentage of positive cells to the total number of cells was determined.
2.9. Real-Time PCR Analysis. The cells were cultured for 7 days with the above four types of media with different osmotic pressures. Then, a TRIzol reagent was used to extract total RNA. Total RNA was reverse transcribed into cDNA using a reverse transcription reagent (TaKaRa, Japan). GAPDH was used as an internal reference gene. A SYBR Premix Ex Taq PCR kit (TaKaRa, Japan) and LightCycler system (Roche, Switzerland) were used to analyze the obtained cDNA for real-time quantitative PCR (RT-qPCR). The primers used for RT-qPCR are listed in Table 1 3 Stem Cells International 2.12. Statistical Analysis. All data in this study are expressed as the mean ± standard error of three independent replicates. The significant difference between groups was analyzed by one-way analysis of variance (ANOVA) using SPSS 19.0 software. A P value < 0.05 was considered statistically significant.
Isolation and Characterization of NPMSCs.
After the primary cells were cultured for 4 to 5 days, the isolated and cultured cells adhered to the bottle. Under a microscope, the cells adhered to the bottle and had a spindle shape. The cells became confluent in approximately 20 days, and the speed of confluence increased after passaging. The cell morphology of the P2 and P3 generations remained uniform, and the cells had a spindle shape (Figure 1). Flow cytometric analysis showed that the cells had high expression of CD73, CD90, and CD105 and low expression of CD34, CD45, and HLA-DR ( Figure 2). After 21 days of osteogenic differentiation, mineralized nodules could be detected by Alizarin Red staining. After 28 days of adipogenic differentiation, Oil Red O staining showed that lipid vesicles were produced in the cells. After 28 days of chondrogenic differentiation, Alcian Blue staining showed the presence of sulfated proteoglycan in the cells (Figure 3).
NPMSCs Cultured under Different Osmotic Pressures.
We cultured NPMSCs in four groups under different osmotic pressures. After five days of culture, morphological changes in the cells were observed, and their morphology was recorded by photographs. As the osmotic pressure changed, the cell morphology also changed. Compared with the isoosmotic pressure group, the cells in the hypertonic group became larger, flatter, and irregular in shape. The morphology of the cells in the hypotonic group also showed the same change. (Figure 4).
Cell Proliferation of NPMSCs.
After the cells in the four groups (1, 2, 3, and 4) were grown for 11 days, the proliferation rate of group 1 was lower than that of group 2, and the cell proliferation rates of groups 3 and 4 decreased and were lower than that of group 2. The cell proliferation rate of group 4 was lower than that of group 3 ( Figure 5).
Apoptosis
Assay of NPMSCs. The cells were cultured in four groups, 1, 2, 3, and 4, for 5 days. The apoptosis rate of group 2 was the lowest. The apoptotic rates of groups 1, 3, and 4 were significantly higher than that of group 2. The apoptosis rate of group 4 was higher than that of group 3 ( Figure 6).
3.5. The Cell Cycle of NPMSCs. We cultured the above 4 groups of cells for 5 days. The results showed that compared with the isotonic pressure group, the hypotonic group and the hypertonic group showed inhibition of the cell cycle, and the higher the osmotic pressure, the more obvious the inhibitory effect on the cells was (Figure 7).
3.6. SA-β-Gal Activity of NPMSCs. The SA-β-Gal activity of the hypotonic group and the hypertonic group was significantly higher than that of the isotonic group. Both hypotonic and hypertonic conditions can promote cell senescence. The greater the osmotic pressure, the more significant the enhancement of cell senescence was (Figure 8).
3.7. Gene Expression of NPMSCs. NPMSCs were cultured under four different osmotic pressures for 7 days. Compared with that in group 2, the expression of stemness genes (Nanog, Notch1, Jag1, and OCT4) and functional genes (Collagen II and Aggrecan) was downregulated in group 1, group 3, and group 4, and the expression in group 4 showed greater downregulation than that in group 3. The expression Stem Cells International of the P16 INK4A and Rb genes was also upregulated, and group 4 showed greater upregulation than group 3 ( Figure 9).
Protein Expression of NPMSCs.
NPMSCs were cultured in groups 1, 2, 3, and 4 for 7 days, and Western blotting results showed that compared with that in group 2, the expression of Collagen II and Aggrecan in group 1, group 3, and group 4 was downregulated, and group 4 showed greater downregulation than group 3. The expression of P16 INK4A and Rb was also upregulated, and group 4 showed greater upregulation than group 3 ( Figure 10).
3.9. The Effect of P16 INK4A Knockdown on the NPMSC Activity. After knockdown of P16 INK4A by siRNA transfection, the effects were verified by PCR and Western blots, which showed that the expression of P16 INK4A was significantly lower than that of the control group ( Figure 11). In this study, we found that group 4 had the greatest effect on the biological activity of the NPMSCs, and then, we selected the osmotic pressure of 600 mOsm/kg H 2 O for negative control analysis ( Figure 12). After knocking down P16 INK4A , we continued to culture the cells under the above 4 osmotic pressure conditions and then tested them. The mRNA and protein levels of Collagen II, Aggrecan, and Rb were detected, and the results showed no significant differences among the groups (Figure 13).
Discussion
The intervertebral disc is a tissue with a special structure. Its biological environment is relatively closed, and blood vessels that directly supply nutrients are lacking, which results in this specific structure. As the degree of IVDD increases, the microenvironment of the intervertebral disc changes, and the osmotic pressure also changes [14]. Moreover, various movements and loads of the spine lead to changes in the osmotic pressure in the NP tissue. A study by Urban [37] showed that the osmotic pressure of NP tissue is between 450 and 550 mOsm/kg H 2 O in a day. However, the exact mechanism of the effect of osmotic pressure changes on NPMSCs is still unclear. This study simulated different osmotic pressure environments to culture human normal NPMSCs from intervertebral discs to study the effect of osmotic pressure on the biological activity of these cells.
Risbud et al. [34], Blanco et al. [19], and Tekari et al. [35] isolated MSC-like progenitor cells from nucleus pulposus by enzymatic digestion, and they fulfill nearly all morphological, immunophenotypic, and differentiation criteria described by the International Association for Stem Cell Therapy. The NP tissue selected in this study was derived from patients with lumbar vertebral fractures and was from normal intervertebral discs (Pfirrmann I or II). We isolated and cultured cells in vitro and found that the cells adhered to the bottle and grew in a radial spiral shape, with high expression of CD73, CD90, and CD105; low expression of HLA-DR, CD34, and CD45; and differentiation towards osteogenic, adipogenic, and chondrogenic lineages. We isolated cells in the same way as Risbud [38]; the isolated and cultured cells were NPMSCs.
Ishihara et al. [39] cultivated NP tissue in vitro and found that increased osmotic pressure could enhance the synthesis of proteoglycans, and the increase in proteoglycan synthesis was related to the osmotic pressure of the culture medium but unrelated to Na + or Cl -. Neidlinger-Wilke et al. [40] increased the osmotic pressure of the medium in bovine NP cells from 300 mOsm/kg H 2 O to 500 mOsm/kg H 2 O, and the proteoglycan expression increased. Spillekom et al. [41] found that the synthesis of proteoglycan in dog NP cells was optimal when the osmotic pressure in the culture medium was 400 mOsm/kg H 2 O. Studies have also found that high osmotic pressure can inhibit the proliferation of NP cells and the synthesis of extracellular matrix Figure 8: Four groups of cell senescence staining cultured at osmotic pressure concentration. Each value is the mean ± SEM; * P < 0:05.
Stem Cells International
and promote the apoptosis of NPMSCs [42][43][44]. The above findings on osmotic pressure on the biological activity of NP cells are different, which may be caused by the method of cell culture, the difference in cell species, and the different exposure conditions. In this study, we simulated different osmotic pressure environments in vitro and cultured NPMSC cells under different osmotic pressure conditions. Experimental results showed that compared with normal osmotic pressure, hypotonic and hypertonic environments inhibited the proliferation of NPMSCs. The higher the osmotic pressure, the more obvious the inhibition of NPMSC proliferation was. High osmotic pressure disrupts the cell cycle, can arrest cells in the G1 phase, and inhibits the cell cycle [45][46][47]. These findings are similar to our research results. We used flow cytometry to detect the cell cycle of the NPMSCs cul-tured under different osmotic pressures and found that compared with the normal osmotic pressure group, the high osmotic pressure group showed arrest of cells in the G1 phase. The higher the osmotic pressure, the stronger the inhibitory effect is. Research shows that not only high osmotic pressure but also low osmotic pressure can impair the cell cycle progression of NPMSCs. Changes in osmotic pressure also affect the expression of extracellular matrix [45][46][47]. The results showed that when the osmotic pressure was decreased or increased, the expression of related genes (Nanog, Nocth1, Jag1, and OCT4) in NPMSCs decreased and the expression of Collagen II and Aggrecan was also reduced compared to that in the normal osmotic pressure group. These results suggested that abnormal changes in osmotic pressure will cause dysfunction of NPMSCs, which will lead to IVDD. Figure 9: (a, b) The expression levels of related genes in four groups cultured under osmotic pressure. Each value is the mean ± SEM; * P < 0:05.
Stem Cells International
The P16 INK4A /Rb signaling pathway plays an important role in cell senescence, and P16 INK4A can cause permanent cell growth arrest and cell senescence [48]. This study also confirmed these findings. We observed the morphological changes of cells cultured in vitro under different osmotic pressures by microscope and found that the cells in the hypotonic and hypertonic groups became larger and flatter, and the cell bodies were irregular in shape, which was consistent with the morphological changes of senescent cells [49,50]. Research shows that compared with the normal osmotic pressure group, the hypotonic and hypertonic groups showed an increased proportion of cell senescence, as demonstrated by galactosidase staining; the expression of the P16 INK4A Figure 11: The results of three-sequence screening; PCR (a) and WB (b) showed that P16 INK4A -676 was effective for transfection. Each value is the mean ± SEM; * P < 0:05. Cell aging was inhibited, and the expression of extracellular matrix components did not decrease. Because of the time limit of siRNA transfection, we did not conduct long-term
12
Stem Cells International cell culture on the knockdown cells and did not perform more in-depth research on cell differentiation and proliferation. We used the fetal bovine serum instead of human serum to culture the human NPMSCs. Differences in serum species may affect the test results, but human serum samples are limited. Second, the study was conducted in a 2D culture medium, and the response of NPMSCs in a 3D culture system may be different. Third, the research on the silencing of the P16 INK4A gene is mainly carried out in monolayercultured NPMSCs. Dedifferentiation and short-term effects are inevitable. Although these effects were biologically important, they may not be relevant to in vivo situations. The long-term effect in the body is very important. Although the exact mechanism of P16 INK4A has yet to be determined, knocking down P16 INK4A in NP cells may have some beneficial biological functions.
Conclusion
In summary, the results showed that changes in osmotic pressure have an important effect on the biological activity of NPMSCs; hypotonic or hypertonic can induce cell apoptosis and promote cell senescence. Studies have shown that this effect may occur through the P16 INK4A /Rb pathway and affect the regeneration and restore functions of NPMSCs. By knocking down the P16 INK4A gene, the adverse effects of changes in osmotic pressure on cells can be reversed. This study provides a useful theoretical basis for the treatment of IVDD in the future.
Data Availability
Data are available on request. | 2022-04-25T15:06:35.208Z | 2022-04-23T00:00:00.000 | {
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237803409 | pes2o/s2orc | v3-fos-license | Pelletizing Analysis on MgO-Fluxed Pellets by DEM
: The Discrete Element Method (DEM) was used to analyze the pelletization process of MgO-fluxed pellets. The effects of the charge ratio and rotational speed of the disc pelletizer on the behavior of MgO-fluxed pellets were investigated by using a simulation. The simulation results show that under the condition of a certain tilt angle of the disc pelletizer (the tilt angle is 49 ◦ ), the suitable parameters of the disc pelletizer are that the charge ratio is 20% and the rotational speedis 0.7 N/NC. This simulation model proposed will be useful to research pellets behavior and for the design of disc pelletizers.
Introduction
Traditional acid pellets, as a kind of burden of blast furnace, have many advantages, such as high ambient temperature compressive strength, uniform size, and high iron grade. The higher the pellet ratio in burden of blast furnace is, the higher the production is. Moreover, the pellet production process also has the characteristics of low energy consumption and low pollution. Therefore, the consumption of acid pellets has been gradually increasing. Nevertheless, compared with sinters, the acid pellets have some deficiencies, such as lower softening and melting temperature, wider softening drop temperature range, poorer permeability of cohesive zone, and higher reduction swelling [1]. The proportion of pellets in BFs in China is approximately 10~25% [2,3]. In order to increase the proportion of pellets in blast furnace burden and improve the metallurgical properties of pellets at high temperature, fluxed pellets have recently been widely developed, especially MgO-fluxed pellets, which have better high-temperature metallurgical properties, such as reduction swelling properties, softening-melting properties, and a high reduction disintegration index. Additionally, it can meet the requirement of MgO content in BF slag. MgO-fluxed pellets can make the blast furnaces have a more reasonable cohesive zone, improve permeability and ensure the reasonable distribution of gas. Moreover, the blast furnaces are stable and run smoothly so that the production of them increases and coke ratio decreases [4][5][6][7][8][9][10][11].
The metallurgical properties of MgO-fluxed pellets have been studied in many literatures. However, there are few studies on the influence of production technical parameters, such as rotational speed and charge ratio of the disc pelletizer, on the pelletizing performance of raw materials. In the current study, efforts have been made to understand the effects of varying the rotational speed and charge ratio of the disc pelletizer on the pelletizing performance of MgO-fluxed pellets raw materials. As we all know, the production process of green pellets can be divided into three stages: the nucleation stage, growing stage, and compact stage. The effect of moisture is mainly reflected in the first two stages. After the formation of green pellets, the improvement of green pellets' strength mainly depends on the mechanical rolling force. This study mainly focused on the effects of the charge ratio and rotational speed of the disc pelletizer on the final green pellets' two stages. After the formation of green pellets, the improvement of green pellets' strength mainly depends on the mechanical rolling force. This study mainly focused on the effects of the charge ratio and rotational speed of the disc pelletizer on the final green pellets' performance, so the effect of moisture on the pelletizing process was ignored. There is no considerable number of papers that report on the effect of production technical parameters for MgO-fluxed pellets. The results can provide a theoretical basis and technical guidance for industrial production of MgO-fluxed pellets.
Principle of the Discrete Element Method (DEM) and Simulation Conditions
The interaction forces in collision between two particles, between particles and boundary, are represented by the normal vibration model, tangential vibration model, and tangential sliding model, as shown in Figure 1 [12]. The assumptions in this simulation model are follows: (1) Pellet diameter is uniform.
(2) Pellet diameter and other parameters are fixed in the simulation.
(3) Effect of moisture is ignored.
Simulation conditions are shown in Tables 1 and 2. Pelletizing is processed in a simulation model with a disc pelletizer of 0.8 m in diameter and 0.2 m rim depth, with different rotational speeds and different charge ratios. According to the actual situation of pellet production in the Anyang Iron and Steel Group Co., Ltd, the disc inclination angle is set at 49° horizontally in this study. N is the rotational Pelletizing is processed in a simulation model with a disc pelletizer of 0.8 m in diameter and 0.2 m rim depth, with different rotational speeds and different charge ratios. According to the actual situation of pellet production in the Anyang Iron and Steel Group Co., Ltd., the disc inclination angle is set at 49 • horizontally in this study. N is the rotational speed of the disc pelletizer and Nc is the critical rotational speed given by following equation [13]: where, Nc and D are the critical rotational speed of the disc pelletizer and the diameter of the disc pelletizer, respectively, f is friction coefficient between raw material and disc pelletizer, K is a regulatory coefficient, α is lean angle of disc pelletizer, and Φ is a repose angle of raw material. In this simulation, the ratio of rotational speed to critical rotational speed (N/Nc) is 0.5, 0.6, 0.7, 0.8, and 0.9, respectively, and the charge ratio, C, is 10%, 15%, 20%, and 25% respectively. speed of the disc pelletizer and Nc is the critical rotational speed given by following equation [13]:
Simulation Results and Discussion
where, Nc and D are the critical rotational speed of the disc pelletizer and the diameter of the disc pelletizer, respectively, f is friction coefficient between raw material and disc pelletizer, K is a regulatory coefficient, α is lean angle of disc pelletizer, and Ф is a repose angle of raw material. In this simulation, the ratio of rotational speed to critical rotational speed (N/Nc) is 0.5, 0.6, 0.7, 0.8, and 0.9, respectively, and the charge ratio, C, is 10%, 15%, 20%, and 25% respectively. When N/Nc is 0.5 or 0.6, the centrifugal force is low due to the low rotational speed, so the pellets only slide in the disc rather than roll. Moreover, the location where the pellets are lifted is low, so the pelletizing process would not take place. When N/Nc is 0.8 or 0.9, the pellets falls more far from the top of the disc to the bottom. The increase of height and the higher rotational speed give a high velocity to the pellets when the pellets move violently; furthermore, they are impacted from other pellets or disc wall, and they would be broken into pieces. Thus, the pelletizing effect is poor. When N/Nc is 0.7, pellets can roll. Seed balls are rotating in fine iron ore powders; the fine powders should adhere to seed balls, and the seed balls then grow into pellets. Figure 3 shows that the motion state of pellets simulated by DEM in the disco pelletizer. At 0.7 N/Nc, the motion state under different charging ratio is 1.2 s. When N/Nc is 0.5 or 0.6, the centrifugal force is low due to the low rotational speed, so the pellets only slide in the disc rather than roll. Moreover, the location where the pellets are lifted is low, so the pelletizing process would not take place. When N/Nc is 0.8 or 0.9, the pellets falls more far from the top of the disc to the bottom. The increase of height and the higher rotational speed give a high velocity to the pellets when the pellets move violently; furthermore, they are impacted from other pellets or disc wall, and they would be broken into pieces. Thus, the pelletizing effect is poor. When N/Nc is 0.7, pellets can roll. Seed balls are rotating in fine iron ore powders; the fine powders should adhere to seed balls, and the seed balls then grow into pellets. Figure 3 shows that the motion state of pellets simulated by DEM in the disco pelletizer. At 0.7 N/Nc, the motion state under different charging ratio is 1.2 s. It can be clearly seen from Figure 3 that when the charge ratio is 10% or 15%, due to the small number of pellets and a small area occupied by pellets in the disc, the utilization rate of the disc is not high, which reduces the efficiency of pelletizing. Moreover, due to the large space between the pellets, all the pellets are too loose, which increases the collision between the pellets, which in turn leads to the fragmentation of the pellets; this is not conducive to the pelletizing process. When the charge ratio is 25%, the pellets near the disc wall move with the disc and do not fall when they reach the highest point. When the other pellets fall down, the phenomenon of multi-layer non-uniform rolling occurs, which is harmful to pelletization. Because the number of pellets is too high and the total kinetic energy is too large, the pellets falling later may crush the pellets falling earlier, which is not conducive to the pelletizing process. When the charge ratio is 20%, the pellets have the best trajectory; that is to say, the seed balls can grow into pellets.
Simulation Results and Discussion
In order to investigate the behavior of pellets, the kinetic and rotational kinetic energies of pellets are calculated by using DEM, as shown in Figure 4. The kinetic energy, Ek, and rotational kinetic energy, Er, are given by the following equations, respectively: where m and v are mass and velocity of pellets, respectively, and I and ω are the moment of inertia and rotational velocity, respectively. It can be clearly seen from Figure 3 that when the charge ratio is 10% or 15%, due to the small number of pellets and a small area occupied by pellets in the disc, the utilization rate of the disc is not high, which reduces the efficiency of pelletizing. Moreover, due to the large space between the pellets, all the pellets are too loose, which increases the collision between the pellets, which in turn leads to the fragmentation of the pellets; this is not conducive to the pelletizing process. When the charge ratio is 25%, the pellets near the disc wall move with the disc and do not fall when they reach the highest point. When the other pellets fall down, the phenomenon of multi-layer non-uniform rolling occurs, which is harmful to pelletization. Because the number of pellets is too high and the total kinetic energy is too large, the pellets falling later may crush the pellets falling earlier, which is not conducive to the pelletizing process. When the charge ratio is 20%, the pellets have the best trajectory; that is to say, the seed balls can grow into pellets.
In order to investigate the behavior of pellets, the kinetic and rotational kinetic energies of pellets are calculated by using DEM, as shown in Figure 4. The kinetic energy, E k , and rotational kinetic energy, E r , are given by the following equations, respectively: where m and v are mass and velocity of pellets, respectively, and I and ω are the moment of inertia and rotational velocity, respectively. From Figure 4, it can be seen that the kinetic energy increases with the increase of the disc rotational speed and the charge ratio. Although the rotational kinetic energy increases with the increase of the disc rotational speed when the charge ratio is too high, such as up to 25%, the rotational kinetic energy actually decreases with the increase of the charge ratio. As the charge ratio increases, it is difficult for each pellet to rotate itself due to being subjected to forces from all directions. This is the reason why the rotational kinetic energy decreases when the charge ratio is too high. Therefore, the proper charge ratio subjects the pellets to the best rotational kinetic energy, which is good for the growth of the pellets. From Figure 4, it can be seen that the kinetic energy increases with the increase of the disc rotational speed and the charge ratio. Although the rotational kinetic energy increases with the increase of the disc rotational speed when the charge ratio is too high, such as up to 25%, the rotational kinetic energy actually decreases with the increase of the charge ratio. As the charge ratio increases, it is difficult for each pellet to rotate itself due to being subjected to forces from all directions. This is the reason why the rotational kinetic energy decreases when the charge ratio is too high. Therefore, the proper charge ratio subjects the pellets to the best rotational kinetic energy, which is good for the growth of the pellets.
Conclusions
(1) The pelletizing process is not only related to the kinetic energy of the pellets, but also to the rotational kinetic energy. When the charge ratio is 20%, the pellet has the best rotational kinetic energy, which is beneficial for pelletization. | 2021-09-28T01:08:51.248Z | 2021-07-15T00:00:00.000 | {
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17481850 | pes2o/s2orc | v3-fos-license | Mentalization-based treatment for psychotic disorder: protocol of a randomized controlled trial
Background Many patients with a non-affective psychotic disorder suffer from impairments in social functioning and social cognition. To target these impairments, mentalization-based treatment for psychotic disorder, a psychodynamic treatment rooted in attachment theory, has been developed. It is expected to improve social cognition, and thereby to improve social functioning. The treatment is further expected to increase quality of life and the awareness of having a mental disorder, and to reduce substance abuse, social stress reactivity, positive symptoms, negative, anxious and depressive symptoms. Methods/design The study is a rater-blinded randomized controlled trial. Patients are offered 18 months of therapy and are randomly allocated to mentalization-based treatment for psychotic disorders or treatment as usual. Patients are recruited from outpatient departments of the Rivierduinen mental health institute, the Netherlands, and are aged 18 to 55 years and have been diagnosed with a non-affective psychotic disorder. Social functioning, the primary outcome variable, is measured with the social functioning scale. The administration of all tests and questionnaires takes approximately 22 hours. Mentalization-based treatment for psychotic disorders adds a total of 60 hours of group therapy and 15 hours of individual therapy to treatment as usual. No known health risks are involved in the study, though it is known that group dynamics can have adverse effects on a psychiatric disorder. Discussion If Mentalization-based treatment for psychotic disorders proves to be effective, it could be a useful addition to treatment. Trial registration Dutch Trial Register. NTR4747. Trial registration date 08-19-2014.
Background and rationale
Non-affective psychotic disorders (NAPD) like schizophrenia are accountable for a substantial part of the total burden of disease, constituting the fifth and sixth leading cause of disability in the world, for men and women respectively [1]. A major contributor to this high level of disability is thought to be the decline in social functioning associated with NAPD. Patients with NAPD experience difficulty communicating [2], and tend to have poor social problem-solving skills [3]. These social deficits are predictive of poor vocational outcome [4] and poor quality of life [5]. It is surprising, therefore, that few treatments have been developed to effectively target them.
Social cognitiondefined as "the ability to construct representations of the relation between oneself and others, and to use those representations flexibly to guide social behavior" [6] has been identified as one of the strongest predictors of social functioning in patients with NAPD [7]. Examples of social cognitive impairments in NAPD include difficulties recognizing emotions [8], empathizing [9] taking another person's perspective [10] and understanding social hints [11]. In recent years just a few treatments, mostly rooted in cognitive/behavioral theory [12][13][14], have been developed that target social cognition. Results, are preliminary, but do suggest that impaired social cognitive deficits are targetable by psychosocial interventions [13][14][15].
Mentalization-based treatment for psychotic disorder (MBT-P) is based on a manualized psychodynamic treatment for Borderline Personality Disorder (BPD) [16]. Recent prospective studies suggest that psychodynamic therapy may improve global functioning in NAPD [17,18], although randomized controlled trials should be conducted to substantiate this claim. Also, metacognitive psychotherapy [19] which is closely related to MBT [20] holds promise as a treatment for patients with schizophrenia [21]. MBT-P was developed to specifically improve social functioning by targeting the social cognitive process called 'mentalizing'. Fonagy and colleagues [22] describe mentalizing as "the process by which we implicitly and explicitly interpret the actions of ourselves and others as meaningful on the basis of intentional mental states". MBT adheres to a few important principles: (i) The therapist focuses on the current mental state of the patient to practice making representations of internal states; (ii) the therapist focuses on the present as opposed to the past; (iii) the therapist avoids talking about mental states that are not linked to subjectively felt reality; (iv) the therapist avoids talking about complex mental states; (v) the therapist focuses on recovering mentalizing, not creating insight [23].
This approach was found to reduce symptoms and interpersonal distress, and improve social functioning in patients with BPD [24]. Although BPD and NAPD may seem qualitatively different disorders, early views assumed borderline psychopathology occupied an area between neurosis and psychosis (e.g. Kernberg [25]) and could involve transient psychotic episodes. Since then, evidence has substantiated psychosis proneness in BPD. Psychotic symptoms, including hallucinatory experiences and delusions, occur regularly in patients with BPD, often persist over time, and are for a large part already present in early childhood [26]. In a recent study that included patients with either BPD or Schizophrenia, 17 % of participants met the criteria for both disorders [27]. Additionally, some NAPD and BPD patients share a tendency to excessively attribute incorrect intentions to others, or to "hypermentalize" [28][29][30]. Furthermore, disturbances in self-awareness and self-representation have been suggested to play an important role in both disorders [31]. Lastly, childhood trauma has been established as an important factor in the origins of both disorders [32]. Thus, as has been suggested earlier [20,33], MBT may be a similarly suitable treatment for NAPD.
Primary research aim
The primary aim of this study is to establish whether mentalization-based treatment for psychotic disorder (MBT-P) improves self-reported social functioning in patients with NAPD. We hypothesize that patients who receive MBT-P will show greater improvements in social functioning compared to patients who have had treatment as usual (TAU) only. We also expect that any difference observed will still be present at a 6 month follow-up.
Secondary research aims
In addition to the self-reported level of social functioning, we will also examine global functioning as rated by researchers. Other outcome measures were chosen with previous research regarding MBT in mind. According to Fonagy and Bateman [34], MBT's mechanism of change is improving patients' mentalizing capacities. We therefore aim to establish whether MBT-P indeed increases mentalizing capacity, measuring several dimensions of social cognition, and whether this increase mediates a potential treatment effect. Furthermore, in earlier studies, Bateman and Fonagy [24] reported a reduction of anxious and depressive symptoms and of substance abuse. We expect similar results regarding NAPD patients. Given the strong relation between social functioning and quality of life, we assume that patients receiving MBT-P will report a higher quality of life. Additionally, based on previous research [35], we predict that improvement of social cognitive capacity will also lead to an increased awareness of having a mental disorder. Furthermore, as Bateman and Fonagy describe [34] that MBT was designed to improve emotion regulation in situations of attachment related (i.e., social) stress. Based on this, we assert that patients will have less aversive emotional reactions to situations of social stress as a result of MBT-P. If MBT-P can reduce patients' emotional reactivity to social stress, they may also become less prone to develop positive psychotic symptoms, as social stress reactivity may be an affective pathway to psychosis [36]. Lastly, because social functioning and mentalizing ability have been found to be strongly related to negative symptoms, we will examine whether MBT-P reduces negative symptoms [37][38][39].
Covariates
Certain potential effect modifiers will be taken into account. First, adverse childhood experiences such as neglect or physical, psychological, and sexual abuse have been associated with social dysfunction later in life [40]. Second, personality organization (PO) has been shown to impact psychotherapy treatment response [41] and the level of social cognition [42]. Five levels of personality organization (PO) are identified, each characterized by different levels of anxiety tolerance and different capacities for impulse inhibition or 'control': Neurotic PO (good anxiety tolerance, over-control); Borderline PO (moderate to poor anxiety tolerance, differing levels of control); Narcissistic Borderline PO (good anxiety tolerance, undercontrol); Latent Psychotic PO (moderate anxiety tolerance, weak control); and Manifest Psychotic PO (poor anxiety tolerance, weak control). Third, the awareness of bodily sensations has been regarded as the first level of emotional awareness [43], which is an essential part of social cognition [44]. Psychopathology usually is accompanied by (vague) physical complaints, called "somatization". A failure to report physical complaints while suffering (severe) psychopathology may be indicative of an absence of bodily awareness and therefore an impaired social cognitive capacity. Fourth, medication use can affect social functioning in patients with NAPD, therefore the type of medication is registered and adherence to medication is measured. Fifth, MBT attendance is taken into account, because it is expected that those who attend more sessions will profit more. Sixth, baseline measurements of the outcome variables will be accounted for. Seventh, the duration of illness, because of its negative impact on functioning; and eighth, adherence to the MBT model by therapists, because it is likely to influence treatment outcome.
Trial design/setting
This study is a rater-blinded, randomized controlled trial. Patients referred to outpatient sites of the Rivierduinen mental health institute are randomly assigned to Treatment as Usual (TAU) plus MBT-P or TAU only. The Rivierduinen mental health institute provides in-and outpatient treatment to thousands of patients with psychiatric disorders in the Dutch province of South-Holland (e.g., Leiden, Gouda). The investigator and patients are aware of treatment allocation, but all measurements are performed by researchers blind to treatment allocation. Social functioning at baseline (t0) and after treatment (t2) will be compared for both treatment conditions. The patients are blind to this primary aim.
Inclusion criteria are: -At least 6 months of prior treatment.
-No more than 10 years of treatment for NAPD.
-Between 18 and 55 years of age.
Exclusion criteria are: -Intellectual disability and/or illiteracy.
-A lack of mastery of the Dutch language.
-Substance abuse to such an extent that it necessitates inpatient detoxification. After detoxification the patient is still eligible for participation in the study. Patients cannot participate in a session while under the influence of drugs.
Sample size calculation
The estimated effect size of the current study is based on two previous studies. A study examining the effect of MBT on BPD [35] [36]. Based on these results, we expect to find a moderate to large effect of MBT-P on social cognition (i.e., a Cohen's d of at least 0.7).
To calculate the required sample size, G*Power [37] was used. To obtain a significant difference with power equal to .80 with an independent samples t-test (for a true effect size of .7 and alpha = .05), 68 participants are needed. However, because repeated measurements (baseline and post-treatment) increase the powerdepending on the test-retest reliability of the outcome measurea smaller sample size is required. A formula has been devised [38] to account for the increased power when using multiple measurements: n repeated measures = (1-ρ 2 )*n t-test ; where ρ is the test-retest reliability of the scale used. Previous research has shown that the test-retest reliability of the social functioning scale after two and a half years is ρ = .40 [39]. Thus we need: (1-0.4 2 )*68 ≈ 58 participants to have 80 % power to find a significant difference.
It is difficult to predict the amount of drop-out in the study. In an unpublished pilot study, conducted at the Rivierduinen mental health institute, the drop-out rate of MBT-P within a period of 1 year was 10 %. Since the current RCT combines treatment and measurements, we estimate that the drop-out rate will be higher. Therefore, to be sure, we will recruit at least 40 (TAU) + 40 (TAU plus MBT-P) = 80 individuals. Even if 25 % of those initial patients (i.e., 20) drop out, there will be 60 patients left to detect an effect. Because patients in the MBT-P condition receive therapy in groups of up to eight persons, five groups will be formed in order to recruit 40 patients in the MBT-P condition.
Procedure
Psychiatrists, psychologists, or psychiatric nurses will check their caseload for patients who meet the requirements for participation and ascertain whether they are interested in participating. If this is the case, the researcher will provide an information letter. The patient will be given 7 days to read the letter and to decide whether he/she wants to participate. At a second appointment, the researcher will check whether the patient has understood the information in the letter. Subsequently, both parties sign an informed consent form in twofold. Randomization is performed by an independent external agency.
Measurements and instruments
There are four moments of assessment over the course of 2 years. The baseline assessment takes place before MBT-P is started. Furthermore, there will be an assessment after 9 months (halfway MBT-P), after 18 months (directly after MBT-P has ended), and after 24 months (6 months after MBT-P has ended). See Table 1 for the specific instruments that are used at each assessment.
Diagnosis
All patients are diagnosed according to DSM-IV criteria [45] by a psychiatrist. Prior to participation, this diagnosis will also be verified using the Comprehensive Assessment of Symptoms and History (CASH). The CASH is a semistructured interview that documents signs, symptoms, and history of psychotic, manic and depressive syndromes as well as substance abuse. The instrument has been extensively tested concerning interrater reliability, test-retest reliability and validity [46].
Social functioning
Social functioning is measured using the Social Functioning Scale (SFS), a self-report questionnaire. The SFS has been found to be reliable, valid, sensitive, and responsive to change [47]. The scale contains seven dimensions of global social functioning that are especially pertinent to patients suffering from psychotic disorders. The dimensions are: social withdrawal, interpersonal communication, independence (competence), independence (performance), recreational activities, social activities, and employment.
Secondly, using the modified GAF scale [48], global functioning will be assessed. The modified GAF scale is a clinician or researcher-rated instrument, which makes it a good addition to the self-reported SFS. It has more detailed criteria and a more structured scoring system than the original GAF, which is underscored by a high interrater reliability.
Social cognitive capacity
It has been pointed out that there is to date no agreement on the assessment of social cognition, but there is a broad consensus that it is a multifaceted construct [49]. In the current study, social cognition is therefore assessed with two instruments that measure different aspects of social cognition: the Thematic Apperception Test (TAT) and the hinting task (HT). The TAT [50], scored with the Social Cognition and Object Relations System (SCORS) [51], is used to assess four dimensions of social cognition: complexity of representations of people and understanding of social causality, which comprise cognitive aspects of social cognition, and the affect-tone of relationships and the capacity for emotional investment, comprising affective aspects of social cognition. Each dimension is scored on a 5-point scale, with higher scores representing higher social cognitive functioning in that dimension. Six pictures of the TAT are used. TAT responses are recorded and transcribed verbatim. TAT responses, when analyzed with the SCORS, have been found to be a valid and reliable way to measure social cognition and object relations [52,53]. According to Luyten and colleagues [54], the TAT is one of the few tests that takes almost all aspects of mentalization into account, including affective and cognitive aspects.
The HT [37] is used to measure the ability to infer intentions from others, or 'Theory of Mind' (ToM). Patients read extracts that describe an interaction between two characters in which one character says something with an implicit message. If the patient infers the implicit message correctly, two points are scored. If a hint is needed, a score of 1 is given. When the answer is incorrect or the participant does not know, 0 points are scored. The test comprise ten short passages. The Hinting Task has very recently been reviewed [55] regarding test-retest reliability, utility as a repeated measure, relation to functional outcome, and internal consistency and was one of two tests shown with strong psychometric properties across all evaluation criteria.
Social stress reactivity
Emotional reactivity to stress in social situations is measured with an electronic diary using the 'Experience Sampling Method' (ESM) [56]. ESM is a repeated selfassessment technique with great ecological validity. Participants carry around an ESM device, which facilitates the monitoring of daily life experiences and behavior. Ten times daily on five consecutive days, it generates an audible signal (beep) at unpredictable moments of the day which participants answer by using the touch screen of the device. Participants are asked whether they are alone or in company of others. Then, both the level of social stress and negative/positive affect are assessed. Social stress is measured with items such as: "I would rather be alone" and "I like the present company" (reverse coded). Negative affect is the averaged score of the mood items "anxious", "lonely", "insecure", "irritated", "down", "guilty", and "gloomy". Positive affect will be measured with the items "happy", "satisfied", "cheerful", "relaxed", and "enthusiastic". All items are scored on a 7-point Likert Scale.
Quality of life
Using the Manchester Short Assessment of quality of life (MANSA) [57], changes in overall quality of life are measured. The MANSA is a 16-item, 7-point Likertscale self-rating instrument.
Psychotic symptom severity
Interviewers use the Positive and Negative Syndrome Scale (PANSS) [58] to assess positive (subscale P), negative (subscale N), anxious (item G2), and depressive symptoms (item G4). The PANSS is a 30-item, 7-point Likert-scale rating instrument developed for the assessment of phenomena associated with schizophrenia. A Dutch version is used [59]. Additionally, the ESM diary is used to measure momentary psychotic experiences. Seven ESM items are used: 'I feel suspicious' , 'I am afraid of losing control' , 'I feel that others don't like me' , 'I feel that others want to hurt me' , 'My thoughts are influenced by other people' , 'I feel unreal' , and 'I hear voices'.
Awareness of having a mental disorder
The PANSS (item G12) is used to assess awareness of having a mental disorder.
Substance abuse
Patients are asked to report substance use on the ESM device at each beep using categorical questions. Patients will report whether they have used any substance since the last beep, including: (1) caffeine, (2) nicotine, (3) medication, (4) alcohol, (5) cannabis, (6) other drugs, or (7) none.
Personality organization/somatization of psychopathology
Assessment of personality organization and the tendency to somatize severe psychopathology is conducted using theory driven profiles of the Dutch short Form of the MMPI (DSFM) [60], an 83-item self-assessment questionnaire. The DSFM measures personality traits on 5 scales: Extraversion, Psychopathology, Shyness, Somatization, and Negativism. Using the theory driven profile approach to the DSFM [61][62][63][64][65], five levels of Personality Organization (PO) are distinguished: Neurotic PO, Borderline PO, Narcissistic Borderline PO, Latent Psychotic PO and Manifest Psychotic PO. To measure bodily awareness, the DSFM "Somatization" subscale (20 items) will be used. This subscale measures the amount and degree of experienced bodily symptoms, and hence, the ability to subjectively report, and be aware of bodily sensations. As described [65] affect regulation through somatization will be expressed as the relative position of scores on the subscale somatization to that on the severe psychopathology subscale.
Childhood trauma
The Childhood Experience of Care and Abuse (CECA) [66] is a semi-structured interview that aims to assess details and the time-sequence of traumatic childhood experiences. It assesses lack of care (neglect, antipathy), physical abuse, sexual abuse, and psychological abuse.
Adherence to drug treatment
Each patient's medical record is consulted to ascertain the pharmacotherapy prescribed at t0, t1, t2, and t3. Adherence to the prescribed medication is measured with the Medication Adherence Questionnaire (MAQ) [67].
Adherence to the MBT-model
Adherence to the MBT model by therapists is rated by an experienced MBT therapist according to the MBT adherence and competence scale [68], using footage of therapy sessions. Therapists are judged on 17 items that characterize proper MBT treatment.
Duration of illness
Assessed using the CASH, refers to the period since first psychosis.
General demographics
The general demographic questionnaire (GDQ) is a standard instrument used in all ESM studies conducted at Maastricht University. It documents treatment history, socio-economic status, educational level, and urbanicity of the place of residence.
Treatment TAU
All patients in the current study receive a multifaceted treatment based on the 'Functional Assertive Community Treatment' (FACT) model. FACT teams consist of psychiatric nurses, welfare workers, psychologists, and at least one psychiatrist. The intervention consists of pharmacotherapy, case-management, psychoeducation, and in some cases cognitive behavioral therapy (CBT). CBT interventions for psychosis usually take around 20 sessions and are based on the work of van der Gaag [69,70]. Whether or not patients have received CBT, and how many sessions, will be registered and taken into account.
Furthermore, all patients will receive supportivestructuring therapy. Sessions focus on problems patients may encounter in their social network, work, daily activities, or medication adherence. Patients meet with a mental health professional for an average of 30 min every 2 weeks, with a minimum of 1 meeting of 30 min per month, over 18 months. The total number of individual sessions is estimated to be around 30 (15 h of individual therapy). Adherence to treatment sessions is monitored by registering patients' presence in the sessions.
MBT-P
Patients in the TAU plus MBT-P condition receive the same treatment mentioned above in combination with individual and group MBT-P. The key elements of MBT, shortly described below, provided the basis for MBT-P [16].
Therapeutic stance
MBT is characterized by the 'not knowing stance' in which the therapist admits to not knowing what the patient experiences. By actively asking questions the therapist cultivates an attitude of sincere interest in the patient. This gives the patient the experience of being 'kept in mind' by someone, but also stimulates curiosity in the patient towards his own mental states.
Interventions
When applying interventions, it should always be kept in mind that arousal tends to diminish the capacity to mentalize. Four stages of intervention can be used in a step-wise manner, depending on the level of arousal. At the first level, interventions are aimed at downregulating arousal in the patient. These include empathic validation of the patient's feelings and complimenting good mentalizing. At the second stage the therapist asks for clarification of a situation or elaboration on the patient's feelings and thoughts. Often the therapist stops or rewinds the patient's narrative to investigate an aspect of the dialogue. This stage is about making implicit mentalizing explicit. Often details are investigated that seem to affect the patient, or should affect the patient, but do not. This then leads to the next stage, called 'mentalized affectivity' , which is the activity of reflecting on emotions, while simultaneously experiencing them. It is considered to be a crucial aspect of emotion regulation. The explicit mentalizing of a primary affective experience gives the patient the opportunity to express (or inhibit) emotions in a non-automatic manner. At the last stage, the relationship between therapist and patient or between patients is mentalized. In this stage, both patient and therapist reflect on and share their affective experience to become aware how their relationship is affecting them. Care should be taken applying this stage of intervention, as it requires a robust level of mentalizing.
Duration and dose
Compared to original MBT, the length of MBT-P, has remained unchanged: 18 months. However, the frequency and length of sessions has been reduced. In our experience, based on a pilot MBT-P intervention, NAPD is associated with more severe mentalizing deficits than BPD, as has also been suggested elsewhere [71]. Given, the danger of overwhelming NAPD patients with mentalizing interventions, group therapy is limited to weekly 1-hour sessions, while individual therapy takes place in biweekly half-hour sessions. We feel this approach is justified by the low drop-out rate of 10 % in the pilot intervention.
Psycho-education
MBT-P starts with two sessions of psycho-education, in which patients are told about the key aspects of MBT, including the meaning of mentalizing and its sensitivity to arousal.
Individual therapy
Individual therapy provides an opportunity for intensive practice in mentalizing. The focus is on establishing a secure relationship that acts as a safe base from which failures in mentalizing can be explored. Treatment goals on the basis of five problem areas are developed and routinely reviewed with the patient, including: commitment to treatment, psychiatric symptoms, social interaction/relationships, destructive behavior, and community functioning.
Group therapy
Since patients with NAPD tend to experience a great deal of stress in social situations, group therapy provides an opportunity to practice mentalizing in a stress-evoking setting. Group therapy can also generate a sense of belonging and attachment that help foster mentalizing. The group size is a maximum of eight patients and there will be one MBT-P therapist and one co-therapist present at each session.
Therapists
Group therapists are experienced and registered MBT therapists. Individual therapists are mental health professionals (psychologists, psychiatric nurses or psychiatrists) who receive training to become MBT-P therapists. Supervision is provided in weekly sessions of up to 1h, during which therapists reflect on their interventions and whether they are faithful to the MBT model.
Statistical analysis Main effect
The main purpose of the statistical analysis is to compare the overall effect of treatment condition on social functioning. For this, an ANCOVA will be used with treatment condition as between-subjects variable, posttreatment social functioning as dependent variable, and baseline social functioning as a covariate. Other potential covariates include childhood trauma, level of PO, somatization of psychopathology, medication use, attendance of MBT-P sessions, duration of illness and adherence to the MBT-model by therapists. Similar analyses will be conducted for the secondary outcome measures (social cognition, quality of life, awareness of having a mental disorder, anxious, depressive, negative and positive symptoms and substance abuse).
Since drop-out will undoubtedly result in missing data, the possibility of attrition-bias is a cause of concern [72]. For example, it is conceivable that those who fare the worst in therapy tend to drop out, thus creating a biased sample. Following the advice of Altman [73], the analyses will therefore be conducted on the basis of 'intention to treat' (ITT), meaning that they will include all patients who sign up, regardless of actual participation in the entire program. Missing data will be handled by means of multiple imputation (MI) [74]. In the current case missingness is most likely to be caused by participants dropping out of their respective treatment programs. Since certain variables have been found to influence drop-out rates in patients with NAPD, we cannot assume that the data are missing at random. A review [75] identified a lack of insight, poor social functioning, positive symptoms, young age, male gender, a history of drug abuse, and unemployment as key predictors of treatment program drop out. Thus in the current study, these variables will be used to predict missingness. Additionally, treatment condition will be used as a predictor as well, because the time investment differs between conditions. For each analysis, a total of 5 imputed datasets will be created using a fully conditional Markov chain Monte Carlo (MCMC) approach, which will be combined using standard procedures [76].
Mediation
We also aim to examine whether changes in various social cognitive dimensions mediate the potential increase in social functioning. In order to test this mediational model, we will carry out a multi-mediator analysis [77]. This allows for a parallel testing of the indirect effects of several social cognitive dimensions, namely: theory of mind, complexity of representations, affect-tone of relationships, capacity for emotional investment and understanding of social causality (Fig. 1).
Multilevel analysis
Social stress reactivity is measured by ESM. This method generates data with a multilevel structure because there are multiple measurements per day for 5 days at a time for each patient. Since observations within patients tend to be more similar than observations from different patients, they are not independent. This necessitates a different (i.e., multilevel) analysis than the one used for the other outcome measures. Similar to Myin-Germeys and colleagues [78], differences in social stress reactivity between groups will be analyzed using mixed-effects regression models with treatment condition, the amount of stress during social situations, and their interaction term as independent variables, and positive and negative affect as dependent variables. The model will include random intercepts and random slopes for the stress predictor at the patient level, which allows for differences in overall levels of positive and negative affect across patients and for differences in the strength of the relationship between stress and these outcomes.
Discussion
There is evidence that MBT improves, among others, social functioning and interpersonal distress in patients with BPD (e.g. [24,79]) and that these effects remain at follow-up [80]. Given the similarities in both origins and symptoms of BPD and NAPD, it has previously been suggested that MBT could be a useful treatment for NAPD [20,33]. This randomized controlled trial, will be the first to examine the effectiveness of MBT as an adjunct therapy for patients with NAPD. Furthermore, we aim to determine a possible mechanism of change by examining social cognitive capacity as a possible mediator. By measuring social cognitive capacity with two instruments, covering a total of five dimensions, we mean to do justice to its complexity. | 2018-04-03T04:13:51.161Z | 2016-06-08T00:00:00.000 | {
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209376407 | pes2o/s2orc | v3-fos-license | Holomorphic Chern-Simons theory and lambda models: PCM case
In this note we consider the symplectic reduction of a four-dimensional holomorphic Chern-Simons theory recently introduced in arXiv:1908.02289 for describing integrable field theories. We work out explicitly the case of the lambda deformed Principal Chiral Model (PCM) and show that the symplectic reduction works as a localization mechanism. The reduced Chern-Simons theory restricts to the set of poles of the twist function underlying the theory, where the known classical integrability of the lambda deformed PCM can be reconstructed from the phase space data associated to this set of points in the spectral space.
Introduction
Integrable deformations of string sigma models have attracted a great deal of attention in recent years. Some of the more prominent examples being the Yang-Baxter (or eta) deformations and the lambda deformations, introduced for the PCM in [3] and 1 [9], respectively. Both types of deformations were further extended to include other kinds of (super)-string backgrounds and formulations in a series of papers, see for instance [4][5][6][7][8] for the eta deformations and [10][11][12][13] for the lambda deformations. Each deformation have the characteristic of covering a different domain in the deformation parameter space, but are related via Poisson-Lie Tduality and analytic continuation, see [16,17]. They are mainly studied for offering a chance to understand the complicated quantum integrable structure of their parent sigma models more efficiently, but as the latter, both types of deformations also belong to the family of the so-called non-ultralocal integrable field theories, were quantization methods like the powerful algebraic Bethe ansatz does not perform well at all. A strategy for eliminating or by-passing this unwanted technical feature, is to to embed the theory into a higher dimensional quantum field theory where the non-ultralocality is absent or emerges under some circumstances. In this note, such a higher dimensional field theory will be the four-dimensional Holomorphic Chern-Simons (CS) theory recently introduced in [1] to formulate integrable field theories 2 .
There are at least three major characteristics present in any lambda model suggesting a relation with a gauge theory of the CS type: (i) The presence of two opposite level, mutually commuting Kac-Moody (KM) algebras [9][10][11][12][13], ii) The factorization (induced by integrability) of the Lagrangian field solution to the equations of motion (eom) in terms of the wave function Ψ [17,18], iii) The form of the Hamiltonian when expressed in terms of the components of the Lax connection L [22,23], The points z ± depending on the deformation parameter λ are poles of the twist function ϕ(z) of the theory. Indeed, (1.1) suggests it in a direct way because of KM algebras rise [19,20], after symplectic reduction (SR) of a Hamiltonian CS theory defined on a solid cylinder, as Poisson structures of a WZW model defined on its boundary. Equation (1.2) mimics the chiral factorization [21] of the solutions to the eom of an ordinary closed string WZW model and each term in (1.3) is identical to the boundary contribution to the canonical Hamiltonian of a CS theory defined on a solid cylinder, if the Lax connection is identified with two of the components of the three-dimensional CS gauge field.
In this note we will focus exclusively on the PCM and consider the problem of how to recover its lambda deformation from the SR of a Hamiltonian CS theory (leaving other models for future work). There are, at least, two possible answers to this question, each one depending fundamentally on the form of the integrand of the symplectic formΩ of the CS theory considered, which is proportional to the two-form with A being the CS gauge field restricted to the constant time manifold M in the decomposition R × M . The key observation being that the restriction of this two-form to the space of flat connections, taken to be of the form A = −dΨΨ −1 , is exact Now we briefly comment on each of the two possibilities for getting a non-trivial reduced symplectic form after integration of the result right above: (I) "Holography". By integrating (1.5) on the disc M = D, we obtain the usual result [19,20] By considering the addition of two CS actions of opposite levels defined on a solid cylinder, one for each pole z ± , it is possible to recover (1.1), (1.2), (1.3) and the lambda deformed PCM action functional as well. This more traditional approach is considered in [22,23], where all the results are presented. The major drawback of this bottom-up approach, is that it is not clear how to include the spectral parameter z in the double CS theory action functional from the very beginning and hence only works partially. In this approach, the SR projects out the degrees of freedom (dof) of the CS theory from the interior of the disc to its boundary inducing some sort of mini-holographic principle and, as a consequence, the reduced theory phase space is determined by the physical data contained on its boundary theory, which turns out to be a lambda model. In this sense, the way the lambda model is recovered is very similar to the way a chiral WZM model is recovered from an usual CS theory. We will not consider this approach in this work.
where ω is a meromorphic differential defined on CP 1 , constructed out of the twist function of the underlying integrable field theory, we get something new from (1.5), i.e. (1.8) The differential d hits ω and the integral is non-trivial as dω is supported at the set of poles p of the twist function in the spectral space CP 1 . The action functional associated to the symplectic form (1.7) is the four-dimensional holomorphic CS theory first presented in [24,25] and subsequently thoroughly studied in a series of papers [1,[26][27][28]. This top-down approach introduce successfully the spectral parameter z into the CS action functional from first principles and everything points towards it is the correct way to do so, as a wide range of known and even new integrable field theories can be described in this way [1,30], not to mention several lattice integrable models as well.
In this approach, and at least for the explicit example to be considered in this note, the SR restricts the degrees of freedom of the CS theory from M = S 1 × CP 1 to M = S 1 × p inducing a localization mechanism and, as a consequence, the information of the reduced theory phase space is determined by the restriction of part of the original CS gauge field to the set of poles p in the spectral manifold, that is identified with the Lax connection of the lambda model in a natural way. This is the approach that we will consider in what follows.
It is the purpose of this note to work out the approach (II) in detail and to show how (1.1), (1.2), (1.3), the lambda deformed PCM action functional and its classical integrability properties can be recovered from the SR of a holomorphic CS theory defined on Σ × CP 1 . We emphasize that in this setup the gauge is not fixed completely but only partially, in contrast to [29,30], where the CS gauge symmetry is fixed in totality. In this regard our results are complementary. In section (2) we gather several relevant results of the lambda deformed PCM case and in section (3) we focus entirely on the approach (II), with the goal of recovering all the results of (2) from this new perspective. We finish with some comments on the relation between approaches (I) and (II) and under which conditions they describe the same physical system. This is done in section (4).
Lambda deformed principal chiral model
In this section we collect some relevant results of the lambda deformed PCM that will facilitate its identification as the reduced field theory obtained by performing a SR on an holomorphic CS theory in the next section. All the results can be found in the literature and are briefly gathered here in order to maintain the text self-contained. The only relatively new detail concerns a differential ω constructed out of the twist function ϕ of the theory that will play a prominent role in the holomorphic CS theory considered in (3).
Action functional and equations of motion
The lambda deformed PCM is defined by the following action functional 3 where * , * = T r( * , * ) is the trace in some faithful representation of the Lie algebra f, Σ = R × S 1 is the closed string world-sheet manifold parameterized by the coordinates (τ , σ), k is the level and is the omega projector defining the deformation with I being the identity operator. Above, we have that and is the Wess-Zumino three-form defined on a manifold M, where Σ = ∂M. The constant κ 2 is the coupling of the un-deformed PCM.
On the one hand, the A ± eom are given by 4 and from this follows that the Maurer-Cartan identity for the flat current F −1 ∂ ± F takes the form where On the other hand, the F eom when combined with (2.6) imply that both terms ξ i in (2.7) vanish separately and Together, (2.6) and (2.9) leads to a system of equations that is formally equivalent to the PCM eom, i.e.
but in terms of the deformed dual currents defined by The pair of equations (2.10) follow from the zero curvature condition of the Lax connection 12) or equivalently, as the compatibility of the associated linear problem where Ψ is the wave function. The latter expression allows to write the Lagrangian fields in the form where are two special points in the complex plane (plus a point at infinity) that will play a prominent role in what follows. They are exchanged when we take λ → λ −1 . From (2.12) we notice that the Lax connection vanishes for z = ∞ and (2.14) is complemented with the wave function boundary condition lim z→∞ Ψ(z) = Id. (2.16) The equations (2.6) also take the alternative form where we have used the Kac-Moody currents expressions (2.24). In this way, the spatial component of the Lax connection satisfy where the J ± are to be taken as the right hand sides in both equations of (2.17).
Using (2.17) and (2.11), we obtain expressions [31] for the space and time components of the Lax connection in terms of the Kac-Moody currents. Namely, where we have defined the functions This condition imply that the variational problem in the holomorphic CS theory is welldefined. In (2.22) the symbol ∼ denotes the z dependence.
Finally, in order to see if the Lax connection (2.12) is flat, not only on-shell but off-shell as well, we must run the Dirac algorithm first. This we do next.
Hamiltonian structure and integrability
The phase space associated to the action functional (2.1) is described by the following data: two currents J ± given by that obey the relations of two opposite levels mutually commuting Kac-Moody algebras 5 and two conjugated pairs of fields (A ± , P ∓ ) with Poisson brackets The time evolution is determined by the canonical Hamiltonian density where f is an arbitrary functional of the phase space variables.
Now we consider the Dirac algorithm. There are two primary constraints By adding them to the canonical Hamiltonian density we construct the total Hamiltonian density where u ± are arbitrary Lagrange multipliers.
The time preservation of the primary constraints under the flow of H T produces two secondary constraints given by which are the gauge field eom (2.17) found above. By adding these secondary constraints to the total Hamiltonian we construct the extended Hamiltonian where µ ± are arbitrary Lagrange multipliers.
Verifying again the preservation of the primary and secondary constraints under the flow of H E , leads to the complete determination of the Lagrange multipliers and no new tertiary constraints produced at this level.
Before we consider the Virasoro constraints, it is useful to separate the constraints we have found so far between first and second class constraints in order to simplify the rest of the analysis. We quickly realize that there are no first class constraints because of the pairs P ± ≈ 0 and C ± ≈ 0 (2.33) form a system of second class pairs of constraints. We impose them strongly by means of a Dirac bracket. However, the Poisson brackets among the currents J ± are not modified [10], so we continue using their usual Kac-Moody Poisson brackets (2.25). As a consequence, the expressions (2.17) and (2.19) are valid in the strong sense. At this point, we can anticipate that no Hamiltonian extension of the Lax connection will be required in contrast to the lambda models on (semi)-symmetric spaces [23]. Now, we are ready to consider the Virasoro constraints which must be imposed by hand in the conformal gauge approach adopted here. After a temporary reintroduction of the 2d world-sheet metric in the action (2.1), we find the stress-tensor components is a covariant derivative and after imposing (2.31) strongly, we find that Above, the currents I ± are given by (2.11). Another expression is given in terms of the Lax connection (2.19) and the points (2.15), i.e.
From these results, it is straightforward to show that confirming that the Lax pair L ± (z) is a strongly flat z-dependent connection and that no Hamiltonian extension is required, i.e. the equation holds on the whole phase space of this lambda model.
Because of the Lax connection is strongly flat, the relations (2.14) are valid off-shell and in terms of the variables Ψ(z ± ), the action (2.1) takes the form [23] As the constraints C ± ≈ 0 have been imposed strongly, the action right above is equivalent to the effective action of the lambda model in the deformed metric and antisymmetric field (i.e. with the field A ± in (2.1) integrated out). In this guise, the action (2.40) is manifestly invariant under the symmetry λ → λ −1 and k → −k or, equivalently, We will return to this symmetry later on.
The remaining constraints left are (the first class) Virasoro's T ±± ≈ 0, whose action on the transport matrix The Hamiltonian and momentum densities are and from this follows that the trace of powers of the monodromy matrix is conserved in time. In terms of the Lax connection, the Hamiltonian and momentum densities take the form As a consequence of the KM algebra structure, the classical exchange algebra of the theory takes the Maillet's algebra form [33] and ϕ −1 (z) is the inverse of the twist function of the model . (2.49) Now, in order to make the connection with the holomorphic CS theory below more transparent, we take a closer look to the twist function.
and introduce a CP 1 spectral space 6 parameterized by the holomorphic coordinate z, with z being the spectral parameter of theory. A very important result involve the differential two-form dω and its corresponding support at the set of poles of the twist function given by To see this explicitly, expand (2.50) locally around the points in p and keep only the singular contributions. We get 7 and from this follows that is the Dirac delta function with the property that for any F ∈ C ∞ (CP 1 ). In this way, we get an useful formula 8 where we have used the definition (2.57) 6 The 2 dimensional complex notation used is: In each term, the coordinate z is to be understood as a local coordinate around the corresponding pole. 8 We have discarded the contribution at ∞ because any F is constructed out of the components of the Lax connection, which vanish at that point.
Armed with these results, we write the Hamiltonian and momentum functions (2.46) in the form Below we will show, that the time evolution in the symplectic reduced CS field theory is dictated by h. There, a clear interpretation of the expression (2.40) will be given as well.
Holomorphic Chern-Simons theory
In this section we recover the results of (2) from the holomorphic CS theory point of view.
It is important to emphasized that this is done without fixing the gauge symmetry of the CS theory completely (as in [29,30]), but rather from a symplectic reduction perspective (as in [22,23,35]). The main result is that in the holomorphic CS theory case, the SR works as a localization mechanism that eliminates the spectral parameter from the reduced CS theory phase space, which is identified as being equivalent to the lambda model. As a consequence, important quantities of the lambda deformed PCM, like the Lax connection, action functional, exchange algebra and so on, are determined by the phase space data associated to the set of poles where the theory localize in the CP 1 spectral space.
Action functional and equations of motion
The holomorphic Chern-Simons theory of our interest is defined by the following four-dimensional action functional 9 where Σ = R × S 1 is the closed string world-sheet manifold, ω is as defined in (2.50), CS(B) is the CS three-form for the gauge field B and CP 1 is the spectral space introduced above. Under certain circumstances, as considered in [30], the action is real.
The gauge field and the exterior derivative decompose in the form where we have ignored the A z dz component of the gauge field A as it completely decouples from the theory. This is because of the 1-form ω already carries the dz factor contribution to the volume form of Σ × CP 1 .
Under the gauge symmetry transformations the CS three-form changes as follows and, in principle, the theory (3.1) will be gauge invariant provided the following two conditions are satisfied, i 8π Σ×CP 1 ω ∧ χ(g) = 2πN and g| p = Id. (3.5) We will analyze these conditions more closely from another perspective below.
In the variables (3.2), the action becomes The Lagrangian of the theory is given by and has an arbitrary variation of the form The eom of the theory follow directly from (3.8). The z-dependent "bulk" eom given by 10 F = 0, ∂ τ A − DA τ = 0 (3.9) must be supplemented with the "boundary" condition (cf. footnote 1) x∈p µν res x ω A µ δA ν = 0 for µ = τ , σ. (3.10) We will refer to these kind of expressions as boundary contributions [1]. The (3.10) is identical to the true geometrical boundary contribution that appear in the double CS theory approach to lambda models of [22,23], so this name is appropriate in both approaches. In contrast, we will refer to the other type of contributions simply as bulk contributions.
The condition (3.10) is equivalent to (2.23) provided we make the identifications In what follows, we will assume this boundary condition is always satisfied and furthermore, we will extend (3.11) to be valid not only at the points z ± but at any other value of z as well, i.e. A µ (z) = L µ (z). (3.12) The proper justification of the key relation (3.12) requires the use of the Hamiltonian analysis, which is our next topic.
Hamiltonian structure and symplectic reduction
The phase space associated to the Lagrangian (3.6) is described by the following data: three conjugate pairs of fields (A i , P i ), i = τ , σ, z obeying the fundamental Poisson bracket relations and a time evolution determined by the canonical Hamiltonian through the relation where f is an arbitrary function of the phase space variables.
Because of the condition (3.10) is assumed to apply, the canonical Hamiltonian has a well-defined functional variation, in the sense that no boundary contributions appear [34], i.e. 16) or more explicitly, where Now, we run the Dirac algorithm. The are three primary constraints given by The constraints φ σ and φ z form a second class pair and it is convenient to impose them strongly through a Dirac bracket before we continue our analysis 11 . We change the Poisson brackets (3.13) by their corresponding Dirac brackets (DB) and, for i = σ, z, both DB brackets boil down to while the Poisson bracket for i = τ remains unaltered. In what follows, we will drop the * and continue referring to them simply as Poisson brackets in order to match common jargon.
Using the remaining primary constraint, we construct the total Hamiltonian where u τ is an arbitrary Lagrange multiplier.
The time preservation of the primary constraint P τ ≈ 0 under the time evolution of h T leads to a secondary constraint F ≈ 0, (3.22) which is the first bulk eom written in (3.9). In order to understand the geometric nature of this constraint, its relation to the gauge symmetry of the theory and its role in the reduction process, it is convenient to invoke the symplectic approach before we continue.
Consider the symplectic form associated to the Poisson brackets (3.20), which is given by a variant of the conventional CS symplectic form. It is given bŷ
whereδ represents the exterior derivative in the symplectic manifold A. Now, using the contractionδ A(X η ) = −Dη, (3.24) where X η is the Hamiltonian vector field induced by the infinitesimal gauge symmetry transformations (3.3) with g = 1 + η, η ∈ g = Ω (0) (S 1 × CP 1 , f), we obtain where is the associated gauge Hamiltonian. Notice that (3.22) constitutes the bulk contribution. A second contraction gives a centrally extended Poisson algebra meaning that the gauge algebra must be centrally extended as well in order to have a morphism of Lie algebras. By equippingĝ = g ⊕ C with the cocycle 12 Two comments are in order: i) the smeared constraint H(η) has a well-defined functional variation for any gauge parameter η, i.e.
δH(η)
or more explicitly and this means that H(η) is identified as the gauge symmetry generator of the theory. However, ii) the constraint algebra (3.27) is first class only when η| p = 0, which is the second condition we found before in (3.5) for the holomorphic CS theory action to be gauge invariant and, as a consequence, true gauge symmetry transformations are generated only by the gauge parameters η that vanish at the poles (2.51) of the twist function. Then, the first class smeared constraint of the theory denoted by is precisely (3.22) and not only the constraint is important but the pair (F, η| p ) is what matters. Considering now the gauge algebra g 0 = {η ⊂ g| η| p = 0}, (3.36) one realize that its corresponding gauge group G 0 is normal. As a consequence, and in complete analogy to the situation considered in [23] (see [35] for further details), after performing a SR and obtaining the space A 0 of flat connections modulo gauge transformation generated by G 0 , there will be a residual gauge symmetry generated by G = G/G 0 acting on it and the reduced space of the theory is actually the space A red = A 0 /G . As we shall see, the space A 0 naturally localizes at the set of poles (2.51), where the lambda deformed PCM starts to emerge. As an abuse of language, in the subsequent subsections we will use the label "red" in all quantities taking values in the space A 0 , the reason being a subtlety related to the residual gauge symmetry G and its role played in the lambda deformed PCM, so care must be taken. We comment on this in the paragraph above equation (3.64) below.
After this digression, we now continue with the Dirac procedure. Adding the secondary constraint (3.26) to the total Hamiltonian we construct the extended Hamiltonian where the test function η plays the role of an arbitrary Lagrange multiplier. It is important to notice that η is not required to vanish on p and that h E has a well-defined functional variation.
The time preservation of the secondary constraint H(η) under the time evolution of h E does not produce any further constraints but rather enforce the condition η| p = 0 and, not surprisingly, only the first class constraint (3.35) is preserved in time. The primary constraint P τ ≈ 0 is also preserved under the time evolution of h E and no tertiary constraints are produced at this level.
The only constraints of the theory are both first class and must be gauge fixed accordingly. However, we will only gauge fix the first one and subsequently perform a SR with the second one and this is quite natural from the symplectic geometry point of view because of (3.22) is not only a Hamiltonian constraint but also a piece of the moment map for the gauge symmetry as well. To see this, let us notice that (3.26) can be written in terms of a pairing 13 between From this, we identify Ω 2 (S 1 ×CP 1 , f)⊕Ω 1 (S 1 ×p, f)⊕C as a subspace ofĝ * and the mapping is an equivariant moment map for the gauge group action because under (3.3), we have that H(η, t) g = H(Ad g −1 (η, t)), (3.41) where The first class constraint P τ ≈ 0 can be gauged fixed via a generic condition of the form The only property we imposed on L τ (A) is that at the points p the boundary conditions (3.10) must be satisfied. This is a good gauge fixing condition whose time preservation determines the Lagrange multiplier u τ but as it couples with the constraint P τ in h E , which is to be imposed strongly at the end anyway, its explicit form is not relevant anymore. Furthermore, the PB (3.20) is not modified by this gauge fixing and we are left only with the second constraint in (3.38).
We are now in the position to perform the SR and to show that the reduced CS theory corresponds to the lambda deformed PCM. We break the proof into three pieces, each one considering a relevant aspect of the reduced theory that is to be compared against the results gathered in (2).
Reduced Poisson structure: Maillet bracket
The moment map for the G 0 -action of the normal gauge subgroup is given by the composition and A 0 = (p • µ) −1 (0, 1)/G 0 is a symplectic reduced space. The A 0 symplectic form of the theory is found by pulling-back (3.23) to the surface defined by the flatness condition F = 0. Equivalently, from (2.56) we find the important result where D σ(±) ( * ) = ∂ σ ( * )+[A σ (z ± ), * ] are covariant derivatives and D −1 σ(±) their formal inverses. At this point we have imposed the boundary condition (2.16) on Ψ above, anticipating the validity of the result (3.12). As a consequence of the SR, the symplectic form localizes at the poles of the twist function where the reduced field theory phase space is now determined by the restricted CS field As mentioned before, there is a residual gauge symmetry action on the space A 0 parameterized now by the coordinates A σ (z ± ). Indeed, using the contractionŝ where η ± ∈ g , we find that − i XηΩred =δH red (η), (3.50) where A second contraction in (3.50) gives their Poisson algebra which is equivalent to two copies of mutually commuting KM algebras of opposite levels, Then, as a consequence of the SR procedure, in the reduced CS theory the z-dependent gauge field A σ (z) naturally interpolates between A σ (z + ) and A σ (z − ) so the obvious expression to be considered is inspired by the Lax connection (2.19) with the functions f ± (z) defined as in (2.20). As showed above around equation (2.47), the KM algebra structure induces the Maillet bracket (2.47) on the component A σ (z) and from (3.55), we obtain the identification justifying equation (3.12) for µ = σ. The spectral parameter in (3.55) now behaves as an auxiliary parameter, which is pretty much its usual interpretation in the classical theory.
Alternatively, if instead of performing the symplectic reduction, we choose to gauge fix the first class constraint H 0 (η) through the gauge fixing condition the resulting Dirac bracket is, as shown in [29], the Maillet algebra bracket again. Hence, by fixing the gauge as right above or by performing the SR we get the same answer, the resulting reduced field theory being independent of the A z component of the original CS gauge field. Also notice that in the SR procedure of the holomorphic CS theory, the component A z behaves quite in the same way as the radius component A r of the gauge field in the SR of the double CS theory defined on the solid cylinder.
Reduced space equations of motion: Lax connection
Let us identify the A red symplectic leaves and the reduced eom on this space.
Start with the symplectic leaves, which are determined by the action of the residual gauge algebraĝ on A 0 . To find them, write (3.51) as a pairing between and identify Ω 1 (S 1 × p, f) ⊕ C as a subspace ofĝ * . From the equivalence The case of interest is z = 1, corresponding to the gauge transformations (3.3) restricted to S 1 × p. Then, the symplectic leaves in the reduced space are in one-to-one correspondence with the co-adjoint orbits (3.60). A clearer picture appears by considering the transport matrix for the fields A(z ± ), denoted generically by Under the co-adjoint action (3.60) with z = 1, we have that T (Ad * g A|σ 2 , σ 1 ) = g(σ 2 )T (A|σ 2 , σ 1 )g(σ 1 ) −1 (3.62) and the action on the monodromy matrix m(A) = T (A|2π, 0) (at each point z = z ± ) is given by showing that the co-adjoint orbits are in one-to-one correspondence with the orbits of the Lie group F acting on itself by conjugation. Thus, the symplectic form (3.23) induces a Poisson structure on A red , the symplectic leaves are then obtained by fixing the conjugacy classes of the monodromy matrices m(A(z ± )) along S 1 . The m(A(z ± )) being related to finite-dimensional quantum groups [31,32].
As A σ (z) is identified with the component L σ (z), an important comment concerning the boundary residual gauge symmetry of the reduced theory is in order. Despite of the fact that (3.51) is interpreted as a gauge symmetry generator from the CS theory point of view, it is not a genuine gauge symmetry generator from the lambda deformed PCM perspective, as can be observed from (2.33), which states that no gauge symmetries are present in the theory and caution must be taken about its interpretation. This apparent enhancement of symmetry is also present in lambda models on semi-symmetric spaces [23], their true gauge symmetry being generated only by a subgroup of the residual CS theory gauge group and this could be understood as a being a consequence of embedding the lambda model phase space into a phase space of bigger dimension. Another issue, is that the gauge symmetry generatedĝ can not [23] be continued outside the poles to act on A σ (z) in the usual way as in (3.34). Now we consider the eom. In the reduced theory the gauge fixing condition (3.43) is now given by the strong expression because of the independence of the reduced theory phase space on the coordinate A z . An explicit form that satisfies the boundary condition (3.10), so far assumed to hold, is clearly inspired by the Lax connection again. Thus, we take with the functions g ± (z) as defined in (2.20), justifying equation (3.12) for µ = τ .
The time evolution in the reduced theory is determined by the reduced Hamiltonian where we have taken η = 0 in (3.37) in order to separate it from the contribution of the boundary gauge generator (3.51) to the extended Hamiltonian. This latter expression is the boundary contribution to the canonical Hamiltonian (3.14) and should be compared with the first equation in 14 (2.58). By considering the generator of translations along the σ direction, given by the second equation in (2.58), we conclude that the pair of components A µ (z) of the 14 The opposite sign when compared with (2.58) is not an issue as the equations (2.28) and (3.15) are reduced CS gauge field is actually a strongly flat z-dependent connection with respect to the KM algebra structure of the reduced phase space and, as a consequence, we have that The z-dependent eom in the reduced space are given by the second expression in (3.9), namely The first equation giving the eom of the lambda deformed PCM (2.39), while the second becomes an identity. Then, two of the components of the CS gauge field B, i.e. (A τ , A σ ), behave as the lambda deformed PCM Lax connection for all intents and purposes.
Once we have understood the time evolution and the integrability of the eom in the reduced theory, we proceed to compute the corresponding action functional from where these quantities can be derived by canonical methods.
Reduced action functional: lambda deformed PCM
In order to construct the reduced action functional having as Euler-Lagrange 15 eom and (3.66) as Hamiltonian function, we take into the action (3.1) and this is because of equations (3.45) and (3.67). We quickly find that This is not an standard WZ term because it involves an integral over the two-dimensional world-sheet manifold Σ rather than on a three-dimensional manifold M with the property that Σ = ∂M. To obtain an expression closer to the usual form, we denote by Ψ the extension of the wave function into the five-dimensional manifold M × CP 1 and write the result as an integral over M. We get which is equal to the action found before in (2.40). This same action was found in [23] starting from the double CS theory. Notice that under the validity of (3.74), the first condition in (3.5) is satisfied giving N = 0. We will assume that equation (3.74) holds, although we will comment about its validity in the final remarks section below.
A more suggestive expression follows from the relations (2.14) found above. Indeed, in terms of the field variables defined on Σ and M, respectively, the reduced action becomes [23] or, alternatively, Both expressions being equivalent under the symmetry (2.41). This symmetry which manifests trivially in the CS theory formulation was first discovered in [36] by working directly on the effective action (3.85) written below, has important implications for the renormalization group structure of the theory, see [36].
Expressions (3.77) and (3.78) can be written in terms of residues over the poles (2.51). In order to do this, we introduce a z-dependent extension of the Lagrangian fields (3.76) defined by and, as a consequence, we have that The expressions (3.78) and (3.77), respectively, now take the compact form where is the strongly flat Lax connection of the theory and from this we obtain the final form for the reduced action functional A similar expression was found in [30] to be valid as well for a wide range of other integrable deformations of string sigma models, after fixing a particular form of the CS gauge field (roughly) enforcing the condition A z = 0 and imposing the so-called archipelago conditions on an analogue to the field F defined above. Here, the CS theory naturally localize at p, as a consequence of the SR.
Finally, an equivalent expression for the reduced action, say (3.77), is given by [23] where A ± depend on F via (2.6). After some algebraic manipulations, we obtain a more familiar form with B 0 denoting the WZ term contribution. The "kernels" G and B are responsible for the deformation of the metric and anti-symmetric background fields. The action (3.85) is the effective action we obtain from (2.1) by integrating out the gauge fields A ± through their equations of motion. Then, showing that the theory localize at the poles of the twist function under the SR of its parent holomorphic CS theory. The symplectic reduced holomorphic CS theory being identified with the lambda deformed PCM.
Final remarks
We have shown how the symplectic reduction applied to a particular holomorphic Chern-Simons theory works as a localization mechanism in the phase space of the theory. The physical data of the reduced theory associated to the points where the theory localize is sufficient to reconstruct the lambda deformation of the Principal Chiral Model and all of its known integrability properties. From the lessons of [23], this opens the possibility of considering lambda models on (semi)-symmetric spaces, like the AdS 5 ×S 5 superstring lambda model, in a direct way and this is because of an analogue of the expression (3.55) in that case is already known to induced the classical exchange (Maillet's) algebra for the theory. Other cases of lambda models from the point of view of the holomorphic CS theory will be considered in a companion work.
Let us comment on the relation between approaches (I) and (II) mentioned in the introduction. By introducing a three-dimensional manifold M with the property that ∂M = Σ and by extending all the quantities (3.2) into this manifold (primed variables), the action (3.1) becomes where F B is the field strength of the gauge field B . Under gauge symmetry transformations, we find the variation (4.2) Notice that this variation is completely localized at the set of poles p. (I) "Holography". When M = R × D is the solid disc and B is chosen to vanish at z = ∞, the first contribution to (4.1) is precisely the double CS theory action functional of [22,23]. In this case, the relation between the physical information contained in the interior of the disc and its geometric boundary is what matters. For instance, gauge invariance in (4.2) leads to two conditions [23] k 4π M [χ(g (z + )) − χ(g (z − ))] = 2πN, g| ∂M = Id. (4. 3) The first condition imposes a quantization condition while the second one imposes a boundary condition on the gauge group elements at the boundary S 1 of the disc. This interpretation is quite close to the known holographic results of [19,20,35], where the SR reduction eliminates the dof in the interior of the disc but retains only those belonging to its geometric boundary. This standard interpretation is what initially inspired the approach (I) and the works [22,23]. (II) "Localization". Because of the action (3.1) is inherently defined on Σ and no geometric boundary terms are present, what matters now is to discriminate the physical information associated to the poles from the rest, i.e. the "bulk". For instance, both contributions in (4.2) vanish simply by imposing the condition on the gauge group elements and the first condition in (3.5) is then unnecessary, in consistency with (3.36). Let us notice as well that the equation (3.74) must hold because of the action (4.1) becomes (3.75) when restricted to the set of flat gauge fields. Fortunately, the results of [35] (dealing purely with surfaces with boundaries) still apply with minor modifications in this scenario giving the same reduced theory. It is the interplay among the objects M, Σ, ∂ andd that allows to easily relate approaches (I) and (II) and to understand why they give the same classical reduced field theory. Both approaches match because of the action (4.1) is to be restricted to the set of flat gauge fields, where the second term is absent.
Finally, as a first quantum test to show that the equivalence between the holomorphic CS theory and the lambda models indeed goes beyond the classical regime, it would be interesting to recover the lambda model dilaton term contribution to the action (3.85), directly from the four-dimensional CS gauge theory (3.1). We expect to consider this issue elsewhere. | 2019-12-17T02:01:14.576Z | 2019-12-16T00:00:00.000 | {
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83626628 | pes2o/s2orc | v3-fos-license | Cys577 Is a Conformationally Mobile Residue in the ATP-binding Domain of the Na,K-ATPase α-Subunit*
2-[4′-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mm ATP or low K+ (<1 mm); the protective effect K+ is reversed at higher concentrations. This biphasic effect was also observed with K+ congeners. In contrast, Na+ ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of ∼5 kDa. N-terminal amino acid sequencing identified the peptide (V545LGFCH … ). This hydrophobic peptide contains only two Cys residues in all sodium pump α-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys577. The cation-dependent change in reactivity of Cys577implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.
2-[4-
Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a timeand concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K ؉ (<1 mM); the protective effect K ؉ is reversed at higher concentrations. This biphasic effect was also observed with K ؉ congeners. In contrast, Na ؉ ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of ϳ5 kDa. N-terminal amino acid sequencing identified the peptide (V 545 LGFCH . . . ). This hydrophobic peptide contains only two Cys residues in all sodium pump ␣-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys 577 . The cation-dependent change in reactivity of Cys 577 implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.
The Na,K-ATPase (EC 3.6.1.37) is a P 2 -type ATPase (1) that is responsible for the maintenance of the sodium and potassium ion gradients in most eukaryotic cells. The integral membrane protein is a heterodimeric enzyme comprised of an ␣-subunit of about 100 kDa and a -subunit, which migrates in SDS-PAGE 1 with an apparent mass of 55 kDa. The ␣-subunit is composed of 10 putative transmembrane segments, several of which are thought to form an intramembrane cation-binding domain and a major cytoplasmic loop between the fourth and fifth trans-membrane segments (2). This intracellular loop of around 45 kDa contains the catalytic phosphorylation site (Asp 369 ) and the ATP-binding domain (for review see Ref. 2). A variety of reagents that have been used to inactivate the Na,K-ATPase in an ATP-protectable fashion have identified several putative contact residues in the ATP-binding domain. These have been predominantly lysine-reactive reagents, and a series of residues have been identified (3). It has been reported that the reactivity of several of these residues is dependent upon cation binding and thus enzyme conformation (4 -6). The dual function of ATP hydrolysis and cation transport, which constitute ion pumping, mechanistically involve the coupled interactions of the ATP-binding domain and the cation-binding domain.
Regions of the ␣-subunit and some specific amino acid residues have been identified as being close to (or at) the ATPbinding region of the enzyme. To date, all of the residues identified lie in the major cytoplasmic loop of ϳ440 amino acids, between transmembrane helices 4 and 5. Recent evidence that bacterial overexpression of the M4M5 loop yields a peptide with the same nucleoside phosphate binding specificity as the intact Na,K-ATPase lends further support to the notion that this segment supplies the ATP-binding residues in the intact sodium pump (7). The majority of the amino acids in the ATP-binding domain identified by direct labeling have been lysine residues, primarily because of the greater availability of lysine-selective reagents. These residues include Lys 480 , Lys 487 , Lys 501 , Lys 589 , Lys 605 , Lys 618 , Lys 622 , and Lys 719 (for review see Refs. 2 and 3).
In addition, cysteine-directed reagents have been used in a variety of transport systems, e.g. P-glycoprotein (8,9) and lac permease (10). Such studies in the gastric H,K-ATPase (11) led to the identification of important extracytosolic cysteine residues unique to this member of the P-type ATPase family. The early use of cysteine-directed reagents in the study of Na,K-ATPase also led to important insights. The consequences of treating the enzyme with N-ethylmaleimide led to inhibition of the major phosphoenzyme transition (E 1 P E 2 P) and resulted in the formulation of the widely accepted Post-Albers scheme for the reaction pathway (12,13). A previous kinetic study of the interactions of MIANS with the Na,K-ATPase reported effects of ATP in modulating the resulting inactivation of the enzyme (14). More recently, studies using fluorescent cysteine reagents on a sided preparation of Na,K-ATPase demonstrated that only two Cys residues (of the possible seven intramembrane cysteines) were exposed to the extracellular medium (15). These observations formed the basis of a site-directed mutagenesis approach to analyze the membrane topology of the Na,K-ATPase ␣-subunit (16).
In the present work, we provide evidence that MIANS inactivates Na,K-ATPase by its covalent attachment to Cys 577 , which results in the elimination of high affinity ATP binding. We also show that the reactivity of Cys 577 is conformationally sensitive and that its reactivity alters in different enzymecation bound states. This indicates that structural changes in the ATP-binding domain are transmitted through at least 100 amino acid residues in the primary structure and suggests that the sequential binding of each of the two transported K ϩ ions produces changes in the ATP-binding site structure.
Na,K-ATPase Purification and Enzyme Activity Assay-Na,K-ATPase was purified from dog kidney as described by Jørgensen (17) with the addition that enzyme was purified through a continuous sucrose gradient (15-45% sucrose) achieved with a zonal rotor. The enzyme was judged greater than 95% pure through SDS-polyacrylamide gel electrophoresis. Protein concentration was determined by the method of Lowry et al. (18) using bovine serum albumin as a standard. Na,K-ATPase activity was determined in a standard assay medium containing 1 mM EGTA, 130 mM NaCl, 20 mM KCl, 3 mM MgCl 2 , 3 mM Na 2 ATP, 50 mM imidazole, pH 7.2, and 0.5 g/ml purified enzyme. The mixture was incubated at 37°C for 15 min, and the amount of P i released through enzyme catalyzed ATP hydrolysis was measured as described by Brotherus et al. (19). The enzyme used in these studies had a specific activity of 18 -30 mol P i mg Ϫ1 min Ϫ1 .
MIANS Inactivation and Labeling of the Na,K-ATPase-The enzyme (0.1-1.0 mg/ml) was treated in buffer (25 mM imidazole, 1 mM EDTA, pH 7.5). MIANS in Me 2 SO was added at the desired concentration and the reaction mixture was incubated at 37°C. The reaction was stopped by diluting 10-fold with ice-cold stopping solution (25 mM imidazole, 1 mM EDTA, 5 mM -mercaptoethanol, pH 7.5). An aliquot was then removed from the stopped reaction mixture, and ATPase activity was determined as described above. The final concentration of Me 2 SO was 10% in the reaction mixture and 0.025% in the assay medium. Enzyme treated with 10% Me 2 SO alone retained normal function.
Ligands were tested for their ability to protect against MIANS inactivation. Purified Na,K-ATPase (0.5 mg/ml) was preincubated for 10 min at room temperature in the imidazole/EDTA buffer with 10 mM KCl, 20 mM NaCl, 3 mM Tris-ATP, or 3 mM magnesium and inorganic phosphate. MIANS was added to a final concentration that resulted in at least 70% inactivation (in the absence of substrates), and the reaction mixture was incubated at 37°C for 30 min. The reaction was stopped by diluting 10-fold with ice-cold stopping solution. An aliquot was removed from the reaction mixture for an ATPase activity measurement, and the remaining protein was pelleted in a Beckman TLX Ultracentrifuge at 436,000 g at 4°C for 40 min. The pellet was resuspended in imidazole/ EDTA to give a protein concentration of 2.5 mg/ml. A sample of this protein (20 g) was then mixed with 8 M urea, 10% SDS, and 125 mM Tris buffer, pH 6.8 (1:1:1 v/v). SDS-PAGE was carried out according to Laemmli (20) in 10% acrylamide gels. Na,K-ATPase ␣-subunit labeled with MIANS was detectable by fluorescence emission on illumination with a hand-held long wavelength (360 nm) UV lamp. After UV detection, protein bands were observed by staining the gel with Coomassie Brilliant Blue R.
Trypsin Digestion of MIANS-treated Enzyme-2 mg of purified enzyme was labeled with MIANS in the presence and absence of 3 mM Tris-ATP for 30 min under the conditions described above. An aliquot was removed for an ATPase activity measurement, and the remaining protein was pelleted at 436,000 g at 4°C for 40 min. The pellet was then resuspended in 0.1 M ammonium bicarbonate, pH 8.0. Tosylphenylalanyl chloromethyl ketone-treated trypsin was added (1:10, w/w, trypsin: enzyme), and the solution was incubated at 37°C for 2 h. The digestion was stopped by the addition of soybean-trypsin inhibitor (1:7, w/w, trypsin:inhibitor), and the soluble fraction was separated from the membrane bound fraction by centrifugation at 436,000 ϫ g.
V8 Protease Digestion of Na,K-ATPase-MIANS-labeled ␣-subunit was isolated from a 7.5% acrylamide gel and eluted from the gel slice with 5 mM Tris-HCl, 0.05% SDS for a minimum of 16 h at room temperature. The protein was concentrated with a Centricon 30 (Amicon) and washed twice with 2 ml of H 2 O. Labeled protein was eluted from the membrane with H 2 O, and the amount of protein recovered was determined by the method of Lowry et al. (18). Proteolytic digestion was performed in 25 mM ammonium bicarbonate with V8 protease (1:15, w/w, protease:protein) for 18 h at room temperature. Protein fragments were precipitated with 10 volumes of acetone (Ϫ20°C for 16 h).
Reverse Phase HPLC Separation of Soluble Peptide Fragments-Soluble peptide fragments from trypsin digestion were separated by linear gradient elution using a Vydac C18 (0.45 ϫ 25 cm) reverse phase column. The gradient was generated with a Beckman System Gold 126 HPLC Dual Pump System Module. For the first purification, pump A delivered 100% H 2 O, 0.1% trifluoroacetic acid, pH 2, and pump B delivered 80% acetonitrile, 20% H 2 O, 0.1% trifluoroacetic acid, pH 2. The flow rate was 0.75 ml/min, and the eluent was monitored with a System Gold 168 photodiode array detector. Fractions were collected in 2-min intervals and those containing the peptide fragments of interest were concentrated with a Savant Automatic AES 1000 Speed Vac System. The volume was brought up to 500 l with H 2 O and further purified on a shallower gradient with the same HPLC system and solvents (pH increased to 6.0 with triethylamine) as described above.
Separation of Labeled Peptide Fragments on Tricine Gels-Soluble peptide fragments were precipitated with 6 volumes of acetone at Ϫ20°C for 16 h. Membrane-associated peptide fragments were resuspended in imidazole/EDTA buffer, and one volume of 10% SDS was added to solubilize the proteins. These solubilized membrane proteins were precipitated by the addition of 10 volumes of methanol at Ϫ20°C for 16 h. The precipitated peptides were pelleted at 5,000 ϫ g and redissolved in sample buffer. Peptides were separated on a 16.5% Tricine gel according to Schagger and von Jagow (21). After electrophoresis protein fragments were transferred to polyvinylidene difluoride membrane (Millipore) by electroblotting in 10 mM CAPS 10% MeOH, pH 11.0 (22) at 180mA for 1 h. Fluorescently labeled bands were sent for N-terminal amino acid sequencing and total amino acids analysis (Dr. Jan Pohl, Microchemistry Facility, Emory University, Atlanta, GA).
Stoichiometry of ADP Binding-ADP binding was determined according to the method of Robinson (23) with slight modifications, in a medium containing 30 mM HEPES, 0.1 mM EDTA, 5 mM NaCl, and 0.5 mg/ml enzyme. Briefly, purified Na,K-ATPase (100 g) was labeled with MIANS (300 M) in the presence of no added ligand, 10 mM NaCl, or 3 mM ATP. A control reaction was incubated without MIANS. Na,K-ATPase activity was determined as described previously. The labeled enzyme was divided into two aliquots, and the protein was pelleted at 436,000 ϫ g for 30 min at 4°C. One aliquot was resuspended in 200 l of buffer A (30 mM HEPES, 0.1 mM EDTA, 5 mM NaCl), and the other was resuspended in 200 l buffer A with 10 mM ATP. The samples were incubated at room temperature for 30 min and then on ice for 1 min. [ 3 H]ADP was added, and the samples were incubated on ice for 30 min. Protein was pelleted at 436,000 ϫ g for 5 min, and the supernatant was removed. Ice-cold buffer A (0.5 ml) was added to the pellet and immediately removed. Protein was resuspended in 0.4 ml of 0.4 M NaOH. The amount of [ 3 H]ADP bound to the enzyme (200 l) was determined in a scintillation counter with EcoLite scintillation fluid. The amount of protein was determined in triplicate by the method of Lowry using bovine serum albumin as a standard.
Kinetic Characterization of MIANS Inactivation and the Relationship of Sodium Pump
Conformation-Treatment of purified enzyme with MIANS at 37°C resulted in a reduction in the Na,K-ATPase activity that was dependent on the concentration of MIANS present (Fig. 1A) and the length of incubation at 37°C (Fig. 1B). The time dependence of MIANS inactivation is not a single exponential, suggesting that more than one class of cysteine residues are labeled, consistent with previous observations (14). However, we believe that the fast initial inactivation reaction is at a single site and that prolonged exposure to MIANS results in indiscriminate labeling of several different cysteines (see Figs. 3B and 6 below). Consequently, subsequent labeling conditions were optimized (e.g. shorter times and lower MIANS concentrations) to determine the specific site of initial inactivation of the Na,K-ATPase.
The presence of enzyme ligands in the reaction mixture significantly altered the extent of Na,K-ATPase inactivation ( Fig. 2A). It seemed that Na ϩ ions facilitated inactivation, whereas the presence of ATP or K ϩ ions protected the enzyme from inactivation (Fig. 3A). The presence of Mg 2ϩ and phosphate ions did not protect the enzyme from inactivation. These results indicate that inactivation of Na,K-ATPase by MIANS is dependent upon the enzyme conformation, i.e. whether E1 or E2, phosphorylated or not, and the cations bound. Labeled sodium pump protein from the experiments shown in Fig. 2A was subjected to SDS-polyacrylamide gel electrophoresis, and MIANS labeling was visualized under UV light (Fig. 2B). The The data in Fig. 2A suggested that Na ϩ and K ϩ ions have different effects on the enzyme reactivity toward MIANS. This appears to be in contrast with previous reports suggesting that all monovalent cations (Na ϩ , K ϩ , and choline) increase the reaction rate of MIANS with the Na,K-ATPase (14). However, Gupta and Lane (14) measured the MIANS fluorescence changes associated with enzyme modification and not enzyme inactivation as performed in our study. Consequently, their measurements would detect all molecules of MIANS that bind to the Na,K-ATPase, whereas our measurements only detect covalent modifications that result in enzyme inactivation. However, it is clear that a low level of binding can occur without significant inactivation (Fig. 3B). It is likely that these experimental differences are the reason for the different interpretations of the effects of cations on MIANS modification.
The protective effect seen with 10 mM K ϩ ( Fig. 2A) appeared to vary slightly between experiments. Therefore, we measured MIANS inactivation in the presence of varying concentrations of several monovalent cations. These data clearly show that the presence of Na ϩ ions increased enzyme inactivation by MIANS, at either low (Ͻ1 mM) or high (Ͼ10 mM) concentrations (Fig. 3). In contrast, low concentrations of K ϩ congeners (Ͻ1 mM K ϩ , Rb ϩ , or Cs ϩ ) protected against MIANS inactivation. However, at higher concentrations of K ϩ and its congeners (Ͼ25 mM), this effect was reversed and enzyme inactivation was not prevented. This biphasic effect was previously observed with di- The reaction was incubated at 37°C, and aliquots were removed at the times indicated. Inactivation was stopped by the addition of 9 volumes of IE buffer with 50 mM -mercaptoethanol. Na,K-ATPase activity was measured as described under "Experimental Procedures." The data were fit to the following equation: Activity ϭ A ϫ exp(Ϫk 1 t) ϩ B ϫ exp(Ϫk 2 t), where A and B are complex functions of the rates of reaction and the degree of inhibition (48) and where k 1 (0.08 Ϯ 0.01) and k 2 (0.009 Ϯ 0.01) are rates for fast and slow inactivation phases, respectively. These data are consistent with MIANS labeling at least two different classes of sulfhydral groups with the initial phase accounting for greater than 80% of the inactivation. hydro-4,4Ј-DIDS (5, 24), 4-acetamido-4Ј-isothiocyanatostilbene-2,2Ј-disulfonate (25), and fluorescein isothiocyanate (6), compounds that also inactivate Na,K-ATPase in an ATP-protectable manner and whose sites of action have been localized to the ATP-binding loop (i.e. K501). It is likely that the variability we had seen with MIANS labeling experiments in the presence of 10 mM K ϩ was because this concentration falls between [K ϩ ] that protect well versus [K ϩ ] that do not. It is interesting that the differences between K ϩ congeners seen with fluorescein isothiocyanate and H 2 DIDS labeling at K501 (6,24) were much less pronounced among the K ϩ congeners protecting Cys 577 from MIANS (Fig. 3A, inset). The differing distances between Lys 501 and Cys 577 from the cation-binding sites may be the reason for this different sensitivity to monovalent cations. A clear explanation for this phenomenon awaits a high resolution structure of the Na,K-ATPase. Fig. 3B shows the extent of MIANS incorporation into the ␣-subunit in the presence of varying [KCl]. The amount of MIANS modification follows the biphasic pattern seen with activity (Fig. 3A), although there is some MIANS incorporation into the ␣-subunit under conditions in which enzyme activity is fully protected, suggesting that some functionally noncritical cysteine residues are modified by MIANS. This labeling that is not associated with inactivation could explain the differences between a previous study (14) and this report (mentioned above).
Evidence Suggesting That Inactivation by MIANS Results from Labeling the Nucleotide-binding Site-Modification of sodium pump by MIANS resulted in a loss of Na,K-ATPase ac-tivity that was prevented by preincubation with 3 mM Tris-ATP ( Fig. 2A). A closer look at the concentration dependence of ATP protection against MIANS inactivation showed that preincubation of sodium pump protein with concentrations of ATP greater than 250 M resulted in the same level of protection observed with 3 mM Tris-ATP in previous experiments (Fig. 4). The apparent K1 ⁄2 for ATP protection was 34 Ϯ 6.3 M. Because reversible ATP binding is protecting against covalent MIANS modification and because the status of the low affinity ATP site in the absence of K ϩ is unclear, it is difficult to identify with confidence this ATP effect as the familiar high or low affinity binding to E 1 or E 2 respectively. ATP protection against inactivation suggests that MIANS binds to a residue in the ATP site. To further test this hypothesis we directly measured high affinity nucleotide binding (Fig. 5). Purified Na,K-ATPase (100 g) was labeled with MIANS in the presence and absence of 3 mM ATP or in the presence of 10 mM Na ϩ . Aliquots of enzyme from each condition were used to determine Na,K-ATPase activity (Fig. 5, gray bars) and [ 3 H]ADP binding (Fig. 5, black bars). The maximal enzyme activity and nucleotide binding capacity were determined in the nonlabeled controls. MIANS-inactivated enzyme does not bind ADP. However, enzyme protected against MIANS inactivation does bind ADP. The amount of ADP bound per mg of protein appears to be proportional to the amount of enzyme activity remaining. Thus it appears that the loss of high affinity ADP (i.e. ATP binding) correlates well with the loss of activity. It seems reasonable to conclude that the loss of ATPase activity is a consequence of the loss of high affinity binding that is necessary for sodium-activated enzyme phosphorylation and ATPase activity.
Purification of the MIANS-labeled Peptide Fragment-Purified Na,K-ATPase (ϳ1 mg) was labeled with MIANS in the presence and absence of 3 mM ATP and digested with trypsin (see "Experimental Procedures"). The soluble tryptic fragments were separated from the membrane-associated fragments via centrifugation and all of the MIANS labeling (i.e. fluorescence) was associated with the soluble fraction. Separation of the soluble tryptic peptides was performed by reverse phase HPLC on a Vydac C18 column using isocratic elution with 0.1% trifluoroacetic acid (10 min) followed by a three-stage linear gradient (0.1% trifluoroacetic acid to 80% acetonitrile/0.1% trifluoroacetic acid) of 3%/min (5 min), 1%/min (15 min), and 0.7%/ min (100 min). Absorbances at 215 nm and 320 nm are shown for sodium pump labeled in the absence (Fig. 6A) and presence (Fig. 6B) of 3 mM Tris-ATP. The complex elution profiles of tryptic peptides monitored at 215 nm (top panels) were essentially identical, demonstrating that the digestion and chromatography were reproducible and not affected by MIANS modification. The chromatograms at 320 nm (bottom panels) represent MIANS-labeled peptides.
Although several peptides contain the MIANS probe, the peptide distribution pattern from enzyme labeled in the presence or absence of ATP differed by a single MIANS peak, clearly visible at ϳ30 min in the 320 nm chromatogram (Fig. 6, cf. A and B, bottom panels). An overlay of the two chromatograms at 215 nm (not shown) also revealed the absence of this peak (at ϳ30 min) in the ATP-protected sample. The elution stage containing the ATP-protectable peak was refined to achieve better separation of the MIANS-labeled peptide. The pH of the elution buffers was altered to 6.0 with ethylamine and a linear gradient (H 2 O to 80% acetonitrile) was run at 0.33%/min (150 min). The MIANS-labeled peptide eluted as a single sharp peak at ϳ98 min (data not shown).
An alternative method employed to isolate the MIANS-labeled tryptic fragment was separation via polyacrylamide gel electrophoresis on a 16.5% Tricine gel (Fig. 7) (21). As was the case with the HPLC experiments, no MIANS fluorescence was associated with the membrane-associated insoluble peptide fraction. However, a single fluorescent band was observed from soluble peptides labeled in that absence of ATP (lane 3), whereas the soluble peptides from the sample protected by ATP (lane 2) contained an equivalent band with much less intensity.
Amino Acid Sequencing of the MIANS-labeled Peptide and Identification of Labeled Residue-Under normal conditions maleimides are selective for cysteine residues. The MIANSlabeled fragment (ϳ5 kDa), which was identified from the Tricine gel (Fig. 7), was sent for N-terminal amino acid sequencing. Sequence data from several experiments revealed a relatively hydrophobic peptide beginning at Val 545 . The short-est peptide fragment that would be produced by trypsin would begin at Val 545 and continue through Arg 589 . This peptide is 45 amino acid residues in length and in most Na,K-ATPase ␣-subunits contains two cysteine residues, Cys 549 and Cys 577 , as potential targets for MIANS attachment.
The protein sequence reported for dog Na,K-ATPase ␣-subunit is the translation from a cDNA clone (GenBank TM accession number L42173; Ref. 26). The amino acid translation in the region of the MIANS-labeled peptide begins V 545 LG-FR 549 HL, whereas all other cloned Na,K-ATPase ␣-subunits contain a cysteine residue at position 549 (or its equivalent, analyzed using BLAST; Ref. 27). If the reported sequence is correct for dog ␣-subunit, our target residue must be Cys 577 ; however, if the reported sequence is in error and residue 549 is in fact cysteine and not arginine, our target could be either Cys 549 or Cys 577 . Indeed, this appeared to be the case because in all of our sequencing attempts we never had a clear amino Purified dog kidney Na,K-ATPase was labeled with MIANS in the absence (A) and presence (B) of ATP (3 mM). Chromatograms at 215 nm (top panels) detect all peptide fragments and at 320 nm (bottom panels) detect MIANS-labeled peptide fragments. The elution gradient is described under "Experimental Procedures." acid signal in the fifth cycle even when the signals in the fourth (Phe 548 ) and sixth (His 550 ) cycle were strong. The fact that amino acid sequencing of the MIANS-labeled peptide did not detect arginine at position 549 is an indication that the protein sequence obtained from translation of the cDNA sequence is incorrect and dog, like all other known Na,K-ATPases, contains a Cys at position 549. Unfortunately, this left unresolved whether MIANS was tethered to Cys 549 or Cys 577 , because free cysteine residues are not identified well in Edman N-terminal amino acid sequencing. Consequently, we digested MIANSlabeled Na,K-ATPase ␣-subunit with protease V8 under conditions that facilitated cleavage after glutamate because there were three glutamate residues between Cys 549 and Cys 577 . After V8 digestion, the peptide fragments were run on a 16.5% Tricine gel, and a single fluorescent band was observed and sent for sequencing. The peptide was identified as G 561 FQFDTDDV . . . , confirming that MIANS was covalently attached to Cys 577 . DISCUSSION In this work we have provided evidence that Cys 577 in the ␣-subunit of canine renal Na,K-ATPase is selectively modified by MIANS. Modification of this cysteine results in inactivation of the enzyme and loss of ATP binding. The presence of ATP during treatment with MIANS prevents both inactivation and labeling of the enzyme. These data provide evidence for the presence of Cys 577 in the ATP-binding pocket of Na,K-ATPase. In addition, the reactivity of this residue changes when different cations are bound providing information about the nature of the conformational changes taking place during active cation transport.
The central cytoplasmic loop of the Na,K-ATPase ␣-subunit has been associated with ATP binding and phosphorylation of the pump during the reaction cycle. Several specific amino acids presumed to be in the ATP-binding pocket have been identified by chemical modification studies (for review see Ref. 2). However, this is the first report identifying Cys 577 ; interestingly, it is not close in the primary sequence to any of the previously identified amino acids (e.g. Lys 501 , Lys 480 , and Asp 369 ) and defines a new region of the M4M5 loop involved in ATP binding. This peptide has previously been thought to be closely associated with the membrane, because of its hydrophobic nature and as a result of chemical modification studies with lipophilic reagents. For example, this peptide was labeled with hydrophobic reagents such as 3- trast, this peptide has been identified in the supernatant after precipitation of the membrane preparation of Na,K-ATPase subjected to limited trypsin hydrolysis (31). A more comprehensive study of the substrate dependence of extensive trypsin digestion of the Na,K-ATPase showed that this peptide could be either associated with the membrane or in the soluble fraction after extensive digestion, depending upon which ligands were present during digestion (32). Such evidence suggests that this peptide is either partially embedded in the membrane or on the surface of the intramembrane moiety of ATPase. It is also possible that this peptide may form a hydrophobic pocket capable of selectively binding lipophilic reagents.
Protein sequences from various P-type ATPases have been aligned with the model of the calcium pump proposed from the limited three-dimensional structural data available (33)(34)(35). These models are essentially identical with the exception of the numbering of the  sheets in the ATP-binding domain. A comparison of Na,K-ATPase with the structural model indicates that the hydrophobic peptide labeled by MIANS is represented by ␣3 and 3 (33). In this model, Cys 549 would lie at the C-terminal portion of ␣3, and Cys 577 would be approximately in the center of 3. The proposed three-dimensional structure from this model places Cys 577 in close proximity to other residues such as Lys 501 and Lys 480 , modification of which has been shown to result in ATP-protectable enzyme inactivation (34). Furthermore, this model places the tail end of the MIANSlabeled peptide (i.e. D 586 PPR 589 ) immediately after 4 and protruding into the ATP-binding cleft (34). The DPPR sequence is highly conserved among P-type ATPases, and recent evidence from mutagenesis studies of the Na,K-ATPase suggests that this region is the Mg 2ϩ ion coordinating site (36,37). It is further hypothesized that this Mg 2ϩ ion enters the enzyme coordinated by the and ␥-phosphates of ATP and remains bound (at least in part to Asp 586 ) after ADP is released (37). In the context of this model, one can easily understand why the introduction of a large molecule such as MIANS at Cys 577 would inhibit the enzyme and how preincubation with ATP would prevent MIANS labeling.
Evidence Consistent with MIANS Labeling the ATP-binding Domain-The observation that the target for inactivation by MIANS, Cys 577 , is in the central loop and can be protected against reaction with MIANS by the presence of ATP suggests that this residue is in the ATP-binding domain. In addition, the inactivation of the enzyme that results from modification is due to a loss of high affinity ATP binding (Fig. 5). However, it is not clear yet whether Cys 577 is a contact site for ATP binding in the nucleotide-binding domain or whether MIANS exerts an inhibitory effect by virtue of its bulk or by alteration of the tertiary structure of this region following modification. It is likely that answers to these questions will only become available when the Na,K-ATPase, or the cytoplasmic loop containing the ATPbinding domain, is crystallized in the presence of substrate. Recent studies suggest that this latter approach may be a productive strategy and a His 6 46-kDa peptide corresponding to the M4M5 loop has recently been shown to exhibit ATPprotectable labeling by MIANS (7).
There is also much suggestive evidence that Cys 577 is in the ATP-binding domain. For example, it has been reported previously that several aromatic isothiocyanates, which modify Lys 501 in the central loop of the ␣-subunit, react much more readily when the enzyme is in the E 1 Na form than in the E 2 (K 2 ) form. These include fluorescein isothiocyanate (6), N-(2-nitro-4-isothiocyanophenyl)-imidazole (4), and DIDS (5,24). This increase in reactivity upon sodium binding is another example where occupancy of the cation-binding region of the protein, believed to be contained within the intramembrane segments, affects the properties of the ATP-binding domain in the cytoplasm. However, since all of these compounds react with Lys 501 , it was unclear whether these cation-induced changes: 1) significantly altered the nucleotide-binding pocket, thus allowing easier access to the site, or 2) were relatively small and localized to the region close to Lys 501 . In the present work, we found that Na ϩ ions also increased the reactivity of Cys 577 toward MIANS. Thus, it appears that cation binding effects are not limited to Lys 501 but rather are transmitted a considerable distance along the primary sequence. It is interesting that Cys 577 has been postulated to be close to Lys 501 in the tertiary structure (see above).
Furthermore, MIANS modification of Cys 577 showed a biphasic dependence on K ϩ ion concentration. That is, at low concentrations, the presence of K ϩ ions protect against modification, whereas at high K ϩ ion concentrations the effect is reversed and Cys 577 retains its reactivity. A similar observation was first reported for DIDS inactivation of the Na,K-ATPase (24) and later with several other arylisothiocyanates reacting with Lys 501 (6). It seems that this biphasic effect has relevance to the normal functioning of the pump as the modification by ATP site probes (i.e. MIANS, DIDS, and fluorescein isothiocyanate) was similar in the presence of K ϩ congeners such as Rb ϩ and Cs ϩ (this work and Refs. 5 and 6). Although several complex models can be proposed to explain this behavior, we believe the simplest model is that the enzyme conformation with a single K ϩ ion bound, E 2 (K), differs from that with two K ϩ ions bound E 2 (K 2 ) (for details see Ref. 6). This difference involves either changes in the environment near Cys 577 and Lys 501 or a change in the overall ATP site accessibility. Nonetheless, Cys 577 (like Lys 501 ) is conformationally mobile, and its reactivity toward MIANS reflects this flexibility.
Previous work on the Na,K-ATPase with MIANS focused on the rate of reaction of Cys residues without identifying the modified residues which caused inactivation (14). These investigators did not observe cation-selective effects in the total labeling but like us saw effects of ATP, which reduced the site and extent of reaction of protein cysteines under somewhat different conditions (e.g. protein/reagent ratios, temperature, and ionic conditions; cf. Ref. 14 and this work).
Domain Interactions and the Mechanism of Cation Pumping-The active transport of Na ϩ and K ϩ ions involves coupling the hydrolysis of ATP to the transmembrane movement of these ions. These separate but coupled functions seem to occur at spatially separate but linked regions of the protein. To understand the mechanism of active cation transport, it is necessary to understand how these separate protein regions interact with each other. Recent work from chemical modification studies or site-directed mutagenesis of the Na,K-ATPase has identified the M5M6 region as being intimately involved in cation binding and occlusion (38 -40). In particular, there are four residues, Ser 775 (38), Glu 779 (38,39,41,42), Asp 804 , and Asp 808 (43), that appear to be involved in cation transport. Furthermore, different experimental approaches suggest that this M5M6 hairpin may move during the catalytic cycle and play an active role in the cation translocation process in both Na,K-ATPase (44) and the H,K-ATPase (45). The recent suggestion that ouabain may interact with the extracellular loop between M5 and M6 is consistent with both ouabain inhibition and K ϩ antagonism of ouabain binding (46).
In a similar way to the identification of the M5M6 hairpin in cation binding, chemical modification studies and expression of the central loop of the ␣-subunit have suggested that most of the ATP-binding residues are in the large cytoplasmic loop between M4 and M5 (7,47). The chemical modification results presented in this paper identify yet another residue that appears to be in a location intimately involved with ATP binding. Indeed, the data show that the environment around Cys 577 changes when either K ϩ ions or Na ϩ ions are bound in the cation-binding domain. Other evidence has also suggested that the position of several residues or segments with respect to the membrane-aqueous interface change as the enzyme assumes its different conformations (32). It is becoming more evident that these cation-induced structural changes are the basis for the long known ATP affinity differences that exist when the enzyme exchanges Na ϩ for K ϩ at its binding sites (13). Therefore, it seems that modification with agents such as MIANS can begin to reveal the regions of the ATP-binding domain that undergo spatial rearrangements during such conformational transitions. | 2019-03-20T13:09:26.935Z | 1999-08-27T00:00:00.000 | {
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13702235 | pes2o/s2orc | v3-fos-license | One Step Forward: Development of a Program Promoting Active School Transportation
Background: Physical activity promotes health and learning. However, up to 80% of the children in industrialized countries do not achieve the recommended level of daily physical activity. By encouraging children to use active school transportation (AST), it is possible to increase their overall physical activity. Objective: The aim of this paper was to present the development of an AST intervention using Intervention Mapping (IM) to promote children’s physical activity. Methods: The principles of IM were applied to guide the development of the intervention. The process was divided into 3 phases. First, a literature review and collection of experiences of stakeholders were carried out to gain a broad perspective on the problem and possible solutions. Thereafter, an analysis of the critical environmental and behavioral factors affecting outcome was conducted, which guided the choice of tangible components of the intervention. Finally, a plan of evaluation and implementation was established. Results: A structured program to increase AST among children was developed, consisting of 3 subsequent phases that are described in detail. Implementation took place, and evaluation of the intervention is being carried out. Conclusions: IM proved to be a valuable method to develop a structured AST intervention for children. By following the steps of the IM process, it became evident that empowerment and gamification are 2 promising avenues to consider when designing AST interventions in a school context. By engaging end users and including important agents, such as parents and teachers, who control the environmental factors, the possibility to design a sustainable program increases. In addition, gamification made it possible to integrate learning into AST, which could motivate schools to devote time and effort to implementing this program.
Introduction
Background Although many studies have illuminated the ways in which physical activity provides children with fundamental health benefits, children are not physically active to the extent that is needed [1]. Among Swedish children aged 11 years, only 21% of males and 13% of females achieve the recommended daily levels of physical activity [2]. This is even more problematic when considering the increasing problem with childhood obesity, which has risen substantially in most high-income countries over the last three decades [3]. To reach the recommended levels of activity, children could be encouraged to walk or bike to school, which are known as forms of active school transportation (AST) [4]. However, many industrial countries have seen a decline in AST among children and adolescents, and an additional decline is likely in the absence of interventions to increase AST [5,6]. Though many studies have focused on the social and environmental factors that influence children's AST, the interventions developed have proven insufficient for promoting AST [7]. Only a few schools implemented these interventions, perhaps as a result of the disconnect between research and schools' needs [8]. To date, several concerns about school interventions for promoting physical activity have been highlighted in reviews. It has been pointed out that research has failed to determine the contextually sensitive attributes that define successful school-based interventions [9], and rigid scientific methodologies and evaluation techniques are incompatible with societally complex issues [10]. Given the disconnect between what was intended and how AST is carried out, there is a need to analyze physical activity interventions in schools to find ways to increase their feasibility and sustainability.
This paper aims to elucidate the logic model for the problem and the solution, as well as the theoretical underpinnings of an intervention promoting AST. Intervention research is defined as the process of creating the elements of an intervention and refining those elements through a series of studies [11]. By using theory and evidence as foundations in intervention research and by presenting multiple theoretical and experiential perspectives to solve a problem [12], other research teams can be inspired to build on this or prevent future mistakes. Intervention Mapping (IM) is both a framework and a structured way of planning, implementing, and evaluating health promotion programs [13]. The theoretical underpinning of IM is the social ecological model, which states that health is a function of the individuals living in an environment. Solving a health promotion problem requires a systematic perspective. The environments include physical, social, and cultural factors, in addition to agents exercising control over these factors at each ecological level, such as interpersonal, organizational, community, or societal environments.
Objective
The aim of this paper was to present the development of an AST intervention using IM to promote children's physical activity.
Intervention Mapping Tool
IM, a 6-step tool that maps the path from recognizing a problem to identifying a solution, was used to develop the program. The process was iterative rather than linear and the planners moved back and forth, as they gained new information and perspectives. The 6 steps include (1) logic model for the problem, (2) logic model for the solution, (3) program design, (4) program production, (5) program implementation plan, and (6) evaluation plan [13]. During program planning and execution, all planning group meetings were documented, as were field notes from every step of the process.
Participants
Program development was driven by a planning group consisting of the 2 authors with research experience concerning children and physical activity, one researcher with research experience concerning AST with a special focus on environmental engineering, 2 teachers, the principal, and the head of the municipality's planning department.
The research project was performed in a municipality of approximately 80,000 inhabitants situated in northern Sweden. Five schools with infrastructure that could allow first-grade children to walk or bike to school were invited to participate in the study, and one school agreed to participate. This primary school had 270 schoolchildren, and the neighborhood included both apartment houses and detached houses. The 2 cohorts consisted of 45 children aged 7 to 8 years (25 males). The parents and the children were informed about the study by the authors, both face-to-face and in writing, and 42 children (23 males and 19 females) agreed to participate. All parents were also invited to participate, and 63 parents (26 fathers and 37 mothers) did so. Of the 63 parents, 46 had a college/university education and the remaining 17 had an upper secondary education. All but 2 were Swedish citizens. The distance between home and school varied between 0.2 and 6.0 km, with an average distance of 1.3 km.
Procedure
Program development began by forming the planning group, which was active during the whole project.
Phase 1
First, a logic model for the problem was formulated, which contained an assessment of the determinants and behavioral and environmental factors. On the basis of this model, a logic model for the solution was created. Gradually, these steps became increasingly intertwined. The phase was informed by a review of the theory and literature concerning children's physical activity interventions (in particular AST, empowerment, and gamification). In addition, several other contacts were made within the municipality, surrounding community, and related scientific areas of research in order to understand the community, its members, and the theoretical models for the problem and the solution. We created a joint logic model for the problem and for the solution, which were inspired by the 2 models of Bartholomew Eldredge [13].
Phase 2
Once the theoretical framework was established, it was used to guide the tangible program components. To be true to an empowerment approach, the program development was a partnership between the teachers, parents, and children that created something that they perceived to be meaningful.
Phase 3
This phase included a plan for evaluation and implementation. Preliminary results were presented and discussed during this phase.
Ethical Considerations
The study was performed in accordance with the principles of the Swedish law for research ethics and the Declaration of Helsinki's Ethical Principles for Medical Research Involving Human Subjects [14]. The study was approved by the Regional Ethical Board in Umeå, Sweden, before the start of the research project (issue date: February 9, 2016; application registration number: 2015/296-31Ö).
Results
The outcomes of the IM process are described in 3 different phases. The process began with the formulation of a logic model for the problem and the solution, and it continued with the program design and production. Ultimately, an evaluation plan was established.
Theory-and Evidence-Based Factors Affecting Active School Transportation Behavior
To identify the factors related to AST behavior, social cognitive theory was used because it has been proven useful for developing effective physical activity behavior change interventions in children [15]. Social cognitive theory conceives of individuals within a collective context, as people do not operate as isolated individuals [16]. Social cognitive theory specifies a set of constructs, including knowledge, perceived self-efficacy, outcome expectations, goals, perceived facilitators, and impediments to change. Self-efficacy is altered by direct mastery experience, vicarious experience, and social persuasion [16]. Thus, self-efficacy might be applicable to this program because it could affect AST performance, modeling, and social support. Previous studies confirmed that self-efficacy mediates the causal pathway between interventions and children's physical activity levels [17]. However, research examining social cognitive theories and physical activity has largely focused on adult populations, and there is limited knowledge regarding children [12].
Research has recognized that gamification has great potential to promote children's physical activity and learning [18,19]. Gamification is defined as the use of game design elements in a nongame context [20]. The use of gamification enhances the possibility of capturing the components that make games engaging, and it can be used to improve the effectiveness of health promotion initiatives [21]. Promising research suggests that gamification can promote AST [22]. By using elements such as recurrent assignments that grant badges to students and allow them to level up to the next challenge, this approach can potentially ignite internal motivation to engage in healthy behavior. Acquiring badges can drive knowledge acquisition and behavior change [23].
The participatory elements of empowerment can improve an intervention's compatibility, and they increase the likelihood that effective programs will be sustained [24]. One study has shown that problems with intervention design and evaluation can be overcome by taking advantage of the end users' involvement and by explaining the connections among programs, policies, and evaluations [25]. We have previously performed promising school-based research using empowerment in order to promote physical activity [15]. According to Tengland [26], using an empowerment approach involves minimizing the influence of professionals, and the individual or group that is in need of support should take responsibility for the change process. Furthermore, empowerment and children's active participation can increase knowledge acquisition and competencies [27]. Yet, critics have claimed that professionals should not reduce their power over projects because professionals are part of the project and must have a say in decisions [26].
Agents Who Control Environmental Factors
In the social ecological model, parents are an important interpersonal factor. Because children in this program were only 7 to8 years of age, their parents are the gatekeepers of their children's AST. Our previous research concluded that parents are important as role models, providing encouragement and tangible support [15]. Therefore, parental attitudes toward AST are a factor that could constitute either a problem or a means of facilitation. Using parental attitudes as a starting point and including them in program development are consistent with Bandura's theories [16] because environmental factors (such as social support) are a central element of both social cognitive theory and previous research [28]. However, few interventions have been based on parents' psychological factors on an intrapersonal level, such as parents' perceived barriers, outcome expectations, and self-efficacy [29].
An organization-level factor that is causally related to successfully increasing AST is school involvement. Promoting health might appear to be an added burden when the primary focus of schools is to meet academic standards. In fact, physical activity is sometimes seen as a competitor to academic studies because the time devoted to physical activity could instead be devoted to academic work [30]. Good health is critical for achieving an optimal education, and studies have found associations between children's physical activity and academic performance [31]. Moreover, research has shown that physical activity improves children's cognition and brain health [32]. This association should be a motivator for schools to be involved in increasing AST.
In this study, teachers were highly involved in the development of the program. For instance, they created special weekly assignments, were responsible for measuring distances for each child's AST, and helped determine how to use these data in their teaching. Therefore, engaging teachers is required for the success of an AST program.
Phase 2: Program Design and Production
Once the theoretical framework was established, the planning group addressed the critical environmental and behavioral factors that were most likely to affect the outcome (Textbox 1).
Textbox 1. Critical environmental and behavioral factors and actions that promote active school transportation (AST).
•
Empowerment of school personnel
• Action: Planning workshops, assignments, and measurement of AST Environmental factors we identified were parental knowledge of AST, attitudes toward AST, and perceived AST barriers. Therefore, during a parenting meeting, we informed parents about the benefits of physical activity for their children in terms of both health and academic performance. We also let parents discuss their doubts and fears around letting their children walk and bicycle to school and discussed solutions. In addition, we assessed parental psychological factors on an intrapersonal level using a questionnaire and discussions during this meeting. We analyzed the results from the questionnaire, determined their specific worries, and suggested solutions that were used as a starting point for program design. The questionnaire was the Modified Integrated model for Children's Active Commuting to School, which has been shown to fit well with this model, and thus, may enable health behavior researchers to design effective interventions to promote AST [29]. Parents highlighted 2 main concerns: (1) having an accident when crossing roads with heavy traffic and (2) meeting a harmful person (stranger danger) while in transit during AST. Examples of suggested solutions used in the program were pairing children according to geographic location and encouraging them to walk or bicycle together to school. Children whose paths included heavily trafficked roads were accompanied by parents or older children. To further enhance the children's motivation to use AST, the program used gamification elements. Every day during the 4-week test period, the children put a sticker on a collective board for every kilometer they walked or bicycled between home and school, and the board's results were integrated into lessons. For example, Mathematics class was integrated with Geography class, and the children summed the total number of kilometers achieved each week, converted kilometers into miles, and identified the locations on a map. In our previous research, we found that measuring physical activity was an effective part of a school-based intervention, and this motivated adolescents to be more physically active [33].
Another gamification element was special assignments (a challenge) that the children were encouraged to solve each week. The teachers selected the assignments, which were directly connected to the curriculum. One of the assignments on the first week was, "Which traffic signs do you encounter on your way to school?" The assignments were integrated throughout the curriculum. For example, in the Art class, the children painted pictures of traffic signs. Another assignment during the week was a security check of the bicycles in which the children followed a checklist concerning legal requirements for bicycle equipment. The second week's special assignment was, "Count how many people you meet when walking or bicycling on your way to school," and this assignment was integrated into the Math class. The assignment for the third week was "Bring a plastic bag and collect some litter on your way to school." This assignment was used in a lesson concerning the environment in which the children learned about and practiced sorting litter into plastic, paper, etc. They also learned about what happens to animals and nature if the litter is left out in the environment. The fourth week's assignment was "Notice which signs of spring you encounter on your way to school," and this assignment was integrated into Biology class. Each Friday, the teachers summarized the assignment for the week, and a badge was awarded to the class for successful achievement.
Phase 3: Program Evaluation Plan and Implementation Plan
Developing a plan for the adoption, implementation, and sustainability of the program in real-life contexts began when the needs assessment was undertaken, as careful examination of the end users' needs contributed to program compatibility.
To evaluate the program, we collected experiences from the children and teachers through focus groups. We used a semistructured interview guide to cover all aspects of the program. The opening question was "Let's pretend I know nothing. Could you please tell me about the program?" To expand upon the answers, follow-up questions were asked. Seven focus groups with 4 to 5 children in each group and one focus group consisting of the teachers were convened. The focus groups' discussions lasted for 16 to 45 min. Collectively, the 42 children walked or biked for 1189 km over 4 weeks, which averaged to 1.4 km for each child per school day. Bicycling contributed to at least 15 min of extra physical activity each day. The preliminary analysis of the focus groups showed that the students got motivated by the gamification elements of the intervention (ie, incorporating learning activities into assignments and measures of the use of AST), and they gained learning outcomes using time outside scheduled school hours. Furthermore, the teachers in this project found it highly rewarding to incorporate learning into AST because it made it possible to use real-life situations to teach various subjects in the curriculum.
In addition, before the intervention, we collected data on parental attitudes using the Modified Integrated model for Children's Active Commuting to School questionnaire, and the parents answered an open letter as well, which was introduced with the following text: "You have answered a questionnaire concerning AST, including obstacles your child might experience while using AST. Describe how these obstacles can be overcome." Two weeks after ending the intervention, a second open letter was introduced with 4 questions: "How have you as a parent experienced your child's participation in the AST project"; "If this project is used in a different class, is there something we should do again, and are there things that should be changed"; "Which attitude towards AST did you have before the intervention, and have your attitudes changed after participating"; and "Has your own choice of travel modes changed during the project?" The knowledge gained from these data will provide important information on key program components, which will enable us to develop sustainable health-promoting programs in a school context. We have planned to implement this program in several schools, and after implementation, feasibility and efficacy studies using cluster randomization are planned.
Principal Findings
In this paper, we describe the systematic development of an intervention program aimed at promoting AST among children. By following the steps of the IM process, it became evident that empowerment and gamification are 2 promising avenues to consider when designing AST interventions in a school context.
Using empowerment and engaging end users was essential. By forming a planning group that included important agents who control the environmental factors, it was possible to create a promising approach for developing a sustainable physical activity program in a school that could yield positive outcomes for children. In young children, parents are the gatekeepers and they decide whether the child will use AST or other means of transportation. Thus, it is essential to involve them in the process. However, this approach is unusual. A common theme that occurs in many school-based research articles is the lack of engagement by end users in planning, implementing, and evaluating health promotion activities [25,34]. Therefore, we anticipate that one success contributor for this program was the use of empowerment.
A second important cornerstone was the use of gamification, which made it possible to integrate learning and AST through the use of school assignments, including ones that incorporated measurements of AST usage. Preliminary finding showed that gamification motivated children to use AST, a finding that is supported by Hamari et al [35]. There is an ongoing debate within gamification research as to whether specific game elements may actually undermine users' intrinsic motivations [36]. A study examining the effects of 3 commonly employed game design elements (points, leaderboard, and levels) on users' performance and intrinsic motivation showed that these game elements significantly increased performance but did not affect intrinsic motivation. These findings suggest that points, levels, and leaderboards by themselves do not make or break users' intrinsic motivation in nongame contexts [36]. However, the use of gamification in schools must be further explored, as the relationship between the engaging aspect of games, learning, and physical activity is still unknown [37].
Future Work
By integrating learning activities into the project, schools may be more motivated to put time and effort into implementing this program. There are complex interactions among socioeconomic, environmental, and ethnic and cultural differences, and these may be important to account for when designing effective programs to promote children's AST. Future evaluation methods need to include a target group that displays a wider range of socioeconomic factors.
Conclusions
IM proved to be a valuable method to develop a structured intervention for an active school transporting intervention for children. By following the steps of the IM process, it became evident that empowerment and gamification are 2 promising avenues to consider when designing AST interventions in a school context. By engaging end users and including important agents, such as parents and teachers, who control the environmental factors, the possibility to design a sustainable program increases. In addition, gamification made it possible to integrate learning into AST, which could motivate schools to devote time and effort to implementing this program. | 2018-05-11T16:40:29.095Z | 2018-05-01T00:00:00.000 | {
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51959907 | pes2o/s2orc | v3-fos-license | Regional Scale Determinants of Nutrient Content of Soil in a Cold-Temperate Forest
The effect of climatic factors on soil nutrients is significant. Identifying whether soil nutrients respond to local climate and how the forest types modulate this responsiveness is critical for forest management. Therefore, six soil nutrients from five main forest types found for a range of sites within the Daxing’an Mountains, China, were investigated. Climatic factors were obtained from the WorldClim dataset. Pearson correlations and stepwise regressions were employed to elucidate and model the response of the six soil nutrients to the four different climatic factors in this study. On the whole, climate was correlated with all the nutrients. Further, from stepwise regressions, climatic factors could affect soil nutrients in distinct forests. Our findings suggest that climatic factors are instrumental in affecting soil nutrients in different forest types. Identifying the relationships between soil nutrients, climatic factors and forest types, as suggested in this research, can provide theoretical foundations to further comprehend nutrient cycling in the forest ecosystem.
Introduction
Climate changes have significant effects on ecosystems.In the present paper, with the primary focus on the links between ecosystems and climate change, gradients of natural climate are noteworthy in studying the interactions between climate and variation in forest ecosystem processes.Terrestrial ecosystems play a dominant and irreplaceable role, due to the functions of releasing and absorbing greenhouse gases in such climate-feedbacks, while storing a great deal of carbon in vegetation and soil, thus serving as the global carbon sink [1].Some studies have shown that there are strong linkages between climate change and soil.The study of Brittany et al. showed that the gradient of climates (precipitation and temperature) has obvious regulating effects on the physical and chemical properties of soil, such as pH, Mg 2+ , N, P and K content [2].The effects of climatic factors on SOC (soil organic carbon) density were obvious and stronger than those of grassland and farmland [3].Furthermore, regression analysis showed that temperature has a negative correlation with SOC content, and precipitation has a positive correlation with SOC content, but using multiple regression analysis, temperature and precipitation explained 43% of total variance in the SOC variables [4,5].Soil organic matter related to SOC, total nitrogen (TN), total phosphorus (TP), total potassium (TK), available P (AP) and available K (AK) has been extensively used to evaluate soil quality [6][7][8][9].Moreover, forest types can impact the cycling and amounts of the nutrients, and nutrients have been confirmed to be influenced by the upper layer [10].However, the impacts of tree species upon soil nutrients varied depending upon the type of bedrock, climate and forest management [11].Therefore, understanding the relationships between soil nutrients and climate change in different forest types will provide more reliable information to prudently manage forest resources and promote sustainable forestry development under climate change in the future.
The Daxing'an Mountains forest area is in the mid-latitude and high-latitude area that is extremely sensitive to global warming [12].The Daxing'an Mountains forest area is the main forest in China.It plays an important role in carbon sequestration management and ecological environment construction.Nevertheless, under the influence of climate change, the edge of the forest has retreated 140 km over the past century in this region [13].Therefore, the soil nutrients of different vegetation types in this region have attracted widespread attention.Jiang et al. [14] studied the soil nutrients of different forests.However, there is less research focused on the soil nutrient characteristics in different forest types in the Daxing'an Mountains forest area.Although the distribution of SOC, N, P, and K in the Liaodong Mountains area [15] and the correlations between SOC, inorganic carbon and soil nutrients in the northeast of China [16] have been studied, studies reporting research related to the comprehensive evaluation of soil nutrients from different forest types in the Daxing'an Mountains forest area are scarce.
In this study, the soil nutrients of a total of 230 sample plots collected from five main forest types were measured from the Daxing'an Mountains, and four bioclimatic variables (mean annual temperature (MAT), temperature seasonality (TS), mean annual precipitation (MAP) and precipitation seasonality (PS)) were obtained from the WorldClim dataset.We hypothesized that climatic factors could affect soil nutrients in different forest types.Thus, identifying whether soil nutrients respond to local climate and how the forest types modulate this responsiveness is critical for forest management.
Site Description
The forest in the Daxing'an Mountains is one of the most important areas in China: the lush natural forest is distributed widely.It is an important production base for forest trees in China, and also an important ecological barrier in northeastern China.The study area comprises about 86,000 km 2 and belongs to the cool coniferous forests.The investigated forest plots are shown in detail in Figure 1
Field Soil Sampling and Preliminary Analysis
The forest-covered area of the Daxing'an Mountains was systematically divided into 30 km × 30 km grids using ArcGIS 10.0 (Esri, Redlands, CA, USA) as the meshing tool.The exact latitude and longitude for each grid were recorded with a GPS system (Google, Mountain, CA, USA) [18,19].Soil sample depth was 0-20 cm [3,4], taken from 3-7 plots (30 m × 30 m each) in each 30 km × 30 km grid (total grids = 52), and 3-7 plots were chosen based on the investigation areas.As much as possible, we chose plots from the central region of the grid; the distance of each plot to the edge of the grid must be more than 15% of the length on the side of the grid.A total of 230 sample plots were included in this study, and the geometric center coordinates for each sample plot were input into Excel, saved in CSV (Comma Separated Value) format, and the ArcGIS 10.0 software was used to extract the climatic data for each sample plot [19].
SOC was determined by external heating with the potassium dichromate oxidation method; TN was determined by the Semi-micro Kjeldahl method; TP and TK were determined by the method of the NaOH melt-Mo-Sb Colorimetry; AP was determined by the method of the HCl-NaOH extracts; AK was determined using the flame photometry method [20,21].
Climatic Data
Climatic data were obtained from the WorldClim database (http://www.worldclim.org/), the accuracy class of which is a spatial resolution of approximately 1 km 2 .The WorldClim data are collected from weather stations across the globe, which include altitude, temperature, and rainfall (period 1950-2000) [22].In the present study, four bioclimatic variables (MAT, TS, MAP and PS) were considered to assess the current climatic conditions.The IPCC 4th assessment data provided information for the future climate projections [23].
Statistical Analyses
The multivariate statistical analysis method has been employed to determine the minimum dataset under the hypothesis that soil nutrients significantly impact forest type.Principal component analysis (PCA) has previously been applied in different research fields to identify nutrients in semiarid soils [24,25] and soil pollutant sources [26] as well as to assess the effect of tillage on soil quality and yield [27][28][29][30].Dimension reduction analysis by using the PCA method to reduce the dimensional data and eliminate the redundant data [31,32].In our research, we built a hypothesis about which principal components (PCs) possess the highest eigenvalues, variables, and absolute eigenvectors and may best express the minimum dataset.
Pearson correlation coefficients were employed to evaluate the correlations between climatic factors and soil nutrients.Analyses of regression are helpful for inspecting differences among group comparisons; therefore, they are suitable for assessing the variation of soil nutrients under diverse climatic factors.To test whether the climatic factors (MAT, TS, MAP, and PS) affected the soil nutrients (SOC, TN, TP, TK, AP, and AK), a simple linear regression was used for each biological element of the 230 sites with the four climatic factors.To study forest types, specifically the response to climate changes, the climate-change response trends were compared among the forest types.The slopes of the regression lines were used to indicate the different responses of forest types to the climate changes.Linear models compared with non-linear models (Spearman Rank Correlation) gave the best regression results.In addition, stepwise regression between climatic factors and soil nutrients in five main forest types was also analyzed (F-to-enter p ≤ 0.05, F-to remove p ≥ 0.10).
The statistical analyses were conducted using SPSS 17.0 software, while the graphs were made using OriginPro 9.0 software (OriginLab Corporation, Northampton, MA, USA).
Variation of Soil Nutrients and Climatic Factors in Different Forest Types
In Figure 2 The averages of the MAT and MAP were −1.74 • C and 534.93 mm, respectively; the ranges of the TS and PS were 14,772-16,885 and 95-116, respectively (Figure 3).MAT and PS were higher in the PQ than in the other four forest types, while TS showed the opposite trend (Figure 3).
Principal Component Analysis of the Different Forest Types and the Soil Nutrients
The results of the PCA showed the variables that characterized the soil nutrients of the different forest types (Figure 4).PCs 1-6 explained 100.0% of the variation, and can be broken down as follows: 95.55%, 2.22%, 1.13%, 0.73%, 0.24% and 0.12%, respectively, as shown in Table 1.Principal component 1 can reflect most of the variation; it includes TN (0.296), TK (0.816), TP (−0.263),AP (0.749), AK (0.447) and SOC (−1.302).As shown in Figure 4, five main forest types showed PQ separated from other types.Extraction Method: Principal Component Analysis.
Correlations between Climatic Factors and Soil Nutrients
The relationships between soil nutrients and climatic factors are shown in Table 2. MAT was negatively correlated with SOC, TN, TK, AK and AP, while it was positively correlated with TP (r = 0.187) (p < 0.01).TS was positively correlated with SOC, TN, TK, AK and AP (r = 0.307-0.417),while it was negatively correlated with TP (r = −0.405)(p < 0.01).In contrast to TS, the relationships between PS and soil nutrients showed the opposite trend (p < 0.01).MAP was positively correlated with SOC, TN, AK and AP, while it was negatively correlated with TK and TP (p < 0.01).Moreover, relationships between soil nutrients and climatic factors in the five main forest types were also observed.In Table 3, p (*, **) value represents whether climatic factors are correlated with soil nutrient contents, so as to determine whether there is statistical significance.The r value indicated the correlation between climate factors and soil nutrient contents.MAT was significantly and positively correlated with nutrients in PL, PB, and MLB, except for SOC in PB.TS had no effect on almost all nutrients but positively correlated with SOC in PB.MAP was similar to MAT in PL, PB, and MLB; in addition, MAP was also significantly correlated with TN, TK, TP, AK, AP in PP and TN, TP in PQ.Although the correlation between PS and nutrients was unimpressive compared with MAT and MAP in five forest types, it was negatively correlated with SOC in PL, TN, TP and TK in MLB, as well as SOC and AK in PP.
Stepwise Regressions between Climatic Factors and Soil Nutrients
Step regression between soil nutrients and climatic factors in the five main forest types is shown in Table 4.In the case of PB, the four climatic factors could affect all the six soil nutrients, and MAP was the first parameter entered into the model.In the case of MLB and PP, MAP and MAT were the key factors for influencing the five soil nutrients (TN, TP, TK, AP and AK).For PL, MAT, MAP and TS mainly affected SOC, TN, TP, TK and AK.However, for PQ, MAP was the key factor for TN and TP, and no parameters were entered into the model of SOC, TK, AP and AK.In all, we found that there were different influencing factors in various forest types.Note: SOC = soil organic carbon, TN = total nitrogen, TP = total phosphorus, TK = total potassium, AP = available phosphorus, and AK = available potassium.PL = pure Larix gmelinii (Rupr.)Kuzen forest, PB = pure Betula platyphylla Suk.Forest, MLB = Larix gmelinii (Rupr.)Kuzen and Betula platyphylla Suk.mixed forest, PQ = pure Quercus mongolica Fisch.ex Ledeb.forest, PP = pure Pinus sylvestris L. var.mongolica Litv.Forest, "-" = there is no value.
Forest Types Influence Soil Nutrient Contents
The relationships between soil nutrients and forest types have been presented previously [3].In addition, northeastern China has been considered as one of the regions with the most abundant soil nutrition [16,33].To confirm the influencing trends of the variation of soil nutrient contents to forest types, PCA was carried out on the collected data.From the distribution of the loading plot in the PCA space, it was found that the forest type influenced the soil nutrients.For instance, SOC, TP, TN, AK, AP and TK were higher in PL, PB, MLB and PP in this study, while these soil nutrients showed the opposite trend.So, we suspect that there were certain correlations between soil nutrients and forest type.In addition, the differences caused by vegetation effects in the responses of the nutrients may be due to slight distinctions in parent material in different forest types [34,35].
From the above results, it was found that the distributions of soil nutrients from different forests were different.These values were influenced by the forest site conditions, advantageous tree species and different amounts of forest litter as well as the composition and decomposition levels, so the differences in the forest soil nutrients are very obvious.For instance, the distribution of SOC was ranged in order PL > MLB > PB > PP > PQ with the SOC average content being 28.68, 28.46, 28.43, 28.28 and 26.68 g•kg −1 respectively.From the viewpoint of succession, PL, MLB and PP were in the top stage of succession-the complexity of the tree species composition increased the possibilities of the accumulation of organic matter [36]-but PB and PQ were the secondary forests which were disturbed more frequently in recent years.So, the SOC content of PL, MLB and PP should be larger than that of PB or PQ [37].However, the SOC content in PP was minimal, even lower than in PB.This phenomenon was unexpected, and it is possible that it is related to the terrain: the slope is large, litter does not accumulate as much, and in addition to the soil acidity, the litter layer was difficult to decompose; therefore, the conditions are not conducive to the formation of organic matter, which means that the SOC content is low [3].This also means that SOC stock will continue to increase if the interference is ended and the forest is developed toward the climax community; otherwise, the forest can turn into PQ and the stock of SOC will decrease, especially in the rich PB forest region.
Statistically, the distributions of TN and SOC were identical.A large number of data analysis results show that the TN was positively correlated with SOC.The order was PL > MLB > PB > PP > PQ with the contents being 4.2, 4.12, 4.11, 4.02 and 3.50 g•kg −1 respectively.The distribution of TN identified in this study was in accordance with that presented by Zu et al. [16] and Jiang et al. [14].The order of the TP content was PP > PL = MLB > PB > PQ and the contents were 1.81, 1.76, 1.76, 1.70 and 1.35 g•kg −1 respectively.The correlations of the AP and SOC were identical but opposite to that of TP.The same phenomenon appeared with AK and TK, and this could be explained by the composition of TP and TK, which is very complex, with the existence of inorganic and organic states, and AP and AK being only part of them.This trend may have been due to the influence of various factors such as the climate.In addition, although AP content decreased with the MAP and MAT increasing, TP content increased [32,33].This confirms that the vegetation type is a key factor that affects the soil nutrients of the Daxing'an Mountains ecosystems.
Soil Nutrient Responses to Climatic Factors
The study of Harradine, F. et al. indicated that climate (especially precipitation and temperature) has significant effects on pedogenesis and macronutrient cycling in soil [38].It is a challenge to isolate each of the individual soil forming factors such as climate, vegetation, parent material and so on, due to the frequent co-variance of many factors [39].For instance, changes in species vary with the climate and location.In the present paper, with the primary focus on the links between ecosystems and climate change, gradients of natural climate are noteworthy in studying the interactions between climate and variation in forest ecosystem processes.
On the whole, it was found that SOC decreased with increasing MAT, and SOC increased with increasing MAP (Table 1).This trend may be due to the hydrothermal conditions of Daxing'an Mountains area.The MAT in Daxing'an Mountains is −3.69 • C and the MAP is 481.2 mm.The region is rich in forest resources, and rainfall is abundant which is conducive to the growth of plants, while the low temperature is beneficial to the accumulation of biomass.Yimer, F. et al. suggested that some other factors, such as erosion, leaching of cations and variations in biomass production may influence soil property [39].Our results were consistent with previous research which showed that increasing temperature leads to the growth of microorganisms [40], thus increasing the decomposition rate of SOC [41,42].Precipitation change will affect the content of plant-available water and the length of the growing season; a reduction in precipitation can limit plant growth [30], and the soil microbial number will surge after rain [43], thus reducing the SOC content in soil.In this study, a similar conclusion can be drawn.
More than half of the soil nutrients were significantly linked with variations in the climatic factors, but PQ had weak correlations between climatic factors and soil nutrients, showing that the soil nutrient distribution characteristics were affected by forest types.TN was affected by MAP, MAT and TS, while TP was susceptible to MAP in PQ (Table 3).However, MAT and MAP were the key factors for most soil nutrients in PB, PL, MLB and PP (Table 3), indicating that MAP and MAT played key roles in the accumulation of biomass matter in this region.In addition, the weak correlations between soil nutrients and the climatic factors (PS, TS) indicated that the changes in temperature and precipitation affected the time scale of soil nutrients.
The wide distribution of the forested land resulted in the higher storage of soil nutrient elements in the Daxing'an Mountains area than in other areas of China, even though the contents vary between the different forest types.Although determining the mechanism through which climate acted on the forest types proved difficult, the spatial distribution of the soil nutrients was related to vegetation in the Daxing'an Mountains.
Conclusions
The results of this study revealed obvious differences in the variation of soil nutrients.The content of each nutrient in PP was minimal, in obvious contrast to other forest types.Correlations between the soil nutrients and climatic factors were found in this paper.Climatic factors could affect soil nutrients in different forest types.We confirmed that climatic factors (MAT and MAP) are instrumental in affecting soil nutrients (SOC, TN, TP, TK, AP and AK) in five main forest types in the Daxing'an Mountains.Identifying the relationships between soil nutrients, climatic factors and forest types, as suggested in this research, can provide theoretical foundations to further comprehend nutrient cycling in the forest ecosystem.
, the contents of SOC, TN, TK, TP, AK and AP in five main forest types averaged at 28.23 g•kg −1 , 4.03 g•kg −1 , 26.23 g•kg −1 , 1.70 g•kg −1 , 158.68 mg•kg −1 and 21.46 mg•kg −1 , respectively.In particular, SOC, TN, TK, TP, AK and AP contents in the PQ were lower than those in the other four forest types.
Table 2 .
Correlation coefficient matrix for soil nutrients and climatic factors.
Table 4 .
Step regressions between soil nutrients and climatic factors in the five main forest types. | 2018-08-09T03:21:09.983Z | 2018-03-30T00:00:00.000 | {
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252406377 | pes2o/s2orc | v3-fos-license | Sleep exerts lasting effects on hematopoietic stem cell function and diversity
Sleep interruption restructures the epigenome of hematopoietic stem and progenitor cells (HSPCs) and increases their proliferation, priming them for exaggerated inflammatory bursts and reducing hematopoietic clonal diversity through accelerated genetic drift. In humans, sleep restriction alters the HSPC epigenome and activates hematopoiesis.
Introduction
Sleep profoundly influences immune and inflammatory responses, protecting against age-associated immune disorders including cardiovascular disease (CVD), cancer, and neurodegenerative diseases (Besedovsky et al., 2019;Irwin, 2019). Despite these associations, more than half of adults do not get sufficient sleep (Ford et al., 2015;Langer and Filer, 2020). Sleep impacts many facets of the immune system including adaptive responses, inflammation, and the synthesis of cytokines and immune mediators (Dimitrov et al., 2004;Lange et al., 2006;Irwin, 2019). Yet, the influence of sleep on hematopoietic stem and progenitor cells (HSPCs) and immune cell production is poorly understood. In humans, poor quality sleep and sleep disorders are associated with increased blood myeloid cells (Ruiz et al., 2012;Geovanini et al., 2018;Vallat et al., 2020), and in murine models, sufficient sleep restricts leukocytosis by limiting HSPC cycling in the bone marrow (BM) through neuroimmune mechanisms (McAlpine et al., 2019). In human and murine atherosclerotic CVD, prolonged sufficient sleep reduces the number of blood monocytes and neutrophils, constraining leukocyte infiltration into the arterial wall and reducing lesion size (McAlpine et al., 2019;Vallat et al., 2020). It has remained unclear, however, how sleep might influence HSPC programming and function.
Sleep abundance and quality vary over an individual's lifespan (Ohayon et al., 2004;Kamel and Gammack, 2006), and the impact of sleep variability on long-term immune-related outcomes is unclear. Sleep declines with age (Unruh et al., 2008), and even young healthy adults are prone to vacillations between periods of adequate and insufficient sleep (Lallukka et al., 2012;Ferguson et al., 2021), which can be shaped by genetically imprinted chronotypes and altered by lifestyle and environments. Bouts of suboptimal sleep, even if followed by periods of sufficient sleep, may have adverse health effects, and recent studies using large human cohorts have indicated that so-called "catch-up" sleep is insufficient at reversing or even neutralizing the adverse effects of sleep debt (Depner et al., 2019;Leger et al., 2020;Dashti et al., 2021). Despite these observations, the impact of sleep and oscillations in sleep quality on future immune function and disease susceptibility are poorly understood, yet critical to uncovering the links between sleep and chronic immune and inflammatory diseases that develop over decades. In this study, we explored how sleep fragmentation (SF) and its recovery shape immunological responses and disease outcomes through epigenetic restructuring of HSPCs. We assess the impact of SF on the clonal diversity of immune cells and the genetic aging of the hematopoietic system. To expand our analysis, we quantify hematopoiesis and HSPC programming in humans participating in a sleep restriction (SR) trial.
Sleep exerts a prolonged influence on hematopoiesis
To explore how sleep might influence prolonged immune function, we first subjected mice to mechanical SF, tactilely rousing mice every 2 min during their rest period (Zeitgeber [ZT] 0-12; McAlpine et al., 2019). We recorded electroencephalography (EEG) signals from the brain and electromyography (EMG) signals from muscle to quantify sleep and wake states and transitions. As expected, SF increased the number of wake bouts (transitions from sleep to wake states) during the rest period ( Fig. 1 A). SF did not alter slow wave sleep (SWS), rapid eye movement sleep (REM), or wake time during the light period ( Fig. 1 B). However, SWS was increased and wake time was reduced during the first 2 h of their active period (ZT13 and 14). We next tested whether fragmented sleep was maintained during long exposures to SF and if it reverts after terminating tactile disruption. We subjected mice to 16 wk of SF followed by 10 wk of undisturbed sleep (recovery sleep [RS]) while continuously monitoring EEG and EMG signals. Wake bouts remained elevated throughout the entire 16 wk of SF ( Fig. 1 C). During RS, wake bouts were elevated for the first 2 wk before returning to baseline for the remaining 8 wk (Fig. 1 C). Together, these findings suggest a lasting if not a permanent influence of SF on sleep quality.
In mice, 16 wk of SF augments hematopoiesis and the proliferation of lineage − Sca1 + cKit + (LSK) cells in the BM, leading to an increased number of blood Ly6C hi monocytes during the rest period (McAlpine et al., 2019). In the BM, SF increases the concentration of myeloid colony-stimulating factor (M-CSF; McAlpine et al., 2019) but does not alter kit ligand, IL-3, or GM-CSF (Fig. S1, A and B). In the plasma, SF raises IL-6 levels while other plasma cytokines, growth factors, corticosterone, and body weight remain unchanged (Fig. S1, C and D). Considering our data on wake bouts during RS, we sought to determine how long increased leukocyte production persists in mice allowed to recover from 16 wk of SF. After 4 wk of RS, at ZT3, blood Ly6C hi monocytes, BM LSKs, and LSK proliferation remained elevated relative to control mice having slept habitually (Fig. 1 D). However, after 10 wk of RS in a separate cohort of animals, hematopoietic activity, monocytosis, and IL-6 levels receded to control levels ( Fig. 1 E and Fig. S1 G). Relative to control mice, measured plasma and BM cytokines and growth factors, plasma corticosterone, and body weight remained unaltered after 10 wk of RS (Fig. S1, E-H). Enhanced hematopoietic activity, therefore, persists for weeks after cessation of SF, raising the possibility that SF exerts a lasting, if not permanent, influence on leukocyte production. To begin to explore this hypothesis, we performed competitive LSK transplantation assays. We transplanted LSKs at a 1:1 ratio from SF (CD45.2) and control (CD45.1) mice into irradiated GFP mice and allowed recipient mice to sleep habitually. 3 wk after transplantation, we measured the proliferation of LSKs derived from SF (GFP − CD45.2 + ) and control (GFP − CD45.1 + ) animals. Despite similar initial engraftment (Fig. S1 I) and inhabiting the same BM microenvironment, LSKs of SF mice incorporated more BrdU than controls (Fig. 1 F). To extend this observation, we tracked CD45.1/2 chimerism in the blood up to 24 wk after transplant. Cells arising from SF mice outcompeted cells from control mice and comprised >60% of the leukocyte and monocyte fractions in the blood ( Fig. 1 G). We made similar observations when LSKs from CD45.2 SF mice were placed in competition with LSKs from UBI-GFP mice (CD45.2) sleeping habitually (Fig. S1 J). Combined, these findings point to stem-intrinsic mechanisms that regulate prolonged SFmediated hematopoietic activity.
Sleep programs the epigenome of hematopoietic progenitor cells and instructs adaptability
To test stem-intrinsic mechanisms of sleep-mediated hematopoiesis, we profiled the epigenome of hematopoietic progenitor cells in the BM. First, we observed increased histone deacetylase (HDAC) activity in hematopoietic progenitor cells of SF mice ( Fig. S1 K). Next, we performed the assay for transposaseaccessible chromatin sequencing (ATAC-seq) on LSKs sorted from three groups of mice: control mice (habitual sleep [HS]), mice subjected to 16 wk of SF, and mice subjected to 16 wk of SF, followed by 10 wk of RS ( Fig. 2 A), a time point when LSK number and proliferation have receded ( Fig. 1 E). Though we found ATAC-seq peaks distributed throughout the genome (Fig. S1 L), we focused our attention on peaks mapping to enhancer elements, which are regions critical to gene expression. Relative to controls, 470 enhancer loci were differentially accessible (DA) in LSKs of SF mice (vs. controls, 65 gained accessibility and 405 lost accessibility, Log 2 FC > 2, P < 0.05, Fig. 2, B and C), revealing broad reprogramming of the epigenome by SF. Next, we compared peaks common to all three groups. We found that 69% (319) of the SF-induced changes in enhancer accessibility reverted to control levels after 10 wk of RS. However, 31% (140 loci) of alterations caused by SF were maintained in the RS group, representing a preserved signature and a potential mark of sustained epigenetic imprinting (Fig. 2 C). Pathway analysis of the preserved signature uncovered elements of cell cycle and cell senescence (Fig. 2 D). These findings reveal that SF restructures the LSK epigenome and that a cluster of epigenetic imprinting is retained even after prolonged sleep recovery.
The differentiation capacity of hematopoietic progenitor cells is tightly regulated by stem-intrinsic epigenetic modifications to lineage commitment genes (Morrison et al., 1996;Rossi et al., 2005;Dorshkind et al., 2020). We found that SF limited accessibility at enhancers important to lymphocyte lineage differentiation (e.g., Bcl11a, Ftl3, Sox4, Icos, Il15, Rag1, Pax5, Ets1, and Btk) but augmented accessibility at enhancers associated with myeloid lineage differentiation (Csf2rb, Irf2bp2, Dyrk3, Erg, Klf9, and Klf3), suggesting SF restricts lymphoid differentiation and drives LSKs toward a myeloid fate (Fig. 2 E). Many changes in myeloid enhancers, but not lymphoid enhancers, were retained after 10 wk of sleep recovery, demonstrating long-term imprinting of sleep on lineage programming. Enhancers associated with cell cycle regulators (Cdk11b, Cdk18, Cdkn1b, and Cdkn2b) were also influenced by SF (Fig. 2, E and F). In agreement with our ATACseq findings, transcript expression of the cell cycle inhibitors Cdkn1b and Cdkn2b was reduced in LSKs of SF mice and remained diminished during recovery, while the cyclin Cdkal1 and the mediators of lineage fate Klf9 and Klf3 were increased in SF mice and remained elevated during recovery (Fig. 2 G). These analyses suggest that SF skews the hematopoietic system toward the myeloid lineage, and the capacity for lymphoid differentiation declines due to stem-intrinsic regulation of lineage commitment genes.
The LSK epigenome instructs secondary recall responses and adaptability (Khan et al., 2020;Divangahi et al., 2021;Rodrigues et al., 2021), so we sought to determine whether the preserved epigenetic signature shapes LSKs' response to a subsequent immunological challenge. We allowed mice to either sleep habitually or exposed them to 16 wk of SF and 10 wk of RS before subjecting both groups to cecal ligation and puncture (CLP), a model of sepsis and systemic bacteremia (Fig. 2 H). 24 h after the CLP, despite equivalent blood bacteremia (Fig. 2 I), mice exposed to SF followed by RS developed an exaggerated inflammatory response that included heightened blood monocytes and BM LSKs, accelerated LSK proliferation ( Fig. 2 I), and augmented plasma IL-6 and TNFα levels ( Fig. 2 J), but unchanged plasma growth factor and corticosterone levels ( Fig. S1 M). This heightened inflammatory response associated with a worsened clinical score and reduced survival ( Fig. 2 K). To test whether the phenomenon was intrinsic to hematopoietic cells, we transplanted BM cells from control or SF mice into irradiated recipients with no history of sleep disruption (Fig. 2 L). 10 wk after the BM transfer, recipients had equivalent blood monocyte counts regardless of whether they received control or SF BM cells (Fig. 2 M). Recipients were then exposed to CLP and tissues were harvested 24 h later. Mice that received BM cells from SF mice responded more aggressively to CLP with increased blood monocytosis (Fig. 2 N), augmented BM hematopoiesis (Fig. 2 O), Figure 1. Sleep exerts a prolonged influence on hematopoiesis. (A) Quantification of wake bouts (transitions from sleep to wake states), during the resting (light) period along with a representative hypnogram in control (C) and SF mice. n = 4 mice per group. (B) Quantification of minutes per hour in SWS, REM sleep, and awake time, along with representative EEG and EMG traces, in control and SF animals over 24 h. n = 4 mice per group. (C) Quantification of resting period wake bouts over 16 wk of SF followed by 10 wk of RS. n = 4 mice per group. (D) Enumeration of blood Ly6C hi monocytes and BM LSKs, along with LSK BrdU incorporation in control mice, SF mice, and mice exposed to 16 wk of SF followed by 4 wk of RS. n = 5-6 mice per group. (E) Enumeration of blood Ly6C hi monocytes and BM LSKs, along with LSK proliferation in a distinct cohort of control mice and mice exposed to 16 wk of SF followed by 10 wk of RS. n = 4 mice per group. (F) BrdU incorporation into LSKs from control (CD45.1) or SF (CD45.2) mice 3 wk after competitive 1:1 LSK transplantation into a UbiGFP mouse sleeping habitually. n = 6-7 mice per group. (G) Competitive 1:1 transplantation of LSKs from control (CD45.1) or SF (CD45.2) mice into UbiGFP recipient mice and quantification of CD45.1/2 chimerism among blood leukocytes and Ly6C hi monocytes up to 24 wk after transplantation. n = 8 mice per group. Mean ± SEM. *, P < 0.05, **, P < 0.01. and increased plasma cytokine levels, leading to a worsened clinical score (Fig. 2 P), indicating these phenotypes are mediated by leukocyte-intrinsic mechanisms. As an alternate rechallenge model, mice subjected to 16 wk of SF and 10 wk of RS were re-exposed to SF. Mice experiencing a second bout of SF developed monocytosis and elevated LSK proliferation more rapidly relative to mice experiencing SF for the first time ( Fig. S1 N). Together, these findings suggest that SF creates memory by priming LSKs, leading to augmented inflammatory responses to subsequent immune challenges.
Given our findings, we hypothesized that modifiers of histone acetylation or epigenetic readers might control these lasting effects. To test this hypothesis, we exposed mice to 4phenylbutyrate (4PBA), a potent HDAC inhibitor, during weeks 8-16 of SF. HDAC inhibition did not influence body weight but did blunt SF-induced LSK expansion and the expression of proliferation-and lineage-related genes (Fig. S2). After HS or SF, with and without 4PBA treatment, mice underwent RS (while drinking regular water) followed by CLP (Fig. 3 A). After CLP, HDAC inhibition attenuated monocytosis, LSK expansion, LSK proliferation, and plasma cytokines (Fig. 3, B and C). Similarly, inhibition of bromodomain and extraterminal reader proteins (Nicodeme et al., 2010;Fig. 3 D) restricted the secondary inflammatory response including monocytosis (Fig. 3 E), BM hematopoiesis ( Fig. 3 F), and plasma cytokine levels ( Fig. 3 G). These observations support the idea that SF primes LSKs through epigenetic reprogramming, rendering adaptability to subsequent inflammatory responses.
Sleep maintains hematopoietic clonal diversity
Having shown that SF has sustained effects on the LSK epigenome, lineage commitment, and turnover, we considered possible implications of these perturbations on somatic evolution of the LSK population. The expansion of hematopoietic clones and the homogenization of the hematopoietic system is a common age-associated premalignant condition known as clonal hematopoiesis (CH; Jaiswal et al., 2014). CH can be driven by accrual of somatic mutations, most commonly in the epigenetic modifiers Tet2 and Dmnt3a (Genovese et al., 2014). CH is associated with accelerated epigenetic aging of HSPCs (Nachun et al., 2021), a twofold increased clinical risk of CVDs (Jaiswal et al., 2017), and all-cause mortality (Jaiswal et al., 2014). Recently, we showed that increased HSPC proliferation caused by SF accelerates the emergence of Tet2-mutant CH (Heyde et al., 2021). Specifically, in mixed chimeric animals exposed to SF, Tet2mutant cells expand at a faster rate relative to animals whose sleep was unaltered, resulting in mutants encompassing a greater proportion of blood myeloid cells and BM HSPCs (Heyde et al., 2021). However, the majority of CH cases arise in the absence of detectable driver mutations (Zink et al., 2017;Poon et al., 2021;Mitchell et al., 2022). We predicted that heightened proliferation of HSPCs would not only accelerate the expansion of mutant clones but would also expedite neutral evolutionrandom fluctuations in mutant frequencies due to stochastic loss and self-renewal of HSPCs (Heyde et al., 2021). Even in the absence of mutant selection, diversity declines over time in stochastically self-renewing stem cell populations due to neutral drift (Snippert et al., 2010), an effect that is accelerated under conditions of increased cell turnover. We set out to investigate experimentally our theoretical prediction that drift would be accelerated in mice subjected to SF (Heyde et al., 2021).
We utilized a multicolor LSK clonal-tracking system (Yu et al., 2016;Shen et al., 2021). Briefly, sorted LSKs were stochastically and permanently tagged ex vivo with up to seven fluorophores and transplanted into lethally irradiated recipient mice ( Fig. 4 A). Flow cytometry and a binary gating strategy allowed us to track 127 clusters that incorporated at least one tag and had a unique fluorophore combination (Fig. 4 B). We evaluated tagged LSK clusters and their descendants, which retained the fluorescent tag of the progenitor cell from which they were derived. First, we confirmed that 126 clusters (99%) were detectable among the tagged LSK cells prior to transplantation into recipient mice (Table S1 and Fig. S3 A). 6 wk after transplantation, 110 ± 3 clusters (87%) were detectable among blood leukocytes ( Fig. S3 B), with minimal variability between recipient mice ( Fig. S3 C). Cluster frequency of blood monocytes and neutrophils correlated remarkably with cluster frequency of BM LSKs ( Fig. S3 D), demonstrating that changes in cluster abundance and dynamics in the blood are reflective of changes among BM hematopoietic progenitors.
We subjected the recipient mice to HS or SF and analyzed cluster dynamics in the blood over time. First, we measured the relative change in frequency of clusters with four or more tags, which represent rare and more homogeneous clusters (Table S1). Among blood monocyte and neutrophil clusters, we found that over time, the range of cluster frequencies, the change in mean cluster frequency, and the cluster variance were substantially elevated in SF mice (Fig. 4,C and D), signifying that SF accelerates the expansion of some clusters and the disappearance of others, consistent with expedited neutral drift. We did not observe changes in relative mean cluster frequency, range, or variance among B and T cell populations, presumably owing to the long half-life of these cells (Fig. S4, A and C; Heyde et al., 2021). To directly quantify the evolution of diversity and neutral drift over time, we computed Simpson's diversity index (SDI). We found that in control animals, the SDI of blood monocytes and neutrophils decreased gradually over time (Fig. 4, E and F). In SF mice, however, monocyte and neutrophil SDI decayed more rapidly (Fig. 4, E and F), consistent with the effects of accelerated neutral drift due to increased cell turnover. We did not observe an influence of SF on lymphocyte SDI (Fig. S4, B and D). Together, these data show that SF increases stochastic fluctuations in the frequencies of neutral clusters, thereby reducing hematopoietic cell diversity over time.
Having observed diversity decay through accelerated neutral drift among circulating myeloid cells of SF mice, we turned our attention to progenitors in the BM. Among BM LSKs, 18 wk of SF increased the mean variance of cluster frequencies and decreased the SDI (Fig. 4 G), providing direct evidence for accelerated neutral drift and reduced diversity amongst hematopoietic progenitors. To garner insights into the impact of sleep on population diversity as LSKs differentiate into mature cells, we assessed relative changes in the SDI of BM LSKs and circulating cells. We found that in both control and SF mice, diversity was higher in BM LSKs than in differentiated blood cells ( Fig. 4 H and Fig. S4 E), reflective of the multipotency and unequal contribution of LSKs to circulating immune cells (Pietras et al., 2015). The reduction in SDI between BM LSKs and blood monocytes was greater in SF animals ( Fig. 4 H and Fig. S4 E), suggesting that SF limits diversity during monocyte generation. These findings demonstrate that sleep maintains the diversity and restricts the neutral drift of BM hematopoietic stem and progenitor cells by limiting cell turnover. Next, we tested whether the rate of SF-induced LSK proliferation inferred from our clonal tracking data was quantitatively consistent with independent experimental observations. We mathematically modeled LSK evolution based on the Moran process of population genetics (see Materials and methods). For this model, we calculated that the LSK SDI decays at a rate proportional to the rate of cellular proliferation divided by the population size. Using the observed LSK population size after 18 wk of SF (Fig. 4 I and Table S2), our model estimated a 1.56 ± 0.26-fold increase in LSK proliferation in SF mice. We confirmed this mathematical prediction experimentally by measuring a 1.52-fold increase in BrdU incorporation in LSKs of SF mice (Fig. 4 J). Using complementary mathematical and experimental methods, our data provide evidence that SF accelerates LSK proliferation and thus neutral drift, rendering the population more homogeneous over time.
Sleep restriction enhances hematopoietic activity and monocytosis in humans
Having established a role for sleep in maintaining the LSK epigenome, immune memory, and clonal diversity in mice, we turned our attention to humans. To test the influence of sleep on hematopoiesis and the epigenome of human hematopoietic progenitor cells, we collected blood samples from humans participating in a randomized crossover study in a free-living setting in which chronic mild insufficient sleep was imposed on healthy young volunteers (mean age 35.7 ± 15.1 yr) with adequate HS duration and free of sleep disorders (Fig. 5 A, study flow chart in Fig. S5 A, cohort characteristics in Table S3). The study comprised a 6-wk HS phase where participants slept an average of 7.4 ± 0.1 h per night and a 6-wk SR phase where participants restricted their sleep to 6.1 ± 0.1 h per night (Fig. 5 B). Participants underwent both study phases, in a random order, which were separated by a washout period of 4-6 wk. As participants completed each phase, we collected blood in the morning (mean clock time 10:54 ± 0:16) and evening (mean clock time 15:21 ± 0:12), and analyzed circulating leukocyte populations by flow cytometry. As expected, we detected a circadian pattern characterized by high circulating leukocyte numbers in the evening and low numbers in the morning (Fig. 5 C). However, we found that after completing SR, participants had more circulating CD14 + CD16 − classical monocytes and CD14 − CD16 + nonclassical monocytes in the evening, but not morning (Fig. 5 C). SR did not change intermediate monocytes or B and T cell populations (Fig. 5 C) and changes in blood monocytes did not depend on energy intake or adiposity (Fig. S5 B). Consistent with our murine findings, these data reveal that sleep protects against To build on these observations, we sought to determine whether sleep affects HSPCs in humans. Human lineage − CD34+ HSPC numbers fluctuated in the blood according to a circadian rhythm (Fig. 5 D). We found that SR increased HSPC number in the blood in the evening (Fig. 5 D), suggesting that, like in the mouse, SR enhances hematopoietic activity in humans. The impact of SR on HSPCs did not depend on energy intake or adiposity (Fig. S5 C). Next, we investigated whether SR influences the epigenome of human HSPCs. We measured histone acetyltransferase (HAT) and HDAC activity in sorted HSPCs retrieved in the evening and found that SR tended to increase HAT activity and significantly augmented HDAC activity (Fig. 5 E), which reduced histone H3 acetylation (Fig. 5 F) and indicated that sleep shapes the epigenome of human HSPCs. As HSPCs differentiate into myeloid cells, they rely on growth factors CSF1 and IL-3 (Mindur and Swirski, 2019), which signal via CD115 and CD123, respectively. Accordingly, we found enhanced expression of CD115 and CD123 on HSPCs retrieved from participants experiencing SR (Fig. 5 G). These findings reveal that human sleep mediates hematopoiesis, shapes the HSPC epigenome, and promotes myeloid differentiation cues.
Discussion
Sleep profoundly influences the immune system and its functioning (Besedovsky et al., 2019;Irwin, 2019). However, sleep's impact on hematopoiesis and the programming and clonality of HSPCs have remained unclear. Here, we report that murine SF augments sleep-wake transitions, increases hematopoiesis, and alters the epigenome of HSPCs through changes in histone acetylation. While hematopoiesis recedes during sleep recovery, HSPCs retain epigenetic imprinting, priming them for myeloidbiased responses and increased inflammatory activation during subsequent immune challenges. Using a multicolor clonaltracking system, we find that SF reduces the diversity of the hematopoietic system by accelerating neutral drift and the emergence and disappearance of HSPC clones. In humans, we show that 6 wk of mild SR increases the number of HSPCs and monocytes in the blood, reduces HSPC histone acetylation, and promotes HSPC myeloid response cues. Together, these findings suggest that oscillations in sleep quality and abundance compromise HSPC epigenetic structure, diversity, and subsequent immune function.
Chronic sleep disruption is pervasive in modern lifestyles (Ford et al., 2015). Sleep disruption encompasses many permutations including, but not limited to, fragmentation, restriction, social jet lag, obstructive sleep apnea (OSA), and insomnia, which substantially increase susceptibility to immuneassociated diseases (Irwin, 2019). Individuals typically do not experience one continuous form of sleep disruption but oscillate between types of sleep disturbances and between periods of inadequate sleep and healthy sleep. To understand the mechanistic links between sleep on chronic age-associated diseases that develop over decades, fluctuations in sleep patterns must be considered. One mechanism by which even short periods of poor sleep might impact future disease pathology is through epigenetic programming. Insufficient sleep, sleep disorders, and shift work markedly alter a cell's epigenome. For example, OSA modifies the epigenome in the cardiovascular system and circulating leukocytes (Chen et al., 2019), and one sleepless night changes DNA methylation in liver and muscle tissue at the transcription start sites of genes associated with metabolism (Cedernaes et al., 2018). Moreover, sleep disorders, including OSA, insomnia, and sleep-disordered breathing, alter DNA methylation and accelerate the epigenetic age of blood leukocytes (Carroll et al., 2017;Li et al., 2019). Together, these data suggest that sleep modifies the epigenome of diverse cell types throughout the body. However, it has remained unclear whether these sleep-mediated epigenetic modifications have a subsequent consequence on future cell function, inflammation, and disease pathology. Moreover, the influence of sleep on the epigenetic status of HSPCs was unknown. We provide evidence for sustained epigenetic imprinting of HSPCs by sleep disruption. Our data show that SF-induced epigenetic programming is partially maintained during prolonged convalescent sleep and predisposes the hematopoietic system to overt inflammatory responses to subsequent immune challenges. The preserved epigenetic signature is not sufficient for sustained LSK proliferation and differentiation during sleep recovery, likely owing to the vast genomic program and alternate epigenetic modifications, like methylation, involved in controlling cellular proliferation. These data have important implications for inflammatory diseases such as sepsis, CVD, and cancer, and may suggest that early life sleep behavior dictates future disease severity. Our findings support the hypothesis that periods of poor sleep, even if followed by sleep recovery, have sustained consequences on immunological health.
The systemic networks that connect sleep with HSPC epigenetic rewiring and adaptability require further study. Neural circuits together with neuropeptidergic, neuroendocrine, and inflammatory pathways are likely involved. For example, hypothalamic hypocretinergic signaling controls sleep-mediated HSPC proliferation (McAlpine et al., 2019), and our data suggest that SF raises, while sleep recovery normalizes systemic IL-6 levels. IL-6 signaling has broad functionality in health and disease (Libby and Rocha, 2018;Ridker and Rane, 2021), and has been linked to sleep and its disruption (Friedman et al., 2005;Hong et al., 2005;Dimitrov et al., 2006) and histone modifications (Samanta et al., 2008). How IL-6 or other immune signaling factors might fit within the sleep-hematopoiesisepigenome interaction and mediate adaptability after sleep recovery should be explored in detail.
The HSPC epigenome dictates fate, division, and clonality, and mediates age-associated changes to the hematopoietic system (Morrison et al., 1996;Buisman and de Haan, 2019;Rodrigues et al., 2021). Clonally expanded HSPCs often harbor mutations in epigenetic modifiers, and CH is strongly associated with epigenetic age acceleration (Genovese et al., 2014;Nachun et al., 2021). Therefore, we hypothesized that epigenetic reprogramming and increased proliferation of HSPCs promoted by sleep disruption may influence their clonality and the neutral evolution of the hematopoietic system. Our prior mathematical framework suggested that the magnitude of HSPC clonal expansion is dependent on their proliferative rate and less on their mutations or conferred fitness advantage (Heyde et al., 2021). For example, increased hematopoiesis induced by SF or high-fatdiet feeding accelerates the emergence and clonal expansion of Tet2-mutant HSPCs (Heyde et al., 2021). The theory predicted that a stochastic elevation in hematopoiesis would accelerate neutral drift and the emergence of clones, even in the absence of mutant selection. Here, we used a fluorescent tagging system to test this theory experimentally. We found that increased proliferation of mutant-free HSPCs caused by SF accelerates neutral drift, which leads to faster expansion and disappearance of HSPC clones, thus limiting hematopoietic diversity. Our observations support the notion of reverse causality-increased HSPC proliferation accelerates the expansion of mutant and neutral clones. These data add to our understanding of the relationship between CH and inflammatory disorders such as CVD whose risk factors, including sleep disruption, augment hematopoiesis and expedite the emergence of mutant and nonmutant HSPC clones, increasing disease susceptibility. Humans experiencing SF (Vallat et al., 2020) and OSA (Geovanini et al., 2018) develop blood monocytosis and neutrophilia. However, the influence of sleep on hematopoiesis, HSPC function, and circadian dynamics in humans had not been explored. We found that 6 wk of chronic mild SR increased the number of blood monocytes and HSPCs in human subjects, but did not influence lymphocyte populations. Whether the elevation in blood HSPCs is due to increased hematopoietic activation through augmented HSPC cycling in the BM and/or increased HSPC release from the BM will require further study. Importantly, these findings were observed in the evening, mirroring our murine data (McAlpine et al., 2019) and suggesting that the time of blood collection is a critical variable when assessing changes in blood leukocyte number. Additionally, our findings reveal that SR alters the intrinsic epigenome of human HSPCs by reducing histone acetylation through increased HDAC activity. Together, our murine and human data propose that different forms of sleep interruption in species with dissimilar sleep architecture perpetuate similar outcomes on hematopoiesis and monocytosis, underscoring the preserved nature of this phenomenon.
Understanding the prolonged influence of sleep on immune function and disease susceptibility is important, particularly given the nearly pandemic prevalence of insufficient sleep (Watson et al., 2015). Our study provides evidence, in mice and humans, that sleep exerts a lasting influence on the immunity and functioning of HSPCs via epigenetic modification, and that SF genetically ages the hematopoietic system by accelerating neutral drift, which collapses clonal diversity and homogenizes the myeloid pool.
Materials and methods
Animal studies Wild-type C57BL/6J, B6.SJL-Ptprca Pepcb/BoyJ (CD45.1), and C57BL/6-Tg(UBC-GFP)30Scha/J (ubiGFP) mice were purchased from The Jackson Laboratory. Age-and sex-matched mice were used. Where appropriate, mice were randomly assigned to interventions. All mice were group-housed with free access to food and water. All animal protocols were approved by the Animal Review Committee at the Massachusetts General Hospital (protocol nos. 2011N000035 and 2015N000044) or the Icahn School of Medicine at Mount Sinai (protocol nos. PROTO202100023 and PROTO202000262) and were in compliance with relevant ethical regulations.
In vivo interventions SF and RS
For SF, studies were performed as previously described (McAlpine et al., 2019). Briefly, mice were placed in an SF chamber (Lafayette Instrument) and the sweep bar moved along the bottom of the cage every 2 min during the light cycle (ZT0-12). The sweep bar automatically shut off and was stationary during the dark cycle (ZT12-24). Control mice whose sleep was unaltered were placed in SF chambers with stationary sweep bars. For recovery experiments, the sweep bar was shut off and animals were transferred to a conventional mouse cage.
Telemetry EEG and EMG monitoring
First, animals were implanted with EEG/EMG transmitters (HDx-02; Data Science International). Briefly, the head of the animal was secured in a stereotactic frame and a 2-3 cm incision was made along the dorsal midline posterior margin of the eyes to midway between the scapulae. A subcutaneous pocket was formed and the implant was inserted with the biopotential leads oriented cranially. The EMG electrodes were inserted into the cervical trapezius muscle in the dorsal region of the neck and the ends were sutured so they remain in place. To place the EEG electrodes, a microdrill was used to perforate the skull above the cortex. The ends of the leads were placed in the hole such that they make contact with the dura of the cortex. The leads were secured in place using dental acrylic. The skin incision was sutured and mice received postsurgical analgesia and were supplemented with warmth for 1 h. Mice were allowed to recover for 1 wk before monitoring began. Mice were placed in SF or control chambers and EEG/EMG data was recorded using the Data Science International (DSI) telemetry system and hardware. Ponemah software (DSI) was used to acquire the data, and Neuroscore 3 (DSI) was used to perform sleep analysis using standard settings.
BM transplantation
For competitive transplantation assays, mice were lethally irradiated (950 cGy) and reconstituted with 50,000 LSKs from each donor along with 2,000,000 BM feeder cells (of the same genotype as the recipient mouse) injected intravenously 18 h after irradiation.
CLP
The CLP procedure was carried out as previously described (Weber et al., 2015). Briefly, the peritoneal cavity was opened during isoflurane anesthesia and the cecum was exteriorized and ligated at the second arterial vessel or third arterial vessel (for survival studies) distal to the end of the cecum. The ligated portion of the cecum was then perforated using a 17-G needle, and a small drop of cecal content was extruded through the puncture. The cecum was relocated into the peritoneal cavity and the peritoneum was closed. Animals were given 1 ml of saline and removed from isoflurane.
Reagent administration
4PBA was dissolved in water at a concentration of 5.75 g/liter, which results in a delivery of ∼1.5 g/kg/d when given as drinking water. 4PBA-containing drinking water was given to mice at week 8 of SF, removed at week 16, and changed weekly. iBet was dissolved in 20%β-cyclodextrin and 11% DMSO and intravenously injected into animals at a concentration of 30 mg/kg four times over 2 d prior to CLP.
Cells
Collection Peripheral blood was collected at ZT3 by retro-orbital bleeding, and red blood cells were lysed in RBC lysis buffer (Biolegend). BM cells were collected at ZT3 by flushing bones with PBS, after which a single-cell suspension was created by passing cells through a 26gauge needle and red blood cells were lysed with RBC lysis buffer. Total viable cell numbers were counted using trypan blue (Cellgro; Mediatech) or counting beads (Thermo Fisher Scientific).
BrdU incorporation
To assess cell proliferation, 1 mg BrdU was injected intraperitoneally 2 h before euthanasia. A BrdU flow kit (BD Biosciences) was used to stain BrdU+ cells.
Cell sorting BM cell suspensions were stained to identify the indicated cell populations, and cells were sorted on a FACS Aria II cell sorter (BD Biosciences) directly into collection medium.
Molecular analysis
Quantitative PCR Total RNA was isolated using the RNeasy Mini Kit (Qiagen) or the NucleoSpin RNA XS kit (Takara Bio) according to the manufacturer's instructions. RNase-free DNase Set (Qiagen) was used for DNase digestion during RNA purification. RNA quantity and quality were assessed by Nanodrop for RNA isolated from tissues and with the Aglient RNA 6000 Pico kit (Aglient Technologies) on the Aglient 2100 Bioanalyzer for RNA of FACS-purified cells. cDNA was generated from 1 μg of total RNA per sample using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time TaqMan PCR was performed using the following TaqMan primers (Applied Biosystems): Mm00438168, Mm00483241, Mm00492956, Mm00495172, and Mm00507441.
Enzyme-linked immunosorbent assay IL-6, TNFα, IL-1β, M-CSF, and G-CSF levels were measured using an ELISA (Boster Biologics) according to the manufacturer's instructions. On sorted cells, HAT and HDAC activity were measured using commercially available kits, HAT kit (cat#-EPl001; Sigma-Aldrich) and HDAC Kit (cat#CS1010; Sigma-Aldrich), respectively, according to the manufacturer's instructions. Corticosterone was measured using an ELISA (Abcam) and according to the manufacturer's instructions.
ATAC-seq ATAC-seq was conducted in technical and biological duplicates. Briefly, 20,000 sorted LSK cells were initially resuspended in lysis buffer and centrifuged. The nuclei pellets were then subjected to transposition reaction using the Nextera Tn5 transposase enzyme (Illumina). Tagmented DNA was purified with the MinElute PCR Purification kit and eluted in 10 μl of elution buffer (Qiagen). Barcoded libraries were prepared and PCR-amplified. Double-sided bead purification was performed using AMPure XP beads to remove primer dimers and large >1,000-bp fragments. Libraries were sequenced as 50-bp paired-end reads on an Illumina HiSeq 2500.
Sequencing reads were first mapped to the mm10 reference genome using BWA-MEM66 with default parameters, followed by calling peaks using HOTSPOT2. We identified 41,000 peaks, which showed high consistency between biological duplicates. Peaks with differential accessibility between conditions were identified using edgeR68, with cutoffs of at least a Log 2 Fold change>1.2 difference in normalized read density and P < 0.05.
Pathway analysis
Kyoto Encyclopedia of Genes and Genomes pathway analysis was done using Enrichr (https://maayanlab.cloud/Enrichr/) with default parameters.
Clinical scoring
Clinical scoring was performed as previously described (Weber et al., 2015). The clinical score of each animal was assessed as follows (points).
Survival analysis CLP were performed as described above. Animals were then monitored until they reached endpoint.
Bacterial cultures
Whole blood was diluted in PBS, plated on trypticase soy agar with 5% horse blood, and incubated at 37°C. The number of bacterial colonies was assessed 12-16 h later.
Generation of fluorescent-tagged mice and tracking of clusters Donor WT mice were sacrificed and 1,000,000 BM LSKs were sorted and pooled. LSKs were cultured in serum-free StemSpan medium (StemCell Technologies) containing penicillin, streptomycin, glutamine, and a combination of cytokines (20 ng/ml IL-3, 100 ng/ml SCF, 100 ng/ml Flt-3L, 50 ng/ml thrombopoietin, all from Peprotech) at a concentration of 106 cells/ml. After 1 h of prestimulation, cells were transduced with a mix of highly concentrated lentiviral vectors encoding for different fluorescent proteins (BFP, Sapphire, Venus, KO, mCherry, iRFP, and Keima) at a dose of 20 transducing units/cell. After tagging, a small aliquot of cells was analyzed by flow cytometry prior to their transplantation (BM baseline). Recipient mice were lethally irradiated (9.5 Gy), and each was i.v. injected with 100,000 tagged LSKs along with 2,000,000 Sca1unlabeled feeder cells. Mice were allowed to recover and sleep habitually for 6 wk. After recovery, mice were bled and circulating leukocytes were analyzed (blood baseline). Mice were then randomly assigned to SF or HS. Animals were bled periodically and sacrificed after 18 wk of SF.
Mathematical calculations of variance Cells that fluoresced above background were considered positive for an incorporated fluorescent tag. Distinguishing dichotomous populations (positive or negative for a fluorophore) allowed the identification of 127 uniquely tagged clusters plus one cluster that did not incorporate any fluorophores and was excluded from analysis. Fold change in relative frequencies was calculated by dividing cluster frequency by the baseline frequency. Variance (s 2 ) of cluster frequency was calculated using the following equation: where x is the mean frequency and n is the number of clusters.
Mathematical calculations of Simpson's diversity and cell division rate estimation For a single mouse, SDI at time t is defined by: where i = 1, .., 127 denote the tagged clusters and X i,t is the frequency of cluster i at time t. To predict the evolution of Simpson's diversity, we model that LSK's divide and die according to the Moran process (Ewens, 2004), which is parameterized by the number of cells N and the per cell division rate a. From the Kolmogorov equations (Durrett, 2012), the expected Simpson's diversity follows the differential equation which has the solution is a consistent estimator for a S /a C , where S is the set of mice in the SF group, C is the set of mice in the control group, D t,m is Simpson's diversity at time t for mouse m, and a S , a C , N S , N C are the division rates and LSK numbers for the SF and control mice. While control vs. SF mice are expected to have similar LSK numbers at the beginning of the experiment, we measure that LSK numbers are 1.4-fold higher in SF vs. control mice at 18 wk. Averaging over the two time points, we take N S /N C = 1.2 ± 0.2. Plugging this into Eq. 2 along with the LSK diversity measurements, an estimate of a S /a C = 1.56 ± 0.26 is obtained. That is, LSKs divide at a rate (1.56 ± 0.26)-fold faster in SF vs. control mice, which is consistent with the BrdU estimate of 1.52. There are caveats to our division rate estimation. For one, the model of LSK evolution is simplistic. A more detailed model could, for example, consider that division rates and LSK numbers may vary over time and between mice in a group. Moreover, there would ideally be more data points to enable improved estimate precision. Nevertheless, the purpose of the modeling is conceptual. We demonstrate that accelerated genetic drift due to increased division rates can explain SF's impact on LSK diversity.
Study design
The human subjects component of this study employed a randomized crossover design to assess the impact of a common model of chronic insufficient sleep on hematopoiesis in 14 participants. This outpatient trial included two sleep conditions: maintenance of habitual adequate sleep (HS) and prolonged mild SR. Each condition was 6 wk in duration. The order of conditions was randomized across participants, and study phases were separated by a 4-6-wk washout. In the fifth and sixth weeks of each study condition, blood samples were collected in the late afternoon/ early evening (hereafter referred to as early evening) and the morning, respectively, for assessment of hematopoietic outcomes. This investigation was a component of an ongoing clinical trial designed to test the impact of prolonged insufficient sleep on energy balance. The trial was prospectively registered on ClinicalTrials.gov (NCT02960776). All procedures were conducted in accordance with the Declaration of Helsinki and approved by the Columbia University Institutional Review Board (protocol number AAAQ7746); all participants provided informed consent prior to participation.
Subjects
Metabolically healthy men and women, aged 20-75 yr, and with an HS duration of ≥7 h/night, were recruited through advertisements placed online and throughout the New York City area. Individuals meeting initial eligibility criteria based on a phone interview were invited to the laboratory for an in-person screening visit. During this visit, height and weight were measured in duplicate using calibrated research-grade stadiometer and scale (Tanita WB-3000), respectively. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. Individuals then completed questionnaires validated for assessment of sleep and circadian rhythms as well as a detailed health history questionnaire. Men and women were deemed potentially eligible if they had overweight or class I obesity (BMI 25.0-34.9 kg/m 2 ) or were at risk for obesity (BMI 20.0-24.9 kg/m 2 with ≥1 parent with BMI >27 kg/m 2 ), had good HS quality (score ≤5 on the Pittsburgh Sleep Quality Index [Buysse et al., 1989]), were at low risk for sleep apnea (<2 positive categories on Berlin Questionnaire [Netzer et al., 1999]), and did not report excessive daytime sleepiness (scores <10 on the Epworth Sleepiness Scale [Johns, 1991]). Those with extreme morning or evening chronotypes (scores ≤30, ≥70 on the Morningness-Eveningness Questionnaire [Horne and Ostberg, 1976]), participating in shift work or daytime napping, or had recent travel across time zones (≤3 wk) were not eligible to participate. Exclusion criteria also included current smoking, dieting, recent weight change >2.5 kg, excessive caffeine intake (>300 mg/d), use of hormonal contraceptives, pregnant or ≤1 y postpartum, as well as presence of cardiovascular, metabolic, neurological, or psychiatric diseases or disorders (including eating and substance abuse disorders). Individuals meeting all eligibility criteria were invited to complete an intensive outpatient sleep screening to ensure habitual adequate sleep duration. Sleep was assessed continuously over 2 consecutive weeks using the Actigraph GT3X + (Actigraph Corporation) and nightly sleep diaries. The Actigraph GT3X+ is a triaxial accelerometer worn on the nondominant wrist and validated against polysomnography (Full et al., 2018), the gold standard objective measurement of sleep. Inclusion in the study required a 14-night average total sleep time (TST) of 7-9 h/night with ≥7 h of sleep on ≥ 10 of the nights and no more than 3 nights with <6 h of sleep. To date, 42 participants met these criteria and have been randomly assigned an order of study conditions. Of the full sample, 16 participants (nine women) were accrued for this ancillary study, which began after initiation of the main study; however, two individuals dropped out of the study. Thus, the analytic sample was 14 (n = 7 women).
Intervention
Participants were studied in a free-living setting over 2 phases of 6 wk each, differing only in sleep duration conditions: HS and SR. In both conditions, habitual patterns of sleep, determined from screening, were used to create fixed personalized sleep schedules in both conditions. In the HS condition, participants were to maintain their average bed and waketimes from screening to achieve their habitual adequate sleep duration of ≥7 h/night. In contrast, during SR, participants were instructed to delay their bedtimes (from screening averages), while maintaining habitual wake times, to achieve a reduction in TST of ∼90 min/night. To promote fidelity to the protocol, bed and wake times were adjusted slightly to the participants' preferences. In addition, sleep was monitored continuously using wrist-actigraphy and nightly sleep diaries, and verified weekly by trained study staff throughout the 6-wk study phases to assess compliance. Study phases were separated by a 4-6-wk washout period; sleep was reassessed over the final 2 wk of this washout period to confirm that participants were achieving average TST ≥7 h/night before beginning the next study phase.
Procedures
During each study phase, participants came to the laboratory for a baseline visit (week 0), during which time they completed a number of procedures and received their sleep assignment. Participants returned on a weekly basis for review of wristactigraphy data and sleep logs, and for additional assessments. Many of these procedures are beyond the scope of this study. At baseline (week 0) and endpoint visits (week 6), participants underwent MRI scans, and peripheral blood was drawn from a vein within the antecubital fossa into an EDTA tube by a trained phlebotomist while participants were in the fasted state. Blood draws at baseline and endpoint visits (week 6) were scheduled for mornings between 09:00 and 11:00. At a separate visit, 5 wk into each condition, whole blood was collected in the evening (15:00-17:00). The amount of blood collected and time of draw was recorded at all visits and the vast majority of draws (86%) occurred within 1 h of the planned ranges of draw times.
Blood samples were processed shortly after collection for extraction and preservation of the viability of blood cells. Whole blood was transferred to a 50 ml conical tube (Corning) and PBS diluted to 1× (Thermo Fisher Scientific) was added to the blood to a final volume of 35 ml. The solution was then carefully underlaid with 15 ml of Ficoll-Paque PLUS (GE Healthcare) and spun for 30 min at 23°C and 1,800 rpm with the centrifuge brake off (Eppendorf 5810R; Eppendorf). The resulting white blood cell layer was aspirated and transferred to a new conical tube; PBS was added to a final volume of 50 ml and then spun for 10 min at 4°C (1800 rpm, brake on). After spinning, the white blood cell pellet was resuspended in freezing media (20% FBS RPMI 1640 [Thermo Fisher Scientific] with 10% DMSO [Sigma Life Sciences]) and transferred into separate 1.5 ml cryovials. The cryovials were frozen at a controlled rate of −1°C/min (Mr. Frosty; Thermo Fisher Scientific) to maintain viability of cells and were then transferred to liquid nitrogen for storage until flow cytometry analysis. Neutrophils are not included in the leukocytes isolated by Ficoll-Plaque PLUS gradient as they are pelleted and discarded.
Human cell sorting Cells suspensions were stained as above and HSPCs were sorted on a FACS Aria II cell sorter (BD Biosciences) directly into collection medium.
Human energy intake
In the human subjects study, which is ongoing, we collect data on food intake using 3-d food records. Participants are asked to record all foods they consume, in real-time, on three nonconsecutive days (2 weekdays, 1 weekend). Participants are asked to provide detailed information on food brands, ingredients, and amounts and are provided with information on the use of common portion estimation tools. Food records are collected at week 0 (baseline), week 3, and week 6 (endpoint) of each phase. Nutrient analyses are completed in Nutrition Data System for Research (UMinn), and daily averages are calculated from the 3-d records.
Human adiposity Total volume of adipose tissue and its distribution was measured at baseline and endpoint with MRI using procedures previously described (Shen et al., 2004).
Statistics
Results are shown as mean ± SEM. Statistical analysis was performed using GraphPad Prism 7 (Graphpad Software). Statistical tests included unpaired, two-tailed nonparametric Mann-Whitney U tests (when Gaussian distribution was not assumed). For multiple comparisons of more than two groups, a nonparametric multiple-comparisons test comparing the mean rank of each group (when Gaussian distribution was not assumed) was used, along with one-or two-way ANOVAs followed by Tukey's test. For analysis of human data, two-way ANOVAs were performed when comparing multiple groups and timepoints, or two-tailed nonparametric Mann-Whitney U tests when comparing two groups. Correlation was computed using linear regression. P values of 0.05 or less were considered to denote significance. Each mouse experiment was repeated independently at least two or more times with similar results.
Description of supplementary information
Supplementary tables and figures accompany this manuscript. They describe the influence of SF and RS on inflammation, LSK fitness, epigenetic rewiring, and adaptability; an analysis of mice treated with HDAC inhibitors; numerous quality control metrics of the clonal tracking system; the clonal frequency and diversity metrics of lymphocytes; and a description of the human trial and its participants along with an assessment of human leukocytosis with energy intake and adiposity.
Online supplemental material Fig. S1 shows the influence of SF and RS on inflammation, LSK fitness, epigenetic rewiring, and adaptability. Fig. S2 is the analysis of mice treated with 4PBA. Fig. S3 shows cluster size and distribution and correlation of cluster frequencies in the BM and blood. Fig. S4 shows the influence of sleep on lymphocyte diversity. Fig. S5 shows the human study. Table S1 provides LSK cluster frequency. Table S2 lists the number of LSKs in recipient control and SF mice. Table S3 provides human subject characteristics at baseline.
Data and materials availability
All data and methods are available and described within. Materials are available upon request. | 2022-09-22T06:16:47.853Z | 2022-09-21T00:00:00.000 | {
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14908696 | pes2o/s2orc | v3-fos-license | Inducible dimerization of FGFR1
To develop an inducible and progressive model of mammary gland tumorigenesis, transgenic mice were generated with a mouse mammary tumor virus–long terminal repeat–driven, conditional, fibroblast growth factor (FGF)–independent FGF receptor (FGFR)1 (iFGFR1) that can be induced to dimerize with the drug AP20187. Treatment of transgenic mice with AP20187 resulted in iFGFR1 tyrosine phosphorylation, increased proliferation, activation of mitogen-activated protein kinase and Akt, and lateral budding. Lateral buds appeared as early as 3 d after AP20187 treatment and initially consisted of bilayered epithelial cells and displayed apical and basolateral polarity appeared after 13 d of AP20187 treatment. Invasive lesions characterized by multicell-layered lateral buds, decreased myoepithelium, increased vascular branching, and loss of cell polarity were observed after 2–4 wk of treatment. These data indicate that acute iFGFR1 signaling results in increased lateral budding of the mammary ductal epithelium, and that sustained activation induces alveolar hyperplasia and invasive lesions.
Introduction
The fibroblast growth factor (FGF)* family consists of Ͼ 20 ligands and four receptor tyrosine kinase genes that have been shown to be important regulators of angiogenesis and embryonic organogenesis (Ornitz, 2000). Loss-of-function experiments have demonstrated that FGFs are critical factors for limb bud outgrowth, lung development, gastrulation, and brain development (Lewandoski et al., 2000). In the adult, aberrant regulation of FGF ligands and their receptors is associated with breast and prostate tumorigenesis (Giri et al., 1999;Valve et al., 2001). Therefore, understanding the function of FGFs and FGF receptors (FGFRs) will be a crucial step in elucidating mechanisms of cellular transformation.
The expression of FGF ligands and FGFRs is developmentally regulated in the mammary gland (Coleman-Krnacik and Rosen, 1994;Chodosh et al., 2000), where they are generally expressed during ductal morphogenesis and decrease throughout pregnancy and lactation. Ductal morphogenesis is a highly proliferative stage during mammary gland development, during which the gland invades the stromal fat pad and establishes a branched ductal network (Williams and Daniel, 1983). FGFR1 is mainly expressed during this stage of development (Chodosh et al., 2000). Additionally, expression of a dominant negative FGFR2 in the mammary gland during pregnancy has been shown to inhibit lobuloalveolar development (Jackson et al., 1997). This expression profile suggests that FGF signaling plays an important role in normal mammary gland development.
The observation that mouse mammary tumor virus (MMTV) proviral insertion can activate FGF-3, FGF-4, and FGF-8 and leads to mammary tumorigenesis has revealed that FGF ligands are potent mammary gland mitogens (van Leeuwen and Nusse, 1995). The direct transforming activity of the same FGFs in the mammary gland was subsequently confirmed using transgenic mouse models (Daphna-Iken et al., 1998). Additionally, inappropriate regulation of FGFs and their receptors has been observed in breast cancer cell lines and in ف 10-20% of primary breast tumors (Penault-Llorca et al., 1995;Marsh et al., 1999). These data suggest that aberrant FGF signaling may exert potent transforming capacity in the mammary gland.
In the present study, the effects of FGFR1 signaling have been investigated using a novel, inducible FGFR oligomerization and activation system. This system was first characterized in fibroblasts and mammary epithelial cells, and then in a transgenic mouse model. Inducible iFGFR1 signaling in mammary epithelium resulted in progressively invasive lesions occurring rapidly within 2-4 wk after iFGFR1 activation. iFGFR1 kinase-induced lesions were examined for cell polarity, hormonal dependence, and invasive properties. This work describes the first application of an inducible dimerization system for studying receptor tyrosine kinase signaling in a transgenic mouse model.
Generation of an inducible iFGFR1 construct
A ligand-independent activation system of the FGFR was employed to study the progressive steps of mammary gland transformation. This system is based on the ability of the FK506 binding protein-12 (FKBP12) to interact with its naturally occurring ligand, FK506 (Spencer et al., 1993;Pruschy et al., 1994). The interaction of FKBP12 with FK506 occurs at a stochiometry of 1:1; thus, the tethering of two FK506 compounds results in a bivalent drug (FK1012) with the capacity to interact with and dimerize two FKBP12 containing proteins. This system has been used to study dimerization-induced signaling by several cell surface receptors, including members of the ErbB family of receptor tyrosine kinases and the T-cell receptor, as well as by intracellular proteins such as caspases (Spencer et al., 1993;Fan et al., 1999;Muthuswamy et al., 1999Muthuswamy et al., , 2001. Using this strategy, chimeric proteins that contain the FGFR kinase domain fused to FKBP12 can be induced to dimerize in the presence of FK1012. A synthetic analogue of FK1012 used in the present studies, AP20187, has been modified to reduce interactions with endogenous FKBPs, and specifically interacts with a variant of FKBP12 containing an F36V mutation (Clackson et al., 1998;Yang et al., 2000). The FGFR-FKBPv fusion proteins (iFGFRs) generated in this study lack the extracellular ligand binding and transmembrane domains, contain an NH 2 -terminal myristylation sequence, an intracellular FGFR kinase domain, two FKBPv domains, and a COOH-terminal hemagglutinin (HA) epitope sequence ( Fig. 1 A). The FGFR1 kinase domain used to generate the iFGFR1 construct contains the intracellular domain of FGFR1 (amino acids 365-822, EMBL/GenBank/DDBJ accession no. U22324) starting three amino acids from the end of the transmembrane domain. Although all four FGFRs were subcloned with the FKBPv domains, only the results obtained with iFGFR1 will be described in detail. A control FKBPv construct also was designed that contains all of the above domains except the FGFR sequences.
AP20187-dependent activation of iFGFR1 signaling in NIH3T3 fibroblasts and HC11 mammary epithelial cells
The phosphorylation levels of iFGFR1, mitogen-activated protein kinase (MAPK), and Akt were determined in both NIH3T3 and HC11 cells transduced with retroviral expression constructs to examine the downstream effects of iFGFR1 dimerization (Fig. 1, A and B). An increase in the tyrosine phosphorylation of iFGFR1 was rapidly detected after AP20187 treatment of iFGFR1-transduced NIH3T3 cells, as determined by immunoprecipitation and immunoblotting using phosphotyrosine and HA epitope antibodies, respectively ( Fig. 1 B). Phosphorylation of both MAPK and Akt was also detected in AP20187-treated NIH3T3 fibroblasts and HC11 mammary epithelial cells (Fig. 1 C). Similar results were obtained with placement of the FKPBv domain NH 2 -terminal to the FGFR1 kinase domain (unpublished data). No increase in MAPK or Akt phosphorylation was observed with cells transduced with FKBPv alone and treated with AP20187 (unpublished data). FGF signaling has been shown to specifically induce tyrosine phosphorylation of the 90-kD adapter protein FRS2/SNT. The tyrosine phosphorylation of FRS2/SNT was examined in AP20187-treated iFGFR1-transduced NIH3T3 fibroblasts to determine if iFGFR1 activation can signal through this pathway. FRS2/SNT was observed to be highly tyrosine phosphorylated in response to AP20187 treatment ( Fig. 1 D), demonstrating that iFGFR1 signaling can activate physiological FGF signaling pathways.
The iFGFR1 and FKBPv constructs were tested in both the fibroblast and mammary epithelial cell lines to determine if they could functionally recapitulate effects mediated by FGF signaling in an AP20187-dependent fashion. One characteristic of NIH3T3 fibroblasts is their dependence on serum growth factors for survival and proliferation. iFGFR1 and FKPBv stably transfected NIH3T3 cells were plated in duplicate plates, grown to subconfluence, placed in serum-free media containing either 30 pM AP20187 or vehicle alone, and then observed for morphological changes over a 72-h time course. When grown in serum-free media in the absence of AP20187, iFGFR1 cells displayed distinct morphological features associated with apoptosis, including membrane blebbing, rounding, and detachment from the surface of the cell culture dish (Fig. 1 E, iFGFR1 Ϫ ; unpublished data). Similar results were obtained with the pBK-neo and FKBPv stably transfected cells in serum-free media treated with and without AP20187 ( Fig. 1 E, pBKneo Ϫ , pBKneo ϩ ; unpublished data). However, iFGFR1 cells treated with AP20187 in the absence of serum remained attached to the cell culture dish, formed distinct multicellular foci and did not display morphological features characteristic of apoptosis ( Fig. 1 E, iFGFR1 ϩ ). A twofold increase in the number of viable cells was observed 48 h after treatment with AP20187 as determined by bioreduction of MTS ( Fig. 1 E, iFGFR ϩ ). These changes were similar to those observed after treatment of NIH3T3 cells with recombinant FGF-8 under the same serum-free conditions ( Fig. 1 F, rFGF8). Thus, treatment of cells stably transfected with iFGFR1 with AP20187 induced a survival response and prevented the contact inhibition that is normally observed in NIH3T3 cells.
Caspase-3 activation was assayed in serum-starved, iFGFR1-transduced NIH3T3 cells in the presence or ab-Figure 1. iFGFR1 dimerization can inhibit apoptosis and induce proliferation. (A) Schematic drawing of FGF-induced dimerization of FGFR and conditional dimerization of the iFGFR1 fusion protein by AP20187. (B) iFGFR1 is phosphorylated in response to AP20187 treatment. Phosphorylated proteins were immunoprecipitated using anti-phospho-tyrosine antibodies and examined by Western analysis with anti-HA epitope antibodies. (C) Western blot of protein extracts isolated from serum-starved NIH3T3 and HC11 cells treated with AP20187 at several time points after treatment (in minutes). Anti-phospho-MAPK and -Akt antibodies show activation of these kinases in response to AP20187 treatment. (D) FRS2/SNT was immunoprecipitated using anti-FRS2 antibodies and tyrosine phosphorylation was analyzed by immunoblotting with anti-phospho-tyrosine antibodies (top). Equal loading of protein on the blot was determined by reprobing the membrane with anti-FRS2 antibodies (bottom). (E) Survival assays were performed using serum-starved NIH3T3 cells. iFGFR1 cells were stably transfected with iFGFR1, whereas pBKneo cells were stably transfected with pBKneo neomycin selection cassette. iFGFR1ϩ and pBKneoϩ cells were treated with 30 pM AP20187, whereas iFGFR1Ϫ and pBKneoϪ cells were treated with ethanol solvent. iFGFR1ϩ cells remained viable when serum starved. (F) The change in cell number from survival assays was quantitated by MTS bioreduction. The fold-change in cell number was determined by normalizing to the untreated control (ϪAP20187 or without rFGF8) for each condition. (G) Caspase-3 fluorometric peptide cleavage assay of serum-starved NIH3T3 cells transduced with iFGFR1 or FKBPv alone and treated with increasing concentrations of AP20187. AP20187-treated iFGFR1 cells showed reduced caspase-3 activity when compared with FKBPv control cells. Data points represent quadruplicate wells. (H) Fold difference in proliferation of AP20187-treated serum-starved NIH3T3 and HC11 cells. Spotted bars represent iFGFR1 and solid black bars FKBPv controls. Data were normalized to cells treated with solvent. Proliferating cells were determined by Ͼ2 N DNA content as measured by propidium iodide staining and FACS analysis. Only iFGFR1-transduced HC11 cells showed an increase in proliferation in response to AP20187. Data points represent at least three independent analyses. Error bars represent standard error of the mean. Bars, 5 m. treatment, the inguinal mammary glands were biopsied for whole-mount and immunofluorescence analyses (Fig. 2). Whole-mount analysis of the mammary epithelium showed no gross morphological differences between agematched nontransgenic littermates treated with AP20187 and transgenic littermates treated with solvent only (Fig. 2, A-D). However, transgenic mice treated with AP20187 displayed increased lateral budding along the ductal epithelium (Fig. 2, E and F). AP20187-induced lateral budding was observed throughout the mammary gland and appeared on primary, secondary, and tertiary ductal branches. Increased lateral budding was also observed at the distal tips of the mammary epithelium (Fig. 2, E and F, arrowhead). Additionally, regional increases in ductal branching were observed in AP20187 treated transgenic mice (Fig. 2, E and F, arrows). Histological analysis revealed extensive lateral buds, with single and multicell-layered epithelium lining the ducts (Fig. 2 L). Although these lateral buds did contain lumens, many of these appeared constricted or convoluted.
AP20187-induced lateral budding does not require ovarian hormones
Transgenic mice were ovariectomized (ovex), and 2 d later were treated with AP20187 every 3 d for 16 d to determine if ovarian hormonal regulation is required for the lateral budding phenotype. Whole-mount analysis of ovex nontransgenic littermates treated with AP20187 showed constriction of ductal epithelium and a loss of terminal-end buds (TEBs), consistent with the loss of the inductive effects from the steroid hormones estrogen and progesterone (Fig. 2,G and H). However, when ovex transgenic mice were treated with AP20187, dilated ductal epithelium and large bloated structures at the distal tips of the ductal network were observed (Fig. 2, I and J, arrowheads). Regions of increased ductal branch points were also observed in ovex AP20187-treated transgenic mice (Fig. 2,I and J,arrows). The ductal network did not completely fill the fat pad in either transgenic or nontransgenic ovex mice treated with AP20187, suggesting that iFGFR1 activation cannot, by itself, rescue normal ductal elongation in the absence of ovarian hormones. However, the observation that iFGFR1-induced lateral budding occurred in the ovex background demonstrates that ovarian hormones are not required for this phenotype.
Transgene expression is localized to ductal and lateral bud epithelium
The localization of transgene expression in the mammary gland of transgenic mice was determined by immunofluorescent detection of the HA-epitope on the iFGFR1 protein. Mammary epithelial cells expressing the HA-epitope were observed in transgenic mice, but not in nontransgenic littermates (Fig. 2,M and N). Expression of the iFGFR1 protein was localized to the periphery of the cell and no nuclear staining was apparent, consistent with membrane targeting by the myristylation sequence. Transgene expression was punctate along the ductal epithelium of untreated 8-wk-old transgenic mice (unpublished data), sence of AP20187 to quantitate the iFGFR1-induced inhibition of apoptosis ( Fig. 1 G). Cells were placed in serum-free medium with or without AP20187 for 24 h. Under these conditions, a significant dose-dependent decrease in caspase-3 activity was detected beginning with a dose of 0.5 pM AP20187 ( Fig. 1 G, solid line). A maximal decrease was achieved with 30 pM AP20187. No significant decrease in caspase-3 activity was detected using the control FKBPv construct ( Fig. 1 G, dashed line). An AP20187-dependent increase in mRNA for the antiapoptotic factor Bcl-xl was also observed (unpublished data). These data suggest that iFGFR1 activation can inhibit caspase-3-mediated apoptosis caused by serum withdrawal.
Cell cycle analysis was employed in these two different cell types to determine if the AP20187-induced iFGFR1 signaling pathway also regulates proliferation. Transduced NIH3T3 fibroblasts and HC11 mammary epithelial cells were treated with AP20187 in serum-free media and cellular DNA content was quantitated by propidium iodide staining and FACS ( Fig. 1 H). NIH3T3 cells expressing iFGFR1 showed no change in proliferation with or without AP20187. However, HC11 cells expressing iFGFR1 showed greater than a twofold increase in proliferation in response to AP20187. In the presence or absence of AP20187, no difference in proliferation was detected in the control FKBPv transduced HC11 or NIH3T3 cells ( Fig. 1 H,
black bars).
These data suggest that iFGFR1-regulated signaling pathways may exert functionally different effects that are cell type dependent. Although iFGFR1 appears to activate the same downstream pathways in fibroblasts and mammary epithelial cells, effects on their proliferation are not equivalent.
Generation of MMTV iFGFR1 transgenic mice
Transgenic mice were generated that express the iFGFR1 construct under the control of the MMTV long-terminal repeat (LTR). iFGFR1 and iFGFR2 constructs were subcloned into an MMTV transgene cassette for the generation of transgenic mice (Leder et al., 1986). Three iFGFR1 and four FGFR2-FKBPv lines were generated that transmitted the transgene through the germline. Of these seven lines, three (one iFGFR1 and two iFGFR2) expressed the transgene in the mammary gland as detected by Western blot analysis of the HA epitope tag (unpublished data). All three expressing lines displayed normal morphology in virgin mice and similar inducible phenotypes described below. A detailed characterization was performed on the iFGFR1 line 4775, which expressed low levels of iFGFR1 mRNA, detectable by RT-PCR, and low levels of protein expression as determined by Western blot analysis (unpublished data).
Activation of iFGFR1 results in increased lateral buds
To analyze the ductal morphology of mammary glands in the expressing transgenic lines, whole-mount analysis of the thoracic and inguinal glands was performed. 6-wk-old transgenic and control mice were treated intraperitoneally with injections (50 g at 0.5 mg ml Ϫ 1 ) of AP20187 every 3 d for 16 d, or by intrascapular implantation of Alzet minipumps containing 100 l of 0.5 mg ml Ϫ 1 AP20187 which delivered 0.25 l h Ϫ 1 for 2 wk. After AP20187 most likely due to an incomplete penetrance of the MMTV LTR-driven transgene, whereas AP20187-induced lateral buds were strongly immunoreactive ( Fig. 2 N). Thus, it is possible that AP20187 treatment results in the expansion of transgene positive cells, as a result changing the expression from a punctate to a multifocal pattern. However, not all cells in lateral buds expressed the HAtagged transgene. Instead, lateral buds contained a mixture of cell types including either transgene positive or progesterone receptor (PR)-positive cells (unpublished data). PR-positive cells within lateral buds and lining the ducts were 5-bromo-2 Ј -deoxyuridine (BrdU) negative, consistent with the observed quiescent nature of PR-expressing cells in the mammary gland (unpublished data) (Seagroves et al., 2000).
Chronic activation of iFGFR1 results in progressively invasive lesions
Whole-mount, histological, and immunofluorescent analyses were utilized to characterize the progressive nature of the iFGFR1-induced lesions. To characterize these lesions morphologically, whole mounts and paraffin-embedded sections were stained with hematoxylin or hematoxylin and eosin (H&E), respectively. In addition, cell polarity was determined by indirect immunofluorescence microscopy using antibodies directed against specific, apical tight junction (ZO-1) and basal, basement membrane (laminin) markers. iFGFR1-induced lesions were classified into three morphologically distinct types. Type I lateral buds were characterized by the presence of a single layer of cubodial epithelium surrounded by myoepithelial cells, and with basally localized laminin and apically localized ZO-1. Type-I lesions appeared within 3 d of AP20187 treatment and were similar to lateral alveolar buds induced in early pregnancy ( Fig. 3 A, type I) (Daniel and Silberstein, 1987). However, unlike lobuloalveolar development during late pregnancy, the iFGFR1-induced lateral buds remained closely associated with their primary ducts and did not completely occupy the interductal stroma.
After ف 2 wk of iFGFR1 activation, multibud, type II lesions appeared along the primary duct. These were the predominant morphology observed in the mammary gland of AP20187 treated transgenic mice (Fig. 3 A, type II). Type II lesions contained multiple cell layers and had collapsed lumens (as determined by ZO-1 localization) (Fig. 3 A). Cells at the periphery of type II lesions still remained organized around the basement membrane (as determined by anti-laminin immunofluorescent staining). Similar to type I lateral buds, these lesions remained closely associated with primary ducts, expressed PR, and were not highly invasive into the surrounding stroma (Fig. 3 A, type II; unpublished data).
Type III lesions were first observed after ف 4 wk of AP20187 treatment and appeared as multifocal lesions along ducts (Fig. 3 A, type III, whole mount). Type III lesions exhibited a marked loss of laminin and ZO-1 expression, and were multicellular, invasive, and highly vascularized (Fig. 3 A, type III, H&E and immunofluorescence). These lesions were often associated with a surrounding leukocyte infiltration and reactive stroma (Fig. 3 A, type III, H&E). Interestingly, analysis of the more invasive type III lesions by FACS suggests that these lesions were predominantly diploid (unpublished data). Thus, type III lesions have some characteristics similar to intraepithelial neoplasia and thus may represent a preneoplasiac growth.
iFGFR1 dimerization induces proliferation and activation of signaling pathways in vivo
Dimerization-induced phosphorylation of the iFGFR1 protein and downstream signaling pathways were analyzed by Western blotting and immunofluorescence in transgenic mice 24 h after the completion of a 2-wk treatment with AP20187. Protein extracts isolated from AP20187-treated and -untreated transgenic littermates showed increased iFGFR1, Akt, and MAPK phosphorylation only in treated mice (Fig. 4, A and B). In transgenic mice treated for 16 d, immunolocalization of the phosphorylated (Thr202/ Tyr204) MAPK correlated with the lateral bud lesions (Fig. 4,C and D). These results suggest that AP20187 treatment induced the phosphorylation and activation of the iFGFR signaling cascade in transgenic mice similar to that observed in NIH3T3 and HC11 cells.
The percentage of cells in S phase was quantitated in lateral buds as determined by BrdU incorporation to assess whether AP20187-induced lateral buds were hyperproliferative in response to activated iFGFR signaling. BrdU was administered to AP20187-treated and untreated transgenic mice 2 h before sacrifice and localized in the mammary epithelium using a FITC-conjugated anti-BrdU antibody (Fig. 4, E and F). In untreated transgenic mice, 0.9% ( Ϯ 0.2 SEM) of mammary epithelial cells were positive for BrdU. However, after 28 d of AP20187 treatment, BrdU was detected in 9.5% ( Ϯ 1.4 SEM) of mammary epithelial cells. The number of BrdU-positive cells found in lateral buds and ductal cells was 8.5% ( Ϯ 1.6 SEM) and 11.7% ( Ϯ 1.9 SEM), respectively. No significant differences were observed in apoptosis between treated and untreated transgenic mice as measured by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining (unpublished data). These results suggest that activation of the iFGFR1 kinase induces proliferation not only in regions of phenotypic lateral buds, but also within the ductal epithelium.
Transgenic mice were treated for 3 d with AP20187, followed by 120 h of drug withdrawal to assess the reversibility of the system. The half-life of AP20187 in the bloodstream has been determined previously to be ف 7 h (Tim Clackson, ARIAD Pharmaceuticals, personal communication). Both the increase in MAPK phosphorylation and proliferation in the Figure 3. Histology of iFGFR1-induced lesions in the mammary gland. Three histologically distinct lesions are observed in AP20187-treated transgenic mouse mammary glands. (A) Panel shows gross mammary gland morphology by whole mounts (10ϫ magnification), cellular detail by H&E stain (20 and 40ϫ), and cellular polarity by immunofluorescence analysis (100ϫ) with anti-ZO-1 (Texas red) and anti-laminin (FITC). Type I lesions initially appeared by day 3 of treatment, and are characterized by the punctate appearance of lateral buds lining the ductal epithelium (Type I, whole mount). Type I lateral buds contain a single layer of polarized mammary epithelial cells with large distinct lumens (Type I, H&E and ZO-1/ Laminin). Type II lesions appear starting at week 2 of AP20187 treatment, and are distinguished by uniform multicellular epithelium with small collapsed lumens (Type II). Type III lesions are multicellular, invasive, well vascularized, and have lost ZO-1 and laminin expression (Type III). Bars, 5 m. iFGFR1-induced mammary lateral budding and hyperplasia | Welm et al. 709 mammary epithelium were not reversible after injections of AP20187 were stopped for 120 h before tissue biopsy (Fig. 4, G and H). Similar results were obtained when mice were treated with AP20187 for 6 wk followed by a withdrawal interval of 2 wk before to tissue biopsy (unpublished data).
iFGFR1 activation alters mammary epithelial cell polarity
Mammary epithelial cells undergo an intrinsic apoptotic response upon detachment from the basement membrane, a process termed anoikis. Confocal microscopy was utilized to determine if the iFGFR1-induced lesions contain cellular layers detached from the basement membrane. Thick frozen sections of mammary glands isolated from AP20187-treated transgenic mice were stained with anti-E-cadherin (Texas red) and laminin (FITC) antibodies (Fig. 4, I and J). Wild-type midpregnant mice were used as a control to contrast alveolar buds with iFGFR1-induced lesions. Confocal microscopic analysis showed that the mammary epithelium forms a single layer of cells contacting both the laminin-rich basement membrane (Fig. 4 I, arrowhead) and lumenal space (Fig. 4 I, asterisk) in wild-type midpregnant mice. Additionally, E-cadherin localization was observed only on lateral sides of epithelial cells, at sites of cell-to-cell contact, and not at the apical or basal membranes (Fig. 4 I, arrow). Transgenic mice treated with AP20187 formed multicellular layers with inner (lumenal) layers detached from the basement membrane (Fig. 4 J, arrow). Additionally, E-cadherin staining was observed on the entire periphery of epithelial cells, suggesting cell-to-cell contact was no longer restricted to lateral membranes (Fig. 4 J, arrow). The loss of attachment to the basement membrane and peripheral contacts with neighboring cells suggests that iFGFR1-induced lesions are no longer responsive to polarity signals, including anoikis. This is consistent with iFGFR1 signaling inhibiting apoptosis and inducing proliferation in the mammary epithelium .
iFGFR1-induced lesions have invasive characteristics
The presence of myoepithelial cells, extracellular matrix (ECM) and the vascular network were analyzed in AP20187treated transgenic mice to assess the invasive characteristics of iFGFR1-induced lesions. The matrix metalloproteinases (MMPs) are a family of related Zn ϩϩ interacting proteases that are increasingly implicated in tumorigenesis (Benaud et al., 1998;John and Tuszynski, 2001). Analysis of MMP activity in conditioned media from iFGFR1-expressing mammary epithelial cells was used to determine if MMPs are regulated through iFGFR1 signaling. Using gelatin zymogra-Anti-BrdU immunofluorescence from wild-type (E) and transgenic (F) mice treated with AP2087 for 4 wk and pulsed with BrdU for 2 h. To determine reversibility, iFGFR mice were treated for 3 d with AP20187, and treatment was stopped 120 h before tissue biopsy. Phosphorylated MAPK (Texas red) and proliferation (anti-BrdU-FITC) were detected in the AP20187-treated (G) and AP20187-withdrawal biopsy (H). Confocal microscopic and immunofluorescence analysis of midpregnant wild-type (I) and 2-wk AP20187-treated transgenic (J) mammary glands using anti-E-cadherin (Texas red, arrows) and antilaminin (FITC, arrowhead) antibodies. AP20187-treated transgenic mice show multi-cell layering (distance between arrowhead and arrow), collapsed lumens (asterisks), and peripheral localization of E-cadherin. Bars, 5 m. Immunoprecipitation with anti-HA epitope antibodies and Western analysis using anti-phospho-tyrosine and HA-epitope antibodies of extracts from transgenic mice treated with AP20187 ( ϩ ) or diluent ( Ϫ ) for 2 wk. iFGFR1 shows increased phosphorylation levels in AP20187-treated transgenic mice. (B) Western blot analysis showing increased phosphorylation levels of MAPK and Akt in AP20187-treated transgenic mice (MMTV-iFGFR1) over wild-type mice treated with diluent (wt). The positive control was iFGFR1-transduced NIH3T3 cells (NIH3T3-iFGFR1) treated with AP20187. Immunofluorescence analysis with anti-phospho-MAPK antibody (Texas red) and DAPI in wild-type (C) and transgenic (D) mice treated with AP20187 for 2 wk. phy, medium from serum-starved iFGFR1-expressing HC11 mammary epithelial cells treated with AP20187 showed increased MMP activity over solvent treated cells (Fig. 5 A). Gelatinase activity was inhibited when the chelating reagent EDTA was added to the incubation buffer (Fig. 5 A). Additionally, increased gel migration of MMP-9 was observed after pretreatment of the protein extracts with p-aminophenylmercuric acetate (APMA), an inducer of autocatalytic cleavage of proMMPs (Fig. 5 A). These data demonstrate that iFGFR1 activation in mammary epithelial cells can induce MMP-2 and MMP-9 activity.
The presence of the myoepithelial cell barrier in mammary glands from AP20187-treated mice was analyzed by confocal microscopy using antibodies against the specific myoepithelial cell markers keratin-14 (K-14) and ␣ -actin. In untreated wildtype mice, a continuous K-14-positive cell layer was observed between the luminal epithelium and stromal cell compartments (Fig. 5 B). However, fewer K-14-positive myoepithelial cells surrounding lateral buds were observed in transgenic mice after 2 wk of AP20187 treatment. Additionally, primary ducts associated with the lateral buds displayed noncontiguous and patchy K-14 staining along the duct (Fig. 5 C). Similar results were obtained using antibodies against smooth muscle ␣ -actin (unpublished data). The absence of myoepthelial cells correlates with a reduction of proteinaceous ECM surrounding the iFGFR1 induced lateral buds, as observed by Masson's trichrome staining (Fig. 5, D and E). However, although collagen IV (unpublished data) and laminin (Fig. 4 H) staining was still observed in the basal lamina surrounding lateral buds, their expression appeared more disorganized than in untreated mice. These data suggest that iFGFR1 activation can initiate the reorganization and breakdown of ECM surrounding the lateral buds.
Proliferation of the epithelium in the mammary gland requires concomitant changes not only in the underlying stroma, but also in the vascular network supporting the new growth. The vascular network surrounding iFGFR1-induced lateral buds was observed in situ using confocal microscopy and FITC-lectin visualization of blood vessels. To visualize blood vessels, 2-wk AP20187-treated transgenic mice were injected into the left ventricle with FITC-Lycopersicon esculentum lectin. FITC-lectin-injected mice were perfused with fixative, and mammary glands were biopsied and frozen in OCT compound. Mammary glands were cryosectioned and stained with Texas red phalloidin to identify the mammary epithelium. Confocal imaging and software-rendered three-dimensional reconstitution revealed a highly branched network of blood vessels surrounding iFGFR1-induced lateral buds (Fig. 5, F and G). Lateral buds were primarily associated with small tortuous vessels branching off of larger vessels lining the ductal epithelium (Fig. 5, F and G, arrows). These data suggest that sprouting angiogenesis from the existing ductal vascular network may be initiated indirectly by iFGFR1 signaling in the mammary epithelium.
Discussion
Several mouse models of breast cancer have been developed, but the utility of these models in studying the early events in transformation of the mammary gland is limited due to the inability to regulate oncogenic events. One exception to this is the use of the tetracycline-inducible system to drive expression of oncogenes in the mammary epithelium (D'Cruz et al., 2001). In this paper we describe a novel inducible mouse model of breast cancer that can be used to study the early progressive steps of tumorigenesis (Fig. 6 A). The iFGFR model is the first use of an inducible-dimerization system of a tyrosine-kinase receptor in transgenic mice.
Activation of iFGFR signaling in the mammary gland results in several distinct stages of transformation, including epithelial hyperproliferation (observable 72 h after AP20187 treatment) and stromal invasion (Fig 6 A). Several factors, including MMP regulation, ECM remodeling, and absence of a myoepithelial cell barrier, may contribute to the invasiveness of these lesions (Fig. 6 B). Myoepithelial cells play a role in the production and maintenance of the ECM barrier that surrounds ductal epithelium, and secrete antiangiogenic factors (Xiao et al., 1999;Nguyen et al., 2000). Loss of myoepithelium and ECM is associated with invasive characteristics in breast cancer (Batsakis and el-Naggar, 1999;Xiao et al., 1999). Moreover, the ECM has been implicated in an active role in the regulation of proliferation, differentiation, and angiogenesis by regulating growth factor bioavailability (Coussens et al., 2000;Silberstein, 2001). Coussens et al. (2000) have demonstrated that neutrophils expressing MMP-9 induce angiogenesis by releasing VEGF from the ECM, thus increasing its availability to endothelial cells (Coussens et al., 2000). Interestingly, we have observed that conditioned media isolated from iFGFR1-transduced mammary epithelial cells treated in culture with AP20187 displayed an increased MMP-9 and MMP-2 activity. Consistent with these data, reduced ECM and increased vascular branching surrounding iFGFR-induced lesions was observed in AP20187-treated transgenic mice. However, it is likely that the invasive nature of type III lesions may be augmented by infiltration of leukocytes and increased secretion of MMP-9 (Coussens et al., 2000). Long-term treatment (3-12 mo) of transgenic mice with AP20187 will be needed to determine if the localized invasive nature of type III lesions are premalignant and can progress to adenocarcinomas with metastatic potential.
The rapid 4-wk time period from the appearance of initial type I to the invasive type III lesions suggests that iFGFR1 signaling in mammary epithelium exerts both potent proliferative and antiapoptotic effects (Fig. 6 A). However, the complex nature of iFGFR1-induced lesions, including the loss of myoepithelium and increased vascular branching, suggest that other indirect effects mediated through stromal interactions also contribute to the invasive characteristics. The conversion Figure 6. Model for FGFR-induced lateral buds and hyperplasia in the mammary gland. (A) Acute iFGFR signaling in the mammary epithelium induces lateral buds (type I) within 72 h of treatment with AP20187. Continuous treatment for 2 wk results in multicellular epithelium (type II) that can progress into invasive lesions (type III) after 4ف wk of treatment. (B) Normal mammary epithelial cells are polarized (box) with apical localization of ZO-1 at tight junctions, and lateral localization of E-cadherin. The ECM and myoepithelium at the basement membrane can regulate the bioavailability of growth factors and inhibit angiogenesis. Acute iFGFR activity in the mammary epithelium induces proliferation and upregulation of ECM proteases resulting in epithelial invasion into the stroma and the formation of lateral buds. Disruption of the ECM, through MMP upregulation (᭹), may induce vascular branching by increasing the bioavailability of endothelial growth factors (᭡), thus supporting epithelial growth. However, chronic iFGFR activation results in disorganized cell polarity including peripheral localization of E-cadherin and loss of ZO-1 localization at tight junctions. Additionally, the loss of anti-angiogenic factors associated with the myoepithelium may contribute to the invasive characteristics of the iFGFRinduced lesions. Through these mechanisms, FGFR signaling during ductal morphogenesis may function in the proliferation and invasion of mammary epithelium to establish a ductal network, whereas its aberrant regulation may play a role in breast cancer. of a single layer ductal epithelium to multicell layers found in type II and type III lesions suggest that iFGFR kinase activity inhibited the apoptosis that is normally associated with detachment of lumenal epithelial cells from the basement membrane. In iFGFR1-expressing mice treated with AP20187, no significant changes in apoptosis were observed by TUNEL staining when compared with untreated mice (unpublished data). Levels of apoptosis in the ductal epithelium of mature virgin mice detected by the TUNEL assay are extremely low ( Ͻ 0.1%), thus limiting the ability to detect a decrease in apoptosis (Humphreys et al., 1996;Rosfjord and Dickson, 1999). Interestingly, recent studies using an MMTV LTRdriven Tet-regulatable system have shown that after c-myc activation, there is an increase in both proliferation and apoptosis, reaching levels of ف 10% in the ductal epithelial cells (D'Cruz et al., 2001). Increased lateral budding and mammary hyperplasia were also observed in this transgenic model, but required a long latency during which other stochastic events, such as Kras2 activation may occur (D'Cruz et al., 2001). In contrast, iFGFR signaling can directly activate both proliferation and survival signals within mammary epithelium to rapidly induce hyperplastic lesions. Moreover, Muthuswamy et al. (1999Muthuswamy et al. ( , 2001, using the same inducible dimerization system, demonstrated that activation of ErbB2 but not ErbB1 in human mammary epithelial MCF10A cells resulted in similar changes in proliferation and a loss of cell polarity demonstrating that highly homologous receptor tyrosine kinases can induce differential effects (Muthuswamy et al., 1999(Muthuswamy et al., , 2001. The FGF and EGF growth factor receptor families share several downstream signaling factors that may explain the similarity in the iFGFR1-and ErbB2-induced effects on mammary epithelial cells. During branching morphogenesis of the Drosophila trachea and mouse lung, signaling between the epithelium and mesenchyme is essential in ductal bifurcation, and FGF ligands and FGFRs have been implicated as critical factors in this process (Metzger and Krasnow, 1999;Sekine et al., 1999). The effects of iFGFR signaling described in this study, and the pattern of FGF ligand and FGFR expression during ductal morphogenesis, are consistent with a possible role for this growth factor family in initiating branching in the mammary gland (Fig. 6 B). In this study, iFGFR1 kinase activation elicited direct effects on mammary epithelium including regulation of proliferation, apoptosis, and MMP secretion. Additional indirect effects, including ECM remodeling and vascular branching, may function in restructuring the stroma to provide support for the developing ductal network (Fig. 6 B). Factors initiating branching morphogenesis and lateral budding should induce similar effects during mammary gland development. Therefore, it is likely that coordinated FGF signaling during ductal morphogenesis contributes to this developmental process. Conditional FGFR knockouts will be important to further substantiate the importance of FGF function during mammary gland development.
Because transgene expression levels may contribute to AP20187-independent dimerization of the iFGFR constructs, the line used in these studies (4775) had the lowest levels of protein expression, as detected by Western blot analysis, and required RT-PCR analysis for mRNA detection (unpublished data). However, all expressing lines dem-onstrated AP20187-independent tumorigenesis in parous mice appearing 6-12 mo after parturition (unpublished data). It is unlikely that integration effects contribute to this phenotype, as all lines exhibit both normal mammary gland morphology in untreated virgin transgenic mice, and tumors after breeding. It is more likely that during pregnancy and lactation, transgene expression is upregulated to levels that favor AP20187-independent activation, resulting in preneoplastic lesions and leading to tumorigenesis. This is consistent with the appearance of multifocal intraepithelial neoplastic lesions in the mammary glands of untreated parous mice and the long latency for tumor development (unpublished data). However, we cannot rule out other initiating factors contributed by pregnancy, lactation, or involution that may cooperate with iFGFR1 signaling to induce tumorigenesis. The use of the MMTV-LTR promoter to drive expression of the transgene may also contribute to the lack of reversibility of the lesions through the initiation of an autoregulatory loop established upon activation of iFGFR signaling. This is consistent with the observed increase in transgene expression after only 3 d of AP20187 treatment as detected by Western blot analyses (unpublished data). Transgenic mice containing iFGFR1 regulated by a tetracycline dependent promoter crossed with mice containing the reverse tetracycline-dependent transcriptional activator under the control of the MMTV promoter (D'Cruz et al., 2001), may provide an improved model to regulate both expression levels of iFGFR1 using doxycycline and iFGFR1 activity using AP20187 resulting in a reversible system in which to study tumor progression. However, the iFGFR constructs described in our study have been shown to elicit reversible effects when expressed downstream of different promoters in transgenic and tumor cell transplantation models (unpublished data).
The iFGFR1 transgenic mice provide a mouse model for the analysis of progression from early hyperproliferative to invasive lesions in the mammary gland. These mice will be useful in defining the steps necessary for transformation. However, these data must be interpreted with the caveat that the iFGFR transgene contains several differences from the endogenous FGFR1 that may alter its activation kinetics and cellular localization. The iFGFR constructs contain only the intracellular domain of the FGFR and eliminate regulatory elements found in the extracellular and transmembrane domains (Rousseau et al., 1996;Burke et al., 1998;Wang et al., 1999). The extracellular domain of the endogenous receptor confers affinity for ligand and heparin-sulfate proteoglycans that regulate signal intensity, duration, and affect cellular response. The iFGFR constructs contain a Src myristylation sequence to anchor the receptor to the plasma membrane. Thus, it is likely that this may influence the localization of the iFGFR, and also, potentially, the duration and nature of iFGFR signaling. The examination of signaling molecules that are activated by differentially localized constructs should reveal whether basolateral versus nonlocalized expression affect the duration and specificity of RTKinduced signaling pathways. Therefore, further comparison between iFGFR and FGFR signaling will be required to assess the capacity of the AP20187/FKBPv system to completely recapitulate the endogenous signaling pathways.
Plasmids and cell culture
The FGFR1 intracellular kinase domain was cloned by PCR amplification of the mouse FGFR1 cDNA plasmid pMo/FR1/IRES, a gift from David Ornitz (Washington University, St. Louis, MO) using the following primers: forward 5 Ј -ATTATAGTCGACATGAAGAGCGGCACCAAGAAGAGC-3 Ј and reverse 5 Ј -CTACTTGTCGACGCGCCGTTTGAGTCCACTGTTGG-3 Ј . The FGFR1 kinase domain was sequenced and subcloned into pSH1/MF v -F vls -E plasmid providing an NH 2 -terminal myristylation sequence, two tandem FKBPv domains (F v -F vls ), and hemagglutinin epitope sequence (Fan et al., 1999). The entire iFGFR1 coding region, including the NH 2 -terminal myristylation and hemagglutinin epitope sequences, was subcloned into pMMP, for retroviral transduction experiments (Riviere et al., 1995). For generating transgenic mice, the iFGFR1 coding sequence was cloned into the MMTV-KCR transgene cassette, pMKBPA, a gift from Steven Chua and Franco DeMayo (Baylor College of Medicine, Houston, TX). NIH3T3 cells were grown in growth media containing DME (GIBCO BRL), 10% bovine calf serum (JRH), and 50 g ml Ϫ1 gentamicin (Sigma-Aldrich). HC11 cells were grown in growth media containing RPMI 1640 medium (RPMI) (JRH) supplemented with 10% fetal bovine serum, 10 ng/ml Ϫ1 EGF (Life Technologies), 5 g ml Ϫ1 insulin (Sigma-Aldrich), and 50 g ml Ϫ1 gentamicin. Serum starvation media for NIH3T3 and HC11 cells contained only DME or RPMI, respectively, supplemented with either 30 pM AP20187 (Ariad Pharmaceuticals) in ethanol or an equal volume of ethanol-diluent alone as control. The 293T cell growth media contained DME, 10% fetal bovine serum (Summit), and 50 g ml Ϫ1 gentamicin. For cell transduction, retroviruses were packaged by cotransfecting pMMP-iFGFR1 or pMMP-FKBPv plasmids with pCLEco (Imgenex) into 293T cells using FuGene (Roche) following the manufacturer's protrocol. 48 h after transfection, retroviruscontaining 293T media was supplemented with 10 g ml Ϫ1 polybrene (Sigma-Aldrich), syringe filtered using a 0.4-m filter unit (Millipore) and 5 ml were added to 100-mm plates of HC11 or NIH3T3 target cells. The target cells with retroviral media were spun in a Marathon centrifuge at 1,800 rpm (500 g) for 30 min with a 1/3 rotation of the plate every 10 min to permit equal distribution of media over the plate.
Survival and proliferation assay
For cell survival assays, the pSH1-iFGFR1 construct was cotransfected with pBKneo into NIH3T3 cells using Fugene. Stable cells were selected by supplementing growth media with 200 g of active G418 ml Ϫ1 for 3 wk. Pooled NIH3T3 cells were placed in serum-free DME media for 72 h with daily media changes for morphological analysis. Images were captured using a Sony CCD-DXC-151A camera. Caspase-3 activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC (PharMingen). Briefly, iFGFR1 and FKBPv-transduced NIH3T3 cells were treated with AP20187 or ethanol diluent in serum-free media for 24 h, followed by cell lysis and incubation with the fluorogenic peptide according to the manufacturer's protocol. Cell proliferation was measured, following the manufacturer's protocol, by propidium iodide (Roche) staining of AP20187 or ethanol diluent-treated cells in serum-free media for 48 h. DNA content was measured by FACS. For in vivo proliferation, a BrdU label retention assay was performed as previously described by Seagroves et al. (1998). Assays were performed in triplicate or quadruplicate and repeated at least two times. BrdU quantitation was performed by counting positive cells from at least five 40ϫ images from two or more mice.
Western blots and zymography
The following antibodies were used for Western blot analysis: phospho-MAPK; phospho-Akt and Akt (Cell Signaling); PY20 (BD Transduction Laboratories); FRS2 (Santa Cruz Biotechnology); and HA-epitope (Santa Cruz Biotechnology). Protein extracts from cultured cells were isolated with Triton X-100 extraction buffer (150 mM NaCl, 1% Triton X-100, 1 g ml Ϫ1 aprotinin, 1 g ml Ϫ1 benzeamidine, 1 g ml Ϫ1 antipain, 10 g ml Ϫ1 soybean trypsin inhibitor, 0.25 g ml Ϫ1 leupeptin, 100 M PMSF, 100 uM NaVanadate, 1 mM NaF) and passed through a 26-G syringe five times followed by incubation on ice for 45 min. Protein extracts were quantitated by Bradford assay (Bio-Rad), and 80 g protein was resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore). Western blot analyses were followed as previously described (Seagroves et al., 1998). Chemiluminsecence was developed following manufacturer's protocol (Pierce Chemical Co.). For zymography, 1.5 ml media from AP20187 or ethanol diluent-treated HC11 cells in serum-free media for 16 h was concentrated with YM-10MW Centricon membranes following the manufacturer's protocol (Millipore), loaded in equal volumes on gelatin zymography gels, and analyzed as described by Talhouk et al. (1991).
Indirect immunofluorescence and histology
The following primary antibodies were used for indirect immunofluorescent detection of specific antigens: rabbit polyclonals were anti-keratin-14 (Convance Research Products); anti-laminin (Dako); and anti-phospho-MAPK (Cell Signaling). Rat monoclonals were anti-E-cadherin (Zymed) and anti-ZO-1 (Chemicon), and mouse monoclonals were anti-HAepitope (Convance Research Products) and anti-BrdU (Becton Dickinson). Secondary antibodies were: anti-mouse Texas red; Texas red anti-rat; FITC anti-rabbit; and Texas red anti-rabbit (Molecular Probes). Tissue was processed and stained as previously described (Seagroves et al., 1998). For confocal microscopic analysis of frozen sections, tissue was fixed in 4% PFA/PBS for 2 h and frozen in OCT. Frozen sections were cut (50 m) and stained overnight with primary antibody in 3%BSA/PBS. Sections were washed in PBS for 10 min and stained with secondary antibodies for 5 h. Sections were then washed for 30 min, mounted with Vectashield, and analyzed using a Zeiss 510 laser scanning confocal microscope. Whole mounts were prepared as previously described (Williams and Daniel, 1983), and images were captured using Olympus dissecting microscope and Sony video camera (#DXC-151A). H&E sections were prepared as previously described (Seagroves et al., 1998). Masson's trichrome staining was performed with Accustain Trichrome Stain following the manufacturer's protocol (Sigma-Aldrich).
FITC-lectin perfusion
Mice were implanted with Alzet minipumps (Alzet) containing 100 l of 0.5 mg ml Ϫ1 AP20187, delivering 0.25 l h Ϫ1 for 2 wk. Mice were anesthetized after 2 wk with Avertin followed by left ventricular injection of Lycopersicon esculentum FITC-lectin (Vector). After 5 min, 5 ml of 1% paraformaldehyde/0.5% gluteraldehyde/PBS was perfused followed by 5 ml of PBS. Tissue was then dissected and frozen in OCT. For confocal microscopy, thick 50-m sections of OCT embedded tissue were cut and postfixed in 4% PFA/PBS for 10 min at room temperature. Sections were then permeablized with 1% Triton X-100 in PBS for 10 min at room temperature, washed in PBS for 5 min, and stained for 5 h with phalloidin-Alexa594 (Molecular Probes). Sections were washed for 20 min in PBS, mounted with Vectashield, and imaged.
Online supplemental material
Supplemental videos are available at http://www.jcb.org/cgi/content/full/ 200107119/DC1. Video 1 shows a midpregnant mouse mammary gland consisting of a single cell layer of polarized mammary epithelium. Threedimensional rotation of the midpregnant mammary gland demonstrates the single-cell epithelial layer between the basement membrane and lumen. Video 2 represents iFGFR1-induced lesions containing multicell-layered mammary epithelium with altered cell polarity. Confocal imaging through the iFGFR1-induced lesion demonstrates the multiple cell layers, reduced lumen size, and peripheral staining of E-cadherin observed throughout these lesions. Videos 3 and 4 show increased vascular branching surrounding iFGFR1-induced lateral buds. Confocal immunofluorescence and threedimensional imaging of the vascular network shows the close proximity and branching of blood vessels surrounding iFGFR1-induced lateral buds. | 2017-06-26T18:49:55.959Z | 2002-05-13T00:00:00.000 | {
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221378331 | pes2o/s2orc | v3-fos-license | Engineering Plant Synthetic Pathways for the Biosynthesis of Novel Antifungals
Plants produce a wealth of biologically active compounds, many of which are used to defend themselves from various pests and pathogens. We explore the possibility of expanding upon the natural chemical diversity of plants and create molecules that have enhanced properties, by engineering metabolic pathways new to nature. We rationally broaden the set of primary metabolites that can be utilized by the core biosynthetic pathway of the natural biopesticide, brassinin, producing in planta a novel class of compounds that we call crucifalexins. Two of our new-to-nature crucifalexins are more potent antifungals than brassinin and, in some instances, comparable to commercially used fungicides. Our findings highlight the potential to push the boundaries of plant metabolism for the biosynthesis of new biopesticides.
■ INTRODUCTION
Engineering of metabolic pathways in plants holds great promise for moving specialized metabolism from one plant to another. 1 Beyond direct pathway transfer there is the opportunity to engineer these pathways to generate new-tonature compounds that are optimized for a particular application. 2 This pathway engineering approach is inspired by the success of natural product analogues and/or derivatives that have been generated for improved activity and/or efficacy in the clinic. Notably, many natural compounds are biosynthesized by plants as biopesticides, protecting them from various pathogens, albeit many times at much lower activity than traditional synthetic pesticides. 3 To explore the limitations of naturally occurring secondary metabolites, we sought to engineer novel biosynthetic pathways for new-tonature biopesticides, inspired in structure by the natural metabolite brassinin.
The antipathogenic properties of cruciferous phytoalexins often have toxicity on plant cells, and so they are only produced in response to stress. However, phytoalexin pathways can be long, and de novo biosynthesis of a phytoalexin from primary metabolism may be too slow to stop pathogens. 4 To circumvent this problem, many plants constitutively accumulate phytoanticipins, nontoxic phytoalexin precursors which are quickly converted to phytoalexins as part of the stress response. In crucifers, the most common phytoanticipins are glucosinolates, biologically inert metabolites with a variable side chain on an imidothioic acid derivative, with the sulfur bound to glucose and the nitrogen bound to sulfate. 5 When the plant is under pathogen stress, or is physically damaged, the sugar of the glucosinolate is cleaved by a specific β-glucosidase enzyme, called myrosinase. At neutral pH, the aglucone forms a thiocyanate (pH > 7) or, more commonly, an isothiocyanate. 6 In Brassica species and a handful of others, indole isothiocyanates can be further modified into the dithiocarbamate containing molecule brassinin. 4,7 Brassinin is an indole-sulfur phytoalexin found in several cruciferous crop species (e.g., Brassica rapa, Brassica oleracea) involved in plant disease resistance against various plant pathogens. 8 As with many plant natural products, brassinin has long been studied for its anticancer properties and has been found to inhibit the immune checkpoint enzyme indoleamine 2,3-dioxygenase. 9 Given its potential application in both agriculture and human health, the minimal set of enzymes necessary to engineer brassinin biosynthesis in planta has recently been elucidated. 4 In this pathway, tryptophan is first oxidized to its oxime via CYP79B2, and then tailored to indole glucosinolate. Subsequently, indole glucosinolate is activated to indole isothiocyanate and ultimately converted to the methyldithiocarbamate, brassinin (Figure 1a, Figure S1). Notably, chemically synthesized variations of brassinin have increased efficacy against cancer cell lines, suggesting that slight structural modifications may enhance the bioactive properties of the molecule. 9 In order to biologically produce brassinin alternatives, we aimed to expand the diversity of substrates used by the brassinin biosynthetic machinery. To unlock the full biochemical diversity of the pathway, we tested various CYP79 homologs known to accept alternative amino acid substrates as the first committed enzyme in the pathway, effectively acting as the gatekeeper for pathway specificity. 10,11 Importantly, other CYP79 homologues have been characterized from disparate plant species for the oxidation of a range of amino acids. 12−14 Since the discovery of glucosinolate genes, researchers have engineered their biosynthesis in the native plant and transferred the biosynthesis to heterologous plant hosts with the eventual goal of improving crop nutrition and defense. The first efforts were in 1999, when Bak et al. engineered Arabidopsis thaliana to produce 4-hyroxybenzyl glucosinolate by expressing CYP79A1 from Sorghum bicolor in the plant, demonstrating the native promiscuity of the glucosinolate pathway. 10 CYP79A1 oxidizes tyrosine to 4-hydroxyphenylaldoxime, which is tailored through the A. thaliana glucosinolate pathway, even though this substrate is not naturally present in the plant. CYP79D2 was later introduced into A. thaliana, causing the plant to produce leucine-and isoleucine-derived glucosinolates. 11 While all of these glucosinolates are found in nature, they were previously unreported in A. thaliana. Researchers soon found that expression of these glucosinolates altered the immunity of A. thaliana. Plants overexpressing CYP79A1/2 were more resistant to common bacterial pathogen Pseudomonas syringae, but more susceptible to the crucifer specialist fungi Alternaria brassicicola. Similarly, isopropyl and methylpropyl glucosinolates produced by expression of CYP79D2 increased resistance to soft rot fungus Erwinia carotovora. 15 Although there has been significant research demonstrating the potential in engineering various 1methylpropyl crucifalexin, select enzymes in the crucifalexin pathway were taken from aliphatic ITC biosynthesis (purple arrows) rather than brassinin biosynthesis. There are several enzymes involved in the conversion of the isothiocyanate to brassinin; notably, there is a Smethyltransferase specific to this pathway. (b) LC/MS extracted ion chromatograph for crucifalexins accumulating in N. benthamiana plants engineered with the core crucifalexin pathway and one of several CYP79 enzymes or no heterologous enzymes (wild-type). Each chromatograph is normalized to its largest peak, with the exception of wild-type which is at the same scale as CYP79D3. The following ions were extracted for all chromatographs: m/z 130.0651, brassinin (dedithiocarbamate ion, molecule fragments in source, parent ion not detectable); 164.0562, 1methylpropyl crucifalexin; 198.0406, benzyl crucifalexin; 214.0355, 4-hydroxybenzyl crucifalexin. (c) Accumulation of multiple crucifalexins in N. benthamiana expressing the core crucifalexin pathway along with CYP79A1, CYP79A2, or CYP79B2 individually, in pairs, or all together. Molecules were detected via LC/MS and quantified using the curve of synthetic standards.
glucosinolates, these approaches have not been well translated to downstream hydrolysis products of glucosinolates, which have novel bioactive properties.
■ RESULTS AND DISCUSSION
To test the capacity of the brassinin pathway for the biosynthesis of new-to-nature methyldithiocarbamates, we heterologously expressed the B. rapa pathway in Nicotiana benthamiana leaves, swapping tryptophan-specific CYP79B2 with phenylalanine-oxidizing CYP79A2 from Setaria italica, tyrosine-oxidizing CYP79A1 from Sorghum bicolor, or isoleucine-and valine-oxidizing CYP79D3 from Lotus japonicus. 12−14 Leaves expressing the engineered biosynthesis accumulated the intended new-to-nature molecules, indicating substantial substrate promiscuity across all the enzymes in the pathway (Figure 1b).
For simplicity, we will refer to this expanded class of cruciferous phytoalexins as "crucifalexins" and describe the crucifalexin pathway as the brassinin pathway without the Rgroup determining CYP79 enzyme. We have accordingly named these new-to-nature molecules benzyl crucifalexin, 4hydroxybenzyl crucifalexin, and 1-methylpropyl crucifalexin. We also observed accumulation of a putative glycosylated version of 4-hydroxybenzyl crucifalexin with expression of CYP79A1 ( Figure S2). Interestingly, we did not observe accumulation of valine-derived propyl crucifalexin with expression of CYP79D3, although we did observe the corresponding intermediate propyl isothiocyanate ( Figure S3). In order to optimize metabolic flux through our various crucifalexin pathways, we tested the effect of various enzymes on overall product titers and found that TGG4, a promiscuous myrosinase from A. thaliana, had higher activity for new-tonature products than the B. rapa myrosinase BABGLU.A ( Figure S4).
As these novel molecules likely have different activities, it could be beneficial to engineer a suite of them into a single plant, mimicking the cocktail of phytoalexins produced simultaneously in crucifers like B. rapa and Nasturtium of f icinale. 7,8 Furthermore, the production of multiple compounds simultaneously may not only provide a defense against different pathogens but also potentially decrease the chances of evolving disease resistance to a single compound. Thus, we attempted to heterologously produce multiple new-to-nature crucifalexins in the same plant by transiently expressing the core pathway with pairwise combinations of BrCYP79B2, SbCYP79A2, and SiCYP79A1, as well as all three together. When two or more CYP79s are expressed, all expected crucifalexins are detected, although their abundance drastically differs from when they are expressed individually ( Figure 1c). This is especially apparent in combinations with CYP79A1. Surprisingly, although brassinin is the only "natural" molecule of the three, when all CYP79s were expressed together, 4hydroxybenzyl crucifalexin production remained relatively fixed, while brassinin and benzyl crucifalexin levels dropped an order of magnitude. This finding suggests that enzyme promiscuity may be high enough to accept non-native substrate at significant rates. However, it is possible that production of all crucifalexins may be limited in this case, especially if the phenol group competitively inhibits one or more enzymes in the pathway.
With the inherent promiscuity of the downstream pathway, it is surprising that more crucifalexins are not naturally made. The lack of CYP79A1 and CYP79D3 homologues in sequenced genomes of brassinin-accumulating plants makes natural 4-hydroxycrucifalexin and 1-methylpropyl crucifalexin unlikely. 16,17 However, CYP79A2 homologues are found in several Brassica genomes, including B. rapa. Our previous work shows that the gene is expressed at low levels in B. rapa leaves accumulating brassinin, but no benzyl glucosinolate or benzyl crucifalexin could be detected in these tissues. 18 pathogen Alternaria brassicicola. 15 The innovation of new metabolic pathways has largely been limited to evolving the existing set of genes found in a given plant genome. Thus, synthetic biology provides a means to bypass these natural constraints and engineer novel biosynthetic pathways that might have never evolved on their own.
Given the promiscuity of the crucifalexin pathway, we expanded our efforts to push the capacity of this pathway beyond canonical amino acids. Notably, synthetically prepared halogenated brassinin analogues have been shown to be more bioactive than brassinin and, thus, may have enhanced antifungal properties. 9 To produce halogenated brassinins in planta, we expressed the brassinin pathway in N. benthamiana along with a combination of bacterial halogenases/reductases: PyrH/RebF which produces 5-chlorotryptophan or RebH/ RebF, which produces 7-chloro and 7-bromotryptophan 19 (Figure 2a). In these plants we observed that halogenated tryptophan was incorporated into the brassinin pathway. Leaves expressing the halogenated PyrH/RebF pathways accumulate 5-chlorobrassinin. Product formed from the RebH/RebF pathway is proposed to be 7-chlorobrassinin based on the precedence for RebH specificity. Notably, no bromobrassinin was observed in leaves. We reasoned that low levels of bromine in the plant would limit incorporation of this halogen onto tryptophan, and eventually brassinin. Since addition of exogenous bromine in hairy root cultures expressing RebH/RebF resulted in 7-bromotryptophan, 19 we watered RebH/RebF + brassinin expressing plants with potassium bromide (KBr), resulting in detectable levels of 7bromobrassinin ( Figure 2b). Although in planta production of chlorinated brassinins is clearly more practical, the accumulation of 7-bromobrassinin demonstrates the versatility of this pathway. The demonstration of engineering halogenated variants opens the door to future studies exploring the broader use of other newly discovered halogenases, such as the highly promiscuous RadH halogenase. 20 Several cruciferous plants that produce brassinin often further tailor it to other bioactive phytoalexins, such as spirobrassinin and cyclobrassinin. There are two known enzymes that tailor brassinin: BrCYP71CR1, which makes spirobrassinin, and BrCYP71CR2, which produces cyclobrassinin. 18 Expression of CYP71CR1 or CYP71CR2 in plants engineered with the halogenated brassinin pathway yielded compounds with masses, MS-MS spectra, and isotope ratios matching 5-and 7-chlorospirobrassinin, 7-bromospirobrassinin ( Figure S5), 5-and 7-chlorocyclobrassinin, and 7-bromocyclobrassinin ( Figure S6). Production of engineered halogenated crucifalexins indicates that halogenated substrates do not severely inhibit any enzymes in the brassinin pathway. The expanded diversity of new substrates that can be utilized by the brassinin pathway underscores the potential to exploit enzyme promiscuity as a means to diversify and engineer novel molecules not found in nature.
Given the expansion of our new engineered crucifalexins, we sought to determine if any of our novel compounds could act as biopesticides against agriculturally relevant fungal pathogens. We chemically synthesized standards of benzyl crucifalexin, 4-hydroxybenzyl crucifalexin, 1-methylpropyl crucifalexin, and 5-chlorobrassinin ( Figure S7) and assayed their inhibitory activity against the agriculturally relevant generalist pathogen Botrytis cinerea. 21 Benzyl crucifalexin, 4hydroxybenzyl crucifalexin, and 1-methylpropyl crucifalexin have a lower inhibitory effect than brassinin ( Figure S8). However, 5-chlorobrassinin outperformed brassinin and is comparable to pyrimethanil (p < 0.05)an active ingredient in commercial pesticidesin rich media. 22 Furthermore, this IC 50 is comparable to estimated levels of 5-chlorobrassinin produced heterologously, even without optimization of the pathway for this product (Figure 3). We estimate ∼20 μM production of 5-chlorobrassinin based on dry weight accumulation of 35 ± 13 μg/g as detected on LC/MS. Furthermore, we are producing similar levels of 7-chlorobrassinin (42 ± 4 μg/g dry weight, based on 5-chlorobrassinin standard) and 7-bromobrassin (37 ± 17 μg/g dry weight). It should be noted that the activity of pyrimethanil has been observed to be reduced in rich media given its function as an anilinopyrimidine inhibitor. 23 For additional context, another antifungal, fludioxonil, inhibits the growth of Botrytis isolates with IC50s on the order of 2.5 μM for many isolates. 24 To demonstrate broad applicability of these novel metabolites, we also assayed their activity against the crucifer specialist Alternaria brassicicola ( Figure S9). Both benzyl crucifalexin and 5-chlorobrassinin outperformed brassinin, with benzyl crucifalexin having the greatest inhibitory effect. Our findings demonstrate a means to produce new-to-nature crucifalexins that have improved efficacies over their natural analogue. Future efforts may optimize the large-scale production of these new compounds or directly engineer our synthetic metabolic pathways into crop plants as a complete or partial substitute to externally applied pesticides. Here, we present proof of concept that these new synthetic pathways can be ported and modified to produce new-to-nature compounds; however, there are several challenges that will be faced in the ultimate goal of being deployed in stable transgenic lines. Specifically, there are a minimum of eight genes that would need to be introduced into heterologous plant systems, requiring optimization of expression levels and accumulation. Moreover, there are several hurdles associated with the production of such glucosinolate-derived compounds which may necessitate proper compartmentalization. Our findings provide new targets for such long-term stable engineering efforts in the future. Modern agricultural practices are heavily dependent on the use of external pesticides, as three billion kilograms of pesticide are utilized annually. 25 Pesticide runoff can result in environmental and human health impacts, 26 which might be addressed by the direct production of biopesticides in crops. By combining pathways from different organisms, we rationally designed and reconstituted synthetic metabolic pathways for the production of a suite of new-to-nature small molecules. The antifungal activity of the engineered crucifalexins highlights the bioactivity of their shared dithiocarbamate group, which is similar to chemical moieties in many synthetic commercial pesticides like mancozeb and metam sodium; 8 however, their differences in activity levels demonstrate that the R-group is extremely important for their potency and has varied effects from pathogen to pathogen. By expanding the structural diversity of this class of molecules, we have engineered new derivatives with improved bioactivity over the natural metabolite, brassinin, with efficacies comparable to commercially used synthetic pesticides. Notably, specialized metabolic pathways differ in plants across phylogeny; our engineering approach enables the combination of diverse mechanisms for pathogen defense that have evolved independently in a way not accessible through breeding.
There are still notable challenges beyond our first proof-ofprinciple biosynthesis of novel compounds, such as how such engineered plants may interact with the environment and how to avoid resistance in pathogen populations, if engineered into stable transgenic plants. Related issues are encountered with the engineering of resistance genes for pathogen defense; here it is thought that stacking multiple genes can help prevent the development of resistance. 27 The development of tissuespecific and targeted approaches in engineered plants may also help address a number of these issues. Moreover, the use of the myrosinase introduces a natural trigger to produce the final molecule on demand in response to pathogen exposure or environmental stimuli. Nonetheless, the biological production of such compounds may provide a cost-effective means to scale the biosynthesis and extraction of new-to-nature products. Many of the halogenated derivatives we have biosynthesized may enhance the bioactive properties of brassinin, not only for disease resistance in plants but also against cancer cell lines, as has been shown in previous work. 9 Thus, our findings also provide a biological means to produce such compounds biologically, avoiding chemical synthesis. This work demonstrates the potential in harnessing synthetic biology to create new compounds with novel or improved bioactive properties in order to enhance the chemical arsenal of crop species for plant disease resistance.
■ METHODS
Materials and General Methods. Synthetic standards were prepared according to procedures documented in the literature: benzyl crucifalexin was synthesized from benzyl amine, 4-hydroxybenzyl crucifalexin from 4-hydroxybenzyl amine, and 1-methylpropyl crucifalexin from sec-butyl amine via a procedure from Pedras et al. 7 5-Chlorobrassinin was synthesized from 5-chloroindole carboxaldehyde using a combined protocol from Griffieon et al. and Pedras et al. 7,28 5-Chloroindole carboxaldehyde (690 mg, 3.76 mmol) was added to a mixture of hydroxylamine hydrochloride (366 mg, 5.27 mmol) and sodium acetate (463 mg, 5.65 mmol) in ethanol (10 mL) and stirred at 40°C with reflux for 3.5 h. The product was concentrated under vacuum, extracted with ethyl acetate and water, and concentrated again under vacuum. The dried product was dissolved in glacial acetic acid (30 mL) with zinc dust (1.48 g) and stirred overnight at room temperature. The resulting suspension was filtered, washed and extracted with ethyl acetate, dried with sodium sulfate, and concentrated under vacuum to yield crude 5-chloroindole-3-methylamine (462 mg, 68% yield). The procedure was repeated to produce additional crude 5chloroindole-3-methylamine (322 mg, 47% yield). 595 mg of crude product was added to trimethylamine (505 μL, 3.62 mmol) in pyridine (1 mL). Carbon disulfide (220 μL, 3.62 mmol) was added dropwise, and the solution was stirred at 0°C for 20 min, at which point methyl iodide (226 μL, 3.62 mmol) was added dropwise, and the solution was stirred at 0°C for another 30 min. The reaction was quenched with 1.5 M sulfuric acid (3 mL), extracted with diethyl ether, dried with sodium sulfate, and concentrated under vacuum. The product was purified via a flash silica column with ethyl acetate, and with another silica column (1:1 ethyl acetate:hexanes) to yield 5-chlorobrassinin (51.9 mg, 6% yield). 7-Bromobrassinin was synthesized via the same procedure using 7-bromoindole carboxaldehyde as the starting material.
All the synthesized compounds showed NMR and highresolution mass spectrometry (HRMS) spectral data consistent with that reported in the literature. 1 H NMR spectra were acquired on a Varian 400 MHz spectrometer. HRMS data were obtained using an Agilent 6520 Q-TOF instrument, as described below. All chemicals were obtained from Sigma-Aldrich or Thermo Fisher Scientific, unless otherwise stated. Ultrapure water was generated by a Milli-Q system (EMD Millipore).
Cloning of the B. rapa Brassinin Pathway and CYP79 Genes. Phusion High-Fidelity DNA polymerase (Thermo Scientific) was used for all PCR amplification steps according to the manufacturer's instructions. All other enzymes used for cloning were purchased from New England Biolabs. Oligonucleotide primers were purchased from Integrated DNA Technologies. DNA excised from agarose gels was purified using the Zymoclean Gel DNA recovery kit (Zymo Research). Escherichia coli TOP10 cells (New England Biolabs) were used for plasmid isolation prior to transformation into other heterologous hosts. Plasmid DNA was isolated from E. Transient Expression in N. benthamiana. pEAQ-HT and constructs were transformed into Agrobacterium tumefaciens (GV3101). Transformants were grown on LB plates containing 50 μg/mL kanamycin and 30 μg/mL gentamicin at 30°C. Cells were removed with a sterile inoculating loop and resuspended in 1 mL of LB medium and centrifuged at 5000g for 5 min, and the supernatant was removed. The pellet was resuspended in 10 mM MES buffer, pH = 5.6, 10 mM MgCl 2 , 150 μM acetosyringone, and incubated at room temperature for 1 h. Agrobacterium suspensions (OD600 = 0.1 for each strain) were infiltrated into the underside of N. benthamiana leaves with a needleless 1 mL syringe. Plants were grown 5−6 weeks under a 16 h light cycle prior to infiltration. Leaves were harvested 5 days postinfiltration, flash frozen, and stored at −80°C for later processing. Three leaves per plant were infiltrated; a single biological replicate consisted of three pooled leaves from a single tobacco plant. Infiltrated leaf areas typically showed some signs of chlorosis or yellowing; leaves expressing GFP as a control also showed a similar phenotype.
Metabolite Extraction and LC/MS Analysis. For metabolite extraction, frozen samples were crushed with a pestle and lyophilized to dryness. The samples were subsequently homogenized on a ball mill (Retsch MM400) using 5 mm diameter steel beads, shaking at 25 Hz for 2 min. To the dry tissue, an 80:20 MeOH/H 2 O (v/v) solution was added (20 μL/mg N. benthamiana tissue). Tissue extracts were heat treated at 65°C for 10 min, spun at 15 000g for 10 min, passed through 0.45 μm PTFE filters, and analyzed (20 μL injection volume) by LC/MS. LC/MS was performed on an Agilent 1260 HPLC instrument using a Gemini NX-C18 column (Phenomenex, 5 μm, 2 × 100 mm). Water and acetonitrile, each supplemented with 0.1% formic acid, were used as the mobile phase components, with a flow rate of 0.4 mL/min. For metabolite analysis of plant extracts, the following 43 min gradient was used (percentages indicate acetonitrile concentration): 3−50% over 30 min; 50−97% over 2 min; 97% for 3 min; 97−3% over 1 min; 3% for 5 min. Column eluent was monitored by an Agilent 1260 diode array detector followed by an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with an ESI source (parameters: mass range, 50−1200 m/z; drying gas, 350°C, 11 L/min; nebulizer, 35 psig; capillary, 3000 V; fragmentor, 150 V; skimmer, 65 V; octupole 1 RF Vpp, 750 V; 1000 ms per spectrum). MS data were collected in either positive or negative ion mode (in the case of glucosinolates) and analyzed using MassHunter Qualitative Analysis software (Agilent). Phytoalexin content was calculated from the integrated peak area of a selected ion chromatogram (extracted using a ± 100 ppm window around the theoretical exact mass) and comparison to a standard curve. The first minute of each run was discarded to avoid salt contamination of the MS apparatus. For tandem mass spectrometry (MS/MS) analysis, 5, 10, 20, and 40 V collision energies were used with an m/z window of 4 centered on the m/z analyzed. Standard curves for brassinin, benzyl crucifalexin, 4-hydroxybenzyl crucifalexin, 1-methylpropyl crucifalexin, 5-chlorobrassinin, and 7-chlorobrassinin based on ion abundance were constructed and used to quantify amounts produced in N. benthamiana.
Fungal Mycelia Growth Inhibition Assay. Fungal growth inhibition assays were performed following a similar protocol to Pedras et al. 7 Spores of Botrytis cinerea SF1 or Alternaria brassicicola FSU218 were grown on PDA at room temperature in the dark for 3 days (B. cinerea) or 7 days (A. brassicicola). Serial dilutions of phytoalexins were made in DMSO and added to warm potato dextrose agar to final concentrations of 20, 50, 100, 200, and 500 μM and poured into 6-well plates (2 mL/well). The same procedure was used for pyrimethanil containing media, but with concentrations of 0.1, 1, 10, 25, 50, and 100 μM. 5 cm circular plugs of growing fungal mycelia were transferred to cooled plates and grown at room temperature in the dark for 24 h (B. cinerea) or 75 h (A. brassicicola). At this time the longest diameter of fungal growth was measured and compared to plugs grown on PDA with 1% DMSO to calculate percent growth inhibition. IC 50 values and significance were calculated from percent growth inhibition of all replicates using a nonlinear regression model in GraphPad Prism.
Safety Statement. No unexpected or unusually high safety hazards were encountered with the reported work.
■ ASSOCIATED CONTENT * sı Supporting Information Demetrix, Constructive Biology, Maple Bio, and Napigen. The research described in this publication is not related to the work of these companies. | 2020-07-23T09:06:47.449Z | 2020-07-20T00:00:00.000 | {
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232138338 | pes2o/s2orc | v3-fos-license | Enhancing the Knowledge of Cervical Cancer Screening among Female Nursing Students: An Interventional Educational Program
Background:Cervical cancer is a growing health risk facing women worldwide with the human papillomavirus (HPV) as the primary underlying cause. Pap smear is a simple screening test that can detect early changes in cervical cells, which might develop into cancer cells. Raising awareness of cervical cancer prevention has a significant impact on decreasing the burden of the disease. The aim of the study is to assess female nursing students’ knowledge on early detection and screening of cervical cancer, and to determine the effectiveness of an educational program. Methods: A quasi-experimental research design (one group for preand post-tests) was utilized with a convenience sample of 130 female nursing students in one of the nursing colleges in Saudi Arabia. The study’s educational intervention included information about anatomy of genital tract and the importance of regular check-ups. The preand post-tests were applied to identify changes after intervention measures. Results: The mean age of the participants were 21.32 years (SD: 1.34). The findings revealed a significant improvement of post-test students’ knowledge in all items related to risk factors, signs and symptoms, occurrence, identification of HPV as causative agent, vaccination against HPV, and finally Pap smear for early detection and screening of cervical cancer. Conclusion: The study results support implementing educational intervention to improve nursing students’ knowledge and awareness about cervical cancer prevention. Furthermore, it is imperative that cervical cancer awareness education modules should be developed and integrated within the nursing curriculum. Further studies with large sample size are recommended to increase generalization of the results.
Introduction
Cervical cancer is one of the diseases that could be avoided by encouraging people who are at risk to have a regular screening. Increasing people's awareness about the importance of disease screening may encourage them to be actively engaged in disease prevention initiatives. According to the World Health Organization (WHO), cervical cancer is the fourth most common cancer among women, with an estimated 570,000 new cases representing 6.6% of all female cancers around the world in 2018. Death from cervical cancer in low-and middle-income countries is about 90% [1]. Factors increasing death in developing countries include lack of awareness and high vaccine costs [2].
From Saudi Arabian context, cervical cancer is not a major contributor to women's health problems in the country. More than 90% of cervical cancer cases are related to human papillomavirus (HPV) infection [3]. The pathogenesis can develop through precancerous lesions to invasive cancer over a period of 10-20 years and ultimately lead to death [4]. Cancer of the cervix is preventable and can be easily managed if identified at early stages through Pap smear [5]. Unfortunately, about 90% of deaths did not have regular Pap smear. A recent report mentioned that about 316 new cases of cervical cancer were diagnosed annually in Saudi Arabia. Cervical cancer is the ninth most common cancer among females aged 15-44 years [6]. Henceforth, studies exploring cervical cancer educational interventions are warranted [7].
Effective health education programs targeting cervical cancer awareness need sophisticated and comprehensive planning and needs assessment of the target groups such as the level of knowledge, beliefs, attitudes, and behavior [8,9]. Comprehensive health education programs are more likely to promote screening, so it is important for health providers including nursing students to provide information about risk factors, early signs of cervical cancer, and encourage women to screen for cervical cancer on a regular basis [3,10].
Raising awareness of cervical cancer prevention has a significant impact on decreasing the burden of the disease. Specifically, evidence has shown that education interventions to raise awareness improve accessibility to services, increase individuals' efficacy, and inspire future choices, which in turn enhance the efficiency of preventive health services [11]. The aim of this study was to assess and enhance female nursing students' knowledge and early detection and screening of cervical cancer utilizing an education program as an intervention. The study hypothesis assumes that educational programs will raise the knowledge and awareness of nursing students about cervical cancer, screening, and early detection.
Methods
A quasi-experimental design (pre-test-post-test design) was utilized to explore a convenient sample of 130 female nursing students the Nursing College at Taibah University in Saudi Arabia. The study included students who were 18 years or older. Those who were younger than 18 years were excluded. Potential candidates were approached and invited to participate in the study.
Instrument
The study instrument consisted of two sections: the first section consisted of the sociodemographic data including age, education level, age of menarche, family history, smoking, and attending an educational session about cervical cancer; while the second section was the pre-post intervention test. As academics, the researchers constructed this section of simple questions recalling knowledge about the importance of genital tract check-ups, diseases that affect the genital tract, definition of cervical cancer, causes and screening, vaccination, HPV, Pap smear definition, importance of test, and the use, technique, periodicity, and precautions before having a Pap smear. This section tested for content validity by five academic nursing experts. Modifications were made according to the experts' assessment. Although the instrument was administered to the same population for more than one session under similar conditions, a pilot study involving 13 participants were undertaken. The final version of the instrument was attained following the pilot study outcome.
Further, the scoring system was adapted from a previous study [12]. In detail, the scoring system classified participants' answers into three categories (wrong answer = 0, incomplete answer = 1, and complete answer = 3). The student with a score below 50% was considered to have poor knowledge. The overall score was classified as follows: low = <50%; accepted knowledge level = 50-75%; and good knowledge = >75%. Data were analyzed using the Statistical Package for Social Sciences software package (SPSS Inc., Chicago, IL, USA) version 20.0. Frequencies, percentages, mean, and median were calculated for the knowledge score. Student t-test and Chi-square test were utilized to analyze the results. Statistical significance was identified at P < 0.05.
Procedure
The aim of the study was explained to the participants followed by assessing the pretest knowledge level (estimated time 10-13 min). The intervention was an educational session delivered for 120 min including the following:
Results
The mean age of the study participants was 21.32 years (SD = 1.34), while the mean age of menarche was 12.93 years (SD = 1.39). Other sociodemographic characteristics (Table 1) showed that 90% of study samples were not married. Neither of the participants smoked nor had a family history of cervical cancer. The majority knew about cervical cancer but did not attend any educational session in this regard. Sources of information about cervical cancer were TV, social media, friends, academic study, and reading newspapers (16.1%, 63.1%, 2.3%, 2.3%, and 16.2%, respectively). As shown in Table 2, high statistically significant improvements were observed in the results of the post-test knowledge of students in the sample regarding the importance of check-up of genital tract, diseases that affect the cervix, risk factors, signs and symptoms of cervical cancer, and the relationship between sexual activity and cervical cancer when compared with the pre-test results, at P < 0.001. It is evident that the complete and correct answers were 22.3%, 27.7%, 6.9%, 3.1%, 2.3%, and 42.3%, respectively in pre-test, while at the post-test, the percentage of complete and correct responses rose, respectively, to 79.2% ,89.2%, 88.5%, 90.0%, and 96.9%. Regarding the students' knowledge about causative organism of cervical cancer (HPV) and its vaccination, there Table 3 shows the comparison of pre-and post-tests knowledge assessment of female students regarding Pap smear definition, associated pain, timing, periodicity, and prior precautions, and statistically significant improvements were observed in all these items, P = 0.000. At the pre-test, the majority of the students had incorrect and wrong answers but this result was reversed to complete and correct at post-test. The results of pre-post total knowledge scores of female students in the study sample are presented in Table 4. It indicates statistically significant improvement in the total students' knowledge scores in the post-test compared to the pre-test, P = 0.001. As seen in the table, the great majority of students (96.9%) had poor knowledge pre-test, which was greatly improved post-test, where 81.5% had good knowledge.
Discussion
Working on preventing cervical cancer through screening is a valuable strategy to combat the increasing rate of the disease. Despite the provision of medical services and availability of screening tests, increasing people's risk awareness and supporting them to be actively engaged in disease prevention could be the best approach to increase screening rate. The current study showed that the majority of the participants despite having a prior knowledge of cervical cancer had not attended any educational programs. While social media was the main source of cervical cancer information, TV and newspapers were the second. These results are in line with a previous study conducted in Egypt that found 95.1% of the participant nurses did not attend any program about cervical cancer, but about two-third of them took their information from work experience followed by academic study and doctors [13]. In addition, similar results were found in India where 20% of the participants identified that reading newspapers is their source of cervical cancer information [10].
The finding of the present study supports previous studies where a significant improvement of post-test students' knowledge in all items of cervical cancer has been reported, which supports the implementation of educational programs especially in mass gathering locations such as universities and schools [13]. Educational programs were a crucial factor in applying cervical cancer awareness campaigns [14]. Furthermore, these programs enhance women's health beliefs, thereby helping in cervical cancer prevention and detection [3,15].
According to the American Cancer Association guideline, Pap smear and the HPV DNA test are significant diagnostic tools [16]. Improvement in participants' awareness about HPV and its vaccine was evident taking into account the difference in the scores of the pre-and post-tests. Similar trend was found in previous studies conducted outside Saudi Arabia [17]. At pre-test, majority of the participants had poor knowledge about Pap smear, importance, procedures, timing, periodicity, and prior precautions. However, the post-test showed great improvement in the students' knowledge regarding all of these items. In the same line of findings, previous studies of similar intervention made significant positive enhancement in participants' knowledge noticing the difference between the pre-and post-tests [18].
In general, the study supports the findings of previous international studies proving overall cervical cancer knowledge improvements [9,11,13]. Therefore, it is imperative to recommend educational programs to attain cervical cancer awareness among target groups [1].
The study results recommend further similar initiatives within the university to raise the general awareness of all students. Furthermore, enhanced educational programs for cervical cancer awareness should be included in the nursing curriculum. From a community stand, community campaigns are warranted to raise public awareness on cervical cancer and Pap smear screening become a routine test for all women in childbearing age.
Conclusion
In this study, the majority of the participants had poor knowledge about cervical cancer pre-test. After delivering the educational intervention, the post-test indicated significant improvements in the participants' level of knowledge for almost all items. Therefore, | 2021-01-07T09:11:26.030Z | 2020-12-31T00:00:00.000 | {
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229337493 | pes2o/s2orc | v3-fos-license | Benevolent Ageism’s Relationship to Self-Compassion and Meta-Memory in Older Adults
Abstract Benevolence directed towards older adults can cross the line between respect and overaccommodation that undermines their physical and cognitive capabilities (Mehrotra & Wagner, 2009); however, little research has examined the subtleties of the influence of benevolent ageism on older adults’ ratings of their own functioning. Because stereotypes about older adults include the decline of mental abilities, this study examined whether their (N= 155) experiences with benevolent ageism, or overaccommodative offers of assistance and protection, influenced their own appraisals of memory abilities through their feelings of self-compassion. Older adults with fewer benevolent ageist experiences had higher rates of self-compassion, which in turn translated into better evaluations of their memory abilities. Future research should consider the potential pernicious influences that benevolent ageism has on older adults’ self-evaluations and performance, consider self-compassion as a buffer in these relationships, and test whether these relationships have downstream consequences on well-being outcomes.
be at the forefront for scientific examination. The long-term effects of ageism, particularly negative self-perceptions, lead to negative health and cognitive outcomes (Chasteen et al., 2015;Levy et al., 2002). One of the intricate components of ageism, however, is that it is often "benevolent". Cuddy and colleagues developed the Stereotype Content Model (SCM) to describe how individuals are categorized based on varying degrees of warmth and competence. Unlike many devalued members of society who are viewed as low on both, older adults are viewed as having high warmth and low competence, leading to more overaccommodative treatment. The goal of the present symposium is to overview the ways in which researchers have dissected this more nuanced type of ageism. Specifically, two of the presenters will cover some of the boundary conditions of understanding age-based stereotypes and their malleability, examining them across ages and across genders. Additionally, one of our presenters will overview the validation of the Ambivalent Ageism Scale on a Chinese sample, lending support to its generalizability. Finally, our last presenter will overview the relationship between benevolent ageism and self-compassion to predict metamemory, given the pervasive stereotype that older adults suffer from severe cognitive decline. Themes and implications of these presentations will be discussed.
BENEVOLENT AGEISM'S RELATIONSHIP TO SELF-COMPASSION AND META-MEMORY IN OLDER ADULTS
Jennifer Sublett, 1 and Toni Bisconti, 2 1. University of Akron, Akron, Ohio, United States, 2. Univesity of Akron, Akron, Ohio, United States Benevolence directed towards older adults can cross the line between respect and overaccommodation that undermines their physical and cognitive capabilities (Mehrotra & Wagner, 2009); however, little research has examined the subtleties of the influence of benevolent ageism on older adults' ratings of their own functioning. Because stereotypes about older adults include the decline of mental abilities, this study examined whether their (N= 155) experiences with benevolent ageism, or overaccommodative offers of assistance and protection, influenced their own appraisals of memory abilities through their feelings of self-compassion. Older adults with fewer benevolent ageist experiences had higher rates of self-compassion, which in turn translated into better evaluations of their memory abilities. Future research should consider the potential pernicious influences that benevolent ageism has on older adults' self-evaluations and performance, consider self-compassion as a buffer in these relationships, and test whether these relationships have downstream consequences on well-being outcomes.
THE MODIFIABILITY OF THE STEREOTYPE CONTENT MODEL TOWARD OLDER ADULT MEN AND WOMEN Michael Vale Toni Bisconti and Jennifer Sublett, University of Akron, Akron, Ohio, United States
Older adults are often stereotyped in a paternalistic manner (warm, but incompetent), deserving of assistance regardless of their need. We have examined the veracity and malleability of this paternalistic stereotype using an experimental vignette with both male and female targets. Younger adults (N = 717) deemed it more necessary and appropriate to offer unnecessary help to older adults in a grocery store scenario. Additionally, competence was malleable for both older adult male and female targets if the older adults denied the offer of help. Interestingly, older women were viewed as warm, which did not change as a function of their response, whereas older men were initially viewed as colder, but their warmth ratings increased. In light of these findings, we will discuss the intersection of age and gender when considering the malleability of the warmth and competence dimensions of the paternalistic older adult stereotype.
THE LINKS BETWEEN AGEISM AND THE AGE-BASED DOUBLE STANDARD
Changrui Li, 1 Sarah Barber, 1 and Gene Brewer, 2 1. Georgia State University, Atlanta,Georgia,United States,2. Arizona State University,Tempe,Arizona,United States There is an age-based double standard in how we evaluate memory failures by younger and older adults. Whereas younger adults' forgetfulness is attributed to lack of effort or attention, older adults' forgetfulness is attributed to lack of ability. Our goal was to replicate this phenomenon, and evaluate its links to benevolent and hostile ageism. To do so, we used a vignette paradigm in which younger and older participants read about a target person (who was a younger or older woman) who left a store without paying for a ring (which varied in price). Results showed that participants were more likely to attribute this to poor memory abilities when the target was an older adult. They were also more lenient in their ascribed punishments for the older adult targets. In addition, reading about an older adult target's mistake was associated with subsequently higher endorsement of benevolent, but not hostile, ageist attitudes.
VALIDATION OF THE AMBIVALENT AGEISM SCALE IN CHINA Xin Zhang, Peking University, Beijing, China
Two studies were conducted to validate the Ambivalent Ageism Scale in China. In the first study, 474 Chinese adults (18-58) were asked to take the Chinese version of the AAS. EFA exhibited a similar factor solution as the original study, with high internal consistency and construct validity. Moreover, in a second study, 372 Chinese adults (18-85) took the AAS and provided their estimations of the similarities between their current and their past/future self via the SIC. Results indicated that all three factors of the SIC positively related to hostile ageism, whereas succession and identity positively related to benevolent ageism and consumption negatively related to it. Additionally, past self-continuity was positively associated with hostile ageism, and future self-continuity was negatively associated with it, but neither form was associated with benevolent ageism. These results further validate the AAS in China and also provide evidence for the uniqueness of benevolent ageism. | 2020-12-22T05:11:59.332Z | 2020-12-16T00:00:00.000 | {
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1039181 | pes2o/s2orc | v3-fos-license | Evaluation of a tobacco prevention programme among teenagers in Sweden
Objective To study the prevalence of tobacco use among teenagers, to evaluate a tobacco prevention programme and to study factors related to participation in the prevention programme. Design and setting Population-based prospective cohort study. Method Within the Obstructive Lung disease in Northern Sweden (OLIN) studies, a cohort study about asthma in schoolchildren started in 2006. All children aged 7–8 years in three municipalities were invited to a questionnaire survey and 2585 (96%) participated. The cohort was followed up at age 11–12 years (n=2612, 95% of invited) and 14–15 years (n=2345, 88% of invited). In 2010, some of the children in the OLIN cohort (n=447) were invited to a local tobacco prevention programme and 224 (50%) chose to participate. Results At the age of 14–15 years, the prevalence of daily smoking was 3.5%. Factors related to smoking were female sex, having a smoking mother, participation in sports and lower parental socioeconomic status (SES). The prevalence of using snus was 3.3% and risk factors were male sex, having a smoking mother, having a snus-using father and non-participation in the prevention programme. In the prevention programme, the prevalence of tobacco use was significantly lower among the participants compared with the controls in the cohort. Factors related to non-participation were male sex, having a smoking mother, lower parental SES and participation in sports. Conclusions The prevalence of tobacco use was lower among the participants in the tobacco prevention programme compared with the non-participants as well as with the controls in the cohort. However, the observed benefit of the intervention may be overestimated as participation was biased by selection.
INTRODUCTION
Smoking is the single most important and preventable risk factor for all respiratory symptoms and a large number of diseases. Although the health consequences are well known, smoking is still common. 1 Daily smoking is usually established during the teen years, most commonly between 14 and 17 years of age, 2 and rarely after the age of 24. 3 Although Sweden is often mentioned as a country with a decreasing prevalence of smokers and high quit rates among adults, 4 the decrease in smoking prevalence among teenagers has been limited. 5 Therefore, reducing smoking in teenagers is an important public health matter.
In the last decades, a wide range of prevention efforts have been carried out in order to reduce smoking among teenagers. [6][7][8][9][10] A key factor for successful prevention is the longterm collaboration between national, regional and local organisations. On the national and regional levels, smoking bans in schools, 11 and combination approaches that include policies, media campaigns and school-based programmes, 12 have been shown to be effective methods to decrease smoking among adolescents. However, many prevention efforts aimed at teenagers are voluntary and participation may be affected by selection bias if those with the greatest need of the intervention choose not to participate. Among adults, it is known that the prevalence of smokers is higher among nonparticipants in questionnaire surveys regarding respiratory conditions [13][14][15] and in health promotion interventions. 16 However, few studies have reported on factors related to non-participation in tobacco intervention Strengths and limitations of this study ▪ This paper presents data from a prospective cohort study with high response rates and few participants lost to follow-up. ▪ A validated questionnaire about asthma and respiratory symptoms was used. ▪ Collaboration with a tobacco prevention programme (Tobacco Free Duo) enabled us to combine the longitudinal data from the Obstructive Lung disease in Northern Sweden (OLIN) studies with intervention data on participation in the prevention programme. ▪ We lack information about the level of activity in the tobacco prevention programme during the follow-up time. ▪ Self-reported smoking was not validated by objective measures.
among teenagers. One available study showed that nonparticipation in a family-directed tobacco and alcohol prevention programme was related to male sex, lower parental education and parental smoking. 17 The aim of the present study was to determine the prevalence of tobacco use among teenagers and to evaluate the outcome of a school-based voluntary tobacco prevention programme for teenagers. Further, factors related to participation in the prevention programme were investigated.
Study population
As a part of the Obstructive Lung Disease in Northern Sweden (OLIN) studies, a population-based paediatric cohort was recruited in 2006. The parents to all children in first and second grade (age 7-8 years) in three municipalities of northern Sweden: Luleå, Piteå and Kiruna, were invited to complete a questionnaire and 2585 participated (96% of invited). 18 19 Four years later, at the age of 11-12 years, the parents were invited to a follow-up questionnaire survey using the same methods, and 2612 completed the questionnaire (95% of invited). At the age of 14-15 years, those who had participated in any of the previous two surveys were reinvited (n=2657) and 2345 participated (88.3%; figure 1). In this latter survey the questionnaire was completed by the teenagers.
The questionnaire
The questionnaire included the International Study of Asthma and Allergies in Childhood (ISAAC) core questionnaire. 20 It was expanded with additional questions about asthma and allergic diseases including physician diagnoses, symptoms, use of medicine and heredity. Other questions included possible risk factors such as living conditions, physical activity, parental smoking and socioeconomic status (SES). 19 In the questionnaire completed by the teenagers at the age of 14-15 years, questions about tobacco use were added.
The tobacco prevention programme Tobacco Free Duo is a long-term school-based tobacco prevention programme with the aim to prevent tobacco use initiation during teen years. 7 At the end of sixth grade, a teenager had the opportunity to team up with an adult. The pair signed a contract to stay tobacco free for the next 3 years. The prevention programme included information to increase knowledge and awareness on tobacco-related issues, to the teenagers as well as to the adults. It also included an annual assurance of fulfilment of the contract after grade 7, 8 and 9, and positive reinforcements to the participants. An evaluation of Tobacco Free Duo in Västerbotten county, Sweden, showed significantly lower prevalence of smoking in the intervention schools compared with control schools. 7 In 2010, Tobacco Free Duo was initiated in several municipalities in Norrbotten county, including Luleå and Kiruna; two of the OLIN study areas. It was up to the schools to decide whether they wanted to participate in the prevention programme. The children in 13 schools in Kiruna (n=360) and 4 schools in Luleå (n=87) were invited to participate by signing the contract at the age of 12 years (figure 1). A collaboration between the OLIN studies and Tobacco Free Duo enabled a joint database with data on participation in the prevention programme and longitudinal questionnaire data from the OLIN studies.
Definitions
Participants: those who attended a school in the study area that was invited to participate in Tobacco Free Duo, and chose to sign the contract to participate at the age of 12 years.
Non-participants: those who attended a school in the study area that was invited to participate in Tobacco Free Duo, but chose not to participate.
Controls: those who attended the schools in the study area that were not invited to Tobacco Free Duo.
Snus: moist ground tobacco which is placed under the upper lip.
Any smoking/snus use: those reporting smoking/snus use daily, weekly or monthly. Classification of SES was based on parental occupation according to a system developed by Statistics Sweden. 21 The highest level of SES among the adults in the household was chosen. In an aggregated form, the classification consisted of six groups of occupationally active persons, and one group of non-active persons as follows: (1) professionals and executives; (2) self-employed; (3) intermediate nonmanual employees; (4) assistant non-manual employees; (5) manual workers in industry; (6) manual workers in service; (7) unemployed, including students, retired and housewives.
Statistical analysis
Analyses were made using the computer software IBM SPSS Statistics (V.22.0; IBM SPSS Statistics, New York, USA). For assessment of differences between groups, χ 2 tests were used and a p value <0.05 was considered statistically significant. Dependent variables were smoking and use of snus, respectively, at the age of 15 years, and Figure 1 Flow chart of the study design and participation in a cohort study about asthma and allergic diseases, and in a tobacco prevention programme. participation in the tobacco prevention programme from the age of 12 years. Independent variables included sex, having smoking or snus-using parents, living conditions, participation in sports and parental SES. Significant and borderline significant factors identified in the bivariate analyses were included in multivariate analyses, which were performed by multiple logistic regression analysis and expressed as ORs with 95% CIs.
RESULTS
Prevalence of tobacco use at the age of 14-15 years The prevalence of any smoking was 5.9%, with no statistical difference by sex. Any snus use was 4.7%, and significantly more common among boys than girls (7.2% and 1.9%, p<0.001). The prevalence of monthly smoking was 1.5%, weekly smoking was 0.9%, monthly use of snus was 0.9% and weekly snus use was 0.5%.
The prevalence of daily smoking in the cohort was 3.5%, and significantly higher among the girls than among boys (table 1). For daily snus use, the overall prevalence was 3.3%, significantly more common among boys. The prevalence of daily smoking and the use of snus were both significantly lower compared with the prevalence in a similar cohort, surveyed 10 years previously in the same study area. At that survey, the prevalence of daily smoking was 5.8% and using snus, 9.9%. 9 In the present study, the prevalence of daily smoking and daily use of snus, respectively, was significantly higher among those with smoking or snus-using parents, living in an apartment, living in a single parent household, not participating in sports and among those with lower parental SES (table 1). There were no significant differences in the prevalence of smoking or snus use related to urban or rural living, having older siblings, having physician-diagnosed asthma or a positive skin prick test.
In a multivariate analysis, daily smoking was related to female sex; having a smoking mother; a smoking father; not participating in sports; and parental SES of selfemployed, assistant non-manual, manual worker in industry and unemployed. Daily use of snus was related to male sex, having a smoking mother and having a father who used snus (table 1).
Participation in the tobacco prevention programme
The children in the participating schools (n=447) were compared with children in non-participating schools (n=2165). There were no statistically significant differences in the prevalence of male sex, parental smoking, living conditions, physician-diagnosis of asthma, participation in sports or parental SES between the sample invited to the tobacco prevention programme and the controls who were not invited. However, among those invited to the prevention programme, the prevalence of urban living (81% vs 58%, p<0.001), and having older siblings (67% vs 62%, p=0.047) was significantly higher compared with the controls. Among the 447 invited to join the prevention programme, 224 (50%) chose to participate by signing a contract. Comparison of baseline characteristics between participants and non-participants in the tobacco prevention programme is presented in table 2. Comparing non-participants with participants, the prevalence of boys (59% vs 43%), having a smoking mother (20% vs 9%) and living in a single parent household (16% vs 8%) was significantly higher among the non-participants, while fewer non-participants were taking part in sports (65% vs 79%). Among the participants, it was more common to have parental SES at the professional and assistant non-manual level, while among non-participants, parental SES from among intermediate non-manual and manual workers, in industry and service level, was more common (test-for-trend p<0.005). There were no significant differences between participants and non-participants regarding living in a house versus living in an apartment, urban versus rural living, having older siblings, or having physiciandiagnosed asthma.
Significant factors related to non-participation in the tobacco prevention programme identified in the bivariate analyses were included in a multivariate analysis. Non-participation was related to male sex (OR 1.8, 95% CI 1.2 to 2.7), having a smoking mother (OR 2.1, 95% CI 1.1 to 3.8) and parental SES of manual workers in service (OR 3.0, 95% CI 1.3 to 6.7). Participation in sports was inversely related to non-participation (OR 0.6, 95% CI 0.3 to 0.9; table 3).
Effect of the intervention
The prevalence of smoking and use of snus was significantly lower among the participants in the prevention programme compared with the non-participants and the controls in the rest of the cohort (figure 2). Of the participants in the programme, only four individuals were daily smokers or snus users at the age of 14-15 years. On the school level, there was no spill-over effect, that is, there was no difference in tobacco use between the children at the invited schools and the control schools.
Among the controls at baseline at the age of 11-12 years, the prevalence of having a smoking mother was 14.4%, and 11.4% had a smoking father. In the follow-up, the corresponding proportions were very similar: 13.4% and 12.4%, respectively. However, among the participants in the intervention, the prevalence of having a smoking mother decreased from 9.0% to 5.8% ( p=0.201), while having a smoking father remained similar, 10.5% and 11.5%. However, none of these differences in prevalence were statistically significant.
DISCUSSION
In this population-based prospective study, we report a low prevalence of tobacco use among Swedish teenagers, especially among the participants in a tobacco prevention programme. Further, we found that participation in the prevention programme was affected by a selection bias, as those in most need of smoking prevention, that is, children having smoking parents and a lower SES, did not participate.
From the 1980s to the 2000s, smoking steadily decreased among Swedish adults, 22 23 while the prevalence of smoking initiation among teenagers remained relatively stable. 5 However, in the last decade there have been some reports of a decrease also among teenagers. In 2003, the prevalence of daily smoking at the age of 14-15 years was 5.8% and using snus 9.9% in a similar cohort in the same study area, 9 compared with 3.5% and 3.3%, respectively, in the present study. Thus, smoking and the use of snus had both decreased, a positive result in accordance with recent nationwide reports among Swedish 5 and Norwegian teenagers. 24 Despite the low prevalence of tobacco use, some teenagers were more likely to be tobacco users than others.
Smoking was related to socioeconomic factors such as parental socioeconomic level, living in a single parent household and living in an apartment, in accordance with other studies. 9 24-26 There are socioeconomic inequalities in health, 27 and the fact that smoking is more common among those with lower socioeconomic level contributes to these inequalities. 24 28 Other factors related to smoking were female sex, having smoking family members and not taking part in sports, as shown in other studies. 9 29 30 Few studies have reported on factors related to snus use among teenagers. 9 Similar to other studies, 29 the risk factors for using snus were male sex and parental tobacco use. Additionally, we found a significant association between snus use and parental SES of manual workers in industry and in service. However, in the multivariate analysis, this association lost significance. It has been shown that lower educational level and income was related to snus use among adults in Sweden. 31 By identification and characterisation of tobacco users, and also populations at risk of becoming tobacco users, prevention efforts might be improved. Because parental tobacco use is an important risk factor for tobacco use among teenagers, 9 29 the study design of the present prevention programme (Tobacco free Duo) included the partnership with a tobacco-free adult. In an evaluation of Tobacco Free Duo in another county in Sweden, it was shown that the prevalence of smoking not only decreased among the teenagers, but also among the adult participants in the programme. 32 This was also seen in our study, but the decrease was not statistically significant. Having tobacco-free role models is an important aspect of tobacco prevention among teenagers.
In studies among adults, non-participation in studies about respiratory conditions is associated with higher prevalence of smoking 13 14 33 and lower SES. 16 Further, non-participation in an alcohol use prevention study among teenagers was related to lower parental socioeconomic level. 34 However, little is known about nonparticipation in smoking prevention programmes among teenagers. In a review of the long-term effects of smoking prevention programmes, the authors noted a selection bias, as most reviewed programmes were based on convenience samples and not random samples. 35 This may impact the external validity of a study because the sample may not be representative of the general population. Further, it has been shown that having smoking family members decreased the efficacy of a school-based smoking prevention programme. 36 Thus, involving the family in smoking prevention seems to be a good idea. However, although the prevention programme in our study involved the child and an adult, and was a collaboration between schools, Norrbotten county council and local organisations, the participation rate was low, as only 50% chose to join the programme. Furthermore, the prevalence of having a smoking mother was twice as high among the nonparticipants compared with the participants. Thus, many of those who would have benefited from the prevention efforts chose not to participate. Another factor related to non-participation was parental SES of manual workers in service. One explanation for this finding may be that manual workers in service as well as smoking are more common among women, in this case the mothers, and both these factors were related to non-participation.
We suggest that in order to avoid this bias and improve the efficacy of smoking prevention, even closer collaboration between policymakers, community, school and the family is needed. If we succeed in reaching and informing these actors, the prevention strategies may target a larger population. Further, as most of the teenagers were at a low risk of becoming smokers, the 'prevention paradox' may apply, similar to smoking cessation intervention among smokers. 37 It states that prevention strategies on the population level are more likely to reduce the smoking-related health problems in the population compared with strategies on the individual level. Although strategies on the individual level may target teenagers at high risk of becoming smokers, these individuals are relatively few and only account for a minority of the overall public health burden. Prevention strategies on the population level are said to be more effective, simply because they reach a higher number of individuals. Promising prevention strategies that have been suggested, aimed at teenagers, include media campaigns, increasing cigarette price and restricting access to tobacco products, and also social environment changes such as reduction of smoking among adult role models. 38 The strengths of this study included the longitudinal study design, with high response rates, few participants lost to follow-up and the use of validated questionnaires. Further, the collaboration with the prevention programme, Tobacco Free Duo, enabled us to combine the longitudinal data from the OLIN studies with intervention data on participation in the prevention programme. A limitation of the study included the lack of information about the level of activity in the tobacco prevention programme during the follow-up time. Another limitation is that self-reported smoking was not validated by objective measures. However, others that compared selfreports of smoking with cotinine levels in saliva found good agreement. 39 In conclusion, prevalence of tobacco use was significantly lower among the participants in the tobacco prevention programme compared with the controls, after 3 years. However, the observed benefit of the intervention may be overestimated as the participation was related to a selection bias, as those in most need of smoking prevention, that is, children having smoking parents and a lower SES, did not participate. One way to improve the efficacy of smoking prevention efforts is to have even closer collaboration between policymakers, community, school and the family. Developing comprehensive strategies for including more high-risk children in prevention efforts at the population level will be an important measure to reduce tobacco use among teenagers.
Contributors LH was responsible for the study design, collected the data, performed the statistical analyses, drafted and revised the manuscript, and approved the final manuscript. MA and CS contributed to the analysis and interpretation of data, reviewed and revised the manuscript, and approved the final manuscript. ER was responsible for study conception and design, contributed to the analysis and interpretation of data, reviewed and revised the manuscript, and approved the final manuscript. Competing interests None declared.
Ethics approval The Regional Ethical Review Board at Umeå University, Sweden.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement No additional data are available.
Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http:// creativecommons.org/licenses/by-nc/4.0/ | 2016-05-12T22:15:10.714Z | 2015-05-01T00:00:00.000 | {
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231711908 | pes2o/s2orc | v3-fos-license | Plasminogen activator inhibitor 1 and venous thrombosis in pancreatic cancer
thrombosis was induced by complete ligation of the inferior vena cava (IVC). Levels of active PAI-1 were independently associated with increased risk of VTE in patients with pancreatic cancer (subdistribution hazard ratio per doubling of levels: 1.39 [95% con fi dence interval, 1.09-1.78], P 5 .007). Mice bearing PANC-1 tumors had increased levels of both active human and active mouse PAI-1 and decreased levels of plasmin activity. Importantly, mice bearing PANC-1 tumors exhibited impaired venous thrombus resolution 8 days after IVC stasis compared with nontumor controls. Our results suggest that PAI-1 contributes to VTE in pancreatic cancer. of venous thrombi 2 days after IVC ligation is not different from that in control mice. 31,32 Mouse models have been developed to investigate the mechanisms of cancer-associated thrombosis. 33 Most of these studies have used mouse and human pancreatic or breast tumors. We showed that tumor-derived human TF contributed to the increased thrombus weight in mice bearing human pancreatic tumors. 34 In addition, we and others found that neutrophils and neutrophil extracellular traps contribute to thrombosis in mice bearing breast or pancreatic tumors. 35-37 suggest
Introduction
Cancer patients have an increased risk of both venous thromboembolism (VTE; 0%-26%) and arterial thromboembolism (0%-3%). [1][2][3][4][5][6] The rate of VTE varies in different types of cancer, with pancreatic cancer and brain cancer having the highest rates. 7,8 This suggests that there may be cancer typespecific pathways that enhance VTE. 9 Many studies have identified biomarkers that are associated with VTE in cancer patients that can be used to improve risk assessment scores. 10 One of the most reliable biomarkers to predict VTE in the general cancer population is the fibrin degradation product D-dimer, which is part of the Vienna risk assessment score. [11][12][13] Recently, some cancer type-specific biomarkers have been reported. For instance, extracellular vesicle tissue factor (TF) activity was associated with VTE in patients with pancreatic cancer. [14][15][16][17][18][19][20] More recently, we found that plasma levels of citrullinated histone H3, which is a biomarker of formation of neutrophil extracellular traps, was associated with VTE in pancreatic and lung cancer patients. 21 Plasminogen activator inhibitor 1 (PAI-1) is associated with thrombosis in a variety of diseases, including obesity, diabetes, and metabolic syndrome. 2223 PAI-1 reduces the generation of plasmin by inhibiting the plasminogen activators tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). PAI-1 circulates in blood in 3 different forms: active; latent inactive; or complexed with either tPA, uPA, or vitronectin. 24,25 Different assays can be used to measure either total or active PAI-1 in plasma. Importantly, active PAI-1 but not PAI-1 antigen is a potential marker of VTE. 26 Pancreatic tumors express high levels of PAI-1 (The Human Protein Atlas, www.proteinatlas.org). Similarly, pancreatic cancer cell lines also express high levels of PAI-1. 27,28 However, there are only 2 studies that have measured plasma levels of PAI-1 in pancreatic cancer patients. One study observed elevated plasma levels of total PAI-1 antigen and PAI-1 activity in 27 patients with pancreatic cancer compared with healthy controls. 29 Another study found that there was no difference in total PAI-1 levels between pancreatic cancer patients with and without VTE. 30 The role of PAI-1 in venous thrombosis has been studied in a mouse model of venous stasis induced by ligation of the inferior vena cava (IVC). PAI-1-deficient mice exhibited more rapid venous thrombus resolution that resulted in significantly smaller thrombi 8 and 21 days after IVC ligation. 31 Conversely, mice overexpressing PAI-1 exhibit impaired thrombus resolution that led to larger thrombi 6 and 14 days after IVC ligation. 32 Importantly, PAI-1-deficient mice and mice overexpressing PAI-1 have normal thrombogenesis because the size of venous thrombi 2 days after IVC ligation is not different from that in control mice. 31,32 Mouse models have been developed to investigate the mechanisms of cancer-associated thrombosis. 33 Most of these studies have used mouse and human pancreatic or breast tumors. We showed that tumor-derived human TF contributed to the increased thrombus weight in mice bearing human pancreatic tumors. 34 In addition, we and others found that neutrophils and neutrophil extracellular traps contribute to thrombosis in mice bearing breast or pancreatic tumors. [35][36][37] In the present study, we investigated the role of PAI-1 in VTE in pancreatic cancer using samples from patients and mice bearing human pancreatic tumors. We measured levels of active PAI-1 in pancreatic cancer patients who were prospectively observed during the disease with regards to the occurrence of VTE. In addition, we measured the weight of venous thrombi in mice with or without human pancreatic tumors that express PAI-1. Our results suggest that PAI-1 contributes to VTE in pancreatic cancer.
Patients
This study included pancreatic cancer patients (n 5 139) who were recruited into the Vienna Cancer and Thrombosis Study, an ongoing prospective and observational cohort study. 38 The study was approved by the Medical University of Vienna Ethics Committee. Details on design, inclusion, and exclusion criteria have been published previously. 38 In brief, adult patients with newly diagnosed or recurrent/progressive cancer were eligible for inclusion. Key exclusion criteria include continuous anticoagulation therapy, diagnosis of VTE within 3 months, chemotherapy within 3 months, and radiotherapy or surgery within 2 weeks before inclusion. 11 Each patient gave a written informed consent. Before initiation of chemotherapy, a baseline blood sample was collected, and patients were prospectively followed for a maximum of 2 years. Blood samples were centrifuged at 1500g for 15 minutes to obtain platelet-poor plasma and aliquots were stored at 280°C until testing was performed in series. The primary outcome of Cancer and Thrombosis Study is occurrence of objectively confirmed, independently adjudicated VTE.
Cell lines
Human pancreatic cancer cell lines BxPC-3, HPAF-II, MIA PaCa-2, and PANC-1 were obtained from American Type Culture Collection (Manassas, VA). AsPC-1 was obtained from Bruce A. Sullenger (Duke University, Durham, NC). Cells were cultured in the recommended medium containing 10% fetal bovine serum (Omega Scientific, Tarzana, CA) and 1% antibiotics-antimycotic (Thermo Fisher Scientific, Waltham, MA) in a 5% CO 2 incubator at 37°C. Cancer cells (1.0 3 10 5 cells/well) were cultured in fetal bovine serum-free recommended medium in a 12-well plate for 24 hours. The culture medium was centrifuged at 3000g for 15 minutes and supernatant was collected. Culture supernatant was frozen and stored at 220°C until use.
Generation of luciferase-expressing PANC-1 cells
Luciferase-expressing PANC-1 cells were generated using the same strategy as described previously. 34
Mouse tumor model
All animal studies were approved by University of North Carolina at Chapel Hill Animal Care and Use Committee and complied with National Institutes of Health guidelines. Crl:NU-Foxn1nu male mice (nude mice) aged 3 to 4 weeks were purchased from the animal service core facility in the University of North Carolina at Chapel Hill. Luciferase expressing PANC-1 cells (2 3 10 6 in phosphatebuffered saline and Matrigel, Corning, Corning, NY) were injected into the pancreas. PANC-1 tumors were grown for 12 to 19 weeks. Luciferase expression was measured weekly with the IVIS Kinetic In Vivo Imaging System (Caliper Life Sciences, Waltham, MA). When the tumors gave a signal count of $1.0 3 10 5 , mice were scheduled for a thrombosis experiment. For biomarker studies, we used mice with tumors weighing from 0.9 to 4.0 g. For thrombosis experiments, we used mice with tumors weighing 2.1 to 2.9 g. We observed 20% of PANC-1 tumor-bearing mice have up to 5 g of ascites. In addition, we observed 13% of PANC-1 tumor-bearing mice have metastasis mainly found in liver. We did not use mice with either ascites or liver metastasis. Blood was collected as described, 39 and plasma was prepared by centrifuging the blood at 4500g for 15 minutes. Plasma samples were frozen and stored at 280°C until use.
Measurement of PAI-1 and thrombin-antithrombin complexes
Levels of active PAI-1 in plasma from pancreatic cancer patients were measured with the TECHNOZYM PAI-1 Actibind enzymelinked immunosorbent assay (ELISA) (#TC16075; Technoclone, Vienna, Austria). This ELISA plate is coated with tPA and detects only active PAI-1. Levels of human total PAI-1 in the culture supernatant of cancer cells were measured using a commercial ELISA (#HPAIKT-TOT; Molecular Innovations, Novi, MI). This ELISA detects all forms of PAI-1, including free, latent, and complexed PAI-1. Commercial ELISAs were used to measure levels of active mouse PAI-1 (#MPAIKT; Molecular Innovations) and active human PAI-1 (#HPAIKT; Molecular Innovations) in plasma from mice with or without tumors. These ELISA plates are coated with uPA and detect free PAI-1, but not latent or complexed PAI-1. Plasma levels of thrombin-antithrombin (TAT) complexes (#OWMG15; Siemens, Munich, Germany) in mice were measured using a commercial assay.
Measurement of circulating neutrophils in mice
Neutrophils, platelets, and red blood cells were counted in whole blood using a Hemavet HV950FS (Drew Scientific, Miami Lakes, FL).
Thrombosis model
We used the IVC stasis venous thrombosis model as described previously. 34 We harvested thrombieither 2 days or 8 days after IVC ligation. Thrombi were fixed with 10% formalin and then stored in 70% ethanol.
Microscopic analysis of thrombi
The fixed thrombi were embedded in paraffin, and 4-mm-thick sections were cut and stained with either hematoxylin and eosin or Martius Scarlet Blue by the Animal Histology and Laboratory Medicine Core Facility at the University of North Carolina at Chapel Hill.
Macrophage staining
Paraffin-embedded thrombi sections were deparaffinized with antigen retrieval. Slides were blocked with 5% nonfat skim milk (AppliChem, Council Bluffs, IA) in phosphate-buffered saline (Thermo Fisher Scientific) for 1 hour at room temperature. Endogenous avidin and biotin were blocked with avidin solution and biotin solution, respectively (Vector Laboratories, Burlingame, CA). Sections were incubated overnight at 4°C with 0.4 mg/mL rabbit anti-mouse Iba1 antibody (#019-19741; FUJIFILM Wako Chemicals, Osaka, Japan). After washing, sections were incubated with biotinylated goat anti-rabbit antibody (#BA-1000; Vector Laboratories) for 1 hour at room temperature. After adding avidin-peroxidase complex solution (#PK-6100; Vector Laboratories), Iba-1 staining (brown) was detected using the DAB substrate (#K3468; Agilent Technology, Santa Clara, CA), which was added for 10 minutes. Sections were counterstaining with hematoxylin (Thermo Fisher Scientific) for 1 minute. Stained sections were evaluated with a 1 to 5 scale by 2 blinded investigators.
Statistics
Cumulative incidence functions of VTE over time were estimated in competing risk analysis, accounting for all-cause mortality as competing outcome event to avoid overestimating VTE risk in this high mortality cohort. 40 Accordingly, uni-and multivariable modeling of the association between PAI-1 per doubling of levels and risk of VTE was studied within a competing risk regression model according to Fine and Gray, 40,41 adjusting for potential confounders (age, sex, body mass index, disease stage, cancer resection as time-dependent covariable). For visualization, levels of PAI-1 were dichotomized at the median of distribution. The proportional subhazards assumption was confirmed by fitting an interaction between predictor variables and follow-up time and by plotting Schoenfeld-like residuals against follow-up time. These statistical analyses were performed using STATA 15.0 (Stata Corp, Houston, TX).
For in vitro and mouse studies, the Shapiro-Wilk test was used for normality test and the F-test was used to compare variances. All data were normally distributed and parametric tests were used for the all data. For the 2-group comparisons, the unpaired 2-tailed Student t test or the Welch t test were used depending on the variances. Data are shown as mean 6 standard deviation. All statistical analyses for mouse experiments were performed with PRISM version 7.03 (GraphPad Software, La Jolla, CA). Values of P , .05 were considered statistically significant for all experiments.
Association of active PAI-1 with VTE in pancreatic cancer patients
To determine if there was an association between active PAI-1 and VTE in pancreatic cancer, we measured the level of active PAI-1 in 139 pancreatic cancer patients ( Table 1). The cumulative 2-year incidence of VTE in patients with PAI-1 levels at or above the median was 25.1% (95% CI, 15.6-35.9) compared with only 12.4% (95% CI, 5.8-21.7) in those with levels below the median (Figure 1).
PAI-1 expression in human pancreatic cancer cell lines
To establish a mouse model to study the effect of tumor-derived PAI-1 on venous thrombosis, we first evaluated PAI-1 expression in human pancreatic cancer cell lines. We used the Cancer Cell Line Encyclopedia database (https://portals.broadinstitute.org/ ccle) to select cell lines that expressed low, medium, and high levels of PAI-1 (Figure 2A). Next, we measured levels of total PAI-1 in the culture supernatant. Consistent with the levels of PAI-1 messenger RNA (mRNA) expression, PANC-1 cells expressed the highest level of PAI-1, BxPC-3, and MIA PaCa-2 cells expressed medium levels and HPAF-II and AsPC-1 cells expressed very low levels of total PAI-1 ( Figure 2B). Previous studies have also reported that PANC-1 cells express a high level of PAI-1. 27,28 In addition, we used the Cancer Cell Line Encyclopedia database to evaluate tPA, uPA, and urokinasetype plasminogen activator receptor (uPAR) mRNA expression ( Figure 2C). PANC-1 cells expressed high uPA and uPAR but low tPA ( Figure 2C). Importantly, we have previously shown that PANC-1 cells express very low levels of TF. 42,43 Therefore, PANC-1 cells can be used to study the role of PAI-1 in thrombosis with minimal interference from tumor-derived TF expression.
Establishment of a human pancreatic tumor model expressing PAI-1 in mice
For the mouse tumor model, we used PANC-1 cells to form tumors because they express high levels of PAI-1. We measured levels of active human and mouse PAI-1 in the plasma of mice bearing PANC-1 tumors using species-specific ELISAs. Mice bearing PANC-1 tumors had increased plasma levels of both active human and mouse PAI-1 ( Figure 3A-B).
We have previously shown that mice bearing human pancreatic tumors with high TF expression (HPAF-II, HPAC, and BxPC-3) but not those expressing low levels of TF (MIA PaCa-2 and PANC-1) have increased levels of circulating tumor-derived human TF and TAT complexes, which are markers of activation of coagulation. 34,42,44 Here, we observed a slightly elevated level of TAT complexes in mice bearing PANC-1 tumors; this was not statistically significant ( Figure 3C). We have also observed increased levels of neutrophils in mice bearing BxPC-3 tumors. 34,37 In contrast, PANC-1 tumor-bearing mice did not have increased numbers of neutrophils compared with controls ( Figure 3D). Similarly, PANC-1 tumor-bearing mice did not have increased number of platelets and red blood cells compared with controls ( Figure 3E; data not shown).
Tumor-bearing mice have impaired venous thrombus resolution in the IVC stasis model
We measured thrombus weight both 2 and 8 days after IVC ligation to study thrombogenesis and thrombus resolution, respectively. We did not observe a difference in thrombus weight 2 days after IVC ligation in mice with or without tumors ( Figure 4A). In contrast, mice bearing PANC-1 tumors had significantly larger venous thrombi 8 days after IVC stasis compared with the weight of thrombi formed in control mice ( Figure 4B), which suggested an impairment of thrombus resolution
Characterization of thrombi from tumor-bearing mice and controls
We stained thrombi from control and tumor-bearing mice with hematoxylin and eosin ( Figure 5A). With this stain, red blood cell areas are red, platelet areas are pink, and inflammatory cell areas are purple. In addition, we stained the thrombi with Martius Scarlet Blue ( Figure 5B). With this stain, red blood cell areas are yellow, platelet areas are gray, and fibrin areas are red. However, no gross differences in composition were observed between thrombi from control (n 5 5) and tumor-bearing (n 5 6) mice ( Figure 5A-B).
A previous study showed that mice overexpressing PAI-1 had significantly reduced numbers of macrophages in thrombi compared with controls. 32 Therefore, we measured the numbers of macrophages in thrombi in control and tumor-bearing mice. Interestingly, macrophages were distributed around the edge of thrombi from both controls and tumor-bearing mice ( Figure 5C
Discussion
In the present study, we found an association between plasma levels of active PAI-1 and VTE in patients with pancreatic cancer (;40% increase per doubling of PAI1-1 levels). In contrast to our result, Kondo and colleagues did not observe an association between total PAI-1 and VTE in patients with pancreatic cancer. 30 However, these 2 studies measured different forms of PAI-1 (active vs total PAI-1) and have different observation times. We believe that levels of active PAI-1 are a better measure of thrombotic risk than levels of total PAI-1 because the latter measures both active and inactive PAI-1. A previous study showed that active PAI-1 but not PAI-1 antigen is a potential marker of VTE. 26 In addition, our study was specifically designed to investigate biomarkers for VTE in patients with cancer. Another study observed increased plasma levels of total PAI-1 protein and activity in pancreatic cancer patients compared with healthy controls, but no difference in patients with or without a prior deep vein thrombosis. 29 However, this study observed intermittent peaks in both PAI-1 antigen and activity over time in the pancreatic cancer patients, which suggests that they may have transient hypercoagulable episodes. 29 We established a new mouse model to study venous thrombosis in mice bearing human pancreatic tumors expressing PAI-1. The important features of our mouse model are that PANC-1 tumors grown in the pancreas release human PAI-1 into the circulation and that tumor-bearing mice have impaired thrombus resolution 8 days after IVC ligation. PANC-1 tumors express low levels of TF; mice bearing PANC-1 tumors do not have an activated coagulation system. These data are consistent with our previous study that showed no difference in plasma TAT complexes in mice bearing PANC-1 tumors and control mice. 42 Human PAI-1 inhibits mouse tPA, albeit with reduced inhibitory activity (;40%) compared with mouse PAI-1. 45 Therefore, human PAI-1 derived from the PANC-1 tumors can inhibit the fibrinolytic system in mice. The presence of the PANC-1 tumors was also associated with an increase in levels of mouse PAI-1 in the plasma. At present, we do not know the source of active mouse PAI-1 in tumor-bearing mice. PAI-1 is expressed in various types of cells, including endothelial cells and platelets. 25 We analyzed the weight of thrombi in mice bearing PANC-1 tumors and controls both 2 and 8 days after IVC ligation to study thrombogenesis and thrombus resolution, respectively. We have shown that tumor-derived TF and elevated levels of circulating neutrophils in mice bearing BxPC-3 tumors both enhance thrombogenesis 2 days after IVC ligation. 34,37 In contrast to these studies with mice bearing BxPC-3 tumors, we did not observe an increase in thrombogenesis in mice bearing PANC-1 tumors 2 days after IVC ligation. This result is consistent with previous studies with PAI-1deficient mice and mice overexpressing PAI-1, showing that PAI-1 does not affect thrombogenesis in this model. 31,32 Furthermore, mice bearing PANC-1 tumors do not increase circulating neutrophils. Importantly, mice bearing PANC-1 tumors had significantly larger venous thrombi 8 days after IVC ligation indicating impaired thrombus resolution. This result is consistent with studies with PAI-1-deficient mice and mice overexpressing PAI-1, which showed a role for PAI-1 in thrombus resolution in this model. 31,32 Therefore, mice bearing human PANC-1 tumors expressing PAI-1 have impaired thrombus resolution that results in larger thrombi 8 days after IVC ligation. We did not observe any difference in the composition of thrombi from controls and tumor-bearing mice. These data are expected because we did not observe a difference in the numbers of circulating red blood cells, platelets, and neutrophils in control and tumor-bearing mice. Mice overexpressing PAI-1 had significantly reduced numbers of macrophages in thrombi compared with controls. 32 In contrast, we did not observe difference of macrophage infiltration in thrombi from controls and tumor-bearing mice. This suggests that the primary mechanism by which the elevated levels of PAI-1 in tumor-bearing mice impair thrombus resolution is by inhibiting plasmin generation.
A recent study showed that mice bearing human lung A549 tumors, which express PAI-1, had larger venous thrombi in an IVC stenosis model. 50 However, thrombus weight was evaluated 3 hours after IVC ligation, which would imply an effect on thrombogenesis. Furthermore, the role of tumor-derived PAI-1 in the increase in thrombus weight was not investigated. Interestingly, treatment of mice with the vascular endothelial growth factor inhibitor bevacizumab increased tumor-derived human PAI-1 expression in A549 tumors and thrombus weight. 50 In addition, administration of an oral PAI-1 inhibitor PAI-039 reduced the thrombus weight to baseline. 50 However, PAI-039 inhibits both human and mouse PAI-1 51,52 and does not inhibit vitronectin-bound PAI-1. 53 A limitation of the current study is that we used platelet-poor plasma. Because platelets are one of the major sources of PAI-1, we cannot exclude the possibility that a low level of contaminating platelets in the plasma could affect the measured levels of PAI-1.
In summary, we have demonstrated an association of active PAI-1 levels and VTE in pancreatic cancer. Mice bearing human pancreatic tumors have increased levels of tumor-derived, human PAI-1 and host-derived, mouse PAI-1 that is associated with impaired thrombus resolution. Our data suggest that PAI-1 is a risk marker of VTE in pancreatic cancer. | 2021-01-27T06:16:52.569Z | 2021-01-21T00:00:00.000 | {
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248795729 | pes2o/s2orc | v3-fos-license | A numerical analysis of the instability in the Taylor Vortex flow by using the visualization of attractors
This study clarifies the critical values of bifurcations from a steady Taylor flow into a wavy Taylor flow by a numerical analysis applying chaos theory. In our previous numerical research, we adopt the amplitude fluctuation of the kinetic energy of the axial velocity component as a standard criterion to identify the critical Reynolds number. However, the amplitude fluctuation of the kinetic energy is very small near the critical value, which affects the judgment of Taylor flow or wavy Taylor flow. In this study, in order to improve this problem, we introduce a new method that visualizes attractors in the three-dimensional embedded space composed from the axial velocity component. The targets are the normal 2-vortex, 4-vortex and 6-vortex modes, which stably present in the range of the aspect ratio from 1.0 to 7.2 in a system with stationary end walls of two cylinders with the radius ratio 0.667. The critical values at which the Taylor flow bifurcates into the wavy Taylor flow are determined based on the converged orbit shapes of the attractors. The analytical result shows that, in each flow mode, the critical Reynolds number for the onset of the wavy flow has its peak and presents a quantitative agreement with the experimental result in the lower range than the peak Reynolds number. This study shows that the instability of the Taylor flow is revealed by determining the structure of the attractor applied by the Hopf bifurcation. In addition, it is represented that this numerical approach using attractors can obtain more accurate results than those obtained by a numerical evaluation method using the amplitude of the kinetic energy of the Taylor flow. The critical value in which Taylor flow with a small aspect ratio bifurcated into the wavy Taylor flow was clarified by a numerical analysis. The analytical results were in agreements with the experimental results, while there were quantitative differences in some areas. This numerical analysis was confirmed that the bifurcation phenomenon from the Taylor flow into the wavy Taylor flow can be clarified. The critical value in which Taylor flow with a small aspect ratio bifurcated into the wavy Taylor flow was clarified by a numerical analysis. The analytical results were in agreements with the experimental results, while there were quantitative differences in some areas. This numerical analysis was confirmed that the bifurcation phenomenon from the Taylor flow into the wavy Taylor flow can be clarified.
Introduction
One of the nonlinear dynamic model that shows nonuniqueness is the Taylor vortex flow that swirls between cylinders. The pioneering study by Taylor [1] showed that the theoretical and experimental results for the instability of a Couette-Taylor flow between infinite length cylinders are in agreement. Grossmann et al. [2] reviewed the progress of recent Taylor-Couette turbulence at higher Reynolds numbers. Coles [3] first clarified the non-linear stability and discovered the critical value that bifurcates from a steady Taylor flow into a wavy Taylor flow. Since then, many studies of the stability of the Taylor flow have been reported. Most of these studies, for examples by Burlhalter et al. [4], Koschmieder [5], assumed that the annulus of the working fluid has infinite length and they identified a phenomenon where the flow bifurcates from a Couette flow to a Taylor flow, a wavy Taylor flow, a modulated wavy Taylor flow and finally to a turbulent Taylor flow as the rotating speed of the cylinder is increased. Jones [6] examined numerically the transition from the steady axisymmetric Taylor flow to the timedependent wavy flow in the infinite-cylinder model. His investigation is the growth of the disturbances added to the two-dimensional fundamental flow and described the behavior of the transition to wavy Taylor flow.
Benjamin [7,8] focused on the new flow states under the condition that the influence of the end walls of the annulus with the small aspect ratio was taken into account. He applied mathematical theory to the bifurcation of the Taylor flow. His work led to subsequent applications of chaos theory in the evaluation of the stability of nonlinear dynamical system phenomena. For the first time, Mullin and Benjamin [9] experimentally revealed the critical Reynolds number at which the Taylor flow bifurcates to the wavy Taylor flow, in the small aspect ratio which the end plates affect the bifurcation between the flows. As a similar visualization experiment with small aspect ratios, Nakamura et al. [10] clarified the instability of Taylor flows having two geometric conditions with axially symmetric and asymmetric systems. Cliffe et al. [11,12] numerically simulated a Taylor flow with a small aspect ratio and compared their results with the experimental results. Watanabe et al. [13,14] reported the mode formation process under the two boundary conditions of axially symmetric and asymmetric systems with small aspect ratios. Toya et al. [15,16] numerically examined the fluctuation of the kinetic energy of vortices and showed the numerical result of the critical Reynolds number at which a steady Taylor flow bifurcates into a wavy Taylor flow. In this analysis, kinetic energy was used as the evaluation standard. However, it was difficult to distinguish whether the difference was due to a kinetic energy fluctuation or a numerical error of the computer. Because the fluctuation range of the kinetic energy was very small near the critical Reynolds number of the bifurcation, the comparison between experimental and numerical results was inadequate.
Several numerical methods have been proposed to determine the instability of the Taylor flow. Razzak et al. [17] used the variation of the magnitude of the averaged axial velocity component to distinguish among the Taylor flow, the transition flow, and the wavy Taylor flow. Martinand et al. [18] performed a weakly nonlinear analysis and examined the mechanism of transition to a wavy Taylor flow when the radius ratio was large. They added exponential developmental perturbations to the stationary solution and investigated the growth of the perturbations in the positive and negative parts of the exponent. Rudman et al. [19] used a new method based on Poincaré section and determined the perturbations of wave formation for the divergence-free interpolation of discrete velocity data. Crowley et al. [20] carried out the experimental visualization and the numerical simulation, and identified the subcritical laminar-turbulent transition in a system of counter-rotating cylinders. Though the flow is not the Taylor flow, Pasche et al. [21] used a bifurcation analysis and clarified the transition process from a helical vortex flow to a chaotic axisymmetric oscillation flow.
In this study, we focus on the attractor drawn from the axial velocity component of the vortex flow. We apply a theory from chaotic time series analysis to perform a numerical analysis on the instability of the Taylor flow. The time series axial velocity component of the vortex on the bottom end wall is converted into a three-dimensional vector by the embedded theorem, and an attractor orbit is drawn in the three-dimensional space. And the critical value at which the Taylor flow bifurcates into the wavy Taylor flow, which shows the Hopf bifurcation from a fixed point to a limit cycle, is determined.
In the rest of this paper, the governing equations and the calculation methods for Taylor flow and their problems of the study that is previously performed are denoted in Sects. 2 and 3, respectively. Section 4 presents details of the numerical calculation and our new analysis procedure to find the bifurcation Reynolds number. In Sect. 5, after explaining the new analysis method, the critical Reynolds number at each aspect ratio will be clarified while the analysis results of typical Taylor flows are exemplified. Also, the analytical result is compared with the experimental results. Finally, Sect. 6 gives some conclusions of the present study.
Flow region and governing equations
We focus on a circulating flow between concentric cylinders with finite length. The inner cylinder rotates and the outer cylinder and the end walls are stationary.
The governing equations are the continuity equation and the Navier-Stokes equations in the cylindrical coordinate system ( r, θ, z) as shown in Fig. 1: where t is the time, u is the velocity vector with its components of u, v and w, p is the pressure, and Re is the Reynolds number. These values are normalized by the reference velocity and the reference length defined by the circumferential velocity of the inner cylinder, the gap width between the inner and outer cylinders, respectively, and the kinematic viscosity of the fluid. The main parameters are the Reynolds number Re and the aspect ratio Γ defined by the fraction of the lengths of the cylinders to the gap between the inner and outer cylinders. In this analysis, the range of the aspect ratio is from 1.0 to 7.2 and the radius ratio is 0.667.
Previous analysis method utilizing kinetic energy amplitude
The authors have conducted numerical analysis on the stability of the Taylor flow, which is representative of nonlinear dynamical systems, and have been conducting research to compare it with the experimental results that have already been clarified. In the numerical calculation, the axial velocity component of the vortex was obtained, and the bifurcation phenomenon was clarified from the time series fluctuation of the spatially averaged kinetic energy. The kinetic energy fluctuation of the Taylor vortex is in the steady state, and that of the wave Taylor flow is in the fluctuation state. In the previous numerical analysis, it is decided to identify by the magnitude of the amplitude fluctuation of the spatially averaged kinetic energy. with the difference between the maximum and minimum values of the amplitude fluctuation being approximately 2.5 × 10 -6 . Although not shown in the figure, the energy value of Re = 620 is 0.57, which is constant. As shown in Fig. 2a and b, the amplitude fluctuation is so small that it is not possible to determine whether this unsteady state is due to flow fluctuations or numerical errors of computer. Therefore, it is difficult to make a clear determination in Fig. 2a and b. Figure 3 shows the amplitude fluctuation of kinetic energy for each Reynolds number. This figure indicates that the amplitude fluctuation increases sharply from Re = 660. From this fact, we decided the threshold of the amplitude fluctuation in which the fluctuation of the kinetic energy of the flow becomes unsteady as 0.001. Figure 4 shows the critical Reynolds number between the Taylor flow and the wavy Taylor flow by comparing the amplitude fluctuation of the kinetic energy in the flow for each aspect ratio with the threshold value of 0.001. It can be seen that the results of the analytical result and the experimental result are qualitatively consistent. On the other hand, it can be seen that there are some variations in the curve of the analytical result. Though it is effective to increase the number of calculations in order to suppress the variation, the number of effective calculations differs depending on the conditions, and the procedure for.
setting the number of calculations is complicated and a large amount of work. This our previous analysis study utilizing kinetic energy amplitude is reported in reference [16]. From this result, another analysis method different from the method using the amplitude of kinetic energy is required.
Numerical calculation method and analysis procedure
The three-dimensional numerical calculation adopts the MAC method for the basic solution: the QUICK method for the convection terms, the central finite difference method for the spatial integration of other terms, and the fractional method for the time integration. The SOR method and the ILUCGS method are used for the Poisson's equation of the pressure. The grid is a staggered grid. The number of grid points in the three-dimensional space (r, θ, z) at Γ = 1.0 is (41, 74, 41). The number of points in the axial direction z is determined to be proportional to the value of Γ . The step value of the dimensionless time is 2.5 × 10 -3 , and the maximum time step number is 10 million TS. The boundary condition is the no-slip condition for the velocity components and the Neuman condition for the pressure Poisson equation. Initially, the fluid is at rest and the inner cylinder is suddenly or linearly accelerated to the prescribed Reynolds number. As, for example, pointed out by Burkhalter et al. [22], Benjamin [23] and Mullin et al. [24], the non-uniqueness indicates that there are multiple stable solutions on a fixed condition. The Taylor flow also has multiple solutions. One of them is called a primary mode that is generated as the Reynolds number is increased in a quasi-stationary manner, and it is unique at each aspect ratio. The others are the secondary modes generated due to the history of the increase in the Reynolds number [9,10]. Figure 5 shows the azimuthal-section views of the flows in the normal 4-vortex mode, the anomalous 4-vortex mode in which the flow direction on the end wall(s) is radially outward and the normal 2-vortex modes at a fixed aspect ratio and a fixed Reynolds number. The normal 4-vortex mode is the primary mode, and the anomalous 4-vortex and the normal 2-vortex modes are the secondary modes at Γ = 3.7 and Re = 660. This study focuses on only the instability of Figure 6 shows the process of the embedded analysis. The mode of a stable flow at each aspect ratio depends on the increase histories of the rotation speed and the final value of the Reynolds numbers as clarified by Toya et al. [25]. At first, a preliminary simulation is carried out to confirm the mode formed at each increasing rate of the rotation speed and each final value of the Reynolds number. In the next step, the time series of the axial velocity component near the outer cylinder side in the bottom vortex at each aspect ratio is sampled. Then the time series fluctuation of the axial velocity component is used to draw a three-dimensional attractor. The attractor requires the three-dimensional embedded process, and the embedded process requires a delay time (τ). The delay time can be obtained from the autocorrelation function r given by Eq. (3): where w i is a value of the axial velocity component, w is an average of w i , and N is a sampling number. In this study, N is set at 1000, and k is a time step difference.
One way of determining the delay time is to adopt the time at which the autocorrelation function is 0. The Hopf bifurcation which bifurcates from the steady Taylor flow to the wavy Taylor flow corresponds to the T1 bifurcation in the Ruelle-Takens-Newhouse scenario [21]. In this analysis, under the assumption that the time series variation of the axial velocity component follows the sine wave, the delay time (τ) is defined as a quarter of one period, where the value of the autocorrelation function becomes 0. This is partly because Abarbanel et al. [26] stated that it may not be misleading to use the first zero crossing of the autocorrelation function as a useful delay time. We identify the Taylor flow or the wavy Taylor flows from the orbit where the attractor converges. When the orbit of the attractor converges to a fixed point, it is determined to be a steady Taylor flow, and when it converges on a limit cycle, it is determined to be a wavy Taylor flow. Figure 7a shows typical sectional vector diagram of the Taylor flow at Re = 1350 and Γ = 4.7. Figure 7b shows the first ten thousand variation of the axial velocity component in the time steps TS from 0 to 10 million. Figure 7c shows the autocorrelation function r given by Eq. (3) from the first 0 to 1000 time steps (TS) created by the axial velocity component.
Numerical calculation
The orbit of the attractor is superposed every 1 million TS up to 10 million TS. The orbit of the attractors shown in Figure 8 shows the orbit of the attractor of a 2-vortex Taylor flow numerically obtained from 7 to 10 million TS at Γ = 2.1 and Re = 1660. In this figure, the three axes show the dimensionless velocities (w(t), w(t + τ), w(t + 2τ)) reconstructed by the embedding process. The calculated values for each axis have a large number of digits, therefore they are processed for easy viewing. For example, in Fig. 8, the number 4 indicates 4 in the 9th decimal place of 2.120160724 × 10-2 as the calculated value on each axis. In the figure, the red colored orbit shows an orbit from 7 to 8 million TS, the green color shows an orbit from 8 to 9 million TS, and the blue color shows an orbit from 9 to 10 million TS. As the time step increases, the orbit shrinks towards a fixed point. According to this result, this state represents a steady Taylor flow. Figure 9 shows the orbit of the attractor of a 2-vortex wavy Taylor flow from 7 to 10 million TS at Γ = 2.1 and Re = 1680. The red, green and blue curves show the orbits from 7 to 8 million, from 8 to 9 million and from 9 to 10 million TS respectively. Pfister and Gerdts [27] measured the time dependence and the final amplitude of the lowest-order wavy Taylor flow. They noted that the slowing down of the time constant at the critical point was clearly seen. In our analysis, the attractor may not always show the final convergence to a fixed point or limit cycle with limited TS, however it is able to show a tendency to converge. A huge number of calculations are required to confirm the convergence point. The way to determine the attractor's convergence at up to 10 million TS is to check the orbit of the attractor with a Reynolds number that is 20 less or greater than the expected critical Reynolds numbers. Therefore, we investigate the orbit of the attractor at Re = 1640, which is 20 smaller than Re = 1660 in Fig. 8, and have confirmed that it clearly converged to a fixed point. Similarly, we check the orbit of the attractor at Re = 1700, which is 20 larger than Re = 1680 in Fig. 9, and have confirmed that it clearly converged on the limit cycle. For example, if we can confirm that the attractor at Re = 1640 which is 20 less than the expected critical Reynolds number Re = 1660, converges to a fixed point, we can estimate that the attractor at Re = 1660 converges at the fixed point as shown in Fig. 8. If we can confirm that the attractor at Re = 1700 which is 20 greater than the another expected critical Reynolds number Re = 1680, converges on the limit cycle, we can estimate that the attractor at Re = 1680 converges on the limit cycle as shown in Fig. 9. By this method, the critical Reynolds number Rec at Γ = 2.1 is determined to be 1680. Therefore, the analysis with the maximum number of time step of 10 million TS, provides sufficient accuracy to detect critical values. Figure 10 shows the orbit of the attractor of a 2-vortex Taylor flow from 7 to 10 million TS at Γ = 2.6 and Re = 960. The attractor does not have a circular orbit and continuously takes an irregular orbit in a quite small range of values. The small variation of these values is estimated as a numerical variation of computer. The size of the orbit indicates quite small like the size that converges at a fixed point. From this result, this state is a Taylor flow. Figure 11 shows the orbit of the attractor of a 2-vortex wavy Taylor flow from 7 to 10 million TS at Γ = 2.6 and Re = 980. Since the attractor converges on a limit cycle, this state is a wavy Taylor flow. From these results of Figs. 10 and 11, and Re = 1340 shows a limit cycle. Therefore the critical Reynolds number Rec is determined to be 1340. The attractor of 6-vortex mode at Γ = 5.9 and Re = 840 gradually shrinked into a fixed point and the attractor at Γ = 5.9 and Re = 860 converged on a limit cycle. From these results, the critical Reynolds number Rec at Γ = 5.9 is determined to be 860. Figure 12 shows the critical Reynolds number in each mode of the Taylor flow obtained by the present numerical analysis and that by the previous experiment by Nakamura et al. [10]. The experimental apparatus has the rotating inner cylinder and the stationary outer cylinder whose aspect ratio is from 1.0 to 7.2 and the radius ratio is 0.667. The numerical analysis and the experiment have the same geometrical condition. The critical value measured in the experiment was determined by the Reynolds number at which the boundary between the vortices began to oscillate. The oscillation of the boundary was observed by a microscope with its accuracy of 0.01 mm. The open symbols denote the numerical results, and the filled symbols represent the experimental results. The circle, square and triangle symbols indicate the 2-vortex, the 4-vortex and the 6-vortex modes, respectively. In the comparison between the numerical and the experimental results, they show an agreement in the smaller aspect ratio range than the aspect ratio at the each peak value of a critical Reynolds number. In the range of the larger aspect ratio than that at the peak value, however, the critical Reynolds number of the analytical result is larger than that of the experimental result. For the experimental values, the Reynolds number at which one of vortices started to oscillate in the Taylor flow was adopted as the critical value. The calculated critical Reynolds number was obtained by the embedding method for the axial velocity component of the vortex at the bottom of the Taylor flow. The reason why the analysis value and the experimental value match on the left side of the peak value of the critical Reynolds number is as follows. On the left side of the peak value, the boundary of vortices near the end walls firstly begins to oscillate, whose axial velocity component is sampled from the numerical results. However, on the right side of the peak value, the first fluctuation of the boundary between vortices firstly observed by the microscope tends to appear around the center of the flow region. While, in the numerical analysis, the determination is performed by the fluctuation of the bottom vortex in all range of the aspect ratio. Therefore difference of the measuring methods may make the higher numerical critical value at downward branches. Regarding this point, it is necessary to confirm by matching the judgment criteria. This will be considered in the future.
The bifurcation of the critical Reynolds number between the steady and wavy Taylor flows
The authors have performed their numerical analysis by using the amplitude of the variation of the kinetic energy of the vortex of the Taylor flow to estimate the Reynolds number of the Hopf bifurcation [15,16]. However, the accuracy of the method was insufficient when the kinetic energy was very small and it is near the critical value and the comparison between the numerical and experimental results was inadequate. Comparing Figs. 4 and 12, it can be said that the results of the new method in Fig. 12 are in good agreement with the results of experimental observations. Especially, the result at smaller aspect ratio gives better agreement. The values of the former method are more variation than the values obtained by the present new method. This study demonstrates that the numerical method using an attractor visualized in three-dimensional phase space gives more accurate results.
Conclusions
The instability of the Taylor flow between two cylinders with both fixed end walls with a small aspect ratio is clarified by a three-dimensional embedded analysis in a chaotic theory. The critical Reynolds number at which the Taylor flow with 2-vortex, the 4-vortex and the 6-vortex bifurcates to the wavy Taylor flow is determined by means of the attractor structure. The attractors are obtained in three-dimensional phase space by embedding the time series of the axial velocity component in the lowest vortex of the flow. The critical Reynolds number, which corresponds to the Hopf bifurcation point where the attractor structure changes from a fixed point to a limit cycle is estimated. The results of the numerical analysis are good agreement with the results of the experiment conducted under the same conditions in the smaller aspect ratio range than the aspect ratio at the each peak value of a critical Reynolds number. In the larger aspect ratio range than that at the each peak value, however, the analytical results are larger than the experimental results. It is presumed the difference of the measured target boundary of vortices to explain why the analytical results differ from the experimental results in the larger aspect ratio range than the aspect ratio at the peak value of the critical Reynolds number. The numerical analysis uses the numerical result of the bottom vortex to determine the critical value, while the experimental method used the boundary of vortices, which firstly began to oscillate, and identified the critical Reynolds number. Regarding this point, it may be helpful to match the criteria to confirm the critical value.
This study shows that the instability of the Taylor flow is revealed by determining the structure of the attractor applied by the Hopf bifurcation. In addition, it is shown that a numerical approach using attractors can obtain more accurate results than those obtained by a numerical evaluation method using the amplitude fluctuation of the kinetic energy of the Taylor flow. Furthermore this study could be extended to the transition to turbulence which is much less well clarification for the instability of the Taylor flow by determining the structure of the attractor applied by the Hopf bifurcation. | 2022-05-15T15:08:00.385Z | 2022-05-13T00:00:00.000 | {
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58038660 | pes2o/s2orc | v3-fos-license | Closed loop Fuzzy Logic Controlled Interleaved DC-to-DC converter Fed DC Drive System
This paper deals with comparison of responses of PI and Fuzzy Logic controlled DC-to-DC converter Fed DC motor (FLCDDCDCM) systems. The DC input is converted into high frequency AC using full bridge inverter. The output is stepped up using a transformer and then it is rectified using voltage doubler rectifier. The open loop system with T filter at the output is simulated. The closed loop PI & FLC based DDCDCM systems are designed, modeled and simulated using Matlab/Simulink. The time domain parameters of FL controlled system are compared with those of PI controlled system. The results indicated that FL controlled DDCDCM system has better response than PI controlled DDCDCM system.
Introduction
SOLAR energy fluctuates during the day and vanishes at night. Therefore, it cannot be considered as a steady energy source for the key load or the grid [1], [2]. However, a photovoltaic (PV)battery hybrid energy system can overcome the intermittent nature of solar energy and provide reliable power. This calls for two DC/DC converters or a three-port converter to interface the PV array, the battery, and the load. The conventional PV-battery hybrid system requires two individual converters. One converter is used to achieve the PV energy conversion and the other one is employed to charge or discharge the battery. This complex configuration contains many component numbers, increases the system volume and cost. Actually, the two individual converters can be replaced by a three-port converter to improve the power density. A parallel structure of several individual bidirectional buck-boost converters is combined for multiport DC/DC conversion systems in [3] and [4]. However, the power devices cannot be shared by different individual converters. By employing the time-sharing control strategy, a multiple-input non isolated buck-boost converter and its isolated counterparts with unidirectional power flow are proposed in [5] and [6]. Furthermore, some improved multiple-input topologies with multidirectional energy conversion are introduced in [7]. These converters can be easily extended to any number of input ports. Unfortunately, the power devices and the magnetic components have to sustain the peak voltage and current stresses. And the output energy of each port is coupled and difficult to manage due to the times sharing control scheme. By integrating the half-bridge converter and the active-clamp forward converter, a tri modal half-bridge converter for a three-port interface is presented in [8] and [9]. The component numbers and the power losses can be saved for the power-harvesting systems.
However, the control variables of two duty cycles are interactional, which increases the control complexity. This concept can also be extended to four-port even higher port converters [10]. In addition, general rules are carried out to derive non isolated and isolated multiple-input converters from the single-input versions in [11]- [13], which are adopted to identify the feasible input cell that complies with some assumptions and conditions. The common feature of the aforementioned multiport converters is that the energy management or the control scheme is part coupled or inter restrictive. Another universal solution to generate multiport converters is to combine the dc-link configuration and adopt the magnetic coupling solution [14]- [19]. Half-bridge structure, full-bridge structure, and their integration can be employed to satisfy some stringent requirements. The clear advantages of the fully coupled multiport converters are zero-voltage switching (ZVS) operation, easy energy management, and flexible configuration. In order to improve the device sharing ratio among different ports, realize soft-switching operation, and achieve decoupled control within a certain operating range, a novel PWM plus phase angle shift (PPAS) control scheme is proposed. In this Phase-shift control scheme or pulse width modulation (PWM) plus phase shift control strategy is usually used in these cases. Furthermore, series resonance control scheme can be also employed [20]. However, the variable frequency operation increases the Electro Magnetic Interference (EMI) filter design difficulty. MLI analyses for various system [28][29][30][31][32][33][34][35][36][37]. The main limitation of the fully coupled multiport converters is that a lot of power devices are required because each port cannot share the same power switches. In order to improve the device sharing ratio among different ports, realize soft-switching operation, and achieve decoupled control within a certain operating range, a novel PWM plus phase angle shift (PPAS) control scheme is used in this paper.
Circuit Description
The full bridge converter are integrated to generate a combined three port converter for the PV-PV hybrid energy system, which is used as an example to show the operation
Simulation Results
Simulation is done for open loop and closed loop systems with PI & FL controllers. The simulation results are presented in this section.
(i) Results of open loop DDCDCM system
Circuit diagram for DC-DC converter system is shown in Fig 4. The output of DDC is applied to the DC motor. The output voltage of solar system is shown in Fig 5 and its value is 12.5 V. The primary Voltage of transformer is shown in Fig. 7 and its peak value is 15V. The output voltage is shown in Fig. 8 and its value is 60 V. The motor speed is shown in Fig. 9 and its value is 400 rpm. The Torque response is shown in Fig. 10 and its value is 2 Nm. The output power is shown in Fig. 11 and its value is 70 W. The summary of output voltage ripple, power and speed for different value of load torque are shown in Table 1. The speed and ripple decrease with the increase in load torque.
(ii). Closed loop DDCDCM with PI Controller
Closed loop system with PI controller is shown in Fig. 12. Actual speed of DC motor is compared with the reference speed and the error is applied to the PI controller. The output of PI is compared with the saw tooth to produce updated pulses for the rectifier. The input voltage is shown in Fig. 13 and its value increases from 12 to 17 V. The output voltage is shown in Fig. 14 and its value is 60 V. The motor speed is shown in Fig. 15 and its value is 382 rpm. It can be seen that the speed is almost equal to the set value. The Torque response is shown in Fig. 16 and its value is 2 Nm. The output power is shown in Fig. 17 and its value is 80 W. Fig. 18. The PI controller is now replaced by FLC. The output voltage is shown in Fig. 19 and its value is 60 V. The motor speed is shown in Fig. 20 and its value is 400 rpm. The Torque response is shown in Fig. 21 and its value is 2 Nm. The output power is shown in Fig. 22
Experimental Results
The hardware for prototype DDCDCM is fabricated and tested. The hardaware Snap shot is shown in Fig. 23. The hareware consists of PV panel, battery, control board, converter board and DC motor. The output voltage of solar system is shown in Fig. 24. Switching pulses for M1 & M3 are shown in Fig. 25. Primary voltage across transformer is shown in Fig. 26 and secondary voltage is shown in Fig. 27. DC output voltage of DDC is shown in Fig. 28. | 2019-01-23T22:16:45.269Z | 2018-04-25T00:00:00.000 | {
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20818946 | pes2o/s2orc | v3-fos-license | Effect of increase in duration of aprepitant consumption from 3 to 6 days on the prevention of nausea and vomiting in women receiving combination of anthracycline/cyclophosphamide chemotherapy: A randomized, crossover, clinical trial
Background: Aprepitant is one of the effective antiemetic drugs that usually used for a period of 3 days for prevention of anthracycline/cyclophosphamide (AC) induced nausea and vomiting. However, many patients still experience nausea and vomiting on days 3–5. The aim of this study was to evaluate the effect of an increase in duration of aprepitant consumption from 3 to 6 days on the prevention of nausea and vomiting in women receiving AC chemotherapy. Materials and Methods: It was a randomized, crossover, controlled clinical trial. Women with breast cancer and scheduled to receive AC regimens were enrolled in this study. Enrolled patients were randomized into two groups. Group I received 3 days regimen of aprepitant in the first course of AC regimen chemotherapy and 6 days regimen of aprepitant in the second course; Group II received 6 days regimen followed by 3 days regimen. For nausea and vomiting assessment, we used Eastern Cooperative Oncology Group questionnaire. Results: Forty-nine patients were enrolled in this study. Sixty-three percent achieved a complete response with 6 days aprepitant regimen compared with 39% with 3 days regimen (P < 0.001). Ten percent had at least one vomiting episode during the 6 days regimen versus 15% with 3 days regimen (P = 0.034). Nausea was significantly more severe in 3 days regimen of aprepitant than in 6 days regimen. Conclusion: Increase in the duration of aprepitant consumption through 6 days resulted in significantly better prevention of nausea and vomiting than 3 days consumption for women receiving AC chemotherapy.
INTRODUCTION
Chemotherapy-induced nausea and vomiting (CINV) can be one of the most distressing problems for patients. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com
Advanced Biomedical Research | 2015 chemotherapy. [1,2] The mechanisms of CINV seems to be depend on the cellular injury induced by chemotherapy, which may release neurotransmitters. [3] Dopamine and serotonin (5-hydroxytryptamine ) are the major excitatory neurotransmitters that are involved in emesis. [3] Several patient-related risk factors for CINV are generally assumed, including gender, age, alcohol use, and history of motion sickness. [4] Patients <50 years old and women are more likely to suffer from CINV. [5] Although, chemotherapy agents are vary in degrees of emetogenic potential and combinations of emetogenic agents may have additive effects on the overall CINV. [6] The combination of anthracycline/cyclophosphamide (AC) chemotherapy is a particularly high risk for inducing nausea and vomiting and considered as being highly emetogenic. [7,8] Strategies for antiemetic prophylaxis have developed in recent years. [9,10] Some antiemetic guidelines for patients receiving highly emetogenic chemotherapy (HEC) recommend the use of the oral neurokinin-1 (NK1) receptor antagonists as part of a routine regimen that also includes a corticosteroid and a selective 5-HT3 receptor antagonist. [11][12][13] The incidence of CINV in patients receiving HEC, including breast cancer patients treated with 5-HT3-receptor antagonists is approximately 50%. [14] Aprepitant is the first commercially available drug of a new class of NK1 receptor antagonists with little or no affinity for other NK receptors. [15,16] Approved by Food and Drug Administration in 2003 for the prevention of chemotherapy-induced emesis and usually administered for a period of 3 days. [17] Recent studies reveal the efficacy of aprepitant in preventing CINV in patients who received AC and nonAC-based HEC regimen. [18][19][20] However, despite these results, many cancer patients still experience CINV; [18,21] also the trend has now been reversed with increasing nausea and vomiting on days 3-5. Hence, there is clearly a need for more effective prevention of CINV in patients receiving HEC on delay phase, especially in a patient, who is particularly susceptible to these symptoms such as women.
Here, we present the study of aprepitant duration for the management of nausea and vomiting associated with AC-containing chemotherapy in women with breast cancer. We hypothesized that addition of aprepitant duration from 3 to 6 days would improve emetic symptoms in women receiving AC -based chemotherapy for breast cancer.
Study design
This study was a randomized, crossover, controlled clinical trial (IRCT2015040121574N1), and designed to evaluate the effect of increase in duration of aprepitant usage from 3 days to 6 days on the prevention of nausea and vomiting in women receiving combination of AC chemotherapy. This trial was conducted in referral university hospital in Isfahan (Iran's third largest city, located in the center of Iran), Iran. The Medical Ethics Committee of Isfahan University of Medical Sciences has approved the study design, protocols, and informed consent procedure (the ethical code was 393449).
Participants
Fifty patients with breast cancer were enrolled in this study through convenience sampling method. We included patients who were under 50 years of age, diagnosed with breast cancer and scheduled to receive four courses AC regimens. The following general exclusion criteria were considered: Previous history of gastritis, diabetes, and a brain tumor. In order to detect two-fifth standard deviation difference in the main outcome (nausea severity score), with α =0.05 and power = 80%. We considered 10% attrition rate and the final sample size was estimated 50 patients (25/group). Patients served as their own controls for study cycles.
Intervention
Enrolled patients were randomized to 1 of 2 groups. According to the crossover design of the study, Group I received Treatment A (3 days regimen) in the first course of 1-day AC regimen chemotherapy and Treatment B (6 days regimen) in the second course of 1-day AC regimen chemotherapy; Group II received Treatment B followed by Treatment A. Treatment A (3 days regimen) consisted of aprepitant (ABITANT, Abidi, Iran) 125 mg orally plus dexamethasone 8 mg injected intravenous (IV) on day 1 followed by 80 mg dexamethasone once per day on days 2 and 3 administered orally (p.o.). Treatment B (6 days regimen) consisted of aprepitant 125 mg orally plus dexamethasone 8 mg injected IV on day 1 followed by 80 mg dexamethasone once per day on days 2 through 6 administered orally (p.o.). There was a 30-day washout between the first and second courses.
Measurement
Patients were followed from the 1 st day of chemotherapy for a total of 7 days.
For nausea and vomiting assessment, we used Eastern Cooperative Oncology Group common toxicity criteria questionnaire, asking patients to answer two specific questions about any nausea and vomiting symptoms. First question was about nausea severity on a 0-3 scale (0 = being no nausea, 1 = able to eat reasonable intake, 2 = intake significantly decreased but can eat, 3 = no significant intake) and second question was about vomiting (0 = none, 1 = 1 episode in 24 h, 2 = 2-5 episodes in 24 h, 3 = 6-10 episodes in 24 h, and 4 = >10 episodes in 24 h, or requiring parenteral support). Complete response (CR) was defined as no nausea and no episodes of vomiting during the study period.
All patients were given this questionnaire on cycle 1 and cycle 2, at baseline (prior to each chemotherapy treatment course), and after 7 days. Only patients with no symptom of nausea and vomiting (score of zero) at baseline (before chemotherapy) were eligible for entering to each cycle of this study.
Statistical analyses
All statistical analysis was performed by using SPSS version 20 (Release 2011, SPSS Inc., Chicago, IL, USA) for windows. Findings had shown as relative frequencies, mean and standard deviation. The comparisons were performed by McNemar's tests, Wilcoxon signed-rank tests, paired Student's t-test. A mixed general linear model was used to adjust for age and order of interventions (Treatment B followed by Treatment A or Treatment A followed by Treatment B) for all comparisons. All tests were two-sided, and P < 0.05 is considered significant. The statistical approach was based on an intention to treat.
RESULTS
A total of 50 patients were enrolled in the study. One patient declined to continue participating in the study on his decision with no reason. A consort diagram illustrates patient flow through each cycle of the study [ Figure 1].
All of the participants were women with breast cancer and were free from nausea and vomiting before entering to each cycle. The mean age of the 49 subjects analyzed was 38.7 ± 6.5 years.
Thirty-one (63%) of 49 patients achieved a (CR was defined as no nausea and no episodes of vomiting) with 6 days regimen of aprepitant compared with 19 (39%) on 3 days regimen of aprepitant (P < 0.001) [ Table 1]. Overall, 19 (38.6%) patients achieved a CR with both of two regimens, 12 (24.4%) experienced CR with 6 days regimen but not attended CR when they cross over to the 3 days regimen and 18 (37%) not achieved CR with both of two regimens [ Table 1].
As present in Figure 2, 90% in the 6 days regimen of aprepitant versus 85% in the 3 days regimen of aprepitant experienced no vomiting episode. In the 6 days regimen, one vomiting episode was experienced for 10.2% of the patient compared with 6.1% in the 3 days regimen. No one experienced more than 1 episode of vomiting with 6 days regimen, but 8.2% of patient experienced 2-5 vomiting episodes with 3 days regimen. The number of vomiting episodes was significantly lower during the 6 days regimen than during the 3 days (P = 0.034) [ Figure 2]. No one experienced more than 5 episode of vomiting during the study.
The proportion of patient that remained free from nausea in 6 days regimen was 63% compare with 39% in 3 days regimen (P < 0.001). Nausea was significantly more severe in 3 days regimen of aprepitant than in 6 days regimen of aprepitant [ Figure 3].
The order of treatment regimens (consumption of each regimen in the first cycle or second cycle) was not affected the results of study.
Both treatment regimens were tolerable for the patient, and no one was complaining of side effects.
DISCUSSION
CINV is strong and dreaded the side effect of chemotherapy that can limit the efficacy of cancer treatments and has a potent negative effect on patient quality of life.
Antiemetic therapy should aim to overcome this problem in all cancer patients receiving chemotherapy. Substantial development has been made in improving the control of CINV, largely because of the introduction of antiemetic agents. However, this trend has now been reversed with increasing nausea and vomiting on days 3-5.
This crossover clinical trial in women with breast cancer addressed the potential efficacy of increase in duration of aprepitant consumption from 3 to 6 days for the prevention of nausea and vomiting induced by the combination of AC chemotherapy. The advantage of this crossover clinical trial design is less need for samples, the similarity of a participant in two intervention groups and the least Bayes.
In the current setting, we found that consumption of aprepitant for 6 days was superior to 3 days consumption in the proportion of patients achieving a CR overall after 1-day AC regimen chemotherapy (63% vs. 39%). As seen in previous trials CINV is well controlled on days 1 and 2 with aprepitant regimen. However, loss of control on days 6 through 8 in the delayed phase remains a challenge, therefore adding aprepitant to the standard antiemetic prophylaxis for 6 days provides a significant improvement in complete control for CINV from 43% to 63%. [22][23][24] Madsen et al. report that a 5-day dosing regimen of aprepitant is highly effective for preventing CINV, although, single doses of oral aprepitant 40 mg or oral aprepitant 125 mg alone were effective for the prevention of postoperative nausea and vomiting. [25] However, other studies demonstrate 60-80% CR to 3 days administration of aprepitant-containing regimen. [19,20] Badar et al. represent that more than75% of patients were free from nausea on day 1 and day 2 after use of aprepitant, but This fraction decreased from day 3 to day 7. [19] This discrepancy may be due to differences in the underlying disease, the patient characteristics, and the chemotherapy regimens the patients received; thus judgments should be made with caution.
We have shown in this study that the number of vomiting episodes is significantly lower during the 6 days aprepitant regimen than during the 3 days regimen. In agreement with our finding, other studies show a significant effect of aprepitant in the episodes of vomiting reduction, in patients treated with HEC regimens. [26,27] The main limitation of our study is a lack of placebo group to identify the placebo effect. We cannot allocate placebo group because not to treat nausea and vomiting in a patient who received chemotherapy is unethical.
The present study provides an experimental evidence of increase duration of aprepitant consumption can decline nausea severity. A patient who cannot significant intake due to severe nausea is 12% with 3 days regimen, versus 2% with 6 days regimen. A similar trend was seen in the previous studies where a patient who received 5 days of aprepitant had less nausea on day 6 and day 7. [28,29] Hence, we can provide superior prevention of CINV in women with breast cancer only with increasing in the duration of aprepitant consumption.
CONCLUSION
The increase in duration of aprepitant consumption through 6 days resulted in significantly better prevention of CINV than 3 days consumption and provides adequate antiemetic therapy for a patient receiving AC chemotherapy.
Financial support and sponsorship
Isfahan University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest. | 2018-04-03T05:01:40.844Z | 2015-10-29T00:00:00.000 | {
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122741116 | pes2o/s2orc | v3-fos-license | Interference in Wireless Networks : Causes , Analyses and Practical Mitigating Techniques
This paper addresses the key characteristics of interference in wireless networks. Moreover, it considers the impact of interference from Code Division Multiple Access base station to Wideband-Code Division Multiple Access one. A detailed discussion of major interference mitigation techniques in wireless networks, in general, and CDMA and W-CDMA systems, in particular is also imparted on. The techniques and findings in this paper can be easily used in the analyses of other wireless technologies.
Introduction
Coexistence of wireless systems in various frequency bands has become a commonplace (Shamsan et al. 2012).In a multi-operator situation and in an area where several network operators are striving to furnish mobile services, placing a cell site without interference is often a hurdle faced by operators (Roke Manor Research Ltd 2008).It is vital to realize the risk of interference between systems when planning in such an environment (Nguyen & Zaghloul, 2007).
In circumstances where several mobile networks of different air interfaces operate in a specific province in a combination of frequency bands (i.e. 900, 1800(i.e. 900, , 1900(i.e. 900, and 2100 MHz) MHz), the probability of developing interference is inevitable (Oudah, 2013).This end-result is blended in territories where there is no site or districting standard and when there are many operators for each of the reserved frequency bands (Li & Tatesh, 2009).In some regions, identical spectrum portions have been licensed to various operators in contiguous geographical domains.Indeed, it is a great obstacle to fight interference when the two systems operate in the same frequency band in neighboring service regions with non-collocated sites.In nations where siting constraints are widespread, particularly in urban and suburban areas, an operator is further restricted as controls are also put on the position of cell sites together with the corresponding antenna masts (Ball et al. 2008).
In occasions where the same frequency band is employed by several operators, interference may still be reduced while respecting siting stipulations, by sharing towers, antennas, etc.; however, additional obstacle is to create a tower that can fit a number of antennas while retaining the overall interference to a lowest (Kazemitabar, 2010).The installation of antennas at distinctive heights (vertical separation) for different frequency bands in disparate clutter types is an additional key consideration that can mitigate interference levels (ITU-R M.2039-2 2010).
The objective of this paper is to describe key features of interference in emerging cellular systems.Furthermore, the interference between Code Division Multiple Access (CDMA) and Wide-CDMA (WCDMA) technologies is analyzed and potential mitigation techniques are also discussed.To serve its purpose, this paper is organized in the following order: section two is an in-depth discussion of major interference causes and measures.Section three embarks on the analysis of interference between two coexisted systems, i.e.CDMA and W-CDMA, where potential interference issues are analyzed and discussed along with possible mitigation techniques.Section four concludes the findings.
Interference Related Factors
Separation between networks is interpreted as the attenuation between the transmitter reference point in the interfering unit, namely Base Station (BS) or Mobile Station (MS) and the receiver reference point (MS or BS).As shown in Fig. 1, it is likely that one BS could potentially disrupt another BS.The separation between the reference points in this case is given below: Figure 1.Transmission metrics between two adjacent systems For gaps more than 10 m, the path loss can be bordered to a free space pathloss model based on the ambience.For short antenna splits, the amount of the separation, i.e., (propagation loss -the combined transmitted and receiver antenna gains) may be approximated by evaluating coupling loss of the typical antennas.For extended separation, particular band pass filters are required.
Employing extra filters in transmit as well as receive paths, one can alleviate or at least lessen the impact of the interference that emerges owing to the coexistence of several wireless networks.The needed filter operation prerequisite can differ hugely relying on the operation frequency, bandwidth, attenuation condition and expense.
Other important elements in interference analysis are the so-called spurious emissions.These mostly give rise to all undesired transmissions from a transmitter.The emissions may incorporate inter-modulation (IM) products stimulated by various frequencies used internally in the Base Station transmitter, the inter-modulation induced by extrinsic carrier frequencies, the harmonics (peaks at double the carrier frequency) and the noise.All of these can result in a co-channel or adjacent channel interference to a neighboring victim receiver.
Interestingly, spurious emissions are sporadic transmissions outside the carrier frequency, but transmitter noise is the weakest level of successive wide-band emission.Transmitter noise cannot be reduced with radio frequency (RF) planning solely, as it is interrelated to the Noise Figure (NF) of the transmit link.This wide-band RF noise is also thought of as sideband noise.The majority of this noise is formed in the exciter section and magnified in following stages and ended at the transmitter output phases.Some transmitters are worse than others in forming this noise.
Furthermore, Inter-modulation (IM) products are established both in transmit and receive links, as two or more frequencies are combined and amplified in non-linear equipment.IM products of order 'n' are the sums and differences in 'n' terms of the primary frequencies.Typically, the greater the order of the IM product, the lesser is its strength.Furthermore, if one of the terms of the product is lower than the rest, it follows that the consequent IM product energy will also decline significantly.The imperfections of the equipment give rise to those unwanted frequencies that will result in a co-channel interference at the victim receiver.Normally, there are two major types of IM products: transmitted and receiver-generated IM(s).On one hand, the transmitter IM products are generated in the BS by mixing of carriers in the same power amplifier, connectors, duplex filter, combiner or the antenna.Transmitter IM can cause co-channel interference, which needs high separation between cells or additional filters to be used to in the intruding cell.Appending filters turns out to be challenging if the intruder cell belongs to a different operator.On the other hand, two or more electromagnetic waves can give rise to inter-modulation in the victim receiver featuring some imperfections.These inter-modulation waves generate interference if they are in the range of the receiver's bandwidth as they are received and amplified in a way similar to that of the wanted transmission.Accordingly, inter-modulation measures are essential in order to diagnose those surrounding transmitters that may impair the operation of the receiver owing to inter-modulation impact.Receiver (Rx) IM is created in the receiver amplifiers.The primary disparity between Tx IM and Rx IM with respect to co-existence is that the amount of co-channel interference from Tx IM is more conceivable than interference from Rx IM.
Remarkably, the intensity of Rx IM3 and IM5 rises 3 and 5 times as fast (in dB) as the received carriers creating this IM.Given that the powers of Rx IM products step up quickly with the power of the strong received (undesired) carriers, Rx IM is often affiliated with a Rx IM rejection level.Carriers higher than the IM rejection level can lead to serious interference while carriers lower than the level are tolerable or harmless.Both IM3 and IM5 ought to be thought about as receiver IM interference.Usually, the IM behavior of a receiver is reported in terms of inter-modulation rejection ratio and is associated with the 3rd Inter-mod intercept point (IIP3) of the receiver interface.For instance, the Pin IM (intermodulation power in dBm) equivalents to 3×Pi -2×IIP3 where Pi is the equal power in the two overlapping signals in dBm and IIP3 is the 3rd order Inter-mod intercept point of the receiver in dBm.An additional filter in front of the influenced receiver stage can solve the dilemma.
Normally, receivers are devised to acknowledge particular forms of electromagnetic waves within the bounds of a fixed frequency band.Nevertheless, receivers additionally respond to unwanted signals featuring different modulation and frequency peculiarities.Occasionally, the interfering signals fall into one of the following fundamental types: • Co-Channel Interference (CCI): This is due to emissions featuring frequencies that present within the bounds of the narrowest pass-band of the receiver.
•
Adjacent Channel Interference (ACI): This is owing to undesired signals possessing frequency parts that exist within or nearby the receiver pass-band.Generally, adjacent channel interference is the interference between two wireless networks whose frequency carrier assignments are adjacent or contiguous, particularly in cases where their cell towers are not collocated.
•
Out-of-band Interference (OOBI): This occurs when signals having frequency parts, that are outside of the receiver pass-band.If this interference overtakes the noise floor of the victim receiver, it weakens the noise figure of the receiver.In order to combat this type of impairment, either the out-of-band signals must be to be decreased (by inserting filters to the interfering transmitter) or the desired signal level at the victim receiver be improved.
The receiver selectivity serves a significant purpose in attenuating adjacent channel interference.Furthermore, the intermediate frequency (IF) selectivity is the ability of a receiver to filter out any adjacent channels interference.The combination of the transmitter emission mask, the RF and IF selectivities together decide the frequency separation requirements.
Usually, receivers are subject to enormous out-of-band signals that give rise to a spurious response in the receiver.A spurious response can be initiated if the frequency of an intruding signal is such that the signal or one of its harmonics can merge with a local oscillator or one of its harmonics to output an IF in the receiver IF pass-band.The main crucial frequency in this respect is the image frequency of the receiver.In order to hold positive spurious response rejection, the sensitivity threshold of the image frequency should be considered.However, this causes detailed image frequency/spurious response qualities of the receiver if interference issues owing to image frequency are found.
A high interference level inside the RF bandwidth of a receiver can lead to interference even if the emission is outside the pass-band of the IF amplifiers resulting in a decrease of gain for the wanted signal owing to imperfections in the receiver front end.Such end-result gives rise to decreased Signal-to-Noise ratio (S/N) of the receiver, if a specific saturation reference power threshold is exceeded.This occurrence is ordinarily named blocking or desensitization.For this goal, the interference level at the front end of the receiver requires to be calculated over the whole RF bandwidth of the receiver.Namely, the receiver blocking or desensitization level is the highest level of a non-cochannel or adjacent channel interference that may be experienced as a low input signal (about three dB above the sensitivity level) without lowering the receiver operation.
Typically, noise, spurious emissions and transmitter IM products outside the allocated frequency block are inherent in any transmitter.These emissions give rise to noise and can stimulate interference to a neighboring receiver that is set to a weak signal from a transmitter (BS or MS) which is far away.As a rule, closer the interfering channel to the wanted channel, more responsive the receiver is to the interference.The interference is a combination of the adjacent channel interference due to deficient suppression in the receiver band-pass filter and the co-channel interference from the wide-band noise of the closer interfering transmitter.Fig. 2 depicts some of the elements explained above.
Figure 2. Key unwanted emissions in wireless systems
Interference from CDMA to W-CDMA Networks
In several countries in Asia, there has always been an argument as to whether Personal Communications Service (PCS) band (1850( -1910( /1930( -1990 MHz) MHz) allocations can coexist (fully or partially) with 2.1 GHz band allocations, also referred to as the UMTS band (1920( -1980( /2100( -2170 MHz) MHz) and if so, what are the acceptable degrees of interference and the needed guard bands between these blocks.There is (60 + 60) MHz of spectrum openness if allocated either for PCS or for UMTS solely.See Fig. 3 for another potential sharing of the spectrum between PCS and 2.1 GHz band allocations (ITU-R M.2116-1 2010).
Figure 3. Potential spectrum sharing scenario between PCS and UMTS bands
In this band sharing plan, if the operators with PCS band allocation are the holders, then the matter will be to protect the holders while assigning more spectrums in the 2.1 GHz band.In some other situations, the conflict has been to rule out PCS allocations to protect forthcoming IMT-2000 core band allocations.In Fig. 3, it is depicted that the border between PCS and UMTS band allocations is 1960 MHz.With 5 MHz protection band (from 1955 to 1960 MHz) at the 1960 MHz margin, one gets (30+30) MHz for PCS and (35+35) MHz for UMTS operations.It is viable to move the border to either side of the spectrum (anywhere, from 1920 to 1980 MHz) to earmark various bandwidth sharing between PCS and UMTS operations.
The next section assesses potential coexistence issues between CDMA2000 network in the PCS band and WCDMA network in the 2.1 GHz band by scrutinizing the smallest guard band necessary between the two networks.
Formulation, Results and Discussion
In this subsection, the interference from CDMA2000 baste station transmitter to the UMTS Node-B receiver is considered.The Thermal Noise Floor at the UMTS Node-B receiver is found as follows: Where TN is the thermal noise of UMTS receiver, K is Boltzman's constant= 1.380 10 T is receiver's temperature and NF Rx is UMTS receiver noise floor.For receiver noise figure of 5 dB, TN= -103dBm /3.84 MHz.
As per 3GPP specification (3GPP TS 25.942), a maximum tolerable level of interference (I RX ) from the intruder base station (Node-B) to the victim Node-B receiver is given as -110 dBm/3.84MHz (3GPP TS 25.942).With TN value of -103 dBm/3.84MHz for WCDMA Node-B receivers, a spurious signal at the maximum tolerable limit of -110 dBm/3.84MHz would be 7 dB below the TN value and its presence would cause a receiver sensitivity degradation of 0.8 dB, which is calculated as per the formula shown below (Oudah et al. 2012): Where S is the degradation in receiver sensitivity, I Rx is maximum tolerable level of interference and TN is thermal noise.Similarly, 3GPP specifies a blocking requirement for WCDMA Node-B at a received spurious signal level of -115 dBm/3.84MHz [10].The processing gain (PG) of the WCDMA signal is 25 dB (i.e., 10×log (3840000/12200)) and for a target Eb/It of 5 dB, the Node-B receiver reference sensitivity would be of the order of -123 dBm/3.84MHz (i.e., TN + Eb/It -PG = -103 + 5 -25 = -123 dBm/3.84MHz).In PCS band, the antenna gains are in the order of 15 dBi and the feeder cable losses would be around 3 dB.Hence, the equivalent isotropically radiated power (EIRP) of CDMA BS Tx would be around 55 dBm .
Assuming that the interfering CDMA2000 BS in PCS band has a vendor specific spurious emission limit of -75 dBc/30 kHz (20 dB better than the standards specified spurious emission limits of -55 dBC/30 kHz) (3GPP TS 25.942) for a carrier-to-carrier separation greater than 1.98 MHz, the amount of required isolation I isolation (dB) achieved through spatial separation of antennas to mitigate the interference due to the out-of-band spurious emissions can be found as follows: Therefore, with an extra filter of realistic complexity and cost in the CDMA BS Tx path with 60 dB attenuation, the required isolation due to physical separation of antennas "I isolation (dB)" = 118 -60 = 58 dB, which can very easily be realized by having about 40 meters of physical separation between CDMA2000 BS Tx and WCDMA Node-B Rx antennas.With some vertical separation of antennas, one can use lesser values for antenna gains and hence the burden on the required isolation further reduces.
As per 3GPP specification (3GPP TS 25.104), there is a 5 dB lower (more stringent) constraint on WCDMA Node-B tolerable blocking level compared to the maximum tolerable level for out-of-band Interference (-115 dBm versus -110 dBm), which translates directly into an extra 5 dB isolation requirement for blocking due to Intermodulation.Therefore, the isolation requirement with physical separation of antennas to mitigate blocking would be around 65 dB.Hence, blocking would be the main issue for WCDMA Node-B.
It is worth mentioning that there are no clear requirements in band class 6 (IMT-2000) to protect WCDMA Node-B receiver in a co-area operation scenario from the spurious emissions caused by a CDMA2000 BS of another operator.But, most of the vendors would supply CDMA2000 BSs with spurious emissions that are most likely be considerably lower than the standards defined values, because a CDMA2000 BS needs to protect its receiver from its own transmitter.
As with CDMA2000 BS system, one can assume that the blocking performance of WCDMA Node-B receiver would most likely be expressively better than the standards stated value, because WCDMA Node-B system also has to protect its receiver from its own transmitter.
Using specified performance values for BS transmitters and receivers to establish isolation requirements also leads to overly pessimistic results, since they are not describing the real equipment performance.Accordingly, around 65 dB of isolation requirement is sufficient to meet the BS to Node-B spurious emission as well as intermodulation interference conditions, and it is quite possible to achieve such isolation requirement through physical separation of antennas with good antenna installation practices.65 dB of antenna isolation is achievable with a site-to-site spacing of around 60 meters.
Conclusions
In this paper, deployment analysis of wireless systems has been discussed in details.The consequences of spurious emissions between two collocated systems and other related side-effects have also been discussed.It has been shown that analyses which looked into the coexistence of collocated CDMA2000 with WCDMA operating in the same geographic area are established on deterministic computations by supposing the worst-case scenario, i.e., BS /Node-B behavior according to minimum prerequisites stipulated in the related standard and maximum transmit power of BS /Node-B.Nevertheless, it is estimated that the real CDMA2000 BS out-of-band spurious emissions and blocking specifications of UMTS Node-B receivers are considerably better than the smallest requirements because CDMA2000 system needs to protect its receiver from its own transmitter.Consequently, the current equipment performance estimated to be at least 22 dB greater than the minimum performance specifications should be used.As a result, a minimum guard band of 5 MHz is adequate assuming actual filters and separations resulting from practical antenna separation.In reality, the guard band between the UMTS uplink and the PCS downlink is constantly more than 5 MHz as described below. | 2018-12-21T15:23:55.333Z | 2014-08-05T00:00:00.000 | {
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254389224 | pes2o/s2orc | v3-fos-license | A qualitative study of blood glucose and side effect self-management among patients with type 2 diabetes undergoing chemotherapy for cancer
Objective This study aimed to identify the process by which patients with type 2 diabetes who are undergoing chemotherapy for cancer personally manage their blood glucose levels and side effects. Methods Semi-structured interviews were conducted with 16 patients with cancer who had completed chemotherapy while taking hypoglycemic drugs. The interview data were analyzed using the modified grounded theory approach proposed by Kinoshita. Results Self-management comprised balancing the management of blood glucose levels and side effects according to physical condition. After commencing chemotherapy, participants experienced confusion regarding the side effects and hyperglycemia they have not previously experienced, started struggling with side effects while paying attention to blood glucose fluctuations, experienced simplification of convalescence based on the diabetes experience, and used trial and error to cope with side effects. When participants learned to understand the changes in blood glucose fluctuations and the pattern of physical recovery, they completed chemotherapy by adjusting their physical condition to the treatment by varying self-control. Conclusions Healthcare providers need to assist patients receiving chemotherapy to promote self-management of both blood glucose levels and side effects of the chemotherapy, depending on their physical condition. It is essential that patients with type 2 diabetes who are undergoing chemotherapy achieve the ability to self-monitor their blood glucose levels and side effects.
Introduction
The numbers of patients with diabetes and those with cancer increase worldwide annually. 1,2 In Japan, the number of patients with diabetes who are co-diagnosed with cancer has recently increased. 3 Thus, the number of patients with diabetes who are undergoing chemotherapy is expected to increase.
When patients with diabetes undergo chemotherapy, they tend to have a higher mortality rate and more severe side effects than patients without diabetes, [4][5][6][7][8] owing to diabetes-related glycemic abnormalities. 9,10 Therefore, it is critical for patients with diabetes to manage blood glucose fluctuations and side effects during chemotherapy because these factors can drastically affect their overall quality of life. However, self-management of type 2 diabetes, which accounts for 90% of all cases of diabetes, is challenging, 1 and even more so when the patient is diagnosed with and is being treated for cancer. This is because self-management of type 2 diabetes involves self-improvement of lifestyle habits, including diet and exercise. 11 Patients with cancer also need to manage their disease; however, the main focus of self-management of cancer is on the side effects of the treatment, physical changes, and psychosocial effects. 12 Considering these, self-management of diabetes alone and self-management of diabetes with cancer must be approached separately.
Patients using hypoglycemic drugs are less likely to routinely selfmonitor their blood glucose levels because the effectiveness of selfmonitoring of the blood glucose level has not been fully established. 13,14 During chemotherapy, hyperglycemia is induced through the administration of steroids to assist patients with monitoring their blood glucose levels. 15 However, it is difficult for the patients to immediately identify and respond to these changes in blood glucose levels. Moreover, chemotherapy is associated with several complex side effects, 16,17 which may further confuse the patients. 18,19 Hence, it is important to assist patients with self-management, specifically patients with diabetes who are undergoing chemotherapy while taking hypoglycemic drugs.
Previous studies have provided information on the blood glucose fluctuations and side effects experienced by patients with diabetes who were diagnosed with cancer and were undergoing chemotherapy. [20][21][22] However, the authors of these studies did not indicate how the patients managed their blood glucose fluctuations and side effects. The support measures for these patients highlighted in existing studies are limited to the introduction of collaborative arrangements among healthcare providers and evaluation of how to manage the support measures using simulated cases. 23,24 Further, it has been reported that medical professionals who specialize in the treatment of cancer or diabetes are not fully familiar with the scope of the expertise of other healthcare professionals; thus, the exchange of patient information between teams is insufficient. [25][26][27][28] Therefore, empirical studies on self-management procedures for patients with type 2 diabetes who are undergoing chemotherapy are essential. In addition, it is important to evaluate the support measures for these patients from the perspectives of both diabetes and cancer. Thus, the aim of this study is to identify the process adopted by patients with type 2 diabetes and cancer in self-managing their blood glucose levels and the accompanying chemotherapy-related side effects upon the simultaneous administration of chemotherapeutic and hypoglycemic drugs.
Definition of terms
Self-management of diabetes while undergoing chemotherapy was defined as the adoption of self-care by patients with diabetes for the prevention of complications and stabilization of their blood glucose levels, which fluctuate due to chemotherapy, and the introduction of coping methods for countering the side effects of chemotherapy on daily life.
Design
This was a qualitative descriptive study conducted using the modified grounded theory approach (M-GTA) proposed by Kinoshita. 29 The M-GTA is a research method that involves the reorganization of methods of analysis based on the GTA developed by Glaser and Strauss. 30 The M-GTA is applicable to research that focuses on the process-related characteristics of phenomena. 29 The M-GTA was deemed appropriate for the present study as it deals with the procedures involved in the self-management process adopted by patients with diabetes who are diagnosed with cancer and are undergoing chemotherapy, from the start of treatment until completion.
Participants
The participants were patients with type 2 diabetes who were diagnosed with cancer and had completed chemotherapy while taking hypoglycemic drugs. The inclusion criteria were as follows: (1) receipt of adjuvant chemotherapy for the first time; (2) an anticancer drug regimen that included steroids; (3) completion of the first chemotherapy treatment no more than 2 years prior to this study; and (4) a performance status of 0-1. The exclusion criteria were as follows: (1) hematologic cancers that required high doses of steroids and complex chemotherapy regimens; (2) cancers of organs that affect insulin secretion; and (3) stage IV cancers.
Data collection
Data were collected through semi-structured interviews conducted using an interview guide prepared by the researcher. The participants were requested to talk freely about their efforts to stabilize their blood glucose levels and to deal with the side effects of chemotherapy from the start to completion of treatment. For some cancer types using a combination of two or three regimens as the standard chemotherapy regimen, completion of chemotherapy was defined as the completion of all regimens; and patients were interviewed thereafter. Each interview was conducted in a private room and lasted approximately 60 min. The interviews were conducted at outpatient visits during the waiting time. Patient information, including demographic information such as age and sex, diabetes history, history of chemotherapy, blood glucose levels, and side effects, were extracted from medical records with informed patient consent. The data collection period was from August 2021 to August 2022.
Data analysis
The data obtained during the interviews were analyzed using the M-GTA developed by Kinoshita. The focus of the analysis was patients with type 2 diabetes diagnosed with cancer and having completed chemotherapy while taking hypoglycemic drugs. The goal of the analysis was to establish how the patients managed their blood glucose levels and side effects from the start to completion of chemotherapy.
For the analysis, the author carefully read the transcribed data of one case that provided a content-rich narrative, extracted the parts that fit the aims of the analysis, and generated the identified concepts. A worksheet with definitions, variations (concrete examples), and theoretical notes was created for each generated concept. Regarding the data obtained from the interviews that followed, continuous comparative analyses were conducted using the previously generated concepts. Further, the concepts were repeatedly modified based on the added variations observed in the records of further interviews. It was determined that theoretical saturation in concept generation had been achieved when there were no new variations to be extracted from the "next" interviews. After completion of the analysis of all participant data, the relationships among the concepts were evaluated, categories and core categories were generated, and a result diagram was created. Finally, storylines that explained the result diagram were created.
Trustworthiness
In this study, Shenton's criteria were used to ensure the trustworthiness of the qualitative research. 31 To increase credibility, the M-GTA, which is suitable for process characteristics research and has a well-established research methodology, was chosen for the analysis. Subsequently, according to the study objectives, the participant selection criteria were set, and four target institutions were selected to collect a wide range of data. The validity of the analysis was enhanced via repeated discussions among several researchers with clinical and qualitative research experience until consensus regarding data analysis was reached. Additionally, the results chart was explained to one participant to ensure that the results were representative of the participant's experience.
Confirmability was ensured by keeping detailed records of the process from data collection to interpretation. To ensure transferability, the study institutions, participant selection criteria, data collection methods, data collection period, and number of participants were described. Dependability was ensured by describing the research process in detail.
Overview of participants
Sixteen outpatients from four medical institutions in two regions of Japan participated in this study. The participants had lung (n ¼ 7), uterine (n ¼ 3), ovarian (n ¼ 3), and breast (n ¼ 3) cancers. Ten patients had diabetes for < 10 years.
The pre-chemotherapy glycated hemoglobin levels of six patients were < 7%. Thirteen patients used insulin during chemotherapy. The chemotherapy regimen of one patient was changed because the patient showed severe symptoms of peripheral neuropathy (Table 1).
All participants had been hospitalized at least once for treatment using anticancer drugs, and their blood glucose levels were measured three or four times a day during hospitalization. The mean blood glucose level of the patients before chemotherapy was (155.0 AE 38.4) mg/dL. Thirteen participants experienced hyperglycemia (> 250 mg/dL), and one participant experienced a fall due to hypoglycemia (< 70 mg/dL) after receiving anticancer drugs ( Table 1). The most common side effects of chemotherapy were anorexia, fatigue, nausea, peripheral neuropathy, and constipation; the severity of the side effects for most participants was Grade 1 as per the Common Terminology Criteria for Adverse Events (CTCAE) (Fig. 1).
Overview of the results
Twenty-five concepts were generated from the interview transcripts and summarized into seven categories, six of which involved one core category. The categories are indicated with single quotation marks (' ') hereafter. The storylines that were created based on results of the analysis are described below. The result diagrams that illustrate the storylines are shown in Fig. 2.
Storylines
Self-management by patients with diabetes who are diagnosed with cancer and are undergoing chemotherapy is a process of "balancing the management of blood glucose level and chemotherapy-related side effects according to the physical condition." Participants constantly try to 'maintain mental balance' during the self-management process from the start until the completion of chemotherapy.
The structure of this core category shows that after the initiation of chemotherapy, when participants experience 'confusion regarding the unprecedented side effects and hyperglycemia,' they begin to 'struggle with the side effects while paying attention to blood glucose fluctuations.' Thereafter, through repeated chemotherapy sessions, participants experience 'simplification of regimens based on the diabetes experience' and engage in 'trial and error to cope with side effects.' These efforts are adjusted depending on the type and severity of the side effects. Further, as the chemotherapy progresses, the participants start to 'understand the changes in blood glucose fluctuations and the pattern of physical recovery' and complete the chemotherapy by 'adjusting their physical condition to the treatment by exercising self-control in various steps' in preparation for the next cycle of anticancer drug administration.
Definitions of categories and concepts
The definitions of the seven categories are outlined in the sections below. The categories are indicated with single quotation marks (' '), whereas participant variations are indicated in italics. Angle brackets (<>) indicate the concept name, whereas supplementary explanations by the researcher are indicated in square brackets ([ ]). The letters in boldface placed after a sentence denote the participant being referred to. The definitions of each concept are outlined in Table 2.
'Confusion regarding the unprecedented side effects and hyperglycemia' The definition of this category indicates that the participants were confused regarding how to cope when the side effects of the treatment were stronger than expected and when their blood glucose levels were higher after receiving anticancer drugs than those recorded previously. This category consists of the concepts <Unsettling feelings about unexpected side effects> and <Fear of blood glucose level elevation>. 'Struggle with side effects while paying attention to blood glucose fluctuations' Participants struggled to alleviate symptoms while paying attention to the possibility of sudden blood glucose level fluctuations due to diabetes; patients wondering if they could cope with both the blood glucose fluctuations and the side effects they experienced for the first time.
This category consists of the concepts <Struggling alone with painful symptoms>, <Preparing for hypoglycemia effects>, <Identifying blood glucose abnormalities>, and <Negotiation with healthcare providers>.
There were times when it was really hard on my body. Thinking it [my blood glucose level] was really high, I checked it. However, it was not the blood glucose level that made me feel sick. I laid down thinking that there may have been another reason. After a while, I got up again and repeated the process. I conducted my household affairs little by little. I cannot do everything at once. A.
'Simplification of convalescence based on the diabetes experience' When it was difficult for participants to continue their diet and exercise regimen owing to side effects such as fatigue, they tried to continue the regimen in a simplified manner based on their knowledge of diabetes.
This category consists of the concepts <Using simple and quick food recipes> and <Opting for easier exercise regimens>.
I need to eat a lot of vegetables, but I cannot cook because it is too hard. If I cook hotpot meals, I can eat a lot of vegetables, tofu, and meat. A.
'Trial and error to cope with side effects' The participants tried, repeatedly and unsuccessfully, to find better ways to reduce the side effects of the treatment and delay the progress of symptoms.
This category consists of the concepts <Taking measures against side effects before they appear>, <Attempts to alleviate symptoms>, and <Determining when to seek help>. Participants gradually began to understand the changes in blood glucose fluctuations and their physical recovery pattern by speculating when blood glucose levels would rise and start to fall after receiving anticancer drugs.
This category consists of the concepts <Assessing the effects of blood glucose levels>, <Predicting the pattern of side effect fluctuations>, and <Estimating the timing of a decrease in blood glucose levels>.
On the days the medication [anticancer drugs] was administered, my blood glucose level sometimes exceeded 200 [mg/dL]. However, it never exceeded 200 at noon the next day, so I just measured [blood glucose level]. F.
After I managed to make it through the first week, the conditions just gradually returned to normal. The joint pains also got better. The anorexia and other symptoms like pain gradually disappeared. D. 'Adjusting their physical condition to the treatment by varying self-control' Participants acquired skills to stabilize blood glucose levels and successfully control side effects and prepared their physical condition to tolerate chemotherapy, aiming to complete the regimen as scheduled.
This category consists of the concepts <Monitoring side effects and blood glucose level>, <Dietary restrictions to control the elevation of blood glucose levels>, <Acquiring skills to deal with side effects>, <Fighting appetite>, <Temporarily relaxing dietary restrictions>, and <Dietary adjustments for physical strength>.
In the second or third week, I sometimes developed an appetite. On such occasions, I asked myself whether I moved around enough to have [an appetite] at that time. I mean I looked back on whether I used physical energy for work. If I made five round trips between the first and second floors on the day, I said to myself, "I can eat enough to replace the consumed energy." I adjusted the amount of food I ate accordingly. F.
'Maintaining mental balance'
Participants tried to maintain their emotional balance in different ways by classifying the information they obtained and changing their moods because they were disheartened upon being diagnosed with cancer and anxious regarding chemotherapy, their response to the treatment, and the various physical symptoms they experienced for the first time.
This category consists of the concepts <Gathering information from others with the same disease>, <Staying away from negative information>, <Positive thinking>, <Shifting of moods>, and <Revealing true feelings>.
I tried not to browse the [inter]net again and again, thinking that there would rarely be very useful information. If I started browsing, there would be no end to it. K.
When I go to work I feel better, as expected. I feel a little different. Time passes and I do not snack while I am on duty. N.
Discussion
The results of this study indicated that patients with diabetes, who were diagnosed with cancer and were undergoing chemotherapy, selfmanaged their conditions by "balancing the management of blood glucose levels and chemotherapy-related side effects according to their physical conditions." Healthcare providers who specialize in the treatment of cancer or diabetes are aware of the challenges related to balancing glycemic control and cancer treatment. Thus, they focus on cancer treatment because most patients with diabetes undergoing chemotherapy experience a feeling of fear regarding their cancer, 32 whereas some lack awareness regarding their diabetes. 33 Patients and healthcare providers have a different understanding of self-management of blood glucose levels and side effects due to chemotherapy. This is important because healthcare providers play a significant role in helping patients manage their blood glucose levels and side effects effectively and successfully. The participants in the present study were bewildered by the unexpectedly high blood glucose levels and strong side effects they experienced when they started chemotherapy. They adopted certain coping methods through trial and error while struggling, <Struggling alone with painful symptoms>, to determine effective methods to resolve the abovementioned problems.
Some of the participants recorded blood glucose levels of 250 mg/dL or higher after receiving anticancer drugs, a finding that is similar to those of previous studies. 8,34 The effects of self-management of diabetes before chemotherapy do not affect the level of expertise shown during chemotherapy. 35 The fact that the participants utilized 'trial and error to cope with side effects' suggests that the instructions provided by nurses regarding methods to cope with the side effects of chemotherapy were inadequate. Oncology nurses tend to provide only general information to patients with diabetes when instructing them on how to deal with the side effects of chemotherapy. 36 It is essential for healthcare providers to provide detailed information and self-management instructions to these patients on how to handle the fluctuations in blood glucose levels that occur after receiving anticancer drugs, the extent of the possible side effects, and how to cope with them. These instructions should be provided early in the pretreatment stage.
Meanwhile, for blood glucose fluctuations, the participants were using 'Simplification of convalescence based on the diabetes experience.' Therefore, it is inferred that the participants were willing to minimize blood glucose fluctuations during chemotherapy and continued blood glucose management by <Using simple and quick food recipes> and <Opting for easier exercise regimens> according to their physical condition. Therefore, healthcare providers are required to provide information on foods, cooking methods, and exercise techniques for patients to continue their diet and exercise regimen in accordance with their physical condition after chemotherapy.
Furthermore, the concept of <Fighting appetite> reveals that maintaining a diet with low blood glucose elevation during the months of chemotherapy can be very stressful for participants. Continuation of this diet is challenging because of the influence of environmental factors, such as the presence of sweet foods, and interpersonal factors, such as the desire to maintain relationships with others. 36 In addition, because steroids have an appetite-enhancing effect, 37 it is anticipated that diet therapy administered during chemotherapy will be more difficult and stressful for patients than that administered before chemotherapy. Participants were able to <Temporarily relax dietary restrictions> and make <Dietary adjustments for physical strength> by understanding the patterns of blood glucose fluctuations and physical recovery after chemotherapy.
The participants in the present study acquired self-monitoring skills while repeatedly receiving anticancer drugs, as illustrated by the category 'understanding the changes in blood glucose fluctuations and the pattern of physical recovery.' Thereafter, the participants may have advanced to the stage of 'adjusting their physical condition to the treatment by exercising self-control in various steps' using the self-monitoring skills they learned experientially. The acquisition of self-monitoring skills is effective in maintaining the motivation to continue treatment and improve lifestyle habits. 38,39 In addition, self-monitoring enables patients to flexibly choose self-management methods to suit their goals and lifestyles. 40,41 Therefore, the acquisition of self-monitoring skills will be a turning point for patients with diabetes with cancer, facilitating flexible management of blood glucose levels and the side effects of chemotherapy.
The participants were confused by the unexpected elevated blood glucose levels and the appearance of side effects after chemotherapy and were anxious about the threat of cancer and the unknown side effects. However, the participants in the present study engaged in selfmanagement by 'maintaining mental balance' while dealing with the threat of cancer and anxiety about the unknown side effects of cancer treatment. Thus, it is apparent that assisting patients in completing chemotherapy while maintaining mental stability and peace of mind is essential.
Based on the study results, it may be necessary to develop a program to promote self-management among patients with diabetes who are diagnosed with cancer and are undergoing chemotherapy. In the present study, patients with diabetes who were undergoing chemotherapy for the first time were confused by the hyperglycemia and side effects that they had not experienced prior. They had to endure a trial and error process of coping strategies during self-management. Therefore, it is pertinent to identify the pattern of blood glucose fluctuations and side effects associated with chemotherapy from an early stage and provide support to enable patients to manage their blood glucose levels as well as take measures to prevent side effects depending on their physical condition. In particular, in a study examining diabetic patients undergoing chemotherapy, diabetes self-management declined 8 weeks after administration of anticancer drugs. 35 Therefore, focused support should be provided before and during the early stages of anticancer drug administration.
Limitations and recommendation
This study has some limitations. First, only participants with cancers in specific locations were included. As such, many cancer types were not covered. Thus, there may be some bias in the selection of the locations of the cancers covered in this study. Future studies should expand on the number of cancer types and participants to determine whether the results revealed in this study can be applied to patients with other cancer types. Second, there were large variations in the number of blood glucose level measurements analyzed, and whether side effects were recorded by participants, making it difficult to collect more meaningful quantitative data. In future, studies should examine whether patients with diabetes who undergo chemotherapy are successfully self-managing by referring not only to blood glucose levels as an indicator but also to HbA1c levels, unscheduled medical visits due to sudden hypo-or hyperglycemia, and emergency hospitalizations.
Conclusions
Self-management by patients with diabetes undergoing chemotherapy for the first time involves a process of "balancing the management of blood glucose level and side effects according to the patient's physical condition," while always striving towards 'maintaining mental balance.' The findings of the present study show that patients with diabetes undergoing chemotherapy make efforts to achieve selfmanagement of both blood glucose levels and chemotherapy-related side effects, while experiencing confusion about their abnormally high blood glucose levels and the new side effects of their treatment. The study results also demonstrate that it is important for patients to acquire selfmonitoring skills that will enable them to understand the patterns of blood glucose fluctuations and the physical recovery process associated with chemotherapy.
Declaration of competing interest
None declared.
Funding
This work was supported by JSPS KAKENHI (Grant No. 20K19054).
Ethics statement
The study was approved by the Ethics Review Committee of Osaka Medical and Pharmaceutical University (2925-5) and the Ethics Review Committee of the facilities from where data were collected. The researcher explained the ethical considerations of the study to the participants orally and in writing, including information regarding voluntary participation, freedom to withdraw, protection of personal information, and data management methods. The author affirms that human research participants provided informed consent for publication of the participant variations. | 2022-12-08T16:11:46.857Z | 2022-12-01T00:00:00.000 | {
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4740645 | pes2o/s2orc | v3-fos-license | Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells
Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium Microcoleus sp. PCC 7113. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.
CBCRs possess heme-derived, linear-tetrapyrrole (bilin)binding GAF domains that can perceive the complete range of the visible light spectrum, extending into the near UV and the near IR (14 -16). All CBCRs utilize phycocyanobilin (PCB) that is spectrally tuned by key residues within the GAF domain scaffold (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). MIC7113_2205 belongs to the DXCF subfamily of CBCRs, characterized by two conserved cysteine residues, each of which can form covalent linkages to the PCB precursor to alter the -conjugated chromophoric system to absorb blue light (18,27). Although CBCRs are typically associated with histidine kinase, methyl chemotaxis, and cyclic di-GMP regulatory GGDEF or EAL output domains, MIC7113_2205 is the first adenylyl cyclase (Fig. S1) to be identified with a putative light-sensing CBCR GAF domain. MIC7113_2205 also contains an N-terminal response regulator (REC) and Per/Arnt/ Sim (PAS) domains, both of which could represent additional input domains for other chemical or physical signaling systems. Atypical for widespread adenylyl cyclases with unrelated GAF domains, this novel adenylyl cyclase-associated CBCR gene appears unique to this Microcoleus species. The observation that the related cyanobacterium Microcoleus chthonoplastes PCC 7420 possesses a flavin-based, B-regulated adenylyl cyclase (10) suggests that light-regulation of cAMP production may be particularly critical for cyanobacteria in this genus.
The ability to manipulate adenylyl cyclase activity with light enables precise spatial and temporal control of cAMP levels within cells, organs, and tissues. Indeed, genetically-encoded photoactivated adenylyl cyclases (PAC) with Blue-Light sensors Using FAD (BLUF) domains, such as EuPAC, bPAC, BgPACs, LiPAC, NaPACs, and OaPAC (11, 28 -38), or with flavin-binding LOV domains, such as mPAC (10), have been successfully deployed in mammalian cells. The utility of bilinbased PACs for similar applications was first documented by Gomelsky and co-workers (39), who fused a biliverdin IX␣ (BV)-dependent bacteriophytochrome to an adenylyl cyclase catalytic domain that could be reversibly controlled with red and far-red light (Fig. 1). As optogenetic tools, bistable phytochrome-based PACs offer the advantage that two wavelengths of light can be used to reversibly regulate cAMP production in cells in real time, rather than relying on thermal reversion pathways to inactivate the photoactivated state, e.g. for flavin-based PACs. Because of the small size of CBCR input domains (ϳ200 amino acids), and extended spectral range, CBCR-based adenylyl cyclases hold particular promise as optogenetic reagents.
The present work was undertaken to test the hypothesis that MIC7113_2205 functions as a light-regulated adenylyl cyclase and to characterize the molecular basis of its light regulation. Our studies show that MIC7113_2205 is a bistable, blue/green (B/G)-regulated adenylyl cyclase that we hereafter refer to as cPAC for cyanobacteriochrome-based Photoswitchable Adenylyl Cyclase. We also test the interchangeability of CBCR GAF domains to alter the wavelength specificity of cPAC for its photoswitchable cAMP regulation in vivo, providing a roadmap for spectral engineering of a versatile new family of CBCRbased optogenetic actuators.
cPAC is a bistable blue/green sensor
To analyze the photochemical properties of cPAC, we expressed a synthetic full-length cPAC gene in an Escherichia coli strain engineered to produce PCB from endogenous heme, and the recombinant holoprotein was purified as described previously (19,40). In the dark-adapted P b state, the PCB chromophore of cPAC adopted a 15Z configuration with an absorbance maximum at 410 nm in the B spectral region. Upon exposure to saturating B light, the chromophore photoconverted to its 15E configuration with an absorbance maximum at 520 nm in the G spectral region ( Fig. 2A). P g could be fully photoconverted to the B-absorbing P b form with saturating G light. The B/G spectral properties of cPAC were consistent with those of other two-cysteine CBCRs in the DXCF subfamily, and its specific absorption ratio value of 0.18 indicated near saturated binding of PCB to the apoprotein (18,19,41). Dark reversion of cPAC was found to be slow on the timescale of biochemical assays, with Յ5% change over 90 min (Fig. S2A). Moreover, the difference spectrum for dark reversion did not match that for photoconversion (Fig. S2B). This difference indicates that other processes are occurring on the same timescale, such as changes in chromophore content (19) or oxidation of the DXCF Cys (42), making it likely that regeneration of the blue-absorbing 15Z dark state is overestimated by this approach. Taken together with the linearity of cPAC in AC assays lasting 60 min (Fig. 2B), it is clear that dark reversion is not a confounding factor in our experiments.
cPAC is a photoswitchable adenylyl cyclase
The C-terminal region of cPAC (MIC7113_2205) is annotated in GenBank TM as an "adenylyl or guanylyl cyclase domain." Further sequence analysis supports designation of cPAC as an adenylyl cyclase as both key adenosine-specific binding residues in the active site as well as all key metal and
Cyanobacterial photoactivatable adenylyl cyclases
transition state residues are present (31,43) (Fig. S1). To resolve whether cPAC is an adenylyl or guanylyl cyclase, the activity of the 15Z-P b and 15E-P g states were compared using Mg-ATP or Mg-GTP as substrate. cPAC was able to convert Mg-ATP to cAMP (Fig. 2B); however, no cGMP was detected by reverse phase-HPLC (RP-HPLC) when Mg-GTP was used as substrate. The 15E-P g state also produced more cAMP than the 15Z-P b state indicating that cPAC is light regulated. In view of the cyanobacterial adenylyl cyclase literature, we next tested whether cPAC activity was affected by addition of calcium or bicarbonate. These studies showed that 5 mM calcium greatly increased the activity of cPAC in both 15Z-P b and 15E-P g states (Fig. S3A). For the more active 15E-P g state, the presence of calcium resulted in a Ͼ10-fold greater cAMP accumulation compared to reaction mixtures lacking added calcium (Fig. 2B). By contrast, cPAC activity was stimulated only slightly (ϳ2.5-fold) by the addition of 50 mM bicarbonate, nor did bicarbonate further stimulate cPAC activity more than calcium alone (Fig. S3B). This indicates that Ca 2ϩ could stimulate cPAC activity in vivo, but probably it is not regulated by bicarbonate.
Initial rate measurements for both E and Z states of cPAC were next performed as a function of Mg-ATP concentration in the optimized buffer medium (Fig. 2C). A modified Michaelis-Menten analysis using a second-order reaction model fit the data slightly better than the standard analysis (44) (Fig. S4A, Table S1). The specific activity of cPAC in the 15Z-P b dark state was estimated to be 3.8 nmol of cAMP min Ϫ1 mg Ϫ1 , whereas that of the 15E-P g active state was estimated to be 11.4 nmol of cAMP min Ϫ1 mg Ϫ1 . These specific activities are comparable with those of flavin-based adenylyl cyclases in the activated state ( Table 1). As has been seen previously for other soluble adenylyl cyclases (45), cPAC turnover was substrate inhibited at high Mg-ATP concentrations (Fig. S4B).
The REC domain is necessary for cPAC activity
To analyze how the domain architecture of cPAC affects its activity, several truncations were constructed. The first two of these, cPAC(⌬R2) and cPAC(⌬R3), lacked the N-terminal REC domain, while retaining two or three predicted helices at the N terminus of the GAF domain, respectively. Two of these helices are typically found necessary for proper GAF domain folding. Two other constructs, cPAC(⌬RG) lacked both the REC and GAF domains, whereas the cPAC(⌬RGP) construct contained only the cyclase domain (Fig. S5A). Truncated cPAC(⌬R2) and cPAC(⌬R3) proteins were both folded and photoactive, exhibiting P b and P g spectra nearly indistinguishable from those of the full-length cPAC ( Fig. 2A and Fig. S5). By contrast, adenylyl cyclase activities of these and all truncated cPAC constructs were less than 5% of WT, and adenylyl cyclase activities of cPAC(⌬RG) and cPAC(⌬RGP) were barely detectable (Fig. 3A). It is also interesting that the two REC-less constructs cPAC(⌬R2) and cPAC(⌬R3) did retain some light-regulated adenylyl cyclase activity although the significance of the differences is unclear due to their low AC activities. Taken together, these data indicated that the REC domain is critical for cPAC activity in either state.
The regulatory activity of REC domains of two-component response regulator proteins is typically modulated by phosphorylation. REC domain phosphorylation could affect the monomer-dimer equilibrium of canonical response regulator proteins to influence, e.g. their DNA-binding affinity, and/or could affect the conformation/dynamics/activity of other associated output domains (46). To test the first possibility, full-length cPAC and all four truncated constructs were analyzed by HPLC-size exclusion chromatography (HPLC-SEC). As seen in Fig. 2D, full-length cPAC migrates as a dimer in both 15Z-P b and 15E-P g states (Table 2). By contrast, all truncated cPAC constructs appear monomeric under these conditions ( Table 2, Fig. S6); these results suggest that the REC domain is critical for homodimer formation, which in turn is necessary for cPAC catalytic activity.
We next examined whether the P b to P g photoconversion could affect the quaternary state of the protein. As shown in Fig. Figure 2. cPAC is blue/green photoswitchable adenylyl cyclase. A, spectra of cPAC displays the distinct B-absorbing (15Z) photostate and the G-absorbing (15E) photostate. B, time course assay of production of cAMP by the G-absorbing 15E state of cPAC with or without calcium present in the media. C, initial rate kinetics of cPAC in the 15E and 15Z photostates. Data are fit to a second order fit derived from the Michaelis-Menten equation: . D, HPLC-SEC trace of full-length cPAC in the 15E and 15Z states. The major peak elutes at 19.6 min. Table 1 Activity comparison of cPAC and flavin binding PACs cPAC specific activity was measured as described under "Experiment procedures." Enzyme activity data points were analyzed in triplicate, and S.D. was calculated.
Adenylyl cyclase
Activated state Dark state Data not reported: estimated value from enzymatic data. b BlaC and bPAC are the same protein, but were analyzed and documented separately (30,31).
Cyanobacterial photoactivatable adenylyl cyclases
2D, the HPLC-SEC elution profiles of both states of full-length cPAC were superimposable. Based on this result, we conclude that photoactivation of cPAC does not lead to a major change in overall protein conformation. This also implies that small lightinduced structural changes are responsible for the regulation of activity of cPAC, a result similar to that reported for BLUF containing PACs (11,37,38). Our HPLC-SEC data also suggests that homodimerization is required for efficient catalytic turnover. However, dimerization is not required for the cPAC photocycle, because both cPAC(⌬R2) and cPAC(⌬R3) constructs retained the same spectral properties as the full-length protein (Fig. S5C). As all truncated proteins are monomeric, the enzyme activity data suggest that REC domain dimerization restricts conformational sampling of CHD domains that disfavor formation of the catalytically competent dimer or, alternatively, that REC-dependent dimerization is necessary to increase the local CDH concentration to support dimerization and catalytic turnover (5).
cPAC activity is modulated by REC domain phosphorylation
Because the REC domain is required for cPAC function, we next performed experiments to test the role of REC domain phosphorylation on its catalytic activity. The transmitter kinase responsible for its phosphorylation is unknown, so we constructed site-directed variants of the conserved predicted phospho-accepting Asp-60 residue of cPAC (as categorized for a REC domain consensus sequence, CDD 238088) to mimic its phosphorylated and nonphosphorylated states (47). To do so, we introduced phosphomimetic D60E "gain-of-function" and D60A "loss-of-function" substitutions; such functional substitutions have been described in the response regulator receiver family previously (48 -50). Both cPAC variants exhibited photochemical properties identical with those of the WT (Fig. S5C), demonstrating that these substitutions did not perturb
Cyanobacterial photoactivatable adenylyl cyclases
the structure or light-sensing function of the CBCR GAF domain of cPAC. Surprisingly, the 15E-P g state of D60E exhibited 150% AC activity of the WT; by comparison, the 15E-P g state of D60A was 30% less active than the WT. However, the activities of the 15Z-P b states of WT and D60E mutant were nearly identical (Fig. 3B), suggesting that phosphorylation selectively enhances the activity of the photoproduct state. This conclusion must be tempered by the possibility that the 15Z-P b state of WT cPAC from E. coli may already be partially phosphorylated when isolated. Taken together, our data suggest that phosphorylation of the REC domain likely plays a regulatory role on cPAC activity in vivo. The interplay between REC phosphorylation and light activation of the GAF domain remains an interesting subject for future study. Taken together, our enzyme data indicate that the maximum signal output from cPAC is achieved following B light treatment, when the REC domain is phosphorylated and calcium levels are elevated (Fig. 3C).
In vivo light-regulated manipulation of cAMP levels by cPAC
To test the efficacy of cPAC as an optogenetic tool in vivo, we co-expressed cPAC with a PCB biosynthetic plasmid in cya Ϫ E. coli strain VJS711 that lacks endogenous adenylyl cyclase activity (51). This strain allows us to measure the cAMP-dependent activation of the endogenous -gal (lacZ) gene via the plasmid-derived cPAC without interference of endogenous CYA-dependent cAMP production. When spread on LB plates containing X-Gal/IPTG, only strains that produce sufficient levels of cAMP will appear blue (Fig. 4A). Indeed, cell lines expressing full-length cPAC were blue on X-Gal plates, revealing the production of endogenous cAMP and its subsequent activation of -gal expression. By contrast, the cya Ϫ strain expressing the truncated cPAC(⌬RGP) construct failed to produce -gal under identical conditions (Fig. S7). As expected from in vitro assay results, WT cPAC-expressing cultures grown under continuous blue light (Bc) possessed significantly more -gal activity than cultures grown under continuous green light (Gc) (Fig. 4B). We expected that cPAC would modulate global cAMP levels in a heterologous system, measured via a direct cAMP assay method, as seen with previous PACs (28,29,31). Similar results were observed with E. coli liquid suspension cultures grown under Bc, which yielded significantly more cAMP (ϳ2.5 times more) than cultures grown under G c (Fig. 4C). Control measurements of the cya Ϫ strain lacking cPAC revealed insignificant amounts of cAMP production. These studies confirm that cPAC retains its photoregulatory properties in vivo.
CBCR domain exchange yields cPAC variants with altered wavelength selectivity
CBCRs have been identified that can sense light from the near UV, throughout the visible spectra, and into the near IR (52). Because 3D structures of CBCRs so far examined are all quite similar, this suggests that substitution of different wavelength-sensing CBCRs for one another will be feasible to provide a broad array of cPAC reagents tailored for potentially any wavelength combination desired (53-55). We therefore sought to create new cPAC-based chimeras by exchanging the B/G CBCR GAF domain of cPAC with GAF domains from two other CBCR lineages. We chose the green/red CBCR RcaE from Fremyella diplosiphon (22) and a newly identified CBCR JSC1_58120g3 from Leptolyngbya sp. JSC1 (Fig. 5A). One of three GAF domains of a multi-GAF methyl-accepting chemotaxis transducer, i.e. JSC1_58120g3, is closely related to the canonical red/green family. JSC1_58120g3 is a previously unde-
Cyanobacterial photoactivatable adenylyl cyclases
scribed CBCR found in the Leptolyngbya sp. JSC1 genome (56), so it was first expressed and purified as its PCB adduct for spectroscopicanalysis.JSC1_58120g3exhibited15Zstatepeakabsorbance of 712 nm and 15E state peak absorbance of 654 nm. Therefore, JSC1_58120g3 can be classified as a far-red/red CBCR similar, albeit unrelated to those recently described (26). JSC1_58120g3 can also utilize biliverdin (BV) as an alternative chromophore, although surprisingly, the spectral properties of the BV adduct of JSC1_58120g3 were quite similar to the PCB adduct (Fig. S8), peaking at 716 nm in its 15Z state and at 646 nm in its 15E state.
For selection of fusion junctions for these chimeras, we chose an N-terminal fusion point between charged residues in a conserved region within the first helix of the cPAC GAF domain, and a C-terminal fusion point in the C-terminal ␣ helix in the GAF domain leading toward a highly conserved region at the N terminus of the downstream PAS domain (Fig. 5B). The resulting fusion proteins, designated cPAC(JSC1) and cPAC(RcaE), retained their donor parent CBCR photocycles (Fig. S8). Because JSC1_58120g3 binds BV as well and PCB, the BV adduct of cPAC (JSC1-BV) was examined spectrophotometrically. These measurements showed that both bilin adducts of cPAC retained the spectroscopic signature of the corresponding bilin-reconstituted JSC1_58120g3 parent, although their 15E photoproduct states of the former reverted to their 15Z dark states more rapidly than those of the parent JSC1_58120g3 constructs.
Finally, the cPAC chimeras were analyzed for adenylyl cyclase activity under similar in vitro assay conditions used for WT cPAC. Although the overall cyclase activity was lower in each construct when compared with WT cPAC, both chimeras displayed differential AC activity for their 15Z and 15E states. For cPAC(RcaE), the 15E state was twice as active as its 15Z state, whereas cPAC(JSC) exhibited only a ϳ33% enhancement in its 15E state (Fig. 5C). A similar result was also observed for cPAC(JSC-BV). In addition, when transformed into the VJS711 cya Ϫ strain, cPAC(RcaE) was able to activate -gal activity under green light, whereas red light suppressed -gal activity on agar plates (Fig. 5D). Taken together, these studies document that re-engineering new color specificity for cPACs by CBCR exchange is a viable and surprisingly facile approach. Such first-generation constructs represent promising subjects for further optimization of spectrally diverse cPAC tools for in vivo regulation of cAMP signaling with light.
Discussion
Cyanobacteria rely on the ability to sense environmental light conditions and therefore utilize photosensory proteins to optimize photosynthesis and regulate motility (37), processes that are influenced by levels of the second messenger cAMP. Microcoleus sp. PCC 7113 has one of the largest complement of adenylyl cyclase enzymes for cyanobacteria; among them is the novel cPAC light sensor described here. The many distinct sensory modules in the Microcoleus adenylyl cyclase family suggest that cAMP signaling participates in many processes critical for adaptation to the complex intertidal and soil sediment environments where Microcoleus is found (57-59). Flavin- ) showing greater X-Gal cleavage and blue coloring in E. coli cells expressing cPAC(RcaE) that are exposed to Gc, as compared with cells exposed to Rc.
Cyanobacterial photoactivatable adenylyl cyclases
and retinal-based light-sensing adenylyl cyclases are notably widespread in cyanobacteria and algae (12, 30, 37, 60 -62), but cPAC is the first example of an adenylyl cyclase with a CBCR input domain.
Our studies show that cPAC is differentially modulated by blue (B) and green (G) lights. Blue light-activated CBCR-based diguanylate cyclases, which trigger rapid settling to avoid photodamage of the photosynthetic apparatus at high fluences, are notably more common in cyanobacteria (23,63). In addition, the need for B light-regulated adenylyl cyclase activity is underscored by the observation that all known PAC sensors are activated by B light. Unlike the bPAC family of BLUF-, LOV-, and retinal-based sensors, the activity of cPAC can be rapidly adjusted/reversed by G light, providing greater temporal control of cAMP signaling. Our in vitro studies document the bistability of cPAC and thus implicate the potential to trigger and reverse cAMP signaling in vivo. Beyond the scope of the present investigation, such studies necessitate construction of cell lines such as mammalian neurons or other eukaryotic cells engineered with more sophisticated reporter systems.
cPAC: An integrator of multiple signal inputs?
Well suited to play a role in light-regulated cAMP production in cyanobacteria, the complex architecture of cPAC suggests that it integrates information from multiple input signals. Indeed, our studies implicate the N-terminal REC domain as an input domain that is also required for the adenylyl cyclase activity of cPAC. cPAC was most active when its REC domain was modified to mimic its phosphorylated state, and was least active in an unphosphorylable REC domain variant. These observations suggest that the REC domain stabilizes the homodimeric quaternary structure of full-length cPAC to enhance formation of the interdomain transition state of Type III adenylyl cyclase enzymes (5). The enhanced activity of the phosphomimic mutant may be due to further constraints on the REC domain that favor catalysis. Because the truncated constructs are essentially inactive, we propose that the REC domain may also minimize conformational sampling of the GAF, PAS, and/or CHD domains possibly by reducing unproductive conformational sampling of the homodimer. Future studies will be needed to address these hypotheses.
The cPAC gene is located immediately downstream of an ORF that encodes a histidine kinase, MIC7113_RS10675, annotated as a PAS-SsrA sensor kinase. SsrA proteins are known components of two-component systems (64), thus we speculate that MIC7113_RS10675 may be responsible for the regulation of the phosphorylation status of cPAC. This locus also has apparent orthologs in other cyanobacteria. It will be interesting to test whether cPAC phosphorylation plays a more dramatic regulatory role of the adenylyl cyclase activity compared with the phosphomimetic variant. With respect to other input signals to cPAC, bicarbonate does not appear to be a major agonist in contrast with its known regulatory role of other cyanobacterial adenylyl cyclase proteins (65)(66)(67)(68)(69). cPAC was greatly stimulated by the presence of calcium ions (Ca 2ϩ ). Because Ca 2ϩ has been shown to help mediate ATP binding in adenylyl cyclase enzymes (69), changes in Ca 2ϩ concentrations may also trigger increased cAMP production by cPAC. The Ca 2ϩ sensitivity of cPAC remains an important consideration for optogenetic applications going forward (see below).
cPAC as a optogenetic tool
The activity of cPAC was sufficient to trigger light-dependent differences in cAMP levels in vivo, as well as trigger cAMPregulated gene expression (lacZ) in E. coli in a B/G-dependent fashion. The successful manipulation of cAMP in vivo by cPAC shows that this protein is a viable platform for optogenetic tool applications in a wide variety of photosynthetic species where reduced bilins, also known as phytobilins, are present. These not only includes cyanobacteria, but also eukaryotic algae and plants (70). We envisage considerable utility of this tool for light-dependent reprogramming of plastid metabolic pathways in chlorophyte algae such as Chlamydomonas reinhardtii, due to its ability to produce PCB despite its lack of bilin-based sensors in the phytochrome family (71).
Known PACs (with BLUF and LOV domains) have previously been shown to be effective in regulating a variety of cAMP-related processes in heterologous organisms with B light (10,11,29,31,72). An advantage of cPAC, and its variants, lies with the extensive color palette of CBCRs and the ability to interchange CBCR GAF domains that sense light from the near UV to the near IR; this contrasts with the more limited colorsensing properties of canonical phytochromes (14). Based on these attributes, CBCRs have garnered considerable interest for use as optogenetic tools in live cells (53)(54)(55)73). The ability of CBCRs to switch rapidly between stable photostates (15E/Z) without relying on a dark or thermal reversion to reset between active and inactive states is another key feature of the cPAC family. This property enables switching between active/inactive states with supplemental light of the appropriate wavelength to trigger rapid changes in cAMP-dependent processes in situ.
Finally, our studies demonstrate that cPAC is a good lead compound for creation of a suite of adenylyl cyclase enzymes that respond to a variety of wavelengths. The successful fusion constructs tested here, especially cPAC(RcaE), establishes the plug-and-play modularity of the cPAC regulator that presage our ability to make robust light-responsive adenylyl cyclases that span the near UV to the NIR. Although intriguing as proofof-concept enzymes, these proteins, along with native cPAC, are good targets for further engineering efforts. We expect that our growing understanding of CBCR structure (22,74) will inform structure-based approaches to design new cPACs with improved signaling dynamics and dynamic range.
Applications beyond photosynthetic species
As various CBCRs bind several types of bilin chromophores, constructs can conceivably be tailored to utilize natural precursors that may be more abundant in the organism of choice. For example, although PCB is not naturally produced in nonphotosynthetic species, BV is widely distributed in nature including yeast and mammals. For this reason, biosynthesis of PCB has also been engineered into eukaryotic "chassis" organisms that naturally do not produce these compounds (75)(76)(77)(78). Increased expression of heme oxygenases has been used to enhance the Cyanobacterial photoactivatable adenylyl cyclases synthesis of BV in bacterial and mammalian cells (79,80). Indeed, bilin availability remains a critical issue to resolve for optogenetic applications because, in all cases tested, chromophore binding is critical for optimizing the dynamic range of two-color regulation by phytochrome and CBCR systems (39,54,81).
As shown previously, CBCRs can also be engineered to alter the dark reversion rate of the photosensory domain, which can be used to create tools that are active at a particular wavelength, and inactive in the dark (55). When combined with light-activated phosphodiesterase proteins (82), the levels of cAMP also can be tailored to precise levels, or to eliminate basal activity, in the 15Z state of cPAC and variants. We envisage a bright future for combination of these enzymes and spectrally diverse PACs for the fine-tuned control of cAMP-regulated processes in living cells using a variety of colors of light.
Protein expression and spectral analysis
The gene Mic7113_2205 (cPAC) was purchased as synthetic DNA from GenScript. cPAC and variants were expressed as intein-chitin-binding domain fusion proteins in E. coli cells engineered to produce PCB using a dual plasmid system, as described previously (27,40). Proteins were purified from E. coli on a chitin column (New England Biolabs) in accordance with the manufacturer's directions and characterized by SDS-PAGE (Fig. S9). Absorption spectra and dark reversion measurements for cPAC and variants were acquired as previously described (27). Photoconversion of cPAC and variants during in vitro calculations was verified in this manner.
In vitro cyclase assay
The BCA assay (Pierce) was used to determine protein concentration for all assays with BSA as a protein standard. The standard cyclase assay buffer was composed of 0.5 M Tris-HCl, pH 7.6, 0.05 M NaCl, 0.01 M MgCl 2 , 0.5 mM EDTA. CaCl 2 was added to buffer at a final concentration of 5 mM, whereas NaHCO 3 was added at a final concentration of 50 mM. The desired concentration of GTP or ATP was added to buffer prior to reaction initiation. Purified protein (10 mM final concentration) in the 15E or 15Z states was added to initiate reaction in the dark. Photostates of protein were achieved using LEE filters (number 071 Tokyo blue and number 090 dark yellow green) as described previously (83), and confirmed via spectroscopic analysis. Assays were terminated after 1 h in the dark with addition of EDTA, pH 8.0, to a final concentration of 0.1 M. An aliquot (15 l) of each reaction solution was injected into an Agilent 1100 series HPLC and passed through a Phenomenex Synergi 4 M Fusion-RP 80 Å column and Security Guard Cartridge (C18, 4 ϫ 2 mm), under conditions previously described (83). The internal standard of cAMP (Sigma) eluted at 12.1 min. A standard curve of cAMP levels using area under peak values correlated with cAMP concentrations of 2.0 to 400 M. All area calculations were performed using Agilent ChemStation software. All cyclase assays are performed in triplicate at room temperature (20°C).
HPLC-SEC
Buffer used for HLPC-SEC assays was 40 mM Tris acetate, pH 8.3, 1 mM EDTA, 0.15 M NaCl. Run time was 40 min with an isocratic flow rate of 0.6 ml/min, using ThermoScientific Ultimate 3000 HPLC and a GE Superdex TM 200 Increase 10/300 GL column. Aliquots (50 l) of purified cPAC and variants (in either 15E or 15Z states) were injected in a 100-l loop. 15E and 15Z states were achieved as described above, and confirmed via spectroscopic analysis. Elution traces and peaks were analyzed using Chromeleon 7 software (Dionex). All runs were performed in darkness to avoid photoconversion prior to elution. Molecular weight calculations were performed via standard curve preparation, seen in supporting Fig. S4. Samples were analyzed in duplicate (n ϭ 2) and the molecular weight measurements were averaged and the plus/minus spread was described.
In vivo cPAC expression in a cya ؊ E. coli strain
cPAC and variants in PCB-producing E. coli K12 strain VJS711 that lacked the endogenous adenylyl cyclase gene (referred to in this paper as strain cya Ϫ ) were grown overnight in LB medium containing 0.2 mM kanamycin and 0.2 mM chloramphenicol in darkness at 30°C before suspension culture or plate assays. For suspension culture assay, aliquots (3 ml) of the overnight suspension cultures were then transferred to NUNC 12-well cell culture plates (catalog number 150628). To generate Bc, Gc, or continuous red light (Rc) conditions, half of each plate was covered with filters (number 071 Tokyo blue (maximum absorbance: 440 nm) or number 090 dark yellow (maximum absorbance: 525 nm) or number 164 flame red (maximum absorbance: 625 nm) respectively; LEE filters, Burbank, CA) and allowed to incubate further for 6 h. The light fluence rate for light incubation was 4 -5 mol m Ϫ2 s Ϫ1 . E. coli liquid suspension cultures were then analyzed for global cAMP levels with the cAMP Direct EIA kit (Arbor Assays LLC, catalog number K019-H1) as directed by manufacturer. All experiments were performed in triplicate with two technical replicates. The limit of detection of the EIA kit was 0.2 pmol/ml with sensitivity of 0.64 pmol/ml. For plate assays, overnight grown cells were plated on LB plates containing 1 mM IPTG, 84 mg/liter of amino levulinic acid, 0.2 mM kanamycin, and 0.2 mM chloramphenicol to which X-Gal (40 l) was freshly added. Plates were incubated at 30°C overnight with light conditions acquired as stated above.
Construction of fusion proteins and site-directed mutagenesis
Synthetic fusion proteins were constructed using a modified protocol for the sequence and ligation-independent cloning method (84). Site-directed mutagenesis was performed using a protocol modified from the QuikChange Site-directed Mutagenesis kit (Agilent Technologies). Proteins were expressed and analyzed as described above.
Alignment of sequences
Alignments for the cPAC enzyme active site and GAF region were analyzed using MUSCLE (default settings), visualized via JalView, and manually edited in Adobe Illustrator. | 2018-04-26T22:59:23.361Z | 2018-04-09T00:00:00.000 | {
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19626294 | pes2o/s2orc | v3-fos-license | Correlations between Gray Matter and White Matter Degeneration in Pure Alzheimer’s Disease, Pure Subcortical Vascular Dementia, and Mixed Dementia
Alzheimer’s disease dementia (ADD) and subcortical vascular dementia (SVaD) both show cortical thinning and white matter (WM) microstructural changes. We evaluated different patterns of correlation between gray matter (GM) and WM microstructural changes in pure ADD, pure SVaD, and mixed dementia. We enrolled 40 Pittsburgh compound B (PiB) positive ADD patients without WM hyperintensities (pure ADD), 32 PiB negative SVaD patients (pure SVaD), 23 PiB positive SVaD patients (mixed dementia), and 56 normal controls. WM microstructural integrity was quantified using fractional anisotropy (FA), axial diffusivity (DA), and radial diffusivity (DR) values. We used sparse canonical correlation analysis to show correlated regions of cortical thinning and WM microstructural changes. In pure ADD patients, lower FA in the frontoparietal area correlated with cortical thinning in the left inferior parietal lobule and bilateral paracentral lobules. In pure SVaD patients, lower FA and higher DR across extensive WM regions correlated with cortical thinning in bilateral fronto-temporo-parietal regions. In mixed dementia patients, DR and DA changes across extensive WM regions correlated with cortical thinning in the bilateral fronto-temporo-parietal regions. Our findings showed that the relationships between GM and WM degeneration are distinct in pure ADD, pure SVaD, and mixed dementia, suggesting that different pathomechanisms underlie their correlations.
independent of GM lesions has been suggested as a possible mechanism for WM microstructural changes 7,8 . In SVaD, secondary axonal and trans-synaptic degeneration following subcortical injury or global ischemia in the cortex, as well as incidentally combined amyloid pathologies, have been proposed as the underlying mechanisms of cortical thinning. Despite these many hypotheses, there is a paucity of information on the correlation between WM and GM degeneration in ADD and SVaD patients.
To evaluate correlation between GM and WM degeneration, we first investigated the patterns of WM microstructural changes (using DTI) and GM degeneration (using cortical thickness analysis) in patients with pure ADD, pure SVaD, and mixed dementia, which were determined by Pittsburgh compound B (PiB) PET result and WM hyperintensities degree as described in the Methods. We have previously reported that both pure ADD and pure SVaD patients showed decreased FA and increased MD 5 . In this study, we further analyzed axial diffusivity (DA) and radial diffusivity (DR), which are diffusivities parallel or perpendicular to the WM fibers that provide more information regarding axonal or myelin damage, respectively 9,10 . Then, we explored the correlation between WM microstructural changes and GM degeneration in each group using sparse canonical correlation analysis (sCCA), which makes it possible to combine different imaging modalities 11 . We hypothesized that in pure SVaD patients, changes in WM would be related to myelin breakdown and subsequent axonal damage. Thus, in pure SVaD, we expected an increase in DR and a decrease in FA to correlate with cortical thinning. On the other hand, we hypothesized that in pure ADD patients, cortical thinning would be the primary change leading to secondary Wallerian degeneration in adjacent WM. Thus, in pure ADD, we expected decreases in both DA and FA to correlate to cortical thinning 12 . Finally, we hypothesized that mixed dementia would show both ADD and SVaD characteristic and that an increase in DR and a decrease in DA would correlate to cortical thinning.
Results
Patient characteristics. Patient characteristics are shown in Table 1. PiB(+) SVaD patients were older than patients with PiB(+) ADD or PiB(−) SVaD. PiB(−) SVaD patients had higher MMSE scores than PiB(+) ADD and PiB(+) SVaD patients. However, there were no differences in Clinical Dementia Rating sum-of-boxes scores among the three groups of dementia patients.
White matter diffusivity properties in patients with PiB(+) ADD, PiB(−) SVaD, and PiB(+)
SVaD. We compared WM microstructural changes of each dementia group to the NC group. FA changes in PiB(+) ADD and PiB(−) SVaD group compared to NC were reported in our previous study 5 and thus, these results are not described here. The PiB(+) ADD group showed a focal higher DR in the left frontal WM region. DA was lower bilaterally in the parietal and occipital subcortical WM regions adjacent to the cortex, while it was higher in small areas including the corona radiata WM regions (Fig. 1A).
The PiB(−) SVaD group showed more extensive WM microstructural changes. DR was higher in all WM regions. DA was bilaterally higher in the centrum semiovale and corona radiata WM regions, while it was bilaterally lower in the parietal and occipital subcortical WM regions adjacent to the cortex (Fig. 1B).
WM microstructural changes of the PiB(+) SVaD group were similar to the PiB(−) SVaD group. FA was lower and DR was higher in all WM regions. DA was bilaterally higher in the centrum semiovale and corona radiata WM regions, while it was bilaterally lower in the parietal and occipital subcortical WM regions adjacent to the cortex (Fig. 1C). Details of the TBSS parameters are described in Table 2.
Cortical thickness in patients with PiB(+) ADD, PiB(−) SVaD, and PiB(+) SVaD. We compared cortical thickness of each dementia group to the NC group. The PiB(+) ADD group showed cortical thinning in diffuse areas including bilateral medial temporal and precuneus regions while the PiB(−) SVaD and the PiB(+) SVaD groups showed cortical thinning mainly in the frontal, inferior parietal, and temporal regions bilaterally, with medial temporal thinning more involved in the PiB(+) SVaD group (Fig. 2). Correlation between cortical thickness and white matter integrity. In the PiB(+) ADD group, FA was the only DTI parameter that was correlated with cortical thickness. Variation of FA, limited to the frontal and parietal WM regions, correlated with variation of cortical thickness in the left inferior parietal lobule and bilateral paracentral lobules (Fig. 3A).
In the PiB(−) SVaD group, there were marked correlations between all DTI indices (especially FA) and cortical thickness. Variation of FA in extensive WM regions correlated with variation of cortical thickness in the bilateral temporo-parietal, medial frontal, and right frontal areas. Variation of DR in the frontal, temporal and parietal WM regions correlated with variation of cortical thickness in the bilateral lateral frontal, left lateral temporo-parietal, left paracentral lobule, and right medial frontal areas. Variation of DA in the bilateral parietal and occipital WM regions correlated with variation of cortical thickness in the inferior temporal, and bilateral anterior and posterior cingulate areas (Fig. 3B).
In the PiB(+) SVaD group, there were marked correlations between all DTI indices (especially DR and DA) and cortical thickness. Variation of FA in the anterior corpus callosum and scattered WM regions including centrum semiovale correlated with variation of cortical thickness in the bilateral frontal areas. Variation of DR in extensive WM regions correlated with variation of cortical thickness in the bilateral temporo-parietal, medial frontal, and right frontal areas. Variation of DA in extensive WM regions correlated with variation of cortical thickness in the bilateral frontal and right parietal areas (Fig. 3C).
Discussion
We report a novel finding regarding correlation between WM microstructural changes and cortical thinning in pure ADD, pure SVaD, and mixed dementia. The first major finding is that in the pure ADD group, disruption of WM integrity was minimal with lower DA in WM adjacent to the cortex. In pure SVaD and mixed dementia, there was extensive disruption of WM integrity with higher DR and overall higher DA, but lower DA in WM adjacent to the cortex. The second major finding is that cortical thinning in pure SVaD strongly correlated with changes in FA and DR, while cortical thinning in mixed dementia strongly correlated with changes in DR and DA. Taken together, these findings suggest that the relationship between GM and WM degeneration differs according to the underlying pathobiology.
In pure ADD, TBSS revealed minimal disruption of WM integrity with lower DA in WM adjacent to the cortex, higher DA in small areas of deep WM, and higher DR in the left frontal WM area. Our finding that DA was especially lower in WM adjacent to the cortex in the pure ADD group is consistent with our hypothesis that Wallerian degeneration following neuronal death in the GM would damage axonal fibers leading to low DA. Meanwhile, we also found higher DA in small areas of deep WM regions as observed in previous studies 8,13,14 . This might be explained by complex pathobiologies: both axonal and myelin loss, subsequent increase in membrane permeability and glial alteration might cause water diffusion in unanticipated directions, leading to an increase in DA.
In pure SVaD and mixed dementia, TBSS showed extensive disruption of WM integrity with higher DR and DA. These results are consistent with previous studies showing that increases in DR and DA were associated with chronic WM degeneration 15,16 . Interestingly, we found a unique pattern of DA change according to the distance from the cortex. DA was lower in the WM adjacent to the cortex in the posterior region, while it was higher in the deep WM regions, which might be related to different underlying pathobiologies. Although a similar pattern was found in the pure ADD group, DA was predominantly increased in the pure SVaD and mixed dementia groups. One possible explanation might be related to vulnerability to chronic ischemia. That is, in both pure SVaD and mixed dementia, deep WM regions, including periventricular WM areas, are more vulnerable to chronic ischemia which is represented by WMH on MRI 17 . Previous pathology studies showed that WMH regions had low myelin content and axonal loss or damage 18,19 . This leads to a decrease in tract volume and an increase in the surrounding extracellular fluid, contributing to increased isotropic diffusivity with correspondingly higher DR and DA 16,20 . On the other hand, WM regions adjacent to the cortex in pure SVaD and mixed dementia are relatively spared from chronic ischemia 17,21 . Lower DA in this region may be due to secondary Wallerian degeneration from combined cortical atrophy 12 . Alternatively, the direction of DA change might reflect the stage of WM degeneration. A previous study showed that DA initially decreased after axonal damage and subsequently increased over time 16 . Therefore, higher DA in deep WM regions might be related to chronic WM degeneration, while lower DA adjacent to cortex might be related to recently developed WM degeneration.
sCCA revealed a disease-specific correlation pattern between GM and WM degeneration. Although the pure ADD group showed cortical thinning and significantly lower FA, there was little correlation between the two. In addition, no correlation was observed between lower DA (as determined by TBSS analysis) and cortical thinning. Although we may not have found significant correlations due to a small sample size, our findings suggest that the overall WM microstructural changes in pure ADD may occur independent of cortical pathology.
In the pure SVaD group, lower FA and higher DR (as determined by TBSS analysis) correlated with cortical thinning, while in the mixed dementia group, higher DR and higher DA (as determined by TBSS analysis) correlated with cortical thinning. In both the pure SVaD and mixed dementia groups, the cortical regions that correlated with WM integrity were mainly located in the lateral temporal, inferior parietal, and the lateral and medial frontal areas, which indicated that neurodegeneration in those areas paralleled WM degeneration. In both groups, chronic ischemia may primarily result in myelin breakdown, which is represented by higher DR. The degeneration process may propagate in a retrograde manner and result in neuronal cell death. Alternatively, disconnection of the cortical-subcortical loop may result in secondary cortical thinning. It is also possible that chronic ischemia may cause GM-WM correlation by simultaneously attacking GM and WM. However, the fact that different DTI indices correlated with cortical thinning in the pure SVaD and mixed dementia groups suggests that different pathomechanisms may underlie the GM-WM correlation in each disease. A higher vascular burden, such as lacunes, in pure SVaD might result in a more pronounced DR increase and FA decrease when compared to mixed dementia. In mixed dementia, the presence of an amyloid burden in addition to the chronic ischemic condition may alter the pattern of GM-WM correlation such that axial (DA) or radial (DR) diffusivity, rather than directionality of diffusion (FA), matters more for the association with widespread cortical thinning. Additionally, there was little correlation between lower FA and cortical thinning in mixed dementia patients, which suggested that these might occur independently in mixed dementia patients. There are several limitations in this study. Firstly, we did not conduct PiB-PET scanning on the NC group. Because previous studies showed that approximately 10 to 30% of cognitively normal subjects are PiB positive 22, 23 , we might have underestimated the degree of WM microstructural changes and cortical thinning in the patient groups. Secondly, the mean ages of the NC and dementia groups were different. However, since the patients were consecutively recruited, we believe this reflected participant characteristics. To minimize the effect of age difference across the groups, we performed the analysis after adjusting for age. In addition, in the GM-WM correlation analysis, we calculated each patient's W score, which is an age-adjusted Z score relative to the control group 24,25 . Finally, sample size was relatively small and the number of subjects was not even across the four groups. Thus, we might have missed some important information because of low statistical power.
In conclusion, we revealed distinct GM-WM relationships in the dementia spectrum of ADD and SVaD (pure ADD, pure SVaD, and mixed dementia). We suggest that the relationship between GM and WM degeneration differs according to the underlying pathobiology of each condition.
Participants.
We prospectively recruited 139 patients who were clinically diagnosed with SVaD (n = 70) or ADD (n = 69). Patients underwent 11 C-PiB-PET scanning and MRI between September 2008 and August 2011. All SVaD patients fulfilled the Diagnostic and Statistical Manual of Mental Disorders -Fourth Edition (DSM-IV) criteria for vascular dementia and had severe WMH on MRI, which was defined as periventricular WMH (caps or band) ≥10 mm and deep WMH ≥25 mm in maximal diameter according to the modified Fazekas criteria 26 . Patients with WMH due to radiation injury, multiple sclerosis, vasculitis, or leukodystrophy (based on clinical history and other information such as blood test results) were excluded. Among the 70 SVaD patients (47 PiB(−) SVaD and 23 PiB(+) SVaD), 15 patients with PiB(−) SVaD were excluded due to preprocessing errors such as failure in brain mask extraction or registration to a common space.
ADD was diagnosed based on the criteria for probable AD proposed by the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association (NINDS-ADRDA) 27 . Among the 69 patients with ADD, we excluded 7 patients who were negative for PiB PET, 14 patients who exceeded mild WMH (periventricular WMH <10 mm and deep WMH <10 mm in maximal diameter), 2 patients who did not have one of the required imaging modalities (DTI or FLAIR), and 6 patients who showed preprocessing errors such as failure in brain mask extraction or registration to a common space. We also recruited 56 normal controls (NC) who had no history of neurologic or psychiatric illnesses and no abnormalities detected during neurological examination. These were classified to be cognitively normal by neuropsychological testing. All the NC had no or mild WMH (periventricular WMH <10 mm and deep WMH <10 mm in maximal diameter) on MRI.
The final patient sample consisted of 32 patients with PiB(−) SVaD (pure SVaD), 23 patients with PiB(+) SVaD (mixed dementia), 40 patients with PiB(+) ADD (pure ADD), and 56 NC. The same study sample of pure SVaD, pure ADD, and NC was used in our previous study 5 . The criteria for a PiB-positive scan are described in the PET acquisition and data analysis section.
We obtained written informed consent from each participant and the Institutional Review Board of Samsung Medical Center approved the study protocol. All methods were carried out in accordance with the approved guidelines.
MRI techniques.
Patients underwent standardized T2, three-dimensional (3D) T1 turbo field echo, 3D fluid-attenuated inversion recovery (FLAIR), and DTI images at Samsung Medical Center using a 3.0T MRI scanner (Philips 3.0T Achieva). For 3D T1 turbo field echo MR images, the following parameters were used: sagittal slice thickness, 1.0 mm, over contiguous slices with 50% overlap; no gap; repetition time (TR) of 9.9 msec; echo time (TE) of 4.6 msec; flip angle of 8°; and matrix size of 240 × 240 pixels, reconstructed to 480 × 480 over a field of view (FOV) of 240 mm. For 3D FLAIR images, we used the following parameters: axial slice thickness of 2 mm; no gap; TR of 11000 msec; TE of 125 msec; flip angle of 90°; and matrix size of 512 × 512 pixels. For whole-brain DT-MRI examinations, sets of axial diffusion-weighted single-shot echo-planar images were obtained: 128 × 128 acquisition matrix, 1.72 × 1.72 × 2 mm 3 voxels; reconstructed to 1.72 × 1.72 × 2 mm 3 ; 70 axial slices; 22 × 22 cm 2 field of view; TE 60 ms, TR 7696 ms; flip angle 90°; slice gap 0 mm; b-factor of 600 smm −2 . We acquired diffusion-weighted images from 45 different directions. We used the baseline image without weighting [0, 0, 0]. PET acquisition and data analysis. All patients completed a 11 C-PiB PET scan at Samsung Medical Center or Asan Medical Center with identical settings using a Discovery STe PET/CT scanner (GE Medical Systems, Milwaukee, WI, USA). To measure PiB retention, we used the cerebellar gray matter as the reference region and calculated the cerebral cortical region to cerebellum uptake ratio. Patients were considered PiB-positive if their global PiB uptake ratio was more than two standard deviations from the mean of the normal controls (PiB retention ratio ≥1.5) 28 . The detailed radio chemistry profiles, scanning protocol and PiB-PET data analysis are described in the supplementary information.
DTI Processing. FMRIB's Software Library (FSL v5.0.2.1, http://www.fmrib.ox.ac.uk/fsl) was used to process DTI data. We corrected motion artifacts and eddy current distortions by normalizing each diffusion-weighted volume to the non-diffusion-weighted volume (b0) by applying the affine registration method in the FMRIB's Linear Image Registration Tool (FLIRT v6.0). Using a general linear-fitting algorithm, diffusion tensor matrices were generated from the sets of diffusion weighted images. Subsequently, we obtained FA, DA, and DR for each voxel according to simple linear fitting algorithm using the DTIFIT Tool (part of FSL).
SCIENTIfIC RePoRTS | 7: 9541 | DOI:10.1038/s41598-017-10074-x Tract-based spatial statistics (TBSS) analysis. The FA, DA and DR maps of DTI pre-processing results were used for TBSS analysis 29 . We aligned all FA images onto a standard FMRIB58 FA template which was provided by the FSL software. In this process, we used a nonlinear registration algorithm implemented in the TBSS package. After alignment, the FA images were averaged to create a skeletonized mean FA image. Aligned FA images from each patient were projected onto the skeleton by filling the skeleton with the highest FA values at the nearest relevant center of fiber tracts. We chose a threshold FA value of 0.2 to exclude voxels of GM or cerebrospinal fluid (CSF). DR and DA images were also processed by implementing the FA nonlinear registration and projecting them onto the skeleton using identical methods to those derived from the original FA data.
Image processing for cortical thickness measurement. The CIVET anatomical pipeline was used to extract cortical thickness (http://mcin-cnim.ca/neuroimagingtechnologies/civet/) 30 . In brief, using a linear transformation, native MRI images were registered to the Montreal Neurological Institute (MNI) 152 standard space 31 . The N3 algorithm was used to correct the images for intensity-based non-uniformities 32 caused by non-homogeneities in the magnetic field. Then, the registered and corrected images were classified into WM, GM, CSF, and background using a 3D stereotaxic brain mask and the Intensity-Normalized Stereotaxic Environment for Classification of Tissues (INSECT) algorithm 33 . The surfaces of the inner and outer cortex were automatically extracted using the Constrained Laplacian-based Automated Segmentation with Proximities (CLASP) algorithm 34 .
Cortical thickness was defined as the Euclidean distance between the linked vertices of the inner and outer surfaces; there were 40,962 vertices in each hemisphere in native space 34 . The thickness value was spatially normalized using surface-based two-dimensional registration with a sphere-to-sphere warping algorithm. Thus, the vertices of each subject were nonlinearly registered to a standard surface template to compare thickness across subjects 35,36 . Cortical thickness was subsequently smoothed using a surface-based diffusion kernel in order to increase the signal-to-noise ratio. We chose a 20-mm full-width at half-maximum kernel size to maximize statistical power while minimizing false positives 37 .
The presence of extensive WMH in the MRI scans made it difficult to completely delineate the inner cortical surface with the correct topology due to tissue classification errors. To overcome this technical limitation, we automatically defined the WMH region using a FLAIR image and substituted it for the intensity of peripheral, normal-appearing tissue on the high-resolution T1 image after affine co-registration, as described in earlier studies 38 . This method was applied to all participants. W-score maps. We carried out W-score mapping to identify the degree of cortical atrophy and WM microstructural changes in each patient using cortical thickness and DTI measures (i.e., FA, DA and DR) with the NC as a reference. Details on the theory and computation of W-scores are available elsewhere 25,39 . In this study, W-score maps were computed vertex-wise for the surface model and voxel-wise for the skeletonized volume of each imaging data set according to the following formula: W-score = [(patient's raw value) − (value expected in the control group for the patient's age, sex, and level of education)]/(SD of the residuals in the control group). W-scores are similar to Z-scores in that they have a mean value of 0 and a SD of 1 in the control group, and values of +1.65 and −1.65 correspond to the 95 th and 5 th percentiles, respectively. However, W-scores are adjusted for specific covariances such as age, sex, and level of education. To avoid confusion with respect to W-score direction in cortical thickness and DTI measures, we used the W-scores as positive values indicating larger cortical thickness and larger values of FA, DA and DR.
Sparse canonical correlation analysis (sCCA). Because traditional canonical correlation analysis is severely limited when the dimensionality of the data is larger than the number of subjects 11 , sCCA was used [40][41][42][43] . sCCA attempts to find relationships between two sets of multi-voxel or multi-vertex dimensional W-scores of DTI measures (i.e., FA,DA, DR) and cortical thickness independently obtained from DTI and T1 imaging modalities. In other words, it simultaneously finds the canonical weight vectors in each imaging modality that maximize the correlation of the projections of each DTI measure and cortical thickness input data onto their canonical weight vectors. sCCA is particularly useful in that it controls the influence of outliers on the computed correlations and automatically locates the most reliable and informative sets in one modality that are related to another modality using a sparse selection process with a regularized energy-minimization approach. sCCA for each patient group was carried out using R packages (http://cran.r-project.org/web/packages/PMA/) with positivity and sparseness constraints. The sparseness parameters, which control the sparsity for either set of the canonical variates, were selected as approximately half of the voxel/vertex dimension of the input data to focus on spatially-distributed patterns. For visualization, canonical weight vectors of each imaging data set were mapped onto an ICBM 152 surface model for cortical thickness and volume template for DTI measures. We used permutation testing of 2,000 iterations to assess sCCA significance. For permutation testing, we randomly reordered the possible pairs of the two input images (one of the DTI measures (i.e., FA, DA, DR) and the cortical thickness). However, the two images were not selected from the same subject. The p-value for canonical variates was estimated as the ratio of the number of the permutations in which correlation value exceeded the original correlation value to the number of total permutations. Significance was defined as p < 0.05.
Statistical analysis.
To analyze demographic and patient characteristic data, one-way ANOVA for continuous variables and Chi-square tests for dichotomous variables were performed. A two-sided p < 0.05 was considered statistically significant.
To test for localized differences in the DTI measures of each dementia group compared with the NC group, voxel-wise statistical analysis of individual skeleton images was performed using a nonparametric permutation test. We included age, sex, and level of education as covariates in the analysis of covariance (ANCOVA), and the null distribution was built up over 5,000 permutations. We used threshold-free cluster-enhancement (TFCE) with the 2D parameter settings 44 to avoid an arbitrary threshold of an initial cluster-formation. To correct for multiple comparisons, the results for DTI measures (i.e., FA, DA and DR) were considered significant at a family-wise error (FWE)-corrected P < 0.05. To estimate the differences in cortical thickness for each dementia group compared with the NC group, general linear modeling and random field theory (RFT) were applied using the SurfStat toolbox (http://www.math.mcgill.ca/keith/surfstat/) 45 . General linear analysis was performed after controlling for age, sex, and level of education. To correct for multiple comparisons, the results for cortical thickness were considered to be significant at RFT corrected p < 0.05.
The results of cortical thickness and DTI measures were projected on an ICBM 152 surface template and volume template for visualization. | 2018-04-03T02:48:13.425Z | 2017-08-25T00:00:00.000 | {
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44354261 | pes2o/s2orc | v3-fos-license | News Analysis : A Case Study of the Thematic Structure of News Texts in National Iranian and American diaries
The objective of this article is to illustrate the structures of some national and international news in the press. Using discourse analysis of the processes and structures of news reports by van Dij k (1988) as a framework, this study refers to an exam in tion and determination of the thematic structure of news texts in Iran and United States’ press based o n a case study of reporting of U.S. and Russia's de al on Syria’s chemical weapons. This study investigate s whether newspapers from different countries and regions of the world and produced in different poli tical and ideological contexts provide equally vari able types of description of such a world event. We hope that the answer to this and other related question s may help us gain a better understanding of the deba te over the perceived imbalance in international ne ws sources, topics, and distribution. Key wordsThematic structure, news texts, national and intern atio al news sources, discourse analysis
INDIA: COURT BANS TOBACCO BOARD FROM TRADE SHOW
The Global Tobacco Networking Forum (GTNF) 2010, billed by its organisers as 'The greatest tobacco talk show on Earth', was held in October in the south Indian city of Bengaluru (formerly Bangalore). The popular Indian slogan, 'India shining', encapsulating the optimistic spirit of India's ongoing economic resurgence, might well have been uttered by conference delegates contemplating the country's thriving tobacco market. However, it is in stark contrast to another aspect of India today: around one million people dying from tobacco related illnesses every year.
The conferencedthe third of its kind, previously held in Brazil and Thailandd offered its delegates (only representatives of the industry) a unique package of experiences. First, a field trip to have a close-up view of tobacco fields, crop varieties, auction platforms, best practices and interaction with tobacco growers. Second, a two-day interactive meeting to present and discuss burning contemporary issues of the industry. The topics at the interaction ranged from routine manufacturing issues ('sustainability', 'ingredients'), to trade regulation and policy ('FCTC: what it is, what it should be', 'taxing to the max'). The event was a closed one where, apart from stringent rules on eligibility to participate, there was also a strict code of conduct limiting note-taking, prohibiting photography and presumably controlling press coverage (which was negligible).
Given the fact that leading Indian and international tobacco companies sponsored the forum, it is reasonable to conclude that much of the agendadapart from 'networking'dwas promotion and celebration of the industry. Unfortunately, the Indian Tobacco Board of India (ITB), a government entity established under the Union (federal) ministry of industry and commerce, was one of the sponsors. By its own admission through a right to information (RTI) act reply, ITB committed 326 630 INR (approximately US$7250) to guide the delegates on the field trip. This was done despite the fact that India ratified the FCTC in 2004 and has a national tobacco control law that prohibits direct or indirect promotion of tobacco in the country.
After spotting the logo of ITB on the GTNF website, civil society organisations mounted concerted advocacy campaigns, including writing to concerned political leaders, gathering signatures, organising a 'walkathon' protest, engaging media and an online petition against the government's sponsorship of this private trade event. Reacting to a question raised in the lower house of parliament, the minister of state for commerce and industry acknowledged concerns expressed by NGOs, but justified the sponsorship in the context of the mandate of ITB to promote Indian tobacco varieties.
With no favourable response from ITB, the Institute of Public Health (IPH), a Bengaluru-based NGO, filed a public interest law suit in the provincial (Karnataka) court. In its third hearing on 17 September, the high court ruled that such sponsorship amounted to indirect promotion of tobacco and hence violated section 5 of the national tobacco control law and article 13 of the WHO's Framework Convention on Tobacco Control (FCTC). The court duly issued an interim order directing ITB to withdraw its sponsorship, its logo and any participation in GTNF 2010.
Although a comprehensive ban on tobacco advertising, promotion and sponsorship is a long way off in India, this verdict is a significant development towards it. It reflects the challenges faced by many countries that have stateowned tobacco operations or a conflicting mandate (controlling and promoting tobacco at the same time). It also highlights the importance of legal measuresd the FCTC and a strong national tobacco control lawdas well as the role of vigilant civil society organisations and the judiciary. and recently ended it with the 'Healthy Shisha', seemingly the water-pipe equivalent of the electronic cigarette. This latest arrival has been heralded as a 'unique Lebanese invention', with 'certification from Swiss laboratories' and registration throughout the world. A mass media marketing campaign has been ongoing through television, radio, online and printed media.
UPENDRA
A dedicated website with a nature theme (www.healthy-shisha.com) describes it as "a new, smart electronic smoking device which provides you an alternative to traditional tobacco smoking (using) advanced technology to provide the user smoking sensations without any fire, flame, tobacco, tar, carbon-monoxide, cancer causing carcinogens, or offensive secondhand smoke found in tobacco". Seven 'natural' flavours are offered in the form of a liquid solution, with ingredients such as chocolate, bread, perfume, honey, coffee and tea, which is why they claim "we call it Healthy Shisha." Interestingly, the website also includes a section on the dangers of both shisha and cigarette smoking, much of which has been copied from the website of the US Centers for Disease Control (CDC). Online purchase of various models of 'Healthy Shishas' is also offered, with prices ranging, depending on size, from the equivalent of US$150 to US$220, with accessories such as car and solar chargers available. More than 50 individual designs of shishas are advertised in different colours and shapes, including one using soccer balls, seemingly for homes where a sports enthusiast rules the roost.
Lebanese health advocates are concerned that this product is being widely marketed to the public despite the absence of evidence of its harmlessness, and while using deceptive claims. They say it will not only appeal to potential smokers as being harmless to consume, India: health advocates protesting about the Global Tobacco Networking Forum in Bengaluru (Bangalore) in October.
All articles written by David Simpson unless otherwise attributed. Ideas and items for News Analysis should be sent to: David.Simpson@ctsu.ox.ac.uk but also that people will be enticed by the manufacturer 's claim that they can now have their freedom back to 'smoke' indoors or wherever they want, including in currently smoke-free places. One version of the television advertisements even portrayed a wife and child who were no longer being exposed to secondhand smoke, as the husband used the 'Healthy Shisha. ' The marketing of this product began shortly after a month-long national media campaign against secondhand smoke, clearly playing into the public's increasing awareness of the dangers of tobacco, particularly during the past year, when new tobacco control legislation was debated.
The case is being followed by the National Tobacco Control Program within the Ministry of Public Health, with a possible ban on the 'Healthy Shisha' being sought. However, the series of photographs was not direct tobacco advertising, which has been prohibited in German periodicals, newspapers and magazines since January 2007, under European Union law. Nevertheless, there was clearly more than a whiff of product placement.
JADE KHALIFE, GEORGES SAADE
First, the tendency of Philip Morris International (PMI), makers of Marlboro, to circumvent regulations is well known from other areasdfor example, even though tobacco companies have been legally bound not to pay for product placement in movies since 1998, research shows that Marlboro is still the leading brand on screen in US motion pictures. Second, PMI is notorious for successfully implementing surreptitious advertising in movies as long ago as 1979, in Superman II. Why should it not be finding a way to do it, even now, in magazines?
Third, there is strong evidence that PMI continues to conduct detailed research on young adults and their lifestyles, in order to create the most effective promotional strategies. In this connection, it is worth noting that the reasons young females read magazines such as Glamour, being highly interested in their appearance and in order to create an up-to-date, stylish image for themselves, are similar to those of girls and women for starting or continuing to smoke, as a perceived status enhancement and as a driving force for staying thin.
It is hard to imagine a better platform than fashion magazines from which to address a young female audience susceptible to promotional messages; reading fashion magazines by adolescents is linked to a higher likelihood of smoking. In addition, the intimate style of presentation of the model in Glamour's series of photographs does nothing to dampen suspicion that the Marlboro cigarette packs have not appeared accidentally in her hands: she gives the impression of being dreamy, uninhibited and perhaps almost 'stoned', and more than one of the pictures shows a glimpse of her underwear. The pictures offer conspicuous accommodation of evidence that sexy smoker stereotypes are associated with smoking susceptibility (see Tob Control 2004;13:308e14).
On PMI's web home page the company claims that children and adolescents do not belong in the target groups of its marketing strategies and therefored among other policiesdit does not employ models under 25 years old in its advertisements. Also, offers for product placement are also said to be declined. However, one statement can be regarded as true without reservation, namely that 'marketing is one of Philip Morris International's great strengths.'
KARIN MARUSKA Institute for Therapy & Health Research,
Kiel, Germany maruska@ift-nord.de INDONESIA: WESTERN BANDS PLAY PIED PIPER AT TOBACCO PROMOTION From 8e10 October, at Jakarta's Ancol Beach, the Gudang Garam cigarette company was the major sponsor of Java Rockin' Land, one of the largest music festivals ever seen in Asia. Among the line-up supporting the lead act, the Smashing Pumpkins, were Wales' Stereophonics, Australia's Wolfmother and The Vines, and bands from several European countries.
Following calls by tobacco control advocates for the bands to either pull out or demand that the tobacco sponsorship be dropped, Wolfmother's frontman Andrew Stockdale posted a notice on the group's fanpage saying, "With the severity of the issue of smoking in Indonesia, I completely understand that bands playing with promo girls handing out cigarettes isn't going to help kids stop smoking in Indonesia. So it is without hesitation I will now announce that we will be cancelling our headline show . We are very regretful to miss this opportunity to play to our Indonesian fans, though hopefully in the not too distant future we can do more shows under different terms." Wolfmother's name disappeared from the festival website for a day but then reappeared after apparent management Germany: one of 11 photographs in a special feature in the German edition of Glamour fashion magazine, three of which showed the model holding a Marlboro cigarette pack.
Lebanon: a scene from one of the television advertisements for the new 'Healthy Shisha' (water-pipe). As in this scene from one of the ads, much of the time of each spot is in splitscreen mode, comparing a regular shisha in black and white, with smokedthe child and her mother can be seen trying to wave it awayd opposite a 'healthy shisha' in colour and without smoke.
intervention: 'This one is for the fans in Indonesia who have parted with their very own cold hard cash to see Wolfmother. We realize their (sic) are sponsors and we neither support or condemn the sponsors affiliated with the festival,' said the new notice.
Had they been born at the time, would the band have played in South Africa 'for the fans' under the then totally legal apartheid? Or would they have played 'for the fans' if they'd have been born in the 19th century and a wealthy, then totally legal slave-trading company had stumped up the sponsorship? But playing to help a totally legal tobacco company promote smoking to a half-price admission for kids gig? Hey . it's 'for the fans'.
By clicking on the festival website and selecting 'international', the terms and conditions tab showed conditions numbered AeS. The final condition, S, was added in the week the criticism began. It read, "One or some stages are sponsored by tobacco company and therefore audience below 18-years-old or pregnant woman are not allowed to enter the show." But to those who clicked on the Indonesian entry portal, condition S was missing. Western sensibilities about the tobacco sponsored festival being adult entry were thus cynically catered for, but in reality, Indonesian kids were welcome and got half-price tickets.
The bands' decisions to play contrast with the action taken by American singer Alicia Keys who in 2008 refused to play a Jakarta tobacco gig, unless all sponsorship and marketing by Philip Morris' Sampoerna brand was stopped. She got her way, as did American Idol winner Kelly Clarkson earlier this year when she sang there.
Indonesia has over 73 million smokers, 95 per cent of them men. Around 66 per cent of men smoke. The country is one of a handful of which have yet to ratify the WHO's Framework Convention on Tobacco Control (FCTC). Indonesia has virtually no tobacco control policies or significant education programmes. British American Tobacco (Bentoel) and Philip Morris (Sampoerna) are both massively engaged in Indonesia, a kind of last frontier of Marlboro Country. Despite repeated unctuous statements from both companies about their corporate social responsibility and not wanting youth to smoke, they are frequent sponsors of youth-oriented music events. Admission often includes free cigarettes.
Earlier this year, pictures of a 2-year-old Indonesian boy, Ardi Rizal, were published around the worlddGoogle shows over a million hits. My Indonesian public health colleagues were very ambivalent about the publicity. While it focused unprecedented attention on smoking in Indonesia, the global conversation that flowed was all about the boy's irresponsible parents and the need for more education. Rizal was depicted as a freak show, with US network crews dispatched to track him down in his village and get him on film to make the world gasp in disbelief. What was missing in any of the coverage was the role of the tobacco industry in lobbying to keep Indonesia a tobacco industry paradise where publicity for international music acts like Java Rockin' Land wallpapers the country's media.
In an interview last year, Wolfmother's Stockdale reflected on his newborn daughter: "And you look at her and you think, she's four months old. What kind of world is she going to live in when she's, you know, 40? That's a scary thought." The scary thought is that nations like Indonesia can still play open host to massive scale tobacco promotions and that international entertainers are lining up to help the companies sell as much tobacco as possible.
In October, 700 delegates from 41 countries who attended the Asia Pacific Conference on Tobacco or Health (APACT) in Sydney, Australia, denounced Indonesia for being the only Asian country not to have signed the FCTC; and urged foreign musicians and athletes performing in Indonesia to boycott tobacco-sponsored events.
SIMON CHAPMAN
University of Sydney, Australia simon.chapman@sydney.edu.au
AMERICAS: WHO WARNS OF INDUSTRY SUBVERSION
Health leaders meeting in September at the Pan American Health Organization (PAHO), the Americas division of the WHO, vowed to counter attempts by the tobacco industry to subvert public health efforts aimed at protecting people from the harmful effects of tobacco use. PAHO's Directing Council, which brings together ministers of health and other high-level delegates from throughout the Americas each year, issued a resolution stating that its members were 'deeply concerned about misinformation campaigns and legal actions' sponsored by cigarette makers and their allies against tobacco control measures. They called on countries to publicise, to the extent legally possible, the activities of the tobacco industry, to expose their strategies and reduce their effectiveness.
The resolution expressed specific support for Uruguay and measures it has implemented that have made it a pioneer in tobacco control in Latin America. Evidence of industry interference in health policy was heard, among other examples, from Uruguay's near neighbour, Paraguay, whose health minister told delegates that tobacco industry opposition was threatening to halt her government's efforts to comply with the WHO's Framework Convention on Tobacco Control (FCTC). She cited recent presidential decrees, banning smoking in enclosed public spaces and regulating labelling and packaging of tobacco, respectively, which had been suspended due to legal injunctions and a challenge to their constitutionality by the tobacco industry, at both the national and international levels.
Twenty-seven countries, three-quarters of all PAHO member countries, have ratified the FCTC. ThreedChile, Cuba and Venezueladtax tobacco products at 75 per cent of the retail price; nine report that they have national or local laws that cover at least 90 per cent of their populations, with bans on smoking in all enclosed public areas and work places without exception; and 17 countries ban the use of misleading labels and require that warnings occupy more than 30 per cent of the main package surfaces. Colombia and Panama have comprehensive laws banning all forms of tobacco advertising, promotion and sponsorship.
USA: PHARMACY TOBACCO SALES BAN
In September, San Francisco expanded its ban on the sale of cigarettes in pharmacies, turning a judicial setback into a legislative triumph.
In 2008, San Francisco became the first US city to mandate tobacco-free pharmacies, but the law came under challenge. The Walgreens retail pharmacy chain argued that it was being discriminated against because tobacco sales remained legal in grocers with pharmacy counters and in 'big box' stores, huge retail outlets that sell everything from toothpaste to car tyres. In June, the city's First District Court of Appeal sided with Walgreens, agreeing that the big box store exemption was arbitrary and unfair.
Rather than allow sales to resume at all pharmacies, however, the San Francisco Board of Supervisorsdthe city's governing councildvoted 7e3 to expand the ban, thus levelling the playing field. As City Supervisor Eric Mar, a co-sponsor of the legislation, noted, 'Cigarettes and pharmacies don't mix. Pharmacies should promote healing and protect our health.' Although the margin of the vote was not high enough to be veto-proof, Mayor Gavin Newsom is expected to sign the measure into lawdit was his idea in the first place.
Prior to the vote, the Safeway grocery chain revealed candour in expressing its opposition to the ban's expansion. Spokeswoman Susan Houghton argued, "For us it's kind of throwing the baby out with the bath water. We do obviously have healthy foods in our stores, and we do sell products that might be less healthy. For us it's about providing an array of products for our customers." Might be less healthy? This begs the question of Safeway's next advertising campaign. Will it appeal to customers to "come to us for all your health -and less healthy -needs"?
Even more intriguing will be Walgreens' own reaction. In attempting to rally customers on its side in advance of its successful court challenge, the chain noted, 'The proposal will force smokers to liquor stores, tobacco shops, gas stations or other retailers that don't carry smoking cessation products and don't have pharmacists available for advice on quitting.' So, deprived of their tobacco sales, will the company that boasts of offering 'specialty pharmaceuticals and wellness services' now drop nicotine replacement therapy (NRT) sales and petulantly instruct its pharmacists to send smokers to gas stations for cessation advice it knows they won't get?
USA: PHARMACY ITEMS AMONG A MILLION IN COLLECTION
The satirical exhibit "Your cancer and drug store"dsee leftdis just one manifestation of the career-long passion of Dr Alan Blum, family physician and founder of the US ginger group DoC -Doctors Ought to Care (and first editor of this journal's News Analysis section). In particular, he has long refused to accept the inconsistency between medical, health and other trusted leaders in society who have an ambivalent attitude to tobacco, including pharmacies which profit from serving the interests of health while at the same time selling tobacco products. In the mid-1990s, Blum saw that a convenience store in Hartford, Connecticut was up for sale, complete with a historic collection: the seller had saved virtually all the cigarette advertising displays and other promotional items used in his shop over the previous 20 years. Blum, long noted for his creative use of satire for education about tobacco, purchased the lot. Thus was born the concept of Your Cancer and Drug Store.
However, the pharmacy ephemera comprises but a fraction of the tobacco related promotional material and other associated items, now numbering more than a million, amassed by Blum. The collection ranges from tobacco-branded advertising signs, sports bags, ash trays, fashion accessories, T-shirts, caps, cameras, CDs, mugs and other give-away items distributed by tobacco companies with the purchase of cartons or packs of cigarettes and spitting tobacco, to a Philip Morris Supports the Arts sweatshirt, an infant-sized Virginia Slims Tennis t-shirt, 1940s get-well cards with a cigarette theme, a Philip Morris sign placed at the bedside of hospital patients reminding them not to smoke in bed, an RJ Reynolds sales representative's book from the 1920s with the same Camel advertisement in more than 50 languages for placement in neighbourhood stores and ethnic newspapersdthe list itself is almost an exhibit of the exploitation of a vast galaxy of promotional tactics to try to ensure that the world smoked as much as possible.
It also includes significant publications that chronicle the history of tobacco control, such as original newspaper headlines on the publication of the Royal College of Physicians of London pioneering report in 1962 and the first Surgeon General's report in 1964. The significance of these early scientific reports is given added context by the collection's magazines from the 1950s and 1960s that include planted articles by tobacco companies debunking the dangers of smoking covered by these early reports. Postage stamps alone, collected from all over the world by Louisiana chest physician Dr Jim Lutschg, form such a large sub-collection that selections have already been exhibited separately (see Tob Control 2010;19:354). Now this unique collection is so large, with attendant access and storage restrictions, that Alan Blum and colleagues are searching for a permanent home for it that will allow it to be used by future generations of tobacco control researchers and advocates, as well as those studying the history of medicine, marketing, tobacco control or any other of the many facets of tobacco and disease history that it illuminates. USA: the satirical exhibit "My Cancer and Drug Store", highlighting the inappropriateness of tobacco being sold in some US pharmacies, is seen on show in Buffalo, New York State in 2009, with its creator, Dr Alan Blum and a health promotion colleague, Rebecca Murphy-Hoefer.
Pakistan: this graphic anti-tobacco advertisement has been appearing on the backs and sides of buses on the busy route between the northern city of Rawalpindi and Islamabad, the capital. They are sponsored by the ministry of health, which hopes to obtain more funds so that the campaign can be expanded. Bus travel is the most common form of public transport in Pakistan, and if coverage could be extended to other city routes and to buses plying the long trunk roads in the country, the eye-catching ads could be seen by a significant proportion of tobacco users in both urban and rural environments. | 2018-04-03T06:05:10.812Z | 2013-01-01T00:00:00.000 | {
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7723357 | pes2o/s2orc | v3-fos-license | ABO phenotypes and malaria related outcomes in mothers and babies in The Gambia: a role for histo-blood groups in placental malaria?
Background Host susceptibility to P.falciparum is critical for understanding malaria in pregnancy, its consequences for the mother and baby, and for improving malaria control in pregnant women. Yet host genetic factors which could influence placental malaria risk are little studied and there are no reports of the role of blood group polymorphisms on pregnancy outcomes in malaria endemic areas. This study analyses the association between ABO blood group phenotypes in relation to placental malaria pathology. Methods A total of 198 mother/child pairs delivering in Banjul and the Kombo-St Mary District (The Gambia) were analysed. ABO blood group was measured by agglutination. Placental malaria parasites wee enumerated and the presence of malaria pigment noted. Birth anthropometry was recorded and placental weight. Maternal and infant haemoglobin was measured. Results 89 (45%) subjects were primiparae and 110 (55%)multiparae. The ABO phenotype distribution was 38(A), 52(B), 6(AB) and 102(O). Placental histo-pathology showed active placental malaria in 74 (37%), past infection in 42 (21%) and no infection in 82 cases (41%). In primiparae blood group O was associated with a higher risk of active infection (OR = 2.99; 95% CI = 1.24–7.25), and a lower risk of past infection (OR = 0.31, 0.10–1.01, p < 0.05). In multiparae the O phenotype was associated with reduced prevalence of active or past placental infection (OR = 0.45; 95% CI 0.21–0.98). The mean feto-placental weight ratio was significantly higher in multiparae with group O women compared to non-O phenotypes (5.74 vs 5.36; p = 0.04). Among primiparae with active placental infection, mean birth weight was higher in children of mothers with the O phenotype (p = 0.04). Conclusion These results indicate that blood group O was significantly associated with increased placental malaria infection in primiparae and reduced risk of infection in multiparae. This parity related susceptibility was not present with other ABO phenotypes. Cell surface glycans, such as ABO and related antigens have special relevance in reproductive biology and could modulate specific cell interactions as those associated with the pathogenesis of placental malaria.
Background
In malaria endemic areas, the increased risk of P. falciparum infection during pregnancy, which is associated with placental parasitaemia, imposes a heavy burden on the health of mothers and newborns [1,2], contributing to maternal anaemia, low birth weight, and infant mortality especially among primiparae [3,4]. At present, available preventive and therapeutic tools can only achieve a partial reduction in the health hazards caused by placental malaria [5,6]. In this context, a better knowledge of host susceptibility to placental P.falciparum infection is central for improving understanding of malaria in pregnancy, as a basis for improved control.
There is increasing evidence that both the risk of acquiring P. falciparum infection, and the risk of developing severe complications are determined by host genetic factors [7]. The protective role of several erythrocytic variants, some of them related to blood groups, is one of the best examples of this genetic modulation [8]. These include haemoglobins S, C and E, α and β thalassaemias, Glucose-6phosphate dehydrogenase deficiency, Southern Asian Ovalocytosis, and Glycophorins A, B and C variants, all of which influence malaria pathogenesis [9].
ABO blood groups are carbohydrate histo-blood antigens that are also expressed in many tissues and which have important roles in modulating protein activities both in infection and in some types of cancer [10]. These antigens are formed by terminal glycosylation of glycoproteins and glycolypid chains present on cell surfaces. Glycosylation modulates all kinds of cell-to-cell interactions and this may be relevant in malaria pathophysiology, where adhesion has been increasingly implicated in disease severity. Studies examining the effects of the ABO blood group phenotype on malaria risk in non-pregnant subjects, have shown inconsistent results [11][12][13]. Blood group A has been reported as a risk factor for severe malaria [14], and as a co-receptor for P.falciparum resetting [15], whereas blood group O may offer some protection against severity of disease [16].
No studies have been identified in the literature assessing associations of blood group types with placental malaria, despite the fact that placental parasites are unable to rosette [17], in contrast to isolates from non-pregnant subjects. Cell surface glycans have an essential role in reproductive biology, and the adhesion and implantation of the blastocyst is partly mediated by carbohydrates with blood group specificity [18]. Each mammalian species has its own glycotype at the feto-maternal interface, and this variation depends on both evolution and the environment [19]. ABO histo-blood groups and related antigens are expressed in the endometrium and are modulated by the hormonal environment [20], but are not expressed in the placenta and fetal endothelium where only other related blood groups can be detected in the interstitial trophoblast directly apposed to the maternal decidua [21]. In contrast, examination of the glycan expression at the fetomaternal interface using lectins, some with ABO determinant specificity have shown binding with placental structures [22,23].
In this analysis we present the first report to describe the association between ABO phenotypes, placental malaria and pregnancy outcomes.
Study sample
Data from a group of 198 mother/child pairs were analysed from a cross-sectional study of placental malaria undertaken in the Gambia. The original descriptive study assessed serum Ig levels in mothers and newborns in relation to placental parasitaemia. The primary outcomes and a detailed description of the methodology are outlined in the original publication [24].
Mothers and their newborn babies were included in the study from September 1967 to May 1968 in Banjul and the Kombo-St Mary District (The Gambia). In this area malaria is endemic with peak transmission during and shortly after the rainy season which lasts from July to October [25]. Mothers and babies were examined soon after delivery at the Royal Victoria Hospital, Banjul, or in one of the maternal and child welfare centres in the Kombo-St Mary District. Basic demographic information was recorded. The weight (g) and length (cm) of the baby were measured by a physician within a few hours of birth. Placentas were transported twice daily to the Medical Research Council Laboratories where they were weighed and examined for placental pathology. Placental blood and maternal and neonatal peripheral venous blood were collected, and blood films were prepared and stained by standard methods. Parasitaemia was counted in the peripheral blood against the number of white cells, and in the placental blood by assuming that 1 parasite per 100 oil-immersion fields represented a density of 10 parasites/ mm 3 blood. Maternal and infant haemoglobin was measured.
Data were entered on punch cards and for all births the following data was available: maternal age, parity, haemoglobin and peripherical parasitaemia; the babies gender, weight, length and haemoglobin. The ABO and Rhesus blood group phenotype of mother and baby were recorded and the placental weight (g), parasite count and presence of malaria pigment. All data was entered into a SPSS data file.
Clinical definitions
Placental malaria definition was based on the pathological classification of Bulmer et al [26], which comprises the following groups: non-infected; acute infection (presence of parasites without pigment); chronic infection (presence of parasites and pigment); past infection (parasites not present; pigment present). Acute and chronic placental malaria were also grouped as 'active' infection.
Statistical analysis
Dichotomous variables were assessed with chi-square or Fisher exact tests, with p values less than 0.05 considered statistically significant. Odds ratios and 95% confidence intervals were estimated. Differences between means were assessed by ANOVA where data was normally distributed, or the Mann-Whitney/Wilcoxon Test. Multiple linear regression was used to analyse factors associated with anthropometric outcomes. Factors included were those significant at p = 0.1 in the univariate analysis. For anthropometric variables the factors included where: maternal blood group (O versus non O), parity, placental infection, maternal haemoglobin, maternal peripheral parasitaemia and maternal height.
Results
Eighty-eight of the 198 women were primiparae (45%) who had a mean age of 17.8 years (SD ± 1.8). The mean age of multiparae was 29.7 years (± 6.1). Placental malaria was present in 116 cases (59%), with active placental malaria infection in 74 (37.4%), of which 26 (13.1%) were acute and 48 (24.2%) chronic infections. Past infection was detected in 42 women (21.2%). In the remaining 82 cases (41%) there was no evidence for current or previous placental malaria. Among primiparae, 56 (63%) had evidence of placental infection of which 41 (47%) were active, 12 (14%) acute and 29 (33%) chronic infections. Among multiparae 60 (55%) had evidence of placental infection with 33 (30%) active, 14 (13%) acute and 19 (17%) chronic ( Table 1). The effect of parity on the risk of placental infection was significant for the O blood group sub-population with lower risk of active infection in multigravidae (OR 0.27, 95% CI 0.12-0.62, p < 0.01) compared with primiparae of the same blood group. For non-O blood groups, there were no significant parity differences in risk of active (OR 1.06, 95%CI 0.44-2.59, p = 0.9) or past placental infection (OR 0.79, 95%CI 0.32-1.98, p = 0.6). The effect of parity on the risk of active placental infection was significantly different for the two sub-populations O and non-O, (Chi-square 4.98, p = 0.03). Table 2 summarises birth outcomes by blood group phenotype. In primiparae, with active placental infection, mean birth weight was significantly higher in babies born to blood group O mothers (2893 g versus 2639 g, p = 0.04).
Maternal haemoglobin (Hb) concentration was higher in O type mothers with active placental malaria (11.65 g/dl, versus 10.53 g/dl) although this difference was not statistically significant (p = 0.48). There were no significant differences in mean infant haemoglobin, infant length, placental weight, ponderal index or placental parasitaemia prevalence by blood group category (table 2).
Mean feto-placental weight ratio was increased in blood group O compared to non-O (p = 0.06) and was significant in multiparae (5.74 versus 5.36; p = 0.04), (Figure 1). Blood group phenotype (O versus non-O) was an independent predictor of the higher feto-placental weight ratio, (linear regression coefficient = 0.26, p = 0.04). The other malaria parameters, did not show any significant associations with blood groups on multivariate analysis.
The Rhesus blood group type showed no significant associations in relation to placental malaria categories or birth outcomes.
Discussion
Blood group O was associated with an increased prevalence of active placental infection in primiparae and with a reduced risk of placental malaria in multiparae. Placental parasitaemia occurs at least twice as frequently in primiparae but only among blood group O women. This effect of parity, that is one of the cardinal features of placental malaria, was not observed in non-O phenotypes for any of the placental malaria histological types.
In multiparae and specifically in mothers with past or non-infected placentae blood group O was also associated with higher mean feto-placental weight ratios compared to non-O individuals. These differences associated with multiparity can be interpreted to represent a parity-specific association of the blood group O phenotype with the protective malarial immunity which occurs in multigravidae [3]. Mean birth weight was also significantly increased in babies of primiparae with the O phenotype, suggesting that despite the greater susceptibility of primigravidae to placental malaria, the O phenotype may be partially protective leading to improved birth anthropometry.
In non-pregnant individuals blood group O has been associated with reduced risk of severe clinical malaria compared to patients with groups A or B [14,16], but this occurred without clear evidence for reduced prevalence of parasitaemia associated with blood group O [12,13]. This would suggest that the ABO phenotypes may be associated more with modification of disease severity than with infection risk. None of these women had HIV infection as the study was conducted in the early 1970s before this human immunodeficiency virus was described or trans- This is the first time an association of birth outcomes with placental malaria and maternal ABO phenotype has been reported. This raises the question of the possible mechanisms underlying the association. A central mechanism in the pathogenesis of placental malaria relates to the cytoadherence of infected red cells to the syncytiotrophoblast which is in contact with maternal blood. The parity related susceptibility to placental malaria is partly dependent on the selection of P.falciparum isolates which bind to trophoblast via chondroitin sulphate A (CSA) [27] and hyaluronic acid [28]. Infected red cells can also bind to the proteoglycan thrombomodulin, present on endothelial cells and placental syncytiotrophoblast, via CSA side chains [29]. Soluble adhesion molecules and endothelial markers (including won Willebrand factor and E-selectin) are associated with ABO phenotypes, especially thrombomodulin which is lower in group O and A than B plasma (P < 0.001) [30].
Duffy binding-like domains (DBL) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) mediate binding to several independent host receptors [31] and in placental isolates it is the DBL-γ3 domain which binds to chondroitin sulfate A (CSA) in syncytiotrophoblast [32,33]. However, there is no published evidence of an association between DBL-γ3 and the ABO phenotype, although it has been shown that the DBL1α domain has an affinity for the blood group A antigen [34].
The interaction between the ABO histo-blood group and placental malaria could relate to generic mechanisms affecting P.falciparum infection. For example the differen-tial affinity of each phenotype for A.gambiae [35], or antigen sharing between ABO phenotypes and P.falciparum leading to changes in immune response [36], or an association with Glycophorin A (GPA) which is an important determinant for P.falciparum entry into the red cell through GPA sialic acid, as ABH antigens have been described in the O-glycans of glycophorin A [37]. In addition ABO phenotypes differ in sialic acid content and composition, with group O showing the highest membrane content, but a lower percentage of sialoglycoproteins [38].
It is well known that host genetic factors modulate the risk and severity of infection. In most cases these are genetic variants with subtle effects on the regulation or function of specific mediators which are often difficult to demonstrate in epidemiological studies [39]. As there is increasing evidence that cell adhesion plays a decisive role in placental malaria pathophysiology, it is clear that cell surface glycans, such as the ABO and related antigens which have special relevance in reproductive biology, could modulate some of those specific cell interactions. Their association with placental malaria risk, birth outcomes and parity related susceptibility, provides a new insight into these interactions and into the role of glycosylation and host-specific genetic factors in placental malaria.
Mean feto-placental weight ratio in primiparae and multi-parae according to blood group Figure 1 Mean feto-placental weight ratio in primiparae and multiparae according to blood group. Shaded blood group O, white non-O groups | 2016-05-15T18:02:21.072Z | 2006-08-17T00:00:00.000 | {
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254124090 | pes2o/s2orc | v3-fos-license | Valproic acid and bladder healing: an experimental study in rats
ABSTRACT Purpose: to recognize the effects of valproic acid (VPA), an epigenetic drug, on the bladder healing process, in rats. Method: twenty male Wistar rats were divided in two groups: experimental (A), treated with VPA (150mg/Kg/day), and control (B) with 0.9% sodium chloridrate. Healing was analyzed on the third and seventh days, evaluating the inflammatory reaction, collagen synthesis and angiogenesis. Results: inflammatory reaction on the third day was minimal and acute in both groups. On the seventh day, it was subacute in both groups, moderate intensity in group A and minimal in group B (p=0.0476). Collagen III intensity, marked by immunohistochemistry, was similar in both groups. Collagen I intensity on the third day was similar in both groups, but on the seventh day it was higher in experimental than control (p=0.0476). Collagen evaluation by picrosiriusred allowed to verify that the presence of collagen III was similar in both groups (p=0.3312) on the third day, and it was higher in control on the seventh day (p=0.0015). Collagen I showed similarity on the third day (p=0.3100), and it was higher in control on the seventh day (p=0.0015). Vessel marked with anti-SMA counting showed fewer vessels on the third (p=0.0034) and seventh day (p=0.0087) in experimental group. The lower intensity of angiogenesis was confirmed with anti-CD34, on the third day (p=0,0006) and on the seventh day (p=0,0072). Conclusion: VPA determined alterations in the bladder healing process, in rats, with lower collagen density and less angiogenic activity, but without compromising the integrity of the organ.
INTRODUCTION
T he self-regenerative capacity is intrinsic and universal to living beings 1 . As the main structural barrier to environmental aggressors (physical, chemical, or biological) we have the skin, pluristratified squamous keratinized tissue endowed with vast cellular communication among themselves and with the body. The loss of cellular interaction triggers immediate activation of homeostatic molecular events, seeking to restore functional integrity 2 .
Thus, healing begins, a complex process that involves migration of inflammatory cells, synthesis of granulation tissue, collagen deposition, and maturation, as well as wound remodeling 3 .
Recent studies on valproic acid (VPA), a drug used to treat epilepsy and bipolar disorder, have shown it to be responsible for the reduction of some types of cancers and their invasive potential, which is classified as an epigenetic.
The epigenetics' mechanism of action lies in the structural change of DNA, resulting in changes in transcription, translation, and replication of genes. This mechanism includes the alteration of methylation (hypermethylation or hypomethylation) of histones and nonhistone proteins, chromatin remodeling, and changes in gene expression by non-coding RNAs (ncRNAs) 4 . In the case of VPA, it acts as a class I selective inhibitor of histone deacetylases enzymes (HDACs) 5 , responsible for removing methyl grouping from histones, thus leaving the S phase, completing G2 phase and cell division. By inhibiting HDACs, specifically HDAC1, HDAC2, HDAC4 overexpressed in bladder cancer 6 , DNA remains hypermethylated, blocking the cell cycle and preventing disordered mitoses 7 .
Biondo-Simões
Valproic acid and bladder healing: an experimental study in rats VPA induces thrombocytopenia mainly in women, which may be due to a greater suppression in the formation of platelets in the bone marrow or because it favors the increase of peripheral destruction by anti-platelet antibodies 9 . The VPA also induces a reduction of secretion of vascular endothelium growth factor (VEGF), interfering in angiogenesis, as well as the decrease in the expression and activity of endothelial nitric oxide synthase (eNOS), reducing the formation of new vessels 10 . Fibrinogen also has its levels altered with VPA, by reducing fibrin precursors and increasing fibrinolysis, facilitating the breakdown of fibrin exudate, and thus reducing the cell adhersion 10 .
Cancer treatment using the association of chemotherapy and radiotherapy with epigenetics has been growing for various types of tumors, such as bladder 11 , and prostata 12 . Bladder tumor is the fifth most frequent neoplasm in men and the twelfth in women. In 2012, 165,000 deaths were recorded worldwide due to this cause 13 . In Brazil, the 2018 estimate was 9.480 new cases Tumor patients often receive preoperative radiotherapy and chemotherapy, and it can be associated with an epigenetic in the future, increasing the rate of recovery and decrease of the tumor population's resistant to certain drugs 15,16 . Considering those patients, many of this group will undergo surgical interventions, therefore it is important to study the influence of these medicines on the wound healing process.
The objective of this work was to analyze the action of valproic acid in the bladder healing process in rats.
Biondo-Simões
Valproic acid and bladder healing: an experimental study in rats CD34 labeling and them with the anti-SMA in order to have greater security of the count, that is, counter-proof.
Statistical analysis was descriptive using graphs and tables. The nonparametric test used was Fisher's test for 2x2 tables, Mann-Whitney, and Student's t-tests, depending on how the data is described in Gaussian curves or not. The level of rejection for the null hypothesis was p<0.05.
RESULTS
Throughout the experiment, there were no deaths.
On the third and seventh day, in both groups the urinary bladder was intact, there was not fluid in the abdominal cavity and the urine was clean and bloodless.
Adhesions were seen in both times evaluated in both groups ( Figure 2 (Figure 3). (Table 3 and Figure 5).
The collagen III intensity, marked by immunohistochemistry, was similar in both groups, in the two times studied, was absent or minimal on the third day (p=1.000) and minimal or moderate on seventh day (p=0,2222). Collagen I was absent on the third day in both groups, and but on the seventh day, the moderate amount predominated in the experimental group and minimal in the control group (p=0.0476).
Collagen evaluation by picrosirius-red allowed to verify that the presence of collagen III was similar in both groups (p=0.3312) on the third day, and it was higher in the control group on the seventh day (p=0.0015) ( Table 1). Collagen I showed similarity on the third day (p=0.3100) and was higher in the control group on the seventh day (p=0.0015) ( Table 2 and Figure 4). The counting of vessels marked by anti-CD34, in ten fields, confirmed a lower number of vessels in the treated group, both on the third day (p=0,0006) and on the seventh day (p=0,0072) ( Table 4). If we consider that the synthesis of the interstitial matrix and fibrous proteins such as collagens is dependent on oxygen supply, this would explain the lower collagen density in the bladders of the animals in the VPA-treated group.
DISCUSSION
In a summary analysis, the inflammatory reaction was very similar in both groups. The marking of collagen by the immunolabel showed a small amount, the highest in the experiment group of collagen type I on the seventh day, in qualitative analysis. However, the picrosirius red analysis, of quantitative bias, allowed the recognition of a higher density of collagen type III and collagen type I, on the seventh day, in the control group. It should be considered that an increase in sample size may give greater confidence to the results.
It is still important to analyze the interference of VPA on angiogenesis, which has been done in parallel to this study by another researcher from the same study group.
However, even though the VPA can decrease the intensity of the healing process, it must be admitted that it does not do so in such a way as to lead to risks.
It was not observed dehiscence of the synthesis of the bladder, an organ that was intact and protected by adhesions, besides the urine that was clear and of normal aspect. This allows us to think that, when the VPA is used to aid in the treatment of neoplasia and surgical intervention is necessary, it can be done safely.
CONCLUSION
VPA determined alterations in the bladder healing process, in rats, with lower collagen density and less angiogenic activity, but without compromising the integrity of the organ. | 2022-12-02T06:17:19.939Z | 2022-11-10T00:00:00.000 | {
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13790376 | pes2o/s2orc | v3-fos-license | Engineered Nanomedicine with Alendronic Acid Corona Improves Targeting to Osteosarcoma
We engineered nanomedicine with the stealth corona made up of densely packed bone seeking ligand, alendronic acid. In a typical nanoconstruct, alendronic acid is conjugated with hydrophilic head moiety of phospholipid that has an ability to self-assemble with hydrophobic polymeric core through its hydrophobic long carbon-chain. Proposed nanomedicine has three distinct compartments namely; poly(l-lactic-co-glycolic acid) polymeric core acting as a drug reservoir and skeleton of the nanoconstruct, phospholipid monolayer covers the core acting as a diffusion barrier, and a densely packed alendronic acid corona acting as a stabilizer and targeting moiety. Thus engineered nanomedicine attain spherical entity with ~90 ± 6 nm having negative zeta potential, −37.7 ± 2 mV, and has an ability to load 7 ± 0.3 wt% of doxorubicin. In-vitro bone targeting efficiency of nanomedicine was studied using hydroxyapatite crystals as a bone model, and found significant accumulation of nanoparticle in the crystals. Moreover, cellular internalization studies with mouse osteosarcoma confirm the selectivity of nanomedicine when compared to its internalization in non-targeted mouse melanoma. This nanomedicine shows prolong stability in serum and deliver the drug into the cell exhibiting an IC50 of 3.7 μM. Given the strong interacting property of alendronic acid with bone, the proposed nanomedicine hold promises in delivering drug to bone microenvironment.
Scientific RepoRts | 6:36707 | DOI: 10.1038/srep36707 Among different types of bone targeting ligands, bisphosphonate has been long emerging as a bone-seeking agent owing to its greatly binding affinity with hydroxyapatite -a major mineral component in bone environment. In addition, with the acidic property and hydrophilic nature, bisphosphonate's permeability through the cellular membrane is insignificant, which in turn makes it more extensively accumulates in skeleton than other organs after the administration 9,10 . Once considering bisphosphonate's distribution within the skeletal system, researches have shown that bisphosphonate accumulates more in bone defect site where high bone turnover is associated 11,12 . High bone turnover occurs when the activity of osteoclasts and osteoblasts are uncontrolled or are aggressive. Taking an advantage of these properties at bone lesion sites, the bisphosphonate conjugation can be a promising approach to design targeted chemotherapy for bone cancer treatment. Moreover, the antiresorptive properties of bisphosphonate make it suitable combination candidate with other drugs to treat cancer at bone 13 .
Recently, studies have been focused on utilizing bisphosphonate to construct bone-homing nanomedicine by either conjugating alendronic acid (a member in bisphosphonate class) with polymeric backbone or chemotherapeutic drugs via polyethylene glycol (PEG) linker [14][15][16][17][18] . These targeted nanocarriers possess common stealth properties provided by well-hydrated PEG moiety decorated on the surface which could evade nanoparticle from reticuloendothelial system (RES). In 2006, Uludag et al. reviewed in an attempt to engineer bone seeking therapeutic agents based on formulating therapeutic agents with bisphosphonates 19 . In this review authors have summarized various classes of bisphosphonate and therapeutic agent conjugates such as: small molecule drugs, protein, and imaging agents capable of targeting bone. In a recent study conducted by Swami et al., bortezomid, a proteasome inhibitor, loaded polymeric nanoparticle was proposed, in which the stealth PEG corona was conjugated with alendronic acid to target bone 17 . Despite promising achievements in enhancing antitumor activity on mice bearing tumor models, a number of limitations and challenges related to those systems need to be considered. For examples, the acceleration on using PEG moiety in both pharmaceutical and non-pharmaceutical products have consequently led to anti-PEG antibody development in human body [20][21][22][23][24] . A recent finding shows that 22-25% occurrence of anti-PEG in healthy blood donors is most probably due to the greater exposure to PEG containing consumer products 24 . Therefore, a substitute material needs to be developed in order to diminish the excessive usage of PEGylated products. Considering the important role of hydrophilic corona layer on the surface of nanoconstruct in stabilizing nanosystem, we hypothesized that the Alendronic acid moiety with hydrophilic phosphate groups could provide favorable environment for water to form hydration layer around the particle protecting it from opsonization when properly structured in nanoconstruct and further sustain the stability of nanosystem under physiological condition.
To this end, we proposed a targeted nanoparticle (TNP) which is made up of Alendronic acid modified lipid and PLGA polymeric core encapsulating chemotherapeutic drug -Doxorubicin (DOX) to simultaneously offer combinatorial actions including targeting and therapy of bone cancer treatment. Proposed nanostructured construct has three distinct layers (1) PLGA core acting as a skeleton of the nanoparticle and drug reservoir, (2) lipophilic phospholipid layer acting as a middle passivating layer and diffusion barrier for the encapsulated drug, and (3) hydrophilic alendronic acid, an outer corona layer, acting as stabilizer and driving of nanoparticle to its target. This densely packed phospholipid conjugated alendronic acid creates a sufficiently thick hydrated shell and prevents nanoparticle from being disassembled. Therefore, the engineered nanomedicine not only has stealth properties providing by bone mineral targeting moiety but also deliver a large quantitative amount of therapeutic agent which could enhance the effectiveness of treatment.
Result and Discussion
Synthesis and characterization of ALE-Lipid conjugate. The synthesis of the lipid conjugate was carried using ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) conjugation chemistry as described in Fig. 1A. The chemical structure of synthesized ALE-Lipid conjugate was confirmed by FT-IR and 1 H-NMR. As shown in Fig. 1B, the FT-IR spectrum of ALE-Lipid (spectrum in black) exhibits all of the characteristic peaks of both unconjugated lipid (spectrum in blue) and alendronic acid (spectrum in red) including strong signal of aliphatic C-H stretch at 2900 cm −1 corresponds to lipid backbone and broad O-H stretch at 3500-3100 cm −1 belongs to ALE moiety (Fig. 1B). It is notable that after the conjugation, C = O acid stretch of lipid at 1750 cm −1 was shifted to 1650 cm −1 due to the formation of C = O amide. Most importantly, broad peak ranges at 1300-750 cm −1 correspond to phosphate functional groups was broaden in ALE-lipid spectrum which could be attributed to the overlap of phosphate ester (1050 cm −1 ) and phosphonate vibration (985 cm −1 , 1050 cm −1 , and 1205 cm −1 ) of lipid and ALE, respectively. In addition, the primary N-H bend (1700 cm −1 ) in ALE spectrum was replaced by secondary N-H bend (1550 cm −1 ) in the spectrum of the conjugated product; thereby confirming the formation of an amide bond. Furthermore, in order to understand the structure of ALE-lipid in detail we conducted the 1 H-NMR study. In the 1 H-NMR spectrum (Fig. 1C), Alendronic acid gave a simple spectrum characterized mainly by a triplet at 2.91 ppm corresponding to N-CH 2 protons and two complicated multiplets arise at 1.9 ppm and 1.88 ppm due to magnetically non-equivalent nature of prochiral CH 2 protons 25 . In the case of synthesized ALE-Lipid, proton signals that relate to lipid were observed at 1.26 ppm owing to the methylene groups of the long hydrocarbon chain and proton signals of terminal methyl group appear as a triplet at 0.88 ppm. The presence of ALE moiety in the conjugated product was confirmed by a small broad peak appears at 1.6 ppm and triplet locates at 2.73 ppm which were assigned for -CCH 2 CH 2 CH 2 NHCO-and CH 2 NHCO-, respectively. With the formation of ALE-Lipid conjugate these proton signals were upfield shifted from their original chemical shift to 1.8 ppm and 2.91 ppm (in ALE spectrum), respectively, due to the emergence of amide bond and the attachment of lipid, which further verifies the presence of covalently conjugated ALE on the fatty acid backbone.
Physicochemical properties of nanoparticles. After successfully attaching ALE with lipid (ALE-lipid), conjugate ALE-lipid was used along with PLGA and DOX for nanofabrication of TNP and DOX-loaded TNP using nanoprecipitation ( Fig. 2A). The physicochemical properties and morphological characterization of TNP and DOX-loaded TNP were determined using dynamic light scattering (DLS), surface zeta potential, and transmission electron microscopy (TEM) as shown in Fig. 2B-D respectively. The hydrodynamic size of the TNP showed a diameter of 69 ± 5 nm whereas DLS size of DOX-loaded TNP was found to be 90 ± 6 nm. Importantly, in both cases, the DLS data showed a unimodal distribution with low polydispersity index (PDI) which demonstrates that the TNP and DOX-loaded TNP are highly monodispersed in aqueous solution. The TEM image of TNP further confirmed the uniformity of nanoparticles with the size is around 50 nm. The significant different between hydrodynamic size and dry stage diameter is likely due to well thick hydrating layer in which the densely packed hydrophilic phosphate moiety acting as a corona of the nanoparticle. This hydrophilic corona layer works in a similar way to that of PEG moiety which could make nanoparticle bypass immune system and prolonged the circulation time. By taking this advantage, we not only avoid overusing of PEG moiety but also creating new material that can give similar stealth property.
In addition, the measurement of the TNP and DOX-loaded TNP surface zeta potential revealed a net charge of − 37.7 ± 2 mV owing to the presence of negatively charged phosphate moiety on the surface. The similarity in zeta potential value of bare and drug loaded nanoparticles further confirmed the unchanged of surface property of TNP after DOX encapsulation. Taking into account that DOX is the cationic drug, if it is nonspecifically absorbed onto the surface of TNP, the surface charge property of this NPs could change, however, after DOX encapsulation, the surface charge of TNPs remains constant. This observation gives strong evident for the localization of DOX into the core of NPs. Besides physicochemical properties, the stability of nanoparticles is another essential parameter that needs to be carefully evaluated in order to translate to biological application stage. Therefore, we conducted stability tests in various in-vitro physiological conditions including ionic and pooled protein milieu at 37 °C using phosphate buffer saline (PBS) and Fetal Bovine Serum (FBS), respectively. After 7 days of incubation in PBS (pH 7.4), there is no noticeable nanoparticles aggregation was observed demonstrating by unchanged in DLS and PDI indexes (Fig. 2E). In addition, TNPs are found to be highly stable in its colloidal state when expose to serum environment as revealed by constant optical density at 560 nm ( Fig. 2F), whereas in the case of bare PLGA NPs, the absorption at 560 nm increase rapidly and reaches to plateau within 15 mins indicating rapid aggregation of PLGA NPs. These kinetic absorption experiments conducted at 560 nm is the measurement of aggregation when NPs aggregated with protein and precipitated to block transparency of the light from the medium 26 . These results implied that the TNPs exhibit robust stability which could enlarge therapeutic window by prolonging circulation time in the bloodstream and enhancing chances of designed nanoparticles to target and accumulate at the bone tumor site. This serum stability further supports our claim that the hydration layer around the NPs due to hydrophilic phosphate moiety is sufficient enough to act as a stealth layer of protection in a manner similar to that of the PEGylated system. Calcium binding affinity. Since the surface of TNPs was decorated with a bisphosphonate, we next investigated in-vitro binding affinity of TNPs with hydroxyapatite (HAp) as a bone-model. Our expectation is that with an increase in NP concentration treated with HAp, the more NPs bound to HAp. However, as the nanoparticle concentration increases, percentage nanoparticle bind to HAp decrease (Fig. 3A). This realized us to optimize the concentration of TNPs that need to be treated with HAp, and the results for optimized concentration is presented in Fig. 3A. With the optimized concentration, we found that nearly 80% of NPs bind to HAp at a concentration of 100 μ g/mL, whereas only 20% of NPs were bound at the concentration of 800 μ g/mL and 1000 μ g/mL. Therefore, we selected minimum and maximum concentration (100 μ g/mL and 1000 μ g/mL, respectively) to elucidate kinetic binding property of TNPs toward HAp. Along with targeted NPs, PEGylated NPs at the same concentrations was used as a control sample. To our expectation, more than 80% TNPs bound to the surface of HAp within 15 mins of incubation time (Fig. 3B). This trend plateaued on a further increase of incubation time. In contrast, in the case of PEGylated NPs, just 20% of NPs bound to HAp after 2 hours of incubation, at this stage when bounded PEGylated NPs with HAp were centrifuged down and the supernatant was measured spectrophotometrically under fluorescence reader which shows sufficient amount of NPs. This non-specific binding of control NPs could be accounted for carboxylic functional groups presence on the surface of PEGylated NPs. The interaction between HAp and TNPs labelled with RhB was further confirmed by imaging of HAp crystal under fluorescent microscope where the presence of TNP highly enhanced fluorescent signal captured on the surface of incubated HAp crystals, while insignificant fluorescent signal was obtained on HAp samples incubated with PEGylated NPs (Fig. 3C,D, respectively).
Drug loading and release study. TNP's drug loading capacity and drug release profile were also evaluated. Figure 4A shows the DOX encapsulation efficiency of TNPs at varying DOX concentrations. There was more than 7 wt% of DOX was successfully encapsulated in 1 mg/mL PLGA. Among these five different formulations, 100 μ g/mL initial input of DOX gave the most effective loading efficiency without changing nanoparticle physicochemical property. Thus, this formulation was chosen to use in further experiments.
With the optimized DOX loaded TNPs sample, the in-vitro drug release was investigated at pH = 7.4 in phosphate buffer saline (PBS). A cumulative drug release study was performed using 10 kDa molecular cut-off dialysis bags. A control experiment was also performed using an aqueous solution of free DOX placed in the dialysis tubing. As can be seen in Fig. 4B, in the case of free DOX, 100% drug was burst released within the period of 6 h, whereas DOX loaded TNP shows the extension of drug release up to 24 h. This result indicates that DOX loaded TNPs exhibit typical sustained drug release profile of nanomedicine over a 24 h time period at 37 °C (Fig. 4B).
Cellular uptake study.
After understanding the drug loading efficiency of the TNPs, we next studied the cellular internalization to evident the targeting capability of TNPs in the in-vitro environment against mouse osteosarcoma K7M2 cells. The cellular internalization of TNPs was first visualized under confocal laser scanning microscope by incubating RhB-labeled TNPs with K7M2 cells for 3 h. Confocal micrographs revealed that a lot of NPs internalized into the cell and localized around the periphery into the cellular compartment (Fig. 5A,B) with some localized into the perinuclear region as evident from merged z-stack CLSM images (Fig. 5B).
A quantitative cellular uptake of RhB-labeled TNPs was then conducted using flow cytometry in compared to conventional PEGylated NPs. Our results showed the cellular uptake of both TNPs and PEGylated NPs displayed time-dependent uptake (Fig. 5C). As the nanoparticle incubation time increased, the enhancement of cellular uptake was observed in both TNPs and PEGylated group. However, at an equal incubation time, the TNPs were taken up by K7M2 cells is higher than that of PEGylated NPs (Fig. 5D), suggesting that conjugation with bisphosphonate plays a significant role in targeting nanoparticles to a bone cancer cell in compare to PEGylated NPs.
As most cancer cells display aggressive profile in the unselectively uptake of substances presence in their growing environment, it is important to set up a comparative experiment in order to assess the specificity of targeting ligands among different cancerous cell types. With this in mind, we have chosen another aggressive non-targeted melanoma (B16-F10) cell line to rationally evaluate the distinctive property of TNPs under cellular environment. As a result, a dramatic increase in K7M2 cellular uptake was observed from 30 min to 3 hours of incubation as evidenced by an increase in fluorescence intensity. This time-dependent cellular uptake pattern was absence in the case B16-F10 melanoma cells where fluorescent intensity remains constant even after 3 hours of incubation. The high cellular uptake of targeted nanoparticle toward K7M2 cells was attributed to cell membrane specific interaction with phosphate moiety of Alendronic acid, in a typical endocytosis-mediated receptor uptake. This observation was in agreement with the previous study conducted by T.-K. Ryu et al. where Alendronic acid conjugated nanodiamond showed extensive accumulation in osteoblastic cells (MC3T3-E1) but not non-targeted HepG2 and NIH3T3 cell types 27 . Specifically, protein tyrosine phosphatases were investigated as one of the possible bisphosphonate binding receptor presence on the surface of osteoblastic cells 28 . Moreover, protein phosphatases were identified to be overexpressed in osteosarcoma in compared to that of normal osteoblast and osteoclast cells which would help to target ligand to target cancerous cells efficiently 29,30 . Also, the same phenomenon was observed by Toledo et al. where researchers were studied in the bone tumor of thirty osteosarcoma patients and found that protein tyrosine phosphatases are over expressed in osteosarcoma 30 . Osteosarcoma cells are of the osteoblastic lineage, which is characterized by cells secreting the osteoclast-inducing factor, receptor activator of nuclear factor-κ B ligand. Receptor activator of nuclear factor-κ B-Fc, osteoprotegerin, bisphosphonates, and Src inhibitor are shown as positive candidates and can control various aspects of osteoclast function 31 . From the results, it could be interpreted that phosphate moiety on the surface of nanoconstruct can selectively facilitate the accumulation of the nanoparticles in the targeted cancerous cells.
Biocompatibility and cellular cytotoxicity study. The biocompatibility of TNPs and in vitro cytotox-
icity of DOX loaded TNPs were studied in K7M2 Osteosarcoma bone cancer cells using MTT assay. As can be seen in Fig. 6A, at low TNPs concentration, no significant toxicity related to TNPs was observed indicating the excellent biocompatible of ALE-lipid at low concentration. However, when this concentration increases up to 150 ug/mL, cell viability decreases to 80%, this reflection could be explained by masking of the cellular surface under 96-well plate environment, hence reduce the cellular accessibility to oxygen and creating an unfavorable growing environment to cell which further induce unexpected cell death.
In a typical cellular cytotoxicity experiment of free DOX and DOX loaded TNPs, the results revealed that both agents exhibit a time-and dose-dependent cytotoxic effect (Fig. 6B) in which at low incubation time (24 hr) the drug loaded NPs showed higher cytotoxicity than free DOX with the IC50 of 3.7 and 6.1 μ M, respectively (Table 1). This enhancement in cytotoxicity of DOX loaded TNPs in lower incubation time is likely due to nanoparticle's internalization mechanism. First, the exterior phosphate groups of targeted NPs were attracted by protein tyrosine phosphatases receptor present on the surface of osteosarcoma cells leading to the acceleration in accumulation and distribution throughout cell membrane. These targeted nanoparticles further internalized into the cell via endocytosis with the core holding drug; thereby, intensely increase intracellular drug concentration and resulted in enhanced cytotoxicity. Whereas, in the case of free DOX, water soluble drug slowly diffuse into the cell limiting intracellular drug concentration. However, when cells were under treatment for a longer period of time (48 hr and 72 hr), both free DOX and DOX loaded TNPs exert similar cytotoxicity effect. This further supports the evidence of rapid uptake of the nanoparticulate system thereby increasing intracellular drug concentration as compared to that of free drug molecules.
Conclusion
Targeted therapy holds great potential for minimizing drug related non-specific toxicity. Towards this end, we have demonstrated the target specific delivery of DOX using a nanoparticulate system consisting of the densely packed alendronic acid corona that strongly binds with bone mineral, hydroxyapatite, aiming to target bone microenvironment. Engineered nano-system shows active accumulation into the osteosarcoma cell exhibiting dose-dependent toxicity similar to that of DOX in in vitro condition. However, in lower incubation time nanomedicine shows higher toxic effect than that of free DOX, which reveals that the nanomedicine delivers a higher dose of DOX into the cells. Most importantly, higher ionic and serum stability of nanoparticle revealed that decorating nanoparticle surface with alendronic acid provides sufficient hydration layer and strong negative surface charge to sterically stabilized NP, which could be an alternative to PEGylated system to design nanocarrier. Overall, alendronic acid decorated proposed nano-system could provide a promising and most effective platform technology in the treatment of osteosarcoma. Phosphate Buffered Saline (PBS) containing 10% triethylamine (TEA) was added and stirred for additional 24 h at room temperature. In order to purify the product, conjugated lipid was placed inside the benzoylated cellulose dialysis bag (MWCO ~500 Da), and dialyzed against water for 24 hours at room temperature. The samples were lyophilized to obtain a dry powder and stored at − 20 °C for further use. FT-IR and 1 H-NMR were used to confirm the formation of ALE-lipid.
Materials and Methods
Nanofabrication of lipid bisphosphonate nanoparticles and multifunctional polymeric nanoparticles. Targeted hybrid nanoparticles were prepared by single step nanoprecipitation. In brief, 400 μ L of PLGA (1 mg) in acetonitrile was added dropwise to 2 mL of 200 μ g ALE-lipid (dispersed in 4% ethanol) under a magnetic stirring condition at 60 °C. To this, 1 mL of Mili-Q water was added to cool down the mixture and stirred continuously for additional 1 hour to facilitate the formation of nanoparticles and evaporation of organic solvent at room temperature. The TNPs were further purified using Amicon Ultra-4 centrifugal filter (Millipore, MA) with a molecular weight cut-off of 10 kDA and stored at 4 °C for further use.
For DOX-loaded TNP preparation, different amount of DOX (10, 25, 50, 100, and 150 μ g) were mixed with 100 μ L of PLGA (10 mg/mL) and dried under vacuum. A polymeric film with the drug was then dissolved in 400 μ L acetonitrile prior to nanoparticle preparation. Controlled PLGA nanoparticles were also prepared by dropwise adding 100 μ L of PLGA (1 mg) in acetonitrile to 1 mL of Milli-Q water and purified following aforementioned protocol.
Characterization of nanoparticles. The hydrodynamic size and zeta potential measurements of the prepared TNPs and DOX-loaded TNPs were analyzed by Dynamic light scattering (DLS) using a Zetasizer Nano ZSP (Malvern, Worcestershire, UK). The Smoluchowski model was used to calculate the zeta potential value. All data represents the average of triplicate measurements of samples prepared in different preparations. The morphology of the prepared TNPs was further analyzed using Transmission Electron Microscope (TEM, Tecnia G2, Spirit Bio TWIN). TEM samples were prepared by drop casting and evaporation technique using formvar coated copper grid (400 mesh). TEM images were analyzed by GATAN digital imaging system (GATAN, Inc.). The amount of encapsulated DOX and the resulting encapsulation efficiency was quantified spectrophotometrically using UV-VIS microplate reader by measuring the absorbance at 490 nm.
Stability study of nanoformulation. The stability of TNPs in physiological ionic condition was investigated at pH 7.4 using PBS. In brief, 500 μ L of 1 mg/mL nanoparticles suspension were added to 500 μ L of 2X PBS and incubated at 37 °C with a rotating motion for 7 days. The stability of nanoparticles was determined by measuring the particle size and PDI every 24 hr. The serum stability of the prepared PLGA NPs and TNPs were carried out as reported 26,32,33 . Specifically, 100 μ L of 1 mg/mL nanoparticles were incubated with 100 μ L of 10% Fetal Bovine Serum at 37 °C and measure its change in absorbance at 560 nm kinetically every 5 s over a period of 1 h, double orbital shaking with slow speed was applied prior to each measurement using Microplate reader (BioTek, Synergy H1 hybrid reader).
Calcium binding affinity. Nanoparticles engineered herein are highly monodispersed and recovered as an aqueous suspension. Under mild centrifugation (1000 to 3000 rpm for 10 mins), they are not pelleted down. We have taken this advantages for Ca 2+ binding assay. The binding affinity of TNPs to Calcium was determined indirectly by measuring the fluorescence intensity of RhB-labelled NPs presence in the supernatant and compare to the initial fluorescence intensity of NPs fluorescent spectrophotometrically. To optimize binding assays, we first used a different concentration of RhB-labelled TNPs (100, 200, 400, 600, 800, and 1000 μ g/mL) to incubate with 5 mg Hydroxyapatite (HAp) for 30 mins at 37 °C. At the end of incubation time, samples were centrifuged at 1,500 rpm for 5 min to spin down HAp aggregates and the nanoparticles that bound to them. 100 μ L of supernatant was used to indirectly quantify the relative amount of nanoparticles bind to HAp. In the case of kinetic binding experiment, 1 mL of RhB-labelled TNPs (100 μ g/mL and 1 mg/mL) were incubated with 5 mg HAp in macro-crystal form for varying periods of time (30 s, 2, 5, 15, 30, 60, 120, and 240 mins) at 37 °C and processed as aforementioned protocol. In addition, RhB-labelled PEGylated NPs (100 μ g/mL and 1 mg/mL) were used as controlled particles followed the same experimental condition.
Drug release study. The cumulative drug release from the DOX loaded TNPs was assessed under a physiological condition at 37 °C. In brief, DOX loaded TNPs (25 μ g/mL, 1 mL) were placed in a dialysis bag membrane (Mw. Cutoff = 10 kDa) and dialyzed against 250 mL of PBS (pH = 7.4). At constant stirring (100 rpm), 200 μ L of the sample was taken at predetermined time intervals. The amount of released DOX was quantified by measuring the DOX fluorescence with the excitation and emission wavelength of 490 nm and 580 nm, respectively. As a control experiment, 25 μ g/mL of Free DOX was placed in a dialysis bag and processed under the same condition.
Intracellular uptake study. In order to verify cellular uptake efficiency of ALE-Lipid decorated NPs, RhB-labeled TNPs was used in mouse Osteosarcoma bone cancer cell line (K7M2). In brief, cells were seeded in Poly-D-lysine coated 8 chamber slide at a density of 50,000 cells per well and incubated for 24 h. Then, the cells were treated with 50 μ g/mL RhB-labeled TNPs suspension prepared in complete DMEM and incubated for 4 h. After incubation, treated cells were washed twice with 1X PBS (pH 7.4), fixed with 4% paraformaldehyde for 30 min at room temperature, stained with DAPI for additional 10 min and imaged under a Confocal Laser Scanning Microscope (Carl Ziess, LSM-700).
Fluorescence-activated cell sorting (FACs) studies.
To quantitatively evaluate the cellular internalization efficiency of ALE-Lipid decorated NPs, a comparative experiment was conducted on targeted Osteosarcoma cell line and non-targeted Melanoma (B16-F10) cells. In brief, K7M2 cells (or B16 cells) were seeded in T25 tissue culture flasks at 4 × 10 6 cells per flask for 24 hr. After incubation, cells treated with 2 mg RhB-labeled TNPs suspended in DMEM. Cells were then incubated at 37 °C at varying periods of time (30 min, 1, and 3 h) at 37 °C. After incubation, cells were washed twice with ice-cold PBS, detached with 0.08% w/v trypsin and analyzed on a flow cytometer. RhB-labeled PEGylated NPs at the same concentration were used as control particles.
In vitro cytotoxicity assays. The in-vitro cytotoxicity of TNPs was conducted on Osteosarcoma K7M2 using MTT assay. In brief, 2 × 10 4 cells per well in DMEM medium were seeded in a 96-well plate and incubated for 24 h. After incubation, the media were replaced with different TNPs concentration (10, 25, 50, 100, 150 and 200 μ g/mL) and DOX loaded TNPs along with free DOX (0.01, 0.05, 0.1, 0.5, 1, 2, 3 and 5 μ M) and incubated for additional 24, 48, and 72 hr. Control cells were also maintained without any TNPs treatment (n = 6). After incubation, MTT was added to each well and further incubated for 3 h according to the manufacturer recommendation. The insoluble formazan crystals were solubilized using DMSO and their absorbance was recorded at 570 nm using a microplate reader (BioTek, Synergy H1 hybrid reader). | 2018-04-03T05:09:23.982Z | 2016-11-08T00:00:00.000 | {
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216084061 | pes2o/s2orc | v3-fos-license | Circular RNA Circ100084 functions as sponge of miR-23a-5p to regulate IGF2 expression in hepatocellular carcinoma
Hepatocellular carcinoma (HCC) has become a major cause of cancer-related mortality worldwide. Circular RNAs (circRNAs) are non-coding RNAs that serve important roles in multiple cancers. However, the role of circRNAs in HCC remains largely unknown. In the present study, a circRNA microarray dataset of HCC samples, GSE97332, was downloaded from the gene expression omnibus database. Following data preprocessing, differentially expressed circRNAs between HCC tissues and normal tissues were determined using GEO2R. The circRNA-miRNA interactions were predicted by the miRanda database. The miRTarbase database was used to search for target genes of the miRNAs. A circRNA-miRNA-mRNA network was constructed using Cytoscape based on the obtained circRNA, miRNA and mRNA. In this network, the upregulated circRNA hsa_circRNA_100084 was found to be involved in a competing endogenous relationship of hsa_circRNA_100084-hsa-miR-23a-5p- insulin-like growth factor 2 (IGF2). The differential expression of hsa_circRNA_100084, hsa-miR-23a-5p and IGF2 in HCC tissues and liver cancer cells was validated by reverse transcription-quantitative PCR. Additionally, the interactions between hsa-miR-23a-5p with hsa_circRNA_100084 and IGF2 were validated by dual-luciferase reporter assays. Knocking down hsa_circRNA_100084 inhibited the proliferation, migration and invasion of liver cancer cells, while the simultaneous overexpression of IGF2 reversed the effects of hsa_circRNA_100084 knockdown. The results show that hsa_circRNA_100084 could promote the expression of IGF2 by acting as a sponge of hsa-miR-23a-5p in liver cancer cells.
Introduction
Hepatocellular carcinoma (Hcc) is the most common type of liver cancer and is the leading cause of cancer-related deaths worldwide (1,2). although surgical resection combined with post-surgery radio-chemotherapy has achieved great progress, the median survival time for patients with Hcc remains unsatisfactory (3). it is estimated that ~700,000 individuals succumb to Hcc each year globally (4). Therefore, further investigations into the molecular basis of Hcc are required to explore innovative targets for its diagnosis and treatment. circular rna (circrna) is a newly discovered class of endogenous noncoding rnas that are characterized by a covalently closed continuous loop (5). due to their special structure, circrnas are highly evolutionarily conserved and stable (6). recent studies have indicated that circrnas may serve important roles in driving cancer initiation and progression, and have the potential to serve as biomarkers for predicting cancer progression (7,8). accumulating evidence has demonstrated that circrnas regulate Hcc progression and serve as potential biomarkers for predicting cancer prognosis (9)(10)(11)(12)(13)(14)(15). However, a considerable number of circrnas remain to be elucidated in Hcc.
insulin-like growth factor 2 (IGF2) is a genomic imprinting gene involved in development and growth, which is located on the short arm of chromosome 11 (16). This gene is a paternally imprinted growth factor regulated by four promoters. during fetal stages, the expression of IGF2 is monoallelically regulated from 3 promoters (P2, P3 and P4) in human liver and in adults, its expression is regulated by both alleles of promoter P1 (17). although IGF2 is highly active during fetal development, it is much less active after birth (18). However, studies have suggested that IGF2 overexpression occurs in numerous types of cancers and is associated with resistance to chemotherapy and a worse prognosis (18,19). This might be partly explained by the reactivation of IGF2 transcription from the fetal-specific promoters or demethylation of its fetal promoter (20,21). However, further studies are still needed to elucidate the mechanisms of IGF2 overexpression in Hcc.
Previous studies have demonstrated that circrnas may function as competing endogenous rnas (cerna) by sponging mirnas to regulate target gene expression (22)(23)(24). in a previous study, Han et al (25) characterized the expression profile of circRNAs in human HCC tissues and paired adjacent liver tissues. They demonstrated that circMTo1 suppresses Hcc progression by acting as a sponge for oncogenic mir-9 to promote p21 expression. in the present study, the sequencing data used in the study of Han et al was downloaded from the Gene expression omnibus (Geo) database and these data were re-analyzed using a series of bioinformatics methods. a cerna network was constructed and IGF2 was found to be involved in a cerna network of hsa_circrna_100084-h sa-mir-23a-5p-IGF2. The present study further validated the cerna association in Hcc tissues and liver cancer cells.
Materials and methods
RNA sequencing data. The expression profile of lncRNAs in human Hcc was downloaded from the Geo database (26) (accession number: GSe97332), which was deposited by Han et al (25). This dataset contained seven pairs of Hcc tumor tissues and matched non-tumor tissues and was based on the agilent-069978 arraystar Human circrna microarray V1 platform. original expression data as well as the platform probes annotation files were downloaded.
Identification of differentially expressed (DE)-circRNAs in HCC.
The original expression profiles of HCC and normal tissues were analyzed using Geo2r (https://www.ncbi.nlm. nih.gov/geo/geo2r/), which is an online tool for processing data using Geo queries and limma packages (27) in r from the Bioconductor project (28). The raw data were preprocessed by background correction and normalization by log2 transformation. The raw P-value was adjusted by the Benjamini and Hochberg method to a false discovery rate (Fdr). The circrnas with thresholds of Fdr <0.05 and |logfold change (Fc)| >2 were considered as de-circrnas. Heatmap for the top 10 upregulated circrnas and the top 10 downregulated circrnas were constructed using pheatmap method in r package (http://finzi.psych.upenn. edu/r/library/pheatmap/html/pheatmap.html).
Construction of a circRNA-miRNA-mRNA network. The top 10 upregulated circrnas and top 10 downregulated circrnas were selected for further analysis. Since circrna nomenclature differs among platforms, the present study first mapped the probe sequences of the top 10 upregulated circrnas and top 10 downregulated circrnas into circBase (http://www.circbase.org/cgi-bin/webBlat). The associations with the highest matching score were selected. The mirnas related to the 20 de-circrnas were predicted by miranda v.3.3a (http://www.microrna.org/microrna/home.do) with the criteria of Score ≥140 and Energy ≤-10 (29).
The Hcc-associated mirnas were searched on the mir2disease (www.mir2disease.org) database using 'hepatocellular carcinoma' as key words. Then, the overlapping mirnas of Hcc-associated mirnas and the predicted mirnas in the previous step were noted for further study.
The circrna-mirna-mrna network was constructed using cytoscape software (31) based on the obtained circrnas, mirnas and mrnas.
Clinical samples and cell culture. The present study was approved by the ethics committee of the lishui Municipal central Hospital (lishui, china) and written informed consent was obtained from all patients included in this study. a total of 37 pairs of Hcc and adjacent normal tissues without preoperative treatment were collected from surgical resections in the hospital between March 2018 and august 2018 and Table i. Human liver cancer cell lines with a stepwise metastatic potential, including MHcc97H with high metastatic potential, and HepG2 (a hepatoblastoma cell line) (32) and Hep3B with very low invasiveness, and the normal human hepatic stellate cell lX2 were purchased from The cell Bank of Type culture collection of the chinese academy of Sciences and icell Bioscience inc. all cells were authenticated via STr profiling. Cells were cultured in Dulbecco's modified Eagle's medium (dMeM; invitrogen; Thermo Fisher Scientific, inc.) with 10% fetal bovine serum (FBS, invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified atmosphere of 5% CO 2 at 37˚C.
RNA extraction and reverse transcription-quantitative (RT-q) PCR. Total rna was extracted from tissue and cells (1x10 6 ) with Trizol ® reagent (invitrogen; Thermo Fisher Scientific, inc.), according to the manufacturer's instructions. The quantity and concentration of total rna were determined by a nanodrop 2000 instrument (Thermo Fisher Scientific, inc.). rT-qPcr was performed as described previously (33). Briefly, total RNA was reverse transcribed to cdna using a PrimeScript rT reagent kit (Takara Biotechnology co., ltd.). qPcr was performed in a 96-well plate on an aBi 7500 system (applied Biosystems; Thermo Fisher Scientific, Inc.) with PowerUp SYBR-Green Master Mix (Thermo Fisher Scientific, Inc.) as per the procedure provided by the manufacturer. For detecting hsa-mir-23a-5p, a hsa-mir-23a-5p-specific stem-loop primer (Guangzhou riboBio co., ltd.) was used for reverse transcription and RT-qPCR amplification was performed using the Bulge-Loop mirna rT-qPcr Starter kit (Guangzhou riboBio co., Ltd.). The thermocycling conditions were 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 60 sec. GaPdH (for circrna and mrna) or u6 (for mirna) was used as reference control. relative expression level was calculated by the 2 -ΔΔcq method (34). Primer sequences are listed in Table ii.
Cell proliferation, migration and invasion assays. cell proliferation was analyzed by a cell counting Kit-8 (ccK-8, cell migration and invasion assays were performed using Transwell assays. For invasion assays, 1x10 5 cells were suspended into 250 µl of serum-free DMEM with 0.1% bovine serum albumin (invitrogen; Thermo Fisher Scientific, inc.) and seeded into the upper chamber of a 24-well Transwell insert (pore size: 8 µm; BD Biosciences) which were precoated with Matrigel at 37˚C for 30 min (BD Biosciences). The lower chamber was filled with DMEM containing 2.5% FBS. For migration assays, 1x10 5 suspended cells were seeded into the upper chambers without a Matrigel coating. after 48 h, the invaded or migrated cells were fixed and stained with 0.5% crystal violet at room temperature (~25˚C) for 15 min and counted under a microscope (olympus corporation). Five images were randomly captured for each sample.
Statistical analysis. all experiments were performed in triplicate and data were analyzed by SPSS 20.0 (iBM corp.). comparisons between two groups were analyzed by Student's t-test or χ 2 test when appropriate, while comparisons among multiple groups were conducted using one-way anoVa with lSd post hoc analysis. P<0.05 was considered to indicate a statistically significant difference. Fig. 1a). The top 10 upregulated circrnas and top 10 downregulated circrnas are shown in Fig. 1B and listed in Table iii.
Identification of DE-circRNAs in
Construction of circRNA-miRNA-mRNA network. By using miranda to predict which mirnas were related to the top 20 de circrnas, 409 mirnas were obtained at the criteria of score ≥140 and Energy ≤-10. Following searching in mir2 disease, 17 circrna-mirna relationships involving 16 mirnas and 8 circrnas were obtained. additionally, a total of 1,669 target genes associated with these mirnas were predicted in 6 of 7 searched databases and 923 target genes were further filtered. The top 10 target genes for each miRNA were used for constructing a cerna network in cytoscape. This cerna network involved 15 circrna-mirna relationships and 140 mirna-mrna relationships (Fig. 2). Dif ferential expression of hsa _ circR NA _ 10 0 0 84, hsa-miR-23a-5p, and IGF2 in HCC tissues and cells. Findings showed that iGF2 was involved in the cerna relationship of hsa_circrna_100084-hsa-mir-23a-5p-iGF2. Therefore, the expression level of hsa_circrna_100084, hsa-mir-23a-5p, and iGF2 in Hcc tissues and liver cancer cells we validated by rT-qPcr, iHc and western blotting. as shown in Fig. 3a, compared with levels in the adjacent normal tissues, the relative expression levels of hsa_circrna_100084 in Hcc tissues were significantly upregulated (P<0.01), while the expression of hsa-miR-23a-5p was significantly downregulated (P<0.01). Then, the expression patterns of hsa_circrna_100084 and hsa-mir-23a-5p were analyzed in three liver cancer cell lines with different metastatic potential, including MHcc97H, Hep3B and HepG2 cells. consistently, the expression of hsa_circRNA_100084 was significantly upregulated in liver cancer cells (Hep3B and MHcc97H) and a hepatoblastoma cell line (HepG2) (P<0.01), while the expression of hsa-miR-23a-5p was significantly downregulated compared with levels in the human normal hepatic cell line lX2 (P<0.01, Fig. 3B). additionally, the mrna and protein expression of iGF2 in Hcc tissues and adjacent tissues were tested by rT-qPcr, iHc and western blotting (Fig. 4). iHc results showed that the number of iGF2-positive cells in Hcc tissues was much higher compared with adjacent normal tissues ( Fig. 4a and B). qPcr and western blotting further confirmed that IGF2 was upregulated in HCC tissues and cells (Fig. 4c-e). Taken together, these data suggested that hsa-mir-23a-5p, hsa_circrna_100084 and IGF2 might be involved in Hcc progression and that there may be competing relationships among them.
Discussion
Hcc is regarded as the most malignant type of liver cancer because of its high incidence rate and poor prognosis (35).
Therefore, it is necessary to investigate the biological basis and identify novel targets for Hcc. due to their special structure, circrnas are evolutionarily conserved and stable. Previous studies have demonstrated that circrnas are disease-, tissue-and stage-specifically expressed, suggesting their particular roles in disease initiation and development (36)(37)(38). The present study re-analyzed the gene expression profile GSE97332 and identified 147 DE-circRNAs, including 50 downregulated circrnas (34.01%) and 97 upregulated circrnas (65.99%). Then, a cerna network was constructed for these de-circrnas. in this cerna network, it was found that IGF2 was involved in a cerna relationship of hsa_circ rna_100084-hsa-mir-23a-5p-IGF2. Further studies demonstrated that hsa_circrna_100084 promoted liver cancer cell proliferation, migration and invasion by competitively binding hsa-mir-23a-5p, leading to the upregulation of IGF2. elucidating the molecular mechanisms of Hcc will be important for the development of therapies to successfully treat Hcc. Several studies have demonstrated that iGF2 is upregulated in a number of cancers, including Hcc, and is associated with resistance to chemotherapy and a worse prognosis (18,19,39). Though loss of imprinting, loss of hetero- Figure 6. Hsa_circ_100084 regulates liver cancer cell proliferation, migration and invasion by modulating iGF2. (a) ccK-8 assay was used to measure cell proliferation in HepG2 cells. (B-D) Transwell assays determined cell migration and invasion in HepG2 cells. Magnification, x200. All data are representative of three independent experiments and expressed as mean ± standard deviation. comparisons between two groups were analyzed by Student's t test, while comparisons among multiple groups were conducted using one-way anoVa with lSd post hoc analysis. ** P<0.01 vs. nc. Figure 5. relationships among hsa_circ_100084, hsa-mir-23a-5p and iGF2. (a) relative expression of hsa_circ_100084 and hsa-mir-23a-5p following transfection with sh-hsa_circ_100084 or hsa-mir-23a-5p mimics. (B) Putative binding sites among hsa_circ_100084, hsa-mir-23a-5p and iGF2. (c) The luciferase activity of luc-circ100084-WT, luc-iGF2-WT, luc-circ100084-mutant, or luc-iGF2-mutant co-transfected with hsa-mir-23a-5p mimics. (d) relative expression of hsa-mir-23a-5p and iGF2 following transfection with sh-hsa_circ_100084 or co-transfection of hsa-mir-23a-5p mimics and sh-hsa_circ_100084. all data are representative of three independent experiments and expressed as mean ± standard deviation. comparisons between two groups were analyzed by Student's t test, while comparisons among multiple groups were conducted using one-way anoVa with lSd post hoc analysis. ** P<0.01 vs. nc. zygosity, or reactivation of IGF2 transcription could partially explain the upregulation of IGF2 in cancer, further studies are necessary to explore these possibilities. The present study found that IGF2 is involved in a cerna relationship of hsa_circrna_100084-hsa-mir-23a-5p-IGF2. consistently, Zhen et al (40) demonstrated that circHMGcS1 promotes hepatoblastoma cell proliferation by regulating IGF2.
The results of the present study demonstrated that knocking down hsa_circrna_100084 could significantly inhibit the proliferation, migration and invasion of liver cancer cells, suggesting that hsa_circrna_100084 might have potential to be used us a promising therapeutic strategy for Hcc. By searching circBase, hsa_circrna_100084 was found to correspond to circeiF4G3, which is located on chr1:21329205-21415706. However, the role of this circrna in cancer initiation and development has not been investigated previously.
The mechanisms of circrnas in cancer initiation and progression have not been clearly elucidated. it has been reported that circrnas can regulate the expression of oncogenes or tumor-suppressive genes in different patterns (41). The most reported pattern is the cerna hypothesis. in this hypothesis, circrnas have been proposed to communicate with mrnas by competing for binding to shared mirna targets (42). This hypothesis has been confirmed in a number of studies. For example, circMTo1 suppresses Hcc progression by acting as a sponge of mir-9 (25). circSMad2 can inhibit the epithelial-mesenchymal transition via targeting mir-629 in Hcc (43). circ_0067934 promotes tumor metastasis and growth in Hcc via the inhibition of mir-1324 (44). it was hypothesized that hsa_circrna_100084 might act as a mirna sponge. Therefore, it was predicted the mirnas related with de-circrnas by bioinformatics analysis. The combination of hsa-mir-23a-5p with hsa_circrna_100084, as well as IGF2, was validated by a dual-luciferase reporter assay. as expected, hsa-miR-23a-5p could diminish the fluorescence of the wildtype of hsa_circrna_100084 and IGF2, but not the mutated forms. in addition, overexpression of hsa-mir-23a-5p could decrease the expression of IGF2 in HepG2 cells. However, sh-hsa_circrna_100084 at the same time could attenuate the effect of hsa-mir-23a-5p overexpression on the expression of IGF2.
However, the present study inevitably possess some limitations. First, although a cerna relationship of hsa_circrna_100084-hsa-mir-23a-5p-IGF2 axis was identified and their relationship confirmed by experiments, the expression downstream of iGF-2, such as the insulin receptor substrate 1/Pi3K/akt axis and sarcomatoid hepatocellular carcinoma/growth factor receptor-bound protein 2/ras/mitogen-activated protein kinase axis has not been examined. Besides, more direct evidence, such as rna immunoprecipitation analysis of the interaction of circrna_100084 and mir-23a-5p was not performed. Therefore, further researches are still needed to make more validate conclusions.
in conclusion, the present study demonstrated that hsa_ circrna_100084 is upregulated in Hcc tissue compared with the matched non-tumor liver tissues and may act as a cerna to increase IGF2 expression by sponging hsa-mir-23a-5p, which consequently contributes to Hcc proliferation, migra-tion and invasion. The deregulated circrnas in Hcc will be the subject of continuing investigation in further studies. | 2020-04-16T09:15:17.643Z | 2020-04-10T00:00:00.000 | {
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260411287 | pes2o/s2orc | v3-fos-license | Dose- and Time-Dependent Effect of Dietary Blueberries on Diabetic Vasculature Is Correlated with Gut Microbial Signature
Evidence from our lab and others indicates the vascular effects of dietary blueberries. In the present study, we determined dietary blueberries’ dose- and time-dependent effects on diabetic vasculature and their association with gut microbes. Seven-week-old db/db diabetic male mice were fed a diet supplemented with ± freeze-dried wild blueberry powder (FD-BB) for 4, 8, or 12 weeks (three cohorts). Diets contained 0%, 1.23%, 2.46%, and 3.7% of FD-BB, equivalent to 0, ½, 1, and 1.5 human servings of wild blueberries, respectively. The non-diabetic db/+ mice fed a standard diet served as controls. Metabolic parameters, vascular inflammation, and gut microbiome were assessed. Dietary supplementation of 3.7% FD-BB improved vascular inflammation in diabetic mice without improving systemic milieu in all three cohorts. Blueberries improved diabetes-induced gut dysbiosis depending on blueberry dosage and treatment duration. Spearman’s correlation indicated that the opportunistic microbes and commensal microbes were positively and negatively associated with indices of vascular inflammation, respectively. Dietary blueberries reduced the opportunistic microbe that was positively associated with vascular inflammation (Desulfovibrio), and increased the commensal microbe that was negatively associated with vascular inflammation (Akkermansia). Dietary blueberries could be a potential adjunct strategy to beneficially modulate gut microbes and improve vascular complications in diabetes.
Introduction
Diabetes has a major impact on cardiovascular diseases such as atherosclerosis.Cardiovascular disease has been associated with a 2-to 4-fold increase in the mortality rate in individuals with diabetes [1].In diabetes, high-glucose-and dyslipidemia-induced monocyte binding to the vascular endothelium is the initial step in developing atherosclerosis [2].The bound monocytes are then transmigrated into the subendothelial space and differentiated into macrophages by the uptake of oxidized LDL [2].These macrophages are further transformed into lipid-laden foam cells, resulting in vascular inflammation and dysfunction, leading to atherosclerosis [2].Hence, reducing monocyte binding to the vascular endothelium is a potential target to prevent diabetes-induced vascular disease., 1, and 1.5 human servings of wild blueberries, respectively.The non-diabetic db/+ mice fed a standard diet served as controls.Metabolic parameters, vascular inflammation, and gut microbiome were assessed.Dietary supplementation of 3.7% FD-BB improved vascular inflammation in diabetic mice without improving systemic milieu in all three cohorts.Blueberries improved diabetes-induced gut dysbiosis depending on blueberry dosage and treatment duration.Spearman's correlation indicated that the opportunistic microbes and commensal microbes were positively and negatively associated with indices of vascular inflammation, respectively.Dietary blueberries reduced the opportunistic microbe that was positively associated with vascular inflammation (Desulfovibrio), and increased the commensal microbe that was negatively associated with vascular inflammation (Akkermansia).Dietary blueberries could be a potential adjunct strategy to beneficially modulate gut microbes and improve vascular complications in diabetes.
Introduction
Diabetes has a major impact on cardiovascular diseases such as atherosclerosis.Cardiovascular disease has been associated with a 2-to 4-fold increase in the mortality rate in individuals with diabetes [1].In diabetes, high-glucose-and dyslipidemia-induced monocyte binding to the vascular endothelium is the initial step in developing atherosclerosis [2].The bound monocytes are then transmigrated into the subendothelial space and differentiated into macrophages by the uptake of oxidized LDL [2].These macrophages are further transformed into lipid-laden foam cells, resulting in vascular inflammation and dysfunction, leading to atherosclerosis [2].Hence, reducing monocyte binding to the vascular endothelium is a potential target to prevent diabetes-induced vascular disease.Diet plays a major role in enhancing or suppressing atherosclerosis by modulating vascular inflammation [3].It is well established that diets rich in fruits and vegetables prevent the development of vascular diseases [3].Blueberries are rich in flavonoids such as anthocyanins and contain more than 18 anthocyanins [2].Anthocyanins are glycosides of anthocyanidins (cyanidin, delphinidin, malvidin, and peonidin) and carbohydrate components (glucose, galactose, arabinose, etc.) [4].Evidence from epidemiological, clinical, and preclinical studies indicates the cardiovascular beneficial effects of consuming blueberries [2].Human studies showed that blueberry intake improves endothelial dysfunction in individuals with metabolic syndrome, increases endothelium-dependent vasodilation in healthy humans, and reduces blood pressure in postmenopausal women [5][6][7].
Gut microbes are essential to host physiology as they produce vitamins, regulate metabolism, modulate the immune system, and metabolize dietary compounds [8,9].Studies suggest that a balanced healthy gut microbiome prevents many chronic diseases, including cardiovascular disease, diabetes, and colon cancer [10,11].The host digestive enzymes are inefficient in metabolizing many phytonutrients, and most phytonutrients (such as anthocyanins in blueberries) reach the colon [4].Gut microbes using microbial enzymes metabolize anthocyanins into small metabolites (such as phenolic acids), which enter the circulation and mediate the biological activities of the dietary blueberries [4].In addition, a two-way relationship exists between dietary blueberries and gut microbes.Gut microbes metabolize the bioactive components of blueberries, and anthocyanins act as prebiotics through their selective use by commensal microbes to improve the host's health and reduce the risk of disease [2].Indeed, gut microbes are essential to mediate the biological activities of the phytonutrients and maintain the host's health.Conversely, an imbalanced gut microbial population (gut dysbiosis) can adversely affect the host [12].
We recently showed that dietary blueberries at nutritional dosage suppress vascular inflammation and improve vascular dysfunction in diabetic mice, possibly through NOX4-mediated mechanisms [2].Our in vitro and ex vivo studies further showed that at physiologically relevant dosages, blueberry metabolites ameliorate lipotoxicity-induced vascular dysfunction and suppress endothelial inflammation in the aortic endothelial cells isolated from individuals with type 2 diabetes.In the present study, we investigated the dose-and time-dependent effects of dietary blueberries on diabetic vasculature and diabetes-induced dysbiosis that are largely unknown.We further identified the role of gut microbes in mediating the vascular effects of dietary blueberries by determining the association between the indices of vascular inflammation and gut microbes.
Experimental Animals
Diabetic db/db mice with C57BLKS/J background (db/db; B6.Cg-m +/+ Lepr db ) are an established model to study diabetes-induced vascular complications [2,13].Six-weekold male diabetic (db/db) mice and control (db/+) mice were obtained from the Jackson Laboratories (Bar Harbor, ME, USA), and the stock number was 000642.Mice were housed at the University of Utah animal facility (5 mice/cage) under humane conditions and acclimated for a week before the experiments.The mice were kept in controlled artificial lighting conditions following a 12 h light and dark cycle.The temperature was maintained at 23 ± 1 • C and the humidity level was set at 45 ± 5%.The Institutional Animal Care and Use Committee at the University of Utah approved the study protocol 18-09003.All procedures were conducted in accordance with the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" published by the US National Institute of Health.
Standard-Diet-and Blueberry-Supplemented Diets
Vaccinium angustifolium is a species of blueberry commonly known as wild lowbush blueberry.The Vaccinium genus belongs to the Ericaceae family and wild blueberries (Vaccinium angustifolium) were used in the present study.The Wild Blueberry Association of North America (Momence, IL, USA) provided the freeze-dried wild blueberry powder.Standard and blueberry-supplemented diets were purchased from Research Diets Inc. (New Brunswick, NJ, USA).This study aimed to identify the time-and dose-dependent effects of dietary blueberries.Hence, blueberry-supplemented diets were prepared with three different freeze-dried blueberry (FD-BB) powder dosages.Diets contained 1.23%, 2.46%, and 3.7% of FD-BBs (w/w), equivalent to the human consumption of ~80 g, 160 g, and 240 g of fresh blueberries per day (Table 1 Diabetes has a major impact on cardiovascular diseases such as atherosclerosis.Cardiovascular disease has been associated with a 2-to 4-fold increase in the mortality rate in individuals with diabetes [1].In diabetes, high-glucose-and dyslipidemia-induced monocyte binding to the vascular endothelium is the initial step in developing atherosclerosis [2].The bound monocytes are then transmigrated into the subendothelial space and differentiated into macrophages by the uptake of oxidized LDL [2].These macrophages are further transformed into lipid-laden foam cells, resulting in vascular inflammation and dysfunction, leading to atherosclerosis [2].Hence, reducing monocyte binding to the vascular endothelium is a potential target to prevent diabetes-induced vascular disease., 1, and 1.5 human servings of wild blueberries).All the diets were matched for sugar and fiber content.The dosage was calculated based on the Food and Drug Administration (FDA) recommendation for extrapolating the doses from humans to animals by normalizing for body surface area.and Integrative Physiology, College of Health, University of Utah, USA; u6024207@utah.edu(A.K.S.B.); chrissa.petersen@utah.edu(C.P.); .B.); miley.nguyen@utah.edu(M.N.); madison.putich@utah.edu(M.P.); .-G.); slarsen@union.utah.edu(S.L.) rition Center, Little Rock, Arkansas, AR 72205, USA., University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA; hapaz-.P.); zhongying@uams.edu(Y.Z.); uwankhade@uams.edu(U.D.W.) .velayutham@utah.edu;Tel.: +1-801-581-8376; Fax: +1-801-585-3874 our lab and others indicates the vascular effects of dietary blueberries.In termined dietary blueberries' dose-and time-dependent effects on diabetic ociation with gut microbes.Seven-week-old db/db diabetic male mice were with ± freeze-dried wild blueberry powder (FD-BB) for 4, 8, or 12 weeks tained 0%, 1.23%, 2.46%, and 3.7% of FD-BB, equivalent to 0, ½ 1, and 1.5 blueberries, respectively.The non-diabetic db/+ mice fed a standard diet bolic parameters, vascular inflammation, and gut microbiome were asentation of 3.7% FD-BB improved vascular inflammation in diabetic mice mic milieu in all three cohorts.Blueberries improved diabetes-induced gut lueberry dosage and treatment duration.Spearman's correlation indicated crobes and commensal microbes were positively and negatively associated inflammation, respectively.Dietary blueberries reduced the opportunistic ely associated with vascular inflammation (Desulfovibrio), and increased the was negatively associated with vascular inflammation (Akkermansia).Diee a potential adjunct strategy to beneficially modulate gut microbes and ications in diabetes.
dose/time-dependent effect; vascular inflammation; cardiovascular; gut ajor impact on cardiovascular diseases such as atherosclerosis.Cars been associated with a 2-to 4-fold increase in the mortality rate iabetes [1].In diabetes, high-glucose-and dyslipidemia-induced he vascular endothelium is the initial step in developing atheroscle-
Measurement of Metabolic Variables and Collection of Tissue Samples
Blood glucose, glucose tolerance, insulin tolerance, and body composition were measured at three different time points (after 4, 8, or 12 weeks of treatment).Food intake and body weight were recorded weekly throughout the study.Contour Next One monitoring system (Bayer, Parsippany, NJ, USA) was used to measure fasting and non-fasting blood glucose concentrations in blood collected from tail veins.Glucose and insulin tolerance tests were performed as we previously described [2,13,14].To perform the intraperitoneal glucose tolerance test (IPGTT), the mice were subjected to an overnight fast, and a single bolus of glucose (2 g/kg body weight) was administered intraperitoneally.Blood glucose concentrations were measured at 0, 15, 30, 60, and 120 min following glucose administration.To perform the intraperitoneal insulin tolerance test (IPGTT), the mice were subjected to a 4 h fast, and insulin was administered intraperitoneally (0.75 U/kg body weight).Blood glucose concentrations were measured at 0, 15, 30, 60, and 120 min following glucose administration.After treatment (4, 8, or 12 weeks), mice were anesthetized using 2-5% isoflurane, and blood samples were collected via cardiac puncture.The organs and cecum contents were collected from the experimental animals, flash-frozen in liquid nitrogen, and stored at −80 • C. The aortic vessels were dissected free from adherent tissues and used to measure vascular inflammation by assessing monocyte binding to the aortic endothelium and inflammatory molecules.Aorta segments were placed in RNAlater for stabilization of RNA and stored at −80 • C for later PCR analysis.The cecum contents were used for microbial profiling.
Measurement of Vascular Inflammation
The effect of blueberry supplementation on vascular inflammation in diabetic mice was assessed by monocyte binding to the aorta and the expression of inflammatory markers in the aortic vessel we have described with a few modifications [2,13,14].Briefly, the segments of the abdominal aortae proximal to the iliac bifurcation were used to measure monocyte binding to the vasculature.The aorta was opened to expose the luminal endothelial layer and covered with EBM medium containing 1% heat-inactivated FBS for 10 min at 37 • C.Then, the medium was gently removed, and the exposed endothelium layer of the aorta was incubated with Calcein-AM-labeled mouse monocytic WEHI78/24 cells for 30 min.The unbound monocytes were removed from the aorta by gentle washing.Confocal microscopy was used to visualize and count the bound monocytes.Five images of five frames from each aorta were captured using an Olympus IX73 fluorescence microscope (Olympus, Tokyo, Japan).The vascular inflammation was also measured by determining the expression of inflammatory chemokines (MCP-1 and IL-8) and adhesion molecules (ICAM1, VCAM1, and E-Selectin) by qPCR using SYBR green as we described previously [2,13,14].Briefly, total RNA was extracted from the aorta using RNeasy Plus mini kit, cDNA was synthesized using an RT-PCR kit, and the expression of inflammatory molecules was measured by qPCR using specific primers and SYBR green.The expression of the housekeeping gene GAPDH was used to normalize the expression.
Microbial Profiling Using 16S rRNA Amplicon Sequencing
Microbial community profiling was carried out using the 16 s rRNA amplification method as we described previously [14].Bacterial DNA was extracted from the cecum contents using DNeasy PowerSoil Kit, and 50 ng of bacterial DNA was used to amplify the V4 variable region of the 16 S rRNA gene using 515F/806R primers.The primers (forward and reverse) were barcoded to achieve the multiplexing of up to 384 samples per run [15].The pooled amplicons were then subjected to paired-end sequencing on the Illumina Miseq platform, utilizing a sequencing configuration of 2 × 250 bp.Approximately 30% of PhiX DNA was included in the sequencing run to improve sequencing quality.Raw sequences are available at the NCBI Sequence Read Archive (SRA) under accession no.PRJNA996159.
Bioinformatics Analysis
Miseq Reporter on the instrument computer was used to automate demultiplexing, adapter trimming, and generating fastq files.QIIME 2 platform was used for the subsequent bioinformatics analysis [16].Denoising was performed through initial quality filtering and applying the Deblur algorithm [17,18].The representative amplicon sequence variants (ASVs) were utilized to generate the phylogenetic tree with FastTree [19].To assign the taxonomy, Naives Bayes classifier trained on the Greengenes 13_8 reference was utilized [20].Rarefaction curves (Supplementary Figure ) based on observed ASV were used to evaluate the adequacy of the sampling depth, which was established at 3628-quality-filtered reads per sample.A total of 136 samples passed the established sampling depth and were used in further analyses.α-Diversity metrics (richness, evenness, and diversity) were assessed using the α-Diversity indices such as observed ASV, Shannon Diversity, Evenness, and Dominance.β-Diversity (microbial community differences) was determined using the weighted Unifrac distances, and the principal coordinate analysis (PCoA) plot was used to visualize the results [21].
Statistical Analysis
Prism 8.0 (GraphPad, CA, USA) or SPSS Version 25 (IBM) was used for statistical analyses.Microbiome data were analyzed using the R programming language (version R-4.2.2).One-way ANOVA was employed to analyze metabolic parameters, and Tukey post hoc tests were performed when the main effects were significant.Where appropriate, all data are presented as mean ± standard error of the mean (SEM) and statistical significance was determined at a p < 0.05.The relationship between the specific taxa and vascular inflammation was assessed by correlation analysis, as we reported previously [14,22].Microbiome data (relative abundance of genera) and indices of vascular inflammation (monocyte binding to the aortic vessel and mRNA expression of vascular inflammatory molecules) were used to identify the association between the abundance of specific genera and vascular inflammation through Spearman's correlation in R [14].
Dietary Blueberries Did Not Improve Diabetes-Induced Metabolic Alterations
The effect of dietary blueberries on metabolic parameters (body weight, blood glucose, body fat, lean body mass, and body fluid) was evaluated after 4, 8, and 12 weeks of treatment.The body weight, blood glucose, and body fat were increased, whereas lean body mass was decreased in diabetic (db/db) mice vs. control (db/+) mice in all three cohorts (Table 2).However, blueberry supplementation (1.23%, 2.46%, or 3.7% in diet) did not improve these metabolic parameters with 4, 8, or 12 weeks of treatment (Table 2).Further, diabetic mice exhibited impaired glucose and insulin tolerance that was not improved with blueberry supplementation (Figure 1).
Dietary Blueberries Exhibit a Dose-Dependent Ability to Suppress Diabetes-Induced Vascular Inflammation
The dose-and time-dependent effects of dietary blueberry supplementation on endothelial inflammation were assessed by monocyte binding to the aortic vessel.As expected, there was an increased binding of mouse monocytic WEHI 78/24 cells to the aortic vessel isolated from diabetic vs. control mice in all three cohorts (4, 8, and 12 weeks).However, a 3.7% blueberry-supplemented diet suppressed the vascular inflammation in diabetic mice, as shown by a reduced monocyte binding to the aortic vessel at 4, 8, and 12 weeks of treatment (Figure 2A).In addition, we assessed inflammatory markers in the 12-week cohort.The mRNA expression of inflammatory chemokines (MCP1 and IL8) and VCAM1 greatly increased in diabetic vs. control mice (Figure 2B).However, 3.7% blueberry supple-mentation reduced the expression of these inflammatory molecules in diabetic mice (D12B3 vs. D12) (Figure 2B).
Dietary Blueberries Dose-and Time-Dependently Improve Diabetes-Induced Gut Dysbiosis
α-Diversity indices such as Observed ASV, Shannon Diversity, Evenness, and Dominance (which measure the richness and evenness of the microbial community) were not significantly different in diabetic vs. control mice at the ASV level (Figure 3).However, β-Diversity (which measures the similarity or dissimilarity of the microbial community) was significantly different between diabetic and control mice (Figure 3).Further, blueberry supplementation to diabetic mice significantly altered the β-Diversity that depended on dose and duration of treatment (Figure 3).In the present study, the microbial community was distributed into six major phyla such as Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Tenericutes, TM7, and Verrucomicrobia (Figure 4).Diabetes affected the relative abundance of several phyla and some of them were improved with blueberry treatment (Figure 4).The diabetic mice exhibited a decreased abundance of Actinobacteria in all three cohorts (4, 8, and 12 weeks).Blueberry supplementation increased the abundance of Actinobacteria at 4 weeks (with 2.46% and 3.7% dose), 8 weeks (with 1.23% and 2.46% dose), and 12 weeks (with 3.7% dose) of treatment.In addition, several taxa (at genus level) were altered in diabetes, and most of them were improved with different dosages of blueberry (Figure 5).The relative abundance of the genera such as Allobaculum and Bifidobacterium was decreased, whereas Anaerotruncus and Clostridium were increased in diabetic mice in the 4-week cohort.Bifidobacterium was decreased, whereas Clostridium and Ruminococcus were increased in diabetic mice in the 8-week cohort.Akkermansia and Dehalobacterium were decreased, whereas Anaerotruncus, Clostridium, and Desulfovibrio were increased in diabetic mice in the 12-week cohort.Blueberry supplementation (1.23%, 2.46%, or 3.7% in diet) improved the abundance of these genera that were altered in diabetes at different time points (Figure 5).
Dietary Blueberries Exhibit a Dose-Dependent Ability to Suppress Diabetes-Induced Vascular Inflammation
The dose-and time-dependent effects of dietary blueberry supplementation on e dothelial inflammation were assessed by monocyte binding to the aortic vessel.As e pected, there was an increased binding of mouse monocytic WEHI 78/24 cells to the aor
Dietary Blueberries Dose-and Time-Dependently Improve Diabetes-Induced Gut Dysbiosis
α-Diversity indices such as Observed ASV, Shannon Diversity, Evenness, and Dominance (which measure the richness and evenness of the microbial community) were not significantly different in diabetic vs. control mice at the ASV level (Figure 3).However, β-Diversity (which measures the similarity or dissimilarity of the microbial community) was
Association between Gut Microbial Signature and Vascular Inflammation
The time-dependent association between gut microbes (genera) and monocyte binding to vasculature was assessed using the data from 4-week (C4, D4, D4B3), 8-week (C8, D8, D8B3), and 12-week (C12, D12, D12B3) cohorts.Several genera were positively or negatively associated with monocyte binding to the vasculature depending on the duration of the treatment (Figure 6A).We also identified the association between the specific genera and indices of vascular inflammation (monocyte binding to the vasculature and expression of inflammatory molecules such as MCP1, IL8, ICAM1, VCAM1, and E-SEL) in the 12-week cohort using the data from C12, D12, and D12B3 mice.Many of the genera positively associated with the inflammatory markers were also positively associated with monocyte binding to the vasculature (Figure 6B).Similarly, many of the genera negatively associated with the inflammatory markers were also negatively associated with monocyte binding to the vasculature (Figure 6B).
ages of blueberry (Figure 5).The relative abundance of the genera such as Allobaculum and Bifidobacterium was decreased, whereas Anaerotruncus and Clostridium were increased in diabetic mice in the 4-week cohort.Bifidobacterium was decreased, whereas Clostridium and Ruminococcus were increased in diabetic mice in the 8-week cohort.Akkermansia and Dehalobacterium were decreased, whereas Anaerotruncus, Clostridium, and Desulfovibrio were increased in diabetic mice in the 12-week cohort.Blueberry supplementation (1.23%, 2.46%, or 3.7% in diet) improved the abundance of these genera that were altered in diabetes at different time points (Figure 5).
Discussion
Emerging evidence from epidemiological, clinal, and preclinical studies indicates the vascular beneficial effects of consuming blueberries.However, dietary blueberries' doseand time-dependent effects on diabetic vasculature and their association with gut mi-
Discussion
Emerging evidence from epidemiological, clinal, and preclinical studies indicates the vascular beneficial effects of consuming blueberries.However, dietary blueberries' doseand time-dependent effects on diabetic vasculature and their association with gut microbes are unknown.In the present study, we determined whether the effect of dietary blueberries on diabetic vasculature is dose/time-dependent and identified whether these effects are associated with specific gut microbes.First, dietary supplementation of blueberries for 4, 8, or 12 weeks suppressed vascular inflammation in diabetic mice at a dosage equivalent to the human consumption of 1.5 cups of fresh blueberries.This effect was not secondary to an improvement in the metabolic milieu, suggesting the direct effect of blueberry bioactives on diabetic vasculature.Second, dietary blueberries ameliorated diabetes-induced gut dysbiosis that depended on the dose and duration of the treatment.Third, opportunistic microbes were positively associated, whereas commensal microbes were negatively associated with indices of vascular inflammation.Finally, dietary blueberries reduced the opportunistic microbe Desulfovibrio that was positively associated with vascular inflammation, and increased the commensal microbe Akkermansia that was negatively associated with vascular inflammation.
Monocyte binding to the inflammatory vasculature is a key initial event involved in atherosclerosis and is a potential target to prevent vascular disease [3].Cardiovascular risk factors such as high glucose and dyslipidemia activate the vascular endothelium to secrete inflammatory chemokines (MCP1 and IL8) and adhesion molecules (ICAM1, VCAM1, and E-Selectin), which leads to monocyte binding to the vasculature and the subsequent vascular inflammation [3,13].In the present study, blueberry supplementation at the nutritional dosage (equivalent to human consumption of ~1.5 cups of fresh blueberries) suppressed monocyte binding to the diabetic vasculature and reduced the mRNA expression of MCP1, IL8, and VCAM1.This is consistent with previous human studies supporting the vascular beneficial effects of dietary blueberries.Blueberry intake was shown to increase endothelium-dependent vessel relaxation in healthy humans in a dose-dependent manner [5].In the present study, the metabolic parameters that were altered in diabetic mice were not improved with blueberry supplementation.We recently showed that blueberry metabolites suppress endothelium inflammation in endothelial cells isolated from individuals with type 2 diabetes and improve lipotoxicity-induced endothelial dysfunction [4,23].Our previous and current data suggest that the observed vascular effects of blueberries could be mediated by the direct effect of their circulating metabolites on the vasculature.
Blueberries act as a prebiotic and improve diabetes-induced gut dysbiosis in a doseand time-dependent manner.A recent study indicated that the effect of flavonoids on the gut microbiome is dosage-dependent, and a significant divergence was observed in the gut microbiome with the habitual intake of high and low flavonoids [24].Blueberry supplementation improved the relative abundance of several taxa in diabetic mice in all three cohorts.Phyla such as Actinobacteria, Tenericutes, and Verrucomicrobia were decreased in diabetic mice.Actinobacteria is a phylum that includes Gram-positive anaerobic bacteria consisting of numerous commensal genera (such as Bifidobacterium, widely used as probiotics) and plays an essential role in gut homeostasis [25,26].Tenericutes improve cardiovascular risk factors as shown by their association with lower triglycerides and higher HDL levels [27].Genera belonging to Verrucomicrobia are associated with reduced hypertension in humans [28].In the present study, blueberry supplementation increased the abundance of Actinobacteria, Tenericutes, and Verrucomicrobia in diabetic mice.Further, commensal genera such as Bifidobacterium and Akkermansia were decreased, whereas opportunistic genera such as Desulfovirbio and Clostridium were increased in diabetic mice.Bifidobacterium is a popular probiotic, and studies indicate its beneficial effects on cardiovascular disease [29].Akkermansia belongs to the phylum Verrucomicrobia and exhibits anti-inflammatory effects in preclinical models [30,31].Akkermansia muciniphila regulates mucin production in the gastrointestinal tract, host metabolism, and immune response, suggesting it could be a potential target in diseases related to gut dysbiosis [31].Akkermansia muciniphila is also considered a next-generation probiotic, and studies indicate its beneficial effect in lowering hypertension [28].In the present study, blueberry supplementation increased the abundance of the commensals (Bifidobacterium and Akkermansia) in diabetic mice, consistent with a previous human study that showed an increase in the abundance of Bifidobacterium following blueberry intake [32].The Desulfovibrio genus is a type of sulfate-reducing bacteria responsible for producing cytotoxic hydrogen sulfite [33].A human study indicated an increased abundance of Desulfovibrio in individuals with type 2 diabetes [34].Desulfovibrio desulfuricans aggravates atherosclerosis by increasing intestinal permeability and circulatory lipopolysaccharide in a mouse model of atherosclerosis (ApoE −/− mice) [35].Gut metabolites produced by Desulfovibrio promote inflammatory molecules such as IL6 and IL8 [33].Taxa belonging to the genera Clostridium are Gram-negative obligate anaerobic pathogens strongly associated with inflammation [36].In the present study, dietary blueberries reduced the abundance of Desulfovibrio and Clostridium.The role of gut microbes is crucial in metabolizing bioactive compounds of blueberries into circulating phenolic metabolites [4,23].Therefore, maintaining a healthy gut microbiome is essential to harness the vascular benefits of consuming blueberries.Our current study suggests that the bioactive compounds found in blueberry may serve as prebiotics, promoting the growth of beneficial microbes.This, in turn, could enhance the production of metabolites derived from blueberries and could be one of the possible reasons for the beneficial effects of dietary blueberries observed in the present study.
Spearman's correlation indicated that several genera are positively or negatively associated with the indices of vascular inflammation.Most importantly, the opportunistic microbes were positively associated with vascular inflammation, whereas commensal microbes were negatively associated with vascular inflammation.The commensal Akkermansia is negatively associated with IL8, MCP1, VCAM1, and monocyte binding to the vasculature.The abundance of Akkermansia is significantly reduced in diabetic mice, but blueberry supplementation increased the abundance of this genera.Similarly, the opportunistic microbe Desulfovibrio is positively associated with IL8, MCP1, and monocyte binding to the vasculature.However, blueberry supplementation suppressed the abundance of Desulfovibrio.These data indicate that dietary blueberries could exert beneficial effects on vasculature by suppressing the opportunistic microbes that contribute to vascular inflammation while simultaneously promoting the growth of commensal microbes that help to counteract vascular inflammation.
Conclusions
Dietary supplementation of blueberries suppressed vascular inflammation and improved gut dysbiosis in diabetic mice that depends on the blueberries' dosage and treatment duration.Dietary blueberries reduced the opportunistic microbe that was positively associated with vascular inflammation (Desulfovibrio), and increased the commensal microbe that was negatively associated with vascular inflammation (Akkermansia).Future research is needed to elucidate the role of gut microbes in the production of blueberry metabolites and identify the influence of blueberry-derived microbial metabolites on the vascular effects of blueberries.In conclusion, dietary blueberries may be a potential strategy to improve diabetes-induced vascular complications by modulating gut microbes.
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104292499 | pes2o/s2orc | v3-fos-license | Nematic superconductivity stabilized by density wave fluctuations: Possible application to twisted bilayer graphene
Nematic superconductors possess unconventional superconducting order parameters that spontaneously break rotational symmetry of the underlying crystal. In this work we propose a mechanism for nematic superconductivity stabilized by strong density wave fluctuations in two dimensions. While the weak-coupling theory finds the fully gapped chiral state to be energetically stable, we show that strong density wave fluctuations result in an additional contribution to the free energy of a superconductor with multicomponent order parameters, which generally favors nematic superconductivity. Our theory shades light on the recent observation of rotational symmetry breaking in the superconducting state of twisted bilayer graphene.
I. INTRODUCTION
Unconventional superconductors can have multicomponent superconducting order parameters which transform in a multidimensional representation of the crystal symmetry group.In such cases, additional symmetries besides the U (1) gauge symmetry -such as timereversal or rotation symmetry -are broken in the superconducting state.Superconductors which break rotation symmetry can be called nematic superconductors (NSC), in analogy with rotational symmetry breaking in liquid crystals, whereas superconductors which break time-reversal symmetry are known as chiral superconductors.Nematic and chiral superconducting order parameters that belong to the same multiplet are degenerate at the superconducting transition temperature, while this degeneracy is lifted at lower temperature.
Recently, NSC have attracted a lot of attention following the discovery of rotation symmetry breaking in superconducting states of doped topological insulators Bi 2 Se 3 in Knight shift 3 , upper critical field [4][5][6][7][8] , specific heat 4,5 , magnetic torque 9 and STM 10 measurements.Importantly, no signatures of rotational symmetry breaking were found in the normal state, indicating that nematicity is a property of the superconducting state itself.The observed features are consistent with a nematic superconductor with a two-component odd-parity SC order parameter 11,12 .
The studies of NSC have mainly focused on strongly spin-orbit-coupled 3D materials and have considered odd-parity pairings [13][14][15][16][17][18][19][20][21][22][23][24][25] .As shown by weak-coupling approach 13,14 , in the presence of strong spin-orbit coupling and odd-parity pairing, the nematic superconducting state can be energetically more favorable than the chiral one due to the difference in their gap structures.In contrast, for two-dimensional (2D) systems without spin-orbit coupling, NSC is not expected from the gap structure: In 2D, the chiral p + ip or d + id SC states generally have a full superconducting gap on the Fermi surface, whereas the nematic p x (p y ) or d x 2 −y 2 (d xy ) states have point nodes.As a result, the chiral state has a lower energy compared to the nematic state within a weak-coupling treatment [26][27][28][29][30] .
In this work, we propose a mechanism for p-or d-wave nematic superconductivity in 2D systems with hexagonal symmetry D 6 .We focus on the vicinity of the superconducting transition temperature, which allows us to treat the problem within the Ginzburg-Landau (GL) theory.By going beyond the weak-coupling approach which only takes into account the energy of Bogoliubov quasiparticles, we show that sufficiently strong fluctuations of a density wave order stabilize the NSC.The energy of such fluctuations is affected by the presence of a pairing potential and can thus distinguish between different SC states.Usually, the corresponding contribution is small compared to the weak-coupling term; however, it becomes more significant as the strength of fluctuations grows.This effect is known as a feedback mechanism and was originally proposed by Anderson and Brinkman to explain the stability of nodal A-phase in superfluid He-3 due to strong ferromagnetic fluctuations [31][32][33] .
We show that strong density wave fluctuations generically favor nematic superconductivity in 2D.Our results are largely independent of microscopic details of such fluctuations.We find that the nematic d-wave state is stabilized more significantly by charge density wave (CDW) fluctuations, while the feedback contribution from spin density wave (SDW) is partially suppressed by the destructive interference in a coherence factor.Interestingly, the conclusion is dual in a case of twocomponent spin-triplet superconductivity, i.e., nematic p-wave SC is more stabilized by SDW.We emphasize that our analysis is valid not far from the superconducting transition point, where GL theory is applicable.
Our work is motivated by recent experiments on twisted bilayer graphene (TBG), which observed superconductivity and strongly correlated insulating state at 'magic' angle θ ≈ 1.1 • 1,34,35 .The mechanism for su-arXiv:1810.04159v2[cond-mat.supr-con]8 Apr 2019 perconductivity in TBG and the pairing symmetry are subject to intense theoretical study . In paticular, Ref. 36 showed that due to the Fermi surface nesting and the proximity to Van Hove singularity, unconventional superconductivity and density wave emerge as the two leading instabilities driven by Coulomb interaction.The most divergent superconducting instabilities are found in the two-component p-wave and d-wave superconducting channels, which are nearly degenerate when the intervalley exchange interaction is small.Related results on superconductivity from density wave/antiferromagnetic fluctuations in graphene superlattices appear in Refs.37-39 (which propose chiral p + ip and d + id pairings).We thus expect that our strong-coupling theory of nematic superconductivity from density wave fluctuations is directly applicable to TBG.The strength of density wave fluctuations, which are important for our theory, may be enhanced by, e.g., changing electron density or tuning the twist angle and the interlayer spacing.We predict that one can observe the transition between chiral and nematic p/d-wave superconducting states upon varying these parameters.
Our result sheds light on the most recent measurements of in-plane upper critical magnetic field in TBG, which shows a pronounced two-fold anisotropy revealing the breaking of rotational symmetry 2 .This experimental finding suggests the possibility of nematic pwave or d-wave superconductivity, instead of chiral p + ip or d + id states which are isotropic.We emphasize that in our study nematicity is an intrinsic property of the anisotropic superconducting state, and we assume there is no primary electronic order that breaks rotation symmetry in the normal state.This should be contrasted with the scenario considered in Ref. 40, where the anisotropy of the superconducting state originates from nematic orbital order that onsets at high temperature in the normal metal.Furthermore, the nematic superconducting state studied in Ref. 40 is fully gapped and breaks time-reversal state, while the p-or d-wave nematic superconductor studied in our work is time-reversal-symmetric and has point nodes in the gap structure.This can be directly probed in future experiments, including thermal transport and tunneling spectroscopy.
II. CDW MODES COUPLED TO SC
To understand the essential physics of strong density wave fluctuations coupled to SC, we begin by considering a phenomenological GL theory.In GL theory the free energy F SC of the superconductor is expanded up to fourth order in the two-component (spin-singlet) d-wave order parameter SC for the hexagonal systems given by φ φ 1. Two lowest-order diagrams describing coupling between CDW fluctuations φi and SC order parameter ∆, see Eq. ( 3).These diagrams are sufficient provided the system is close to the superconducting transition (∆ is small) and the CDW fluctuations are massive.
where < 0, the order parameter is real and given by (d 1 , d 2 ) ∼ (cos θ, sin θ).This state defines a nematic superconductor, owing its name to the nonzero subsidiary nematic order which transforms as a nematic director; this state has nodes in the excitation spectrum.As shown below, calculating α 1,2 within weak-coupling gives α 2 > 0, selecting the chiral state.Next, we introduce the coupling to density wave fluctuations.For the sake of definiteness, we consider CDW fluctuations, but note that the argument is similar for SDW fluctuations.In hexagonal systems CDW order is described by a three-component complex order parameter φ = (φ 1 , φ 2 , φ 3 ), where the fields φ i correspond to CDW modes at ordering wave vectors Q i .These three wave vectors are related by sixfold rotation.To the lowest order, the coupling of the SC order parameter d to the CDW modes φ, shown diagrammatically in Fig. 1, can be expressed as (3) ) is a subsidiary nematic order parameter quadratic in the fields φ i and describes anisotropic CDW fluctuations which transform as partners under rotations, and d = (d 1 , d 2 ).Since it has the same symmetry as (N 1 , N 2 ) in (2) it couples linearly.
We further assume that the fields φ i are massive and can be described by a Gaussian contribution F φ , the precise form of which is immaterial for present purpose.The free energy of the superconductor coupled to the CDW fluctuations can thus be expressed as Since the fields φ i are massive they can be integrated out, which leads to an effective free energy for the superconductor given by F SC = F ∆ + δF ∆ ; at fourth order, the correction δF ∆ is given by Using the identity | 2 , we observe that (4) implies a lowering of the energy of the nematic superconducting state relative to the chiral state.This effect is enhanced as the fluctuations become stronger, thus exceeding the weak-coupling or any other fourth-order contribution and eventually leading to NSC.Remarkably, the argument leading to Eq. ( 4) is general and does not rely on the nature of the fluctuating field.In particular, as mentioned, it also applies to SDW fluctuations, which can be described by a vectorial order parameter φ = ( φ 1 , φ 2 , φ 3 ).Finally, a similar argument was applied to demonstrate the existence of s + d-wave SC state in the presence of nematic fluctuations in systems with tetragonal symmetry 63 .
III. GENERAL MODEL FOR CDW FLUCTUATIONS AND SC
While the above argument is physically compelling and correctly captures the physical mechanism of fluctuation-induced NSC, it is based on a simplified approach which neglects the contribution of modes with nonzero momentum or frequency.To develop a theory of NSC which takes this into account, we now consider a more general model for a two-component d-wave superconductor in the presence of CDW fluctuations.The Hamiltonian of such a system is given by H , where describe the normal state electronic excitations ψ kα with dispersion ξ k and spin α =↑, ↓, and the (bosonic) CDW fluctuations φ q governed by the bare propagator Ṽ0 (q), respectively.The propagator Ṽ0 (q) is peaked at the six symmetry-related CDW ordering vectors ±Q i=1,2,3 .The coupling of the fermions to the superconducting pair potential and the CDW fluctuations is given by respectively.On the Fermi surface, the pairing potential of the two-component d-wave SC is given by ∆ where, again, ∆ is the overall pairing strength and d = (d 1 , d 2 ), which satisfies |d| 2 = 1, captures the structure of the two-component order parameter.This form of the pairing potential corresponds to the E 2 representation of the point group D 6 .
The Hamiltonian H of Eqs. ( 5) and ( 6) defines a general model for a d-wave superconductor coupled to CDW fluctuations.To demonstrate how the (gapped) fluctuations can induce NSC via the so-called feedback mechanism, we proceed in two main steps: First, we integrate out the fermions and then the fluctuation fields φ q .In this way, we obtain an effective free energy functional for the superconducting order parameter which includes the effect of CDW fluctuations and renormalizes the weak-coupling result.The latter is directly obtained from H by neglecting the effect of CDW fluctuations altogether.More precisely, within weak-coupling the BCS free energy of the superconductor is given by After straightforward evaluation, we find F ∆ in hexagonal systems up to fourth order as (see Appendix A for details) where r ∼ (T − T c ), , and ω n = πT (2n + 1) are fermionic Matsubara frequencies.Since K 0 > 0, we find that within weak-coupling the chiral state indeed has lower energy below T c , in agreement with Ref. 29.This applies very generally to systems with hexagonal symmetry and does not depend on microscopic details of Fermi surface.
IV. FLUCTUATION-INDUCED NSC
We now proceed to calculate the correction to the weak-coupling free energy originating from the CDW fluctuations.As an intermediate step, we consider the normal state electronic structure described by ξ k of (5) in more detail.At low energies, the most important electronic excitations are located in the vicinity of those points on the Fermi surface which are connected by the CDW ordering vectors, the so-called hot spots.The hexagonal symmetry dictates that there are six such hot spots.Following the results of Ref. 36, we focus on CDWs with wavevectors that connect all adjacent hot spots, as shown in Fig. 2(b), though this assumption is not important for our analysis.This hot spot model, which introduces six flavors of low-energy fermions ψ ikα (i = 1, . . ., 6) with corresponding ξ ik , also establishes a natural connection with TBG 36,41 .Note that within the hot spot model all momenta are measured with respect to the hot spots.
To calculate the feedback correction, we integrate out the CDW fluctuations, which we assume to be massive, and determine their contribution to the free energy.Importantly, this contribution depends on the SC order parameter and can be expanded in ∆ to obtain renormalized GL coefficients.Since the full calculation is tedious but straightforward, we discuss only the final result and present the details in Appendix A. We find the correction to the free energy due to CDW fluctuations as where V is an effective propagator of the CDW fluctuations, and δ χ is the correction to the CDW susceptibility due to the coupling to ∆.Note that in (8) Tr implies summation over frequencies, momenta, as well as patches.Near T c , δ χ can be expanded in powers of ∆, with the lowest-order term proportional to ∆ 2 .This term is diagrammatically shown in Fig. 1 and in the limit of zero frequency and momentum has exactly the form of Eq. ( 3).As a result, the term in Eq. ( 8) proportional to Tr( V δ χ) 2 gives rise to a quartic correction δF ∆ to the free energy of the superconductor and shifts the energetic balance towards the nematic state, in agreement with Eq. ( 4).As we discuss in Appendix A, the effect of the term proportional to Tr( V δ χ) is less important and can be neglected as the strength of fluctuations increases.
Remarkably, we find that δF ∆ always favors nematic SC, irrespective of the precise form of V (Ω, q) or ξ k , provided CDW fluctuations are sufficiently strong.This result solely relies on the form of δ χ, and is a direct generalization of Eq. ( 4) for the case when CDW modes with nonzero Ω and q are taken into account.Specifically, for the model described by Eqs. ( 5) and ( 6), the leading contribution to the fourth-order free energy feedback correction δF (4) ∆ reads as with and the functions K 1 and K 2 are defined as Here ξ 1,2k are the dispersions of the hot spot fermions, see Fig. 2, which are related by the mirror symmetry and Ω m = 2πT m are fermionic and bosonic Matsubara frequencies, respectively, and we have suppressed Ω m in (10) for brevity.Since V (Ω, q) in ( 10) is the CDW propagator within the hot spot model it is peaked at q = 0; V (Ω, q) is related to the (effective) propagator of the full model Ṽ as V (Ω, q) ≡ Ṽ (Ω, Q 1 + q), where Q 1 connects hot spots 1 and 2, see Fig. 2. Our claim that the feedback effect of CDW fluctuations always favors NSC now follows from Eq. ( 9): It is easily demonstrated that the coefficient of |d 2 1 + d 2 2 | 2 is always positive, i.e., Y 1 + 2Y 2 + 2Y 3 − Y 4 > 0, thus proving our statement.Importantly, Eq. ( 9) does not require any assumptions about the explicit form of V (Ω, q) or ξ ik , and only relies on the (rotation and mirror) symmetries relating the hot spots.Furthermore, when V (Ω, q) is strongly peaked at Ω = q = 0, we exactly recover Eqs. ( 3)-( 4) with The overall prefactor in Eq. ( 4) is proportional to q V 2 (0, q), implying that the feedback contribution becomes more significant as the strength of CDW fluctuations grows.As such, Eq. ( 9) in combination with (4) represents the central result of this paper.
Finally, we consider a specific model for twisted bilayer graphene to exemplify our results.We assume that the effective normal-state CDW propagator is given by V (q) = χ 0 /c 2 (q 2 0 + q 2 ).We use the Fermi surface reproduced from the band structure calculation for TBG with filling factor close to the filling of two electrons/holes per supercell 64 , see Fig. 2(a).The dispersion near the hot spots can be approximated as ξ ik = v(k • n i ), where n i is the unit vector in the direction ΓM i .The feedback correction to free energy then equals (13) As the strength of the fluctuations increases, i.e., as q 0 becomes smaller, this correction becomes more significant, eventually exceeding the weak-coupling contribution and leading to NSC.We emphasize that while the numerical prefactors in (13) depend on the particular model chosen, the results given by Eqs. ( 9)-( 11) were derived without specifying the normal-state dispersion ξ ik and normal-state CDW propagator V (Ω, q), and thus apply very generally.In particular, it can be used in the case when the Fermi energy is close to Van Hove singularity, and the hot spot dispersion (in proper coordinates) is given by ξ k = Ak 2 x − Bk 2 y with some constants A, B > 0. In the latter case, we reach the same qualitative conclusion that sufficiently strong density wave fluctuations stabilize NSC (see Appendix A 6 for details).
The analysis for the case of strong SDW fluctuations in a d-wave superconductor is similar.The important difference, however, is the relative minus sign between two diagrams in Fig. 1.This leads to the destructive interference for the coherence factor in the expression for δ χ65 .It is straightforward to show that all results for SDW fluctuations, apart from a possible overall numerical prefactor, can be obtained from the CDW case simply by changing K 2 (q, Ω) → −K 2 (q, Ω), which leads to the partial (but not complete) suppression of the feedback contribution to free energy (see Appendix B).
V. CONCLUSION
In conclusion, by going beyond weak-coupling and including fluctuations of a density wave order we have presented a general mechanism for nematic multicomponent superconductivity in 2D.The theory we develop can be directly applied to twisted bilayer graphene, where density wave fluctuations are strong due to Fermi surface nesting and intertwined with d/p-wave superconductivity 36 .Together with the recent observation of the upper critical magnetic field anisotropy in twisted bilayer graphene 2 , this serves as a main experimental motivation for our work.
As mentioned in the introduction, in our theory the onset of nematicity is tied to pairing.Since nematicity appears as the composite order parameter of Eq. ( 2), we expect that the two transitions can be separated, i.e., the nematic transition can occur at a higher temperature than the superconducting transition, giving rise to a vestigial nonsuperconducting phase with broken rotation symmetry 23,55,66 .To assess the possibility of a vestigial nematic phase one must go beyond the theory developed here and consider the fluctuations of superconducting order parameter.We leave this as a direction for the future.
Finally, we notice that, alternatively to the stronglycorrelated scenario considered in this paper, NSC may, in principle, originate from the internal strain.Indeed, symmetry allows the coupling between the nematic subsidiary order (2) and the components of the strain tensor u ij of the form 20 It is clear from this expression that in the presence of uniaxial strain, u 2 xx − u 2 yy = 0, one of the superconducting components develops order at higher temperature than the other one, thus resulting in nematic superconductivity.If strain is not too strong, the effect of nematicity becomes weaker as one lowers the temperature 20 , and eventually NSC transits into a chiral superconductor at sufficiently small temperature.To distinguish between the scenarios for NSC from density wave fluctuations and from internal strain, as well as from the strain-induced nematicity in s-wave superconductor, a detailed study of the upper critical field behavior in different regimes is required.
VI. ACKNOWLEDGMENTS
We greatly acknowledge the discussions with Pablo Jarillo-Herrero, Yuan Cao, Daniel Rodan-Legrain, and Oriol Rubies-Bigorda, who shared with us their unpublished experimental data.We also thank Rafael Fernandes, Jonathan Ruhman, and Max Metlitski for numerous valuable discussions.This work is supported by DOE Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award DE-SC0018945.LF is partly supported by the David and Lucile Packard Foundation.J.W.F.V. was supported by the National Science Foundation MRSEC Program, under Grant No. DMR-1720530.In this appendix, we present a detailed calculation of the feedback correction to the free energy of a two-component superconductor due to the presence of charge density wave (CDW) fluctuations.For definiteness, we focus on a d-wave superconductor.First, we derive the effective imaginary time action that describes the interplay between pairing potential and the density wave fluctuations.Next, we calculate the very general expression for the feedback contribution to free energy.Finally, we apply our results to particular models which are relevant to twisted bilayer graphene.
The model description and the effective action
As was discussed in the main text, the presence of density wave fluctuations allows us to focus on the vicinities of the points on the Fermi surface connected by the density wave ordering wave vectors, so-called hot spots.The corresponding regions in momentum space near hot spots are called patches.We consider a 2D model with six nonequivalent hot spots in the Brillouin zone, with the CDW wavevectors connecting adjacent hot spots, see Fig. 3.Then, Hamiltonian ( 5)-( 6) of the main text in the low-energy limit translates into the imaginary time action for six patches The first term describes the noninteracting electrons near hot spots: Here, ω n = 2πT (n + 1/2) are fermionic Matsubara frequencies, index i = 1, .., 6 numerates hot spots, ξ i (k) is a dispersion near the i-th hot spot, and the summation over repeated spin indices α =↑, ↓ is implied.We assume the presence of sixfold rotational symmetry in the system, which implies that the dispersions near adjacent hot spots are related as is the π/3 rotation matrix.This results in an additional relation that we will actively use, ξ i (−k) = ξ i+3 (k), where the hot spot index i is defined mod 6.Finally, we assume that different hot spots with indices i and j are related by a mirror symmetry M ij , which leads to another useful equality ξ i (k) = ξ j (M ij k).
The second term in Eq. (A1) is a quadratic action for CDW fluctuations: where Ω m = 2πT m are bosonic Matsubara frequencies and index i = 1, .., 6 numerates CDW fluctuations with different ordering wavevectors.The fields φ i (q) should be viewed as 'shifted' with respect to the global CDW field φ(q) introduced in Eqs. ( 5)-( 6) of the main text, i.e., φ i (q) ≡ φ(Q i + q), where CDW wavevectors Q i connect hot spots with indices i and i + 1 as shown in Fig. 3(a).We assume that the relevant bosonic momenta are much smaller than the distance between the hot spots, q Q.Even though the propagator for the global field φ(q), Ṽ0 (q), is peaked at Q i , the propagators of fields φ i (q) are apparently peaked at q = 0, so V 0i (q) can be thought of as, e.g., Lorentzians with the maximum at q = 0.The particular form of V 0i (q), however, is not important for us at this moment.Finally, since the global field φ(r) is real, the different 'shifted' components φ i (Ω, q) are not independent, but related according to φ i+3 (−Ω, −q) = [φ i (Ω, q)] * .The third term in Eq. (A1) describes the two-component d-wave pairing, where ε αβ is the Levi-Civita tensor in spin space, and the pairing matrix ∆ is given by Here (d 1 , d 2 ) plays the role of a two-component superconducting order parameter, and, again, summation over repeated spin indices α, β =↑, ↓ and patch indices i, j = 1, .., 6 is implied.Finally, the coupling between electrons and CDW fluctuations is described by the last term in Eq. (A1): with Σ(q, Ω) defined as To proceed, we introduce Nambu particle-hole space according to Then, using the identity ξ i (−k) = ξ i+3 (k), the action (A1) can be conveniently rewritten as with Here, Σ is given by Eq. (A7), τ i are Pauli matrices in Nambu space, and ∆ equals 1 0 0 0 0 0 0 −1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 −1 0 0 0 0 0 0 0 We note that ∆ has matrix structure different from ∆, Eq. (A5), because Nambu space introduced in Eq. (A8) mixes different patch indices.Combining Eqs.(A3) and (A9), action (A1) takes simple form where G 0 = diag{G 0i } patches , and Tr implies trace over spin, Nambu, and patch indices, as well as summation over momenta and frequencies.
To derive the effective theory that describes interplay between CDW fluctuations φ i and SC order parameter d i , we integrate out fermions: Neglecting the normal-state electronic part of the partition function, Det −T G −1 0 1/2 (which does not depend on φ or ∆), we find that CDW fluctuations in the presence of pairing potential are described by the effective partition function Z eff given by From this expression, we can extract the effective action: All transformations so far have been exact.Now we make some assumptions that allow us to proceed with our calculation.First, we consider the vicinity of the superconducting transition temperature T c , so we expand the effective action in powers of small pairing potential ∆ (equivalently, in powers of Σ ∆ ) up to fourth order.Second, we assume that CDW fluctuations, though strong, remain massive.Hence, we expand the effective action up to second order in φ (equivalently, in Σ φ ).
Expanding the logarithm in Eq. (A15), we find Here, S ∆ is a weak-coupling part of Ginzburg-Landau free energy of a superconductor.Terms S φ and δS φ are bare bosonic propagator, Eq. (A3), and the normal-state CDW susceptibility, respectively.Finally, S φ−∆ describes the interplay between CDW fluctuations and superconductivity, which eventually results in the feedback correction to free energy.The different terms in S φ−∆ are shown diagrammatically in Fig. 4 and will be explicitly calculated below.
Weak-coupling analysis
In this section, we calculate the fourth-order weak-coupling contribution to free energy.The corresponding part of the effective action is given by where ξ ik is a dispersion near any of the hot spots, and no summation over i is needed here.The weak-coupling free energy then equals This result is a 'hot spots' version of Eq. ( 7) of the main text.We see that, since X 0 > 0, the weak-coupling approximation favors the fully gapped chiral state, (d 1 , d 2 ) ∼ (1, i).
Coupling between pairing potential and CDW fluctuations
In this section, we present the general expressions for the terms that describe the interplay between CDW fluctuations φ i and SC order parameter d i , see Eq. (A16).After straightforward calculation, we find: where, again, R 6 is a six-fold rotation matrix, M ≡ M 12 is a mirror symmetry operation between hot spots 1 and 2, and we used the equalities ξ i (k) = ξ i+1 (R 6 k) and ξ 1 (k) = ξ 2 (M k) in our derivation.Functions K 1 (Ω, q), .., K 5 (Ω, q) are defined as hyperlattice constant is absorbed into the definition of t.While we consider sufficiently strong CDW fluctuations, we stay away from the immediate vicinity of the transition into the CDW-ordered state.Hence, we assume that the CDW propagator is given by where, again, k max is an effective ultraviolet momentum cutoff.The relevant bosonic momenta and frequencies then satisfy q T /t and Ω ∼ T , respectively.In this range of frequencies and momenta, the asymptotic behavior of functions K i (Ω, q) defined in Eq. (A20) is given by The main contribution to K i comes from the 'nesting' direction, k y ≈ 0. Frequency-dependent functions f i (Ω) are given by where, again, ω n = 2πT (n+1/2), Ω m = 2πT m, ζ(x) is the Riemann zeta function, and ψ 0 (x) is the digamma function.
As a consequence of a different structure of Σ φ in Nambu space, the expressions for Tr[(G 0 Σ ∆ G 0 Σ φ ) 2 ] and Tr[(G 0 Σ ∆ ) 2 (G 0 Σ ∆ G 0 Σ φ ) 2 ] in Eqs.(A19b) and (A19d) contain an overall extra minus sign compared to the case of CDW fluctuations, which can be absorbed by redefining K 2 → −K 2 and K 4 → −K 4 .This leads, in particular, to an extra minus sign in front of terms in free energy containing X 2 , X 4 , and Y 3 .More explicitly, the feedback corrections in case of SDW fluctuations are given by δF where, again, X i ≡ Ω,q V (Ω, q)K i (Ω, q), and K i , Y i are defined in Eqs.(A20) and (A27).
Similarly to the case of CDW fluctuations, the contribution δF ∆ always favors the nematic superconducting state, hence, the main result of our paper remains valid for SDW fluctuations as well.The absolute value of this correction, however, is smaller because of a minus sign in front of Y 3 .The quartic term in δF (2) ∆ , on the other hand, changes its sign within the specific models for twisted bilayer graphene we considered in Appendices A 5 and A 6.This happens because of an additional minus sign in front of X 4 .Hence, δF ∆ favors chiral state in case of SDW fluctuations.At sufficiently strong fluctuations, however, the fourth-order term in δF (2) ∆ is parametrically smaller than δF (4) ∆ and thus can be neglected again, justifying our conclusion about the stability of nematic superconductivity.
Appendix A: Nematic d-wave superconductivity in presence of charge density wave fluctuations
FIG. 3 .
FIG. 3. (a) Six inequivalent hot spots in the Brillouin zone are connected by CDW wavevectors Qi.All Qi connect hot spots with numbers i and i + 1, and can be obtained from Q1 by sixfold rotations.(b) Fermi surface in twisted bilayer graphene above the Van Hove singularity.(c) Fermi surface of twisted bilayer graphene at Van Hove energy.This scenario is realized when the filling factor is close to n = 2 electrons/holes per supercell.Blue and red parts of the Fermi surface originate from different valleys.
FIG. 4 .
FIG. 4. Diagrams describing the coupling between pairing potential ∆(d1, d2) and density wave fluctuations φi.Diagrams (a)-(b) schematically represent the coupling φ 2 d 2 and describe the ∆ 2 correction to CDW susceptibility, while diagrams (c)-(e) correspond to the coupling φ 2 d 4 contributing the ∆ 4 correction to CDW susceptibility.The contribution to the feedback free energy from diagrams (a)-(b) becomes dominant when fluctuations become sufficiently strong. | 2019-04-08T20:48:59.000Z | 2018-10-09T00:00:00.000 | {
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255839836 | pes2o/s2orc | v3-fos-license | Hypermethylation of the GATA binding protein 4 (GATA4) promoter in Chinese pediatric acute myeloid leukemia
Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature of AML. GATA4 has been suggested to be a tumor suppressor gene regulated by promoter hypermethylation in various types of human cancers although the expression and promoter methylation of GATA4 in pediatric AML is still unclear. Transcriptional expression levels of GATA4 were evaluated by semi-quantitative and real-time PCR. Methylation status was investigated by methylation-specific PCR (MSP) and bisulfate genomic sequencing (BGS). The prognostic significance of GATA4 expression and promoter methylation was assessed in 105 cases of Chinese pediatric acute myeloid leukemia patients with clinical follow-up records. MSP and BGS analysis showed that the GATA4 gene promoter is hypermethylated in AML cells, such as the HL-60 and MV4-11 human myeloid leukemia cell lines. 5-Aza treatment significantly upregulated GATA4 expression in HL-60 and MV4-11 cells. Aberrant methylation of GATA4 was observed in 15.0 % (3/20) of the normal bone marrow control samples compared to 56.2 % (59/105) of the pediatric AML samples. GATA4 transcript levels were significantly decreased in AML patients (33.06 ± 70.94; P = 0.011) compared to normal bone marrow/idiopathic thrombocytopenic purpura controls (116.76 ± 105.39). GATA4 promoter methylation was correlated with patient leukocyte counts (WBC, white blood cells) (P = 0.035) and minimal residual disease MRD (P = 0.031). Kaplan-Meier survival analysis revealed significantly shorter overall survival time in patients with GATA4 promoter methylation (P = 0.014). Epigenetic inactivation of GATA4 by promoter hypermethylation was observed in both AML cell lines and pediatric AML samples; our study implicates GATA4 as a putative tumor suppressor gene in pediatric AML. In addition, our findings imply that GATA4 promoter methylation is correlated with WBC and MRD. Kaplan-Meier survival analysis revealed significantly shorter overall survival in pediatric AML with GATA4 promoter methylation but multivariate analysis shows that it is not an independent factor. However, further research focusing on the mechanism of GATA4 in pediatric leukemia is required.
Background
Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells, which lose the ability to differentiate normally and to respond to normal regulators of proliferation [1]. Pediatric AML comprises up to 20 % of all childhood leukemias. Epigenetic disturbances have been implicated in the development and pathogenesis of leukemia [2]. These include aberrations in methylation, which is a key epigenetic event responsible for enhanced proliferation and self-renewal, differentiation arrest, and impaired apoptosis of leukemic cells [3]. Several studies have evaluated genome-wide methylation in acute myeloid leukemia [4]. In AML, the presence of common methylation patterns in a few genes such as p15 and E-cadherin has been described independently by several groups across large patient cohorts [5,6]. Progression from myelodysplastic syndrome to AML has also been associated with increased aberrant DNA methylation [7]. Identifying these aberrantly methylated genes may provide a better understanding of AML, thereby paving the way for the development of novel tumor markers and therapeutic targets.
In vertebrates, the existence of a covalent modification of the base cytosine in the context of CpG dinucleotides by addition of a methyl group to C-5 has been appreciated since the mid-70s [8]. The promoter regions of approximately 50 % of human genes contain regions of DNA with a cytosine and guanine content greater than expected (so-called CpG islands) that, once hypermethylated, mediate transcriptional silencing. The human genome consists of approximately 28 million CpGs, of which 60-80 % are normally 5-C methylated [9]. Approximately 10 % of CpGs occur in the context of CpG islands: CpG-rich regions which are on average 1 kilobase in size [9]. The following distinct roles in genomic methylation have been reported for DNMT isoforms: DNMT1 preferentially replicates already existing methylation patterns; DNMT3A and 3B are responsible for establishing de novo methylation. Abnormal expression of these methylation-related enzymes may disturb DNA methylation in pediatric AML. In cancer, aberrantly occurring DNA hypermethylation of these CpG islands, especially in tumor suppressor genes, is a well-established phenomenon, which occurs alongside a global loss of methylation, which in turn is associated with genomic instability [10,11]. A common approach to the study of DNA methylation is to treat cells with 5-aza-2'-deoxycytidine (5-Aza) demethylation reagent. This epigenetic modifier inhibits DNA methyltransferase activity, resulting in DNA demethylation (hypomethylation); as such, treatment with 5-Aza can identify the genes that are inactivated by methylation.
Transcription factors of the GATA family are essential regulators of the specification and differentiation of numerous tissues. GATA factors typically bind to the element A/T GATA A/G to coordinate cellular maturation with proliferation arrest and cell survival. GATA4 is a member of the GATA family of zinc finger transcription factor, which regulates gene transcription by binding to GATA elements [12]. GATA4 was originally discovered as a regulator of cardiac development and subsequently identified as a major regulator of adult cardiac hypertrophy. GATA4 works in combination with other essential cardiac transcription factors as well, such as Nkx2-5 and Tbx5 [13]. GATA4 is expressed in both embryo and adult cardiomyocytes where it functions as a transcriptional regulator for many cardiac genes, and also regulates hypertrophic growth of the heart [14]. Mutations or defects in the GATA4 gene can lead to a variety of cardiac problems including congenital heart disease, abnormal ventral folding, and defects in the cardiac septum separating the atria and ventricles, and hypoplasia of the ventricular myocardium [15]. In addition to the heart, GATA4 plays important roles in the reproductive system, gastrointestinal system, respiratory system and cancer [16].
Numerous studies gave implicated GATA4 as a tumor suppressor gene involved in tumorigenesis in various types of human cancers. A previous investigation of the methylation status of GATA4 promoters by methylation-specific PCR in 99 glioblastoma patients showed that GATA4 was aberrantly methylated in 23.2 % of glioblastoma tumors, but not in normal brain [17]. In endometrioid carcinoma, GATA4 promoter methylation was detected in 81.5 % (44/54) of the carcinoma group and in none of the control group [18]. In ovarian cancer, methylation-specific PCR revealed GATA4 promoter methylation in 31.3 % (21/67) of specimens with ovarian cancer, and in none of the control ovarian tissue samples [19]. Furthermore, methylation of GATA4 is significantly higher in the ovarian cancer group compared with the control group [20]. Methylation of GATA4 was found in human gastric mucosa samples, including normal gastric biopsies, gastric dysplasia (low-grade gastric intraepithelial neoplasia) and paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues.GATA4 methylation was frequently observed in SGCs (53.8 %) by MSP. Moreover, a high frequency of GATA-4 methylation was found in both gastric low-grade GIN (57.1 %) and indefinite for dysplasia (42.9 %). However,GATA4 methylation was detected only in 4/32 (12.5 %) of normal gastric biopsies. Epigenetic inactivation of GATA4 by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection [21]. Promoter methylation of GATA4 was analyzed in colorectal tissue and fecal DNA from colorectal cancer patients and healthy controls using methylation-specific PCR. GATA4 methylation was observed in 70 % (63/90) of colorectal carcinomas and was independent of clinicopathologic features [22]. In glioblastoma multiforme (GBM), loss of GATA4 was observed in 58 % (94/163) of GBM operative samples and was found to be a negative survival prognostic marker [23]. Furthermore,GATA4 promoter methylation was detected in 67 % (42/63) of primary lung cancers [24]. In diffuse large B-cell lymphoma (DLBCL) GATA4 showed significant methylation in over 85 % of tumors [25].
Currently, the expression of GATA4 and the methylation status of its promoter in pediatric acute myeloid leukemia have not been reported. In this study, we have provided the first evidence of GATA4 methylation in two AML cell lines and pediatric myeloid leukemia samples. These data suggest that GATA4 may function as a tumor suppressor in pediatric acute myeloid leukemia.
Patients and samples
Bone marrow specimens were obtained at the time of diagnosis during routine clinical assessment of 105 pediatric patients with AML, who presented at the Department of Hematology and Oncology, Children's Hospital of Soochow University between 2006 and 2011. Research involving human subjects, human material, or human data, have been performed in accordance with the Declaration of Helsinki. Ethical approval was provided by the Children's Hospital of Soochow University Ethics Committee (No.SUEC2006-011 and No.SUEC2000-021), and informed consent was obtained from the parents or guardians. AML diagnosis was made in accordance with the revised French-American-British (FAB) classification. Additionally, bone marrow samples from 12 healthy donors and 8 patients with Idiopathic thrombocytopenic purpura (ITP) were analyzed as controls. Bone marrow mononuclear cells (BMNCs) were isolated using Ficoll solution within 2 h after bone marrow samples harvested and immediately subjected for the extraction of total RNA and genomic DNA.
CD34 + cell purification
For CD34 + cell selection, the Miltenyi immunoaffinity device (VarioMACS 130-046-703) was used according to the manufacturer's instructions (Miltenyi Biotech, Auburn, CA). Briefly, the CD34 + cells are magnetically labeled with CD34 MicroBeads. Then, the cell suspension is loaded onto a MACSR Column which is placed in the magnetic field of a MACS Separator. The magnetically labeled CD34 + cells are retained within the column. The unlabeled cells run through; this cell fraction is thus depleted of CD34 + cells. After removing the column from the magnetic field, the magnetically retained CD34 + cells can be eluted as the positively selected cell fraction.
Analysis of promoter methylation in pediatric AML by NimbleGen Human DNA Methylation arrays
Analysis of the methylation status of genes in five pediatric AML samples (M1, M2, M3, M4 and M5) and three NBM samples (N1, N2, and N3) using NimbleGen Human DNA Methylation arrays. NimbleGen Human DNA Methylation arrays Protocol: Step 1, Genomic DNA Extraction and Fragmentation, Genomic DNA (gDNA) was extracted from 8 samples using a DNeasy Blood & Tissue Kit (Qiagen, Fremont, CA). The purified gDNA was then quantified and quality assessed by nanodrop ND-1000. Step 2, Immunoprecipitation, 1 μg of sonicated genomic DNA was used for immunoprecipitation using a mouse monoclonal anti-5-methylcytosine antibody (Diagenode). For this, DNA was heatdenatured at 94°C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4°C with rocking agitation in 400 μL immunoprecipitation buffer (0.5 % BSA in PBS). To recover the immunoprecipitated DNA fragments, 200 μL of anti-mouse IgG magnetic beads were added and incubated for an additional 2 h at 4°C with agitation. After immunoprecipitation, a total of five immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25 % SDS and 0.25 mg/mL proteinase K for 2 h at 65°C and then allowed to cool down to room temperature. MeDIP DNA were purified using Qiagen MinElute columns (Qiagen).
Step 4, DNA Labelling and Array Hybridization, the purified DNA was quantified using a nanodrop ND-1000. For DNA labelling, the NimbleGen Dual-Color DNA Labeling Kit was used according to the manufacturer's guideline detailed in the NimbleGen MeDIP-chip protocol (Nimblegen Systems, Inc.,
Sodium bisulphite modification of genomic DNA
High-molecular-weight genomic DNA was extracted from cell lines and biopsies by a conventional phenol/ chloroform method. The sodium bisulphite modification procedure was as described with slight modification [26][27][28]. In brief, 600 ng of genomic DNA was denatured in 3 M NaOH for 15 min at 37°C, then mixed with 2 volumes of 2 % low-melting-point agarose. Agarose/DNA mixtures were then pipetted into chilled mineral oil to form agarose beads. Aliquots of 200 μl of 5 M bisulphite solution (2.5 M sodium metabisulphite, 100 mM hydroquinone, both Sigma, USA) were added into each tube containing a single bead. The bisulphite reaction was then carried out by incubating the reaction mixture for 4 h at 50°C in the dark. Treatments were stopped by equilibration against 1 ml of TE buffer, followed by desulphonation in 500 μl of 0.2 M NaOH. Finally, the beads were washed with 1 ml of TE buffer and directly used for PCR.
Each PCR reaction contained 20 ng of sodium bisulphite-modified DNA, 250 pmol of each primer, 250 pmol deoxynucleoside triphosphate, 1 × PCR buffer, and one unit of ExTaq HS polymerase (Takara, Tokyo) in a final reaction volume of 20 μl. Cycling conditions were initial denaturation at 95°C for 3 min, 40 cycles of 94°C for 30 s, 65°C (M) or 63°C (U) for 30 s, and 72°C for 30 s. For each set of methylation-specific PCR reactions, in vitro-methylated genomic DNA treated with sodium bisulphite served as a positive methylation control. PCR products were separated on 4 % agarose gels, stained with ethidium bromide and visualized under UV illumination. For cases with borderline results, PCR analyses were repeated.
Leukemia cell cells treated with 5-aza-2'-deoxycytidine
De-methylation was induced with 5-aza-dC (5-Aza, Sigma-Aldrich, St Louis, MO, USA) treatment at a concentration that induced de-methylation of the DNA without killing the cells. Culture media for HL-60 and MV4-11 cells contained 5 μM 5-Aza. DNA and RNA were extracted after 72 h of 5-Aza treatment for the following analysis.
Quantitative reverse-transcription PCR for GATA4
Quantitative real-time PCR was performed to determine the expression levels of GATA4 genes. Total RNA was reverse transcribed using the Reverse Transcription Kit, according to the manufacturer's protocol (Applied Biosystems Inc., Foster City, CA). The real time PCR primers used to quantify GAPDH expression were: F: 5′-AGAAGGCTGGGGCTCATTTG-3′ and R: 5′-AGG GGCCATCCACAGTCTTC-3′ and for GATA4 were: F: (See figure on previous page.) Fig. 1 Promoter methylation analysis of pediatric AML with NimbleGen Human DNA Methylation Arrays. a Analysis of the methylation status of genes in four pediatric AML samples (M1, M2, M3, M4 and M5) and three NBM samples (N1, N2, and N3) using NimbleGen Human DNA Methylation Arrays. Each red box represents the number of methylation peaks (PeakScore) overlapping the promoter region for the corresponding miRNA. The PeakScore is defined as the average -log10 (P-value) from probes within the peak. The scores reflect the probability of positive methylation enrichment. b DNA methylation array analysis showing significant methylation of the GATA4 promoter in AML samples (4/5), and unmethylated in NBM samples (0/3) 5′-TAGCCCCACAGTTGACACAC-3′ and R: 5′-GTCCTGCACAGCCTGCC −3′. Real-time PCR analysis was according to the MIQE Guidelines and performed in a total volume of 20 μl including 1 μl of cDNA, primers (0.2 mM each) and 10 μl of SYBR Green mix (Roche). Reactions were run on an Lightcycler 480 (Roche) using universal thermal cycling parameters (95°C for 5 min, 45 cycles of 10 s at 95°C, 20 s at 60°C and 15 s at 72°C; followed by a melting curve: 10 s at 95°C, 60 s at 60°C and continued melting). The results were obtained using the sequence detection software of the Lightcycler 480 and analyzed using Microsoft Excel. For quality control purposes, melting curves were acquired for all samples. The comparative Ct method was used to quantify gene expression. The target gene expression level was normalized to expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) within the same sample (−⊿Ct), the relative expression of GATA4 was calculated with 10 6 *Log2(-⊿Ct ).
Western blot analysis
Western blot analysis was introduced before [29]. Cellular proteins were extracted in 40 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and 1 % (v/v) Triton X-100, supplemented with protease inhibitors. Equal amounts of protein were resolved on 12 % SDS-PAGE gels, and then transferred to a PVDF membrane (Millipore, Bedford, MA). Blots were blocked and then probed with Polyclonal Goat IgG antibodies against GATA4 (1:1000, R&D. Minneapolis, MN) and GAPDH (1:5000, Sigma, St. Louis, MO). After three times' washing, blots were then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies and visualized by enhanced chemiluminescence kit (Pierce, Rockford, IL). Protein bands were visualized after exposure of the membrane to Kodak X-ray film.
Statistical analysis SPSS v11.5 (SPSS Inc., Chicago, IL) was used for statistical analysis. Data are presented as means ± standard deviation. Group t-test was used to compare the expression of GATA4 between DMSO group and 5-Aza group. Statistical significance between methylated sample data and clinical pathological features of AML patients were analyzed by Pearson chi-square test or Fisher's exact test. Statistical significance of GATA4 expression among NBM and pediatric AML groups was determined using one-way ANOVA. A p <0.05 was considered statistically significant.
Results and discussion
The GATA4 promoter is hypermethylated in AML cells The correlation between aberrant methylation and downregulation of GATA4 has been extensively documented in numerous cancers and cell lines; these are discussed in the Background. However, the methylation status of GATA4 in the blood system, particular in pediatric AML, has not been reported to date. Our analyses of promoter methylation in pediatric AML, using NimbleGen Human DNA Methylation 385 K Promoter plus CpG Island arrays, indicated that the GATA4 promoter is hypermethylated in AML (Fig. 1a). The GATA4 promoter was hypermethylated in 80 % (4/5) of pediatric AML samples and 0 % (0/3) of normal bone marrow samples (Additional file 1) .
Subsequent analyses of the GATA4 promoter sequence identified four CpG islands (Fig. 2a). Methylationspecific PCR (MSP) assays were performed to detect the methylation status of the GATA4 promoter in 11 leukemia cell lines. The MSP primers were designed using MethPrimer (http://www.urogene.org/cgi-bin/ methprimer/methprimer.cgi ) to encompass the CpG islands of the GATA4 promoter identified in Fig. 2a. Our results showed that the GATA4 promoter was hypermethylated in five leukemia cell lines, especially in SHI-1, HL-60, MV4-11,U937 and K562 cells); and unmethylated in the other cell lines (Fig. 2b). The results of RT-PCR analysis of the expression of GATA4 is presented in Fig. 2b; GATA4 expression was detected in only three cell lines (THP-1, Raji and U937), indicating that downregulation of GATA4 in AML cells is a common phenomenon. Figure 2c showed that expression of GATA4 in leukemia cell lines is significantly lower than NBM. 6/9 NBM samples with obvious expression of GATA4 and in leukemia cell lines GATA4 only can be detected in THP-1 and Raji cells. To confirm methylation of the GATA4 promoter, we treated the leukemia cell lines with the demethylation reagent 5-Aza. Our results showed that 5-Aza treatment significantly upregulated GATA4 expression. As shown in Fig. 2d which also showed a change in the methylation status of the GATA4 promoter after 5-Aza treatment. In summary, these results showed that the GATA4 promoter was consistently and significantly methylated in the HL-60, MV4-11, SHI-1, U937 and K562 human myeloid leukemia cell lines. Based on these findings, we hypothesized that the promoter of GATA4 is methylated in pediatric AML patients.
The GATA4 promoter is methylated in pediatric AML patients
We next examined the GATA4 promoter methylation status in pediatric AML samples and NBM/ITP (normal bone marrow/idiopathicthrombocytopenic purpura) control samples. Aberrant GATA4 promoter methylation was observed in 15.0 % (3/20) of the NBM control samples compared to 56.2 % (59/105) of the pediatric AML samples (Fig. 3a). Three NBM samples and three AML samples were further analyzed by BSG (Fig. 3b). The results showed that the CpG islands in the GATA4 promoter were methylated in the AML samples (69.4, 58.8, and 62.4 % in AML7#, AML10#, and AML11#, respectively). In contrast, the CpG islands of the GATA4 promoter in the NBM samples were unmethylated (24.7, 14.1, and 10.6 % in NBM4#, NBM5#, and NBM9#, respectively). These results were supported by MSP assays.
Expression of GATA4 is downregulated with promoter methylation in Chinese pediatric acute myeloid leukemia
The transcript levels of GATA4 were examined in 105 pediatric AML patients by real-time PCR (Fig. 3c). As shown in Fig. 3d, GATA4 expression was significantly decreased in 105 AML patients (33.06 ± 70.94; P = 0.011) compared to that in 20 NBM/ITP controls (116.76 ± 105.39). Figure 3d shows that patients with GATA4 promoter methylation (16.02 ± 17.59, n = 59) exhibited lower GATA4 transcript levels compared to those without GATA4 promoter methylation (54.92 ± 101.80, P < 0.01; n = 46). Furthermore, AML patients with and without GATA4 promoter methylation showed significantly lower GATA4 transcript levels compared to those in controls.
The prognostic significance of GATA4 expression was assessed in 105 cases of Chinese pediatric acute myeloid leukemia patients with clinical follow-up records. There was no significant association with GATA4 expression and patient age, sex, FAB (French-American-British classification) or cytogenetics (Table 1). Kaplan-Meier survival analysis of 105 pediatric acute myeloid leukemia patients revealed almost identical survival times for patients with GATA4 high or low expressing tumors (P = 0.769, Table 3 and Fig. 4b). Furthermore, multivariate analysis revealed that GATA4 expression was not an independent prognostic factor in pediatric AML (P = 0.096, Table 4).
(See figure on previous page.) Fig. 3 GATA4 is inactivated by promoter hypermethylation in pediatric AML. a MSP analysis of the methylation status of GATA4 shows aberrant methylation in pediatric AML samples compared to NBM/ITP control samples. Aberrant methylation of GATA4 was observed in 15.0 % (3/20) of the NBM control samples compared to 56.2 % (59/105) of the pediatric AML samples. b Three NBM samples and three AML samples were analyzed by BSG. The results showed that the CpG islands in the GATA4 promoter were methylated in the AML samples (69.4, 58.8, and 62.4 % in AML7#, AML10#, and AML11#, respectively). In contrast, the CpG islands of the GATA4 promoter in the NBM samples were unmethylated (24.7, 14.1, and 10.6 % in NBM4#, NBM5#, and NBM9#, respectively). c The transcript levels of GATA4 were examined in 105 pediatric AML patients by real-time PCR. d GATA4 expression was significantly decreased in 105 AML patients (33.06 ± 70.94; P =0.011) compared to 20 NBM/ITP controls (116.76 ± 105.39); AML patients with GATA4 promoter methylation (16.02 ± 17.59, n = 59) showed lower GATA4 transcript levels compared to those without GATA4 promoter methylation (54.92 ± 101.80, P <0.001; n = 46) GATA4 promoter methylation correlates with poor survival in Chinese pediatric acute myeloid leukemia The prognostic significance of GATA4 promoter methylation was also assessed in 105 cases of Chinese pediatric acute myeloid leukemia patients with clinical follow-up records. Table 2 shows GATA4 promoter methylation was correlated with leukocyte counts (P = 0.035) and MRD (P = 0.031). Table 2 also shows there were no significant differences in clinical features, such as sex, age, FAB or cytogenetics between patients with and without GATA4 promoter methylation. Kaplan-Meier survival analysis revealed significantly shorter overall survival times in patients with GATA4 promoter methylation (P = 0.014, Table 3 and Fig. 4a). Furthermore, multivariate analysis revealed that GATA4 promoter methylation was not an independent prognostic factor in pediatric AML (P = 0.170, Table 4). In summary, our results showed firstly that the GATA4 promoter was consistently significantly methylated in leukemia cells, such as HL-60, MV4-11, SHI-1, U937, and K562 human myeloid leukemia cell lines; the expression of GATA4 was significantly lower in pediatric AML compared to NBM control samples, patients with methylated GATA4 showed lower GATA4 transcript levels compared to those without methylated; GATA4 promoter methylation was correlated with leukocyte and MRD, Kaplan-Meier survival analysis revealed a significantly shorter overall survival times in pediatric AML with GATA4 promoter methylation.
In this study, promoter methylation in Chinese pediatric AML was analyzed using NimbleGen Human DNA Methylation 385 K Promoter plus CpG Island arrays. This approach revealed significant differences in the methylation status of genes between pediatric AML and normal bone marrow samples. Previous studies have demonstrated that promoters of TFPI-2 [30] and miR-663 [31,32] were hypermethylated in Chinese pediatric acute myeloid leukemia. Our results showed significantly greater GATA4 promoter hypermethylation in pediatric AML samples and 0/3 (0 %) in normal bone marrow samples. indicating that the GATA4 promoter is hypermethylated in AML.
GATA4 was suggested to be a tumor suppressor gene with promoter hypemethylation in various types of human cancers. The GATA4 promoter is methylated in glioblastoma [17], endometrioid carcinoma [18], ovarian cancer [19], gastric mucosa [21], colorectal carcinomas [22] and lung cancers [24]. To our knowledge, this is the first report describing the expression of GATA4 and promoter methylation status in pediatric AML. In this study, methylation-specific PCR (MSP) assays showed that the GATA4 promoter was hypermethylated in five leukemia cell lines, especially in SHI-1, HL-60, MV4-11, U937 and K562 cells GATA4 promoter hypermethylation is an important prognostic marker in several tumors. Kaplan-Meier analysis revealed that high methylation levels of the GATA4 promoter were significantly correlated with patient survival in oropharyngeal squamous cell carcinoma (OPSCC) [33]. In high grade serous ovarian carcinoma (HGSOC), GATA4 promoter methylation was associated with disease recurrence [34]. In this study, the prognostic significance of GATA4 promoter methylation was assessed in 105 cases of Chinese pediatric AML patients with clinical follow-up records. GATA4 promoter methylation was correlated with leukocyte counts and MRD. Kaplan-Meier survival analysis revealed significantly shorter overall survival in patients with GATA4 promoter methylation. These observations demonstrate that GATA4 promoter methylation correlates with poorer survival in Chinese pediatric AML.
The molecular function of GATA4 has been studied in certain tumors. Re-expression of GATA4 in human glioblastoma multiforme (GBM) cell lines, primary cultures, and brain tumor-initiating cells suppressed tumor growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21 (CIP1). Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. GATA4 reduced expression of APNG (alkylpurine-DNA-N-glycosylase), a DNA repair enzyme which is poorly characterized in GBM-mediated temozolomide resistance [23]. The potential function of GATA4 as a tumor suppressor was studied by inducing GATA4-overexpression in human colorectal cancer cell lines.GATA4 overexpression suppressed colony formation, proliferation, migration, invasion, and anchorage-independent growth of colorectal cancer cells [22].GATA4 can control expression of the anti-apoptotic factor Bcl-2 and the cell cycle regulator cyclin D2 in normal and neoplastic granulosa cells.-GATA4 expression correlated with Bcl-2 and cyclin | 2023-01-16T14:11:19.942Z | 2015-10-21T00:00:00.000 | {
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